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

Abstract

To provide non-human animals having humanized a Programmed cell death 1 gene.SOLUTION: Non-human animals, and methods and compositions for making and using the same, are provided, where the non-human animals comprise a humanization of a Programmed cell death 1 (Pdcd1) gene. The non-human animals may be described, in some embodiments, as having a genetic modification to an endogenous Pdcd1 gene so that the non-human animals express a PD-1 polypeptide that includes a human portion and an endogenous portion (e.g., a non-human portion). For example, the present invention provides a rodent that expresses a PD-1 polypeptide, where the PD-1 polypeptide comprises a human portion and an endogenous portion.SELECTED DRAWING: None

Description

[Technical field]

[0001] (CROSS REFERENCE TO RELATED APPLICATIONS) This application is a continuation of U.S. Provisional Patent Application No. 62 / 014,181, filed June 19, 2014. No. 62 / 086,518 filed December 2, 2014, and No. 62 / 086,518 filed March 25, 2015 This application claims the benefit of priority to provisional patent application Ser. No. 62 / 138,221, filed on Dec. 13, 2003 ... and is hereby incorporated by reference in its entirety. the entire contents of which are incorporated herein by reference.

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

[0003] Medical research and development has focused on cancer immunotherapy, and significant improvements have been made; however, This remains a major challenge for the medical industry worldwide. This is due in part to the ability of cancer cells to evade monitoring mechanisms. This is the result of inhibition and / or downregulation of tumor immunity. Optimally determine the therapeutic potential of new cancer treatments designed to promote and / or improve , how cancer cells provide inhibitory signals to immune cells (e.g., T cells) No in vivo systems have been developed to determine the specific aspects of the , a source of assays to evaluate the therapeutic efficacy of candidate agents that promote an anti-tumor environment in vivo to provide. Summary of the Invention [Means for solving the problem]

[0004] The present invention provides an improved method for identifying and developing new therapeutic agents that can be used to treat cancer. This includes the recognition that it is desirable to design non-human animals that tolerate such systems. The present invention relates to a novel compound that can be used to treat an autoimmune (or inflammatory) disease, disorder, or condition. Engineering non-human animals that allow improved systems for identifying and developing new therapeutics The present invention also encompasses the recognition that it is desirable to improve, for example, anti-tumor immunity. To identify and develop cancer drugs that correct the humanized Pdcd1 gene, and / or otherwise express, contain, or comprise a human or humanized PD-1 polypeptide. In some embodiments, the present invention also encompasses the recognition that non-human animals that have or produce the The non-human animals of the invention are useful for identifying and developing combination therapies that involve targeting PD-1. The present invention provides an improved in vivo system.

[0005] In some embodiments, the present invention provides a method for the detection of HIV-1 infections from two different species (e.g., human and non-human). A non-human animal is provided having a genome comprising a Pdcd1 gene that includes genetic material. In one embodiment, the Pdcd1 gene of the non-human animal described herein is a human or non-human Pdcd1 gene. encoding a PD-1 polypeptide comprising a human portion, wherein the human and non-human portions are homologous to each other In some embodiments, the PD-1 polypeptides described herein are linked to form a functional PD-1 polypeptide. The non-human Pdcd1 gene encodes the entire extracellular domain of the human PD-1 polypeptide. or a portion thereof.

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

[0007] 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 comprises the mouse PD-1 polypeptide shown in FIG. At least 50%, at least 60%, at least 70% identical to the corresponding amino acid sequence of having an amino acid sequence that is at least 80%, at least 90%, or at least 95% identical In some embodiments, the endogenous portion is the sequence of the mouse PD-1 polypeptide shown in FIG. The amino acid sequence is substantially identical to the corresponding amino acid sequence. The amino acid sequence of the polypeptide is identical to the corresponding amino acid sequence of the mouse PD-1 polypeptide shown in FIG. It has an amino acid sequence.

[0008] In some embodiments, the human portion comprises amino acids 35-145 of a human PD-1 polypeptide; 27 to 145, 27 to 169, 26 to 169, or 21 to 170. In one embodiment, the human portion has the corresponding amino acid sequence of the human PD-1 polypeptide shown in FIG. and at least 50%, at least 60%, at least 70%, at least 80%, In some embodiments, the amino acid sequence of each of the sequences is 90%, or at least 95% identical to each other. , the human portion corresponds substantially to the corresponding amino acid sequence of the human PD-1 polypeptide shown in FIG. In some embodiments, the human portion has an amino acid sequence substantially identical to that of the human It has an amino acid sequence identical to the corresponding amino acid sequence of the PD-1 polypeptide.

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

[0010] In some embodiments, the PD-1 polypeptide expressed by a non-human animal described herein is The peptide has a structure that is at least 50%, at least 60%, or at least 70% identical to SEQ ID NO:6. , having an amino acid sequence that is at least 80%, at least 90%, or at least 95% identical. In some embodiments, the PD-1 polypeptide expressed by a non-human animal described herein is The peptide has an amino acid sequence substantially identical to SEQ ID NO:6. The PD-1 polypeptide expressed by the non-human animals described herein is set forth in SEQ ID NO: : It has the same amino acid sequence as 6.

[0011] In some embodiments, the present invention provides a method for the detection of all or part of one or more exons of the human Pdcd1 gene. or a portion thereof, In some embodiments, the humanized Pdcd1 locus is The locus contains 5' and 3' non-human P sequences flanking one or more exons of the human Pdcd1 gene. In some embodiments, the humanized Pdcd1 further comprises a dcd1 untranslated region (UTR). The locus is under the control of a rodent promoter and, in certain embodiments, an endogenous rodent promoter. It is under the control of a promoter similar to the one described above.

[0012] In some embodiments, the humanized Pdcd1 locus operably links to human Pdcd1 exon 2. It contains non-human Pdcd1 exons 1, 3, 4 and 5 linked together as desired. In embodiments, the humanized Pdcd1 locus comprises non-human Pdcd1 exons 1, 4 and 5, Human Pdcd1 exon 2, Pdcd1 exon 3, exon 3 comprises a human portion and a non-human portion, wherein the non-human and human exons are In some embodiments, the human portion of Pdcd1 exon 3 is operably linked to In some embodiments, the Pdcd1 gene comprises nucleotides encoding a PD-1 stalk sequence. The human portion of exon 3 comprises approximately 71 bp of human Pdcd1 exon 3. In this study, the non-human portion of Pdcd1 exon 3 contains nucleotides encoding a transmembrane sequence. In some embodiments, the non-human portion of Pdcd1 exon 3 is about 91 bp of the rodent Pd Includes cd1 exon 3.

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

[0014] In some embodiments, the endogenous portion comprises endogenous Pdcd1 exons 1, 4 and 5. In some embodiments, the endogenous portion comprises all or a portion of endogenous Pdcd1 exon 3. In some embodiments, the endogenous Pdcd1 gene is further provided with all or part of exons 1 and 3. 4 and 5 correspond to the corresponding exon 1 of the endogenous Pdcd1 gene shown in Fig. 8. , all or part of 3, 4 and 5 and at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 95% identical. In some embodiments, all or part of exons 1, 3, 4 and 5 of the endogenous Pdcd1 gene are and 5 are the whole or the whole of the corresponding exons 1 and 3 of the endogenous Pdcd1 gene shown in FIG. In some embodiments, the endogenous Pdcd1 gene is substantially identical to 4 and 5. Exons 1, all or part of 3, 4 and 5 of the Pdcd1 gene are expressed in the endogenous Pdcd1 gene as shown in Figure 8. It is identical to all or part of the corresponding exons 1, 3, 4 and 5 of the gene.

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

[0016] In some embodiments, the human portion comprises exon 2 of the human Pdcd1 gene. In embodiments, the human portion further comprises all or a portion of human Pdcd1 exon 3. In some embodiments, all or part of human Pdcd1 exons 2 and 3 are and at least part of the corresponding exons 2 and 3 of the human Pdcd1 gene, At least 50%, at least 60%, at least 70%, at least 80%, at least 90% or at least 95% identical. and 3 are all or part of the corresponding exons of the human Pdcd1 gene shown in FIG. 2 and 3. In some embodiments, the human Pdcd 1 All or part of exons 2 and 3 correspond to the human Pdcd1 gene shown in Figure 8. In some embodiments, the human portion is identical to all or part of exons 2 and 3. comprises a sequence that is codon-optimized for expression in non-human animals, and in some embodiments, in rodents. In certain embodiments, expression in a mammalian species, in certain embodiments, expression in a mouse, and in certain embodiments, expression in a rat The gene has been codon-optimized for expression in vitro.

[0017] In some embodiments, the human portion is at least 50% identical to SEQ ID NO:23, at least 6 0%, at least 70%, at least 80%, at least 90%, or at least 95% % identical to SEQ ID NO:23. In some embodiments, the human portion comprises a sequence substantially identical to SEQ ID NO:23. In some embodiments, the human portion comprises a sequence identical to SEQ ID NO:23. In some embodiments, the human portion comprises SEQ ID NO:23.

[0018] In some embodiments, the present invention relates to a method for producing a non-human animal as described herein. In certain embodiments, the present invention provides a PD-1 polypeptide (or a polypeptide generated by the PD-1 polypeptide generating process). The PD-1 polypeptide produced by the non-human animals described herein has the structure set forth in SEQ ID NO:6. and at least 50%, at least 60%, at least 70%, at least 80%, In some particular embodiments, the amino acid sequence of The PD-1 polypeptide produced by the non-human animals described herein is represented by SEQ ID NO: In certain embodiments, the amino acid sequence of any one of the sequences described herein is substantially the same as that of: The PD-1 polypeptide produced by the engineered non-human animal has an amino acid sequence identical to SEQ ID NO:6. It has an amino acid sequence.

[0019] In some embodiments, the present invention relates to isolated cells or cells derived from a non-human animal as described herein. In some embodiments, the present invention provides tissues comprising the Pdcd1 gene described herein. In some embodiments, the cell is a lymphocyte. In some embodiments, the cells are B cells, dendritic cells, macrophages, monocytes (e.g., activated monocytes), NK cells, and T cells (e.g., activated T cells). In an embodiment, the tissue is adipose, bladder, brain, breast, bone marrow, eye, heart, intestine, kidney, liver, lung. , lymph nodes, muscles, pancreas, plasma, serum, skin, spleen, stomach, thymus, testes, eggs, and The combination is selected from the above.

