Non-human animals with humanized programmed cell death 1 gene

Abstract

The present invention provides non-human animals and compositions and methods for preparing the non-human animals, wherein the non-human animals comprise a humanized programmed cell death 1 (Pdcd1) gene. In some embodiments, the non-human animal can be described as having a genetic modification of an endogenous Pdcd1 gene such that the non-human animal expresses a PD-1 polypeptide comprising a human portion and an endogenous portion (e.g., a non-human portion).

Description

[0001] This application is a divisional application of Chinese invention patent application No. 201580033075.2, whose application date is June 19, 2015 and whose invention name is “Non-human animals having humanized programmed cell death 1 gene”.

[0002] Cross-references to related patent applications

[0003] This application claims the benefit of priority to U.S. Provisional 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.

[0004] Sequence Listing Incorporated by Reference

[0005] The Sequence Listing is an ASCII text file named 31969_SEQ.txt, 23 KB in size, created on June 4, 2015, submitted to the U.S. Patent and Trademark Office via EFS-Web, and is incorporated herein by reference. Background Art

[0006] Although the focus of medical research and development has been devoted to cancer immunotherapy and significant improvements have been achieved, cancer remains a major challenge in the global health care industry. This major challenge is partly due to the ability of cancer cells to evade the monitoring mechanisms of the immune system, which leads to the suppression and / or downregulation of anti-tumor immunity to a certain extent. There is still a lack of development of such in vivo systems, which are used to optionally determine the therapeutic potential of new cancer therapies designed to activate and / or promote anti-tumor immunity and to determine how cancer cells provide inhibitory signals to immune cells (e.g., T cells) at the molecular level. Such systems provide a source of assays for evaluating the therapeutic efficacy of candidate agents that promote an anti-tumor environment in vivo. Summary of the invention

[0007] The present invention encompasses the recognition that it is desirable to engineer non-human animals to allow for improved systems for identifying and developing new therapeutic agents that can be used to treat cancer. The present invention also encompasses the recognition that it is desirable to engineer non-human animals to allow for improved systems for identifying and developing new therapeutic agents that can be used to treat autoimmune (or inflammatory) diseases, disorders or conditions. In addition, the present invention also encompasses the recognition that it is desirable to engineer non-human animals with humanized Pdcd1 genes and / or otherwise express, contain or produce human or humanized PD-1 polypeptides, for example, for identifying and developing cancer therapeutic agents 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 combined therapies including targeted PD-1.

[0008] In some embodiments, the present invention provides a non-human animal having a genome comprising a Pdcd1 gene, the Pdcd1 gene comprising genetic material from two different species (e.g., human and non-human). In some embodiments, the Pdcd1 gene of a non-human animal as described herein encodes a PD-1 polypeptide comprising a human and a non-human portion, wherein the human and the non-human portions are connected together and form a functional PD-1 polypeptide. In some embodiments, the Pdcd1 gene of a non-human animal as described herein encodes a PD-1 polypeptide comprising all or part of the extracellular domain of a human PD-1 polypeptide.

[0009] In some embodiments, the present invention provides a non-human animal expressing a PD-1 polypeptide comprising a human portion and an endogenous portion. In some embodiments, the PD-1 polypeptide of the present invention is translated from a non-human signal peptide in a cell of a non-human animal; in certain embodiments, it is translated from a rodent signal peptide.

[0010] In some embodiments, the endogenous portion comprises an intracellular portion of an endogenous PD-1 polypeptide. In some embodiments, the endogenous portion further comprises a transmembrane portion of an endogenous PD-1 polypeptide. In some embodiments, the endogenous portion has Figure 8 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 presented in . Figure 8 In some embodiments, the endogenous portion has an amino acid sequence substantially identical to the corresponding amino acid sequence of the mouse PD-1 polypeptide presented in Figure 8 The amino acid sequence is identical to the corresponding amino acid sequence of the mouse PD-1 polypeptide presented in .

[0011] 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 Figure 8 The amino acid sequence of the human PD-1 polypeptide presented in the present invention 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 human PD-1 polypeptide presented in the present invention. In some embodiments, the human portion comprises Figure 8 In some embodiments, the human portion comprises an amino acid sequence substantially identical to the corresponding amino acid sequence of the human PD-1 polypeptide presented in Figure 8 The amino acid sequence is identical to the corresponding amino acid sequence of the human PD-1 polypeptide presented in .

[0012] In some embodiments, the PD-1 polypeptide comprising a human portion and an endogenous portion is encoded by an endogenous Pdcd1 gene. In certain embodiments, the endogenous Pdcd1 gene comprises endogenous Pdcd1 exons 1, 4, and 5. In certain embodiments, the endogenous Pdcd1 gene further comprises all or part of endogenous Pdcd1 exon 3. In certain embodiments, the endogenous Pdcd1 gene comprises SEQ ID NO: 21. In certain embodiments, the endogenous Pdcd1 gene comprises SEQ ID NO: 22. In certain embodiments, the endogenous Pdcd1 gene comprises SEQ ID NO: 21 and SEQ ID NO: 22.

[0013] In some embodiments, the PD-1 polypeptide expressed by the non-human animal as described herein 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 SEQ ID NO: 6. In some embodiments, the PD-1 polypeptide expressed by the non-human animal as described herein has an amino acid sequence that is substantially identical to SEQ ID NO: 6. In some embodiments, the PD-1 polypeptide expressed by the non-human animal as described herein has an amino acid sequence that is identical to SEQ ID NO: 6.

[0014] In some embodiments, the present invention provides a humanized Pdcd1 locus comprising one or more exons of a non-human Pdcd1 gene, wherein the one or more exons of the non-human Pdcd1 gene are effectively connected to one or more exons of all or part of a human Pdcd1 gene. In some embodiments, the humanized Pdcd1 locus further comprises 5' and 3' non-human Pdcd1 untranslated regions (UTRs) flanking one or more exons of a human Pdcd1 gene. In some embodiments, the humanized Pdcd1 locus is controlled by a rodent promoter; in certain embodiments, it is controlled by an endogenous rodent promoter.

[0015] In some embodiments, the humanized Pdcd1 locus comprises non-human Pdcd1 exons 1, 3, 4, and 5, which are 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, which comprises a human portion and a non-human portion, and 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 handle 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 about 91 bp of rodent Pdcd1 exon 3.

[0016] In some embodiments, the present 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.

[0017] In some embodiments, the endogenous portion comprises endogenous Pdcd1 exons 1, 4, and 5. In some embodiments, the endogenous portion further comprises all or part of endogenous Pdcd1 exon 3. In some embodiments, exon 1, all or part of exon 3, exons 4, and 5 of the endogenous Pdcd1 gene are Figure 8 The corresponding exon 1, all or part of the corresponding exon 3, and the corresponding exons 4 and 5 of the endogenous Pdcd1 gene represented in the expression vector are at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 95% identical to the corresponding exon 1, all or part of the corresponding exon 3, and the corresponding exons 4 and 5 of the endogenous Pdcd1 gene represented in the expression vector. In some embodiments, the exon 1, all or part of the exon 3, exon 4, and 5 of the endogenous Pdcd1 gene are identical to the corresponding exon 1, all or part of the corresponding exon 3, and exons 4 and 5 of the endogenous Pdcd1 gene represented in the expression vector are at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 95% identical to the corresponding exon 1, all or part of the corresponding exon 3, and exons 4 and 5 of the endogenous Pdcd1 gene represented in the expression vector. Figure 8 The corresponding exon 1, all or part of the corresponding exon 3, and the corresponding exons 4 and 5 of the endogenous Pdcd1 gene presented in are substantially identical. In some embodiments, exon 1, all or part of exon 3, exon 4 and 5 of the endogenous Pdcd1 gene are substantially identical to Figure 8 The corresponding exon 1, all or part of the corresponding exon 3, and the corresponding exons 4 and 5 of the endogenous Pdcd1 gene presented in are identical.

[0018] 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.

[0019] In some embodiments, the human portion comprises exon 2 of the human Pdcd1 gene. In some embodiments, the human portion further comprises all or part of human Pdcd1 exon 3. In some embodiments, human Pdcd1 exon 2 and all or part of human Pdcd1 exon 3 are Figure 8 The corresponding exon 2 and all or part of the corresponding exon 3 of the human Pdcd1 gene presented in the sequence of the human Pdcd1 gene are at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 95% identical. In some embodiments, the human Pdcd1 exon 2 and all or part of the human Pdcd1 exon 3 are identical to Figure 8 The corresponding exon 2 and all or part of the corresponding exon 3 of the human Pdcd1 gene presented in are substantially identical. In some embodiments, human Pdcd1 exon 2 and all or part of human Pdcd1 exon 3 are identical to Figure 8 The corresponding exon 2 and all or part of the corresponding exon 3 of the human Pdcd1 gene presented in are identical. In some embodiments, the human portion comprises a sequence that is codon-optimized for expression in non-human animals; in some embodiments, in rodents; in certain embodiments, in mice; in certain embodiments, in rats.

[0020] In some embodiments, the human portion comprises a sequence that is 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 that is substantially identical to SEQ ID NO: 23. In some embodiments, the human portion comprises a sequence that is identical to SEQ ID NO: 23. In some embodiments, the human portion comprises SEQ ID NO: 23.

[0021] In some embodiments, the present invention provides a PD-1 polypeptide produced (or generated) by a non-human animal as described herein. In certain embodiments, the PD-1 polypeptide produced by a non-human animal as described herein 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 SEQ ID NO: 6. In certain embodiments, the PD-1 polypeptide produced by a non-human animal as described herein comprises an amino acid sequence that is substantially identical to SEQ ID NO: 6. In certain embodiments, the PD-1 polypeptide produced by a non-human animal as described herein comprises an amino acid sequence that is identical to SEQ ID NO: 6.

[0022] In some embodiments, the present invention provides cells or tissues separated from non-human animals as described herein. In some embodiments, the present invention provides cells or tissues separated comprising Pdcd1 genes as described herein. In some embodiments, the cell is a lymphocyte. In some embodiments, the cell is 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, ovum, and combinations thereof.

[0023] In some embodiments, the present invention provides non-human embryonic stem cells, whose genome comprises the Pdcd1 gene as described herein. In some embodiments, the non-human embryonic stem cells are mouse embryonic stem cells and are from 129 strains, C57BL / 6 strains or BALB / c strains. In some embodiments, the non-human embryonic stem cells are mouse embryonic stem cells and are from 129 strains, C57BL / 6 strains or mixtures thereof. In some embodiments, the non-human embryonic stem cells are mouse embryonic stem cells and are from a mixture of 129 and C57BL / 6 strains.

[0024] In some embodiments, the non-human embryonic stem cell has 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.

[0025] In some embodiments, the present invention provides the use of non-human embryonic stem cells as described herein for preparing non-human animals. In certain embodiments, the non-human embryonic stem cells are mouse embryonic stem cells and are used to prepare mice comprising the Pdcd1 gene as described herein. In certain embodiments, the non-human embryonic stem cells are rat embryonic stem cells and are used to prepare rats comprising the Pdcd1 gene as described herein.

[0026] In some embodiments, the present invention provides a non-human embryo comprising, prepared by, obtained by, or produced by a non-human embryonic stem cell containing a Pdcd1 gene as described herein. In certain embodiments, the non-human embryo is a rodent embryo; in some embodiments, a mouse embryo; in some embodiments, a rat embryo.

[0027] In some embodiments, the present invention provides the use of non-human embryos as described herein for preparing non-human animals. In certain embodiments, the non-human embryos are mouse embryos and are used to prepare mice comprising the Pdcd1 gene as described herein. In certain embodiments, the non-human embryos are rat embryos and are used to prepare rats comprising the Pdcd1 gene as described herein.

[0028] In some embodiments, the present invention provides a targeting vector (or nucleic acid construct) as described herein. In some embodiments, the present invention provides a targeting vector (or nucleic acid construct) comprising a humanized Pdcd1 gene as described herein. In some embodiments, the present invention provides a targeting vector (or nucleic acid construct) comprising a Pdcd1 gene, wherein the Pdcd1 gene encodes a PD-1 polypeptide comprising all or part of a human extracellular domain; in certain embodiments, a PD-1 polypeptide comprising amino acids 21-170, 26-169, 27-169, 27-145, or 35-145 of a human PD-1 polypeptide.

[0029] In some embodiments, the targeting vector (or nucleic acid construct) comprises one or more exons of all or part of a non-human Pdcd1 gene, which are effectively connected to one or more exons of all or part of a 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 a human Pdcd1 gene. In some embodiments, the targeting vector (or nucleic acid construct) comprises one or more selection 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 human Pdcd1 exon 2 and all or part of human Pdcd1 exon 3.

[0030] In some embodiments, the present invention provides the purposes of the targeting vector (or nucleic acid construct) as described herein for preparing modified non-human embryonic stem cells. In some embodiments, the present invention provides the purposes of the targeting vector (or nucleic acid construct) as described herein for preparing modified non-human embryos. In some embodiments, the present invention provides the purposes of the targeting vector (or nucleic acid construct) as described herein for preparing non-human animals.

[0031] In some embodiments, the present invention provides a method for preparing a non-human animal expressing a PD-1 polypeptide from an endogenous Pdcd1 gene, wherein the PD-1 polypeptide comprises a human sequence, the method comprising (a) inserting a genomic fragment into an endogenous Pdcd1 gene in a rodent embryonic stem cell, the genomic fragment comprising a nucleotide sequence encoding all or part of a human PD-1 polypeptide; (b) obtaining the rodent embryonic stem cells produced in (a); and forming a rodent using the rodent embryonic stem cells of (b).

[0032] 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.

[0033] In some embodiments, the nucleotide sequence comprises human Pdcd1 exon 2. In some embodiments, the nucleotide sequence further comprises all or part of human Pdcd1 exon 3. In some embodiments, the nucleotide sequence comprises one or more selection markers. In some embodiments, the nucleotide sequence comprises one or more site-specific recombination sites.

[0034] In some embodiments, the present invention provides a method for preparing a non-human animal, the genome of the non-human animal comprising a Pdcd1 gene encoding a PD-1 polypeptide having a human portion and an endogenous portion, the portion being operably linked to a rodent Pdcd1 promoter, the method comprising modifying the genome of the non-human animal so that it comprises a Pdcd1 gene encoding a PD-1 polypeptide having a human portion and an endogenous portion, the portion being operably linked to a rodent Pdcd1 promoter, thereby preparing the non-human animal.

[0035] In some embodiments, the rodent Pdcd1 promoter is an endogenous rodent Pdcd1 promoter.

[0036] 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.

[0037] 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.

[0038] In some embodiments, the modification of the genome of a non-human animal is performed in a non-human embryonic stem cell, and a non-human animal having the non-human embryonic stem cell is then produced. In certain embodiments, the non-human embryonic stem cell is a rodent embryonic stem cell; in some embodiments, a mouse embryonic stem cell; in some embodiments, a rat embryonic stem cell.

[0039] In some embodiments, the invention provides a non-human animal obtainable by a method as described herein.

[0040] In some embodiments, the present invention provides a method for reducing tumor growth in a non-human animal, the method comprising the step of administering a drug targeting human PD-1 to a non-human animal, the genome of the non-human animal comprising a Pdcd1 gene encoding a PD-1 polypeptide having a human portion and an endogenous portion, the portions being operably linked to a rodent Pdcd1 promoter; the administration is performed under conditions and for a duration sufficient to reduce tumor growth in the non-human animal.

[0041] In some embodiments, the present invention provides a method for killing tumor cells in a non-human animal, the method comprising the step of administering a drug targeting human PD-1 to the non-human animal, the genome of the non-human animal comprising a Pdcd1 gene encoding a PD-1 polypeptide having a human portion and an endogenous portion, the portions being operably linked to a rodent Pdcd1 promoter; the administration is performed under conditions and for a duration sufficient to allow the drug to mediate the killing of tumor cells in the non-human animal.

[0042] In some embodiments, the present invention provides a method for evaluating the pharmacokinetic properties of a drug targeting human PD-1, the method comprising the following steps: administering the drug to a non-human animal whose genome comprises a Pdcd1 gene encoding a PD-1 polypeptide having a human portion and an endogenous portion, the portions being operably linked to a rodent Pdcd1 promoter; and performing an assay to determine one or more pharmacokinetic properties of the drug targeting human PD-1.

[0043] In many embodiments, the non-human animal as described herein is a rodent, the genome of which comprises a Pdcd1 gene encoding a PD-1 polypeptide having a human portion and an endogenous portion, the portions being 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.

[0044] In some embodiments, the drug targeting human PD-1 is a PD-1 antagonist. In some embodiments, the drug targeting human PD-1 is a PD-1 agonist. In some embodiments, the drug targeting human PD-1 is an anti-PD-1 antibody. In some embodiments, the drug targeting human PD-1 is administered intravenously, intraperitoneally, or subcutaneously.

[0045] In some embodiments, the present invention provides a non-human animal tumor model that expresses a PD-1 polypeptide comprising a human portion and an endogenous portion.

[0046] In some embodiments, the present invention provides a non-human animal tumor model having a genome 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 non-human animal Pdcd1 promoter.

[0047] In some embodiments, the present invention provides a non-human animal tumor model obtained by the following steps: (a) providing a non-human animal, the genome of which 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) implanting one or more tumor cells into the rodent of (a); thereby providing the non-human animal tumor model.

[0048] In some embodiments, the non-human animal tumor model of the present invention is a rodent tumor model. In some embodiments, the non-human animal Pdcd1 promoter is a rodent Pdcd1 promoter.

[0049] In some embodiments, the present invention provides a method for identifying or validating a drug or vaccine, the method comprising the steps of: delivering a drug or vaccine to a non-human animal, the genome of the non-human animal comprising a Pdcd1 gene encoding a PD-1 polypeptide, the PD-1 polypeptide comprising a human portion and an endogenous portion; and monitoring one or more of an immune response to the drug or vaccine, a safety feature of the drug or vaccine, or an effect on a disease, disorder, or condition. In some embodiments, monitoring safety features comprises determining whether a non-human animal exhibits side effects or adverse reactions due to the delivery of a drug or vaccine. In some embodiments, side effects or adverse reactions are selected from morbidity, mortality, changes in body weight, changes in the levels of one or more enzymes (e.g., liver), changes in the weight of one or more organs, loss of function (e.g., sensation, movement, organs, etc.), increased susceptibility to one or more diseases, changes in the genome of non-human animals, increases or decreases in food consumption, and complications of one or more diseases. In some embodiments, a disease, disorder, or condition is induced in a non-human animal. In some embodiments, a disease, disorder, or condition induced in a non-human animal is associated with a disease, disorder, or condition suffered by one or more patients in need of treatment. In certain embodiments, the drug is an antibody.

[0050] In some embodiments, the invention provides for use of a non-human animal as described herein in developing a drug or vaccine for use in medicine, such as for use as a medicament.

[0051] In some embodiments, the invention provides use of a non-human animal as described herein in the preparation of a medicament for treating cancer, a tumor, an infectious disease, an inflammatory disease, disorder or condition, or an autoimmune disease, disorder or condition.

[0052] In various embodiments, the Pdcd1 gene of the present invention includes a Pdcd1 gene as described herein. In various embodiments, the Pdcd1 gene of the present invention encodes a PD-1 polypeptide having a human portion and an endogenous portion, the 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 part of human Pdcd1 exon 3.

[0053] In various embodiments, the PD-1 polypeptides of the invention include PD-1 polypeptides as described herein. In various embodiments, the non-human animals of the invention do not detectably express full-length endogenous non-human PD-1 polypeptides. In various embodiments, the non-human animals of the invention do not detectably express the extracellular portion of an endogenous PD-1 polypeptide. In various embodiments, the non-human animals of the invention do not detectably express the N-terminal immunoglobulin V domain of an endogenous PD-1 polypeptide.

[0054] In various embodiments, the non-human animals of the present invention are rodents; in some embodiments, mice; in some embodiments, rats. In some embodiments, the mice of the present invention are selected from the 129 strain, the BALB / C strain, the C57BL / 6 strain, and the mixed 129xC57BL / 6 strain; in certain embodiments, selected from 50% 129 and 50% C57BL / 6; in certain embodiments, selected from 25% 129 and 75% C57BL / 6.

