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

Abstract

To provide non-human animals having humanized a Programmed cell death 1 gene.SOLUTION: Non-human animals, and methods and compositions for making and using the same, are provided, where 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). For example, the present invention provides a rodent that expresses a PD-1 polypeptide, where the PD-1 polypeptide comprises a human portion and an endogenous portion.SELECTED DRAWING: None

Description

[Technical Field]

[0001] (CROSS-REFERENCE TO RELATED APPLICATIONS) This application claims the benefit of priority to U.S. Provisional Patent Application Nos. 62 / 014,181, filed June 19, 2014, 62 / 086,518, filed December 2, 2014, and 62 / 138,221, filed March 25, 2015, the entire contents of which are incorporated herein by reference.

[0002] (Incorporated by reference to the sequence listing) The Sequence Listing, in the form of a 23 KB ASCII text file entitled 31969_SEQ.txt, created on June 4, 2015, and submitted to the U.S. Patent and Trademark Office via EFS-Web, is incorporated herein by reference. [Background technology]

[0003] Although significant improvements have been made in cancer immunotherapy, cancer remains a major challenge for the global healthcare industry. This challenge is due in part to the ability of cancer cells to evade immune system surveillance, which in part results in the inhibition and / or downregulation of antitumor immunity. Yet, no in vivo system has been developed to optimally determine the therapeutic potential of novel cancer therapies designed to activate and / or promote antitumor immunity and to determine the molecular aspects of how cancer cells deliver inhibitory signals to immune cells (e.g., T cells). Such a system would provide a source of assays for evaluating the therapeutic efficacy of candidate agents that promote an antitumor environment in vivo. Summary of the Invention [Means for solving the problem]

[0004] The present invention encompasses the recognition that it is desirable to design non-human animals that permit improved systems for identifying and developing new therapeutics that can be used to treat cancer. The present invention also encompasses the recognition that it is desirable to design non-human animals that permit improved systems for identifying and developing new therapeutics that can be used to treat autoimmune (or inflammatory) diseases, disorders, or conditions. Furthermore, the present invention encompasses the recognition that non-human animals that harbor a humanized Pdcd1 gene and / or otherwise express, contain, or produce human or humanized PD-1 polypeptides are desirable for use in, for example, identifying and developing cancer therapeutics that upregulate anti-tumor immunity. In some embodiments, the non-human animals of the present invention provide improved in vivo systems for identifying and developing combination therapies that involve targeting PD-1.

[0005] In some embodiments, the invention provides non-human animals having a genome comprising a Pdcd1 gene that comprises genetic material from two different species (e.g., human and non-human). In some embodiments, the Pdcd1 gene of the non-human animals described herein encodes a PD-1 polypeptide comprising human and non-human portions, wherein the human and non-human portions are linked to each other to form a functional PD-1 polypeptide. In some embodiments, the Pdcd1 gene of the non-human animals described herein encodes a PD-1 polypeptide that comprises all or a portion of the extracellular domain of a human PD-1 polypeptide.

[0006] In some embodiments, the invention provides a non-human animal that expresses a PD-1 polypeptide, where the PD-1 polypeptide comprises a human portion and an endogenous portion. In some embodiments, the PD-1 polypeptide of the invention is translated in the cells of the non-human animal with a non-human signal peptide, which in some embodiments is a rodent signal peptide.

[0007] In some embodiments, the endogenous portion comprises the intracellular portion of an endogenous PD-1 polypeptide. In some embodiments, the endogenous portion further comprises the transmembrane portion of an endogenous PD-1 polypeptide. In some embodiments, 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 the corresponding amino acid sequence of the mouse PD-1 polypeptide found in Figure 8. In some embodiments, the endogenous portion has an amino acid sequence that is substantially identical to the corresponding amino acid sequence of the mouse PD-1 polypeptide found in Figure 8. In some embodiments, the endogenous portion has an amino acid sequence that is identical to the corresponding amino acid sequence of the mouse PD-1 polypeptide found in Figure 8.

[0008] In some embodiments, the human portion comprises amino acids 35-145, 27-145, 27-169, 26-169, or 21-170 of a human PD-1 polypeptide. In some embodiments, 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 the corresponding amino acid sequence of a human PD-1 polypeptide found in Figure 8. In some embodiments, the human portion has an amino acid sequence that is substantially identical to the corresponding amino acid sequence of a human PD-1 polypeptide found in Figure 8. In some embodiments, the human portion has an amino acid sequence that is identical to the corresponding amino acid sequence of a human PD-1 polypeptide found in Figure 8.

[0009] In some embodiments, the PD-1 polypeptide comprising a human portion and an endogenous portion is encoded by an endogenous Pdcd1 gene. In some specific embodiments, the endogenous Pdcd1 gene comprises endogenous Pdcd1 exons 1, 4, and 5. In some specific embodiments, the endogenous Pdcd1 gene further comprises all or a portion of endogenous Pdcd1 exon 3. In some specific embodiments, the endogenous Pdcd1 gene comprises SEQ ID NO:21. In some specific embodiments, the endogenous Pdcd1 gene comprises SEQ ID NO:22. In some specific embodiments, the endogenous Pdcd1 gene comprises SEQ ID NO:21 and SEQ ID NO:22.

[0010] In some embodiments, the PD-1 polypeptide expressed by the non-human animals described herein has an amino acid sequence at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 95% identical to SEQ ID NO: 6. In some embodiments, the PD-1 polypeptide expressed by the non-human animals described herein has an amino acid sequence substantially identical to SEQ ID NO: 6. In some embodiments, the PD-1 polypeptide expressed by the non-human animals described herein has an amino acid sequence identical to SEQ ID NO: 6.

[0011] In some embodiments, the invention provides a humanized Pdcd1 locus comprising one or more exons of a non-human Pdcd1 gene operably linked to all or a portion of one or more exons of the human Pdcd1 gene. In some embodiments, the humanized Pdcd1 locus further comprises 5' and 3' non-human Pdcd1 untranslated regions (UTRs) flanking the one or more exons of the human Pdcd1 gene. In some embodiments, the humanized Pdcd1 locus is under the control of a rodent promoter, and in certain embodiments, is under the control of an endogenous rodent promoter.

[0012] In some embodiments, the humanized Pdcd1 locus comprises non-human Pdcd1 exons 1, 3, 4, and 5 operably linked to human Pdcd1 exon 2. In some embodiments, the humanized Pdcd1 locus comprises non-human Pdcd1 exons 1, 4, and 5, human Pdcd1 exon 2, and further comprises Pdcd1 exon 3, wherein Pdcd1 exon 3 comprises a human portion and a non-human portion, wherein the non-human and human exons are operably linked. In some embodiments, the human portion of Pdcd1 exon 3 comprises nucleotides encoding a PD-1 stalk sequence. In some embodiments, the human portion of Pdcd1 exon 3 comprises approximately 71 bp of human Pdcd1 exon 3. In some embodiments, the non-human portion of Pdcd1 exon 3 comprises nucleotides encoding a transmembrane sequence. In some embodiments, the non-human portion of Pdcd1 exon 3 comprises approximately 91 bp of rodent Pdcd1 exon 3.

[0013] In some embodiments, the invention provides a non-human animal comprising a Pdcd1 gene comprising an endogenous portion and a human portion, wherein the endogenous portion and the human portion are operably linked to a rodent Pdcd1 promoter. In some embodiments, the rodent Pdcd1 promoter is an endogenous rodent Pdcd1 promoter.

[0014] In some embodiments, the endogenous portion comprises endogenous Pdcd1 exons 1, 4, and 5. In some embodiments, the endogenous portion further comprises all or a portion of endogenous Pdcd1 exon 3. In some embodiments, all or a portion of exons 1, 3, 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 all or a portion of exons 1, 3, 4, and 5 of the endogenous Pdcd1 gene seen in Figure 8. In some embodiments, all or a portion of exons 1, 3, 4, and 5 of the endogenous Pdcd1 gene are substantially identical to the corresponding all or a portion of exons 1, 3, 4, and 5 of the endogenous Pdcd1 gene seen in Figure 8. In some embodiments, all or part of exons 1, 3, 4 and 5 of the endogenous Pdcd1 gene are identical to the corresponding all or part of exons 1, 3, 4 and 5 of the endogenous Pdcd1 gene seen in FIG.

[0015] In some embodiments, the human portion encodes amino acids 21-170, 26-169, 27-169, 27-145, or 35-145 of a human PD-1 polypeptide.

[0016] In some embodiments, the human portion comprises exon 2 of the human Pdcd1 gene. In some embodiments, the human portion further comprises all or a portion of human Pdcd1 exon 3. In some embodiments, all or a portion of human Pdcd1 exons 2 and 3 are at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 95% identical to all or a portion of the corresponding exons 2 and 3 of the human Pdcd1 gene seen in Figure 8. In some embodiments, all or a portion of human Pdcd1 exons 2 and 3 are substantially identical to all or a portion of the corresponding exons 2 and 3 of the human Pdcd1 gene seen in Figure 8. In some embodiments, all or a portion of human Pdcd1 exons 2 and 3 are identical to all or a portion of the corresponding exons 2 and 3 of the human Pdcd1 gene seen in Figure 8. In some embodiments, the human portion comprises a sequence that is codon-optimized for expression in a non-human animal, and in some embodiments is codon-optimized for expression in a rodent, in some particular embodiments, in a mouse, and in some particular embodiments, in a rat.

[0017] In some embodiments, the human portion comprises a sequence at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 95% identical to SEQ ID NO: 23. In some embodiments, the human portion comprises a sequence substantially identical to SEQ ID NO: 23. In some embodiments, the human portion comprises a sequence identical to SEQ ID NO: 23. In some embodiments, the human portion comprises SEQ ID NO: 23.

[0018] In some embodiments, the invention provides PD-1 polypeptides produced (or generated) by the non-human animals described herein. In some specific embodiments, the PD-1 polypeptides produced by the non-human animals described herein comprise an amino acid sequence at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 95% identical to SEQ ID NO:6. In some specific embodiments, the PD-1 polypeptides produced by the non-human animals described herein comprise an amino acid sequence substantially identical to SEQ ID NO:6. In some specific embodiments, the PD-1 polypeptides produced by the non-human animals described herein comprise an amino acid sequence identical to SEQ ID NO:6.

[0019] In some embodiments, the present invention provides isolated cells or tissues from the non-human animals described herein. In some embodiments, the present invention provides isolated cells or tissues comprising the Pdcd1 gene described herein. In some embodiments, the cells are lymphocytes. In some embodiments, the cells are selected from B cells, dendritic cells, macrophages, monocytes (e.g., activated monocytes), NK cells, and T cells (e.g., activated T cells). In some embodiments, the tissue is selected from fat, bladder, brain, breast, bone marrow, eye, heart, intestine, kidney, liver, lung, lymph node, muscle, pancreas, plasma, serum, skin, spleen, stomach, thymus, testis, egg, and combinations thereof.

[0020] In some embodiments, the present invention provides non-human embryonic stem cells whose genome comprises a Pdcd1 gene described herein. In some embodiments, the non-human embryonic stem cells are mouse embryonic stem cells and are from the 129 strain, the C57BL / 6 strain, or the BALB / c strain. In some embodiments, the non-human embryonic stem cells are mouse embryonic stem cells and are from the 129 strain, the C57BL / 6 strain, or a mixture thereof. In some embodiments, the non-human embryonic stem cells are mouse embryonic stem cells and are from a mixture of the 129 strain and the C57BL / 6 strain.

[0021] In some embodiments, the non-human embryonic stem cells have a genome comprising a Pdcd1 gene comprising SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, or a combination thereof.

[0022] In some embodiments, the invention provides methods of using the non-human embryonic stem cells described herein to generate non-human animals. In some specific embodiments, the non-human embryonic stem cells are mouse embryonic stem cells and are used to generate mice comprising the Pdcd1 gene described herein. In some specific embodiments, the non-human embryonic stem cells are rat embryonic stem cells and are used to generate rats comprising the Pdcd1 gene described herein.

[0023] In some embodiments, the invention provides a non-human embryo comprising, made from, obtained from, or generated from a non-human embryonic stem cell comprising a Pdcd1 gene described herein, hi some specific embodiments, the non-human embryo is a rodent embryo, in some embodiments, a mouse embryo, and in some embodiments, a rat embryo.

[0024] In some embodiments, the invention provides methods of using the non-human embryos described herein to generate non-human animals. In some specific embodiments, the non-human embryonic stem cells are mouse embryonic stem cells and are used to generate mice comprising the Pdcd1 gene described herein. In some specific embodiments, the non-human embryo is a rat embryo and is used to generate rats comprising the Pdcd1 gene described herein.

[0025] In some embodiments, the present invention provides a targeting vector (or nucleic acid construct) described herein. In some embodiments, the present invention provides a targeting vector (or nucleic acid construct) comprising a humanized Pdcd1 gene described herein. In some embodiments, the present invention provides a targeting vector (or nucleic acid construct) comprising a Pdcd1 gene encoding a PD-1 polypeptide comprising all or a portion of the human extracellular domain, which in some specific embodiments is a PD-1 polypeptide comprising amino acids 21-170, 26-169, 27-169, 27-145, or 35-145 of a human PD-1 polypeptide.

[0026] In some embodiments, the targeting vector (or nucleic acid construct) comprises all or a portion of one or more exons of a non-human Pdcd1 gene operably linked to all or a portion of one or more exons of the human Pdcd1 gene. In some embodiments, the targeting vector (or nucleic acid construct) comprises 5' and 3' non-human Pdcd1 untranslated regions (UTRs) flanking one or more exons of the human Pdcd1 gene. In some embodiments, the targeting vector (or nucleic acid construct) comprises one or more selectable markers. In some embodiments, the targeting vector (or nucleic acid construct) comprises one or more site-specific recombination sites. In some embodiments, the targeting vector (or nucleic acid construct) comprises human Pdcd1 exon 2. In some embodiments, the targeting vector (or nucleic acid construct) comprises all or a portion of human Pdcd1 exon 2 and human Pdcd1 exon 3.

[0027] In some embodiments, the invention provides methods of using the targeting vectors (or nucleic acid constructs) described herein to generate modified non-human embryonic stem cells. In some embodiments, the invention provides methods of using the targeting vectors (or nucleic acid constructs) described herein to generate modified non-human embryos. In some embodiments, the invention provides methods of using the targeting vectors (or nucleic acid constructs) described herein to generate modified non-human animals.

[0028] In some embodiments, the invention provides methods for producing a non-human animal that expresses a PD-1 polypeptide from an endogenous Pdcd1 gene, where the PD-1 polypeptide comprises a human sequence, the method comprising: (a) inserting a genomic fragment into the endogenous Pdcd1 gene of a rodent embryonic stem cell, wherein the genomic fragment comprises a nucleotide sequence encoding all or part of a human PD-1 polypeptide; (b) obtaining the rodent embryonic stem cells produced in (a); and producing a rodent using the rodent embryonic stem cells of (b).

[0029] In some embodiments, the human sequence comprises amino acids 35-145, 27-145, 27-169, 26-169, or 21-170 of a human PD-1 polypeptide.

[0030] In some embodiments, the nucleotide sequence comprises human Pdcd1 exon 2. In some embodiments, the nucleotide sequence further comprises all or a portion of human Pdcd1 exon 3. In some embodiments, the nucleotide sequence comprises one or more selectable markers. In some embodiments, the nucleotide sequence comprises one or more site-specific recombination sites.

[0031] In some embodiments, the invention provides methods for making a non-human animal whose genome comprises a Pdcd1 gene encoding a PD-1 polypeptide having a human portion and an endogenous portion, said portion operably linked to a rodent Pdcd1 promoter, the method comprising modifying the genome of a non-human animal such that the genome of the non-human animal comprises a Pdcd1 gene encoding a PD-1 polypeptide having a human portion and an endogenous portion, said portion operably linked to a rodent Pdcd1 promoter, thereby making said non-human animal.

[0032] In some embodiments, the rodent Pdcd1 promoter is an endogenous rodent Pdcd1 promoter.

[0033] In some embodiments, the human portion comprises amino acids 35-145, 27-145, 27-169, 26-169, or 21-170 of a human PD-1 polypeptide.

[0034] In some embodiments, the Pdcd1 gene is modified to include human Pdcd1 exon 2. In some embodiments, the Pdcd1 gene is modified to include human Pdcd1 exon 2 and all or part of human Pdcd1 exon 3.

[0035] In some embodiments, modification of the genome of the non-human animal is performed in non-human embryonic stem cells, and then generating the non-human animal from said non-human embryonic stem cells. In some specific embodiments, the non-human embryo is a rodent embryonic stem cell, in some embodiments, a mouse embryonic stem cell, and in some embodiments, a rat embryonic stem cell.

[0036] In some embodiments, the invention provides a non-human animal obtained by the methods described herein.

[0037] In some embodiments, the invention provides a method of reducing tumor growth in a non-human animal, the method comprising administering an agent that targets human PD-1 to a non-human animal whose genome comprises a Pdcd1 gene encoding a PD-1 polypeptide having a human portion and an endogenous portion, said portion operably linked to a rodent Pdcd1 promoter, wherein the administration is under conditions and for a time sufficient for tumor growth to be reduced in the non-human animal.

[0038] In some embodiments, the invention provides methods for killing tumor cells in a non-human animal, the method comprising administering an agent that targets human PD-1 to a non-human animal whose genome comprises a Pdcd1 gene encoding a PD-1 polypeptide having a human portion and an endogenous portion, said portion operably linked to a rodent Pdcd1 promoter, under conditions and for a time sufficient for the agent to mediate killing of the tumor cells in the non-human animal.

[0039] In some embodiments, the invention provides methods for evaluating the pharmacokinetic properties of an agent that targets human PD-1, the method comprising administering the agent to a non-human animal whose genome comprises a Pdcd1 gene encoding a PD-1 polypeptide having a human portion and an endogenous portion, said portion being operably linked to a rodent Pdcd1 promoter, and performing an assay to determine one or more pharmacokinetic properties of the agent that targets human PD-1.

[0040] In many embodiments, the non-human animals described herein are rodents whose genomes comprise a Pdcd1 gene encoding a PD-1 polypeptide having a human portion and an endogenous portion, said portion operably linked to a rodent Pdcd1 promoter. In many embodiments, the rodent Pdcd1 promoter is an endogenous rodent Pdcd1 promoter. In many embodiments, the human portion comprises amino acids 35-145, 27-145, 27-169, 26-169, or 21-170 of a human PD-1 polypeptide.

[0041] In some embodiments, the agent that targets human PD-1 is a PD-1 antagonist. In some embodiments, the agent that targets human PD-1 is a PD-1 agonist. In some embodiments, the agent that targets human PD-1 is an anti-PD-1 antibody. In some embodiments, the agent that targets human PD-1 is administered intravenously, intraperitoneally, or subcutaneously.

[0042] In some embodiments, the invention provides non-human animal models in which the non-human animal expresses a PD-1 polypeptide comprising a human portion and an endogenous portion.

[0043] In some embodiments, the invention provides a non-human animal, wherein the non-human animal comprises a Pdcd1 gene comprising an endogenous portion and a human portion, wherein the endogenous portion and the human portion are operably linked to a rodent Pdcd1 promoter.

[0044] In some embodiments, the present invention provides a non-human animal tumor model obtained by: (a) providing a non-human animal whose genome comprises a Pdcd1 gene comprising an endogenous portion and a human portion, wherein the endogenous portion and the human portion are operably linked to a non-human animal Pdcd1 promoter; and (b) transplanting one or more tumor cells of the rodent of (a), thereby providing said non-human animal tumor model.