[0020] In some embodiments, the present invention relates to a method for the detection of inflammatory bowel disorders ... In some embodiments, the non-human embryonic stem cells are mouse embryonic stem cells. Stem cells, derived from 129, C57BL / 6, or BALB / c strains. In some embodiments, the non-human embryonic stem cells are mouse embryonic stem cells, including lineage 129, C57B L / 6 lineage, or a mixture thereof. The cells are mouse embryonic stem cells, derived from a mixture of 129 and C57BL / 6 strains. be.

[0021] In some embodiments, the non-human embryonic stem cells are selected from the group consisting of SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO: SEQ ID NO:21, SEQ ID NO:22, or a combination thereof Has a genome.

[0022] In some embodiments, the present invention provides a method for producing a non-human animal comprising the steps of: Methods for using embryonic stem cells are provided. In certain embodiments, the non-human embryonic stem cells are mouse embryonic stem cells. The cells are then used to generate mice carrying the Pdcd1 gene described herein. In certain embodiments, the non-human embryonic stem cells are rat embryonic stem cells, The resulting mouse model is used to generate rats carrying the Pdcd1 gene as described herein.

[0023] In some embodiments, the present invention relates to a non-human ovarian cancer comprising the Pdcd1 gene described herein. comprising, made from, derived from, or produced from human embryonic stem cells In some specific embodiments, the non-human embryo is a rodent embryo, and some In embodiments, the embryo is a mouse, and in some embodiments, a rat embryo.

[0024] In some embodiments, the present invention provides a method for producing a non-human animal comprising the steps of: Methods for using the embryos are provided. In certain embodiments, the non-human embryonic stem cells are mouse embryonic stem cells. and used to generate mice comprising the Pdcd1 gene described herein. In certain embodiments, the non-human embryo is a rat embryo and is treated with the Pdc Used to generate rats with the d1 gene.

[0025] In some embodiments, the present invention relates to a targeting vector (or nucleic acid construct) described herein. In some embodiments, the present invention provides a humanized Pdc construct as described herein. A targeting vector (or nucleic acid construct) comprising the d1 gene is provided. In some embodiments, The present invention provides a method for the production of a PD-1 polypeptide comprising the entire or a portion of the human extracellular domain. The present invention provides a targeting vector (or nucleic acid construct) comprising a Pdcd1 gene that is capable of targeting certain specific In certain embodiments, amino acids 21-170, 26-169, 27-29, 30-31 of the human PD-1 polypeptide are 7 to 169, 27 to 145, or 35 to 145.

[0026] In some embodiments, the targeting vector (or nucleic acid construct) comprises a targeting vector encoding the human Pdcd1 gene. a non-human Pdcd1 gene operably linked to all or part of one or more exons of In some embodiments, the targeting vector comprises all or a portion of one or more exons of a gene. The target (or nucleic acid construct) comprises five nucleotides adjacent to one or more exons of the human Pdcd1 gene. In some embodiments, the target comprises a 3' and 3' non-human Pdcd1 untranslated region (UTR). The targeting vector (or nucleic acid construct) comprises one or more selectable markers. In the present invention, a targeting vector (or nucleic acid construct) is provided with one or more site-specific recombination sites. In some embodiments, the targeting vector (or nucleic acid construct) comprises a human Pdcd1 exon. In some embodiments, the targeting vector (or nucleic acid construct) comprises human Pdc d1 exon 2 and all or part of human Pdcd1 exon 3.

[0027] In some embodiments, the present invention relates to a method for producing modified non-human embryonic stem cells, comprising the steps of: In some embodiments, the present invention provides methods for using the targeting vector (or nucleic acid construct). The invention provides a method for producing a modified non-human embryo using the targeting vectors (or nucleic acid constructs) described herein. In some embodiments, the present invention provides methods of using the constructs to generate modified non-human animals. The present invention also provides methods of using the targeting vectors (or nucleic acid constructs) described herein.

[0028] In some embodiments, the present invention provides a method for producing a PD-1 polypeptide from an endogenous Pdcd1 gene. The present invention provides a method for producing a non-human animal expressing a PD-1 polypeptide, wherein the PD-1 polypeptide is a human sequence. The method comprises: (a) inserting a genomic fragment into an endogenous Pdcd1 gene of a rodent embryonic stem cell; wherein the genomic fragment contains the entire human PD-1 polypeptide. (b) providing a nucleotide sequence encoding a portion or part of the nucleic acid sequence produced in (a); (b) obtaining rodent embryonic stem cells; and (b) generating a rodent using the rodent embryonic stem cells. The method includes the steps of:

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

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

[0031] In some embodiments, the present invention provides a PD gene whose genome has human and endogenous portions. a Pdcd1 gene encoding a Pdcd1 polypeptide, The present invention provides a method for producing a non-human animal operably linked to a human promoter, the method comprising: The Pdcd1 gene, which encodes a PD-1 polypeptide having both endogenous and endogenous portions, was The genome of the human animal is provided such that the portion is operable in the rodent Pdcd1 promoter. and modifying the genome of the non-human animal so that the non-human animal is linked to the The method includes the step of:

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

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

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

[0035] In some embodiments, modification of the genome of the non-human animal is performed in a non-human embryonic stem cell, The non-human embryonic stem cells are then used to generate a non-human animal. are rodent embryonic stem cells, in some embodiments mouse embryonic stem cells, in some embodiments These are rat embryonic stem cells.

[0036] In some embodiments, the present invention relates to a non-human animal obtained by the methods described herein. Provide something.

[0037] In some embodiments, the present invention provides a method of reducing tumor growth in a non-human animal, the method comprising: The genome encodes a PD-1 polypeptide having a human portion and an endogenous portion. Pdcd1 gene, said portion being operable with a rodent Pdcd1 promoter. administering to the linked non-human animal an agent that targets human PD-1; is carried out under conditions and for a time sufficient for tumor growth to be reduced in the non-human animal.

[0038] In some embodiments, the invention provides a method of killing tumor cells in a non-human animal, the method comprising: The genome of Pdc encodes a PD-1 polypeptide that has both human and endogenous portions. d1 gene, said portion being operably linked to a rodent Pdcd1 promoter. The method includes administering to a non-human animal having a human PD-1-targeting agent, the administration comprising administering to the non-human animal an agent that targets human PD-1 ... The agent is administered under conditions and for a time sufficient to mediate killing of the tumor cells in the non-human animal.

[0039] In some embodiments, the present invention provides a method for evaluating the pharmacokinetic properties of agents that target human PD-1. The present invention provides a method for the production of a PD-1 polypeptide comprising: administering a drug to a non-human animal comprising a Pdcd1 gene encoding a polypeptide. wherein said portion is operably linked to a rodent Pdcd1 promoter. Assays to determine one or more pharmacokinetic properties of agents targeting human PD-1 and performing a step of:

[0040] In many embodiments, the non-human animals described herein comprise a genome that contains human portions and A rodent that contains the Pdcd1 gene encoding the PD-1 polypeptide with an endogenous portion. and the portion is operably linked to a rodent Pdcd1 promoter. In embodiments, the rodent Pdcd1 promoter is an endogenous rodent Pdcd1 promoter. In many embodiments, the human portion comprises amino acids 35-14 of a human PD-1 polypeptide. 5, 27-145, 27-169, 26-169 or 21-170.

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

[0042] In some embodiments, the present invention provides a method for the production of PD in a non-human animal comprising a human portion and an endogenous portion. A non-human animal model expressing the -1 polypeptide is provided.

[0043] In some embodiments, the present invention provides a method for the production of Pd A non-human animal is provided comprising a cd1 gene, wherein the endogenous portion and the human portion are from a rodent. It is operably linked to the Pdcd1 promoter.

[0044] In some embodiments, the invention relates to a method for the detection of a mutated human genome comprising: (a) detecting a mutated human genome comprising an endogenous portion and a human portion; Providing a non-human animal comprising a Pdcd1 gene comprising an endogenous portion and a human (b) operably linking the moiety to a non-human animal Pdcd1 promoter; ) implanting one or more tumor cells of the rodent of (a) in a non-human animal. The present invention provides a tumor model, thereby providing said non-human animal tumor model.

[0045] In some embodiments, the non-human animal tumor models of the present invention are rodent tumor models. In one embodiment, the non-human Pdcd1 promoter is a rodent Pdcd1 promoter. be.

[0046] In some embodiments, the present invention provides methods for identifying or validating a drug or vaccine, the methods comprising: The method comprises the steps of: determining whether or not a PD-1 polypeptide is expressed in a non-human animal, the genome of which contains the Pdcd1 gene encoding the PD-1 polypeptide; A process for delivering a drug or vaccine to a subject, the process comprising: and endogenous moieties, and immune responses to drugs or vaccines, monitor one or more of the safety profile of the drug or its effect on a disease, disorder or condition In some embodiments, monitoring the safety profile includes administering a non-human immunization Whether the subject animal exhibits side effects or adverse reactions as a result of drug or vaccine delivery In some embodiments, the side effects or adverse reactions include determining morbidity, mortality, , change in body weight, change in one or more enzyme levels (e.g., liver), change in the weight of one or more organs Changes or loss of function (e.g., sensory, motor, organ, etc.) or susceptibility to one or more diseases Increase, modification of the genome of a non-human animal, increased food intake and one or more disease complications or In some embodiments, the disease, disorder, or condition is induced in a non-human animal. In some embodiments, the disease, disorder or condition induced in the non-human animal is in need of treatment. The subject may be a patient suffering from one or more diseases, disorders or conditions. In certain embodiments, the agent is an antibody.

[0047] In some particular embodiments, the present invention provides a pharmaceutical agent for use in medicine, such as for use as a drug. Alternatively, the present invention provides the use of a non-human animal as described herein in the development of a vaccine.

[0048] In some embodiments, the present invention relates to a method for treating a cancer, neoplastic, infectious, or inflammatory disease, disorder, or condition. in the manufacture of a drug for treating a disease, disorder or condition of Uses of the non-human animals described herein are provided.

[0049] In various embodiments, the Pdcd1 gene of the present invention is a Pdcd1 gene as described herein. In various embodiments, the Pdcd1 gene of the present invention comprises a human portion and an endogenous portion. The present invention relates to a PD-1 polypeptide having an inhibitory portion that is capable of binding to a rodent Pdcd1 promoter. In various embodiments, the rodent promoter is operably linked to an endogenous promoter. In various embodiments, the human portion is a human Pdcd1 exon. In various embodiments, the human portion comprises human Pdcd1 exon 2, It further comprises all or part of human Pdcd1 exon 3.