[0055] As used in this application, the terms "about" and "approximately" are used equivalently. Any numerals used in this application with or without about / approximately are intended to encompass any normal fluctuations understood by a person of ordinary skill in the relevant art.

[0056] Other features, purposes and advantages of the present invention are apparent in the detailed description of certain embodiments below. However, it should be understood that the detailed description, although indicating certain embodiments of the present invention, is provided only by way of example and is not restrictive. According to the detailed description, various changes and modifications within the scope of the present invention will become apparent to those skilled in the art. Sequence Listing <110> BUROVA, Elena MUJICA, Alexander O. LAI, Ka-Man Venus O. MURPHY, Andrew J. <120> Non-human animals with humanized programmed cell death 1 gene <130> 31969 (10121US1) <150> 62 / 086,518 <151> 2014-12-02 <150> 62 / 138,221 <151> 2015-03-25 <150> 62 / 014,181 <151> 2014-06-19 <160> 29 <170> PatentIn Version 3.5 <210> 1 <211> 1972 <212> DNA <213> Mus musculus <400> 1 tgagcagcgg ggaggaggaa gaggagactg ctactgaagg cgacactgcc aggggctctg 60 ggcatgtggg tccggcaggt accctggtca ttcacttggg ctgtgctgca gttgagctgg 120 caatcagggt ggcttctaga ggtccccaat gggccctgga ggtccctcac cttctaccca 180 gcctggctca cagtgtcaga gggagcaaat gccaccttca cctgcagctt gtccaactgg 240 tcggaggatc ttatgctgaa ctggaaccgc ctgagtccca gcaaccagac tgaaaaacag 300 gccgccttct gtaatggttt gagccaaccc gtccaggatg cccgcttcca gatcatacag 360 ctgcccaaca ggcatgactt ccacatgaac atccttgaca cacggcgcaa tgacagtggc 420 atctacctct gtggggccat ctccctgcac cccaaggcaa aaatcgagga gagccctgga 480 gcagagctcg tggtaacaga gagaatcctg gagacctcaa caagatatcc cagcccctcg 540 cccaaaccag aaggccggtt tcaaggcatg gtcattggta tcatgagtgc cctagtgggt 600 <h2 style=";text-align:left;direction:ltr">atccctgtat tgctgctgct ggcctgggcc ctagctgtct tctgctcaac aagtatgtca 660<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> gaggccagag gagctggaag caaggacgac actctgaagg aggagccttc agcagcacct 720<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> gtccctagtg tggcctatga ggagctggac ttccagggac gagagaagac accagagctc 780<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> cctaccgcct gtgtgcacac agaatatgcc accattgtct tcactgaagg gctgggtgcc 840<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> tcggccatgg gacgtagggg ctcagctgat ggcctgcagg gtcctcggcc tccaagacat 900<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> gaggatggac attgttcttg gcctctttga ccagattctt cagccattag catgctgcag 960<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> accctccaca gagagcaccg gtccgtccct cagtcaagag gagcatgcag gctacagttc 1020<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> agccaaggct cccagggtct gagctagctg gagtgacagc ccagcgcctg caccaattcc 1080<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> agcacatgca ctgttgagtg agagctcact tcaggtttac cacaagctgg gagcagcagg 1140<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> cttcccggtt tcctattgtc acaaggtgca gagctggggc ctaagcctat gtctcctgaa 1200<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> tcctactgtt gggcacttct agggacttga gacactatag ccaatggcct ctgtgggttc 1260<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> tgtgcctgga aatggagaga tctgagtaca gcctgctttg aatggccctg tgaggcaacc 1320<h2 style=";text-align:left;direction:ltr"> ccaaagcaag ggggtccagg tatactatgg gcccagcacc taaagccacc cttgggagat 1380 gatactcagg tgggaaattc gtagactggg ggactgaacc aatcccaaga tctggaaaag 1440 ttttgatgaa gacttgaaaa gctcctagct tcgggggtct gggaagcatg agcacttacc 1500 aggcaaaagc tccgtgagcg tatctgctgt ccttctgcat gcccaggtac ctcagttttt 1560 ttcaacagca aggaaactag ggcaataaag ggaaccagca gagctagagc cacccacaca 1620 tccagggggc acttgactct ccctactcct cctaggaacc aaaaggacaa agtccatgtt 1680 gacagcaggg aaggaaaggg ggatataacc ttgacgcaaa ccaacactgg ggtgttagaa 1740 tctcctcatt cactctgtcc tggagttggg ttctggctct ccttcacacc taggactctg 1800 aaatgagcaa gcacttcaga cagtcagggt agcaagagtc tagctgtctg gtgggcaccc 1860 aaaatgacca gggcttaagt ccctttcctt tggtttaagc ccgttataat taaatggtac 1920 caaaagcttt aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aa 1972 <210> 2 <211> 288 <212> PRT <213> Mus musculus <400> 2 Met Trp Val Arg Gln Val Pro Trp Ser Phe Thr Trp Ala Val Leu Gln 1 5 10 15 Leu Ser Trp Gln Ser Gly Trp Leu Leu Glu Val Pro Asn Gly Pro Trp 20 25 30 Arg Ser Leu Thr Phe Tyr Pro Ala Trp Leu Thr Val Ser Glu Gly Ala 35 40 45 Asn Ala Thr Phe Thr Cys Ser Leu Ser Asn Trp Ser Glu Asp Leu Met 50 55 60 Leu Asn Trp Asn Arg Leu Ser Pro Ser Asn Gln Thr Glu Lys Gln Ala 65 70 75 80 Ala Phe Cys Asn Gly Leu Ser Gln Pro Val Gln Asp Ala Arg Phe Gln 85 90 95 Ile Ile Gln Leu Pro Asn Arg His Asp Phe His Met Asn Ile Leu Asp 100 105 110 Thr Arg Arg Asn Asp Ser Gly Ile Tyr Leu Cys Gly Ala Ile Ser Leu 115 120 125 His Pro Lys Ala Lys Ile Glu Glu Ser Pro Gly Ala Glu Leu Val Val 130 135 140 Thr Glu Arg Ile Leu Glu Thr Ser Thr Arg Tyr Pro Ser Pro Ser Pro 145 150 155 160 Lys Pro Glu Gly Arg Phe Gln Gly Met Val Ile Gly Ile Met Ser Ala 165 170 175 Leu Val Gly Ile Pro Val Leu Leu Leu Leu Ala Trp Ala Leu Ala Val 180 185 190 Phe Cys Ser Thr Ser Met Ser Glu Ala Arg Gly Ala Gly Ser Lys Asp 195 200 205 Asp Thr Leu Lys Glu Glu Pro Ser Ala Ala Pro Val Pro Ser Val Ala 210 215 220 Tyr Glu Glu Leu Asp Phe Gln Gly Arg Glu Lys Thr Pro Glu Leu Pro 225 230 235 240 Thr Ala Cys Val His Thr Glu Tyr Ala Thr Ile Val Phe Thr Glu Gly 245 250 255 Leu Gly Ala Ser Ala Met Gly Arg Arg Gly Ser Ala Asp Gly Leu Gln 260 265 270 Gly Pro Arg Pro Pro Arg His Glu Asp Gly His Cys Ser Trp Pro Leu 275 280 285 <210> 3 <211> 2115 <212> DNA <213> Homo sapiens <400> 3 agtttccctt ccgctcacct ccgcctgagc agtggagaag gcggcactct ggtggggctg 60 ctccaggcat gcagatccca caggcgccct ggccagtcgt ctgggcggtg ctacaactgg 120 gctggcggcc aggatggttc ttagactccc cagacaggcc ctggaacccc cccaccttct ccccagccct gctcgtggtg accgaagggg acaacgccac cttcacctgc agcttctcca 240 acacatcgga gagcttcgtg ctaaactggt accgcatgag ccccagcaac cagacggaca agctggccgc cttccccgag gaccgcagcc agcccggcca ggactgccgc ttccgtgtca 360 420. cacaactgcc caacgggcgt gacttccaca tgagcgtggt caggcccgg cgcaatgaca gcggcaccta cctctgtggg gccatctccc tggcccccaa ggcgcagatc aaagagagcc 480 tgcgggcaga gctcagggtg acagaga gggcagagt gcccacagcc caccccagcc 540 cctcacccag gccagccggc cagttccaaa ccctggtggt tggtgtcgtg ggcggcctgc 600 tgggcagcct ggtgctgcta gtctgggtcc tggccgtcat ctgctcccgg gccgcacgag 660 ggacaatagg agccaggcgc accggccagc ccctgaagga ggacccctca gccgtgcctg 720 tgttctctgt ggactatggg gagctggatt tccagtggcg agagaagacc ccggagcccc 780 ccgtgccctg tgtccctgag cagacggagt atgccaccat tgtctttcct agcggaatgg 840 gcacctcatc ccccgcccgc aggggctcag ctgacggccc tcggagtgcc cagccactga 900 ggcctgagga tggacactgc tcttggcccc tctgaccggc ttccttggcc accagtgttc 960 tgcagaccct ccaccatgag cccgggtcag cgcatttcct caggagaagc aggcagggtg 1020 caggccattg caggccgtcc aggggctgag ctgcctgggg gcgaccgggg ctccagcctg 1080 cacctgcacc aggcacagcc ccaccacagg actcatgtct caatgcccac agtgagccca 1140 ggcagcaggt gtcaccgtcc cctacaggga gggccagatg cagtcactgc ttcaggtcct 1200 gccagcacag agctgcctgc gtccagctcc ctgaatctct gctgctgctg ctgctgctgc 1260 tgctgctgcc tgcggcccgg ggctgaaggc gccgtggccc tgcctgacgc cccggagcct 1320 cctgcctgaa cttgggggct ggttggagat ggccttggag cagccaaggt gcccctggca 1380 gtggcatccc gaaacgccct ggacgcaggg cccaagactg ggcacaggag tgggaggtac 1440 atggggctgg ggactcccca ggagttatct gctccctgca ggcctagaga agtttcaggg 1500 aaggtcagaa gagctcctgg ctgtggtggg cagggcagga aacccctcca cctttacaca 1560 tgcccaggca gcacctcagg ccctttgtgg ggcagggaag ctgaggcagt aagcgggcag 1620 gcagagctgg aggcctttca ggcccagcca gcactctggc ctcctgccgc cgcattccac 1680 cccagcccct cacaccactc gggagaggga catcctacgg tcccaaggtc aggagggcag 1740 ggctggggtt gactcaggcc cctcccagct gtggccacct gggtgttggg agggcagaag 1800 tgcaggcacc tagggccccc catgtgccca ccctgggagc tctccttgga acccattcct 1860 gaaattattt aaaggggttg gccgggctcc caccagggcc tgggtgggaa ggtacaggcg 1920 ttcccccggg gcctagtacc cccgccgtgg cctatccact cctcacatcc acacactgca 1980 cccccactcc tggggcaggg ccaccagcat ccaggcggcc agcaggcacc tgagtggctg 2040 ggacaaggga tcccccttcc ctgtggttct attatattat aattataatt aaatatgaga 2100 gcatgctaag gaaaa 2115 <210> 4 <211> 288 <212> PRT <213> Homo sapiens <400> 4 Met Gln Ile Pro Gln Ala Pro Trp Pro Val Val Trp Ala Val Leu Gln 1 5 10 15 Leu Gly Trp Arg Pro Gly Trp Phe Leu Asp Ser Pro Asp Arg Pro Trp 20 25 30 Asn Pro Pro Thr Phe Ser Pro Ala Leu Leu Val Val Thr Glu Gly Asp 35 40 45 Asn Ala Thr Phe Thr Cys Ser Phe Ser Asn Thr Ser Glu Ser Phe Val 50 55 60 Leu Asn Trp Tyr Arg Met Ser Pro Ser Asn Gln Thr Asp Lys Leu Ala 65 70 75 80 Ala Phe Pro Glu Asp Arg Ser Gln Pro Gly Gln Asp Cys Arg Phe Arg 85 90 95 Val Thr Gln Leu Pro Asn Gly Arg Asp Phe His Met Ser Val Val Arg 100 105 110 Ala Arg Arg Asn Asp Ser Gly Thr Tyr Leu Cys Gly Ala Ile Ser Leu 115 120 125 Ala Pro Lys Ala Gln Ile Lys Glu Ser Leu Arg Ala Glu Leu Arg Val 130 135 140 Thr Glu Arg Arg Ala Glu Val Pro Thr Ala His Pro Ser Pro Ser Pro 145 150 155 160 Arg Pro Ala Gly Gln Phe Gln Thr Leu Val Val Gly Val Val Gly Gly 165 170 175 Leu Leu Gly Ser Leu Val Leu Leu Val Trp Val Leu Ala Val Ile Cys 180 185 190 Ser Arg Ala Ala Arg Gly Thr Ile Gly Ala Arg Arg Thr Gly Gln Pro 195 200 205 Leu Lys Glu Asp Pro Ser Ala Val Pro Val Phe Ser Val Asp Tyr Gly 210 215 220 Glu Leu Asp Phe Gln Trp Arg Glu Lys Thr Pro Glu Pro Pro Val Pro 225 230 235 240 Cys Val Pro Glu Gln Thr Glu Tyr Ala Thr Ile Val Phe Pro Ser Gly 245 250 255 Met Gly Thr Ser Ser Pro Ala Arg Arg Gly Ser Ala Asp Gly Pro Arg 260 265 270 Ser Ala Gln Pro Leu Arg Pro Glu Asp Gly His Cys Ser Trp Pro Leu 275 280 285 <210> 5 <211> 1972 <212> DNA <213> Artificial Sequence <220> <223> Humanized Pdcd1 <400> 5 tgagcagcgg ggaggaggaa gaggagactg ctactgaagg cgacactgcc aggggctctg 60 ggcatgtggg tccggcaggt accctggtca ttcacttggg ctgtgctgca gttgagctgg 120 caatcagggt ggcttctaga ctccccagac aggccctgga acccccccac cttctcccca 180 gccctgctcg tggtgaccga aggggacaac gccaccttca cctgcagctt ctccaacaca 240 tcggagagct tcgtgctaaa ctggtaccgc atgagcccca gcaaccagac ggacaagctg 300 gccgccttcc ccgaggaccg cagccagccc ggccaggact gccgcttccg tgtcacacaa 360 ctgcccaacg ggcgtgactt ccacatgagc gtggtcaggg cccggcgcaa tgacagcggc 420 acctacctct gtggggccat ctccctggcc cccaaggcgc agatcaaaga gagcctgcgg 480 gcagagctca gggtgacaga gagaagggca gaagtgccca cagcccaccc cagcccctca 540 cccaggccag ccggccagtt ccaaaccctg gtcattggta tcatgagtgc cctagtgggt 600 atccctgtat tgctgctgct ggcctgggcc ctagctgtct tctgctcaac aagtatgtca 660 gaggccagag gagctggaag caaggacgac actctgaagg aggagccttc agcagcacct 720 gtccctagtg tggcctatga ggagctggac ttccagggac gagagagac accagagctc 780 cctaccgcct gtgtgcacac agaattgcc accattgtct tcactgaagg gctgggtgcc 840 tcggccatgg gacgtagggg ctcagctgat ggcctgcagg gtcctcggcc tccaagacat 900 gaggatggac attgttcttg gcctctttga ccagattctt cagccattag catgctgcag accctccaca gagagcaccg gtccgtccct cagtcaagag gagcatgcag gctacagttc agccaaggct cccagggtct gagctagctg gagtgacagc ccagcgcctg caccaattcc agcacatgca ctgttgagtg agagctcact tcaggtttac cacaagctgg gagcagcagg cttcccggtt tcctattgtc acaaggtgca gagctggggc ctaagcctat gtctcctgaa tcctactgtt gggcacttct agggacttga gacactatag ccaatggcct ctgtgggttc tgtgcctgga aatggagaga tctgagtaca gcctgctttg aatggccctg tgaggcaacc 1320 ccaaagcaag ggggtccagg tatactatgg gcccagcacc taaagccacc cttgggagat gatactcagg tgggaaattc gtagactggg ggactgaacc aatcccaaga tctggaaaag ttttgatgaa gacttgaaaa gctcctagct tcgggggtct gggaagcatg agcacttacc 1500 aggcaaaagc tccgtgagcg tatctgctgt ccttctgcat gcccaggtac ctcagttttt 1560 ttcaacagca aggaaactag ggcaataaag ggaaccagca gagctagagc cacccacaca 1620 tccagggggc acttgactct ccctactcct cctaggaacc aaaaggacaa agtccatgtt 1680 gacagcaggg aaggaaaggg ggatataacc ttgacgcaaa ccaacactgg ggtgttagaa 1740 tctcctcatt cactctgtcc tggagttggg ttctggctct ccttcacacc taggactctg 1800 aaatgagcaa gcacttcaga cagtcagggt agcaagagtc tagctgtctg gtgggcaccc 1860 aaaatgacca gggcttaagt ccctttcctt tggtttaagc ccgttataat taaatggtac 1920 caaaagcttt aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aa 1972 <210> 6 <211> 288 <212> PRT <213> Artificial Sequence <220> <223> Humanized PD-1 <400> 6 Met Trp Val Arg Gln Val Pro Trp Ser Phe Thr Trp Ala Val Leu Gln 1 5 10 15 Leu Ser Trp Gln Ser Gly Trp Leu Leu Asp Ser Pro Asp Arg Pro Trp 20 25 30 Asn Pro Pro Thr Phe Ser Pro Ala Leu Leu Val Val Thr Glu Gly Asp 35 40 45 Asn Ala Thr Phe Thr Cys Ser Phe Ser Asn Thr Ser Glu Ser Phe Val 50 55 60 Leu Asn Trp Tyr Arg Met Ser Pro Ser Asn Gln Thr Asp Lys Leu Ala 65 70 75 80 Ala Phe Pro Glu Asp Arg Ser Gln Pro Gly Gln Asp Cys Arg Phe Arg 85 90 95 Val Thr Gln Leu Pro Asn Gly Arg Asp Phe His Met Ser Val Val Arg 100 105 110 Ala Arg Arg Asn Asp Ser Gly Thr Tyr Leu Cys Gly Ala Ile Ser Leu 115 120 125 Ala Pro Lys Ala Gln Ile Lys Glu Ser Leu Arg Ala Glu Leu Arg Val 130 135 140 Thr Glu Arg Arg Ala Glu Val Pro Thr Ala His Pro Ser Pro Ser Pro 145 150 155 160 Arg Pro Ala Gly Gln Phe Gln Thr Leu Val Ile Gly Ile Met Ser Ala 165 170 175 Leu Val Gly Ile Pro Val Leu Leu Leu Leu Ala Trp Ala Leu Ala Val 180 185 190 Phe Cys Ser Thr Ser Met Ser Glu Ala Arg Gly Ala Gly Ser Lys Asp 195 200 205 Asp Thr Leu Lys Glu Glu Pro Ser Ala Ala Pro Val Pro Ser Val Ala 210 215 220 Tyr Glu Glu Leu Asp Phe Gln Gly Arg Glu Lys Thr Pro Glu Leu Pro 225 230 235 240 Thr Ala Cys Val His Thr Glu Tyr Ala Thr Ile Val Phe Thr Glu Gly 245 250 255 Leu Gly Ala Ser Ala Met Gly Arg Arg Gly Ser Ala Asp Gly Leu Gln 260 265 270 Gly Pro Arg Pro Pro Arg His Glu Asp Gly His Cys Ser Trp Pro Leu 275 280 285 <210> 7 <211> twenty three <212> DNA <213> Artificial sequence <220> <223> Synthetic oligonucleotide: 7106 hTU forward <400> 7 cccagcagag acttctcaat gac 23 <210> 8 <211> 20 <212> DNA <213> Artificial sequence <220> <223> Synthetic oligonucleotide: 7106 hTU probe <400> 8 tggcccttcc agagcccttg 20 <210> 9 <211> 18 <212> DNA <213> Artificial sequence <220> <223> Synthetic oligonucleotide: 7106 hTU reverse <400> 9 cggccacctg ctcacatc 18 <210> 10 <211> twenty one <212> DNA <213> Artificial sequence <220> <223> Synthetic oligonucleotide: 7106 hTD forward <400> 10 ggcatctctg tcctctagct c 21 <210> 11 <211> twenty four <212> DNA <213> Artificial sequence <220> <223> Synthetic oligonucleotide: 7106 hTD probe <400> 11 aagcacccca gcccctctag tctg 24 <210> 12 <211> 18 <212> DNA <213> Artificial sequence <220> <223> Synthetic oligonucleotide: 7106 hTD reverse <400> 12 gggctgtggg cacttctg 18 <210> 13 <211> twenty two <212> DNA <213> Artificial sequence <220> <223> Synthetic oligonucleotide: 7106 TU forward <400> 13 ccttcctcac agctctttgt tc 22 <210> 14 <211> 25 <212> DNA <213> Artificial sequence <220> <223> Synthetic oligonucleotide: 7106 TU probe <400> 14 tctgcatttc agaggtcccc aatgg 25 <210> 15 <211> 19 <212> DNA <213> Artificial sequence <220> <223> Synthetic oligonucleotide: 7106 TU reverse <400> 15 gagccaggct gggtagaag 19 <210> 16 <211> 25 <212> DNA <213> Artificial sequence <220> <223> Synthetic oligonucleotide: 7106 TD forward <400> 16 cggtgtccta gaactctatt ctttg 25 <210> 17 <211> 27 <212> DNA <213> Artificial sequence <220> <223> Synthetic oligonucleotide: 7106 TD probe <400> 17 tcctggagac ctcaacaaga tatccca 27 <210> 18 <211> 19 <212> DNA <213> Artificial sequence <220> <223> Synthetic oligonucleotide: 7106 TD reverse <400> 18 tgaaaccggc cttctggtt 19 <210> 19 <211> 156 <212> DNA <213> Artificial sequence <220> <223> Synthetic oligonucleotides <400> 19 tcaaaggaca gaatagtagc ctccagaccc taggttcagt tatgctgaag gaagagccct 60 ctcgagataa cttcgtataa tgtatgctat acgaagttat atgcatggcc tccgcgccgg 120 gttttggcgc ctcccgcggg cgcccccctc ctcacg 156 <210> 20 <211> 236 <212> DNA <213> Artificial sequence <220> <223> Synthetic oligonucleotides <400> 20 ctggaataac ttcgtataat gtatgctata cgaagttatg ctagtaacta taacggtcct 60 aaggtagcga gctagcaaga ggctctgcag tggaggccag tgcccatccc cgggtggcag 120 aggccccagc agagacttct caatgacatt ccagctgggg tggcccttcc agagcccttg 180 ctgcccgagg gatgtgagca ggtggccggg gaggctttgt ggggccaccc agcccc 236 <210> twenty one <211> 160 <212> DNA <213> Artificial sequence <220> <223> Synthetic oligonucleotides <400> twenty one cccttccaga gagaagggca gaagtgccca cagccccaccc cagcccctca cccaggccag 60 ccggccagtt ccaaaccctg gtcattggta tcatgagtgc cctagtgggt atccctgtat 120 tgctgctgct ggcctgggcc ctagctgtct tctgctcaac 160 <210> twenty two <211> 232 <212> DNA <213> Artificial sequence <220> <223> Synthetic oligonucleotides <400> twenty two tcaaaggaca gaatagtagc ctccagaccc taggttcagt tatgctgaag gaagagccct 60 ctcgagataa cttcgtataa tgtatgctat acgaagttat gctagtaact ataacggtcc 120 taaggtagcg agctagcaag aggctctgca gtggaggcca gtgcccatcc ccgggtggca 180 gaggccccag cagagacttc tcaatgacat tccagctggg gtggcccttc ca 232 <210> twenty three <211> 883 <212> DNA <213> Artificial sequence <220> <223> Synthetic oligonucleotide: human 883 bp DNA fragment <400> twenty three aagaggctct gcagtggagg ccagtgccca tccccgggtg gcagaggccc cagcagagac 60 ttctcaatga cattccagct ggggtggccc ttccagagcc cttgctgccc gagggatgtg 120 agcaggtggc cgggggaggct ttgtggggcc acccagcccc ttcctcacct ctctccatct 180 ctcagactcc ccagacaggc cctggaaccc ccccaccttc tccccagccc tgctcgtggt 240 gaccgaaggg gaacgcca ccttcacctg cagcttctcc aacacatcgg agagcttcgt 300 gctaaactgg taccgcatga gccccagcaa ccagacggac aagctggccg ccttccccga 360 ggaccgcagc cagcccggcc aggactgccg cttccgtgtc acacaactgc ccaacgggcg 420 tgacttccac atgagcgtgg tcagggcccg gcgcaatgac agcggcacct acctctgtgg 480 ggccatctcc ctggccccca aggcgcagat caaagagagc ctgcgggcag agctcagggt 540 gacaggtgcg gcctcggagg ccccggggca ggggtgagct gagccggtcc tggggtgggt 600 gtcccctcct gcacaggatc aggagctcca gggtcgtagg gcagggaccc cccagctcca 660 gtccagggct ctgtcctgca cctggggaat ggtgaccggc atctctgtcc tctagctctg 720 gaagcacccc agcccctcta gtctgccctc acccctgacc ctgaccctcc accctgaccc 780 cgtcctaacc cctgaccttt gtgcccttcc agagagaagg gcagaagtgc ccacagccca 840 ccccagcccc tcacccaggc cagccggcca gttccaaacc ctg 883 <210> twenty four <211> twenty two <212> DNA <213> Artificial sequence <220> <223> Synthetic oligonucleotides <400> twenty four gctctggctg gtcttcagta tg 22 <210> 25 <211> twenty two <212> DNA <213> Artificial sequence <220> <223> Synthetic oligonucleotides <400> 25 ttgccgtatg gttggtttga ac 22 <210> 26 <211> 17 <212> DNA <213> Artificial sequence <220> <223> Synthetic oligonucleotides <400> 26 agcagctctg ccctcat 17 <210> 27 <211> 18 <212> DNA <213> Artificial sequence <220> <223> Synthetic oligonucleotides <400> 27 acttccacat gagcgtgg 18 <210> 28 <211> 18 <212> DNA <213> Artificial sequence <220> <223> Synthetic oligonucleotides <400> 28 gggctgtggg cacttctg 18 <210> 29 <211> 20 <212> DNA <213> Artificial sequence <220> <223> Synthetic oligonucleotides <400> 29 gcagatcaaa gagagcctgc 20 BRIEF DESCRIPTION OF THE DRAWINGS