[0045] In some embodiments, the non-human animal tumor model of the invention is a rodent tumor model. In some embodiments, the non-human animal Pdcd1 promoter is a rodent Pdcd1 promoter.

[0046] In some embodiments, the invention provides methods for identifying or validating a drug or vaccine, the method comprising delivering the drug or vaccine to a non-human animal whose genome comprises a Pdcd1 gene encoding a PD-1 polypeptide, where the PD-1 polypeptide comprises a human portion and an endogenous portion, and monitoring one or more of the immune response to the drug or vaccine, the safety profile of the drug or vaccine, or its effect on the disease, disorder, or condition. In some embodiments, monitoring the safety profile comprises determining whether the non-human animal experiences any side effects or adverse reactions as a result of drug or vaccine delivery. In some embodiments, the side effects or adverse reactions are selected from morbidity, mortality, changes in body weight, changes in one or more enzyme levels (e.g., liver), changes in the weight of one or more organs, loss of function (e.g., sensory, motor, organ, etc.), increased susceptibility to one or more diseases, alterations to the non-human animal's genome, increased or decreased food intake, and one or more disease complications. In some embodiments, the disease, disorder, or condition is induced in the non-human animal. In some embodiments, the disease, disorder, or condition induced in the non-human animal is associated with a disease, disorder, or condition suffered by one or more human patients in need of treatment. In some particular embodiments, the agent is an antibody.

[0047] In some particular embodiments, the present invention provides for the use of the non-human animals described herein in the development of drugs or vaccines for use in medicine, such as for use as pharmaceuticals.

[0048] In some embodiments, the invention provides the use of a non-human animal described herein in the manufacture of a medicament for the treatment of cancer, a neoplasm, an infectious disease, an inflammatory disease, disorder or condition, or an autoimmune disease, disorder or condition.

[0049] In various embodiments, the Pdcd1 gene of the invention comprises a Pdcd1 gene described herein. In various embodiments, the Pdcd1 gene of the invention encodes a PD-1 polypeptide having a human portion and an endogenous portion, said portion being operably linked to a rodent Pdcd1 promoter. In various embodiments, the rodent promoter is an endogenous rodent promoter. In various embodiments, the human portion comprises human Pdcd1 exon 2. In various embodiments, the human portion comprises human Pdcd1 exon 2 and further comprises all or a portion of human Pdcd1 exon 3.

[0050] In various embodiments, the PD-1 polypeptides of the invention include the PD-1 polypeptides described herein. In various embodiments, the non-human animals of the invention do not detectably express the full-length endogenous non-human PD-1 polypeptide. In various embodiments, the non-human animals of the invention do not detectably express the extracellular portion of the endogenous non-human PD-1 polypeptide. In various embodiments, the non-human animals of the invention do not detectably express the N-terminal immunoglobulin V region of the endogenous non-human PD-1 polypeptide.

[0051] In various embodiments, the non-human animals of the invention are rodents, and in some embodiments, mice, and in some embodiments, rats. In some embodiments, the mice of the invention are selected from the group consisting of 129, BALB / C, C57BL / 6, and mixed 129xC57BL / 6 strains, and in some particular embodiments, are 50% 129 and 50% C57BL / 6, and in some particular embodiments, are 25% 129 and 75% C57BL / 6. In particular embodiments, for example, the following items are provided: (Item 1) A rodent expressing a PD-1 polypeptide, wherein the PD-1 polypeptide comprises a human portion and an endogenous portion. (Item 2) 2. The rodent of item 1, wherein the PD-1 polypeptide is translated in cells of the rodent with a rodent signal peptide. (Item 3) 3. The rodent of item 1 or 2, wherein the endogenous portion comprises an intracellular portion of an endogenous PD-1 polypeptide. (Item 4) 4. The rodent of item 3, wherein the endogenous portion further comprises a transmembrane portion of an endogenous PD-1 polypeptide. (Item 5) 8. The rodent of item 4, wherein the endogenous portion has an amino acid sequence at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 95% identical to the corresponding amino acid sequence of the mouse PD-1 polypeptide shown in FIG. (Item 6) 8. The rodent of item 4, wherein the endogenous portion has an amino acid sequence identical to the corresponding amino acid sequence of the mouse PD-1 polypeptide shown in FIG. (Item 7) 7. The rodent of any one of items 1 to 6, wherein the human portion comprises amino acids 35 to 145 of a human PD-1 polypeptide. (Item 8) 7. The rodent of any one of items 1 to 6, wherein the human portion comprises amino acids 27 to 145 of a human PD-1 polypeptide. (Item 9) 7. The rodent of any one of items 1 to 6, wherein the human portion comprises amino acids 26 to 169 of a human PD-1 polypeptide. (Item 10) 7. The rodent of any one of paragraphs 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 the corresponding amino acid sequence of a human PD-1 polypeptide found in Figure 8. (Item 11) 7. The rodent of any one of items 1 to 6, wherein the human portion comprises an amino acid sequence identical to the corresponding amino acid sequence of a human PD-1 polypeptide found in FIG. 8. (Item 12) 12. The rodent of any one of paragraphs 1 to 11, wherein the PD-1 polypeptide comprising a human portion and an endogenous portion is encoded by an endogenous Pdcd1 gene. (Item 13) 13. The rodent of item 12, wherein the endogenous Pdcd1 gene comprises endogenous Pdcd1 exons 1, 4 and 5. (Item 14) 14. The rodent of item 13, wherein the endogenous Pdcd1 gene further comprises all or part of endogenous Pdcd1 exon 3. (Item 15) A rodent comprising a Pdcd1 gene comprising an endogenous portion and a human portion, said endogenous and human portions operably linked to a rodent Pdcd1 promoter. (Item 16) 16. The rodent of item 15, wherein the rodent Pdcd1 promoter is an endogenous rodent Pdcd1 promoter. (Item 17) 17. The rodent of item 15 or 16, wherein the endogenous portion comprises endogenous Pdcd1 exons 1, 4 and 5. (Item 18) 18. The rodent of item 17, wherein the endogenous portion further comprises all or part of endogenous Pdcd1 exon 3. (Item 19) 19. The rodent of item 17 or 18, wherein all or part of exons 1, 3, 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 corresponding all or part of exons 1, 3, 4, and 5 of the endogenous Pdcd1 gene shown in Figure 8. (Item 20) 20. The rodent of any one of items 15 to 19, wherein the human portion encodes amino acids 26 to 169 of a human PD-1 polypeptide. (Item 21) 20. The rodent of any one of items 15 to 19, wherein the human portion comprises exon 2 of the human Pdcd1 gene. (Item 22) 22. The rodent of item 21, wherein the human portion further comprises all or part of human Pdcd1 exon 3. (Item 23) 23. The rodent of item 22, wherein all or a portion of human Pdcd1 exons 2 and 3 are at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 95% identical to all or a portion of corresponding exons 2 and 3 of the human Pdcd1 gene shown in FIG. 8. (Item 24) 23. The rodent of item 22, wherein the human portion comprises a sequence that is codon-optimized for expression in the rodent. (Item 25) 23. The rodent of item 22, wherein the human portion comprises SEQ ID NO:23. (Item 26) 26. The rodent of any one of items 1 to 25, wherein the rodent is a mouse or a rat. (Item 27) 27. A PD-1 polypeptide produced by the rodent of any one of items 1 to 26. (Item 28) 28. The PD-1 polypeptide of paragraph 27, wherein the PD-1 polypeptide comprises an amino acid sequence at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 95% identical to the humanized PD-1 polypeptide shown in Figure 8. (Item 29) An isolated rodent cell or tissue, the genome of which comprises a Pdcd1 gene encoding a PD-1 polypeptide having a human portion and an endogenous portion, said portion operably linked to a rodent Pdcd1 promoter. (Item 30) 30. The rodent cell or tissue of item 29, wherein the rodent Pdcd1 promoter is an endogenous rodent Pdcd1 promoter. (Item 31) 31. The isolated cell or tissue of item 29 or 30, wherein the human portion comprises human Pdcd1 exon 2. (Item 32) 32. The isolated cell or tissue of item 31, wherein the human portion further comprises all or part of human Pdcd1 exon 3. (Item 33) 33. The isolated rodent cell or tissue of any one of items 29 to 32, wherein the rodent cell or tissue is a mouse cell or tissue, or a rat cell or tissue. (Item 34) A rodent embryonic stem cell, the genome of which comprises a Pdcd1 gene encoding a PD-1 polypeptide having a human portion and an endogenous portion, said portion operably linked to a rodent Pdcd1 promoter. (Item 35) 35. The rodent embryonic stem cell of item 34, wherein the rodent Pdcd1 promoter is an endogenous rodent Pdcd1 promoter. (Item 36) 36. The rodent embryonic stem cell of item 34 or 35, wherein the human portion comprises human Pdcd1 exon 2. (Item 37) 37. The rodent embryonic stem cell of item 36, wherein the human portion further comprises all or part of human Pdcd1 exon 3. (Item 38) 38. The rodent embryonic stem cell according to any one of items 34 to 37, wherein the rodent embryonic stem cell is a mouse embryonic stem cell and is a 129 strain, a C57BL strain, or a mixture thereof. (Item 39) 39. The rodent embryonic stem cells of item 38, wherein the rodent embryonic stem cells are mouse embryonic stem cells and are a mixture of 129 and C57BL strains. (Item 40) 40. A rodent embryo generated from the embryonic stem cell of any one of items 34 to 39. (Item 41) 1. A method of producing 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 the endogenous Pdcd1 gene of a rodent embryonic stem cell, wherein the genomic fragment comprises a nucleotide sequence encoding all or part of a human PD-1 polypeptide; (b) obtaining the rodent embryonic stem cells produced in (a); (c) generating a rodent using the rodent embryonic stem cells of (b). (Item 42) 42. The method of claim 41, wherein the human sequence comprises amino acids 35 to 145 of the human PD-1 polypeptide. (Item 43) 42. The method of claim 41, wherein the human sequence comprises amino acids 27 to 145 of the human PD-1 polypeptide. (Item 44) 42. The method of claim 41, wherein the human sequence comprises amino acids 26 to 169 of the human PD-1 polypeptide. (Item 45) 45. The method of any one of items 41 to 44, wherein the nucleotide sequence comprises human Pdcd1 exon 2. (Item 46) 46. The method of item 45, wherein the nucleotide sequence further comprises all or part of human Pdcd1 exon 3. (Item 47) 47. The method of any one of items 41 to 46, wherein the nucleotide sequence comprises one or more selectable markers. (Item 48) 48. The method of any one of items 41 to 47, wherein the nucleotide sequence comprises one or more site-specific recombination sites. (Item 49) 1. A method of producing a rodent whose genome comprises a Pdcd1 gene encoding a PD-1 polypeptide having a human portion and an endogenous portion, said portion operably linked to a rodent Pdcd1 promoter, said method comprising: 1. A method comprising modifying the genome of a rodent so that it comprises a Pdcd1 gene encoding a PD-1 polypeptide having a human portion and an endogenous portion, said portion being operably linked to a rodent Pdcd1 promoter, thereby making said rodent. (Item 50) 50. The method of claim 49, wherein the rodent Pdcd1 promoter is an endogenous rodent Pdcd1 promoter. (Item 51) 50. The method of item 49, wherein the human portion comprises amino acids 35 to 145 of a human PD-1 polypeptide. (Item 52) 50. The method of item 49, wherein the human portion comprises amino acids 27 to 145 of a human PD-1 polypeptide. (Item 53) 50. The method of item 49, wherein the human portion comprises amino acids 26 to 169 of a human PD-1 polypeptide. (Item 54) 54. The method of any one of items 49 to 53, wherein the Pdcd1 gene is modified to contain human Pdcd1 exon 2. (Item 55) 55. The method of item 54, wherein the Pdcd1 gene is modified to include all or part of human Pdcd1 exon 2 and human Pdcd1 exon 3. (Item 56) 56. The method of any one of items 41 to 55, wherein the rodent is a mouse or a rat. (Item 57) 57. A rodent obtainable by the method according to any one of items 41 to 56. (Item 58) 58. The rodent of item 57, wherein the rodent is a mouse or a rat. (Item 59) 1. A method of reducing tumor growth in a rodent, said method comprising: administering an agent that targets human PD-1 to a rodent whose genome comprises a Pdcd1 gene encoding a PD-1 polypeptide having a human portion and an endogenous portion, said portion operably linked to a rodent Pdcd1 promoter; The method wherein said administering is carried out under conditions and for a time sufficient to reduce tumor growth in said rodent. (Item 60) 1. A method of killing tumor cells in a rodent, said method comprising: administering an agent that targets human PD-1 to a rodent whose genome comprises a Pdcd1 gene encoding a PD-1 polypeptide having a human portion and an endogenous portion, said portion operably linked to a rodent Pdcd1 promoter; The method wherein said administering is carried out under conditions and for a time sufficient to mediate killing of said tumor cells. (Item 61) 1. A method for evaluating the pharmacokinetic properties of a drug targeting human PD-1, the method comprising: administering the agent to a rodent whose genome comprises a Pdcd1 gene encoding a PD-1 polypeptide having a human portion and an endogenous portion, said portion operably linked to a rodent Pdcd1 promoter; and performing an assay to determine one or more pharmacokinetic properties of the agent that targets PD-1. (Item 62) 62. The method of any one of items 59 to 61, wherein the human portion comprises amino acids 35 to 145 of a human PD-1 polypeptide. (Item 63) 62. The method of any one of items 59-61, wherein the human portion comprises amino acids 27-145 of human PD-1 polypeptide. (Item 64) 62. The method of any one of items 59 to 61, wherein the human portion comprises amino acids 26 to 169 of a human PD-1 polypeptide. (Item 65) 65. The method of any one of items 59 to 64, wherein the agent targeting human PD-1 is a PD-1 antagonist. (Item 66) 65. The method of any one of items 59 to 64, wherein the agent targeting human PD-1 is a PD-1 agonist. (Item 67) 65. The method of any one of items 59 to 64, wherein the agent targeting human PD-1 is an anti-PD-1 antibody. (Item 68) 68. The method of any one of items 59 to 67, wherein the agent targeting human PD-1 is administered intravenously to the rodent. (Item 69) 68. The method of any one of items 59 to 67, wherein the agent targeting human PD-1 is administered intraperitoneally to the rodent. (Item 70) 68. The method of any one of items 59 to 67, wherein the agent targeting human PD-1 is administered subcutaneously to the rodent. (Item 71) 71. The method of any one of items 59 to 70, wherein the rodent Pdcd1 promoter is an endogenous rodent Pdcd1 promoter. (Item 72) 72. The method of any one of items 59 to 71, wherein the rodent is a mouse or a rat. (Item 73) A rodent tumor model, wherein the rodent expresses a PD-1 polypeptide comprising a human portion and an endogenous portion. (Item 74) A rodent tumor model, wherein the rodent comprises a Pdcd1 gene comprising an endogenous portion and a human portion, the endogenous and human portions being operably linked to a rodent Pdcd1 promoter. (Item 75) 1. A rodent tumor model comprising: a) providing a rodent comprising a Pdcd1 gene comprising an endogenous portion and a human portion, said endogenous and human portions operably linked to a rodent Pdcd1 promoter; b) implanting one or more tumor cells of said rodent of (a); thereby providing said rodent tumor model. (Item 76) 76. The rodent tumor model of any one of items 73 to 75, wherein the rodent is a mouse or a rat. (Item 77) 77. The rodent tumor model of item 76, wherein the rodent is a mouse. (Item 78) 78. The rodent tumor model of item 77, wherein the mice are selected from the group consisting of 129 strain, C57BL / 6 strain, and mixed 129 x C57BL / 6 strain. (Item 79) 79. The rodent tumor model of item 78, wherein the mice are 25% 129 and 75% C57BL / 6.

[0052] As used herein, the terms "about" and "approximately" are used interchangeably. Any numbers used herein, with or without about / approximately, are intended to cover any normal variations understood by one of ordinary skill in the art.

[0053] Other features, objects, and advantages of the present invention will become apparent from the following detailed description of specific embodiments. It should be understood, however, that the detailed description, while illustrating specific embodiments of the present invention, is given by way of example only and is not meant to be limiting. Various changes and modifications within the scope of the present invention will become apparent to those skilled in the art from the detailed description.

[0054] The drawings contained herein, consisting of the following figures, are for illustrative purposes only and not for limitation: [Brief explanation of the drawings]

[0055] [Figure 1] A diagram (not to scale) of the genomic structure of the non-human (e.g., mouse) and human programmed cell death 1 (Pdcd1) gene is shown. Exons and untranslated regions (UTRs) are numbered below each exon and above each UTR. [Figure 2]

[0023] Figure 1 shows a diagram (not to scale) of an exemplary method for humanizing the non-human programmed cell death 1 (Pdcd1) gene. The locations of selected nucleotide junctions are indicated by lines below each junction. The sequences of these selected nucleotide junctions are indicated by SEQ ID NOs. [Figure 3] FIG. 1 shows a diagram (not to scale) of the genomic structure of the mouse and human programmed cell death 1 (Pdcd1) gene, indicating the approximate locations of the probes used in the assays described in Example 1. [Figure 4] 1 shows exemplary histograms gated on CD19 and CD8 isolated from wild-type mice and mice heterozygous for humanization of the endogenous Pdcd1 gene as described in Example 1 expressing mouse and / or humanized PD-1. Stimulated and unstimulated cell populations are shown, as are cells stained with an isotype control. [Figure 5] Figure 1 shows exemplary tumor growth curves over 21 days for mice homozygous for humanization of the endogenous Pdcd1 gene as described in Example 1. Control: antibody not specific for PD-1; a-hPD-1 Ab: antibody specific for human PD-1. Arrows indicate days of antibody treatment. The number of tumor-free mice at day 21 is shown for each treatment group. [Figure 6]Figure 1 shows exemplary real-time PCR analysis of splenic CD8b, CD3, IFN-g, and PD-1 mRNA in mice homozygous for humanization of the endogenous Pdcd1 gene as described in Example 1 after treatment with an anti-PD-1 antibody. A, Average of five mice per group. B, Expression levels of individual mice in each treatment group. Control: antibody not specific for PD-1; α-PD-1: anti-PD-1 antibody. [Figure 7] Figure 1 shows exemplary tumor growth curves over 60 days in mice homozygous for humanization of the endogenous Pdcd1 gene as described in Example 1, administered 0.3 to 25 mg / kg of anti-hPD-1 antibody or 25 mg / kg of a control antibody (an antibody not specific for PD-1). Arrows indicate days of antibody treatment. The number of tumor-free mice at day 60 is shown for each treatment group. [Figure 8-1] Exemplary mouse, human, and humanized Pdcd1 and PD-1 sequences are shown, as well as exemplary human nucleic acid sequences for humanizing non-human Pdcd1 genes. For mRNA sequences, bold indicates the coding sequence and consecutive exons, separated by alternating underlined letters where indicated. For humanized mRNA sequences, the human sequence is in parentheses. For protein sequences, the signal protein is underlined, the extracellular sequence is in bold, the immunoglobulin V region sequence is in parentheses, and the intracellular sequence is in italics. For humanized protein sequences, the non-human sequence is in regular text, and the human sequence is in bold. [Figure 8-2] Exemplary mouse, human, and humanized Pdcd1 and PD-1 sequences are shown, as well as exemplary human nucleic acid sequences for humanizing non-human Pdcd1 genes. For mRNA sequences, bold indicates the coding sequence and consecutive exons, separated by alternating underlined letters where indicated. For humanized mRNA sequences, the human sequence is in parentheses. For protein sequences, the signal protein is underlined, the extracellular sequence is in bold, the immunoglobulin V region sequence is in parentheses, and the intracellular sequence is in italics. For humanized protein sequences, the non-human sequence is in regular text, and the human sequence is in bold. [Figure 8-3]Exemplary mouse, human, and humanized Pdcd1 and PD-1 sequences are shown, as well as exemplary human nucleic acid sequences for humanizing non-human Pdcd1 genes. For mRNA sequences, bold indicates the coding sequence and consecutive exons, separated by alternating underlined letters where indicated. For humanized mRNA sequences, the human sequence is in parentheses. For protein sequences, the signal protein is underlined, the extracellular sequence is in bold, the immunoglobulin V region sequence is in parentheses, and the intracellular sequence is in italics. For humanized protein sequences, the non-human sequence is in regular text, and the human sequence is in bold. [Figure 8-4] Exemplary mouse, human, and humanized Pdcd1 and PD-1 sequences are shown, as well as exemplary human nucleic acid sequences for humanizing non-human Pdcd1 genes. For mRNA sequences, bold indicates the coding sequence and consecutive exons, separated by alternating underlined letters where indicated. For humanized mRNA sequences, the human sequence is in parentheses. For protein sequences, the signal protein is underlined, the extracellular sequence is in bold, the immunoglobulin V region sequence is in parentheses, and the intracellular sequence is in italics. For humanized protein sequences, the non-human sequence is in regular text, and the human sequence is in bold. DETAILED DESCRIPTION OF THE INVENTION

[0056] (Definition of terms) The present invention is not limited to the particular methods and experimental conditions described herein, as such methods and conditions may vary. It is also understood that the terminology used herein is used for the purpose of describing particular embodiments only, and is not intended, since the scope of the present invention is defined by the claims.