[0050] In various embodiments, the PD-1 polypeptides of the invention are PD- In various embodiments, the non-human animals of the invention comprise a full-length endogenous non-human 1 polypeptide. In various embodiments, the subject invention provides a method for the detection of PD-1 polypeptide in a subject cell. The disclosed non-human animals detectably express the extracellular portion of the endogenous non-human PD-1 polypeptide. In various embodiments, the non-human animals of the invention do not express endogenous non-human PD-1 polypeptide. There is no detectable expression of the N-terminal immunoglobulin V region of the polypeptide.

[0051] In various embodiments, the non-human animals of the present invention are rodents, and in some embodiments, mice. In some embodiments, the mouse of the present invention is a mouse, and in some embodiments a rat. 129, BALB / C, C57BL / 6, and mixed 129xC57BL / 6 and in some particular embodiments, 50% 129 and 50% C57B. L / 6, and in some particular embodiments, 25% 129 and 75% C57BL / 6. be. In certain embodiments, for example, the following items are provided: (Item 1) A rodent expressing a PD-1 polypeptide, wherein the PD-1 polypeptide is a human polypeptide. rodents with endogenous and endogenous portions. (Item 2) The PD-1 polypeptide is translated in a cell of the rodent with a rodent signal peptide. , A rodent as described in item 1. (Item 3) Item 1 or 2, wherein the endogenous portion comprises an intracellular portion of an endogenous PD-1 polypeptide. A rodent as described in. (Item 4) 4. The method of claim 3, wherein the endogenous portion further comprises a transmembrane portion of an endogenous PD-1 polypeptide. The described rodent. (Item 5) The endogenous portion has the corresponding amino acid sequence of the mouse PD-1 polypeptide shown in FIG. Column and at least 50%, at least 60%, at least 70%, at least 80%, at least 5. The rodent according to claim 4, having an amino acid sequence which is at least 90% identical, or at least 95% identical, to said kind. (Item 6) The endogenous portion has the corresponding amino acid sequence of the mouse PD-1 polypeptide shown in FIG. 5. A rodent according to item 4, having an amino acid sequence identical to that of item 4. (Item 7) Item 1 - The human portion comprises amino acids 35 to 145 of a human PD-1 polypeptide. 7. A rodent according to any one of claims 6 to 6. (Item 8) Item 1 - The human portion comprises amino acids 27 to 145 of a human PD-1 polypeptide. 7. A rodent according to any one of claims 6 to 6. (Item 9) Item 1 - The human portion comprises amino acids 26 to 169 of a human PD-1 polypeptide. 7. A rodent according to any one of claims 6 to 6. (Item 10) The human portion has a sequence similar to the corresponding amino acid sequence of the human PD-1 polypeptide shown in FIG. At least 50%, at least 60%, at least 70%, at least 80%, at least 7. Any of items 1 to 6, having an amino acid sequence that is at least 90% or at least 95% identical to A rodent according to claim 1. (Item 11) The human portion has a sequence similar to the corresponding amino acid sequence of the human PD-1 polypeptide shown in FIG. 7. The rodent according to any one of items 1 to 6, comprising an identical amino acid sequence. (Item 12) The PD-1 polypeptide comprising a human portion and an endogenous portion is derived from an endogenous Pdcd1 gene. 12. A rodent according to any one of items 1 to 11, encoded by a progeny. (Item 13) The endogenous Pdcd1 gene comprises endogenous Pdcd1 exons 1, 4 and 5. A rodent according to the order 12. (Item 14) the endogenous Pdcd1 gene further comprises all or a portion of endogenous Pdcd1 exon 3 Item 14. A rodent according to item 13, comprising: (Item 15) A rodent comprising a Pdcd1 gene comprising an endogenous portion and a human portion, said endogenous portion comprising A rodent gene encoding a human Pdcd1 promoter having a human and human portion operably linked to the rodent Pdcd1 promoter. . (Item 16) The rodent Pdcd1 promoter is an endogenous rodent Pdcd1 promoter. A rodent according to the order 15. (Item 17) Item 15. The endogenous portion comprises endogenous Pdcd1 exons 1, 4 and 5; or 16. A rodent according to claim 16. (Item 18) the endogenous portion further comprises all or a portion of endogenous Pdcd1 exon 3. 17. A rodent according to claim 17. (Item 19) Exons 1, all or part of 3, 4 and 5 of the endogenous Pdcd1 gene are shown in Figure 8. The corresponding exons 1, 3, 4 and 5 of the endogenous Pdcd1 gene and at least 50%, at least 60%, at least 70%, at least 80%, 19. The rodent according to item 17 or 18, which is 90%, or at least 95% identical to said (Item 20) the human portion encodes amino acids 26 to 169 of a human PD-1 polypeptide. 20. A rodent according to any one of claims 15 to 19. (Item 21) 20. Any of items 15 to 19, wherein the human portion comprises exon 2 of the human Pdcd1 gene. A rodent according to any one of claims 1 to 4. (Item 22) Item 21. The human portion further comprises all or a portion of human Pdcd1 exon 3. A rodent as described in. (Item 23) All or part of human Pdcd1 exons 2 and 3 are identified in human Pdcd1 shown in FIG. 1 gene and at least 50%, or at least At least 60%, at least 70%, at least 80%, at least 90%, or at least 23. The rodent according to item 22, which is 95% identical. (Item 24) Item 22. The human portion comprises a sequence that is codon-optimized for expression in the rodent. A rodent as described in. (Item 25) 23. The rodent of item 22, wherein the human portion comprises SEQ ID NO:23. (Item 26) 26. The rodent according to any one of items 1 to 25, wherein the rodent is a mouse or a rat. . (Item 27) 27. A PD-1 polypeptide produced by the rodent of any one of items 1 to 26. (Item 28) The PD-1 polypeptide has at least the same structure as the humanized PD-1 polypeptide shown in FIG. At least 50%, at least 60%, at least 70%, at least 80%, at least 90% 28. The PD-1 polypeptide according to claim 27, which has an amino acid sequence identical thereto or at least 95% identical thereto. peptide. (Item 29) Pd, whose genome encodes a PD-1 polypeptide having human and endogenous portions. cd1 gene, said portion operably linked to a rodent Pdcd1 promoter. , isolated rodent cells or tissues. (Item 30) The rodent Pdcd1 promoter is an endogenous rodent Pdcd1 promoter. A rodent cell or tissue as described in order 29. (Item 31) 31. The isolated method according to item 29 or 30, wherein the human portion comprises human Pdcd1 exon 2. Cells or tissues. (Item 32) Item 31. The human portion further comprises all or part of human Pdcd1 exon 3. 2. The isolated cell or tissue according to claim 1 . (Item 33) The rodent cells or tissues are mouse cells or mouse tissues, or rat cells or 33. The isolated rodent cell or tissue according to any one of items 29 to 32, wherein the isolated rodent cell or tissue is a rat tissue. . (Item 34) Pd, whose genome encodes a PD-1 polypeptide having human and endogenous portions. cd1 gene, said portion operably linked to a rodent Pdcd1 promoter. , rodent embryonic stem cells. (Item 35) The rodent Pdcd1 promoter is an endogenous rodent Pdcd1 promoter. Rodent embryonic stem cells according to the method 34. (Item 36) 36. The rodent of claim 34 or 35, wherein the human portion comprises human Pdcd1 exon 2. Embryoid stem cells. (Item 37) Item 36. The human portion further comprises all or part of human Pdcd1 exon 3. 2. A rodent embryonic stem cell according to claim 1 . (Item 38) The rodent embryonic stem cells are mouse embryonic stem cells, and may be 129, C57BL, or similar. 38. The rodent embryonic stem cell according to any one of items 34 to 37, which is a mixture thereof. (Item 39) The rodent embryonic stem cells are mouse embryonic stem cells, a mixture of 129 and C57BL strains. 39. The rodent embryonic stem cell according to Item 38. (Item 40) 40. A rodent embryo generated from the embryonic stem cell according to any one of items 34 to 39. (Item 41) The method was to generate rodents that express the PD-1 polypeptide from the endogenous Pdcd1 gene. wherein the PD-1 polypeptide comprises a human sequence, and the method comprises: (a) Insertion of a genomic fragment into the endogenous Pdcd1 gene of rodent embryonic stem cells. wherein the genomic fragment is a whole or part of a human PD-1 polypeptide. and providing a nucleotide sequence encoding (b) obtaining the rodent embryonic stem cells produced in (a); (c) generating a rodent using the rodent embryonic stem cells of (b). (Item 42) Item 41. The human sequence comprises amino acids 35 to 145 of a human PD-1 polypeptide. The method according to (Item 43) Item 41. The human sequence comprises amino acids 27 to 145 of a human PD-1 polypeptide. The method according to (Item 44) Item 41. The human sequence comprises amino acids 26 to 169 of a human PD-1 polypeptide. The method according to (Item 45) 45. Any of items 41 to 44, wherein the nucleotide sequence comprises human Pdcd1 exon 2. 3. The method according to claim 1 . (Item 46) the nucleotide sequence further comprises all or a portion of human Pdcd1 exon 3; The method according to item 45. (Item 47) 47. Any of items 41 to 46, wherein the nucleotide sequence comprises one or more selection markers. 13. The method according to claim 1. (Item 48) 47. The nucleotide sequence comprises one or more site-specific recombination sites. 2. The method according to claim 1 . (Item 49) Pd, whose genome encodes a PD-1 polypeptide having human and endogenous portions. cd1 gene, said portion operably linked to a rodent Pdcd1 promoter. 16. A method of producing a rodent comprising: Pdcd, which encodes a PD-1 polypeptide having a human portion and an endogenous portion. 1 gene, said portion being operably linked to a rodent Pdcd1 promoter. and modifying said genome of a rodent to thereby produce said rodent. method. (Item 50) The rodent Pdcd1 promoter is an endogenous rodent Pdcd1 promoter. The method according to item 49. (Item 51) Item 49. The human portion comprises amino acids 35 to 145 of a human PD-1 polypeptide. The method according to (Item 52) Item 49. The human portion comprises amino acids 27 to 145 of a human PD-1 polypeptide. The method according to (Item 53) Item 49. The human portion comprises amino acids 26 to 169 of a human PD-1 polypeptide. The method according to (Item 54) Item 49. The Pdcd1 gene is modified to contain human Pdcd1 exon 2. A method according to any one of claims 1 to 53. (Item 55) The Pdcd1 gene is a human Pdcd1 exon 2 and a human Pdcd1 exon 3 55. The method of claim 54, modified to include all or part of (Item 56) 56. The method according to any one of items 41 to 55, wherein the rodent is a mouse or a rat. . (Item 57) 57. A rodent obtainable by the method according to any one of items 41 to 56. (Item 58) 58. The rodent of item 57, wherein the rodent is a mouse or a rat. (Item 59) 1. A method of reducing tumor growth in a rodent, said method comprising: A P whose genome encodes a PD-1 polypeptide having human and endogenous portions. dcd1 gene, said portion being operably linked to a rodent Pdcd1 promoter. administering to the ligated rodent an agent that targets human PD-1; The administration is performed under conditions and for a time sufficient to reduce tumor growth in the rodent. How it is done. (Item 60) 1. A method of killing tumor cells in a rodent, the method comprising: A P whose genome encodes a PD-1 polypeptide having human and endogenous portions. dcd1 gene, said portion being operably linked to a rodent Pdcd1 promoter. administering to the ligated rodent an agent that targets human PD-1; The administration is performed under conditions and for a time sufficient to mediate killing of the tumor cells. How to do it. (Item 61) 1. A method for evaluating the pharmacokinetic properties of an agent targeting human PD-1, the method comprising: A P whose genome encodes a PD-1 polypeptide having human and endogenous portions. dcd1 gene, said portion being operably linked to a rodent Pdcd1 promoter. administering the agent to the rodent; Assays to determine one or more pharmacokinetic properties of the agent that targets PD-1. and performing the steps of: (Item 62) Item 59. The human portion comprises amino acids 35 to 145 of a human PD-1 polypeptide. A method according to any one of claims 1 to 61. (Item 63) Item 59. The human portion comprises amino acids 27 to 145 of a human PD-1 polypeptide. A method according to any one of claims 1 to 61. (Item 64) Item 59. The human portion comprises amino acids 26 to 169 of a human PD-1 polypeptide. A method according to any one of claims 1 to 61. (Item 65) Any of items 59 to 64, wherein the drug targeting human PD-1 is a PD-1 antagonist. 3. The method according to claim 1 . (Item 66) Any of items 59 to 64, wherein the drug targeting human PD-1 is a PD-1 agonist. 3. The method according to claim 1 . (Item 67) Any of items 59 to 64, wherein the drug targeting human PD-1 is an anti-PD-1 antibody. 3. The method according to claim 1 . (Item 68) Items 59 to 67, wherein the agent targeting human PD-1 is administered intravenously to the rodent. 2. The method according to claim 1 . (Item 69) Items 59 to 67, wherein the agent targeting human PD-1 is administered intraperitoneally to the rodent. 2. The method according to claim 1 . (Item 70) 68. The method according to claim 59, wherein the agent targeting human PD-1 is administered subcutaneously to the rodent. 2. The method according to any one of claims 1 to 11. (Item 71) The rodent Pdcd1 promoter is an endogenous rodent Pdcd1 promoter. 71. The method according to any one of items 59 to 70. (Item 72) 72. The method according to any one of items 59 to 71, wherein the rodent is a mouse or a rat. . (Item 73) A rodent tumor model, wherein the rodent is a PD- Rodent tumor models expressing the .ALPHA.-.1 polypeptide. (Item 74) A rodent tumor model, wherein the rodent is a mouse model of Pdcd comprising an endogenous portion and a human portion. 1 gene, wherein the endogenous and human portions are operable with a rodent Pdcd1 promoter. A similarly linked rodent tumor model. (Item 75) 1. A rodent tumor model comprising: a) Providing a rodent comprising a Pdcd1 gene comprising an endogenous portion and a human portion wherein the endogenous and human portions are operably linked to a rodent Pdcd1 promoter. The steps are linked as follows: b) implanting one or more tumor cells in said rodent of (a); thereby providing said rodent tumor model. Dell. (Item 76) 76. The rodent according to any one of items 73 to 75, wherein the rodent is a mouse or a rat. Tumoroid model. (Item 77) 77. The rodent tumor model of claim 76, wherein the rodent is a mouse. (Item 78) The mice were selected from 129, C57BL / 6, and mixed 129×C57BL / 6 strains. 78. The rodent tumor model of item 77, selected from the group consisting of: (Item 79) 79. The method of claim 78, wherein the mice are 25% 129 and 75% C57BL / 6. Rodent tumor models.