[0057] The drawings consisting of the following figures included herein are for illustration purposes only and are not intended to be limiting.

[0058] Figure 1 Schematic diagram not to scale showing the genomic organization of non-human (eg, mouse) and human programmed cell death 1 (Pdcd1) genes. Exons and untranslated regions (UTRs) are numbered below each exon and above each UTR.

[0059] Figure 2 A schematic diagram not to scale of an exemplary method for humanizing a non-human programmed cell death 1 (Pdcd1) gene is shown. The positions of selected nucleotide junctions are marked with lines below each junction. The sequences of these selected nucleotide junctions are indicated by SEQ ID NOs.

[0060] Figure 3 Schematic diagram not to scale showing the genomic organization of the mouse and human programmed cell death 1 (Pdcd1) genes, indicating the approximate locations of the probes used in the assays described in Example 1.

[0061] Figure 4 Shown are exemplary histograms of T cells gated on CD19 and CD8 isolated from wild-type mice and mice expressing humanized heterozygous for the endogenous Pdcd1 gene and / or humanized PD-1 as described in Example 1. Stimulated and unstimulated cell populations are indicated, as are cells stained with an isotype control.

[0062] Figure 5An exemplary tumor growth curve over 21 days for mice heterozygous for the endogenous Pdcd1 gene as described in Example 1 is shown. Control: antibody not specific for PD-1; a-hPD-1Ab: antibody specific for human PD-1. Arrows indicate the days of antibody treatment. The number of tumor-free mice at 21 days is shown for each treatment group.

[0063] Figure 6 An exemplary real-time PCR analysis of CD8b, CD3, IFN-g and PD-1 mRNA expression in the spleen of mice heterozygous for humanization of the endogenous Pdcd1 gene as described in Example 1 after treatment with anti-PD-1 antibodies is shown. A: Mean of five mice per group. B: Single mouse expression level in each treatment group. Control: antibody not specific for PD-1; α-PD-1: anti-PD-1 antibody.

[0064] Figure 7 An exemplary tumor growth curve over 60 days is shown for mice that are humanized heterozygous for the endogenous Pdcd1 gene as described in Example 1, to which 0.3-25 mg / kg anti-hPD-1 antibody or 25 mg / kg control antibody (antibody not specific for PD-1) is administered. Arrows indicate the days of antibody treatment. The number of tumor-free mice at 60 days is shown for each treatment group.

[0065] Figure 8 Exemplary mouse, human and humanized Pdcd1 and PD-1 sequences are shown, as well as exemplary human nucleic acid sequences for humanization of non-human Pdcd1 genes. For mRNA sequences, boldface indicates that the coding sequence and consecutive exons (where indicated) are separated by alternating underlined text; for humanized mRNA sequences, the human sequence is contained in parentheses. For protein sequences, the signal peptide is underlined, the extracellular sequence is in boldface, the immunoglobulin V domain sequence is in parentheses, and the intracellular sequence is in italic font; for humanized protein sequences, non-human sequences are indicated in regular font, and human sequences are indicated in bold font.

[0066] definition

[0067] The present invention is not limited to the specific methods and experimental conditions described herein, as such methods and conditions may vary. It should also be understood that the terminology used herein is for the purpose of describing specific embodiments only, and is not intended to be limiting, as the scope of the present invention is limited only by the claims.

[0068] Unless otherwise defined, all terms and phrases used herein include the meanings that the terms and phrases have acquired in the art, unless expressly indicated to the contrary or clearly contrary from the context in which the term or phrase is used. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, specific methods and materials are now described. All publications mentioned are hereby incorporated by reference.

[0069] The term "approximately" as used herein for one or more target values ​​refers to a value similar to the reference value. In certain embodiments, unless otherwise indicated or obviously different according to context, the term "approximately" or "about" refers to 25%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1% or a smaller range of values ​​(except that such a number will exceed 100% of a possible value) falling on the reference value in either direction (greater than or less than).

[0070] As used herein, the term "biological activity" includes the property of any agent that is active in a biological system in vitro or in vivo (e.g., in an organism). For example, an agent that has a biological effect in vivo when present in an organism is considered to have biological activity. In specific embodiments, when a protein or polypeptide has biological activity, a portion of the protein or polypeptide that shares at least one biological activity of the protein or polypeptide is generally referred to as a "biologically active" portion.

[0071] The term "comparable" includes two or more agents, entities, conditions, sets of conditions, etc., which may be different from each other but are sufficiently similar to allow comparisons to be made between them so that conclusions can be reasonably drawn based on the observed differences or similarities. One of ordinary skill in the art will understand in context what degree of identity is required for two or more such agents, entities, conditions, sets of conditions, etc. to be considered comparable in any given case.

[0072] The term "conservative" as used herein in conservative amino acid substitutions includes substitution of an amino acid residue by another amino acid residue having a side chain R group with similar chemical properties (e.g., charge or hydrophobicity). Generally speaking, conservative amino acid substitutions do not substantially change the target functional properties of a protein, e.g., the ability of a receptor to bind a ligand. Examples of groups of amino acids with side chains with similar chemical properties include: aliphatic side chains such as glycine, alanine, valine, leucine, and isoleucine; aliphatic-hydroxy 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 substitution groups 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 may be substitutions of alanine to any native residue in a protein, such as used in, for example, alanine scanning mutagenesis. In some embodiments, conservative substitutions are substitutions with positive values ​​in the PAM250 log-likelihood matrix disclosed in Gonnet et al. (1992) Exhaustive Matching of the Entire Protein Sequence Database, Science 256: 1443-45 (incorporated by reference). In some embodiments, a substitution is considered to be "moderately conservative" if it has a non-negative value in the PAM250 log-likelihood matrix.

[0073] The term "control" includes the meaning of "control" as understood in the art, as a standard for result comparison. Typically, the control is used to enhance experimental integrity by separating variables to draw conclusions about such variables. In some embodiments, the control is a reaction or determination carried out simultaneously with a test reaction or determination to provide a contrast. As used herein, a "control" may include a "control animal." A "control animal" may have a modification as described herein, a modification different from the modification described herein, or no modification (i.e., a wild-type animal). In an experiment, a "test" (i.e., a variable tested) is applied. In a second experiment "control", the variable tested is not applied. In some embodiments, the control is a historical control (i.e., a test or determination previously performed, or an amount or result previously known). In some embodiments, the control is or includes a record printed or otherwise preserved. The control may be a positive control or a negative control.

[0074] The term "disruption" includes the result of homologous recombination with a DNA molecule (e.g., with an endogenous homologous sequence such as a gene or locus). In some embodiments, disruption can achieve or represent insertion, deletion, substitution, replacement, missense mutation, or DNA sequence frameshift or a combination thereof. Insertion can include insertion of an entire gene or gene fragment, such as an exon, which can be derived from other sources (e.g., heterologous sequences) other than an endogenous sequence. In some embodiments, disruption can increase the expression and / or activity of a gene or gene product (e.g., a protein encoded by a gene). In some embodiments, disruption can reduce the expression and / or activity of a gene or gene product. In some embodiments, disruption can change the sequence of a gene or a gene product (e.g., an encoded protein). In some embodiments, disruption can truncate a gene or a gene product (e.g., an encoded protein) or fragment it. In some embodiments, disruption can extend a gene or a gene product that is encoded; in some such embodiments, disruption can achieve the assembly of a fusion protein. In some embodiments, disruption can affect the level of a gene or a gene product but not its activity. In some embodiments, disruption can affect the activity of a gene or a gene product but not its level. In some embodiments, the destruction may have no significant effect on the level of a gene or a gene product. In some embodiments, the destruction may have no significant effect on the activity of a gene or a gene product. In some embodiments, the destruction may have no significant effect on the level or activity of a gene or a gene product.

[0075] The terms "determine," "measure," "evaluate," "assess," "measure," and "analyze" are used interchangeably to refer to any form of measurement, and include determining whether an element is present or not. These terms include quantitative and / or qualitative measurements. Measurements can be relative or absolute. "Determining the presence of" includes determining the amount of something present and / or determining whether it is present or not.

[0076] The term "dosage regimen" or "treatment regimen" includes a group of unit doses, and in some embodiments, more than one group of unit doses, which are administered to a subject individually, usually separated by a certain period of time. In some embodiments, a given therapeutic agent has a recommended dosing regimen that can provide one or more doses. In some embodiments, the dosing regimen comprises multiple doses, each of which is separated from each other by a period of the same length; in some embodiments, the dosing regimen comprises multiple doses and at least two different time periods separating the individual doses.

[0077] The phrase "endogenous locus" or "endogenous gene" includes a locus present in a parent or reference organism. In some embodiments, the endogenous locus has a naturally occurring sequence. In some embodiments, the endogenous locus is a wild-type locus. In some embodiments, the reference organism is a wild-type organism. In some embodiments, the reference organism is an engineered organism. In some embodiments, the reference organism is a laboratory-grown organism (whether wild-type or engineered).

[0078] The phrase "endogenous promoter" includes promoters naturally associated with an endogenous gene, such as in a wild-type organism.

[0079] The term "heterologous" includes agents or entities from different sources. For example, when referring to a polypeptide, gene, or gene product used or present in a particular cell or organism, the term illustrates that the relevant polypeptide, gene, or gene product: 1) is artificially engineered; 2) is artificially (e.g., via genetic engineering) introduced into a cell or organism (or a precursor thereof); and / or 3) is non-naturally produced by a related cell or organism (e.g., a related cell type or organism type) or is non-naturally present in the cell or organism.

[0080] The term "host cell" includes cells into which heterologous (e.g., exogenous) nucleic acids or proteins have been introduced. Those skilled in the art will appreciate, after reading this disclosure, that such terms refer not only to specific subject cells, but also to the progeny of the cells. Because some modifications may occur in subsequent generations due to mutations or environmental influences, such progeny may not in fact be equivalent to the parental cell, but are still included in the scope of the term "host cell" used herein. In some embodiments, the host cell is or comprises a prokaryotic or eukaryotic cell. Generally speaking, a host cell is any cell suitable for receiving and / or producing heterologous nucleic acids or proteins, regardless of the kingdom of life to which the cell is designated. Exemplary cells include cells of prokaryotes and eukaryotes (single or multicellular), bacterial cells (e.g., strains of E. coli, Bacillus spp., Streptomyces spp., etc.), mycobacterial cells, fungal cells, yeast cells (e.g., S. cerevisiae, S. pombe, P. pastoris, P. methanolica, etc.), plant cells, insect cells (e.g., SF-9, SF-21, baculovirus-infected insect cells, Trichoplusia ni, etc.), non-human animal cells, human cells, or cell fusions such as hybridomas or quadromas. In some embodiments, the cells are human, monkey, ape, hamster, rat, or mouse cells. In some embodiments, the cell is a eukaryotic cell and is 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, 293EBNA, MSR293, 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, MMT060562, 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, such as retinal cells expressing viral genes (e.g., PER.C6TM cells). In some embodiments, the host cell is an isolated cell or comprises an isolated cell. In some embodiments, the host cell is a part of a tissue. In some embodiments, the host cell is a part of an organism.

[0081] The term "humanization" includes such nucleic acids or proteins: the structure of the nucleic acid or protein (i.e., nucleotide or amino acid sequence) includes a portion that substantially or completely corresponds to the structure of a specific gene or protein naturally present in a non-human animal, and also includes a portion that is different from the portion present in the relevant specific non-human gene or protein but more closely corresponds to the comparable structure present in the corresponding human gene or protein. In some embodiments, a "humanized" gene is a gene encoding a polypeptide having an amino acid sequence substantially identical to the amino acid sequence of a human polypeptide (e.g., a human protein or a portion thereof-e.g., a characteristic portion thereof). Just to give an example, in the case of a membrane receptor, a "humanized" gene may encode all or part of a polypeptide having an extracellular portion, the amino acid sequence of the extracellular portion being identical to the amino acid sequence of the human extracellular portion, and the remaining sequence being identical to the sequence of a non-human (e.g., mouse) 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 complete DNA sequence of a human gene. In some embodiments, a humanized protein comprises a sequence of a portion that appears in a human protein. In some embodiments, a humanized protein comprises the entire sequence of a human protein and is expressed from an endogenous locus of a non-human animal corresponding to a homolog or ortholog of the human gene.

[0082] As used in relation to sequence comparisons, the term "identity" includes identity as determined by any of a number of different algorithms known in the art that can be used to measure nucleotide and / or amino acid sequence identity. In some embodiments, alignments are made using ClustalW v.1.83 (slow) (with an open gap penalty of 10.0, an extension gap penalty of 0.1) and using the Gonnet similarity matrix (MACVECTOR TM 10.0.2, MacVector Inc., 2008) to determine the identities described herein.

[0083] The term "isolated" includes substances and / or entities that (1) have been separated from at least some of the components with which they were associated when originally produced (whether naturally or in an experimental setting), and / or (2) have been artificially designed, produced, prepared and / or manufactured. An isolated substance and / or entity 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 more than about 99% of other components with which they were originally associated. In some embodiments, the 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 more than about 99% pure. A substance is "pure" if it is substantially free of other components. In some embodiments, as will be understood by those skilled in the art, a substance may still be considered "isolated" or even "pure" after being combined with certain other components, such as one or more carriers or excipients (e.g., buffers, solvents, water, etc.); in such embodiments, the percentage of separation or purity of the substance is calculated without including such carriers or excipients. To give just one example, in some embodiments, a naturally occurring biopolymer such as a polypeptide or polynucleotide is considered "isolated" a) when, by virtue of the origin or source from which it is derived, it is not associated with some or all of the components that naturally accompany it in its native state; b) when it is substantially free of other polypeptides or nucleic acids of the same species as the species in which it is naturally produced; c) when expressed by or otherwise associated with components from a cell or other expression system of a species other than the species in which it is naturally produced. Thus, for example, in some embodiments, a polypeptide that is chemically synthesized or synthesized in a cellular system different from the cellular system in which it is naturally produced is considered an "isolated" polypeptide. Alternatively or additionally, in some embodiments, a polypeptide that has been subjected to one or more purification techniques may be considered an "isolated" polypeptide to the extent that it has been separated from other components with which it is a) associated in nature; and / or b) associated when originally produced.