[0057] Unless otherwise specified, all terms and phrases used herein include the meaning that the term and phrase has acquired in the art unless expressly stated otherwise or is clearly apparent from the context in which the term or phrase is used. Although any methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the present invention, particular methods and materials are now described. All publications mentioned herein are incorporated by reference.

[0058] As used herein, the term "approximately," when applied to one or more subject values, refers to a value similar to a specified reference value. In certain embodiments, the term "approximately" or "about" refers to a value within a range of 25%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1% or less in either direction of the specified reference value, unless otherwise specified or apparent from the context (except where such number would exceed 100% of the possible values).

[0059] The term "biologically active" includes the characteristic of any agent that has activity in a biological system, in vitro, or in vivo (e.g., in an organism). For example, an agent is considered to be biologically active if, when present in an organism, it has a biological effect in that organism. In certain embodiments, if a protein or polypeptide is biologically active, a portion of that protein or polypeptide that shares at least one biological activity of the protein or polypeptide is generally referred to as a "biologically active" portion.

[0060] The term "equivalent" includes two or more agents, entities, situations, sets of conditions, etc. that may not be identical to one another, but are sufficiently similar to permit a comparison between them, such that reasonable conclusions can be drawn based on the observed differences or similarities. Those of skill in the art will understand the degree of identity required in a given situation for two or more such agents, entities, situations, sets of conditions, etc. to be considered equivalent, within the context.

[0061] For example, the term "conservative," as in conservative amino acid substitution, includes the substitution of an amino acid residue with another amino acid residue having a side chain R group with similar chemical properties (e.g., charge or hydrophobicity). Generally, conservative amino acid substitutions do not substantially alter the functional properties of the protein of interest (e.g., the ability of a receptor to bind to a ligand). Examples of amino acids with 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 acid substitutions include, for example, valine / leucine / isoleucine, phenylalanine / tyrosine, lysine / arginine, alanine / valine, glutamic acid / aspartic acid, and asparagine / glutamine. In some embodiments, conservative amino acid substitutions can be for any native residue in a protein, including alanine, such as those used in alanine-scanning mutagenesis. In some embodiments, conservative substitutions are made that have 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, which is incorporated herein by reference. In some embodiments, substitutions are moderately conservative, where the substitution has a non-negative value in the PAM250 log-likelihood matrix.

[0062] The term "control" includes the art-recognized meaning of "control" as a standard against which results are compared. Generally, controls are used to increase the integrity of an experiment by isolating a variable in order to draw conclusions about such variable. In some embodiments, a control is a reaction or assay performed simultaneously with a test reaction or assay to provide a comparator. As used herein, "control" may include a "control animal." A "control animal" may have a modification described herein, a modification different from those described herein, or be unmodified (i.e., a wild-type animal). In one experiment, the "test" (i.e., the variable being tested) is administered. In a second experiment, the "control," the variable being tested, is not administered. In some embodiments, a control is a historical control (i.e., of a previously performed test or assay, or of a previously known quantity or result). In some embodiments, a control is or includes a printed or otherwise kept record. A control may be a positive or negative control.

[0063] The term "disruption" includes the result of a homologous recombination event in a DNA molecule (e.g., at an endogenous homologous sequence such as a gene or locus). In some embodiments, the disruption may achieve or represent an insertion, deletion, substitution, exchange, missense mutation, or frameshift of a DNA sequence, or any combination thereof. The insertion may include the insertion of an entire gene or a fragment of a gene (e.g., an exon), which may be of a source other than the endogenous sequence (e.g., a heterologous sequence). In some embodiments, the disruption may increase the expression and / or activity of a gene or gene product (e.g., of a protein encoded by the gene). In some embodiments, the disruption may decrease the expression and / or activity of a gene or gene product. In some embodiments, the disruption may alter the sequence of a gene or encoded gene product (e.g., the encoded protein). In some embodiments, the disruption may truncate or fragment a gene or encoded gene product (e.g., the encoded protein). In some embodiments, the disruption may extend a gene or encoded gene product, and in some embodiments, the disruption may achieve the assembly of a fusion protein. In some embodiments, the disruption may affect the level of the gene or gene product but not its activity. In some embodiments, the disruption may affect the activity of the gene or gene product but not its level. In some embodiments, the disruption may have no significant effect on the level of the gene or gene product. In some embodiments, the disruption may have no significant effect on the activity of the gene or gene product. In some embodiments, the disruption may have no significant effect on either the level or activity of the gene or gene product.

[0064] The terms "determining," "measuring," "evaluating," "assessing," "assaying," and "analyzing" are used interchangeably and refer to any form of measurement, including determining whether an element is present. These terms include both quantitative and / or qualitative determinations. Assays can be relative or absolute. "Assaying for the presence of" can be determining the amount of something present and / or determining whether it is present or absent.

[0065] The term "dosing regimen" or "treatment regimen," in some embodiments, includes one or more unit doses administered individually to a subject, typically spaced apart over time. In some embodiments, a given therapeutic agent has a recommended dosing regimen, which may involve one or more doses. In some embodiments, a dosing regimen includes multiple doses, each spaced apart by the same length of time from each other; in some embodiments, a dosing regimen includes multiple doses, with at least two different time periods separating the individual doses.

[0066] The phrase "endogenous locus" or "endogenous gene" includes a locus found in a parent organism or indicator organism. In some embodiments, an endogenous locus has a sequence found in nature. In some embodiments, an endogenous locus is a wild-type locus. In some embodiments, an indicator organism is a wild-type organism. In some embodiments, an indicator organism is a genetically engineered organism. In some embodiments, an indicator organism is an organism (wild-type or genetically engineered) bred in a laboratory.

[0067] The phrase "endogenous promoter" includes a promoter naturally associated with an endogenous gene, e.g., in a wild-type organism.

[0068] The term "heterologous" includes an agent or entity from a different origin. For example, when used in reference to a polypeptide, gene, or gene product, or present in a particular cell or organism, the term clarifies that the relevant polypeptide, gene, or gene product 1) has been genetically manipulated by the hand of man, 2) has been introduced into the cell or organism (or its precursor) by the hand of man (e.g., by genetic manipulation), and / or 3) is not naturally produced by or present in the relevant cell or organism (e.g., the relevant cell type or organism type).

[0069] The term "host cell" includes a cell into which a heterologous (e.g., exogenous) nucleic acid or protein has been introduced. Those skilled in the art will understand upon reading this disclosure that such terms are used to refer not only to the particular subject cell, but also to the progeny of such a cell. Because certain modifications may occur in successive generations due to mutation or environmental influences, such progeny may not, in fact, be identical to the parent cell, but are still included within the scope of the term "host cell." In some embodiments, a host cell is or comprises a prokaryotic or eukaryotic cell. Generally, a host cell is any cell suitable for receiving and / or producing a heterologous nucleic acid or protein, regardless of the species to which the cell is designated. Exemplary cells include prokaryotic and eukaryotic (unicellular or multicellular) cells, bacterial cells (e.g., strains of E. coli, Bacillus, Streptomyces, etc.), mycobacterial cells, fungal cells, yeast cells (e.g., Saccharomyces cerevisiae, Schizosaccharomyces pombe, Pichia methanolica, etc.), plant cells, insect cells (e.g., SF-9, SF-21, baculovirus-infected insect cells, Triticum aureus, etc.), non-human animal cells, human cells, or cell fusions such as, for example, hybridomas or quadromas. In some embodiments, the cells are human, monkey, ape, hamster, rat, or mouse cells. In some embodiments, the cells are prokaryotic and selected from the following cells: CHO (e.g., CHO K1, DXB-11 CHO, Veggie-CHO), COS (e.g., COS-7), retinal cells, Vero, CV1, kidney (e.g., HEK293, 293 EBNA, MSR 293, MDCK, HaK, BHK), HeLa, HepG2, WI38, MRC 5, Colo205, HB 8065, HL-60, (e.g., BHK21), Jurkat, Daudi, A431 (epidermal), CV-1, U937, 3T3, L cells, C127 cells, SP2 / 0, NS-0, MMT 060562, Sertoli cells, BRL 3A cells, HT1080 cells, myeloma cells, tumor cells, and cell lines derived from the foregoing cells. In some embodiments, the cell comprises one or more viral genes, e.g., a retinal cell (e.g., a PER.C6™ cell) that expresses a viral gene. In some embodiments, the host cell is or comprises an isolated cell.In some embodiments, the host cell is part of a tissue. In some embodiments, the host cell is part of an organism.

[0070] The term "humanized" includes nucleic acids or proteins whose structure (i.e., nucleotide or amino acid sequence) includes portions that correspond substantially or identically to the structure of a particular gene or protein found naturally in a non-human animal, and also includes portions that differ from that found in the relevant particular non-human gene or protein and instead correspond more closely to the equivalent structure found in the corresponding human gene or protein. In some embodiments, a "humanized" gene is a gene (e.g., a human protein or portion thereof—e.g., a characteristic portion thereof) that encodes a polypeptide with an amino acid sequence substantially like that of a human polypeptide. By way of example, in the case of a membrane receptor, a "humanized" gene can be a gene that encodes a polypeptide with all or part of an intracellular portion having an amino acid sequence like that of a human extracellular portion and the remaining sequence like that of a non-human (e.g., murine) polypeptide. In some embodiments, a humanized gene comprises at least a portion of the DNA sequence of a human gene. In some embodiments, a humanized gene comprises the entire DNA sequence of a human gene. In some embodiments, a humanized protein comprises a sequence with a portion found in a human protein. In some embodiments, the humanized protein comprises the entire sequence of a human protein and is expressed from an endogenous locus in the non-human animal that corresponds to a homolog or ortholog of the human gene.

[0071] For example, the term "identity" in the context of sequence comparisons includes identity determined by many different algorithms known in the art that can be used to measure nucleotide and / or amino acid sequence identity. In some embodiments, identity as described herein is determined using ClustalW v. 1.83 (slow) alignment using a Gonnet similarity matrix (MACVECTOR™ 10.0.2, MacVector Inc., 2008), with a gap opening penalty of 10.0 and a gap extension penalty of 0.1.

[0072] The term "isolated" includes substances and / or entities that are (1) separated from at least some of the components with which it was associated when originally produced (either in nature and / or in an experimental setting) and / or (2) designed, produced, prepared, and / or manufactured by the hand of man. Isolated substances and / or entities may be separated from about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or greater than about 99% of the other components with which they were originally associated. In some embodiments, an isolated agent is about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or greater than about 99% pure. A substance is "pure" if it is substantially free of other components. In some embodiments, as one of skill in the art will understand, a substance may still be considered "isolated" or even "pure" even after being combined with certain other components, such as, for example, one or more carriers or excipients (e.g., buffers, solvents, water, etc.), and in such embodiments, the percent isolation or purity of the substance is calculated without including such carriers or excipients. By way of example, in some embodiments, a naturally occurring biopolymer, such as a polypeptide or polynucleotide, is considered "isolated" when: a) its origin or source is free from association with some or all of the components that accompany it in its natural state in nature; b) it is substantially free of other polypeptides or nucleic acids of the same species that produces it in nature; or c) it is expressed by or is otherwise associated with components from a cell or other expression system other than the species that produces it in nature. Thus, for example, in some embodiments, a polypeptide that is chemically synthesized or synthesized in a cellular system different from that which produces it in nature is considered an "isolated" polypeptide. Alternatively, or additionally, in some embodiments, a polypeptide that has been subjected to one or more purification techniques is considered to be an "isolated" polypeptide if it: a) is free from association with some or all of the components that accompany it in its natural state; A polypeptide can be considered "isolated" so long as it a) is separated from other components with which it is associated in nature and / or b) is associated when originally produced.

[0073] The phrase "non-human animal" includes any vertebrate organism that is not a human. In some embodiments, the non-human animal is a cyclostome, a bony fish, a cartilaginous fish (e.g., a shark or a ray), an amphibian, a reptile, a mammal, or a bird. In some embodiments, the non-human mammal is a primate, a goat, a sheep, a pig, a dog, a cow, or a rodent. In some embodiments, the non-human animal is a rodent such as a rat or a mouse.

[0074] The phrase "nucleic acid" includes any compound and / or substance that is or can be incorporated into an oligonucleotide chain. In some embodiments, a "nucleic acid" is an oligonucleotide chain or a compound and / or substance that can be incorporated into an oligonucleotide chain via phosphodiester bonds. As the context makes clear, in some embodiments, a "nucleic acid" comprises individual nucleic acid residues (e.g., nucleotides and / or nucleosides), and in some embodiments, a "nucleic acid" comprises an oligonucleotide chain comprising individual nucleic acid residues. In some embodiments, a "nucleic acid" is or comprises RNA, and in some embodiments, a "nucleic acid" is or comprises DNA. In some embodiments, a "nucleic acid" comprises or consists of one or more naturally occurring nucleic acid residues. In some embodiments, a "nucleic acid" comprises or consists of one or more nucleic acid analogs. In some embodiments, nucleic acid analogs differ from "nucleic acids" in that they do not utilize a phosphodiester backbone. For example, in some embodiments, a "nucleic acid" is or comprises one or more "peptide nucleic acids," which are known in the art and have peptide bonds instead of phosphodiester bonds in the backbone, and are considered within the scope of the present invention. Alternatively or additionally, in some embodiments, a "nucleic acid" has one or more phosphorothioate and / or 5'-N-phosphoramidite linkages rather than phosphodiester linkages. In some embodiments, a "nucleic acid" is or is composed of one or more naturally occurring nucleosides (e.g., adenosine, thymidine, guanosine, cytidine, uridine, deoxyadenosine, deoxythymidine, deoxyguanosine, and deoxycytidine).In some embodiments, a "nucleic acid" is or consists of one or more nucleoside analogs (e.g., 2-aminoadenosine, 2-thiothymidine, inosine, pyrrolo-pyrimidine, 3-methyladenosine, 5-methylcytidine, C-5 propynyl-cytidine, C-5 propynyl-uridine, 2-aminoadenosine, C5-bromouridine, C5-urouridine, C5-iodouridine, C5-propynyl-uridine, C5-propynyl-cytidine, C5-methylcytidine, 2-aminoadenosine, 7-deazaadenosine, 7-deazaguanosine, 8-oxoadenosine, 8-oxoguanosine, O(6)-methylguanine, 2-thiocytidine, methylated bases, intercalating bases, and combinations thereof). In some embodiments, a "nucleic acid" comprises one or more modified sugars (e.g., 2'-fluororibose, ribose, 2'-deoxyribose, arabinose, and hexose) compared to those in naturally occurring nucleic acids. In some embodiments, a "nucleic acid" has a nucleotide sequence that encodes a functional gene product, such as RNA or a protein. In some embodiments, a "nucleic acid" comprises one or more introns. In some embodiments, a "nucleic acid" is prepared by one or more of isolation from a natural source, enzymatic synthesis by polymerization based on a complementary template (in vivo or in vitro), replication in a recombinant cell or system, and chemical synthesis. In some embodiments, a "nucleic acid" is at least 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 20, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, 500, 600, 700, 800, 900, 1000, 1500, 2000, 2500, 3000, 3500, 4000, 4500, 5000 or more residues in length. In some embodiments, a "nucleic acid" is single-stranded, and in some embodiments, a "nucleic acid" is double-stranded. In some embodiments, a "nucleic acid" has a nucleotide sequence that encodes a polypeptide or has at least one element that is the complement of a sequence that encodes a polypeptide.In some embodiments, the "nucleic acid" has enzymatic activity.

[0075] The phrase "operably linked" includes a juxtaposition where the described elements are in a relationship permitting them to function in their intended manner. A control sequence "operably linked" to a coding sequence is ligated such that expression of the coding sequence is achieved under conditions compatible with the control sequences. "Operatively linked" sequences include both expression control sequences adjacent to a gene of interest and expression control sequences that act in trans or at a distance to regulate the gene of interest. The term "expression control sequences" includes polynucleotide sequences necessary to cause the expression and processing of coding sequences to which they are linked. "Expression control sequences" include appropriate transcription initiation, termination, promoter and enhancer sequences, efficient RNA processing signals such as splicing and polyadenylation signals, sequences that stabilize cytoplasmic mRNA, sequences that enhance translation efficiency (i.e., Kozak sequences), sequences that enhance protein stability, and, if desired, sequences that enhance protein secretion. The nature of such control sequences will vary depending on the host organism. For example, in prokaryotes, such control sequences generally include promoters, ribosomal binding sites, and transcription termination sequences, while in eukaryotes, such control sequences generally include promoters and transcription termination sequences. The term "control sequence" is intended to include elements whose presence is essential for expression and processing, and can also include additional elements whose presence is advantageous, for example, leader sequences and fusion partner sequences.

[0076] The term "patient" or "subject" includes any organism to which a provided composition is or may be administered, for example, for experimental, diagnostic, prophylactic, cosmetic, and / or therapeutic purposes. Typical patients include animals (e.g., mammals such as mice, rats, rabbits, non-human primates, and / or humans). In some embodiments, the patient is a non-human animal. In some embodiments, the patient (e.g., a non-human animal patient) may have a modification described herein, a modification different from those described herein, or no modification (i.e., a wild-type non-human animal patient). In some embodiments, the non-human animal suffers from or is susceptible to one or more disorders or conditions. In some embodiments, the non-human animal exhibits one or more symptoms of a disorder or condition. In some embodiments, the non-human animal has been diagnosed with one or more disorders or conditions.