[0052] As used herein, the terms "about" and "approximately" are used interchangeably. Any numbers used herein, whether about / approximate or not, will be understood by one of ordinary skill in the art to be within the true range of values. It is intended to encompass any normal variations that are understood in the art.

[0053] Other features, objects and advantages of the present invention will become apparent from the following detailed description of specific embodiments. However, the detailed description indicating specific embodiments of the present invention is given by way of example only. It should be understood that the present invention is not limited to the above embodiments. Changes and modifications will become apparent to those skilled in the art from the detailed description.

[0054] The drawings contained herein, which consist of the following figures, are for illustrative purposes only and not for limiting purposes: . [Brief description of the drawings]

[0055] [Figure 1] A diagram (not to scale) of the genomic structure of the non-human (e.g., mouse) and human programmed cell death 1 (Pdcd1) genes is shown. Exons and untranslated regions (UTRs) are numbered below each exon and above each UTR. [Diagram 2] FIG. 1 shows a diagram (not to scale) of an exemplary method for humanization of the non-human Programmed Cell Death 1 (Pdcd1) gene. The locations of selected nucleotide junctions are indicated by lines under each junction. The sequences of these selected nucleotide junctions are shown in SEQ ID NO: [Diagram 3] FIG. 1 shows a diagram (not to scale) of the genomic structure of the mouse and human programmed cell death 1 (Pdcd1) genes, indicating the approximate locations of the probes used in the assay described in Example 1. [Figure 4]1 shows exemplary histograms gated on CD19 and CD8 isolated from wild-type mice heterozygous for humanization of the endogenous Pdcd1 gene and mice expressing mouse and / or humanized PD-1 as described in Example 1. Stimulated and unstimulated cell populations are shown, as are cells stained with an isotype control. [Diagram 5] 1 shows exemplary tumor growth curves over 21 days for mice homozygous for humanization of the endogenous Pdcd1 gene as described in Example 1. Control: antibody not specific for PD-1, a-hPD-1 Ab: antibody specific for human PD-1. Arrows indicate days of antibody treatment. The number of tumor-free mice at day 21 is shown for each treatment group. [Figure 6] 1 shows exemplary real-time PCR analysis of splenic CD8b, CD3, IFN-g, and PD-1 mRNA in mice homozygous for humanization of the endogenous Pdcd1 gene described in Example 1 after treatment with anti-PD-1 antibodies. A, average of 5 mice per group. B, Expression levels of individual mice in each treatment group. Control: antibody not specific for PD-1, α-PD-1: anti-PD-1 antibody. [Figure 7] FIG. 1 shows exemplary tumor growth curves over 60 days in mice homozygous for humanization of the endogenous Pdcd1 gene as described in Example 1 administered 0.3-25 mg / kg anti-hPD-1 antibody or 25 mg / kg control antibody (antibody not specific for PD-1). Arrows indicate days of antibody treatment. The number of tumor-free mice at day 60 is shown for each treatment group. [Figure 8-1]Exemplary mouse, human and humanized Pdcd1 and PD-1 sequences, as well as exemplary human nucleic acid sequences for humanization of non-human Pdcd1 genes are shown. For mRNA sequences, bold indicates coding sequences and consecutive exons, where indicated, separated by alternating underlined letters. For humanized mRNA sequences, human sequences are in brackets. For protein sequences, signal proteins are underlined, extracellular sequences are in bold, immunoglobulin V region sequences are in brackets, intracellular sequences are in italics, and for humanized protein sequences, non-human sequences are in normal letters and human sequences are in bold. [Figure 8-2] Exemplary mouse, human and humanized Pdcd1 and PD-1 sequences, as well as exemplary human nucleic acid sequences for humanization of non-human Pdcd1 genes are shown. For mRNA sequences, bold indicates coding sequences and consecutive exons, where indicated, separated by alternating underlined letters. For humanized mRNA sequences, human sequences are in brackets. For protein sequences, signal proteins are underlined, extracellular sequences are in bold, immunoglobulin V region sequences are in brackets, intracellular sequences are in italics, and for humanized protein sequences, non-human sequences are in normal letters and human sequences are in bold. [Figure 8-3] Exemplary mouse, human and humanized Pdcd1 and PD-1 sequences, as well as exemplary human nucleic acid sequences for humanization of non-human Pdcd1 genes are shown. For mRNA sequences, bold indicates coding sequences and consecutive exons, where indicated, separated by alternating underlined letters. For humanized mRNA sequences, human sequences are in brackets. For protein sequences, signal proteins are underlined, extracellular sequences are in bold, immunoglobulin V region sequences are in brackets, intracellular sequences are in italics, and for humanized protein sequences, non-human sequences are in normal letters and human sequences are in bold. [Figure 8-4]Exemplary mouse, human and humanized Pdcd1 and PD-1 sequences, as well as exemplary human nucleic acid sequences for humanization of non-human Pdcd1 genes are shown. For mRNA sequences, bold indicates coding sequences and consecutive exons, where indicated, separated by alternating underlined letters. For humanized mRNA sequences, human sequences are in brackets. For protein sequences, signal proteins are underlined, extracellular sequences are in bold, immunoglobulin V region sequences are in brackets, intracellular sequences are in italics, and for humanized protein sequences, non-human sequences are in normal letters and human sequences are in bold. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

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

[0057] Unless otherwise specified, all terms and phrases used herein have the same meaning as they do not. It is clearly indicated or is clearly evident from the context in which the term or phrase is used. Except where expressly stated otherwise, the terms and phrases include the meanings they have acquired in the art. Any methods and materials similar or equivalent to those described may also be used in the practice or testing of the present invention. The specific methods and materials are now described. The publications are incorporated herein by reference.