[0084] The phrase "non-human animal" includes any non-human vertebrate organism. In some embodiments, the non-human animal is a cyclostomite vertebrate, a bony fish, a cartilaginous fish (e.g., a shark or 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.

[0085] The phrase "nucleic acid" includes any compound and / or substance that is or can be incorporated into an oligonucleotide chain. In some embodiments, "nucleic acid" is a compound and / or substance that is or can be incorporated into an oligonucleotide chain through a phosphodiester linkage. As will be clear from the context, in some embodiments, "nucleic acid" comprises a single nucleic acid residue (e.g., nucleotide and / or nucleoside); in some embodiments, "nucleic acid" comprises an oligonucleotide chain containing a single nucleic acid residue. In some embodiments, "nucleic acid" is RNA or comprises RNA; in some embodiments, "nucleic acid" is DNA or comprises DNA. In some embodiments, "nucleic acid" is one or more natural nucleic acid residues, comprises one or more natural nucleic acid residues, or consists of one or more natural nucleic acid residues. In some embodiments, "nucleic acid" is one or more nucleic acid analogs, comprises one or more nucleic acid analogs, or consists of one or more nucleic acid analogs. In some embodiments, the difference between nucleic acid analogs and "nucleic acids" is that they do not utilize a phosphodiester backbone. For example, in some embodiments, "nucleic acid" is one or more "peptide nucleic acids", comprises one or more peptide nucleic acids, or consists of one or more peptide nucleic acids, which are known in the art and have peptide bonds in the backbone instead of phosphodiester bonds, which are considered to be 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 bonds. In some embodiments, a "nucleic acid" is one or more natural nucleosides (e.g., adenosine, thymidine, guanosine, cytidine, uridine, deoxyadenosine, deoxythymidine, deoxyguanosine, and deoxycytidine), comprises one or more natural nucleosides, or consists of one or more natural nucleosides. In some embodiments, a "nucleic acid" is one or more nucleoside analogs (e.g., 2-aminoadenosine, 2-thiothymidine, inosine, pyrrolopyrimidine, 3-methyladenosine, 5-methylcytidine, C-5 propynyl-cytidine, C-5 propynyl-uridine, 2-aminoadenosine, C5-bromouridine, C5-fluorouridine, 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, intercalated bases, and combinations thereof), comprises one or more nucleoside analogs, or consists of one or more nucleoside analogs. In some embodiments, a "nucleic acid" comprises one or more modified sugars (e.g., 2'-fluororibose, ribose, 2'-deoxyribose, arabinose, and hexose) compared to sugars in natural nucleic acids. In some embodiments, a "nucleic acid" has a nucleotide sequence encoding a functional gene product such as an RNA or a protein. In some embodiments, a "nucleic acid" includes one or more introns.In some embodiments, a "nucleic acid" is prepared by one or more of the following: isolation from a natural source, enzymatic synthesis by complementary template-based polymerization (in vivo or in vitro), replication in a recombinant cell or system, chemical synthesis, and combinations thereof. 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, the "nucleic acid" is single-stranded; in some embodiments, the "nucleic acid" is double-stranded. In some embodiments, the "nucleic acid" has a nucleotide sequence comprising at least one element that encodes a polypeptide or is complementary to a sequence encoding a polypeptide. In some embodiments, the "nucleic acid" has enzymatic activity.

[0086] The phrase "operably linked" includes juxtaposition, wherein the components described are in a relationship that allows them to function in the intended manner. A control sequence that is "operably linked" to a coding sequence is connected in such a way that expression of the coding sequence is obtained under conditions compatible with the control sequence. An "operably linked" sequence includes an expression control sequence adjacent to a target gene and an expression control sequence that acts in a trans manner or acts remotely to control the target gene. The term "expression control sequence" includes polynucleotide sequences that are necessary to achieve expression and processing of the coding sequence to which it is connected. "Expression control sequences" include suitable transcription initiation, termination, promoter, and enhancer sequences; effective RNA processing signals such as splicing and polyadenylation signals; sequences that stabilize cytoplasmic mRNA; sequences that enhance translation efficiency (i.e., Kozak consensus sequences); sequences that enhance protein stability and qualitative properties; and sequences that enhance protein secretion when necessary. The nature of such control sequences varies with the host organism. For example, in prokaryotes, such control sequences generally include promoters, ribosome binding sites, and transcription termination sequences, while in eukaryotes, such expression control sequences typically include promoters and transcription termination sequences. The term "control sequence" is intended to include components whose presence is essential for expression and processing, and may also include additional components whose presence is advantageous, for example, leader sequences and fusion partner sequences.

[0087] The term "patient" or "subject" includes any organism to which the provided composition is administered or can be administered, for example, for experimental, diagnostic, preventive, 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 modifications as described herein, modifications different from the modifications described herein, or no modifications (i.e., wild-type non-human animal patients). In some embodiments, non-human animals suffer from or are susceptible to one or more disorders or conditions. In some embodiments, non-human animals display one or more symptoms of disorders or conditions. In some embodiments, non-human animals have been diagnosed with one or more disorders or conditions.

[0088] The term "polypeptide" includes any amino acid polymeric chain. In some embodiments, the polypeptide has a naturally occurring amino acid sequence. In some embodiments, the polypeptide has a non-naturally occurring amino acid sequence. In some embodiments, the polypeptide has an amino acid sequence that includes naturally occurring portions (i.e., from two or more different organisms, e.g., human and non-human portions) that are separate from each other. In some embodiments, the polypeptide has an engineered amino acid sequence that is designed and / or produced by artificial behavior.

[0089] The term "recombinant" refers to a polypeptide (e.g., a PD-1 polypeptide described herein) that is designed, engineered, prepared, expressed, produced, or isolated by recombinant means, such as a polypeptide expressed using a recombinant expression vector transfected into a host cell, a polypeptide 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), an antibody isolated from an animal (e.g., a mouse) transgenic for a human immunoglobulin gene (see, 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 by any other means involving splicing selected sequence elements to each other to prepare, express, produce or isolate polypeptides. In some embodiments, one or more of such selected sequence elements are naturally occurring. In some embodiments, one or more of such selected sequence elements are computer designed (in silico). In some embodiments, one or more such selected sequence elements are generated by mutagenesis (e.g., in vivo or in vitro) of known sequence elements, such as from natural sources or synthetic sources. For example, in some embodiments, the recombinant polypeptide is composed of a sequence present in the genome of a target source organism (e.g., human, mouse, etc.). In some embodiments, the recombinant polypeptide has an amino acid sequence produced by mutagenesis (e.g., in vitro or in vivo, such as in a non-human animal) so that the amino acid sequence of the recombinant polypeptide is a sequence that, while derived from and related to the polypeptide sequence, may not naturally exist in the genome of the non-human animal.

[0090] The term "replacement" includes a process by which a "replaced" nucleic acid sequence (e.g., a gene) present in a host locus (e.g., in a genome) is removed from the 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 comparable to each other because, for example, 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 comprises one or more of a promoter, an enhancer, a splice donor site, a splice acceptor site, an intron, an exon, an untranslated region (UTR); in some embodiments, the replacement nucleic acid sequence comprises 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 a human nucleic acid sequence or comprises a human nucleic acid sequence. In some embodiments, including when the replacement nucleic acid sequence is a human nucleic acid sequence or comprises a human nucleic acid sequence, the replaced nucleic acid sequence The nucleic acid sequence is a rodent sequence or comprises a rodent sequence (e.g., a mouse or rat sequence). The nucleic acid sequence so placed may comprise one or more regulatory sequences (e.g., a promoter, an enhancer, a 5'- or 3'-untranslated region, etc.) that are part of the source nucleic acid sequence used to obtain the sequence so placed. For example, in various embodiments, the replacement is the replacement of an endogenous sequence by a heterologous sequence, which results in the production of a gene product from the nucleic acid sequence so placed (comprising the heterologous sequence), but without the expression of the endogenous sequence; the replacement is the replacement of an endogenous genomic sequence with a nucleic acid sequence encoding a protein having a similar function to that 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 by a corresponding human gene or fragment thereof. The corresponding human gene or fragment thereof is a human gene or fragment that is a direct homolog of the replaced endogenous gene or fragment thereof, or a human gene or fragment that is substantially similar or identical in structure and / or function to the replaced endogenous gene or fragment thereof.

[0091] The phrase "programmed cell death 1 protein" or "PD-1 protein" includes a type I transmembrane protein that belongs to the CD28 / CTLA-4 family of T cell regulatory factors. The protein structure of the PD-1 protein includes an extracellular amino-terminal immunoglobulin V domain, a transmembrane domain, and a carboxyl-terminal intracellular tail, which contains an immunoreceptor tyrosine-based inhibitory motif (ITIM) and an immunoreceptor tyrosine-based 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, in activated T cells, B cells, macrophages, monocytes, mast cells, and many tumor cells. It has been shown that PD-1 is involved in the negative regulation of immune responses, and in particular, in the negative regulation of T cell responses. By way of example only, the nucleotide and amino acid sequences of the mouse and human Pdcd1 genes encoding the PD-1 protein are in Figure 8 A skilled artisan will recognize after reading this disclosure that one or more endogenous Pdcd1 genes (or all) in the genome may be replaced by one or more heterologous Pdcd1 genes (e.g., polymorphic variants, isoforms or mutants, genes from another species, humanized forms, etc.).

[0092] "PD-1-expressing cells" include cells that express PD-1 type I membrane proteins. In some embodiments, PD-1-expressing cells express PD-1 type I membrane proteins on their surfaces. In some embodiments, PD-1 proteins are expressed on the cell surface in an amount sufficient to mediate cell-to-cell interactions. Exemplary PD-1-expressing cells include B cells, macrophages, and T cells. PD-1-expressing cells regulate cell processes via PD-1 expressed on the surface of immune cells (e.g., T and B cells), and play a role in determining the differentiation and fate of such cells. In some embodiments, non-human animals of the present invention exhibit regulation of multiple cell processes (as described herein) via humanized PD-1 proteins expressed on the surface of one or more cells of non-human animals. In some embodiments, non-human animals of the present invention exhibit negative regulation of signal transduction through T cell receptors (TCRs) via humanized PD-1 proteins expressed on the surface of one or more cells of non-human animals. In some embodiments, non-human animals exhibit negative regulation of immune responses via humanized PD-1 proteins expressed on the surface of one or more cells of non-human animals.

[0093] The term "reference" includes a standard or control reagent, queue, individual, colony, sample, sequence or value compared to a target reagent, animal, queue, individual, colony, sample, sequence or value. In some embodiments, the test or determination of a reference reagent, queue, individual, colony, sample, sequence or value is substantially performed simultaneously with the test or determination of a target reagent, queue, individual, colony, sample, sequence or value. In some embodiments, the reference reagent, queue, individual, colony, sample, sequence or value is a historical reference, which is optionally embodied in a tangible medium. In some embodiments, reference may refer to control. As used herein, "reference" may include "reference animals". "Reference animals" may have modifications as described herein, modifications different from the modifications described herein or no modifications (i.e., wild-type animals). Generally, those skilled in the art will understand that the reference reagent, animal, queue, individual, colony, sample, sequence or value are measured or characterized under certain conditions, and the conditions are similar to the conditions used to measure or characterize the target reagent, animal (e.g., mammal), queue, individual, colony, sample, sequence or value.

[0094] The term "substantially" includes a qualitative condition that exhibits all or nearly all of the extent or degree of a target characteristic or property. One of ordinary skill in the biological field will appreciate that biological and chemical phenomena are rarely, if ever, carried to completion and / or carried to completeness, or achieve or avoid absolute results. Thus the term "substantially" is used to capture the potential lack of completeness inherent in many biological and chemical phenomena.

[0095] The phrase "substantially homologous" includes comparisons between amino acid or nucleic acid sequences. As will be appreciated by one of ordinary skill 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 having appropriately similar structural and / or functional properties. For example, as is well known to one of ordinary skill in the art, certain amino acids are generally classified as "hydrophobic" or "hydrophilic" amino acids, and / or as having "polar" or "non-polar" side chains. The substitution of one amino acid for another amino acid of the same type may generally be considered a "homologous" substitution. Typical amino acid classes are summarized in Tables 1 and 2.

[0096] Table 1

[0097]

[0098] Table 2

[0099]

[0100]

[0101] 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 commercial computer programs such as BLASTN for nucleotide sequences and BLASTP, 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, if at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more of the corresponding residues of the two sequences are homologous on the relevant residue sequence stretch, then the two sequences are considered to be substantially homologous. In some embodiments, the relevant sequence stretch is a complete sequence. In some embodiments, the relevant sequence stretch is at least 9, 10, 11, 12, 13, 14, 15, 16, 17 or more residues. In some embodiments, the relevant sequence stretch includes continuous residues along the complete sequence. In some embodiments, the relevant sequence stretch includes discontinuous residues along the complete sequence. In some embodiments, the coherent stretch is at least 10, 15, 20, 25, 30, 35, 40, 45, 50 or more residues.

[0102] The phrase "substantially identical" includes comparisons between amino acid or nucleic acid sequences. As will be appreciated by one of ordinary skill 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 commercial computer programs such as BLASTN for nucleotide sequences and BLASTP, GAP 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 generally also 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 the corresponding residues of the two sequences are identical over the relevant residue sequence segment. In some embodiments, the relevant sequence segment is a complete sequence. In some embodiments, the relevant segment is at least 10, 15, 20, 25, 30, 35, 40, 45, 50 or more residues.

[0103] The phrase "targeting vector" or "targeting construct" includes a polynucleotide molecule comprising a targeting region. The targeting region comprises a sequence that is identical or substantially identical to a sequence in a target cell, tissue, or animal and provides for integration of the targeting construct into a position in the genome of the cell, tissue, or animal via homologous recombination. Also included are targeting regions targeted using site-specific recombinase recognition sites (e.g., loxP or Frt sites). In some embodiments, the targeting construct of the present invention further comprises a specific target nucleic acid sequence or gene, a selectable marker, a control and / or regulatory sequence, and other nucleic acid sequences that allow for the reorganization mediated by exogenous addition of proteins that assist or promote the reorganization of such sequences. In some embodiments, the targeting construct of the present invention further comprises all or part of a target gene, wherein the target gene is a heterologous gene encoding all or part of a protein having a function similar to that of a protein encoded by an endogenous sequence. In some embodiments, the targeting construct of the present invention further comprises all or part of a target humanized gene, wherein the target humanized gene encodes all or part of a protein having a function similar to that of a protein encoded by an endogenous sequence.

[0104] The phrase "therapeutically effective amount" includes an amount that produces the expected effect of administration. In some embodiments, the term refers to an amount sufficient to treat the disease, disorder and / or condition when administered to a subject (e.g., animal) suffering from or susceptible to a disease, disorder and / or condition according to a therapeutic agent dosing regimen. In some embodiments, a therapeutically effective amount is an amount that reduces the incidence and / or severity of one or more of the disease, disorder and / or condition, and / or delays their onset. One of ordinary skill in the art will understand that the term "therapeutically effective amount" does not actually require successful treatment in a particular individual. On the contrary, a therapeutically effective amount can be an amount that provides a specific desired pharmacological response when administered to a large number of subjects who need such treatment. In some embodiments, reference to a therapeutically effective amount can be a reference to the amount measured in one or more specific tissues (e.g., tissues affected by a disease, disorder or condition) or fluids (e.g., blood, saliva, serum, sweat, tears, urine, etc.). One of ordinary skill in the art will understand that in some embodiments, a therapeutically effective amount of a specific agent or therapy can be formulated and / or administered in a single dose. In some embodiments, a therapeutically effective agent may be formulated and / or administered in multiple doses (eg, as part of a dosing regimen).

[0105] The term "treatment" in its broadest sense includes partial or complete alleviation, improvement, relief, inhibition of one or more symptoms, features and / or causes of a particular disease, disorder and / or condition; any administration of a substance (e.g., a provided composition) that delays its onset, reduces its severity and / or reduces its incidence. In some embodiments, such treatments may be applied to subjects who do not show signs of the relevant disease, disorder and / or condition and / or subjects who only show early signs of the disease, disorder and / or condition. Alternatively or additionally, in some embodiments, treatment may be applied to subjects who show one or more established signs of the relevant disease, disorder and / or condition. In some embodiments, treatment can be used for subjects who have been diagnosed with the relevant disease, disorder and / or condition. In some embodiments, treatment can be used for subjects who are known to have one or more susceptibility factors that are statistically associated with an increased risk of developing the relevant disease, disorder and / or condition.

[0106] The term "variant" includes entities that show significant structural identity to a reference entity but are structurally different from the reference entity in the presence or level of one or more chemical moieties compared to the reference entity. In many embodiments, a "variant" is also functionally different from its reference entity. Generally speaking, whether a particular entity is properly considered to be a "variant" of a reference entity is based on the degree of structural identity between it and the reference entity. As will be appreciated by those skilled in the art, any biological or chemical reference entity has certain characteristic structural elements. By definition, a "variant" is a different chemical entity that shares one or more such characteristic structural elements. Just to give some examples, a small molecule may have a characteristic core structural element (e.g., a macrocyclic core) and / or one or more characteristic side chain moieties, such that variants of a small molecule are molecules that share the core structural element and the characteristic side chain moieties, but differ in other side chain moieties and / or in the type of bonds present in the core (single bonds versus double bonds, E versus Z, etc.), a polypeptide may have a characteristic sequence element comprising a plurality of amino acids that have specified positions relative to each other in linear or three-dimensional space and / or contribute to a specific biological function, a nucleic acid may have a characteristic sequence element comprising a plurality of nucleotide residues that have specified positions relative to each other in linear or three-dimensional space. For example, a "variant polypeptide" may differ from a reference polypeptide due to one or more differences in the amino acid sequence and / or one or more differences in the chemical moieties (e.g., carbohydrates, lipids, etc.) covalently attached to the polypeptide backbone. In some embodiments, a "variant polypeptide" shows an overall sequence identity of at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97% or 99% with a reference polypeptide. Alternatively or additionally, in some embodiments, a "variant polypeptide" does not share at least one characteristic sequence element with a reference polypeptide. In some embodiments, a reference polypeptide has one or more biological activities. In some embodiments, a "variant polypeptide" shares one or more biological activities of a reference polypeptide. In some embodiments, a "variant polypeptide" lacks one or more biological activities of a reference polypeptide. In some embodiments, a "variant polypeptide" shows one or more biological activities that are reduced in level compared to a reference polypeptide. In many embodiments, if a target polypeptide has an amino acid sequence that is identical to the amino acid sequence of a parent but has an amino acid sequence with a few sequence changes at a specific position, the target polypeptide is considered to be a "variant" of a parent or reference polypeptide. Typically, less than 20%, 15%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2% of the residues in the variant are replaced compared to the parent. In some embodiments, a "variant" variant has 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 replaced residues compared to the parent.Typically, a "variant" has a very small number (e.g., less than 5, 4, 3, 2, or 1) of substituted functional residues (i.e., residues involved in a particular biological activity). In addition, a "variant" typically has no more than 5, 4, 3, 2, or 1 additions or deletions, and typically has no additions or deletions, compared to the parent. In addition, any additions or deletions are typically 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 residues, and typically less than about 5, about 4, about 3, or about 2 residues. In some embodiments, the parent or reference polypeptide is a naturally found polypeptide. As will be appreciated by one of ordinary skill in the art, multiple variants of a particular target polypeptide may typically occur in nature, particularly when the target polypeptide is an infectious agent polypeptide.

[0107] The term "vector" includes a nucleic acid molecule capable of transporting another nucleic acid associated therewith. In some embodiments, vectors are capable of extrachromosomal replication and / or expression of nucleic acids connected thereto in host cells such as eukaryotic and / or prokaryotic cells. Vectors capable of directing the expression of operably connected genes are referred to herein as "expression vectors."

[0108] The term "wild type" includes an entity having a structure and / or activity as naturally occurring in a "normal" (as opposed to mutant, diseased, altered, etc.) state or environment. One of ordinary skill in the art will appreciate that wild-type genes and polypeptides typically exist in a variety of different forms (e.g., alleles).