[0077] The term "polypeptide" includes any polymeric chain of amino acids. In some embodiments, a polypeptide has an amino acid sequence that occurs in nature. In some embodiments, a polypeptide has an amino acid sequence that does not occur in nature. In some embodiments, a polypeptide has an amino acid sequence that includes portions that occur in nature separately from each other (i.e., from two or more different organisms, e.g., human and non-human portions). In some embodiments, a polypeptide has an amino acid sequence that is engineered, in that it is designed and / or produced through the action of man.

[0078] The term "recombinant" is intended to include polypeptides (e.g., the PD-1 polypeptides described herein) that are designed, engineered, prepared, expressed, produced, or isolated by recombinant means, including, for example, polypeptides expressed using a recombinant expression vector transfected into a host cell, polypeptides isolated from a recombinant combinatorial human polypeptide library (Hoogenboom HR, (1997) TIB Tech. 15:62-70; Azzazy H., and Highsmith WE, (2002) Clin. Biochem. 35:425-445; Gavilondo JV, and Larrick JW (2002) BioTechniques 29:128-145; Hoogenboom H., and Chames P. (2000) Immunology Today 21:371-378), antibodies isolated from animals (e.g., mice) transgenic for human immunoglobulin genes (e.g., Taylor, LD, et al. (1992) Nucl. Acids Res. 20:6287-6295; Kellermann SA., and Green LL (2002) Current Opinion in Biotechnology 13:593-597; Little M. et al. (2000) Immunology Today 21:364-370; Murphy, AJ, et al. (2014) Proc. Natl. Acad. Sci. USA 111(14):5153-5158), or any other means involving splicing selected sequence elements together. In some embodiments, one or more of such selected sequence elements occur in nature. In some embodiments, one or more of such selected sequence elements are designed in silico. In some embodiments, one or more such selected sequence elements result, for example, from mutagenesis (e.g., in vivo or in vitro) of known sequence elements of natural or synthetic origin.For example, in some embodiments, a recombinant polypeptide consists of a sequence found in the genome of a source organism of interest (e.g., human, mouse, etc.). In some embodiments, a recombinant polypeptide has an amino acid sequence that results from mutagenesis (e.g., in vitro or in vivo in a non-human animal), and thus the amino acid sequence of the recombinant polypeptide is derived from a polypeptide sequence that does not naturally occur in the genome of the non-human animal in vivo.

[0079] The term "replacement" includes a process through which a "replacement" nucleic acid sequence (e.g., a gene) found in a host locus (e.g., in a genome) is removed from that locus and a different "replacement" nucleic acid is placed in its place. In some embodiments, the replaced nucleic acid sequence and the replacement nucleic acid sequence are equivalent to each other, e.g., in that they are homologous to each other and / or contain corresponding elements (e.g., protein-coding elements, regulatory elements, etc.). In some embodiments, the replaced nucleic acid sequence includes one or more of a promoter, enhancer, splice donor site, splice acceptor site, intron, exon, untranslated region (UTR), and in some embodiments, the replacement nucleic acid sequence includes one or more coding sequences. In some embodiments, the replacement nucleic acid sequence is a homolog of the replaced nucleic acid sequence. In some embodiments, the replacement nucleic acid sequence is an ortholog of the replaced sequence. In some embodiments, the replacement nucleic acid sequence is or comprises a human nucleic acid sequence. In some embodiments, the replaced nucleic acid sequence is or comprises a rodent sequence (e.g., a mouse or rat sequence), including when the replacement nucleic acid sequence is or comprises a human nucleic acid sequence. The nucleic acid sequence so positioned may include one or more regulatory sequences (e.g., promoters, enhancers, 5'- or 3'-untranslated regions, etc.) that were part of the source nucleic acid sequence used to obtain the sequence so positioned. For example, in various embodiments, the substitution is of an endogenous sequence with a heterologous sequence resulting in production of a gene product from the nucleic acid sequence so positioned (comprising the heterologous sequence), but not substitution for expression of the endogenous sequence. The substitution is of an endogenous genomic sequence with a nucleic acid sequence encoding a polypeptide having a similar function to the polypeptide encoded by the endogenous sequence (e.g., the endogenous genomic sequence encodes a PD-1 polypeptide and the DNA fragment encodes one or more human PD-1 polypeptides). In various embodiments, an endogenous gene or fragment thereof is replaced with a corresponding human gene or fragment thereof.The corresponding human gene or fragment thereof is an ortholog of, or a human gene or fragment substantially similar or identical in structure and / or function to, the endogenous gene or fragment being replaced.

[0080] The term "programmed cell death 1 protein" or "PD-1 protein" includes a type I transmembrane protein belonging to the CD28 / CTLA-4 family of T cell regulatory factors. The PD-1 protein structure includes an intracellular amino-terminal immunoglobulin V domain, a transmembrane domain, and a carboxy-terminal extracellular tail, the latter of which contains an immunoreceptor tyrosine-dependent inhibitory motif (ITIM) and an immunoreceptor tyrosine-dependent switch motif. PD-1 is expressed on the cell surface and interacts with PD-L1 and PD-L2, members of the B7 family of immunoregulatory ligands (Collins, M. et al. (2005) Genome Biol. 6:223). PD-1 is expressed, inter alia, on activated T cells, B cells, macrophages, monocytes, and mast cells, as well as in many tumors. PD-1 has been shown to be involved in the negative regulation of immune responses, particularly T cell responses. By way of example, the nucleotide and amino acid sequences of the mouse and human Pdcd1 genes, which encode the PD-1 protein, are provided in Figure 8. One of skill in the art, upon reading this disclosure, will recognize that one or more Pdcd1 genes (or all) in a genome can be replaced with one or more heterologous Pdcd1 genes (e.g., polymorphic variants, subtypes or mutants, genes from other species, humanized forms, etc.).

[0081] "PD-1-expressing cells" include cells that express the PD-1 type I membrane protein. In some embodiments, PD-1-expressing cells express the PD-1 type I membrane protein on their surface. In some embodiments, the PD-1 protein is expressed on the surface of the cell in an amount sufficient to mediate cell-cell interactions. Exemplary PD-1-expressing cells include B cells, macrophages, and T cells. PD-1-expressing cells regulate various cellular processes through the interaction of PD-1 expressed on the surface of immune cells (e.g., T and B cells) and play a role in the differentiation and fate determination of such cells. In some embodiments, the non-human animals of the invention exert regulation of various cellular processes (as described herein) through a humanized PD-1 protein expressed on the surface of one or more cells of the non-human animal. In some embodiments, the non-human animals of the invention exert negative regulation of signaling through the T cell receptor (TCR) through a humanized PD-1 protein expressed on the surface of one or more cells of the non-human animal. In some embodiments, the non-human animal exerts negative regulation of the immune response via a humanized PD-1 protein expressed on the surface of one or more cells of the non-human animal.

[0082] The term "reference" includes a standard or control agent, cohort, individual, population, sample, sequence, or value to which a subject agent, animal, cohort, individual, population, sample, sequence, or value is compared. In some embodiments, the reference agent, cohort, individual, population, sample, sequence, or value is tested and / or determined substantially contemporaneously with the testing or determination of the subject agent, cohort, individual, population, sample, sequence, or value. In some embodiments, the reference agent, cohort, individual, population, sample, sequence, or value is a historical reference, optionally embodied in a tangible medium. In some embodiments, a reference may refer to a control. As used herein, "reference" may include a "reference animal." A "reference animal" may have a modification described herein, a modification different from those described herein, or may be unmodified (i.e., a wild-type animal). Generally, as will be understood by one of skill in the art, a reference agent, animal, cohort, individual, population, sample, sequence, or value is determined or characterized under conditions equivalent to those used to determine or characterize the agent, animal (e.g., mammal), cohort, individual, population, sample, sequence, or value of interest.

[0083] The term "substantially" includes the qualitative condition of indicating the complete or near complete extent or degree of a characteristic or property of interest. Those skilled in the art of biology will understand that biological and chemical phenomena rarely, if ever, proceed to completion and / or to a perfect state or achieve or avoid an absolute result. Thus, the term "substantially" is used herein to capture the possible lack of completeness inherent in many biological and chemical phenomena.

[0084] The phrase "substantial homology" includes comparisons between amino acid or nucleic acid sequences. As will be appreciated by those skilled in the art, two sequences are generally considered to be "substantially homologous" if they contain homologous residues at corresponding positions. Homologous residues may be identical residues. Alternatively, homologous residues may be non-identical residues with appropriately similar structural and / or functional characteristics. For example, as will be appreciated by those skilled in the art, certain amino acids are commonly classified as "hydrophobic" or "hydrophilic" amino acids, and / or as having "polar" or "non-polar" side chains. Substitution of one amino acid with another amino acid of the same type is often considered a "homologous" substitution. Common amino acid classifications are summarized in Tables 1 and 2. [Table 1] [Table 2]

[0085] As is well known in the art, amino acid or nucleic acid sequences can be compared using any of a variety of algorithms, including those available in commercially available computer programs such as BLASTN and BLASTP for nucleotide sequences, gapped BLAST, and PSI-BLAST for amino acid sequences. Exemplary such programs are described in Altschul et al. (1990) Basic local alignment search tool, J. Mol. Biol., 215(3): 403-410; Altschul et al. (1997) Methods in Enzymology; Altschul et al., "Gapped BLAST and PSI-BLAST: a new generation of protein database search programs", Nucleic Acids Res. 25:3389-3402; Baxevanis et al. (1998) Bioinformatics: A Practical Guide to the Analysis of Genes and Proteins, Wiley; and Misener et al. (eds.) (1999) Bioinformatics Methods and Protocols (Methods in Molecular Biology, Vol. 132), Humana Press. In addition to identifying homologous sequences, the above-mentioned programs generally provide an indication of the degree of homology. In some embodiments, two sequences are considered to be substantially homologous if at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more of their corresponding residues are homologous over the relevant stretch of residues. In some embodiments, the relevant stretch is a complete sequence. In some embodiments, the relevant stretch is at least 9, 10, 11, 12, 13, 14, 15, 16, 17 or more residues. In some embodiments, the relevant stretch includes adjacent residues along the complete sequence.In some embodiments, the relevant section comprises non-contiguous residues along the entire sequence, hi some embodiments, the relevant section is at least 10, 15, 20, 25, 30, 35, 40, 45, 50 or more residues.

[0086] The phrase "substantial identity" includes comparisons between amino acid or nucleic acid sequences. As will be understood by those skilled in the art, two sequences are generally considered to be "substantially identical" if they contain identical residues at corresponding positions. As is well known in the art, amino acid or nucleic acid sequences can be compared using any of a variety of algorithms, including those available in commercially available computer programs, such as BLASTN and BLASTP for nucleotide sequences, gapped BLAST, and PSI-BLAST for amino acid sequences. Exemplary such programs are described in Altschul et al. (1990) Basic local alignment search tool, J. Mol. Biol., 215(3): 403-410; Altschul et al., Methods in Enzymology; Altschul et al. (1997) Nucleic Acids Res. 25:3389-3402; Baxevanis et al. (1998) Bioinformatics: A Practical Guide to the Analysis of Genes and Proteins, Wiley; and Misener et al., (eds.) (1999) Bioinformatics Methods and Protocols (Methods in Molecular Biology, Vol. 132), Humana Press. In addition to identifying identical sequences, the above-mentioned programs typically provide an indication of the degree of identity. In some embodiments, two sequences are considered to be substantially identical if at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more of their corresponding residues are identical over the relevant stretch of residues. In some embodiments, the relevant stretch is the complete sequence. In some embodiments, the relevant stretch is at least 10, 15, 20, 25, 30, 35, 40, 45, 50 or more residues.

[0087] The phrase "targeting vector" or "targeting construct" includes a polynucleotide molecule comprising a targeting region. The targeting region comprises a sequence identical or substantially identical to a sequence in a target cell, tissue, or animal, allowing the targeting construct to integrate into a location within the genome of the cell, tissue, or animal by homologous recombination. Also included are targeting regions that are targeted using site-specific recombinase recognition sites (e.g., loxP or Frt sites). In some embodiments, the targeting constructs of the present invention further comprise other nucleic acid sequences that allow for recombination mediated through the exogenous addition of a nucleic acid sequence or gene of particular interest, a selectable marker, control and / or regulatory sequences, and proteins that support or promote recombination involving such sequences. In some embodiments, the targeting constructs of the present invention further comprise all or a portion of a gene of interest, where the gene of interest is a heterologous gene that encodes all or a portion of a protein with a similar function to that encoded by the endogenous sequence. In some embodiments, the targeting construct of the present invention further comprises all or a portion of a humanized gene of interest, wherein the humanized gene of interest encodes all or a portion of a protein with a similar function to the protein encoded by the endogenous sequence.

[0088] The phrase "therapeutically effective amount" includes an amount that produces a desired effect in a subject to which it is administered. In some embodiments, the term refers to an amount sufficient to treat a disease, disorder, and / or condition when administered to a subject (e.g., an animal) suffering from or susceptible to the disease, disorder, and / or condition in accordance with a therapeutic dosing regimen. In some embodiments, a therapeutically effective amount is one that reduces the incidence and / or severity of, and / or delays the onset of, one or more symptoms of the disease, disorder, and / or condition. Those skilled in the art will appreciate that the term "therapeutically effective amount" does not necessarily require that successful treatment be achieved in a particular individual. Rather, a therapeutically effective amount may be an amount that, when administered to subjects in need of such treatment, provides a particular desired pharmacological response in a significant number of subjects. In some embodiments, reference to a therapeutically effective amount may be a reference to the amount measured in one or more specific tissues (e.g., tissues affected by a disease, disorder, or condition) or bodily fluids (e.g., blood, saliva, serum, sweat, tears, urine, etc.). Those skilled in the art will appreciate that in some embodiments, a therapeutically effective amount of a particular agent or treatment may be formulated and / or administered in a single dose, while in some embodiments, a therapeutically effective amount may be formulated and / or administered in multiple doses, e.g., as part of a dosing regimen.

[0089] The term "treatment" (also "treat" or "treating"), in its broadest sense, includes any administration of a substance (e.g., a provided composition) that partially or completely alleviates, ameliorates, mitigates, inhibits, delays the onset of, and / or reduces the incidence of, one or more symptoms, characteristics, and / or causes of a particular disease, disorder, and / or condition. In some embodiments, such treatment may be administered to subjects who do not exhibit signs of the associated disease, disorder, and / or condition and / or who exhibit only early signs of the disease, disorder, and / or condition. Alternatively, or in addition, in some embodiments, treatment may be administered to subjects who exhibit one or more established signs of the associated disease, disorder, and / or condition. In some embodiments, treatment may be administered to subjects who have been diagnosed as suffering from the associated disease, disorder, and / or condition. In some embodiments, treatment may be administered to subjects who are known to have one or more susceptibility factors that statistically correlate with an increased risk of developing the associated disease, disorder, and / or condition.

[0090] The term "variant" includes an entity that exhibits substantial structural identity with a reference entity, but differs structurally from the reference entity by the presence of one or more chemical moieties relative to the reference entity. In many embodiments, a "variant" also differs functionally from the reference entity. Generally, whether a particular entity is properly considered a "variant" of a reference entity is based on its degree of structural identity with the reference entity. As one of ordinary skill in the art will appreciate, all biological or chemical reference entities possess certain characteristic structural elements. A "variant," by definition, is a distinct chemical entity that shares one or more such characteristic structural elements. To give some examples, small molecules may have a characteristic core structural element (e.g., a macrocyclic core) and / or one or more characteristic pendant moieties; variants of small molecules may therefore share the core structural element and characteristic pendant moieties but differ in the type of linkages (single versus double, E versus Z, etc.) present in the other pendant moieties and / or core; polypeptides may have characteristic sequence elements consisting of multiple amino acids that have designated positions relative to each other in linear or three-dimensional space and / or that contribute to a particular biological function; and nucleic acids may have characteristic sequence elements consisting of multiple nucleotide residues that have designated positions relative to each other in linear or three-dimensional space. For example, a "variant polypeptide" may differ from a reference polypeptide as a result of one or more differences in amino acid sequence and / or one or more differences in chemical moieties (e.g., carbohydrates, lipids, etc.) covalently attached to the polypeptide backbone. In some embodiments, a "variant polypeptide" exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 99% overall sequence identity with a reference polypeptide. Alternatively, or in addition, in some embodiments, a "variant polypeptide" does not share at least one characteristic sequence element with a reference polypeptide. In some embodiments, the reference polypeptide possesses one or more biological activities. In some embodiments, a "variant polypeptide" shares one or more biological activities of the reference polypeptide.In some embodiments, a "variant polypeptide" lacks one or more biological activities of a reference polypeptide. In some embodiments, a "variant polypeptide" exhibits a reduced level of one or more biological activities compared to the reference polypeptide. In many embodiments, a subject polypeptide is considered a "variant" of a parent or reference polypeptide if the subject polypeptide has an amino acid sequence identical to that of the parent, except for minor sequence changes at specific positions. Typically, less than 20%, 15%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, or 2% of the variant's residues are substituted compared to the parent. In some embodiments, a "variant" has 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 substituted residue compared to the parent. Often, a "variant" has very few (e.g., less than 5, 4, 3, 2, or 1) functional residues (i.e., residues that participate in a particular biological activity) substituted. Furthermore, a "variant" typically has no more than 5, 4, 3, 2, or 1 additions or deletions, and often no additions or deletions compared to the parent. Furthermore, any additions or deletions will generally be less than about 25, about 20, about 19, about 18, about 17, about 16, about 15, about 14, about 13, about 10, about 9, about 8, about 7, about 6, and usually about 5, about 4, about 3, or about 2 residues. In some embodiments, the parent or reference polypeptide is one found in nature. As one of skill in the art will appreciate, multiple variants of a particular polypeptide of interest may typically be found in nature, particularly when the polypeptide of interest is an infectious agent peptide.

[0091] The term "vector" includes a nucleic acid molecule capable of transporting another nucleic acid to which it is associated. In some embodiments, vectors are capable of extrachromosomal replication and / or expression of nucleic acids to which they are linked in host cells, such as eukaryotic and / or prokaryotic cells. Vectors capable of directing the expression of genes to which they are operatively linked are referred to herein as "expression vectors."

[0092] The term "wild-type" includes entities that have a structure and / or activity found in nature in a "normal" (as opposed to mutant, diseased, altered, etc.) state or condition. Those skilled in the art will understand that wild-type genes and polypeptides often exist in multiple alternative forms (e.g., alleles).