[0058] As used herein, when applied to one or more subject values, the term "approximately" means In certain embodiments, the term "approximately" or "about" refers to a value similar to a reference value. Unless otherwise specified or the context makes it clear that Either direction of the specified reference value, except when it exceeds 100% of the possible values 25%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12% , 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1% or more It refers to a range of values ​​less than.

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

[0060] The term "equivalent" means that although they may not be identical to each other, they allow for a comparison between them. are sufficiently similar to allow reasonable conclusions to be drawn based on the observed differences or similarities. This includes a set of two or more agents, entities, conditions, states, etc. that can be generated by a single agent. If, within the context, two or more such agents, entities, Understanding how much identity is required in a given situation, set of situations, etc. It will be.

[0061] For example, the term "conservative" in conservative amino acid substitutions refers to similar chemical properties (e.g. The substitution of an amino acid residue by another amino acid residue having a side chain R group with a specific molecular weight (e.g., charge or hydrophobicity) Generally, conservative amino acid substitutions are made to improve the functional properties (e.g., linker properties) of the protein of interest. Side chains with similar chemical properties Examples of amino acids having the following structure include: glycine, alanine, valine, leucine, and and isoleucine; and aliphatic hydroxyl groups such as serine and threonine. side chains, amide-containing side chains such as asparagine and glutamine, phenylalanine, tyrosine aromatic side chains such as tryptophan, lysine, arginine, and histidine The side chains include basic acids such as α- and β-amino acids, acidic acids such as aspartic acid and glutamic acid, and cysteine. Conservative amino acid substitutions include, for example, valine / Leucine / Isoleucine, Phenylalanine / Tyrosine, Lysine / Arginine, Alanine / valine, glutamic acid / aspartic acid, and asparagine / glutamine In some embodiments, conservative amino acid substitutions can be made using techniques such as, for example, alanine scanning mutagenesis. It may be a substitution for any native residue in a protein, including alanine, used in In some embodiments, the Gonnet et al. (1992) Exhaust ive Matching of the Entire Protein Seque nce Database, Science 256:1443-45 (which is referenced (which is incorporated herein by reference) In some embodiments, the substitutions are moderately conservative substitutions, Here the permutations have non-negative values ​​in the PAM250 log-likelihood matrix.

[0062] The term "control" refers to a standard against which results are compared. Generally, controls are used to guide conclusions about such variables. It is used to increase the completeness of an experiment by isolating variables in order to obtain In some embodiments, a control may be a test reaction or assay to provide a comparator. As used herein, a "control" is a reaction or assay that is performed simultaneously with a A "control animal" may include a "control animal." A "control animal" is an animal that has been modified, have modifications different from those described in the document, or are unmodified (i.e., wild-type animals). In one experiment, the "test" (i.e., the variable being tested) is applied. In the second experiment, the "control," the variable being tested is not applied. In this study, the control was a historical control (i.e., a control of a previously performed test or assay, or In some embodiments, the control is a printed or otherwise The control may be a positive or negative control. May be gender-symmetric.

[0063] The term "disruption" refers to a disruption (e.g., in an endogenous homologous sequence, such as a gene or locus). In some embodiments, the disruption is the result of a homologous recombination event in a DNA molecule. Insertion, deletion, substitution, exchange, missense mutation, or frameshift of the A sequence, or Any combination of these may be achieved or demonstrated. Insertions may involve the entire gene or may contain the insertion of a fragment (e.g., an exon) of a gene other than the endogenous sequence. In some embodiments, the disruption may be of a gene or a sequence of a heterologous origin. or expression of a gene product (e.g., of a protein encoded by a gene) and / or In some embodiments, the disruption may increase the activity of a gene or gene product. In some embodiments, the disruption may reduce expression and / or activity of a gene or may alter the sequence of the encoded gene product (e.g., the encoded protein). In some embodiments, the disruption is in a gene or an encoded gene product (e.g., In some embodiments, the disruption may cleave or fragment the genetically modified protein. In some embodiments, the disruption may extend the gene or the encoded gene product. In some embodiments, the disruption may involve a gene or may affect the level but not the activity of the gene product. In some cases, the disruption affects the activity but not the level of the gene or gene product. In some embodiments, the disruption has no significant effect on the level of the gene or gene product. In some embodiments, the disruption is not evident on the activity of the gene or gene product. In some embodiments, the disruption may have no significant effect on the expression of a gene or gene product. may have no significant effect on either levels or activity.

[0064] "Determine," "Measure," "Evaluate," "Assess," "Assay," and The term "analyze" is used interchangeably and refers to any form of measurement, These terms include quantitative and / or qualitative determinations. Assays may be relative or absolute. "Determining the amount of something that exists and / or determining whether it is present or absent" It is possible that this is the case.

[0065] The term "dosing regimen" or "treatment regimen" refers, in some embodiments, to one or more The term includes a set of unit doses that are administered separately to a subject, typically at intervals of time. In embodiments, a given therapeutic agent has a recommended dosing regimen, which may involve one or more doses. In some embodiments, the dosing regimens are of equal length to each other. In some embodiments, the dosing regimen includes multiple doses spaced apart over a period of time. at least two distinct periods of time separate the individual doses.

[0066] The phrase "endogenous locus" or "endogenous gene" refers to a gene that is present in a parent or indicator organism. In some embodiments, an endogenous locus comprises a sequence found in nature. In some embodiments, the endogenous locus is a wild-type locus. In some embodiments, the indicator organism is a wild-type organism. In some embodiments, the indicator organism is a genetically engineered organism. In some embodiments, the indicator organism is a laboratory-bred organism (wild type or genetically modified organism). (child operation was performed).

[0067] The phrase "endogenous promoter" refers to a promoter that is naturally associated with an endogenous gene, e.g., in a wild-type organism. Including the associated promoter.

[0068] The term "heterologous" includes agents or entities from different origins. For example, related to a peptide, gene, or gene product, or to its presence in a particular cell or organism When used in conjunction, the term refers to a polypeptide, gene, or gene product that is: 1) derived from a human 2) by human hands (e.g., by genetic manipulation); ) introduced into a cell or organism (or a precursor thereof), and / or 3) into a relevant cell or is not naturally produced by an organism (e.g., a relevant cell type or organism type). , or make it clear that it does not exist there.

[0069] The term "host cell" refers to a cell in which a heterologous (e.g., foreign) nucleic acid or protein is present. A skilled artisan reading this disclosure will understand that such terms refer to particular subject cells. It will be understood that the term "cell" refers not only to the individual cells but also to the progeny of such cells. Certain modifications may occur in subsequent generations due to mutations or environmental influences. Although such progeny may not be identical in fact to the parent cell, they are still referred to as "host cells." In some embodiments, the host cell is a prokaryotic or eukaryotic cell. Generally, the host cell is a heterologous nucleus, regardless of the species to which the cell is designated. The cell may be any cell suitable for receiving and / or producing an acid or protein. Vacuoles include prokaryotic and eukaryotic (unicellular or multicellular) cells, bacterial cells (e.g. Enterobacteriaceae, Bacillus, Streptomyces, etc.), mycobacterial cells, fungal cells , yeast cells (e.g., Saccharomyces cerevisiae, Schizosaccharomyces pombe, Pichia methanolica, etc.), plant cells, insect Cells (e.g., SF-9, SF-21, baculovirus-infected insect cells, Urticaria ginseng Waba, etc.), non-human animal cells, human cells, or e.g., hybridomas or quads. In some embodiments, the cells are human, monkey, ape, hamster, or other cell fusion. In some embodiments, the cell is a prokaryotic cell, such as a human, rat, or mouse cell. Cells selected from: CHO (e.g., CHO K1, DXB-11 CHO, Vegg i.e.-CHO), COS (e.g., COS-7), retinal cells, Vero, CV1, kidney ( For example, HEK293, 293 EBNA, MSR 293, MDCK, HaK, BHK ), HeLa, HepG2, WI38, MRC 5, Colo205, HB 8065, HL-60, (e.g., BHK21), Jurkat, Daudi, A431 (epidermis), CV-1, U937, 3T3, L cells, C127 cells, SP2 / 0, NS-0, MMT 060562, Sertoli cells, BRL 3A cells, HT1080 cells, myeloma cells, tumor In some embodiments, the cells include one or more virus-specific markers. retinal cells expressing a viral gene, e.g., a viral gene (e.g., PER.C6 ( In some embodiments, the host cell comprises or is an isolated cell. In some embodiments, the host cell is part of a tissue. In some embodiments, the host cell is part of a Part of it.

[0070] The term "humanized" refers to a humanized polypeptide whose structure (i.e., nucleotide or amino acid sequence) is , which may have a structure substantially or completely similar to that of a particular gene or protein found naturally in a non-human animal. Contains identical corresponding portions found in specific related non-human genes or proteins. instead, they are compared with the equivalent structures found in the corresponding human genes or proteins. In some embodiments, the nucleic acid or protein includes a more closely corresponding portion. A "humanized" gene is a polypeptide having an amino acid sequence substantially similar to that of a human polypeptide. A gene encoding a human protein or a portion thereof, such as a characteristic portion thereof For example, in the case of membrane receptors, the "humanized" gene may be a human extracellular domain. and amino acid sequences such as those of non-human (e.g., mouse) polypeptides. A gene that codes for a polypeptide that has all or part of its intracellular portion with the remaining sequence In some embodiments, a humanized gene comprises at least one identical DNA sequence to a human gene. In some embodiments, the humanized gene comprises a portion of the entire DNA sequence of a human gene. In some embodiments, the humanized protein comprises a portion of a human protein. In some embodiments, the humanized protein comprises a sequence having the sequence of a human protein. The endogenous genes of non-human animals that correspond to the homologs or orthologs of human genes, with complete sequences It is expressed from a gene locus.

[0071] For example, the term "identity" in sequence comparisons refers to the nucleotide and / or amino acid sequence identity. There are many different algorithms known in the art that can be used to measure amino acid sequence identity. In some embodiments, the identity as described herein may be determined by the uses a gap opening penalty of 10.0 and a gap extension penalty of 0.1, and Go nnet similarity matrix (MACVECTOR™ 10.0.2, MacVec ClustalW v. 1.83 (sl tor Inc., 2008) was used. ow) alignment.