[0109] DETAILED DESCRIPTION OF CERTAIN EMBODIMENTS

[0110] The present invention particularly provides improved and / or engineered non-human animals having humanized genetic material encoding programmed cell death 1 (Pdcd1) genes, which are used to determine the therapeutic efficacy of Pdcd1 modulators (e.g., anti-PD-1 antibodies) for cancer treatment, as well as assays in T cell responses and signal transduction. It is conceivable that such non-human animals provide improvements in determining the therapeutic efficacy of PD-1 modulators and their potential for PD-1 blockade. Therefore, the present invention is particularly useful for developing anti-PD-1 therapies for treating a variety of cancers, as well as for enhancing immune responses to treat and / or eliminate viral infections in non-human animals. Specifically, the present invention includes humanization of the mouse Pdcd1 gene, which results in expression of humanized PD-1 proteins on the surface of non-human animal cells. Such humanized PD-1 proteins can provide human PD-1 for determining the efficacy of anti-PD-1 therapeutic agents in promoting anti-tumor immune responses. +Source of cells. In some embodiments, the non-human animals of the present invention exhibit an enhanced immune response by blocking PD-1 signaling through humanized PD-1 proteins expressed on the surface of non-human animal cells. In some embodiments, the humanized PD-1 protein has a sequence corresponding to the N-terminal immunoglobulin V domain of all or part 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; in some embodiments, it has a sequence corresponding to the transmembrane domain 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 present invention comprise an endogenous Pdcd1 gene containing genetic material from non-human animals and heterologous species (e.g., humans). In some embodiments, the non-human animal of the present invention comprises a humanized Pdcd1 gene, wherein the humanized Pdcd1 gene comprises exon 2 of the human PDCD1 gene and all or part of exon 3. In certain embodiments, the non-human animal of the present invention comprises a humanized Pdcd1 gene, wherein the humanized Pdcd1 gene comprises 883 bp of the human PDCD1 gene corresponding to exon 2 and the first 71 bp of exon 3 (i.e., encoding the handle) of the human PDCD1 gene.

[0111] Various aspects of the present invention are described in detail in the following sections. The use of sections is not meant to limit the present invention. Each section may be applicable to any aspect of the present invention. In this application, unless otherwise stated, the use of "or" means "and / or".

[0112] Programmed cell death 1 (Pdcd1) gene

[0113] Pdcd1 (also known as CD279) was originally 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 5 exons encoding PD-1, a type I membrane protein (referred to as PD-1) that includes an N-terminal immunoglobulin V (IgV) domain, a stalk (~20 amino acids in length), a transmembrane domain, and an intracellular tail that contains both an immunoreceptor tyrosine-based inhibitory motif (ITIM) and an immunoreceptor tyrosine-based switch motif (ITSM). PD-1 is expressed on many cell types, such as 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 been reported and vary based on which exon is missing (Nielsen, C. et al. (2005) Cell. Immunol. 235: 109-116). In fact, certain splice variants have been observed to be causative factors in autoimmune diseases (Wan, B. et al. (2006) J. Immunol. 177(12): 8844-8850). In addition, it has been reported that Pdcd1-deficient mice can develop autoimmune disorders (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), which leads to the fixation of PD-1 as a negative regulator of activated lymphocytes that prevent the development of autoimmune diseases. Interestingly, tumors have been found to use PD-1 signaling to evade the surveillance of the immune system. Therefore, PD-1 and at least one of its ligands (i.e., PD-L1) are currently being explored as targets for cancer therapy by promoting anti-tumor activity in the tumor microenvironment via PD-1 blockade (see, e.g., Pedoeem, A. et al. (2014) Clin. Immunol. 153: 145-152; and Philips, GK and Atkins, M. (2014) Intern. Immunol. 8 p.).

[0114] A fuller and detailed understanding of PD-1-mediated functions and the PD-1 pathway is needed to develop viable targeted therapies for future cancer treatment.

[0115] Pdcd1 and PD-1 sequences

[0116] Exemplary mouse, human and humanized Pdcd1 and PD-1 sequences are Figure 8 An exemplary human nucleic acid sequence for humanization of a non-human Pdcd1 gene is also shown in Figure 8 Shown in.

[0117] Humanized Pdcd1 non-human animals

[0118] A non-human animal is provided, which expresses a humanized PD-1 protein on the cell surface of the non-human animal, wherein the humanized PD-1 protein is produced by genetically modifying an endogenous locus (e.g., a Pdcd1 locus) of the non-human animal encoding the PD-1 protein. Suitable examples described herein include rodents, in particular, mice.

[0119] In some embodiments, the humanized Pdcd1 gene comprises genetic material from a heterologous species (e.g., a human), wherein the humanized Pdcd1 gene encodes a PD-1 protein comprising an encoded portion of genetic material from a heterologous species. In some embodiments, the humanized Pdcd1 gene of the present invention comprises genomic DNA from a heterologous species, the genomic DNA encoding the extracellular portion of the PD-1 protein expressed on the surface of the cell plasma membrane. Non-human animals, embryos, cells, and targeting constructs for preparing non-human animals, non-human embryos, and cells comprising the humanized Pdcd1 gene are provided.

[0120] In some embodiments, the endogenous Pdcd1 gene is deleted. In some embodiments, the endogenous Pdcd1 gene is changed, wherein a part of the endogenous Pdcd1 gene is replaced by a heterologous sequence (e.g., all or part of the human PDCD1 sequence). In some embodiments, all or substantially all of the endogenous Pdcd1 genes are replaced by heterologous genes (e.g., human PDCD1 genes). In some embodiments, a part of the heterologous Pdcd1 gene is inserted into the endogenous Pdcd1 locus of the endogenous non-human Pdcd1 gene. In some embodiments, the heterologous gene is a human gene. In some embodiments, one of the two copies of the endogenous Pdcd1 gene is modified or humanized, thereby producing a non-human animal heterozygous for the humanized Pdcd1 gene. In other embodiments, a non-human animal heterozygous for the humanized Pdcd1 gene is provided.

[0121] In many aspects, non-human animals include all or part of the human PDCD1 gene at the endogenous non-human Pdcd1 locus. Therefore, such non-human animals can be described as having a heterologous Pdcd1 gene. The replaced, inserted, modified or changed Pdcd1 gene at the endogenous Pdcd1 locus or the protein expressed from such genes can be detected using a variety of methods (including, for example, PCR, Western blots, Southern blots, restriction fragment length polymorphisms (RFLPs) or alleles to gain or lose assays). In some embodiments, the non-human animal is heterozygous with respect to the humanized Pdcd1 gene.

[0122] In various embodiments, the humanized Pdcd1 gene according to the present invention includes a Pdcd1 gene having a second exon having a sequence similar to Figure 8 The invention relates to 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 presented in the human PDCD1 gene.

[0123] In various embodiments, the humanized Pdcd1 gene according to the present invention includes a Pdcd1 gene having a second exon having Figure 8 The sequence is substantially identical to the second exon presented in the human PDCD1 gene.

[0124] In various embodiments, the humanized Pdcd1 gene according to the present invention includes a Pdcd1 gene having a second exon having Figure 8 The sequence is identical to the second exon presented in the human PDCD1 gene.

[0125] In various embodiments, the humanized Pdcd1 gene according to the present invention includes a Pdcd1 gene having a third exon having Figure 8 The invention relates to a humanized Pdcd1 mRNA sequence comprising a third exon 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 presented in the humanized Pdcd1 mRNA sequence.

[0126] In various embodiments, the humanized Pdcd1 gene according to the present invention includes a Pdcd1 gene having a third exon having Figure 8 The humanized Pdcd1 mRNA sequence presents a sequence essentially identical to the third exon.

[0127] In various embodiments, the humanized Pdcd1 gene according to the present invention includes a Pdcd1 gene having a third exon having Figure 8 The humanized Pdcd1 mRNA sequence presents the same sequence as the third exon.

[0128] In various embodiments, a humanized Pdcd1 gene according to the present invention includes a Pdcd1 gene comprising 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 SEQ ID NO:21 or SEQ ID NO:23.

[0129] In various embodiments, the humanized Pdcd1 gene according to the present invention includes a Pdcd1 gene comprising a sequence substantially identical to SEQ ID NO:21 or SEQ ID NO:23.

[0130] In various embodiments, the humanized Pdcd1 gene according to the present invention includes a Pdcd1 gene comprising a sequence identical to SEQ ID NO:21 or SEQ ID NO:23.

[0131] In various embodiments, the humanized Pdcd1 gene according to the present invention includes a Pdcd1 gene having a second exon and a portion of a third exon, wherein the second exon and the portion of the third exon each have Figure 8 The invention relates to 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 and a portion of the third exon presented in the human PDCD1 gene.

[0132] In various embodiments, the humanized Pdcd1 gene according to the present invention includes a Pdcd1 gene having a first exon, a fourth exon, and a fifth exon, wherein the first, fourth, and fifth exons each have a Figure 8 The first exon, the fourth exon and the fifth exon presented in the mouse Pdcd1 gene 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 sequences.

[0133] In various embodiments, the humanized Pdcd1 gene according to the present invention includes a Pdcd1 gene having a first exon, a portion of a third exon, a fourth exon, and a fifth exon, each of which has a Figure 8 The invention relates to a mouse Pdcd1 gene comprising a first exon, a portion of a third exon, a fourth exon and a fifth exon 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 in sequence to the first exon, a portion of the third exon, the fourth exon and the fifth exon presented in the mouse Pdcd1 gene.

[0134] In various embodiments, the humanized Pdcd1 gene according to the present invention includes a Pdcd1 gene having a 5' untranslated region and a 3' untranslated region, wherein the 5' untranslated region and the 3' untranslated region each have Figure 8 The invention relates to a mouse Pdcd1 gene comprising a 5' untranslated region and a 3' untranslated region 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 in sequence to each other.

[0135] In various embodiments, the humanized Pdcd1 gene according to the present invention includes a Pdcd1 gene having a nucleotide coding sequence (eg, a cDNA sequence) that is Figure 8 The nucleotide coding sequence presented in the humanized Pdcd1 nucleotide coding sequence 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.

[0136] In various embodiments, the humanized Pdcd1 mRNA sequence according to the present invention comprises Figure 8 The invention relates to a humanized mRNA 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 presented in the present invention.

[0137] In various embodiments, the humanized Pdcd1 gene according to the present invention encodes a PD-1 polypeptide having an amino acid sequence similar to Figure 8The amino acid sequence presented in the PD-1 polypeptide sequence 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 that presented in the PD-1 polypeptide sequence.

[0138] In various embodiments, the humanized PD-1 protein produced by the non-human animal of the present invention has an extracellular portion having Figure 8 The extracellular portion of the human PD-1 protein represented by the present invention 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 amino acid sequence.

[0139] In various embodiments, the humanized PD-1 protein produced by the non-human animal of the present invention has an extracellular portion comprising Figure 8 The invention relates to an amino acid 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 amino acid residues 21-170 presented in a human or humanized PD-1 protein.

[0140] In various embodiments, the humanized PD-1 protein produced by the non-human animal of the present invention has an extracellular portion comprising Figure 8 The invention further comprises an amino acid sequence that is substantially identical to amino acid residues 21-170 present in a human or humanized PD-1 protein.

[0141] In various embodiments, the humanized PD-1 protein produced by the non-human animal of the present invention has an extracellular portion comprising Figure 8 The amino acid sequence is identical to amino acid residues 21-170 present in human or humanized PD-1 protein.

[0142] In various embodiments, the humanized PD-1 protein produced by the non-human animal of the present invention has an extracellular portion comprising Figure 8 The invention further comprises an amino acid 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 amino acid residues 26-169 presented in a human or humanized PD-1 protein.

[0143] In various embodiments, the humanized PD-1 protein produced by the non-human animal of the present invention has an extracellular portion comprising Figure 8 The invention relates to an amino acid sequence that is substantially identical to amino acid residues 26-169 present in a human or humanized PD-1 protein.

[0144] In various embodiments, the humanized PD-1 protein produced by the non-human animal of the present invention has an extracellular portion comprising Figure 8 The amino acid sequence is identical to amino acid residues 26-169 present in human or humanized PD-1 protein.

[0145] In various embodiments, the humanized PD-1 protein produced by the non-human animal of the present invention has an extracellular portion comprising Figure 8 The invention further comprises an amino acid 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 amino acid residues 27-169 presented in a human or humanized PD-1 protein.

[0146] In various embodiments, the humanized PD-1 protein produced by the non-human animal of the present invention has an extracellular portion comprising Figure 8 The invention relates to an amino acid sequence that is substantially identical to amino acid residues 27-169 present in a human or humanized PD-1 protein.

[0147] In various embodiments, the humanized PD-1 protein produced by the non-human animal of the present invention has an extracellular portion comprising Figure 8 The amino acid sequence is identical to amino acid residues 27-169 present in human or humanized PD-1 protein.

[0148] In various embodiments, the humanized PD-1 protein produced by the non-human animal of the present invention has an extracellular portion comprising Figure 8 The invention further comprises an amino acid 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 amino acid residues 27-145 presented in a human or humanized PD-1 protein.

[0149] In various embodiments, the humanized PD-1 protein produced by the non-human animal of the present invention has an extracellular portion comprising Figure 8The invention further comprises an amino acid sequence that is substantially identical to amino acid residues 27-145 present in a human or humanized PD-1 protein.

[0150] In various embodiments, the humanized PD-1 protein produced by the non-human animal of the present invention has an extracellular portion comprising Figure 8 The amino acid sequence is identical to amino acid residues 27-145 present in human or humanized PD-1 protein.

[0151] In various embodiments, the humanized PD-1 protein produced by the non-human animal of the present invention has an extracellular portion comprising Figure 8 The invention further comprises an amino acid 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 amino acid residues 35-145 presented in a human or humanized PD-1 protein.

[0152] In various embodiments, the humanized PD-1 protein produced by the non-human animal of the present invention has an extracellular portion comprising Figure 8 An amino acid sequence that is substantially identical to amino acid residues 35-145 present in a human or humanized PD-1 protein.

[0153] In various embodiments, the humanized PD-1 protein produced by the non-human animal of the present invention has an extracellular portion comprising Figure 8 The amino acid sequence is identical to amino acid residues 35-145 present in human or humanized PD-1 protein.

[0154] In various embodiments, the humanized PD-1 protein produced by the non-human animal of the present invention has an N-terminal immunoglobulin V domain having a Figure 8 The N-terminal immunoglobulin V domain of the human or humanized PD-1 protein presented in the present invention 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 amino acid sequence.

[0155] In various embodiments, the humanized PD-1 protein produced by the non-human animal of the present invention has an N-terminal immunoglobulin V domain having a Figure 8 The amino acid sequence is substantially the same as the N-terminal immunoglobulin V domain presented in the human or humanized PD-1 protein.

[0156] In various embodiments, the humanized PD-1 protein produced by the non-human animal of the present invention has an N-terminal immunoglobulin V domain having a Figure 8 The amino acid sequence is identical to the N-terminal immunoglobulin V domain presented in the human or humanized PD-1 protein.

[0157] In various embodiments, the humanized PD-1 protein produced by the non-human animal of the present invention has a transmembrane domain having a Figure 8 The invention further 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 transmembrane domain of the mouse PD-1 protein presented in the present invention.

[0158] In various embodiments, the humanized PD-1 protein produced by the non-human animal of the invention has an intracellular tail having a Figure 8 The intracellular tail of the mouse PD-1 protein presented in the present invention 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 sequence.

[0159] In various embodiments, the humanized PD-1 protein produced by the non-human animal of the invention has Figure 8 The invention relates to an amino acid 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 amino acid residues 27-169 (or 26-169) presented in a human PD-1 protein.

[0160] In various embodiments, the humanized PD-1 protein produced by the non-human animal of the invention has Figure 8 An amino acid sequence substantially identical to amino acid residues 27-169 (or 26-169) present in the human PD-1 protein.

[0161] In various embodiments, the humanized PD-1 protein produced by the non-human animal of the invention has Figure 8 The amino acid sequence is identical to amino acid residues 27-169 (or 26-169) present in the human PD-1 protein.

[0162] In various embodiments, the humanized PD-1 protein produced by the non-human animal of the invention has Figure 8 The amino acid sequence of the humanized PD-1 protein presented in the above 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 amino acid sequence of the humanized PD-1 protein presented in the above.

[0163] In various embodiments, the humanized PD-1 protein produced by the non-human animal of the invention has Figure 8 The amino acid sequence of the humanized PD-1 protein presented in is substantially the same as the amino acid sequence of the humanized PD-1 protein presented in.

[0164] In various embodiments, the humanized PD-1 protein produced by the non-human animal of the invention has Figure 8 The amino acid sequence is identical to the amino acid sequence of the humanized PD-1 protein presented in .

[0165] Compositions and methods for preparing non-human animals are provided, and the non-human animals express humanized PD-1 proteins, including specific polymorphic forms or allelic variants (e.g., single amino acid differences) or optional splicing variants, including compositions and methods for preparing non-human animals expressing such proteins from human promoters and human regulatory sequences. In some embodiments, compositions and methods for preparing non-human animals expressing such proteins from endogenous promoters and endogenous regulatory sequences are also provided. In certain embodiments, the endogenous promoter and endogenous regulatory sequence are endogenous rodent promoters and endogenous rodent regulatory sequences. The method includes inserting genetic material encoding all or part of the human PD-1 protein into the non-human animal genome at a precise position corresponding to the endogenous Pdcd1 gene, thereby producing a humanized Pdcd1 gene expressing all or part of the human PD-1 protein. In some embodiments, the method comprises inserting genomic DNA corresponding to exon 2 and all or part of exon 3 of the human PDCD1 gene into a non-human animal, thereby generating a humanized gene encoding a PD-1 protein comprising a human portion containing amino acids encoded by the inserted exons.

[0166] Where appropriate, the coding region of the genetic material or polynucleotide sequence encoding all or part of the human (or humanized) PD-1 protein may be modified to include codons optimized for expression from cells in non-human animals (e.g., see U.S. Pat. Nos. 5,670,356 and 5,874,304). The codon-optimized sequence is a synthetic sequence and preferably encodes a polypeptide identical to the polypeptide encoded by the non-codon-optimized parent polynucleotide (or a biologically active fragment of a full-length polypeptide having substantially the same activity as the full-length polypeptide). In some embodiments, the coding region of the genetic material encoding all or part of the human (or humanized) PD-1 protein may include a sequence for optimizing the alteration of codon usage for a specific cell type (e.g., rodent cells). For example, the codons of the genomic DNA corresponding to exon 2 and part of exon 3 (e.g., 71 bp) of the endogenous Pdcd1 gene of a non-human animal (e.g., a rodent) to be inserted into the non-human animal (e.g., rodent) may be optimized for expression in cells of the non-human animal. This sequence may be described as a codon-optimized sequence.

[0167] The humanized PD-1 gene method uses relatively minimal endogenous gene modifications and produces natural PD-1-mediated signal transduction in non-human animals. In multiple embodiments, because the genomic sequence of the Pdcd1 sequence is modified in a single fragment and thus maintains normal functionality by including the necessary regulatory sequences. Therefore, in such embodiments, the PD-1 gene modification will not affect other surrounding genes or other endogenous Pdcd1-interacting genes (e.g., PD-L1, PD-L2, etc.). In addition, in multiple embodiments, the modification will not affect the assembly of the functional PD-1 transmembrane protein on the cell membrane, and maintains normal effector function by binding and subsequent signal transduction through the cytoplasmic portion of the protein that is not affected by the modification.

[0168] A schematic diagram (not drawn to scale) of the genomic organization of the endogenous mouse Pdcd1 gene and the human PDCD1 gene is provided in Figure 1 An exemplary method for humanizing the endogenous murine Pdcd1 gene using a genomic fragment comprising exon 2 and part of exon 3 of the human PDCD1 gene is provided in Figure 2 As shown in the figure, a 883bp genomic DNA fragment containing exon 2 and part of exon 3 (e.g., the first 71bp) of the human PDCD1 gene is inserted into a 900bp sequence of the endogenous mouse Pdcd1 locus by a targeting construct. The 883bp human DNA fragment can be directly cloned from human DNA or synthesized from a source sequence (e.g., Genbank accession number NM_005018.2). This genomic DNA includes a gene portion encoding substantially all of the extracellular portion (e.g., amino acid residues 27-169 or 26-169) of the human PD-1 protein responsible for ligand attachment.