[0093] (Mode for Carrying Out the Invention) The present invention provides, inter alia, improved and / or genetically engineered non-human animals with humanized genetic material encoding programmed cell death 1 (Pdcd1) for determining the therapeutic efficacy of Pdcd1 modulators (e.g., anti-PD-1 antibodies) for the treatment of cancer and for assaying T cell responses and signaling. Such non-human animals provide improved determination of the therapeutic efficacy of PD-1 modulators and their ability to inhibit PD-1. Thus, the present invention is particularly useful for the development of anti-PD-1 therapies for the treatment of various cancers and for enhancing immune responses to treat and / or eliminate viral infections in non-human animals. In particular, the present invention encompasses the humanization of the mouse Pdcd1 gene, resulting in the expression of a humanized PD-1 protein on the surface of cells in the non-human animal. Such humanized PD-1 proteins can be used in assays of human PD-1 to determine the efficacy of anti-PD-1 therapeutics in promoting anti-tumor immune responses. +The non-human animals of the invention are capable of providing a source of human PD-1 cells. In some embodiments, the non-human animals of the invention exhibit an enhanced immune response via inhibition of PD-1 signaling through a humanized PD-1 protein expressed on the surface of the non-human animal's cells. In some embodiments, the humanized PD-1 protein has a sequence corresponding to all or a portion of the N-terminal immunoglobulin V region of the human PD-1 protein. In some embodiments, the humanized PD-1 protein has a sequence corresponding to the intracellular tail of the mouse PD-1 protein, and in some embodiments, the transmembrane region and intracellular tail of the mouse PD-1 protein. In some embodiments, the humanized PD-1 protein has a sequence corresponding to amino acid residues 21-170 (or 26-169, 27-169, or 27-145, or 35-145) of the human PD-1 protein. In some embodiments, the non-human animals of the invention comprise an endogenous Pdcd1 gene comprising genetic material from the non-human animal and a heterologous species (e.g., human). In some embodiments, non-human animals of the invention comprise a humanized Pdcd1 gene, wherein the humanized Pdcd1 gene comprises all or part of exon 2 and exon 3 of the human PDCD1 gene. In some embodiments, non-human animals of the invention comprise a humanized Pdcd1 gene, wherein the humanized Pdcd1 gene comprises 883 bp of human PDCD1 corresponding to exon 2 and the first 71 bp of exon 3 of the human PDCD1 gene (i.e., encoding the stalk).

[0094] Various aspects of the present invention are described in detail in the following sections. The use of the sections is not intended to limit the invention. Each section may be applicable to any aspect of the present invention. As used herein, the use of "or" means "and / or" unless otherwise specified.

[0095] Programmed cell death 1 (Pdcd1) gene Pdcd1 (also known as CD279) was first discovered as an upregulated gene in T cell hybridomas undergoing apoptosis (Ishida, Y. et al. (1992) EMBO J. 11(11):3887-3895). The Pdcd1 gene consists of five exons encoding PD-1, a type I membrane protein (termed PD-1) containing an N-terminal immunoglobulin V (IgV) region, a stalk (~20 amino acids long), a transmembrane region, and an intracellular tail containing both immunoreceptor tyrosine-dependent inhibitory motifs (ITIMs) and immunoreceptor tyrosine-dependent switch motifs (ITSMs). PD-1 is expressed on many cell types, including B cells, dendritic cells, activated monocytes, natural killer (NK) cells, and activated T cells (Keir, ME, et al. (2008) Annu. Rev. Immunol. 26:677-704). Various splice variants of PD-1 have also been reported, differing based on which exons are missing (Nielsen, C. et al. (2005) Cell. Immunol. 235:109-116). Indeed, certain splice variants have been observed as causative factors in autoimmune diseases (Wan, B. et al. (2006) J. Immunol. 177(12):8844-8850). Furthermore, Pdcd1-deficient mice have been reported to develop autoimmune conditions (Nishimura, H. et al. (1998) Intern. Immunol. 10(10):1563-1572; Nishimura, H. et al. (1999) Immunity 11:141-151; Nishimura, H. et al. (2001) Science 291:319-322), suggesting that PD-1 signaling may play a role in preventing the development of autoimmune diseases by consolidating PD-1 as a negative regulator of activated lymphocytes. Interestingly, tumors have been found to use PD-1 signaling to evade immune system surveillance.Accordingly, PD-1 and at least one of its ligands (i.e., PD-L1) are currently being investigated as targets for cancer therapy by promoting the anti-tumor activity of the tumor microenvironment through PD-1 inhibition (see, e.g., Pedoeem, A. et al. (2014) Clin. Immunol. 153:145-152; and Phillips, GK and Atkins, M. (2014) Intern. Immunol. p. 8).

[0096] A more thorough and detailed understanding of PD-1-mediated functions and the PD-1 pathway is needed to develop practical targeted therapies for future cancer treatment.

[0097] Pdcd1 and PD-1 sequences Exemplary mouse, human, and humanized Pdcd1 and PD-1 sequences are set forth in Figure 8. Exemplary human nucleic acid sequences for humanization of non-human Pdcd1 genes are also set forth in Figure 8.

[0098] Humanized Pdcd1 non-human animals Non-human animals are provided that express a humanized PD-1 protein on the cell surface of the non-human animal resulting from genetic modification of the non-human animal's endogenous locus (e.g., the Pdcd1 locus) that encodes the PD-1 protein. Suitable examples described herein include rodents, particularly mice.

[0099] In some embodiments, the humanized Pdcd1 gene comprises genetic material from a heterologous species (e.g., human), and the humanized Pdcd1 gene encodes a PD-1 protein comprising a coding portion of the genetic material from the heterologous species. In some embodiments, the humanized Pdcd1 gene of the invention comprises genomic DNA from the heterologous species that encodes the extracellular portion of the PD-1 protein that is expressed on the plasma membrane of a cell. Non-human animals, embryos, cells, and targeting constructs for producing non-human animals, non-human embryos, and cells comprising the humanized Pdcd1 gene are also provided.

[0100] In some embodiments, the endogenous Pdcd1 gene is defective. In some embodiments, the endogenous Pdcd1 is altered, such that a portion of the endogenous Pdcd1 gene is replaced with a heterologous species sequence (e.g., all or a portion of the human PDCD1 sequence). In some embodiments, all or substantially all of the endogenous Pdcd1 gene is replaced with a heterologous gene (e.g., a human PDCD1 gene). In some embodiments, a portion of a heterologous Pdcd1 gene is inserted into the endogenous Pdcd1 locus of an endogenous non-human Pdcd1 gene. In some embodiments, the heterologous gene is a human gene. In some embodiments, modification or humanization is performed on one of the two copies of the endogenous Pdcd1 gene, resulting in a non-human animal heterozygous for the humanized Pdcd1 gene. In other embodiments, non-human animals homozygous for the humanized Pdcd1 gene are provided.

[0101] In various embodiments, the non-human animal comprises all or part of a human PDCD1 gene at the endogenous non-human Pdcd1 locus. Thus, such non-human animals can be described as having a heterologous Pdcd1 gene. A replaced, inserted, modified, or altered Pdcd1 gene at the endogenous Pdcd1 locus, or a protein expressed from such a gene, can be detected using a variety of methods, including, for example, PCR, Western blot, Southern blot, restriction fragment length polymorphism (RFLP), or allele gain or loss assay. In some embodiments, the non-human animal is heterozygous for the humanized Pdcd1 gene.

[0102] In various embodiments, a humanized Pdcd1 gene according to the present invention comprises a Pdcd1 gene having a second exon with a sequence that is at least 50% (e.g., 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) identical to the second exon found in the human PDCD1 gene of Figure 8.

[0103] In various embodiments, a humanized Pdcd1 gene according to the present invention comprises a Pdcd1 gene having a second exon with a sequence substantially identical to the second exon found in the human PDCD1 gene of FIG.

[0104] In various embodiments, a humanized Pdcd1 gene according to the present invention comprises a Pdcd1 gene having a second exon with a sequence identical to the second exon found in the human PDCD1 gene of FIG.

[0105] In various embodiments, a humanized Pdcd1 gene according to the present invention comprises a Pdcd1 gene having a third exon with a sequence that is at least 50% (e.g., 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) identical to the third exon found in the humanized Pdcd1 mRNA sequence of Figure 8.

[0106] In various embodiments, a humanized Pdcd1 gene according to the present invention comprises a Pdcd1 gene having a third exon with a sequence substantially identical to the third exon found in the humanized Pdcd1 mRNA sequence of FIG.

[0107] In various embodiments, a humanized Pdcd1 gene according to the present invention comprises a Pdcd1 gene having a third exon with a sequence identical to the third exon found in the humanized Pdcd1 mRNA sequence of FIG.

[0108] In various embodiments, the humanized Pdcd1 gene according to the present invention comprises a Pdcd1 gene having a sequence at least 50% (e.g., 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) identical to SEQ ID NO:21 or SEQ ID NO:23.

[0109] In various embodiments, a humanized Pdcd1 gene according to the present invention comprises a Pdcd1 gene with a sequence substantially identical to SEQ ID NO:21 or SEQ ID NO:23.

[0110] In various embodiments, a humanized Pdcd1 gene according to the present invention comprises a Pdcd1 gene with a sequence identical to SEQ ID NO:21 or SEQ ID NO:23.

[0111] In various embodiments, a humanized Pdcd1 gene according to the present invention comprises a Pdcd1 gene having a portion of a second exon and a portion of a third exon, each of which has a sequence that is at least 50% (e.g., 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) identical to a portion of the second exon and the third exon found in the human PDCD1 gene of Figure 8.

[0112] In various embodiments, a humanized Pdcd1 gene according to the present invention comprises a Pdcd1 gene having first, fourth, and fifth exons, each of which has a sequence that is at least 50% (e.g., 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) identical to the first, fourth, and fifth exons found in the mouse Pdcd1 gene of Figure 8.

[0113] In various embodiments, a humanized Pdcd1 gene according to the present invention comprises a Pdcd1 gene having a first, a portion of a third, a fourth and a fifth exon, each of which has at least 50% (e.g., 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) sequence identity to the first, a portion of a third, a fourth and a fifth exon found in the mouse Pdcd1 gene of Figure 8.

[0114] In various embodiments, a humanized Pdcd1 gene according to the present invention comprises a Pdcd1 gene having 5' untranslated and 3' untranslated regions that have sequences that are at least 50% (e.g., 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more), identical to the 5' and 3' untranslated regions found in the mouse Pdcd1 gene of Figure 8.

[0115] In various embodiments, a humanized Pdcd1 gene according to the present invention comprises a Pdcd1 gene having a nucleotide coding sequence (e.g., a cDNA sequence) that is at least 50% (e.g., 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) identical to the nucleotide coding sequence found in the humanized Pdcd1 nucleotide coding sequence of Figure 8.

[0116] In various embodiments, a humanized Pdcd1 mRNA sequence according to the present invention comprises a sequence that is at least 50% (e.g., 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) identical to the humanized mRNA sequence found in Figure 8.

[0117] In various embodiments, a humanized Pdcd1 gene according to the present invention encodes a PD-1 polypeptide having an amino acid sequence that is at least 50% identical (e.g., 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) to the amino acid sequence found in the PD-1 polypeptide sequence of Figure 8.

[0118] In various embodiments, the humanized PD-1 protein produced by the non-human animals of the invention has an extracellular portion with an amino acid sequence that is at least 50% identical (e.g., 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) to the extracellular portion of the human PD-1 protein shown in Figure 8.

[0119] In various embodiments, the humanized PD-1 protein produced by the non-human animals of the invention has an extracellular portion that comprises an amino acid sequence at least 50% (e.g., 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) identical to amino acid residues 21-170 found in the human or humanized PD-1 protein of Figure 8.

[0120] In various embodiments, the humanized PD-1 protein produced by the non-human animals of the invention has an extracellular portion that comprises an amino acid sequence substantially identical to amino acid residues 21-170 found in the human or humanized PD-1 protein of Figure 8.

[0121] In various embodiments, the humanized PD-1 protein produced by the non-human animals of the invention has an extracellular portion that comprises an amino acid sequence identical to amino acid residues 21-170 found in the human or humanized PD-1 protein of Figure 8.

[0122] In various embodiments, the humanized PD-1 protein produced by the non-human animals of the invention has an extracellular portion that comprises an amino acid sequence at least 50% (e.g., 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) identical to amino acid residues 26-169 found in the human or humanized PD-1 protein of Figure 8.

[0123] In various embodiments, the humanized PD-1 protein produced by the non-human animals of the invention has an extracellular portion that comprises an amino acid sequence substantially identical to amino acid residues 26-169 found in the human or humanized PD-1 protein of Figure 8.

[0124] In various embodiments, the humanized PD-1 protein produced by the non-human animals of the invention has an extracellular portion that comprises an amino acid sequence identical to amino acid residues 26-169 found in the human or humanized PD-1 protein of Figure 8.

[0125] In various embodiments, the humanized PD-1 protein produced by the non-human animals of the invention has an extracellular portion that comprises an amino acid sequence at least 50% (e.g., 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) identical to amino acid residues 27-169 found in the human or humanized PD-1 protein of Figure 8.

[0126] In various embodiments, the humanized PD-1 protein produced by the non-human animals of the invention has an extracellular portion that comprises an amino acid sequence substantially identical to amino acid residues 27-169 found in the human or humanized PD-1 protein of Figure 8.

[0127] In various embodiments, the humanized PD-1 protein produced by the non-human animals of the invention has an extracellular portion that comprises an amino acid sequence identical to amino acid residues 27-169 found in the human or humanized PD-1 protein of Figure 8.

[0128] In various embodiments, the humanized PD-1 protein produced by the non-human animals of the invention has an extracellular portion that comprises an amino acid sequence at least 50% (e.g., 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) identical to amino acid residues 27-145 found in the human or humanized PD-1 protein of Figure 8.

[0129] In various embodiments, the humanized PD-1 protein produced by the non-human animals of the invention has an extracellular portion that comprises an amino acid sequence substantially identical to amino acid residues 27-145 found in the human or humanized PD-1 protein of Figure 8.

[0130] In various embodiments, the humanized PD-1 protein produced by the non-human animals of the invention has an extracellular portion that comprises an amino acid sequence identical to amino acid residues 27-145 found in the human or humanized PD-1 protein of Figure 8.

[0131] In various embodiments, the humanized PD-1 protein produced by the non-human animals of the invention has an extracellular portion that comprises an amino acid sequence at least 50% (e.g., 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) identical to amino acid residues 35-145 found in the human or humanized PD-1 protein of Figure 8.

[0132] In various embodiments, the humanized PD-1 protein produced by the non-human animals of the invention has an extracellular portion that comprises an amino acid sequence substantially identical to amino acid residues 35-145 found in the human or humanized PD-1 protein of Figure 8.

[0133] In various embodiments, the humanized PD-1 protein produced by the non-human animals of the invention has an extracellular portion that comprises an amino acid sequence identical to amino acid residues 35-145 found in the human or humanized PD-1 protein of Figure 8.

[0134] In various embodiments, the humanized PD-1 proteins produced by the non-human animals of the invention have an N-terminal immunoglobulin V region with an amino acid sequence at least 50% identical (e.g., 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) to the N-terminal immunoglobulin V region of a human or humanized PD-1 protein shown in Figure 8.

[0135] In various embodiments, the humanized PD-1 proteins produced by the non-human animals of the invention have an N-terminal immunoglobulin V region with substantially identical amino acid sequence to the N-terminal immunoglobulin V region found in the human or humanized PD-1 protein of Figure 8.

[0136] In various embodiments, the humanized PD-1 proteins produced by the non-human animals of the invention have an N-terminal immunoglobulin V region with an amino acid sequence identical to the N-terminal immunoglobulin V region found in the human or humanized PD-1 protein of Figure 8.

[0137] In various embodiments, the humanized PD-1 protein produced by the non-human animals of the invention has a transmembrane region with at least 50% (e.g., 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) sequence identity to the transmembrane region of the murine PD-1 protein shown in Figure 8.

[0138] In various embodiments, the humanized PD-1 proteins produced by the non-human animals of the invention have an intracellular tail with at least 50% (e.g., 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) sequence identity to the intracellular tail of the mouse PD-1 protein shown in Figure 8.

[0139] In various embodiments, the humanized PD-1 protein produced by the non-human animals of the invention has an amino acid sequence that is at least 50% identical (e.g., 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) to amino acid residues 27-169 (or 26-169) found in the human PD-1 protein of Figure 8.

[0140] In various embodiments, the humanized PD-1 protein produced by the non-human animals of the invention has an amino acid sequence substantially identical to amino acid residues 27-169 (or 26-169) found in the human PD-1 protein of Figure 8.

[0141] In various embodiments, the humanized PD-1 protein produced by the non-human animals of the invention has an amino acid sequence identical to amino acid residues 27-169 (or 26-169) found in the human PD-1 protein of Figure 8.

[0142] In various embodiments, the humanized PD-1 protein produced by the non-human animals of the invention has an amino acid sequence that is at least 50% identical (e.g., 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) to the amino acid sequence of the humanized PD-1 protein found in Figure 8.

[0143] In various embodiments, the humanized PD-1 protein produced by the non-human animals of the present invention has an amino acid sequence substantially identical to the amino acid sequence of the humanized PD-1 protein found in FIG.

[0144] In various embodiments, the humanized PD-1 protein produced by the non-human animals of the invention has an amino acid sequence identical to the amino acid sequence of the humanized PD-1 protein found in FIG.

[0145] Compositions and methods are provided for generating non-human animals that express humanized PD-1 proteins, including specific polymorphic forms, allelic variants (e.g., single amino acid differences), or alternatively spliced isoforms, including compositions and methods for generating non-human animals that express such proteins from a human promoter and human regulatory sequences. In some embodiments, compositions and methods are also provided for generating non-human animals that express such proteins from an endogenous promoter and endogenous regulatory sequences. In some specific embodiments, the endogenous promoter and endogenous regulatory sequences are endogenous rodent promoter and endogenous rodent regulatory sequences. The methods include inserting genetic material encoding all or part of the human PD-1 protein into the genome of the non-human animal at a precise location that corresponds to the endogenous Pdcd1 gene, thereby generating a humanized Pdcd1 gene that expresses a PD-1 protein that is all or partly human. In some embodiments, the method comprises inserting genomic DNA corresponding to all or part of exon 2 and exon 3 of the human PDCD1 gene into the endogenous Pdcd1 gene of the non-human animal, thereby generating a humanized gene encoding a PD-1 protein that includes a human portion containing the amino acids encoded by the inserted exons.

[0146] Where appropriate, the coding region or polynucleotide sequence of genetic material encoding all or a portion of a human (or humanized) PD-1 protein may be modified to contain codons optimized for expression from cells of a non-human animal (see, e.g., U.S. Patent Nos. 5,670,356 and 5,874,304). A codon-optimized sequence is preferably a synthetic sequence that encodes the same polypeptide (or a biologically active fragment of a full-length polypeptide that is substantially the same biologically active as the full-length polypeptide) encoded by a non-codon-optimized parent polynucleotide. In some embodiments, the coding region of genetic material encoding all or a portion of a human (or humanized) PD-1 protein may contain a sequence that has been altered to optimize codon usage for a particular cell type (e.g., rodent cells). For example, the codons of genomic DNA corresponding to a portion (e.g., 71 bp) of exon 2 and exon 3 of the human PDCD1 gene, inserted into the endogenous Pdcd1 gene of a non-human animal (e.g., rodent), may be optimized for expression in cells of the non-human animal. Such sequences are sometimes described as codon-optimized sequences.

[0147] The humanized Pdcd1 gene approach utilizes relatively minimal modification of the endogenous gene to provide natural PD-1-mediated signaling in non-human animals, because, in various embodiments, the genomic sequence of the Pdcd1 sequence is modified in a single fragment, thus maintaining normal function by including necessary regulatory sequences. Thus, in such embodiments, the Pdcd1 gene modification does not affect other surrounding genes or other endogenous Pdcd1-interacting genes (e.g., PD-L1, PD-L2, etc.). Furthermore, in various embodiments, the modification does not affect the assembly of functional PD-1 transmembrane protein on the cell membrane, maintaining normal effector function via binding and subsequent signaling through the cytoplasmic portion of the protein that is unaffected by the modification.