[0072] The term "isolated" means (1) the most suitable organism (either in its natural and / or experimental environment); separated from at least some of the components with which it was associated when originally produced; and / or or (2) any substance designed, produced, formulated, and / or manufactured by human hands; Separated substances and / or entities are those that are not related to the first About 10%, about 20%, about 30%, about 40%, about 50%, about 60% of other ingredients , about 70%, about 80%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95% , about 96%, about 97%, about 98%, about 99%, or greater than about 99%. 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 greater than about 99% pure. A substance is substantially free of other components. In some embodiments, as will be appreciated by those of skill in the art, a substance is "pure" if it is free of any of the above-mentioned impurities. , one or more carriers or excipients (e.g., buffers, solvents, water, etc.), Even after being combined with ingredients, a substance is still considered "isolated" or even "pure." In such embodiments, the percent isolation or purity of a substance may be determined in such a manner. The formula is calculated without including such carriers or excipients. In the present invention, a biological polymer such as a polypeptide or polynucleotide existing in nature is a The origin or source of the derivation is some or all of the components that accompany it in their natural state in nature. b) when it is not associated with other polymorphs of the same species that naturally produces it; or c) is substantially free of peptides or nucleic acids in a species that naturally produces it. expressed by or otherwise derived from cells or other expression systems not When associated with a component, it is considered to be "isolated." Thus, for example, in some embodiments So what is a protein that is chemically synthesized or synthesized in a cell system different from the one that produces it in nature? A polypeptide that has been isolated is considered an "isolated" polypeptide. Specifically, in some embodiments, a polypeptide that has been subjected to one or more purification techniques is one that is a) it is associated in nature, and / or b) it is associated when first produced A polypeptide is considered "isolated" so long as it is separated from other components that were present in the polypeptide. It is possible.

[0073] The phrase "non-human animal" includes any vertebrate organism that is not a human. The non-human animals include cyclostomes, bony fish, cartilaginous fish (e.g., sharks or rays), amphibians, and worms. In some embodiments, the non-human mammals are primates, goats, hamsters, and birds. In some embodiments, the non-human animal is a rat, a porcine, a canine, a bovine, or a rodent. The animal may be a rodent, such as a rat or a mouse.

[0074] The term "nucleic acid" refers to an oligonucleotide chain or In some embodiments, the present invention includes any compound and / or substance that can be incorporated into the A "nucleic acid" is an oligonucleotide chain or a phosphodiester-linked oligonucleotide. The term "octide chain" refers to a compound and / or substance capable of incorporating an octide chain. Thus, in some embodiments, a "nucleic acid" refers to individual nucleic acid residues (e.g., nucleotides and / or or nucleosides), and in some embodiments, a "nucleic acid" comprises individual nucleic acid residues. In some embodiments, the "nucleic acid" is RNA or In some embodiments, the "nucleic acid" is or comprises DNA. In embodiments, a "nucleic acid" comprises or consists of one or more naturally occurring nucleic acid residues. In some embodiments, a "nucleic acid" comprises or consists of one or more nucleic acid analogs. In some embodiments, the nucleic acid analogs are nucleic acid analogs in that they do not utilize a phosphodiester backbone. For example, in some embodiments, a "nucleic acid" is a nucleic acid that is known in the art. It is made up of one or more "peptides" that have peptide bonds instead of phosphodiester bonds in the backbone. "peptide nucleic acid" and are considered to be within the scope of the present invention. Alternatively or additionally, in some embodiments, a "nucleic acid" is a nucleic acid having a phosphodiester bond. without one or more phosphorothioate and / or 5'-N-phosphoramidite linkages In some embodiments, a "nucleic acid" may contain one or more naturally occurring nucleosides (e.g., adenosines). thymidine, guanosine, cytidine, uridine, deoxyadenosine, deoxythymidine deoxyguanosine, deoxyguanosine, and deoxycytidine) or one or more of the naturally occurring nucleic acids In some embodiments, a "nucleic acid" is a nucleic acid that is composed of one or more nucleoside analogs. (e.g., 2-aminoadenosine, 2-thiothymidine, inosine, pyrrolo-pyrimidine, 3-Methyladenosine, 5-Methylcytidine, C-5 Propynyl-cytidine, C-5 Propynyl-uridine, 2-aminoadenosine, C5-bromouridine, C5-fluorouridine Uridine, C5-iodouridine, C5-propynyl-uridine, C5-propynyl-succinimide Cytidine, C5-methylcytidine, 2-aminoadenosine, 7-deazaadenosine, 7-de Azaguanosine, 8-oxoadenosine, 8-oxoguanosine, O(6)-methylguanosine N-methylcytidine, 2-thiocytidine, methylated bases, intercalated bases, and combinations thereof In some embodiments, a "nucleic acid" is a naturally occurring nucleic acid. compared to the original, one or more modified sugars (e.g., 2'-fluororibose, ribose, 2'-deoxyribose, arabinose, and hexose). In the present context, a "nucleic acid" refers to a nucleic acid that encodes a functional gene product, such as RNA or a protein. In some embodiments, a "nucleic acid" includes one or more introns. In some embodiments, "nucleic acid" refers to a nucleic acid that is isolated from a natural source, polymerized on a complementary template, or Enzymatic synthesis (in vivo or in vitro), replication in recombinant cells or systems, and chemical synthesis In some embodiments, the "nucleic acid" is prepared by one or more of the following: , 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, 350 0, 4000, 4500, 5000 or more residues in length. In some embodiments, the "nucleic acid" is single-stranded, and in some embodiments, the "nucleic acid" is double-stranded. In this embodiment, a "nucleic acid" encodes a polypeptide or is a polypeptide-encoding molecule. A nucleotide sequence that contains at least one element that is the complement of a sequence that In embodiments, the "nucleic acid" has enzymatic activity.

[0075] The phrase "operably linked" means that the elements described are linked together in such a way that they are not "Operably linked" includes a juxtaposition in which the coding sequences are in a relationship such that they can function in a coordinated manner. The "controlled" control sequence is designed to be linked such that expression of the coding sequence is achieved under conditions compatible with the control sequence. "Operably linked" sequences include sequences that are adjacent to the gene of interest and are in Regulatory sequences and expression regulators that act in trans or at a distance to regulate genes of interest The term "expression control sequence" includes both polynucleotide sequences, which necessary to bring about the expression and processing of the coding sequences to which they are linked "Expression control sequences" include appropriate transcription initiation, termination, promoter and enhancer sequences. Efficient RNA processing signals, such as splicing and polyadenylation signals sequences that stabilize cytoplasmic mRNA and sequences that enhance translation efficiency (i.e., Kozak sequences) sequences), sequences that enhance protein stability, and, if desired, sequences that enhance protein secretion. The nature of such control sequences will vary depending upon the host organism. In organisms, such control sequences generally include promoters, ribosome binding sites, and transcription factors. In eukaryotes, such control sequences generally include promoters and transcription termination sequences. The term "control sequence" refers to a sequence whose presence regulates the expression and processing of a gene. It is intended to include elements that are essential for the design and additional elements whose presence is advantageous. For example, leader sequences and fusion partner sequences can also be included.

[0076] The term "patient" or "subject" refers to, for example, experimental, diagnostic, prophylactic, cosmetic, and / or or for therapeutic purposes, to which the provided compositions are or may be administered. Typical patients include animals (e.g., mice, rats, rabbits, non-human In some embodiments, the patient is a non-human primate, a mammal, such as a primate, a human, or a human. In some embodiments, the patient (e.g., a non-human animal patient) is a human animal, The modifications described herein may be different from those described herein, or may not have any modifications. In some embodiments, the non-human animal may be a wild-type non-human animal patient. suffers from or is predisposed to one or more disorders or conditions. In some embodiments, the non-human animal exhibits one or more symptoms of the disorder or condition. The non-human animal has been diagnosed with one or more disorders or conditions.

[0077] The term "polypeptide" includes any polymeric chain of amino acids. In some embodiments, a polypeptide has an amino acid sequence that occurs in nature. In some embodiments, a polypeptide has an amino acid sequence that is not found in nature. have amino acid sequences that contain naturally occurring portions that are distinct from each other (i.e., e.g. from two or more different organisms, such as human and non-human portions. The polypeptide is said to be designed and / or produced through the action of the human hand. At this point, it has a genetically engineered amino acid sequence.

[0078] The term "recombinant" means any substance that is designed, engineered, prepared, expressed, or produced by recombinant means. A synthesized or isolated polypeptide (e.g., a PD-1 polypeptide described herein). ), e.g., recombinant vectors transfected into host cells. Polypeptides expressed using expression vectors, recombinant human recombinant polypeptides Polypeptides isolated from the library (Hoogenboom HR, (19 97) TIB Tech. 15:62-70; Azzazy H., and H ighsmith WE, (2002) Clin. Biochem. 35 :425-445; Gavilondo JV, and Larrick J W. (2002) BioTechniques 29:128-145; ogenboom H., and Chames P. (2000) Immuno logy Today 21:371-378), and Antibodies isolated from transgenic animals (e.g., mice) (e.g., Taylor, LD, et al. (1992) Nucl. Acids Res. 2 0:6287-6295; Kellermann SA., and Green LL (2002) Current Opinion in Biotechn 13:593-597; Little M. et al. (2000 ) Immunology Today 21:364-370; Murphy, A .J., et al. (2014) Proc. Natl. Acad. Sci USA 111(14):5153-5158) or selected It may also be prepared, expressed, or produced by any other means that involves splicing sequence elements together. In some embodiments, such selected sequences include synthesized or isolated polypeptides. One or more of the sequence elements are naturally occurring. In some embodiments, such a selected sequence One or more of the elements are designed in silico. In some embodiments, one or more of such Selection sequence elements can be determined, for example, by mutagenesis (e.g., mutation) of known sequence elements of natural or synthetic origin. For example, in some embodiments, recombinant polypeptides are produced from The sequences consist of sequences found in the genome of the source organism of interest (e.g., human, mouse, etc.). In some embodiments, the recombinant polypeptide is prepared by mutagenesis (e.g., in a non-human animal). The amino acid sequence is derived from a recombinant polypeptide (in vitro or in vivo) and is therefore The amino acid sequence of the peptide is derived from a polypeptide sequence, but is expressed in vivo in a non-human animal genome. may not occur naturally within the