[0169] A non-human animal (eg, mouse) having a humanized Pdcd1 gene at the endogenous Pdcd1 locus can be prepared by any method known in the art. For example, a targeting vector can be prepared that introduces all or part of the human Pdcd1 gene with a selectable marker gene. Figure 2 A targeting vector is shown comprising the endogenous Pdcd1 locus of the mouse genome comprising an insert of an 883 bp human DNA fragment including exon 2 and the first 71 bp of exon 3 of the human PDCD1 gene. As shown, the targeting construct comprises a 5' homology arm comprising a sequence upstream of exon 2 of the endogenous murine Pdcd1 gene (~61.7 Kb), followed by a genomic DNA fragment (883 bp) comprising exon 2 and the first 71 bp of exon 3 of the human Pdcd1 gene comprising a drug selection cassette (e.g., a neomycin resistance gene flanked by loxP sequences; ~5 Kb), and a 3' homology arm comprising the remaining sequence of endogenous murine exon 3 (i.e., the portion encoding the transmembrane portion of the PD-1 protein), exon 4 and exon 5 (~84 Kb) of the endogenous murine Pdcd1 gene. The targeting construct comprises a self-deleted drug selection cassette (e.g., a neomycin resistance gene flanked by loxP sequences; see U.S. Patent Nos. 8,697,851, 8,518,392, and 8,354,389, all of which are incorporated herein by reference). When electroporation is performed in embryonic stem cells, a modified endogenous Pdcd1 gene is formed, which exchanges the 900bp endogenous wild-type Pdcd1 gene for the 883bp human PDCD1 gene (i.e., exon 2 and the first 71bp of exon 3) contained in the targeting vector. The formation of a humanized Pdcd1 gene results in the production of a cell or non-human animal expressing a humanized PD-1 protein, which comprises amino acids encoded by an 883bp human DNA fragment (i.e., exon 2 and 71bp 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 whose germline cells contain the humanized Pdcd1 gene described above will shed the selectable marker from differentiated cells during development (see Figure 2 bottom).

[0170] Although the humanized Pdcd1 gene is widely discussed in the text for application to mice (i.e., mice having a Pdcd1 gene encoding a PD-1 protein, the PD-1 protein comprising a human portion and a mouse portion), other non-human animals comprising a humanized Pdcd1 gene are also provided. In some embodiments, such non-human animals comprise a humanized Pdcd1 gene operatively linked to a rodent Pdcd1 promoter. In some embodiments, such non-human animals comprise a humanized Pdcd1 gene operatively linked to an endogenous Pdcd1 promoter; in some embodiments, operatively linked to 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 a human PD-1 protein. Such non-human animals include any animals that can be genetically modified to express the PD-1 protein as described herein, including, for example, mammals, such as mice, rats, rabbits, pigs, cattle (e.g., cows, bulls, buffaloes), deer, sheep, goats, chickens, cats, dogs, minks, primates (e.g., marmosets, macaques), etc. For example, for non-human animals for which appropriate ES cells that can be genetically modified are not readily available, other methods are used to prepare non-human animals comprising genetic modifications. Such methods include, for example, modifying a non-ES cell genome (e.g., a fibroblast or an induced pluripotent cell) and using somatic cell nuclear transfer (SCNT) to transfer the genetically modified genome to a suitable cell, such as an enucleated oocyte, and gestating the modified cell (e.g., a modified oocyte) in a non-human animal under conditions suitable for forming an embryo.

[0171] Methods for modifying non-human animal genomes (eg, pig, cow, rodent, chicken, etc. genomes) 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.

[0172] In some embodiments, the non-human animal of the present invention is a mammal. In some embodiments, the non-human animal of the present invention is a small mammal, such as a mammal of the superfamily Jerboidea or Muroidea. In some embodiments, the genetically modified animal of the present invention is a rodent. In some embodiments, the rodent of the present invention is selected from mice, rats and hamsters. In some embodiments, the rodent of the present invention is selected from the superfamily Muroidea. In some embodiments, the genetically modified animal of the present invention is from a family selected from the following: Cricetidae (e.g., hamsters of mouse-like hamsters), Cricetidae (e.g., hamsters, New World rats and mice, voles), Muridae (true mice and rats, gerbils, spiny mice, crown mice), Isla Muscicapidae (climbing mice, rock mice, rats with tails (with-tailed rat), Madagascar rats and mice), Spiny Mountain Muscicapidae (e.g., spiny rod dormice) and Mole-shaped Muscicapidae (e.g., moles, bamboo rats and zokors). In certain embodiments, the genetically modified rodent of the present invention is selected from true mice or rats (Muridae), gerbils, spiny mice and crown mice. In certain embodiments, the genetically modified mouse of the present invention is a member from the family Muridae. In some embodiments, the non-human animal of the present invention is a rodent. In certain embodiments, the rodent of the present invention is selected from mice and rats. In some embodiments, the non-human animal of the present invention is a mouse.

[0173] In some embodiments, the non-human animal of the present invention is a rodent, which is a mouse of the following C57BL strains: 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 certain embodiments, the present invention The mouse is a 129 strain selected from the following strains: 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 certain embodiments, the genetically modified mouse of the present invention is a mixture of the above-mentioned 129 strain and the aforementioned C57BL / 6 strain. In certain embodiments, the mouse of the present invention is a mixture of the above-mentioned 129 strains, or a mixture of the aforementioned BL / 6 strains. In certain embodiments, the 129 strain of the mixture as described herein is a 129S6 (129 / SvEvTac) strain. In some embodiments, the mouse of the present invention is a BALB strain, such as a BALB / c strain. In some embodiments, the mouse of the present invention is a mixture of a BALB strain and another of the above-mentioned strains.

[0174] In some embodiments, the non-human animal of the present invention is a rat. In certain 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 certain embodiments, the rat strain described herein is a mixture of two or more strains selected from Wistar, LEA, Sprague Dawley, Fischer, F344, F6 and Dark Agouti.

[0175] Methods using non-human animals with a humanized Pdcd1 gene

[0176] Studies on PD-1 function have used a variety of Pdcd1 mutants or transgenic non-human animals (see, for example, 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; US Patent No. 7,414,171; and European Patent No. 1 334 659B1; these references are incorporated herein by reference). Such mutants and transgenic animals have been successfully used to determine the molecular aspects of PD-1 expression, function and regulation of various cellular processes. However, they are not without limitation. For example, PD-1-deficient mice generated by knocking human PD-1 cDNA into exon 1 of the mouse Pdcd1 gene do not express human PD-1 even after stimulation with PMA (Carter, LL et al., above). In addition, the considerable phenotypic differences between PD-1 mutant animals of different genetic backgrounds complicate the study, especially when trying to assign multiple functions and / or regulatory activities to PD-1. Still other transgenic animals that overexpress PD-1 have been formed (Chen, L. et al., above). Such animals have shown different transgenic expression patterns, which can be reasonably attributed to the construct design. In addition, since the same source genetic material (i.e., mice) is used, PD-1 overexpression may correspond to endogenous PD-1 rather than transgenic PD-1 due to possible transgenic position effects. Although PD-1 transgenic mice have proven useful in elucidating some PD-1-mediated biological functions, they have demonstrated variability in the results obtained, based at least in part on the different methods used to generate them. Thus, current in vivo systems that exploit PD-1-mediated biology are imperfect. The potential at the molecular level of PD-1-mediated biological functions and signaling pathways has not yet been fully exploited in transgenic mice.

[0177] The non-human animals of the present invention provide improved in vivo systems and sources of biological materials (e.g., cells) expressing human (or humanized) PD-1, which are useful for a variety of assays. In multiple embodiments, the non-human animals of the present invention are used to develop therapeutic agents that target PD-1 and / or mediate PD-1 signaling (e.g., interfere with interactions with PD-L1 and / or PD-L2). In multiple embodiments, the mice of the present invention are used to identify, screen, and / or develop candidate therapeutic agents (e.g., antibodies) that bind to human PD-1. In multiple embodiments, in multiple embodiments, the non-human animals of the present invention are used to screen and develop therapeutic agents (e.g., antibodies) that block interactions between human PD-1 and human PD-L1 and / or human PD-L2. In multiple embodiments, the non-human animals of the present invention are used to determine the binding pattern of antagonists and / or agonists of humanized PD-1 on the cell surface of the non-human animals described herein; in some embodiments, the non-human animals of the present invention are used to determine one or more epitopes of one or more candidate therapeutic antibodies that bind to human PD-1.

[0178] In various embodiments, non-human animals of the present invention are used to determine the pharmacokinetic properties of anti-PD-1 antibodies. In various embodiments, one or more non-human animals of the present invention and one or more control or reference non-human animals are exposed to multiple 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 / mg, 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) of one or more candidate therapeutic anti-PD-1 antibodies. Candidate therapeutic antibodies can be administered via various desired routes of administration (including parenteral and non-parenteral routes of administration). Parenteral routes include, for example, intravenous, intraarterial, intraportal, intramuscular, subcutaneous, intraperitoneal, intraspinal, intrathecal, intraventricular, intracranial, intrapleural or other injection routes. Non-parenteral routes include, for example, oral administration, nasal administration, transdermal administration, pulmonary administration, rectal administration, buccal administration, vaginal administration, and ocular administration. Administration can also be performed by continuous infusion, topical administration, sustained release from implants (gels, films, etc.), and / or intravenous injection. Blood is separated from non-human animals (humanized and control groups) at multiple time points (e.g., 0 hr, 6 hr, 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 or more days). Various assays can be used to determine the pharmacokinetic properties of the candidate therapeutic antibodies administered, including but not limited to total IgG, anti-therapeutic antibody responses, agglutination, etc.

[0179] In various embodiments, the non-human animals of the present invention are used to measure the therapeutic effects of blocking or modulating PD-1 signaling and the effects of cellular changes on gene expression. In various embodiments, the non-human animals of the present invention or cells isolated therefrom are exposed to candidate therapeutic agents that bind to humanized PD-1 proteins (or human portions of PD-1 proteins) on the surface of non-human animal cells, and then, after a period of time, the effects on PD-1-dependent processes, such as adhesion, apoptosis, cytokine production, inflammation, proliferation, self-tolerance, and viral infection (or response) are analyzed.

[0180] The non-human animals of the present invention express humanized PD-1 proteins, and thus cells, cell lines, and cell cultures can be generated to serve as sources of humanized PD-1 for binding and functional assays, such as for binding and functional assays of PD-1 antagonists or agonists, particularly where the antagonists or agonists are specific for human PD-1 sequences or epitopes, or alternatively, for PD-1 sequences or epitopes associated with human PD-L1 and / or PD-L2. In various embodiments, the PD-1 epitopes that bind to candidate therapeutic antibodies can be determined using cells isolated from the non-human animals of the present invention. In various embodiments, the humanized PD-1 proteins expressed by the non-human animals described herein may comprise variant amino acid sequences. Variant human PD-1 proteins (e.g., polymorphisms) associated with autoimmunity and infectious diseases have been reported (e.g., see 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 the SNPs of NCBI. Those listed in the GeneView webpage and summarized in Table 3. In various embodiments, the non-human animals of the present invention express humanized PD-1 protein variants. In various embodiments, the variants have polymorphisms at amino acid positions associated with ligand binding. In various embodiments, the non-human animals of the present invention are used to determine the effect of ligand binding by interaction with polymorphic variants of human PD-1. In certain embodiments, the non-human animals of the present invention express the human PD-1 variants presented in Table 3.

[0181] Table 3

[0182]

[0183]

[0184]

[0185]

[0186] Cells from non-human animals of the invention can be isolated and used immediately, or can be maintained in culture for many generations. In various embodiments, cells from non-human animals of the invention are immortalized and maintained in culture indefinitely (eg, in continuous culture).

[0187] In multiple embodiments, the cells and / or non-human animals of the present invention are used in various immunization schemes to determine the PD-1-mediated functions in the immune response to the antigen. In some embodiments, candidate therapeutic agents that bind to human (or humanized) PD-1 or block one or more functions of human (or humanized) PD-1 are characterized in the non-human animals of the present invention. Suitable measurements include various cell assays, proliferation assays, serum immunoglobulin analysis (e.g., antibody titers), cytotoxicity assays, characterization of ligand-receptor interactions (e.g., immunoprecipitation assays). In some embodiments, the non-human animals of the present invention are used to characterize the PD-1-mediated functions that regulate the immune response to the 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 suffered by one or more patients in need of treatment.

[0188] In various embodiments, the non-human animals of the invention are used in serum assays to determine the titer of autoantibody production for testing drug toxicology aspects of candidate therapeutic agents targeting human PD-1. In some embodiments, autoantibody production in the non-human animals of the invention is caused by one or more autoimmune diseases, disorders or conditions induced in the non-human animals.

[0189] In various embodiments, non-human animals of the invention are used to challenge with one or more antigens to determine the therapeutic potential of compounds or biologics to modulate PD-1-dependent modulation of immune responses, including but not limited to specific T cell-dependent and B cell-dependent responses to a given antigen.

[0190] In multiple embodiments, the cells and / or non-human animals of the present invention are used for survival and / or proliferation assays (e.g., using B or T cells) to screen and develop candidate therapeutic agents that regulate human PD-1 signaling. The activation or loss of PD-1 can play an important role in regulating cell proliferation, and the self-tolerance regulation of PD-1 can be caused by the activation of specific epitopes of the extracellular domain of PD-1. Therefore, the cells of the non-human animals of the present invention and / or the non-human animals as described in the text can be used to identify, characterize and develop candidate PD-1 regulators (e.g., antagonists or agonists). In some embodiments, the cells and / or non-human animals of the present invention are used in survival or death assays to determine the effects of proliferation and apoptosis on specific cells (e.g., cancer cells) in the presence or absence of PD-1.

[0191] In multiple embodiments, the cells and / or non-human animals of the present invention are used for xenotransplantation of allogeneic (e.g., human) cells or tissues to determine the PD-1-mediated functions in the physiological (e.g., immune) response to transplanted human cells or tissues. In some embodiments, candidate therapeutic agents that bind or block one or more functions of human PD-1 are characterized in the non-human animals of the present invention. Suitable measurements include various cell 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 present invention are used to characterize the PD-1-mediated functions that regulate immune responses to antigens. In some embodiments, antigens are associated with tumors. In some embodiments, antigens are associated with autoimmune diseases, disorders, or conditions. In some embodiments, antigens are associated with inflammatory diseases, disorders, or conditions. In some embodiments, antigens are targets associated with diseases or conditions suffered by one or more patients in need of treatment.

[0192] In various embodiments, the non-human animals of the present invention are used in transplantation or adoptive transfer experiments to determine the therapeutic potential of compounds or biologics for PD-1-dependent regulation of new lymphocytes and their immune functions. In various embodiments, the non-human animals of the present invention are transplanted with human T cells; in some embodiments, naive T cells; in some embodiments, activated T cells.

[0193] In various embodiments, cells of non-human animals of the invention are used in T cell assays to determine the therapeutic potential of compounds or biologics for PD-1 dependent modulation of regulatory T cell dependent responses and functions. Exemplary T cell assays include, but are not limited to, ELISpot, intracellular cytokine staining, major histocompatibility complex (MHC) restriction, viral inhibition assays, cytotoxicity assays, proliferation assays, and regulatory T cell inhibition assays.

[0194] In various embodiments, cells of non-human animals of the invention are used in cell transmigration assays to screen and develop candidate therapeutic agents that modulate human PD-1. Cell transmigration involves the migration of cells across the endothelium, and transmigration assays allow for the measurement of the interaction of leukocytes or tumor cells with the endothelium and their transmigration.

[0195] In various embodiments, cells of the non-human animals of the invention are used in tumor growth (proliferation) assays to determine the therapeutic potential of compounds or biologics to modulate PD-1-dependent regulation and / or tumor cell apoptosis.

[0196] In multiple embodiments, cells of non-human animals of the present invention are used in cytokine production assays to determine the therapeutic potential of compounds or biologics for PD-1-dependent regulation of cytokines from T cells. In some embodiments, cells of non-human animals of the present invention are used to detect (and / or measure) intracellular cytokine release caused by the interaction of humanized PD-1 with drugs targeting human PD-1 or PD-1 ligands (e.g., PD-L1 or PD-L2).

[0197] 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 to determine the therapeutic potential of a compound or biologic to modulate PD-1-dependent modulation of one or more functions of an 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.

[0198] The non-human animal of the present invention provides an in vivo system for analyzing and testing drugs or vaccines. In multiple embodiments, candidate drugs or vaccines can be delivered to one or more non-human animals of the present invention, then the non-human animals are monitored to determine one or more immune responses to the drugs or vaccines, the safety characteristics of the drugs or vaccines, or the effect of a disease or illness. In some embodiments, vaccine targeting viruses, such as human immunodeficiency virus or hepatitis virus (such as HCV). Exemplary methods for determining safety characteristics include measuring toxicity, optimal dose concentration, efficacy of drugs or vaccines and possible risk factors. Such drugs or vaccines can be improved and / or developed in such non-human animals.

[0199] The non-human animals of the present invention provide an in vivo system for evaluating the pharmacokinetic properties of drugs targeting human PD-1. In multiple embodiments, drugs targeting human PD-1 can be delivered or administered to one or more non-human animals of the present invention, followed by monitoring the non-human animals (or cells isolated therefrom) or performing one or more assays to determine the effect of the drug on non-human animals. Pharmacokinetic properties include, but are not limited to, the way in which animals process drugs into various metabolites (or one or more drug metabolites, including detection of the presence or absence of toxic metabolites), drug half-life, circulating levels of drugs after administration (e.g., serum concentrations of drugs), anti-drug responses (e.g., anti-drug antibodies), drug absorption and distribution, routes of administration, excretion routes, and / or drug clearance. In some embodiments, the pharmacokinetic and pharmacodynamic properties of drugs (e.g., PD-1 modulators) are monitored in non-human animals of the present invention or by using non-human animals of the present invention.

[0200] The non-human animals of the present invention provide an in vivo system for evaluating the on-target toxicity of drugs targeting human PD-1. In multiple embodiments, drugs targeting human PD-1 can be delivered or administered to one or more non-human animals of the present invention, followed by monitoring the non-human animals (or cells isolated therefrom) or performing one or more assays to determine the on-target toxicity effects of the drugs on non-human animals. Typically, drugs are intended to regulate the functions of one or more of their targets. Just to give an example, PD-1 is intended to regulate PD-1-mediated functions (e.g., PD-1 signal transduction) by interacting with PD-1 molecules on one or more cell surfaces in some way. In some embodiments, such modulators may have the adverse effect of amplifying the desired pharmacological effects of modulators. Such effects are referred to as on-target effects. Exemplary on-target effects include too high a dose, chronic activation / inactivation, and the correct effects in incorrect tissues. In some embodiments, previously unknown PD-1 functions are determined using the on-target effects of drugs targeting human PD-1 identified in the non-human animals of the present invention or by using the non-human animals of the present invention.

[0201] The non-human animals of the present invention provide an in vivo system for evaluating the off-target toxicity of drugs targeting human PD-1. In multiple embodiments, drugs targeting human PD-1 can be delivered or administered to one or more non-human animals of the present invention, followed by monitoring the non-human animals (or cells isolated therefrom) or performing one or more assays to determine the off-target toxic effects of the drug on non-human animals. Off-target effects may occur when a drug interacts with an unexpected target (e.g., cross-reactivity to a common epitope). Such interactions may occur in expected or unexpected tissues. Just to give an example, the mirror image isomer (enantiomer) of a drug may lead to off-target toxic effects. In addition, the drug may not be suitable for interacting with different receptor subtypes or is not intended to activate different receptor subtypes. Exemplary off-target effects include incorrect activation / inhibition of incorrect targets, regardless of the tissue in which the incorrect target is found. In some embodiments, the off-target effects of drugs targeting human PD-1 are determined by comparing the effects of the drug administered to the non-human animals of the present invention with the effects administered to one or more reference non-human animals.

[0202] In some embodiments, the determination includes determining the effect on the phenotype (e.g., weight change) and / or genotype of the non-human animal to which the drug is administered. In some embodiments, the determination includes determining the batch-to-batch variability of PD-1 modulators (e.g., antagonists or agonists). In some embodiments, the determination includes determining the difference between the effects of drugs targeting human PD-1 administered to non-human animals of the present invention and reference non-human animals. In multiple embodiments, the reference non-human animal may have modifications as described herein, modifications different from the modifications described herein, or no modifications (e.g., modifications with destruction, deletion, or otherwise non-functional Pdcd1 genes) or no modifications (i.e., wild-type non-human animals).