[0148] A schematic diagram (not to scale) of the genomic structure of the endogenous mouse Pdcd1 gene and the human PDCD1 gene is provided in Figure 1. An exemplary method for humanizing an endogenous mouse Pdcd1 gene using a genomic fragment containing a portion of exon 2 and exon 3 of the human PDCD1 gene is provided in Figure 2. As shown, an 883 bp genomic DNA fragment containing a portion of exon 2 and exon 3 (e.g., the first 71 bp) of the human PDCD1 gene is inserted into the endogenous mouse Pdcd1 locus at a 900 bp sequence by a targeting construct. The 883 bp human DNA fragment may be cloned directly from human DNA or synthesized from a source sequence (e.g., GenBank accession number NM_005018.2). The genomic DNA includes a portion of the gene encoding substantially all of the extracellular portion of the human PD-1 protein involved in ligand binding (e.g., amino acid residues 27-169 or 26-169).

[0149] Non-human animals (e.g., mice) carrying a humanized Pdcd1 gene at the endogenous Pdcd1 locus can be generated by any method known in the art. For example, a targeting vector can be generated that introduces all or part of the human Pdcd1 gene with a selectable marker gene. Figure 2 shows a targeting vector containing the endogenous Pdcd1 locus of the mouse genome with an insert of an 883 bp human DNA fragment containing exon 2 and the first 71 bp of exon 3 of the human PDCD1 gene. As shown, the targeting construct contains a 5' homology arm containing sequences upstream of exon 2 of the endogenous mouse Pdcd1 gene (~61.7 Kb), followed by a drug selection cassette (e.g., a neomycin resistance gene flanked on both sides by loxP sequences; ~5 Kb), a genomic DNA fragment (883 bp) containing exon 2 and the first 71 bp of exon 3 of the human Pdcd1 gene, and the remaining sequence of endogenous mouse exon 3 (i.e., the portion encoding the transmembrane portion of the PD-1 protein), and a 3' homology arm (~84 Kb) containing exon 4 and exon 5 of endogenous mouse Pdcd1. The targeting construct contains an auto-excision drug selection cassette (e.g., a neomycin resistance gene flanked on both sides by loxP sequences; see U.S. Patent Nos. 8,697,851, 8,518,392, and 8,354,389, all of which are incorporated by reference herein). Embryonic stem cells are electroporated to create a modified endogenous Pdcd1 gene that replaces 900 bp of the endogenous wild-type Pdcd1 gene with 883 bp (i.e., exon 2 and the first 71 bp of exon 3) of the human PDCD1 gene, contained in a targeting vector. A humanized Pdcd1 gene is created, resulting in cells or non-human animals that express a humanized PD-1 protein containing amino acids encoded by the 883 bp human DNA fragment (i.e., exon 2 and 71 bp of exon 3 of the human PDCD1 gene). The drug selection cassette is removed in a developmentally dependent manner; i.e., progeny derived from mice containing the above-described humanized Pdcd1 gene in their germline cells shed the selectable marker from differentiated cells during development (see the bottom of Figure 2).

[0150] While embodiments using a humanized mouse Pdcd1 gene (i.e., a mouse having a Pdcd1 gene encoding a PD-1 protein comprising a human protein portion and a mouse portion) are broadly contemplated herein, other non-human animals comprising a humanized Pdcd1 gene are also provided. In some embodiments, such non-human animals comprise a humanized Pdcd1 gene operably linked to a rodent Pdcd1 promoter. In some embodiments, such non-human animals comprise a humanized Pdcd1 gene operably linked to an endogenous Pdcd1 promoter, which in some embodiments is an endogenous rodent Pdcd1 promoter. In some embodiments, such non-human animals express a humanized PD-1 protein from an endogenous locus, wherein the humanized PD-1 protein comprises amino acid residues 21-170 (or 26-169, or 27-169, 27-145, or 35-145) of the human PD-1 protein. Such non-human animals include any of the animals genetically modified to express the PD-1 protein disclosed herein, including, for example, mammals such as mice, rats, rabbits, pigs, cattle (e.g., cows, bulls, buffalo), deer, sheep, goats, chickens, cats, dogs, ferrets, and primates (e.g., marmosets and rhesus monkeys). For example, for non-human animals for which suitable genetically modifiable ES cells are not readily available, other methods are employed to generate non-human animals containing genetic modifications. Such methods include, for example, modifying the genome of a non-ES cell (e.g., a fibroblast or an induced pluripotent cell) and using somatic cell nuclear transfer (SCNT) to introduce the genetically modified genome into a suitable cell, such as an enucleated oocyte, and gestation of the modified cell (e.g., the modified oocyte) into a non-human animal under conditions suitable for the formation of an embryo.

[0151] Methods for modifying non-human animal genomes (e.g., the genomes of pigs, cows, rodents, chickens, etc.) include, for example, using zinc finger nucleases (ZFNs) or transcription activator-like effector nucleases (TALENs) to modify the genome to include a humanized Pdcd1 gene.

[0152] In some embodiments, the non-human animals of the present invention are mammals. In some embodiments, the non-human animals of the present invention are small mammals, for example, of the Jerboidea or Murine superfamily. In some embodiments, the genetically modified animals of the present invention are rodents. In some embodiments, the rodents of the present invention are selected from mice, rats, and hamsters. In some embodiments, the rodents of the present invention are selected from the Murine superfamily. In some embodiments, the genetically modified animals of the present invention are from a family selected from the family Odontoidea (e.g., mouse-like hamsters), Cricetidae (e.g., hamsters, New World rats and mice, and voles), Muridae (pure-breed mice and rats, gerbils, spiny mice, and maned mice), Tetragnathidae (tree mice, rock mice, white-tailed rats, Madagascar rats and mice), Dormiceidae (e.g., spiny dormice), and Moleratidae (e.g., mole rats, bamboo rats, and plateau mole rats). In some embodiments, the genetically modified rodent of the present invention is selected from a pure breed mouse or rat (Muridae), a gerbil, a spiny mouse, and a maned mouse. In some embodiments, the genetically modified mouse of the present invention is from a member of the Muridae family. In some embodiments, the non-human animal of the present invention is a rodent. In some embodiments, the rodent of the present invention is selected from a mouse and a rat. In some embodiments, the non-human animal of the present invention is a mouse.

[0153] In some embodiments, the non-human animal of the invention is a rodent that is a mouse of the C57BL strain 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 some embodiments, the mouse of the present invention is a 129 strain selected from the group consisting of strains that are 129P1, 129P2, 129P3, 129X1, 129S1 (e.g., 129S1 / SV, 129S1 / SvIm), 129S2, 129S4, 129S5, 129S9 / SvEvH, 129 / SvJae, 129S6 (129 / SvEvTac), 129S7, 129S8, 129T1, 129T2 (see, e.g., Festing et al., 1999, Mammalian Genome 10:836; Auerbach, W. et al., 2000, Biotechniques 29(5):1024-1028, 1030, 1032). In some embodiments, the genetically modified mice of the present invention are a mix of the aforementioned 129 strain and the aforementioned C57BL / 6 strain. In some embodiments, the genetically modified mice of the present invention are a mix of the aforementioned 129 strains, or a mix of the aforementioned BL / 6 strains. In some embodiments, the mixed 129 strain described herein is a 129S6 (129 / SvEvTac) strain. In some embodiments, the mice of the present invention are a BALB strain (e.g., a BALB / c strain). In some embodiments, the mice of the present invention are a mix of a BALB strain and another of the aforementioned strains.

[0154] In some embodiments, the non-human animal of the present invention is a rat. In some embodiments, the rat of the present invention is selected from Wistar rats, LEA strains, Sprague Dawley strains, Fischer strains, F344, F6, and Dark Agouti. In some embodiments, the rat strains described herein are a mixture of two or more strains selected from the group consisting of Wistar, LEA, Sprague Dawley, Fischer, F344, F6, and Dark Agouti.

[0155] Methods using non-human animals carrying a humanized Pdcd1 gene Investigations of PD-1 function have employed the use of various Pdcd1 mutants and transgenic non-human animals (e.g., Nishimura, H. et al. (1998) Intern. Immunol. 10(10):1563-1572; Nishimura, H. et al. (1999) Immunity 11:141-151; Nishimura, H. et al. (2001) Science 291:319-322; Iwai, Y. et al. (2004) Intern. Immunol. 17(2):133-144; Keir, ME et al. (2005) J. Immunol. 175:7372-7379; Keir, ME et al. (2007) J. Immunol. 179:5064-5070; Carter, LL et al. (2007) J. Neuroimmunol. 182:124-134; Chen, L. et al. (2007) Europ. Soc. Organ Transplant. 21:21-29; Okazaki, T. et al. (2011) J. Exp. Med. 208(2):395-407; U.S. Patent No. 7,414,171, and European Patent No. 1 334 659 B1, which are incorporated herein by reference.) Such mutant and transgenic animals have been useful in determining the molecular aspects of PD-1 expression, function, and regulation of various cellular processes. However, they have limitations. For example, PD-1-deficient mice generated by knocking in human PD-1 cDNA into exon 1 of the mouse Pdcd1 gene did not express human PD-1 even after stimulation with PMA (Carter, LL et al., supra). Furthermore, considerable phenotypic differences in PD-1 mutant animals of different genetic backgrounds have complicated investigations, especially when attempting to assign various functions and / or regulatory activities to PD-1. Nevertheless, other transgenic animals overexpressing PD-1 have been generated (Chen, L. et al., supra). Such animals exhibit different expression patterns of the transgene, which can reasonably be attributed to the design of the construct.Furthermore, because the same source genetic material (i.e., mice) was used, PD-1 overexpression may have corresponded to endogenous PD-1 rather than transgenic PD-1 due to potential position effects of the transgene. Although PD-1 transgenic mice have proven useful in elucidating some PD-1-mediated functions, they have shown variability in the results obtained, at least in part due to the different approaches used to generate the mice. Thus, current in vivo systems exploiting PD-1-mediated biology are incomplete. Molecular aspects of PD-1-mediated functions and signaling pathways have not been exploited to their full potential in transgenic mice.

[0156] The non-human animals of the invention provide improved in vivo systems and sources of biological material (e.g., cells) expressing human (or humanized) PD-1 useful for various assays. In various embodiments, the non-human animals of the invention are used to develop therapeutics that target PD-1 and / or modulate PD-1 signaling (e.g., interfere with its interaction with PD-L1 and / or PD-L2). In various embodiments, the non-human animals of the invention are used to identify, screen, and / or develop candidate therapeutics (e.g., antibodies) that bind human PD-1. In various embodiments, the non-human animals of the invention are used to screen and develop candidate therapeutics (e.g., antibodies) that inhibit the interaction of human PD-1 with human PD-L1 and / or human PD-L2. In various embodiments, the non-human animals of the invention are used to determine the binding profile of antagonists and / or agonists of humanized PD-1 on the surface of cells of the non-human animals described herein; in some embodiments, the non-human animals of the invention are used to determine the epitope or epitopes of one or more candidate therapeutic antibodies that bind to human PD-1.

[0157] In various embodiments, non-human animals of the invention are used to determine the pharmacokinetic profile of anti-PD-1 antibodies. In various embodiments, one or more non-human animals of the invention and one or more control or reference non-human animals are each exposed to one or more candidate therapeutic anti-PD-1 antibodies at various doses (e.g., 0.1 mg / kg, 0.2 mg / kg, 0.3 mg / kg, 0.4 mg / kg, 0.5 mg / kg, 1 mg / kg, 2 mg / kg, 3 mg / kg, 4 mg / kg, 5 mg / kg, 7.5 mg / kg, 10 mg / kg, 15 mg / kg, 20 mg / kg, 25 mg / kg, 30 mg / kg, 40 mg / kg, or 50 mg / kg or more). Candidate therapeutic antibodies may be administered by any desired route of administration, including parenteral and non-injectable routes of administration. Parenteral routes include, for example, intravenous, intraarterial, intraportal, intramuscular, subcutaneous, intraperitoneal, intrathecal, intrathecal, intraventricular, intracranial, intrapleural, or other routes of infusion. Non-injection routes include, for example, oral, nasal, transdermal, pulmonary, rectal, buccal, vaginal, and ocular. Administration may also be by continuous infusion, topical administration, sustained release from implants (gels, membranes, etc.), and / or intravenous injection. Blood is isolated from non-human animals (humanized and control) at various time points (e.g., 0 hours, 6 hours, 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, or up to 30 days or more). Various assays may be performed to determine the pharmacokinetic profile of the administered candidate therapeutic antibody using samples obtained from the non-human animals described herein, including, but not limited to, total IgG, anti-therapeutic antibody response, agglutination, etc.

[0158] In various embodiments, the non-human animals of the invention are used to measure the therapeutic effect of PD-1 signaling inhibition or modulation and the effect on gene expression as a result of cellular changes. In various embodiments, the non-human animals of the invention, or cells isolated therefrom, are exposed to candidate therapeutic agents that bind to a humanized PD-1 protein (or a human portion of a PD-1 protein) on the cell surface of the non-human animals, and after a subsequent period of time, are analyzed for effects on PD-1-dependent processes, such as adhesion, apoptosis, cytokine production, inflammation, proliferation, self-tolerance, and viral infection (or response).

[0159] The non-human animals of the invention can express humanized PD-1 proteins, and thus cells, cell lines, and cell cultures can be generated to serve as a source of humanized PD-1 for use in binding and functional assays, e.g., to assay the binding or function of PD-1 antagonists or agonists, particularly where the antagonists or agonists are specific for a human PD-1 sequence or epitope, or alternatively, for a human PD-1 sequence or epitope associated with PD-L1 and / or PD-L2. In various embodiments, cells isolated from the non-human animals of the invention can be used to determine the PD-1 epitope bound by a candidate therapeutic antibody. In various embodiments, the humanized PD-1 proteins expressed by the non-human animals described herein may comprise a variant amino acid sequence. Variant human PD-1 proteins (e.g., polymorphisms) associated with autoimmune and infectious diseases have been reported (e.g., Lee, YH et al. (2014) Z. Rheumatol. PMID: 24942602; Mansur, A. et al. (2014) J. Investig. Med. 62(3):638-643; Nasi, M. et al. (2013) Intern. J. Infect. Dis. 17:e845-e850; Piskin, IE et al. (2013) Neuropediatrics 44(4):187-190; Carter, LL et al. (2007) J. Neuroimmunol. 182(1-2):124-134; Wan, B. et al. (2006) J. Immunol. 177(12):8844-8850). Exemplary human PD-1 variants include those listed on the SNP GeneView webpage from NCBI and are summarized in Table 3. In various embodiments, the non-human animals of the invention express humanized PD-1 protein variants. In various embodiments, the variants are polymorphisms at amino acid positions associated with ligand binding. In various embodiments, the non-human animals of the invention are used to determine the effect of ligand binding through interaction with polymorphic variants of human PD-1.In some embodiments, the non-human animals of the invention express a human PD-1 protein variant found in Table 3. [Table 3-1] [Table 3-2] [Table 3-3]

[0160] Cells from the non-human animals of the invention can be isolated and used ad hoc or maintained in culture for many generations, hi various embodiments, cells from the non-human animals of the invention are immortalized (e.g., by the use of a virus) and maintained in culture (e.g., in continuous culture) indefinitely.

[0161] In various embodiments, the cells and / or non-human animals of the invention are used in various immunization regimens to determine PD-1-mediated functions of the immune response to an antigen. In some embodiments, candidate therapeutic agents that bind to or inhibit one or more functions of human (or humanized) PD-1 are characterized in the non-human animals of the invention. Suitable assays include various cellular assays, proliferation assays, serum immunoglobulin analysis (e.g., antibody titers), cytotoxicity assays, and characterization of ligand-receptor interactions (e.g., immunoprecipitation assays). In some embodiments, the non-human animals of the invention are used to characterize PD-1-mediated modulation of functions of the immune response to an antigen. In some embodiments, the antigen is associated with an autoimmune disease, disorder, or condition. In some embodiments, the antigen is associated with an inflammatory disease, disorder, or condition. In some embodiments, the antigen is a test antigen (e.g., ovalbumin or OVA). In some embodiments, the antigen is a target associated with a disease or condition afflicting one or more human patients in need of treatment.

[0162] In various embodiments, the non-human animals of the invention are used in serum assays to determine the titer of autoantibody production to test the pharmacological and toxicological profiles of candidate therapeutics targeting human PD-1. In some embodiments, the autoantibody production in the non-human animals of the invention results from one or more autoimmune diseases, disorders, or conditions induced in the non-human animals.

[0163] In various embodiments, the non-human animals of the present invention are used to challenge with one or more antigens to determine the therapeutic potential of compounds or biologics that modulate PD-1-dependent regulation of immune responses, including, but not limited to, specific T cell-dependent and B cell-dependent responses to certain antigens.

[0164] In various embodiments, the cells and / or non-human animals of the invention are used in survival and / or proliferation assays (e.g., using B or T cells) to screen and develop candidate therapeutics that modulate human PD-1 signaling. Activation or loss of PD-1 plays an important role in regulating T cell responses, and PD-1 regulation of self-tolerance may result from activation of specific epitopes in the extracellular domain of PD-1. Thus, candidate PD-1 modulators (e.g., antagonists or agonists) may be identified, characterized, and developed using the cells of the non-human animals of the invention and / or the non-human animals described herein. In some embodiments, the cells and / or non-human animals of the invention are used in survival or mortality assays to determine the effect on proliferation or apoptosis of specific cells (e.g., cancer cells) in the presence and absence of PD-1.

[0165] In various embodiments, the cells and / or non-human animals of the invention are used in xenotransplantation of heterologous (e.g., human) cells to determine PD-1-mediated functions in physiological (e.g., immune) responses to transplanted human cells or tissues. In some embodiments, candidate therapeutic agents that bind to or inhibit one or more functions of human PD-1 are characterized in the non-human animals of the invention. Suitable assays include various cellular assays, proliferation assays, serum immunoglobulin analysis (e.g., antibody titers), cytotoxicity assays, and characterization of ligand-receptor interactions (immunoprecipitation assays). In some embodiments, the non-human animals of the invention are used to characterize PD-1-mediated functional modulation of immune responses to antigens. In some embodiments, the antigen is associated with a neoplasm. In some embodiments, the antigen is associated with an autoimmune disease, disorder, or condition. In some embodiments, the antigen is associated with an inflammatory disease, disorder, or condition. In some embodiments, the antigen is a target associated with a disease or condition afflicting one or more human patients in need of treatment.

[0166] In various embodiments, the non-human animals of the invention are used in transplantation or adoptive transfer experiments to determine the therapeutic potential of compounds or biologics that modulate PD-1-dependent regulation of new lymphocytes and their immune function. In various embodiments, the non-human animals of the invention are transplanted with human T cells, in some embodiments naive T cells, and in some embodiments, activated T cells.

[0167] In various embodiments, cells of the non-human animals of the invention are used in T cell assays to determine the therapeutic potential of compounds or biologics that modulate PD-1-dependent regulation of T cell-dependent responses and function. Exemplary T cell assays include, but are not limited to, ELISpot, intracellular cytokine staining, major histocompatibility complex (MHC) restriction, viral suppression assays, cytotoxicity assays, proliferation assays, and regulatory T cell suppression assays.

[0168] In various embodiments, cells of the non-human animals of the invention are used in cell migration assays to screen and develop candidate therapeutics that modulate human PD-1. Cell migration involves the movement of cells across the endothelium, and migration assays allow for the measurement of endothelial interaction and migration by leukocytes or tumor cells.

[0169] In various embodiments, the cells of the non-human animals of the invention are used in tumor cell growth (or proliferation) assays to determine the therapeutic ability of compounds to modulate PD-1-dependent regulation and / or apoptosis of tumor cells.