[0079] The term "substitution" refers to a "substitution" that occurs at a host locus (e.g., in a genome) through which A "permuted" nucleic acid sequence (e.g., gene) is removed from its locus and a different "permuted" nucleic acid sequence is generated. In some embodiments, the replaced nucleic acid is The sequence and the replacement nucleic acid sequence may be, for example, selected so that they are homologous and / or complementary to each other. each other in that it contains elements that correspond to In some embodiments, the replaced nucleic acid sequence includes a promoter, an enhancer, a -, splice donor site, splice acceptor site, intron, exon, untranslated region (U TR), and in some embodiments, the replacement nucleic acid sequence includes one or more In some embodiments, the replacement nucleic acid sequence comprises a homolog of the replaced nucleic acid sequence. In some embodiments, the replacement nucleic acid sequence is an ortholog of the replaced sequence. In some embodiments, the replacement nucleic acid sequence is or comprises a human nucleic acid sequence. In embodiments, including where the replacement nucleic acid sequence is or comprises a human nucleic acid sequence: The substituted nucleic acid sequence may be a rodent sequence (e.g., a mouse or rat sequence) or may be a The nucleic acid sequences so arranged are It may contain one or more regulatory sequences that are part of the source nucleic acid sequence used (e.g., promoters, enhancers, 5'- or 3'-untranslated regions, etc.). For example, In certain embodiments, the substitution is a genetic change from the nucleic acid sequence (with the heterologous sequence) so arranged. Substitution of an endogenous sequence with a heterologous sequence resulting in the production of a product, but not the expression of the endogenous sequence. Substitutions are not replacements. Substitutions have a similar function to the polypeptide encoded by the endogenous sequence. It is an endogenous genomic sequence having a nucleic acid sequence encoding a polypeptide (e.g., The genomic sequence encodes a PD-1 polypeptide, and the DNA fragment is one or more human In various embodiments, the endogenous gene or its The fragment is replaced with the corresponding human gene or a fragment thereof. The target gene or fragment thereof is a nucleotide sequence similar to that of the endogenous gene or fragment thereof to be replaced. orthologs or have substantial similarity in structure and / or function or the same human gene or fragment.

[0080] The term "programmed cell death 1 protein" or "PD-1 protein" refers to the A type I transmembrane protein belonging to the CD28 / CTLA-4 family of cell regulators The protein structure of PD-1 protein includes the intracellular amino-terminal immunoglobulin V domain. The immunoreceptor tyrosine kinase domain (TKI) is a cytoplasmic domain that encodes a cytoplasmic receptor that encodes a cytoplasmic receptor. Immune receptor tyrosine-dependent inhibitory motifs (ITIMs) and immunoreceptor tyrosine-dependent switch motifs PD-1 is expressed on the cell surface and is a member of the B7 family of immunoregulatory ligands. Interacts with D-L1 and PD-L2 (Collins, M. et al. ( 2005) Genome Biol. 6:223). PD-1, in particular, mediates activation It is expressed in T cells, B cells, macrophages, monocytes, and mast cells, and is also expressed in many tumors. PD-1 has been shown to be involved in the negative regulation of immune responses, particularly T cell responses. As an example, mouse and human Pdcd, which encodes the PD-1 protein, The nucleotide and amino acid sequences of the gene are provided in FIG. Upon reading the disclosure, one or more Pdcd1 genes (or all) of the genome may be Heterologous Pdcd1 genes (e.g., polymorphic variants, subtypes or mutants, It will be appreciated that the present invention can be substituted with a gene from a humanized form, such as a humanized form of the gene.

[0081] "PD-1 expressing cells" includes cells that express the PD-1 type I membrane protein. In some embodiments, PD-1 expressing cells express the PD-1 type I membrane protein on their surface. In some embodiments, the PD-1 protein is sufficient to mediate cell-cell interactions. PD-1 is expressed on the cell surface in measurable amounts. Exemplary PD-1 expressing cells include B cells, macrophages, and and T cells. PD-1 expressing cells are expressed on the surface of immune cells (e.g., T and B cells). It regulates various cellular processes through the interaction of PD-1 expressed on the In some embodiments, the non-human leukocytes of the present invention play a role in cell differentiation and fate determination. The subject animal is a mammalian animal having a humanized PD-1 protein expressed on the surface of one or more cells of the non-human animal. Through these, the cellular processes (as described herein) are regulated. In an embodiment, the non-human animal of the present invention comprises a non-human animal having a marker gene that is expressed on the surface of one or more cells of the non-human animal. The humanized PD-1 protein inhibits signaling through the T cell receptor (TCR). In some embodiments, the non-human animal comprises one or more cells of the non-human animal. It exerts negative regulation of immune responses through humanized PD-1 protein expressed on the surface of do.

[0082] The term "reference" includes the drug, animal, cohort, individual, population, or subgroup against which it is A sample, sequence or value to be compared with a standard or control agent, cohort, individual, population, sample In some embodiments, the reference agent, cohort, individual, population, subpopulation, or value. A sample, sequence, or value is a drug, cohort, individual, population, sample, sequence, or value of interest. In some embodiments, , reference agent, cohort, individual, population, sample, sequence or value may optionally be embodied in a tangible medium. In some embodiments, the reference may refer to a contrast. As used herein, "reference" may include "reference animal." The modifications described in the document, modifications different from those described in this specification, or unmodified. (i.e., wild-type animals). Generally, as will be understood by those of skill in the art, Thus, a reference agent, animal, cohort, individual, population, sample, sequence or value is a Determine a drug, animal (e.g., mammal), cohort, individual, population, sample, sequence, or value or is determined or characterized under conditions equivalent to those used to characterize the subject matter.

[0083] The term "substantially" includes the complete or nearly complete extent or characteristic of the subject matter. Those skilled in the art of biology will understand that biological and chemical The phenomenon, if any, proceeds to completion and / or progresses to a state of perfection, or It will be understood that it is rare to achieve or avoid absolute results. The term "generally" is used herein to refer to the lack of completeness inherent in many biological and chemical phenomena. It is used to capture possibilities such as:

[0084] The phrase "substantial homology" includes comparisons between amino acid or nucleic acid sequences. As will be appreciated, if two sequences contain homologous residues at corresponding positions, Generally, it is considered to be "substantially homologous." Homologous residues may be identical residues. Alternatively, homologous residues may be non-identical residues that have suitably similar structural and / or functional characteristics. For example, as will be appreciated by those of skill in the art, certain amino acids may be "hydrophobic" or as "hydrophilic" amino acids and / or as having "polar" or "non-polar" side chains. Generally, the substitution of one amino acid for another of the same type is called "homologous." These are often considered substitutions. Common amino acid classifications are summarized in Tables 1 and 2. do. [Table 1] [Table 2]

[0085] As is well known in the art, an amino acid or nucleic acid sequence can be expressed as a B LASTN and BLASTP, gapped BLAST, and for amino acid sequences and those available in commercially available computer programs such as PSI-BLAST, which Any of a variety of algorithms can be used for the comparison. A suitable program is Altschul et al. (1990) Basic location al 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 gene ration of protein database search program ms”, Nucleic Acids Res. 25:3389-3402; xevanis et al. (1998) Bioinformatics: A Practical Guide to the Analysis of Genes and Proteins, Wiley; and Misener et al. (eds.) (1999) Bioinformatics Methods an d Protocols (Methods in Molecular Biolog The homologous sequences are described in Humana Press. In addition to identifying, the above programs generally provide an indication of the degree of homology. In some embodiments, the two sequences have at least two identical sequences with respect to their corresponding residues over the relevant stretch of residues. 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91% %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher are considered to be substantially homologous. In some embodiments, the relevant sections are In some embodiments, the relevant interval is at least 9, 10, 11, 12 In some embodiments, the amino acid sequence is 13, 14, 15, 16, 17 or more residues. A related section includes adjacent residues along the entire sequence. In some embodiments, the relevant section comprises at least 10 , 15, 20, 25, 30, 35, 40, 45, 50 or more residues.

[0086] The phrase "substantial identity" includes comparisons between amino acid or nucleic acid sequences. As will be appreciated, if two sequences contain identical residues at corresponding positions, As is well known in the art, the term "substantially identical" is generally used to refer to a sequence having an amino acid sequence similar to that of the corresponding sequence. or nucleic acid sequences are searched using BLASTN and BLASTP for nucleotide sequences, gapp ed BLAST, and PSI-BLAST for amino acid sequences. Use any of a variety of algorithms, including those available in computer programs. An exemplary such program can be found in Altschul et al. al. (1990) Basic local alignment search tool, J. Mol. Biol., 215(3): 403-410; A ltschul et al., Methods in Enzymology; ltschul et al. (1997) Nucleic Acids Res. 25:3389-3402; Baxevanis et al. (1998) B ioinformatics: A Practical Guide to the Analysis of Genes and Proteins, Wiley; nd Misener et al., (eds.) (1999) Bioinfo rmatics Methods and Protocols (Methods i n Molecular Biology, Vol. 132), Humana P ress. In addition to identifying identical sequences, the above programs In some embodiments, the two sequences are similar in that the residues are related. Over the span of the sequence, at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, Products that are 97%, 98%, 99% or more identical are considered substantially identical. In some embodiments, the relevant interval is a complete sequence. The interval is at least 10, 15, 20, 25, 30, 35, 40, 45, 50 or more The residue above.

[0087] The phrase "targeting vector" or "targeting construct" refers to a polypeptide comprising a targeting region. The targeting region comprises a nucleotide molecule that is identical or similar to a sequence in a target cell, tissue or animal. and a targeting construct that comprises substantially the same sequence and that is capable of being introduced into a cell, tissue or animal by homologous recombination. The site-specific recombinase recognition site (e.g., loxP) is inserted into the genome of the target organism. In some embodiments, the present invention also includes targeting regions that are targeted using the Frt site. The targeting construct comprises a nucleic acid sequence or gene of particular interest, a selectable marker, a control and and / or regulatory sequences, as well as sequences that support or promote recombination involving such sequences. Further, other nucleic acid sequences that allow recombination mediated through the exogenous addition of proteins are also included. In some embodiments, the targeting constructs of the present invention target all or a portion of a gene of interest. and wherein the gene of interest has a similar function as the protein encoded by the endogenous sequence. In some embodiments, the heterologous gene is a heterologous gene encoding all or part of a protein having a function of The targeting construct of the present invention further comprises all or a portion of a humanized gene of interest. The humanized gene produces a protein with a similar function to the protein encoded by the endogenous sequence. The humanized gene of interest encodes all or part of a protein.