[0203] Exemplary parameters that can be measured in non-human animals (or in cells isolated therefrom and / or using cells isolated therefrom) to assess the pharmacokinetic properties, on-target toxicity and / or off-target toxicity of drugs targeting human PD-1 include, but are not limited to, aggregation, autophagy, cell division, cell death, complement-mediated hemolysis, DNA integrity, drug-specific antibody titer, drug metabolism, gene expression arrays, metabolic activity, mitochondrial activity, oxidative stress, phagocytosis, protein biosynthesis, protein degradation, protein secretion, stress response, target tissue drug concentration, non-target tissue drug concentration, transcriptional activity, etc. In various embodiments, the non-human animals of the present invention are used to determine the pharmaceutically effective dose of the PD-1 modulator.

[0204] The non-human animal system of the present invention provides an in vivo system for developing and characterizing candidate therapeutic agents for cancer. In multiple embodiments, the non-human animal of the present invention can be implanted with a tumor, followed by administration of one or more candidate therapeutic agents. In some embodiments, the candidate therapeutic agent may include a multispecific antibody (e.g., a bispecific antibody) or an antibody mixture; in some embodiments, the candidate therapeutic agent system includes a combination therapy, such as the administration of a monospecific antibody administered sequentially or simultaneously. Allow the tumor enough time to establish in one or more locations within the non-human animal. Tumor cell proliferation, growth, survival, etc. can be measured before and after administration of the candidate treatment. The cytotoxicity of the candidate therapeutic agent can also be measured in non-human animals.

[0205] The non-human animals of the present invention can be used to develop one or more disease models to evaluate or assess candidate therapeutic agents and / or treatment regimens (e.g., monotherapy, combination therapy, dose range testing, etc.), thereby effectively treating diseases, disorders, or conditions that affect people. A variety of disease conditions can be established in the non-human animals of the present invention, and then one or more candidate molecules (e.g., drugs targeting PD-1) are administered so that the efficacy of one or more candidate molecules in disease conditions can be determined. In some embodiments, disease models include autoimmune, inflammatory, and / or neoplastic diseases, disorders, or conditions.

[0206] Just to give an example, the non-human animals of the present invention provide an improved animal model for the preventive 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 and then administered with one or more candidate therapeutic agents (e.g., antibodies). In some embodiments, the administration of one or more candidate therapeutic agents is performed after the implantation of one or more tumor cells (e.g., minutes or hours thereafter, but usually on the same day therewith), and the efficacy of one or more candidate therapeutic agents in preventing the establishment of solid tumors and / or tumor cell growth in the non-human animals of the present invention is evaluated. In some embodiments, the administration of one or more candidate therapeutic agents is performed after the implantation of one or more tumor cells (e.g., several days thereafter), and in certain embodiments, after a period of time sufficient for one or more implanted tumor cells to have reached a predetermined size (e.g., volume) in the non-human animals of the present invention; and the efficacy of one or more candidate therapeutic agents in treating one or more established tumors is evaluated. Non-human animals can be placed in different treatment groups according to the dose, so that the optimal dose or dose range associated with the effective treatment of established tumors can be determined.

[0207] Candidate molecules can be administered to non-human animal disease models using any administration method including parenteral and non-parenteral administration routes. Parenteral routes include, for example, intravenous, intraarterial, intraportal, intramuscular, subcutaneous, intraperitoneal, intraspinal, intrathecal, intraventricular, intracranial, intrapleural or other injection routes. Non-parenteral routes include, for example, oral, nasal, transdermal, pulmonary, rectal, buccal, vaginal, and ocular. Administration can also be performed by continuous infusion, topical administration, sustained release from implants (gels, films, etc.) and / or intravenous injection. When evaluating combination therapy in non-human animals of the present invention, candidate molecules can be administered via the same administration route or via different administration routes. When evaluating the dosing regimen in non-human animals of the present invention, candidate molecules can be administered once every two months, once a month, once every three weeks, once every two weeks, once a week, once a day, at variable intervals and / or with gradually increasing concentrations to determine a dosing regimen that exhibits the desired therapeutic or preventive effect in non-human animals where one or more disease models have been established.

[0208] The non-human animal system of the present invention provides an in vivo system for developing and characterizing candidate therapeutic agents for infectious diseases. In multiple embodiments, the non-human animals of the present invention can be infected by injecting viruses (e.g., MHV, HIV, HCV, etc.) or pathogens (e.g., bacteria), and then administering one or more candidate therapeutic agents. In some embodiments, candidate therapeutic agents may include multispecific antibodies (e.g., bispecific antibodies) or antibody mixtures; in some embodiments, candidate therapeutic agents include combination therapies, such as the administration of monospecific antibodies administered sequentially or simultaneously; in some embodiments, candidate therapeutic agents may include vaccines. Allow the virus or pathogen to have enough time to establish in one or more locations or cells in the non-human animal so that one or more symptoms associated with the virus or pathogen develop in the non-human animal. T cell proliferation and growth can be measured before and after the candidate treatment is administered. In addition, survival, serum and / or intracellular cytokine analysis, liver and / or spleen tissue pathology can be measured in non-human animals infected with viruses or pathogens. In some embodiments, the non-human animals of the present invention are used to determine the extent of organ damage associated with viral infection. In some embodiments, non-human animals of the invention are used to determine the cytokine expression profiles in various organs of non-human animals infected with a specific virus.

[0209] The non-human animals of the present invention can be used to evaluate the efficacy of therapeutic drugs targeting human cells. In multiple embodiments, the non-human animals of the present invention are transplanted with human cells, and the candidate drugs targeting such human cells are administered to such non-human animals. The therapeutic efficacy of the drug is then determined by monitoring the human cells in the non-human animals after the drug is administered. The drugs that can be tested in non-human animals include small molecule compounds, i.e., compounds with molecular weights less than 1500kD, 1200kD, 1000kD or 800 daltons, and macromolecular compounds (e.g., proteins, such as antibodies), which have an expected therapeutic effect for treating human diseases and illnesses by targeting human cells (e.g., combining and / or acting thereon).

[0210] In some embodiments, the drug is an anticancer drug, and the human cell is a cancer cell, which may be a cell of a primary cancer or a cell of a cell line established by a primary cancer. In these embodiments, the non-human animal of the present invention is transplanted with human cancer cells, and the anticancer drug is administered to the non-human animal. The efficacy of the drug can be determined by evaluating whether the growth or metastasis of human cancer cells in the non-human animal is inhibited by the administration of the drug.

[0211] In a specific embodiment, the anticancer drug is an antibody molecule that binds to an antigen on a human cancer cell. In a specific embodiment, the anticancer drug is a bispecific antibody that binds to an antigen on a human cancer cell and to an antigen on other human cells, such as cells of the human immune system (or "human immune cells"), such as B cells and T cells. Example

[0212] The following examples are provided to describe to those of ordinary skill in the art how to make and use the methods and compositions of the 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 pressure.

[0213] Example 1 Humanization of the endogenous programmed cell death 1 (Pdcd1) gene

[0214] This example illustrates an exemplary method for humanizing the endogenous Pdcd1 gene encoding the programmed cell death protein 1 (PD-1) in 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 human sequences (or sequence fragments) as described. In this example, an 883 bp human DNA fragment comprising exon 2, intron 2, and the first 71 bp of exon 3 of the human PDCD1 gene presented in GenBank Accession No.: NM_005018.2 (SEQ ID NO: 23) was used to humanize the endogenous Pdcd1 gene. The targeting vector used to humanize the genetic material encoding the extracellular N-terminal IgV region of the endogenous Pdcd1 gene is used. The technology is constructed (see, for example, US Pat. No. 6,586,251 and Valenzuela et al., 2003, Nature Biotech. 21(6):652-659; incorporated herein by reference).

[0215] In brief, the mouse bacterial artificial chromosome (BAC) clone RP23-93N20 (Invitrogen) was modified to delete exon 2, intron 2, and partial exon 3 sequences containing the endogenous Pdcd1 gene, and ~883bp human DNA fragments were used to insert exon 2, intron 2, and partial exon 3 of the human PDCD1 gene, which encodes amino acids 26-169 of the human PD-1 polypeptide. Endogenous DNA containing exon 1, partial exon 3 (ie, it encodes a transmembrane domain), exons 4 and 5, and 5' and 3' untranslated regions (UTRs) was retained. Sequence analysis of the ~883bp human DNA fragment confirmed all human PDCD1 exons (ie, exon 2 and 71bp exon 3) and splicing signals. Sequence analysis showed that the sequence matched the reference genome and PDCD1 transcript NM_005018.2.

[0216] In more detail, first, a small bacterial homologous recombination donor was constructed by a synthetic DNA fragment comprising the following: [(HindIII)-(mouse upstream 78bp)-(XhoI / NheI restriction endonuclease site)-(human PDCD1883bp)-(mouse downstream 75bp)-(HindIII)]. This fragment was synthesized by Genescript Inc. (Piscataway, NJ) and cloned into an ampicillin-resistant plasmid vector. The XhoI-NheI site was used to connect a 4,996bp self-deleting neomycin cassette (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). Neomycin was subsequently selected. The targeting vector was linearized using flanking HindIII sites prior to homologous recombination with the mouse BAC clone RP23-93N20. Intentionally, the junction between the human PDCD1 883 bp fragment and the mouse downstream 75 bp allowed the open reading frame in exon 3 to be retained ( 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, a self-deleting neomycin cassette flanked by loxP sites, an 883 bp human genomic DNA fragment (comprising exon 2 to the first 71 bp of exon 3 of the human Pdcd1 gene), and ∼84 kb of the mouse genome from BAC clone RP23-93N20.

[0217] As described above, modified RP23-93N20 BAC clones are used to electroporate mouse embryonic stem (ES) cells to form modified ES cells, wherein the modified ES cells comprise endogenous Pdcd1 genes humanized from exon 2 to part of exon 3 (i.e., 900bp of endogenous Pdcd1 gene deletions, and 883bp of human sequences are inserted). ES cells containing forward targeting of humanized Pdcd1 genes are identified by detecting the presence of human PDCD1 sequences (e.g., exon 2 and part of exon 3) and confirming the loss and / or retention of mouse Pdcd1 sequences (e.g., exon 2 and part of exon 3, and / or exons 1, 4, and 5) (Valenzuela et al., described above). Table 4 shows primers and probes for confirming the humanization of endogenous Pdcd1 genes as described above ( Figure 3 ). The nucleotide sequence spanning the upstream insertion site includes the following sequence, which indicates that the endogenous mouse sequence upstream of the 5' end of the self-deleting neomycin cassette of the insertion site (enclosed in parentheses below, with the XhoI restriction site in italics) is contiguously linked to the loxP site (in bold) and cassette sequence present at the insertion site: The nucleotide sequence spanning the downstream insertion point located 3' of the self-deleting neomycin cassette includes the following sequence, which indicates that the cassette sequence (enclosed in parentheses below, with loxP sequence in bold and NheI restriction site in italics) is adjacent to the human Pdcd1 genomic sequence downstream of the insertion point: The nucleotide sequence spanning the downstream insertion point located at the 3' end of the human PDCD1 genomic sequence includes the following sequence, which indicates that the human PDCD1 sequence is adjacent to the mouse Pdcd1 genomic sequence (enclosed in parentheses below): The nucleotide sequence spanning the upstream insertion point after deletion of the neomycin cassette (remaining 77 bp) includes the following sequence, which shows the mouse and human genomic sequences juxtaposed with the remaining cassette sequence loxP sequence (enclosed in parentheses below, XhoI and NheI restriction sites in italics, loxP sequence in bold):

[0218] Positive ES cell clones were then used to implant female mice. (See, e.g., U.S. Pat. No. 7,294,754 and Poueymirou et al., 2007, Nature Biotech. 25(1):91-99) to produce a litter of pups containing human PDCD1 exon 2 and a portion of human PDCD1 exon 3 inserted into the mouse endogenous Pdcd1 gene. Allele assay modification (Valenzuela et al., above) detecting the presence of human PDCD1 gene sequences was then used to confirm and identify mice in which exon 2 and a portion of exon 3 of the endogenous Pdcd1 gene were humanized (i.e., 883 bp human DNA fragments) by DNA genotyping isolated from the tail. The pups were genotyped and a cohort of animals heterozygous for the humanized Pdcd1 gene construct was selected for characterization.

[0219] Table 4

[0220]

[0221] Example 2 Expression of humanized PD-1 on activated T cells

[0222] This example demonstrates that a non-human animal (e.g., rodent) modified to include a humanized Pdcd1 gene according to Example 1 expresses a 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 as described in Example 1 were stained with an anti-PD-1 antibody to determine the expression of PD-1 in stimulated T cells isolated from wild-type and humanized mice.

[0223] Briefly, spleens were obtained from wild-type mice and mice heterozygous for the endogenous Pdcd1 gene humanized 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 plated at 1×10 6 / mL resuspended, 200 μL (200,000 cells) were plated in 96-well plates. The cells in the selected cells were stimulated with anti-CD3 and anti-CD28 antibodies (both at 1 μg / mL) for 72 hours. The cells were stained for FACS according to the manufacturer's instructions with antibodies recognizing CD4, CD8, CD19, and human (clone MIH4, BD Biosciences) or mouse (clone J43, eBioscience) PD1. The stained cells were run on an LSRII flow cytometer and the data were analyzed using Flowjo software. + T cell gating (CD19 - CD8 + ) to express human and mouse PD1. Exemplary results are shown in Figure 4 Shown in.

[0224] like Figure 4 As shown in , mice carrying a humanized Pdcd1 gene as described in Example 1 express a PD-1 polypeptide comprising a human portion and an endogenous mouse portion. The human portion can be detectably expressed via recognition by an antibody that recognizes the intact human PD-1 polypeptide.

[0225] Example 3 In vivo efficacy of PD-1 modulators

[0226] This example demonstrates that non-human animals (e.g., rodents) modified to include 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 properties, e.g., inhibition of tumor growth and / or killing of tumor cells. In this example, several anti-PD-1 antibodies were screened in mice heterozygous for the humanization of the endogenous Pdcd1 gene as described in Example 1 to determine the optimal antibody dose for inhibition of tumor growth and the extent to which the anti-PD-1 antibodies mediated tumor cell killing.

[0227] Briefly, mice were evenly divided into five treatment or control groups for Study 1 (n=5 / group), eight treatment or control groups for Study 2 (n=5 / group), and five treatment or control groups for Study 3 (n=7 / group) according to body weight. On day 0, mice were anesthetized by isoflurane inhalation, and then a suspension of MC38. oocytes in 100 μL DMEM was injected subcutaneously into the right flank (Study 1: 5×10 5 ; Research 2 / 3: 1×10 6 ). MC38.OV (mouse cloned adenocarcinoma) cells were engineered to express chicken ovalbumin to improve tumor immunogenicity. For Study 1, the treatment groups were injected intraperitoneally with 200 μg of any of the 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. For Study 2, the treatment groups were injected intraperitoneally with 10 mg / kg or 5 mg / kg / dose of any of the three anti-PD-1 antibodies, 10 mg / kg / dose of one anti-PD-1 antibody (Ab B, IgG4), or 10 mg / kg of an isotype control antibody of irrelevant specificity on days 3, 7, 10, 14, and 17 of the experiment. For Study 3, the treatment group was injected intraperitoneally with either 5 mg / kg or 2.5 mg / kg / dose of two anti-PD-1 antibodies, or 5 mg / kg of a control antibody not specific for PD-1 (control) on days 3, 7, 10, 14, and 17 of the experiment. Table 5 shows the experimental dosing and treatment regimens for each group of mice.

[0228] For each study, the mean tumor volume and percent survival determined by caliper measurements on days 14 or 17 and 23 or 24 were recorded for each treatment group. The number of tumor-free mice was 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) was calculated for each study. 3 ) (± SD), percent survival, and number of tumor-free mice (Tables 7-9). Exemplary tumor growth curves are Figure 5 Available in.

[0229] As shown in Table 6, for Study 1, mice treated with Ab A did not develop any detectable tumors during the study. On the 17th and 24th days of the study, mice treated with Ab C showed a tumor volume that continued to decrease compared to the control; and 3 mice in 5 mice had no tumor at the end of the experiment. In contrast, in this study, the treatment carried out with Ab B did not show significant efficacy in reducing tumor volume compared to the control. On the 23rd day of the study, 1 mouse died in 5 mice in the group receiving Ab B, and 2 mice died in 5 mice in the isotype control treatment group. In the non-treated group and the isotype control group, some mice showed spontaneous regression of tumors (respectively: 1 mouse in 5 mice; 2 mice in 5 mice).

[0230] As shown in Table 7, for Study 2, mice treated with 10 mg / kg of Ab A did not develop detectable tumors during the study. Mice treated with 10 mg / kg of Ab C or Ab D exhibited significantly reduced tumor volume on days 17 and 24 of the study compared to controls. Four of the five mice in each group treated with 10 mg / kg of Ab C or Ab D were tumor-free on day 31, while only one of the five animals in the isotype control treatment group was tumor-free due to natural tumor regression. Ab B tested at 10 mg / kg exhibited significantly reduced tumor volume on days 17 and 24 of the study compared to controls, but this antibody was the least potent anti-PD1 antibody, with only two of the five mice surviving at the end of the experiment.

[0231] A dose-dependent response in tumor inhibition at the tested doses (5 mg / kg and 10 mg / kg) was observed in the groups treated with Ab A, Ab C, and Ab D. The 5 mg / kg Ab A or Ab C therapy was less effective, with 4 of 5 mice being tumor-free at the end of the experiment on day 31, while 5 of 5 mice were tumor-free in the 10 mg / kg Ab A dose group. Dunnett's test for multiple comparisons of two-way ANOVA showed that the difference in tumor growth between the groups treated with 10 mg / kg of isotype control antibody as a reference and the groups treated with 10 mg / kg of Ab A, Ab C, or Ab D was statistically significant, with a p value of <0.005. The difference in tumor growth between the groups treated with 10 mg / kg of isotype control antibody and the groups treated with 5 mg / kg of Ab A, Ab C, or Ab D was also statistically significant, with a p value of <0.05.

[0232] As shown in Table 8, for Study 3, 6 of 7 mice treated with 5 mg / kg of Ab A or Ab C were tumor-free at the end of the experiment, while there were no tumor-free animals in the isotype control group. One tumor-bearing mouse in the IgG4 control group died on day 17 after implantation. 4 of 7 mice treated with 2.5 mg / kg of Ab C were 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, as measured by one-way ANOVA with Dunnett's multiple comparison post hoc test, p<0.01. The efficacy of all four anti-PD-1 antibodies tested at the 5 mg / kg dose was also stronger than that at the 2.5 mg / kg dose.

[0233] exist Figure 5 As shown in, anti-PD-1 antibodies significantly inhibited tumor growth in the preventive MC38. egg tumor growth model in PD-1 humanized mice prepared according to Example 1. 10mg / kg of anti-PD-1Ab therapy promoted tumor regression in all mice (5 out of 5 mice) throughout the experiment, while only 1 of 5 mice in the control group remained tumor-free due to natural tumor regression. The efficacy of 5mg / kg anti-PD-1 therapy was slightly lower, with 4 out of 5 mice being tumor-free at the end of the experiment. One-way ANOVA with Dunnett's multiple comparison post hoc test showed significant tumor volume differences between anti-PD-1 and control antibody treatments, with p values ​​<0.05 (5mg / kg) and p values ​​<0.01 (10mg / kg).

[0234] In a similar experiment, we measured CD8 + T cells and CD3 + T cell responses and IFNγ production were used to investigate the intact functional PD-1 signaling in the PD-1 humanized mice prepared according to Example 1.

[0235] Briefly, splenocytes were obtained from PD-1 humanized mice (75% C57BL / 6 / 25% 129) (described above) treated with anti-PD-1 or control antibodies at the end of the experiment on day 21. Total RNA was isolated and reverse transcribed cDNA was subjected to real-time PCR using a mixture of oligonucleotides and taqman probes for mouse CD8b (forward primer: GCTCTGGCTGGTCTTCAGTA 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) are specific. Samples were normalized to the expression of mouse cyclophilin B. Exemplary results are shown in Figure 6 Available in.