[0170] In various embodiments, the cells of the non-human animals of the invention are used in cytokine production assays to determine the therapeutic potential of compounds or biologics that modulate PD-1-dependent regulation of cytokines released from T cells. In some embodiments, the cells of the non-human animals of the invention are used to detect (and / or measure) intracellular cytokine release resulting from the interaction of humanized PD-1 with a human PD-1 or PD-1 ligand (e.g., PD-L1 or PD-L2)-targeted agent.

[0171] In various embodiments, an autoimmune disease, disorder, or condition is induced in one or more non-human animals of the invention to provide an in vivo system for determining the therapeutic potential of a compound or biological agent that modulates PD-1-dependent regulation of one or more functions of the autoimmune disease, disorder, or condition. Exemplary autoimmune diseases, disorders, or conditions that can be induced in one or more non-human animals of the invention include diabetes, experimental autoimmune encephalomyelitis (e.g., a model of multiple sclerosis), rheumatoid arthritis, and systemic lupus erythematosus.

[0172] One or more non-human animals of the present invention provide an in vivo system for the analysis and testing of drugs or vaccines. In various embodiments, a candidate drug or vaccine may be delivered to one or more non-human animals of the present invention, and the non-human animals may then be monitored to determine one or more of the immune response to the drug or vaccine, the safety profile of the drug or vaccine, or its effect on a disease or condition. In some embodiments, the vaccine targets a virus, such as human immunodeficiency virus or hepatitis virus (e.g., HCV). Exemplary methods used to determine the safety profile include measuring toxicity, optimal dose concentrations, drug or vaccine efficacy, and potential risk factors. Such drugs or vaccines may be improved and / or developed in such non-human animals.

[0173] The non-human animals of the present invention provide an in vivo system for evaluating the pharmacokinetic properties of human PD-1-targeting agents. In various embodiments, a human PD-1-targeting agent is delivered or administered to one or more non-human animals of the present invention, and the non-human animal (or cells isolated therefrom) may then be monitored or one or more assays performed to determine the effect of the agent on the non-human animal. Pharmacokinetic properties include, but are not limited to, how the animal processes the agent into various metabolites (or detecting the presence or absence of one or more drug metabolites, including toxic metabolites), drug half-life, circulating levels of the agent after administration (e.g., serum concentration of the agent), anti-drug responses (e.g., anti-drug antibodies), drug absorption and distribution, route of administration, and drug excretion and / or clearance routes. In some embodiments, the pharmacokinetic and pharmacodynamic properties of an agent (e.g., a PD-1 modulator) are monitored in or through the use of the non-human animals of the present invention.

[0174] The non-human animals of the present invention provide an in vivo system for assessing the target toxicity of human PD-1-targeting agents. In various embodiments, a human PD-1-targeting agent may be delivered or administered to one or more non-human animals of the present invention, and the non-human animals (or cells isolated therefrom) may then be monitored or one or more assays performed to determine the target toxicity effects of the agent on the non-human animals. Generally, agents are intended to modulate one or more functions of a target. By way of example, a PD-1 modulator is intended to modulate PD-1-mediated functions (e.g., PD-1 signaling) by interacting in some way with PD-1 molecules on the surface of one or more cells. In some embodiments, such modulators may have adverse effects that are an exaggeration of the modulator's desired pharmacological action. Such effects are referred to as target effects. Exemplary target effects include excessively high doses, chronic activation / inactivation, and the correct action in the correct tissue. In some embodiments, the target effects of PD-1-targeting agents identified in or through use with the non-human animals of the present invention are used to determine previously unknown functions of PD-1.

[0175] The non-human animals of the present invention provide an in vivo system for assessing the off-target toxicity of human PD-1-targeting drugs. In various embodiments, a human PD-1-targeting drug is delivered or administered to one or more non-human animals of the present invention, and the non-human animals (or cells isolated therefrom) can then be monitored or one or more assays performed to determine the off-target toxic effects of the drug on the non-human animal. Off-target effects can occur when a drug interacts with an unintended target (e.g., cross-reactivity to a common epitope). Such interactions can occur in intended or unintended tissues. For example, a drug's enantiomers can result in off-target toxic effects. Additionally, a drug may inappropriately interact with and unintendedly activate a different receptor subtype. Exemplary off-target effects include erroneous activation / inhibition of the wrong target, regardless of the tissue in which the wrong target is located. In some embodiments, the off-target effects of a human PD-1 targeted agent are determined by comparing the effects of administering the agent to a non-human animal of the invention with the effects of administering the agent to one or more reference non-human animals.

[0176] In some embodiments, performing the assay involves determining the phenotypic and / or genotypic effect of the non-human animal to which the agent is administered. In some embodiments, performing the assay involves determining lot-to-lot variation of the PD-1 modulator (e.g., antagonist or agonist). In some embodiments, performing the assay involves determining the difference in effect of a PD-1-targeted agent administered to a non-human animal of the invention compared to the effect in a reference non-human animal. In various embodiments, the reference non-human animal may have a modification described herein, a modification different from those described herein (e.g., one having a disrupted, deleted, or otherwise non-functional Pdcd1 gene), or no modification (i.e., a wild-type non-human animal).

[0177] Exemplary parameters that may be measured in non-human animals (or in and / or using cells isolated therefrom) to assess the pharmacokinetic properties, target toxicity, and / or off-target toxicity of human PD-1-targeted agents include, but are not limited to, aggregation, autophagy, cell division, cell death, complement-mediated hemolysis, DNA integrity, drug-specific antibody titers, drug metabolism, gene expression arrays, metabolic activity, mitochondrial activity, oxidative stress, phagocytosis, protein biosynthesis, protein degradation, protein secretion, stress response, target tissue drug concentrations, off-target tissue drug concentrations, transcriptional activity, etc. In various embodiments, the non-human animals of the invention are used to determine a pharmaceutically effective amount of a PD-1 modulator.

[0178] The non-human animals of the present invention provide an improved in vivo system for the development and characterization of candidate therapeutic agents for use in cancer. In various embodiments, the non-human animals of the present invention may be implanted with tumors, followed by administration of one or more candidate therapeutic agents. In some embodiments, the candidate therapeutic agents may include multispecific antibodies (e.g., bispecific antibodies) or antibody cocktails, and in some embodiments, the candidate therapeutic agents include combination therapies, such as the administration of monospecific antibodies administered side-by-side or simultaneously. The tumor may be allowed sufficient time to establish at one or more locations within the non-human animal. Tumor cell proliferation, growth, survival, etc. may be measured both before and after administration of the candidate therapeutic agent. The cytotoxicity of the candidate therapeutic agent may also be measured in the non-human animal, if desired.

[0179] The non-human animals of the invention may be used to develop one or more disease models to evaluate or assess candidate therapeutics and / or treatment regimens (e.g., monotherapy, combination therapy, dose-ranging studies, etc.) for effectively treating diseases, disorders, or conditions that affect humans. Various disease states may be established in the non-human animals of the invention, after which one or more candidate molecules may be administered so that the effectiveness of one or more molecules (e.g., PD-1-targeted agents) in the disease state can be determined. In some embodiments, disease models include autoimmune, inflammatory, and / or neoplastic diseases, disorders, or conditions.

[0180] For example, the non-human animals of the present invention provide improved animal models for the prophylactic and / or therapeutic treatment of tumors or tumor cells. In various embodiments, the non-human animals of the present invention may be implanted with one or more tumor cells, followed by administration of one or more candidate therapeutic agents (e.g., antibodies). In some embodiments, administration of the one or more candidate therapeutic agents is performed (e.g., minutes or hours, but typically the same day) after implantation of the one or more tumor cells and evaluation of the one or more candidate therapeutic agents in the non-human animals of the present invention for their effectiveness in preventing the establishment of solid tumors and / or tumor cell growth in the non-human animals. In some embodiments, administration of the one or more candidate therapeutic agents is performed (e.g., days after implantation of the one or more tumor cells), and in some specific embodiments, after a sufficient time for the one or more implanted tumor cells to reach a predetermined size (e.g., volume) in the non-human animals of the present invention, and evaluation of the one or more candidate therapeutic agents for their effectiveness in treating the one or more established tumors. The non-human animals may be placed into treatment groups with different doses to allow for determination of an optimal dose or dose range that correlates with effective treatment of established tumors.

[0181] Candidate molecules can be administered to non-human animal disease models using any administration method, including parenteral and non-injectable routes. Parenteral routes include, for example, intravenous, intraarterial, intraportal, intramuscular, subcutaneous, intraperitoneal, intrathecal, intrathecal, intraventricular, intracranial, intrapleural, or other routes of infusion. Non-injectable routes include, for example, oral, nasal, transdermal, pulmonary, rectal, buccal, vaginal, and ocular. Administration may also be by continuous infusion, topical administration, sustained release from an implant (gel, membrane, etc.), and / or intravenous injection. When combination therapies are evaluated in non-human animals of the invention, candidate molecules can be administered by the same or different routes of administration. When dosing regimens are evaluated in non-human animals of the invention, candidate molecules may be administered bimonthly, monthly, every three weeks, every two weeks, weekly, daily, at variable intervals, and / or at increasing concentrations to determine a dosing regimen that exhibits the desired therapeutic or prophylactic effect in non-human animals in which one or more disease models have been established.

[0182] The non-human animals of the present invention provide an improved in vivo system for the development and characterization of candidate therapeutics for use in infectious diseases. In various embodiments, the non-human animals of the present invention may be infected by injection with a virus (e.g., MHV, HIV, HCV, etc.) or pathogen (e.g., bacteria) and then administered one or more candidate therapeutics. In some embodiments, the candidate therapeutics may include a multispecific antibody (e.g., a bispecific antibody) or antibody cocktail; in some embodiments, the candidate therapeutics include combination therapies, such as the administration of monospecific antibodies administered concomitantly or simultaneously; and in certain embodiments, the candidate therapeutics may include a vaccine. The virus or pathogen may be allowed sufficient time to establish itself in one or more locations or cells within the non-human animal such that the non-human animal develops one or more symptoms associated with the virus or pathogen infection. T cell proliferation and growth may be measured both before and after administration of the candidate therapeutic. Additionally, survival, serum and / or intracellular cytokine analysis, and liver and / or spleen histopathology may be measured in non-human animals infected with the virus or pathogen. In some embodiments, the non-human animals of the invention are used to determine the extent of organ damage associated with a viral infection, hi some embodiments, the non-human animals of the invention are used to determine the cytokine expression profile of various organs of a non-human animal infected with a particular virus.

[0183] The non-human animals of the present invention can be used to evaluate human cell-targeted therapeutic drugs. In various embodiments, human cells are implanted into the non-human animals of the present invention, and drug candidates that target human cells or the like are administered to the non-human animals. The therapeutic efficacy of the drug is then determined by monitoring the human cells in the non-human animals after administration of the drug. Drugs that can be tested in non-human animals include both small molecule compounds, i.e., compounds with a molecular weight of less than 1500 kD, 1200 kD, 1000 kD, or 800 daltons, and large molecule compounds (such as proteins, e.g., antibodies), which have their intended therapeutic effect in treating human diseases and conditions by targeting (e.g., binding to and / or acting on) human cells.

[0184] In some embodiments, the agent is an anti-cancer agent and the human cells are cancer cells, which may be cells of a primary cancer or cells of a cell line established from a primary cancer. In these embodiments, a non-human animal of the invention is implanted with human cancer cells and an anti-cancer agent is administered to the non-human animal. The efficacy of the agent can be determined by assessing whether the growth or metastasis of human cancer cells in the non-human animal is inhibited as a result of administration of the agent.

[0185] In certain embodiments, the anti-cancer agent is an antibody molecule that binds to an antigen on a human cancer cell, hi certain embodiments, the anti-cancer agent is a bispecific antibody that binds to an antigen on a human cancer cell and to an antigen on other human cells (e.g., cells of the human immune system (or "human immune cells"), such as B cells and T cells). [Example]

[0186] The following examples are provided to illustrate to those of ordinary skill in the art how to make and use the methods and compositions of the present invention, and are not intended to limit the scope of what the inventors regard as their invention. Unless otherwise indicated, temperatures are given in degrees Celsius and pressures are at or near atmospheric.

[0187] Example 1: Humanization of the endogenous programmed cell death 1 (Pdcd1) gene This example shows an exemplary method for humanizing the endogenous Pdcd1 gene, which encodes programmed cell death 1 (PD-1), of a non-human mammal, such as a rodent (e.g., a mouse). The method described in this example can be used to humanize the endogenous Pdcd1 gene of a non-human animal using any human sequence, or combination of desired human sequences (or sequence fragments). In this example, an ∼883 bp human DNA fragment containing exon 2, intron 2, and the first 71 bp of exon 3 of the human PDCD1 gene found in GenBank accession number NM_005018.2 (SEQ ID NO: 23), is used to humanize the endogenous Pdcd1 gene of a mouse. A targeting vector for humanization of the genetic material encoding the extracellular N-terminal IgV region of the endogenous Pdcd1 gene was generated using VELOCIGENE® technology (see U.S. Pat. No. 6,586,251 and Valenzuela et al., 2003, Nature Biotech. 21(6):652-659, which are incorporated herein by reference).

[0188] Briefly, mouse bacterial artificial chromosome (BAC) clone RP23-93N20 (Invitrogen) was modified using an ~883 bp human DNA fragment encoding amino acids 26–169 of the human PD-1 polypeptide to delete exon 2, intron 2, and part of exon 3 of the endogenous Pdcd1 gene and insert exon 2, intron 2, and part of exon 3 of the human PDCD1 gene. Endogenous DNA, including exons 1, part of exon 3 (i.e., encoding the transmembrane domain), 4, and 5, and the 5' and 3' untranslated regions (UTRs), was retained. Sequence analysis of the ~883 bp human DNA fragment confirmed the presence of all human PDCD1 exons (i.e., 71 bp of exon 2 and exon 3) and splicing signals. Sequence analysis revealed that the sequence matched the reference genome and the PDCD1 transcript NM_005018.2.

[0189] More specifically, a small bacterial homologous recombination donor was first generated from a synthetic DNA fragment containing the following: [(HindIII)-(mouse upstream 78 bp)-(XhoI / NheI restriction enzyme sites)-(human PDCD1 883 bp)-(mouse downstream 75 bp)-(HindIII)]. This fragment was synthesized by Genescript Inc. (Piscataway, NJ) and cloned into an ampicillin-resistant plasmid vector. The XhoI-NheI sites were used to ligate a ~4,996 bp auto-deleted neomycin cassette flanked by recombinase recognition sites (loxP-hUb1-em7-Neo-pA-mPrm1-Crei-loxP; see U.S. Patent Nos. 8,697,851, 8,518,392, and 8,354,389, which are incorporated herein by reference). Subsequent selection was performed using neomycin. The flanking HindIII sites were used to linearize the targeting vector prior to homologous recombination with mouse BAC clone RP23-93N20. The junction between the human PDCD1 883-bp fragment and the mouse downstream 75 bp intentionally maintained an open reading frame in exon 3 (Figure 2). The resulting targeting vector contained, from 5' to 3', a 5' homology arm containing ~61.7 kb of mouse genomic DNA from BAC clone RP23-93N20, an auto-deleted neomycin cassette flanked by loxP sites, an 883-bp human genomic DNA fragment (including exon 2 through the first 71 bp of exon 3 of the human Pdcd1 gene), and ~84 kb of mouse genomic DNA from BAC clone RP23-93N20.

[0190] The modified RP23-93N20 BAC clone described above was used to electroporate mouse embryonic stem (ES) cells to generate modified ES cells with endogenous Pdcd1 humanized from exon 2 through part of exon 3 (i.e., a 900-bp deletion of the endogenous Pdcd1 gene and an 883-bp insertion of human sequence). Specifically targeted ES cells containing the humanized Pdcd1 gene were identified by an assay (Valenzuela et al., supra) that detects the presence of human Pdcd1 sequences (e.g., exon 2 and part of exon 3) and confirmed the loss and / or retention of mouse Pdcd1 sequences (e.g., exons 2 and part of exon 3, and / or 1, 4, and 5). Table 4 shows the primers and probes used to confirm the humanization of the endogenous Pdcd1 gene described above (Figure 3). The nucleotide sequence spanning the upstream insertion point includes the following, which shows the endogenous mouse sequence upstream of the 5' end of the auto-deletion neomycin cassette at the insertion point linked adjacent to a loxP site (bold) (included in brackets below, with the XhoI restriction site shown in italics) and the cassette sequence at the deletion point: (TCAAAGGACA GAATAGTAGC CTCCAGACCC TAGGTTCAGT TATGCTGAAG GAAGAGCCCT CTCGAG)ATAACTTCGT ATAATGTATG CTATACGAAG TTATATGCAT GGCCTCCGCG CCGGGTTTTG GCGCCTCCCG CGGGCGCCCC CCTCCTCACG (SEQ ID NO: 19).The nucleotide sequence spanning the downstream insertion point at the 3' end of the auto-excision neomycin cassette includes the following, which shows the cassette sequence (included in brackets below, with the loxP sequence in bold and the NheI restriction site in italics) flanking human Pdcd1 genomic sequence downstream of the insertion point: (CTGGAATAAC TTCGTATAAT GTATGCTATA CGAAGTTATG CTAGTAACTA TAACGGTCCT AAGGTAGCGA GCTAGC) AAGAGGCTCT GCAGTGGAGG CCAGTGCCCA TCCCCGGGTG GCAGAGGCCC CAGCAGAGAC TTCTCAATGA CATTCCAGCT GGGGTGGCCC TTCCAGAGCC CTTGCTGCCC GAGGGATGTG AGCAGGTGGC CGGGGAGGCT TTGTGGGGCC ACCCAGCCCC (SEQ ID NO: 20). The nucleotide sequence spanning the insertion point downstream of the 3' end of the human PDCD1 genomic sequence includes the following, which represents the human PDCD1 sequence adjacent to the mouse Pdcd1 genomic sequence (included in brackets below): CCCTTCCAGA GAGAAGGGCA GAAGTGCCCA CAGCCCACCC CAGCCCCTCA CCCAGGCCAG CCGGCCAGTT CCAAACCCTG (GTCATTGGTA TCATGAGTGC CCTAGTGGGT ATCCCTGTAT TGCTGCTGCT GGCCTGGGCC CTAGCTGTCT TCTGCTCAAC) (SEQ ID NO: 21).After deletion of the neomycin cassette (remaining 77 bp), the nucleotide sequence spanning the upstream insertion point contains the following, which shows the mouse and human genomic sequence juxtaposed with the remaining cassette sequence loxP sequences (included in brackets below, with XhoI and NheI restriction sites in italics and the loxP sequences in bold): TCAAAGGACA GAATAGTAGC CTCCAGACCC TAGGTTCAGT TATGCTGAAG GAAGAGCCCT (CTCGAG ATAACTTCGT ATAATGTATG CTATACGAAG TTATGCTAGT AACTATAACG GTCCTAAGGT AGCGA GCTAGC) AAGAG GCTCTGCAGT GGAGGCCAGT GCCCATCCCC GGGTGGCAGA GGCCCCAGCA GAGACTTCTC AATGACATTC CAGCTGGGGT GGCCCTTCCA (SEQ ID NO: 22).

[0191] Positive ES cell clones were then used to implant female mice using the VELOCIMOUSE® method (see, e.g., U.S. Patent No. 7,294,754 and Poueymirou et al., 2007, Nature Biotech. 25(1):91-99) to generate litters containing an insertion of human PDCD1 exon 2 and part of human PDCD1 exon 3 into the mouse endogenous Pdcd1 gene. Mice carrying humanized exons 2 and 3 (i.e., an 883-bp human DNA fragment) of the endogenous Pdcd1 gene were again confirmed and identified by genotyping DNA isolated from tail fragments using a modified version of the allele assay (Valenzuela et al., supra) that detected the presence of human PDCD1 gene sequences. Pups were genotyped, and a cohort of animals heterozygous for the humanized Pdcd1 gene construct was selected for characterization. [Table 4]

[0192] Example 2: Expression of humanized PD-1 on activated T cells This example demonstrates that non-human animals (e.g., rodents) modified to contain a humanized Pdcd1 gene according to Example 1 express humanized PD-1 protein on the surface of activated lymphocytes. In this example, activated T cells from mice heterozygous for humanization of the endogenous Pdcd1 gene described in Example 1 were stained with an anti-PD-1 antibody to determine the expression of PD-1 on stimulated T cells isolated from wild-type and humanized mice.

[0193] Briefly, spleens were harvested from wild-type mice and mice heterozygous for humanization of the endogenous Pdcd1 gene as described in Example 1 and processed into single-cell suspensions by mechanical dissociation. Cells were washed in culture medium (RPMI supplemented with 10% FBS) and diluted to 1 × 10 6 The cells were resuspended at 1 μg / mL and 200 μL (200,000 cells) were plated in a 96-well plate. Cells in selected wells were stimulated with anti-CD3 and anti-CD28 antibodies (both at 1 μg / mL) for 72 hours. For FACS, the cells were stained with antibodies recognizing CD4, CD8, CD19, and human (clone MIH4, BD Biosciences) or mouse (clone J43, eBioscience) PD1 according to the manufacturer's specifications. The stained cells were run on an LSRII flow cytometer, and data were analyzed using Flowjo software. CD8 + T cells were gated for expression of human and mouse PD1 (CD19 - CD8 + ). Exemplary results are shown in FIG.

[0194] As shown in Figure 4, mice carrying the humanized Pdcd1 gene described in Example 1 express a PD-1 polypeptide comprising a human portion and an endogenous mouse portion, the human portion being detectably expressed via recognition by an antibody that recognizes the entire human PD-1 polypeptide.

[0195] Example 3: In vivo efficacy of PD-1 modulators This example demonstrates that non-human animals (e.g., rodents) modified to contain a humanized Pdcd1 gene according to Example 1 can be used in in vivo assays to screen PD-1 modulators (e.g., anti-PD-1 antibodies) and determine various characteristics, such as, for example, inhibition of tumor growth and / or tumor cell killing. In this example, several anti-PD-1 antibodies are screened in mice homozygous for the humanization of the endogenous Pdcd1 gene described in Example 1 to determine the optimal antibody dose that inhibits tumor growth and the extent to which the anti-PD-1 antibody mediates tumor cell killing.

[0196] Briefly, mice were evenly divided by body weight into five treatment or control groups (n = 5 / group) in Study 1, eight treatment or control groups (n = 5 / group) in Study 2, and five treatment or control groups (n = 7 / group) in Study 3. On day 0, mice were anesthetized with isoflurane inhalation and then injected subcutaneously into the right flank with MC38.ova cells suspended in 100 μL of DMEM (Study 1: 5 × 10 5 , Test 2 / 3: 1 x 10 6 MC38.ova (murine colon adenocarcinoma) cells were genetically engineered to express chicken ovalbumin to increase tumor immunogenicity. In Study 1, treatment groups were intraperitoneally injected with 200 μg of one of three anti-PD-1 antibodies or an isotype control antibody of irrelevant specificity on days 3, 7, 10, 14, and 17 of the experiment, while one group of mice was left untreated. In Study 2, treatment groups were intraperitoneally injected with one of three anti-PD-1 antibodies at 10 mg / kg or 5 mg / kg per dose, one anti-PD-1 antibody (Ab B, IgG4) at 10 mg / kg per dose, or an isotype control antibody of irrelevant specificity at 10 mg / kg per dose on days 3, 7, 10, 14, and 17 of the experiment. In Study 3, treatment groups were injected intraperitoneally with one of two anti-PD-1 antibodies at a dose of 5 mg / kg or 2.5 mg / kg, or a control antibody not specific for PD-1 (control) at 5 mg / kg, on days 3, 7, 10, 14, and 17 of the experiment. Table 5 shows the experimental doses and treatment protocols for the groups of mice.

[0197] For each study, the mean tumor volume determined by caliper measurement and percent survival were recorded on days 14 or 17 and 23 or 24 of each experiment for each treatment group. The number of tumor-free mice was also assessed at the end of the study (day 42 for study 1 and day 31 for studies 2 and 3). The mean tumor volume (mm 3 ) (±SD), percent survival, and number of tumor-free mice were calculated for each study (Tables 7-9). Exemplary tumor growth curves are provided in Figure 5.

[0198] As shown in Table 6 for Study 1, mice treated with Ab A did not develop detectable tumors during the course of the study. Mice treated with Ab C showed a sustained reduction in tumor volume compared to controls on days 17 and 24 of the study, with 3 of 5 mice remaining tumor-free by the end of the experiment. In contrast, treatment with Ab B did not demonstrate significant efficacy in reducing tumor volume compared to controls in this study. By day 23 of the study, 1 of 5 mice in the group receiving Ab B had died, and 2 of 5 mice in the isotype control antibody-treated group had died. In the untreated and isotype control groups, some mice showed spontaneous regression of tumors (1 of 5 and 2 of 5 mice, respectively).

[0199] As shown in Table 7 for Study 2, mice treated with 10 mg / kg Ab A did not develop detectable tumors during the course of the study. Groups of mice treated with either 10 mg / kg Ab C or Ab D showed significant reductions in tumor volume compared to controls on days 17 and 24 of the study. Four of five mice in each group treated with either 10 mg / kg Ab C or Ab D were tumor-free on day 31, whereas only one of five mice in the isotype control-treated group was tumor-free as a result of spontaneous tumor regression. Ab B, tested at 10 mg / kg, showed significant reductions in tumor volume compared to controls on days 17 and 24 of the study, but this antibody was the least effective anti-PD1 antibody, with only two of five mice surviving at the end of the experiment.

[0200] A dose-dependent response of tumor inhibition at the test doses (5 mg / kg and 10 mg / kg) was observed in the Ab A, Ab C, and Ab D treatment groups. Ab A or Ab C therapy at 5 mg / kg was less effective, with 4 of 5 mice tumor-free at the end of the experiment on day 31, whereas 5 of 5 mice in the 10 mg / kg Ab A dose group remained tumor-free. A two-way ANOVA with multiple comparisons, Dunnett's test, revealed that the difference in tumor growth between the group treated with 10 mg / kg isotype control antibody as reference and the group treated with 10 mg / kg Ab A, Ab C, or Ab D was statistically significant with a p-value of <0.005. The difference in tumor growth between the group treated with 10 mg / kg isotype control antibody as reference and the group treated with 5 mg / kg Ab A, Ab C, or Ab D was also statistically significant with a p-value of <0.05.

[0201] As shown in Table 8 for Study 3, 6 of 7 mice treated with 5 mg / kg Ab A or Ab C were tumor-free at the end of the experiment, compared with no tumor-free mice in the isotype control group. One tumor-bearing mouse in the IgG4 control group died 17 days after implantation. Only 4 of 7 mice treated with 2.5 mg / kg Ab C remained tumor-free at the end of the experiment. The difference in tumor volume at day 21 between the tested anti-PD-1 antibodies and the isotype control group was statistically significant, p<0.01, as determined by one-way ANOVA with post-test Dunnett's multiple comparison. All four tested anti-PD-1 antibodies were similarly more effective at the 5 mg / kg dose than at the 2.5 mg / kg dose.

[0202] As shown in Figure 5, anti-PD-1 antibodies significantly inhibited tumor growth in a prophylactic MC38.ova tumor growth model in PD-1-humanized mice generated according to Example 1. Anti-PD-1 Ab therapy at 10 mg / kg promoted tumor regression in all mice (5 of 5) throughout the course of the experiment, whereas only 1 of 5 mice in the control group remained tumor-free as a result of spontaneous tumor regression. Anti-PD-1 therapy at 5 mg / kg was slightly less effective, with 4 of 5 mice remaining tumor-free at the end of the experiment. One-way ANOVA with post-test Dunnett's multiple comparison revealed significant differences between anti-PD-1 and control antibody treatments, with p values < 0.05 (5 mg / kg) and < 0.01 (10 mg / kg).

[0203] In a similar experiment, intact and functional PD-1 signaling in PD-1-humanized mice generated according to Example 1 was significantly correlated with CD8 expression in tumor-bearing mice treated with anti-PD-1 antibodies. + T cells and CD3 + This was investigated by measuring T cell responses and splenic IFNγ production.

[0204] Briefly, splenocytes were collected from PD-1 humanized mice (75% C57BL / 6 / 25% 129) treated with anti-PD-1 or control antibodies on day 21, the end of the experiment. Total RNA was isolated, and real-time PCR was performed on reverse-transcribed cDNA using oligonucleotides and a Taqman probe mix specific for mouse CD8b (forward primer: GCTCTGGCTG GTCTTCAGTA TG, SEQ ID NO: 24; reverse primer: TTGCCGTATG GTTGGTTTGA AC, SEQ ID NO: 25; probe: AGCAGCTCTG CCCTCAT, SEQ ID NO: 26), mouse CD3ζ (Mm00446171_m1, Applied Biosystems), mouse IFN-γ (Mm01168134_m1, Applied Biosystems), human PD-1 (forward primer: ACTTCCACAT GAGCGTGG, SEQ ID NO: 27; reverse primer: GGGCTGTGGG CACTTCTG, SEQ ID NO: 28; probe: GCAGATCAAA GAGAGCCTGC, SEQ ID NO: 29), and mouse PD-1 (Mm01285676_m1, Applied Biosystems). Samples were normalized to the expression of mouse cyclophilin B. Exemplary results are provided in FIG.

[0205] As shown in Figure 6, administration of anti-hPD-1 antibody significantly increased CD8 expression in the spleen of humanized mice bearing MC38.ova tumors (produced according to Example 1). + and CD3 + The results showed that the anti-hPD-1 antibody induced increased production of T cells. Furthermore, the activity of anti-hPD-1 antibodies in tumor-bearing PD-1-humanized mice was dependent on IFNγ, confirming proper signaling through humanized PD-1 on the cell surface. Overall, increases in T cells and IFNγ were observed in both treatment groups compared to control-treated mice.

[0206] Human PD-1 mRNA expression was measured with a human-specific probe designed against the extracellular portion of the PD-1 protein, confirming proper expression of the humanized PD-1 protein on the cell surface. Furthermore, measurement of mouse PD-1 mRNA expression with primers designed to detect the extracellular portion of mouse PD-1 failed to produce a product.

[0207] Taken together, this example demonstrates that the non-human animals of the invention can be used to evaluate the in vivo efficacy of agents (e.g., antibodies) that target PD-1, and that such animals are useful in distinguishing the therapeutic effects of anti-PD-1 antibodies. Furthermore, the non-human animals described herein can be used to evaluate the extent to which agents that target PD-1 inhibit tumor growth and / or mediate tumor cell killing. The non-human animals (e.g., mice) of the invention demonstrate functional PD-1 signaling through humanized PD-1 and appropriate PD-1-dependent immune responses, as evidenced by T cell and cytokine expression (e.g., IFN-γ). [Table 5] [Table 6] [Table 7] [Table 8]

[0208] Example 4: Rodent Model of Anti-PD-1 Tumor Therapy This example demonstrates that non-human animals (e.g., rodents) modified to contain a humanized Pdcd1 gene according to Example 1 can be used in tumor models to determine the optimal therapeutic dose of a PD-1 modulator (e.g., an anti-PD-1 antibody). In this example, an anti-PD-1 antibody is administered to mice homozygous for the humanization of the endogenous Pdcd1 gene described in Example 1 to determine the optimal therapeutic dose for treatment of established tumors.

[0209] Briefly, mice containing the humanized Pdcd1 gene (described in Example 1) were inoculated with 1 x 10 6 MC38.Ova cells were implanted subcutaneously, and tumor volumes subsequently increased to 80–120 mm 3 At the time of reaching 100 mg / kg, mice were randomized into six treatment groups (n=8-9 per group) (day 0). Mice were intraperitoneally administered anti-hPD-1 antibody at an escalating dose range of 0.3-25 mg / kg (i.e., 0.3, 1, 3, 10, or 25 mg / kg) or an isotype control antibody at 25 mg / kg. Antibodies were administered on days 0, 3, 7, 10, and 13. Tumor volume was monitored by caliper measurement twice weekly for the duration of the experiment (60 days). An exemplary tumor growth curve is provided in Figure 7.

[0210] As shown in Figure 7, none of the mice administered the control antibody were tumor-free at the end of the experiment. In contrast, a dose range of 3-25 mg / kg of anti-hPD-1 antibody resulted in approximately 44-55% tumor-free mice across the different treatment groups. Collectively, this example demonstrates that the non-human animals of the present invention can be used as rodent tumor models to determine the optimal dose and / or dose range of PD-1-targeting agents (e.g., antibodies) for effectively treating established tumors.

[0211] equivalent It should be apparent to those skilled in the art that various modifications, changes, and improvements of at least one embodiment of this invention will readily occur to those skilled in the art. Such modifications, changes, and improvements are intended to be part of this disclosure and are intended to be within the spirit and scope of the invention. Accordingly, the foregoing description and drawings are by way of example only, and the invention is particularly described by the following claims.

[0212] The use of ordinal terms such as "first," "second," "third," etc. to modify the claims does not, in itself, imply any priority, precedence, or order of one claim element over another, or the chronological order in which acts of a method are performed, but is used solely as a marker to distinguish one claim element with a particular name from another element with the same name (other than the use of ordinal terms).

[0213] As used in this specification and claims, the articles "a" and "an" should be understood to include plural reference unless clearly indicated otherwise. A claim or statement including "or" between one or more elements of a group is satisfied when one, more than one, or all of the group members are present in, employed in, or otherwise relevant to a given product or process, unless otherwise indicated or otherwise clear from the context. The invention includes embodiments in which exactly one member of the group is present in, employed in, or otherwise relevant to a given product or process. The invention also includes embodiments in which two or more, or the entire group member, is present in, employed in, or otherwise relevant to a given product or process. Furthermore, the invention should be understood to cover all variations, combinations, and permutations of one or more limitations, elements, phrases, descriptive terms, etc. from one or more of the claims described herein that are introduced into another claim (or any other related claim) relying on the same base claim, unless otherwise specified or unless a contradiction or inconsistency would arise apparent to one of ordinary skill in the art. Where elements are present as recited (e.g., in a Markush group or similar format), each subgroup of elements is also disclosed, and any element can be removed from the group. It will be understood that, generally, when the invention, or aspects of the invention, are referred to as including particular elements, features, etc., that particular embodiment of the invention or aspect of the invention consists of, or consists essentially of, such elements, features, etc. For the sake of brevity, these embodiments will not in all instances be specifically described in so many words herein. It will also be understood that any embodiment or aspect of the invention may be explicitly excluded from the claims, regardless of whether a specific exclusion is recited in the specification.

[0214] Those of ordinary skill in the art will understand the typical standard deviation or error attributable to values obtained in an assay or other process described herein. The publications, websites, and other references herein that describe the background of the invention and provide additional details regarding its practice are incorporated herein by reference.

Claims

1. a mouse, the genome of which comprises a humanized programmed cell death 1 (Pdcd1) gene at the endogenous Pdcd1 locus; the mouse expresses a humanized programmed cell death 1 (PD-1) polypeptide from the humanized Pdcd1 gene, the humanized PD-1 polypeptide having an amino acid sequence at least 95% identical to SEQ ID NO:6 and comprising a human portion and an endogenous portion; A mouse, wherein the human portion comprises amino acids 26-169 of a human PD-1 polypeptide, and the endogenous portion comprises an intracellular portion of an endogenous mouse PD-1 polypeptide.

2. the humanized PD-1 polypeptide is (a) is translated in the cells of said mouse with a mouse signal peptide; or (b) the mouse of claim 1, further comprising a transmembrane portion of the endogenous mouse PD-1 polypeptide.

3. an isolated mouse cell or tissue, the genome of which comprises a humanized Pdcd1 gene at the endogenous Pdcd1 locus; the humanized Pdcd1 gene encodes a humanized PD-1 polypeptide, the humanized PD-1 polypeptide having an amino acid sequence at least 95% identical to SEQ ID NO:6 and comprising a human portion and an endogenous portion; the human portion comprises amino acids 26-169 of a human PD-1 polypeptide, and the endogenous portion comprises an intracellular portion of an endogenous mouse PD-1 polypeptide; the humanized Pdcd1 gene is operably linked to a mouse Pdcd1 promoter; The isolated mouse cell or tissue, wherein said mouse cell is optionally a mouse embryonic stem cell.

4. A mouse embryo generated from the mouse embryonic stem cell of claim 3.

5. 1. A method of making a mouse, comprising: (A) the mouse genome comprises a Pdcd1 gene encoding a PD-1 polypeptide having a human portion and an endogenous portion, the Pdcd1 gene being operably linked to a mouse Pdcd1 promoter; and the method comprises: modifying the genome of the mouse such that the genome comprises a humanized Pdcd1 gene at the endogenous Pdcd1 locus; the mouse expresses a humanized PD-1 polypeptide from the humanized Pdcd1 gene, the humanized PD-1 polypeptide having an amino acid sequence at least 95% identical to SEQ ID NO:6 and comprising a human portion and an endogenous portion; the human portion comprises amino acids 26-169 of a human PD-1 polypeptide, and the endogenous portion comprises the intracellular portion of an endogenous mouse PD-1 polypeptide; or (B) the mouse expresses a PD-1 polypeptide from an endogenous Pdcd1 gene, wherein the PD-1 polypeptide comprises a human sequence; and the method comprises: (a) inserting a human genomic fragment of the human Pdcd1 gene into the endogenous mouse Pdcd1 gene at the endogenous Pdcd1 locus in mouse embryonic stem cells to form a humanized Pdcd1 gene operably linked to the endogenous mouse Pdcd1 promoter and encoding a humanized PD-1 polypeptide; the humanized PD-1 polypeptide has an amino acid sequence at least 95% identical to SEQ ID NO:6, and comprises a human portion and an endogenous portion, the human portion comprising amino acids 26-169 of human PD-1 polypeptide, and the endogenous portion comprising an intracellular portion of an endogenous mouse PD-1 polypeptide; (b) obtaining the mouse embryonic stem cells produced in (a); (c) producing a mouse using the mouse embryonic stem cells of (b).

6. The method according to claim 5 (A) or (B), The method, wherein the humanized PD-1 polypeptide comprises the transmembrane sequence of the endogenous murine PD-1 polypeptide.

7. A method for evaluating the pharmacokinetic properties of a drug that targets human PD-1, comprising: Administering the agent to the mouse of claim 1 or claim 2; conducting an assay to determine one or more pharmacokinetic properties of the agent that targets human PD-1, wherein the agent is optionally an anti-PD-1 antibody.

8. A mouse tumor model comprising: a) providing a mouse according to claim 1 or claim 2; b) implanting one or more tumor cells into the mouse of (a); thereby obtaining the mouse tumor model.

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