[0236] like Figure 6 As shown in Figure 2, administration of anti-hPD-1 antibodies induced CD8 + and CD3 + The production of T cells was increased. In addition, the activity of anti-hPD-1 antibodies in tumor-bearing PD-1 humanized mice was dependent on IFNγ, which confirmed the correct signaling through humanized PD-1 on the cell surface. In summary, an increase in T cells and IFNγ was observed in both treatment groups compared to control-treated mice.

[0237] Human PD-1 mRNA expression was measured with a human-specific probe designed for the extracellular portion of the PD-1 protein and confirmed the correct expression of the humanized PD-1 protein on the cell surface. In addition, measurement of mouse PD-1 mRNA expression with primers designed to detect the extracellular portion of mouse PD-1 failed to produce results.

[0238] Taken together, this example demonstrates that the non-human animals of the present invention can be used to evaluate the in vivo efficacy of drugs (e.g., antibodies) targeting PD-1, and such animals can be used to discern the therapeutic effects of anti-PD-1 antibodies. In addition, the non-human animals described herein can be used to evaluate the extent to which drugs targeting PD-1 can inhibit tumor growth and / or mediate tumor cell killing. The non-human animals (e.g., mice) of the present invention exhibit functional PD-1-signaling and correct PD-1-dependent immune responses via humanized PD-1, as demonstrated by T cell expression and cytokine expression (e.g., IFN-γ).

[0239] Table 5

[0240]

[0241] Table 6

[0242]

[0243] Table 7

[0244]

[0245] Table 8

[0246]

[0247] Example 4 Rodent Model of Anti-PD-1 Tumor Therapy

[0248] 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 a tumor model to determine the optimal therapeutic dose of a PD-1 modulator (e.g., an anti-PD-1 antibody). In this example, anti-PD-1 antibodies were administered to mice heterozygous for the endogenous Pdcd1 gene as described in Example 1 to determine the optimal therapeutic dose for treating established tumors.

[0249] Briefly, mice harboring the humanized Pdcd1 gene (as described in Example 1) were implanted subcutaneously with 1×10 6 MC38. oocytes (described above) and once the tumor volume reaches 80-120 mm 3 (Day 0) they were randomized into six treatment groups (n=8-9 / group). Mice were intraperitoneally administered an increasing dose range of 0.3-25 mg / kg (i.e., 0.3, 1, 3, 10, or 25 mg / kg) of anti-hPD-1 antibody or 25 mg / kg of isotype control antibody. Antibodies were administered on days 0, 3, 7, 10, and 13. Tumor volume was monitored by caliper measurements twice weekly for the duration of the experiment (60 days). Exemplary tumor growth curves are shown in Figure 7 Available in.

[0250] like Figure 7 As shown in , at the end of the experiment, no mice administered the control antibody were tumor-free. In contrast, anti-hPD-1 antibodies at doses ranging from 3-25 mg / kg resulted in approximately 44-55% tumor-free mice in the different treatment groups. Taken together, 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 drugs (e.g., antibodies) targeting PD-1 to effectively treat established tumors.

[0251] The present invention relates to the following embodiments:

[0252] 1. A rodent expressing a PD-1 polypeptide, wherein the PD-1 polypeptide comprises a human portion and an endogenous portion.

[0253] 2. The rodent of embodiment 1, wherein the PD-1 polypeptide is translated from a rodent signal peptide in cells of the rodent.

[0254] 3. The rodent of embodiment 1 or embodiment 2, wherein the endogenous portion comprises an intracellular portion of an endogenous PD-1 polypeptide.

[0255] 4. The rodent of embodiment 3, wherein the endogenous portion further comprises a transmembrane portion of an endogenous PD-1 polypeptide.

[0256] 5. The rodent of embodiment 4, wherein the endogenous portion has Figure 8 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 presented in.

[0257] 6. The rodent of embodiment 4, wherein the endogenous portion has Figure 8 The amino acid sequence is identical to the corresponding amino acid sequence of the mouse PD-1 polypeptide presented in .

[0258] 7. The rodent of any one of embodiments 1-6, wherein the human portion comprises amino acids 35-145 of the human PD-1 polypeptide.

[0259] 8. The rodent of any one of embodiments 1-6, wherein the human portion comprises amino acids 27-145 of the human PD-1 polypeptide.

[0260] 9. The rodent of any one of embodiments 1-6, wherein the human portion comprises amino acids 26-169 of the human PD-1 polypeptide.

[0261] 10. The rodent of any one of embodiments 1-6, wherein the human portion comprises Figure 8 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 human PD-1 polypeptide presented in .

[0262] 11. The rodent of any one of embodiments 1-6, wherein the human portion comprises Figure 8 The amino acid sequence is identical to the corresponding amino acid sequence of the human PD-1 polypeptide presented in .

[0263] 12. The rodent of any one of the preceding embodiments, wherein the PD-1 polypeptide comprising a human portion and an endogenous portion is encoded by an endogenous Pdcd1 gene.

[0264] 13. The rodent of embodiment 12, wherein the endogenous Pdcd1 gene comprises endogenous Pdcd1 exons 1, 4, and 5.

[0265] 14. The rodent of embodiment 13, wherein the endogenous Pdcd1 gene further comprises all or part of endogenous Pdcd1 exon 3.

[0266] 15. A rodent 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.

[0267] 16. The rodent of embodiment 15, wherein the rodent Pdcd1 promoter is an endogenous rodent Pdcd1 promoter.

[0268] 17. The rodent of embodiment 15 or embodiment 16, wherein the endogenous portion comprises endogenous Pdcd1 exons 1, 4, and 5.

[0269] 18. The rodent of embodiment 17, wherein the endogenous portion further comprises all or part of endogenous Pdcd1 exon 3.

[0270] 19. The rodent of embodiment 17 or embodiment 18, wherein exon 1, all or part of exon 3, exon 4 and exon 5 of the endogenous Pdcd1 gene are Figure 8 The corresponding exon 1, all or part of the corresponding exon 3, the corresponding exons 4 and 5 of the endogenous Pdcd1 gene represented in are at least 50%, at least 60%, at least 70%, at least 80%, at least 90% or at least 95% identical.

[0271] 20. The rodent of any one of embodiments 15-19, wherein the human portion encodes amino acids 26-169 of a human PD-1 polypeptide.

[0272] 21. The rodent of any one of embodiments 15-19, wherein the human portion comprises exon 2 of the human Pdcd1 gene.

[0273] 22. The rodent of embodiment 21, wherein the human portion further comprises all or part of human Pdcd1 exon 3.

[0274] 23. The rodent of embodiment 22, wherein human Pdcd1 exon 2 and all or part of human Pdcd1 exon 3 are Figure 8 The corresponding exon 2 and all or part of the corresponding exon 3 of the human Pdcd1 gene presented in are at least 50%, at least 60%, at least 70%, at least 80%, at least 90% or at least 95% identical.

[0275] 24. The rodent of embodiment 22, wherein the human portion comprises a sequence that is codon-optimized for expression in the rodent.

[0276] 25. The rodent of embodiment 22, wherein the human portion comprises SEQ ID NO: 23.

[0277] 26. The rodent of any one of embodiments 1-25, wherein the rodent is a rat or a mouse.

[0278] 27. A PD-1 polypeptide produced by the rodent of any one of embodiments 1-26.

[0279] 28. The PD-1 polypeptide according to embodiment 27, wherein the PD-1 polypeptide comprises Figure 8 The humanized PD-1 polypeptide represented in the present invention is at least 50%, at least 60%, at least 70%, at least 80%, at least 90% or at least 95% identical to the amino acid sequence of the humanized PD-1 polypeptide represented in the present invention.

[0280] 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, the portions being operably linked to a rodent Pdcd1 promoter.

[0281] 30. The isolated rodent cell or tissue of embodiment 29, wherein the rodent Pdcd1 promoter is an endogenous rodent Pdcd1 promoter.

[0282] 31. The isolated cell or tissue of embodiment 29 or embodiment 30, wherein the human portion comprises human Pdcd1 exon 2.

[0283] 32. The isolated cell or tissue of embodiment 31, wherein the human portion further comprises all or part of human Pdcd1 exon 3.

[0284] 33. An isolated rodent cell or tissue as described in any one of embodiments 29-32, wherein the rodent cell or tissue is a mouse cell or mouse tissue, or a rat cell or rat tissue.

[0285] 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, the portions being operably linked to a rodent Pdcd1 promoter.

[0286] 35. The rodent embryonic stem cell of embodiment 34, wherein the rodent Pdcd1 promoter is an endogenous rodent Pdcd1 promoter.

[0287] 36. The rodent embryonic stem cell of embodiment 34 or embodiment 35, wherein the human portion comprises human Pdcd1 exon 2.

[0288] 37. The rodent embryonic stem cell of embodiment 36, wherein the human portion further comprises all or part of human Pdcd1 exon 3.

[0289] 38. The rodent embryonic stem cell of any one of embodiments 34-37, wherein the rodent embryonic stem cell is a mouse embryonic stem cell and is from the 129 strain, the C57BL strain, or a mixture thereof.

[0290] 39. The rodent embryonic stem cell of embodiment 38, wherein the rodent embryonic stem cell is a mouse embryonic stem cell and is a mixture of the 129 strain and the C57BL strain.

[0291] 40. A rodent embryo produced from the embryonic stem cells of any one of embodiments 34-39.

[0292] 41. A method for preparing 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

[0293] (a) inserting a genomic fragment into the endogenous Pdcd1 gene in rodent embryonic stem cells, wherein the genomic fragment comprises a nucleotide sequence encoding all or part of a human PD-1 polypeptide;

[0294] (b) obtaining the rodent embryonic stem cells produced in (a);

[0295] (c) forming a rodent using the rodent embryonic stem cell of (b).

[0296] 42. The method of embodiment 41, wherein the human sequence comprises amino acids 35-145 of the human PD-1 polypeptide.

[0297] 43. The method of embodiment 41, wherein the human sequence comprises amino acids 27-145 of the human PD-1 polypeptide.

[0298] 44. The method of embodiment 41, wherein the human sequence comprises amino acids 26-169 of the human PD-1 polypeptide.

[0299] 45. The method of any one of embodiments 41-44, wherein the nucleotide sequence comprises exon 2 of human Pdcd1.

[0300] 46. ​​A method as described in embodiment 45, wherein the nucleotide sequence further comprises all or part of human Pdcd1 exon 3.

[0301] 47. The method of any one of embodiments 41-46, wherein the nucleotide sequence comprises one or more selection markers.

[0302] 48. A method as described in any of embodiments 41-47, wherein the nucleotide sequence comprises one or more site-specific recombination sites.

[0303] 49. A method of preparing a rodent, the genome of which comprises a Pdcd1 gene encoding a PD-1 polypeptide having a human portion and an endogenous portion, the portions being operably linked to a rodent Pdcd1 promoter, the method comprising

[0304] The genome of the rodent is modified so that the genome comprises a Pdcd1 gene encoding a PD-1 polypeptide having a human portion and an endogenous portion, wherein the portions are operably linked to a rodent Pdcd1 promoter, thereby preparing the rodent.

[0305] 50. The method of embodiment 49, wherein the rodent Pdcd1 promoter is an endogenous rodent Pdcd1 promoter.

[0306] 51. The method of embodiment 49, wherein the human portion comprises amino acids 35-145 of the human PD-1 polypeptide.

[0307] 52. The method of embodiment 49, wherein the human portion comprises amino acids 27-145 of the human PD-1 polypeptide.

[0308] 53. The method of embodiment 49, wherein the human portion comprises amino acids 26-169 of the human PD-1 polypeptide.

[0309] 54. The method of any one of embodiments 49-53, wherein the Pdcd1 gene is modified to comprise human Pdcd1 exon 2.

[0310] 55. The method of embodiment 54, wherein the Pdcd1 gene is modified to comprise human Pdcd1 exon 2 and all or part of human Pdcd1 exon 3.

[0311] 56. The method of any one of embodiments 41-55, wherein the rodent is a mouse or a rat.

[0312] 57. A rodent obtainable by the method of any one of embodiments 41-56.

[0313] 58. The rodent of embodiment 57, wherein the rodent is a mouse or a rat.

[0314] 59. A method of reducing tumor growth in a rodent, the method comprising the steps of

[0315] administering a drug targeting human PD-1 to a rodent, wherein the genome of the rodent comprises a Pdcd1 gene encoding a PD-1 polypeptide having a human portion and an endogenous portion, wherein the portions are operably linked to a rodent Pdcd1 promoter;

[0316] The administering is performed under conditions and for a duration sufficient to reduce tumor growth in the rodent.

[0317] 60. A method for killing tumor cells in rodents, the method comprising the following steps

[0318] administering a drug targeting human PD-1 to a rodent, wherein the genome of the rodent comprises a Pdcd1 gene encoding a PD-1 polypeptide having a human portion and an endogenous portion, wherein the portions are operably linked to a rodent Pdcd1 promoter;

[0319] The administration is performed under conditions and for a duration sufficient for the drug to mediate the killing of tumor cells.

[0320] 61. A method for evaluating the pharmacokinetic properties of a drug targeting human PD-1, the method comprising the following steps

[0321] administering the drug to a rodent, the genome of which comprises a Pdcd1 gene encoding a PD-1 polypeptide having a human portion and an endogenous portion, the portions being operably linked to a rodent Pdcd1 promoter; and

[0322] Assays are performed to determine one or more pharmacokinetic properties of the drug targeting human PD-1.

[0323] 62. The method of any one of embodiments 59-61, wherein the human portion comprises amino acids 35-145 of the human PD-1 polypeptide.

[0324] 63. The method of any one of embodiments 59-61, wherein the human portion comprises amino acids 27-145 of the human PD-1 polypeptide.

[0325] 64. The method of any one of embodiments 59-61, wherein the human portion comprises amino acids 26-169 of the human PD-1 polypeptide.

[0326] 65. A method as described in any one of embodiments 59-64, wherein the drug targeting human PD-1 is a PD-1 antagonist.

[0327] 66. A method as described in any one of embodiments 59-64, wherein the drug targeting human PD-1 is a PD-1 agonist.

[0328] 67. A method as described in any one of embodiments 59-64, wherein the drug targeting human PD-1 is an anti-PD-1 antibody.

[0329] 68. A method as described in any one of embodiments 59-67, wherein the drug targeting human PD-1 is administered intravenously to the rodent.

[0330] 69. A method as described in any one of embodiments 59-67, wherein the drug targeting human PD-1 is administered to the rodent intraperitoneally.

[0331] 70. A method as described in any one of embodiments 59-67, wherein the drug targeting human PD-1 is administered subcutaneously to the rodent.

[0332] 71. The method of any one of embodiments 59-70, wherein the rodent Pdcd1 promoter is an endogenous rodent Pdcd1 promoter.

[0333] 72. The method of any one of embodiments 59-71, wherein the rodent is a mouse or a rat.

[0334] 73. A rodent tumor model, said rodent expressing a PD-1 polypeptide comprising a human portion and an endogenous portion.

[0335] 74. A rodent tumor model, the rodent having a genome 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.

[0336] 75. A rodent tumor model obtained by the following steps

[0337] a) providing a rodent, the genome of which comprises a Pdcd1 gene comprising an endogenous portion and a human portion, the endogenous portion and the human portion being operably linked to a rodent Pdcd1 promoter; and

[0338] b) implanting one or more tumor cells into the rodent of (a);

[0339] Thereby providing the rodent tumor model.

[0340] 76. The rodent tumor model of any one of embodiments 73-75, wherein the rodent is a mouse or a rat.

[0341] 77. The rodent tumor model of embodiment 76, wherein the rodent is a mouse.

[0342] 78. The rodent tumor model of embodiment 77, wherein the mouse is selected from the 129 strain, the C57BL / 6 strain, and a mixed 129xC57BL / 6 strain.

[0343] 79. The rodent tumor model of embodiment 78, wherein the mice are 25% 129 and 75% C57BL / 6.

[0344] Equivalent

[0345] Having thus described several aspects of at least one embodiment of the present invention, it will be appreciated by those skilled in the art that various changes, modifications and improvements will be readily made by those skilled in the art. Such changes, modifications and improvements are intended to be a part of the present disclosure and are intended to be within the spirit and scope of the present invention. Therefore, the above description and accompanying drawings are intended only as examples, and the present invention is described in detail by the following claims.

[0346] The use of ordinal terms such as "first", "second", "third", etc. in the claims to modify claim elements does not in itself imply any priority, precedence or order of one claim element relative to another element or the temporal order in which method acts are performed, but is merely used as a mark to distinguish one claim element with a certain name from another element with the same name (but using ordinal terms) to distinguish the claim elements.

[0347] Unless expressly indicated to the contrary, the articles "a" and "an" used in the specification and claims herein should be understood to include plural referents in the specification and claims. Claims or descriptions that include "or" between one or more members of a group should be deemed to satisfy the following situation, that is, one, more than one, or all of the group members are present in, applied to, or otherwise related to a given product or method, unless otherwise indicated or clearly different from the context. The present invention includes such embodiments in which an exact member of the group is present in, applied to, or otherwise related to a given product or method. The present invention also includes such embodiments in which more than one group member or all group members are present in, applied to, or otherwise related to a given product or method. In addition, it should be understood that the present invention includes and covers all variations, combinations and permutations in which one or more limitations, elements, clauses, descriptive terms from one or more of the listed claims are introduced into another claim (or any other claim related thereto) that is subordinate to the same basic claim, unless otherwise indicated or unless it is obvious to a person of ordinary skill in the art that it would cause a contradiction or inconsistency. When elements are presented in the form of a list (e.g., in Markush groups or similar forms), it should be understood that each subgroup of these elements is also disclosed, and any element can be removed from the group. It should be understood that, in general, when the present invention or aspects of the present invention are referred to as comprising specific elements, features, etc., certain embodiments of the present invention or aspects of the present invention are composed of such elements, features, etc. or are essentially composed of them. For the purpose of simplification, these embodiments are not specifically described in each case with so many words stated herein. It should be understood that any embodiment or aspect of the present invention can be explicitly excluded from the claims, regardless of whether such specific exclusions are described in the specification.

[0348] Those skilled in the art will appreciate the typical standard deviations or errors attributable to the values ​​obtained in the assays or other methods described herein.Publications, websites, and other reference materials cited herein to describe the background of the present invention and to provide additional details related to its implementation are hereby incorporated by reference.

Claims

1. An isolated mouse cell or tissue, the genome of which comprises a humanized programmed cell death 1 (Pdcd1) gene at the endogenous mouse programmed cell death 1 (Pdcd1) locus, wherein the humanized Pdcd1 gene encodes a humanized programmed cell death 1 (PD-1) polypeptide comprising the amino acid sequence shown in SEQ ID NO: 6, and wherein the mouse cell or tissue is not an animal species.

2. The isolated mouse cell or tissue according to claim 1, wherein the humanized PD-1 polypeptide is translated together with the mouse signal peptide.

3. The isolated mouse cell or tissue according to claim 1, wherein the humanized Pdcd1 gene is operably linked to a mouse Pdcd1 promoter.

4. The isolated mouse cell or tissue according to claim 3, wherein the Pdcd1 promoter is an endogenous mouse Pdcd1 promoter.

5. Use of isolated mouse embryonic stem (ES) cells for preparing mice, wherein the genome of the isolated mouse ES cells comprises a humanized Pdcd1 gene at the endogenous mouse programmed cell death 1 (Pdcd1) locus, The humanized Pdcd1 gene encodes a humanized programmed cell death 1 (PD-1) polypeptide comprising the amino acid sequence shown in SEQ ID NO:

6.

6. The use according to claim 5, wherein the humanized PD-1 polypeptide is translated together with a mouse signal peptide.

7. The use according to claim 5, wherein the humanized Pdcd1 gene is operably linked to a mouse Pdcd1 promoter.

8. The use according to claim 7, wherein the Pdcd1 promoter is an endogenous mouse Pdcd1 promoter.

9. A nucleic acid vector comprising a humanized Pdcd1 gene encoding a humanized PD-1 polypeptide, The humanized PD-1 polypeptide comprises the amino acid sequence shown in SEQ ID NO:

6.

10. A PD-1 polypeptide, wherein the PD-1 polypeptide comprises the amino acid sequence shown in SEQ ID NO: 6.

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