[0088] The phrase "therapeutically effective amount" refers to an amount that produces a desired effect in the subject to which it is administered. In some embodiments, the term refers to a method for treating diseases, disorders, and conditions in accordance with a therapeutic administration regimen. and / or when administered to a subject (e.g., an animal) suffering from or susceptible to a condition. refers to an amount sufficient to treat a disease, disorder, and / or condition. A therapeutically effective amount is one that reduces the incidence and / or severity of one or more symptoms of a disease, disorder, and / or condition. or reduce the severity and / or delay its onset. For example, the term "therapeutically effective amount" actually refers to the amount of a compound required to achieve therapeutic success in a particular individual. It will be understood that a therapeutically effective amount does not necessarily mean the amount of An amount that, when administered to a subject, provides a specific desired pharmacological response in a significant number of subjects. In some embodiments, reference to a therapeutically effective amount refers to an amount that is sufficient to treat one or more specific tissues (e.g., tissue affected by a disease, disorder or condition) or bodily fluids (e.g., blood, saliva, In some cases, the reference is to the amount measured in a sample (such as serum, sweat, tears, urine, etc.). In some embodiments, a therapeutically effective amount of a particular agent or treatment is formulated and / or administered in a single dose. It will be appreciated that a therapeutically effective amount may be administered in some embodiments, e.g. For example, multiple doses may be prescribed and / or administered as part of a dosing regimen. .

[0089] The term "treatment" (also "treat" or "treating") is used broadly to refer to: Identify one or more symptoms, characteristics, and / or causes of a particular disease, disorder, and / or condition Partially or completely alleviate, ameliorate, mitigate, inhibit, delay the onset, and / or reduce the incidence In some embodiments, this includes any administration of a substance (e.g., a provided composition) that Such treatment is provided to subjects who are free of symptoms of the relevant disease, disorder and / or condition, and and / or performed on subjects who exhibit only early signs of a disease, disorder, and / or condition. Alternatively or additionally, in some embodiments, the treatment is directed to treating an associated disease, disorder, and / or may be performed on subjects who exhibit one or more established symptoms of the condition. In some embodiments, treatment includes treating a patient suffering from an associated disease, disorder, and / or condition. In some embodiments, the treatment is for a subject diagnosed with an associated One or more characteristics that are statistically correlated with an increased risk of developing a disease, disorder, and / or condition This may be in subjects known to have susceptibility factors.

[0090] The term "variant" includes variants that exhibit significant structural identity with a reference entity, but do not necessarily represent a variant of the reference entity. Includes entities that differ structurally from a reference entity by the presence of one or more chemical moieties compared to the entity. In many embodiments, a "variant" also differs functionally from its reference entity. In general, whether a particular entity is properly considered a "variant" of a reference entity is a matter of reference. As will be appreciated by those skilled in the art, all A biological or chemical reference entity has certain characteristic structural elements. By definition, they are distinct chemical entities that share one or more such characteristic structural elements. To give some examples, small molecules have characteristic core structural elements (e.g., macrocycle cores). a) and / or may have one or more characteristic pendant moieties, thus providing a barrier to small molecules. The components share the core structural elements and characteristic pendant portions, but may differ in other pendant portions and / or or different types of bonds present in the core (single vs double, E vs Z, etc.) and the polypeptides have designated positions relative to each other in linear or three-dimensional space. and / or a characteristic sequence element consisting of multiple amino acids that contributes to a specific biological function. Nucleic acids may have designated positions relative to one another in linear or three-dimensional space. They may have characteristic sequence elements consisting of multiple nucleotide residues. For example, A "synonymous polypeptide" refers to a polypeptide that has one or more differences in amino acid sequence and / or in the polypeptide backbone. As a result of one or more differences in covalently attached chemical moieties (e.g., carbohydrates, lipids, etc.) In some embodiments, a "variant polypeptide" may differ from a reference polypeptide. A "tide" is a polypeptide that is identical to the reference polypeptide by at least 85%, 86%, 87%, 88%, 89%, or 90%. 0%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 99% overall Alternatively or additionally, in some embodiments, "variant polypeptides" refer to A "peptide" does not share at least one characteristic sequence element with the reference polypeptide. In embodiments, the reference polypeptide has one or more biological activities. A "variant polypeptide" shares one or more of the biological activities of the reference polypeptide. In embodiments, a "variant polypeptide" retains one or more of the biological activities of a reference polypeptide. In some embodiments, a "variant polypeptide" lacks a unique amino acid sequence as compared to a reference polypeptide. In many embodiments, the antibody exhibits a reduced level of biological activity of at least one sequence change at a particular position. If the target polypeptide has an identical amino acid sequence to the parent, then the target polypeptide A peptide is considered to be a "variant" of a parent or reference polypeptide. 20%, 15%, 10%, 9%, 8%, 7%, 6%, and 5% of the variant residues compared to the parent In some embodiments, less than 2%, 4%, 3%, or 4% of a sequence is substituted relative to the parent. All have 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 substituted residue. A "variant" is one that has very few (e.g., less than 5, 4, 3, 2, or 1) substitutions. They also have specific functional residues (i.e., residues that participate in a particular biological activity). A "parent" generally has no more than 5, 4, 3, 2, or 1 addition or deletion compared to the parent. Furthermore, any additions or deletions are generally About 25, about 20, about 19, about 18, about 17, about 16, about 15, about 14, about 13, about 10, It is less than about 9, about 8, about 7, about 6, and usually about 5, about 4, about 3, or about 2 residues. In some embodiments, the parent or reference polypeptide is one found in nature. As will be appreciated, particularly when the polypeptide of interest is an infectious agent peptide, Multiple variants of a particular polypeptide may typically be found in nature.

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

[0092] The term "wild type" includes the meaning of "normal" (as opposed to mutant, diseased, altered, etc.) This includes entities that have a structure and / or activity found in nature under certain conditions or circumstances. If so, wild-type genes and polypeptides often exist in multiple different forms (e.g., alleles). You will understand that it exists in a universal form.

[0093] (Mode for carrying out the invention) The present invention relates, inter alia, to Pdcd1 modulators (e.g., anti-PD-1 antibodies) for the treatment of cancer. to determine therapeutic efficacy of the antibody (antibody), and for assays of T cell responses and signaling Improved cell death assay with humanized genetic material encoding programmed cell death 1 (Pdcd1) for and / or genetically engineered non-human animals. Such non-human animals are The present invention provides improved determination of the therapeutic efficacy of PD-1 modulators and their ability to inhibit PD-1. Thus, the present invention provides a novel method for the development of anti-PD-1 therapeutics for the treatment of various cancers and for the immunotherapy of cancers. Particularly useful for enhancing immune responses to treat and / or eliminate viral infections in non-human animals. In particular, the present invention relates to the expression of a humanized PD-1 protein on the surface of a cell of a non-human animal. Such humanized PD-1 proteins include humanized mouse Pdcd1 genes, which result in The protein is a human target for determining the efficacy of anti-PD-1 therapeutics in promoting anti-tumor immune responses. PD-1 + In some embodiments, the non-human animal of the present invention is capable of providing a source of cells. The object is to inhibit PD-1 through humanized PD-1 protein expressed on the surface of non-human animal cells. In some embodiments, the humanized P The D-1 protein is the entire or partial N-terminal immunoglobulin V region of the human PD-1 protein. In some embodiments, the humanized PD-1 protein has a sequence similar to that of mouse PD-1. and in some embodiments, a mouse PD-1 protein having a sequence corresponding to the intracellular tail of the mouse PD-1 protein. The sequences correspond to the transmembrane domain and intracellular tail of the PD-1 protein. In one embodiment, the humanized PD-1 protein comprises amino acid residues 21-1 of the human PD-1 protein. 70 (or 26-169, 27-169, or 27-145, or 35-145) In some embodiments, the non-human animals of the invention have a sequence corresponding to the non-human animal and It comprises an endogenous Pdcd1 gene that contains genetic material from a heterologous species (e.g., human). In one embodiment, the non-human animal of the invention comprises a humanized Pdcd1 gene, wherein the humanized P The dcd1 gene is composed of all or part of exon 2 and exon 3 of the human PDCD1 gene. In some embodiments, the non-human animal of the invention comprises a humanized Pdcd1 gene, Here, the humanized Pdcd1 gene is a clone derived from exon 2 and exon 3 of the human PDCD1 gene. 883 of human PDCD1, which corresponds to the first 71 bp (i.e., encoding the stalk) Equipped with bp.

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

[0095] Programmed cell death 1 (Pdcd1) gene Pdcd1 (also known as CD279) is a T cell hybrid undergoing apoptosis. It was first discovered as an upregulated gene in Doma (Ishida, Y. et al. al. (1992) EMBO J. 11(11):3887-3895). Pdc The d1 gene encodes a type I membrane protein (P Five exons code for PD-1, the stalk (~20 amino acids), length), transmembrane domain, and immunoreceptor tyrosine-dependent inhibitory motif (ITIM) and It consists of an intracellular tail that contains both immunoreceptor tyrosine-dependent switch motifs (ITSMs) PD-1 is involved in the regulation of, for example, B cells, dendritic cells, activated monocytes, and natural killer (NK) cells. It is expressed on many cell types, including cytoplasmic reticulum and activated T cells (Keir, ME , et al. (2008) Annu. Rev. Immunol. 26:6 Various splice variants of PD-1 have been reported, and They differ based on whether they lack chthon or not (Nielsen, C. et al. (2005 ) Cell. Immunol. 235:109-116). In fact, Is variants have been observed as causative factors of autoimmune diseases (Wan, B. et al. (2006) J. Immunol. 177(12):8844-8 850) Furthermore, it has been reported that Pdcd1-deficient mice develop autoimmune conditions. Nishimura, H. et al. (1998) Intern. Im munol. 10(10):1563-1572; Nishimura, H. e (1999) Immunity 11:141-151; ura, H. et al. (2001) Science 291:319-32 2) This may help consolidate PD-1 as a negative regulator of activated lymphocytes and may contribute to autoimmune disease. Interestingly, tumors use PD-1 signaling to , which have been found to evade immune system surveillance. At least one of its ligands (i.e., PD-L1) inhibits tumor It is currently being investigated as a target for cancer therapy by promoting the antitumor activity of the microenvironment ( For example, Pedoeem, A. et al. (2014) Clin. Immunology Nol. 153:145-152; and Philips, G.K. and A. See tkins, M. (2014) Intern. Immunol. p. 8 see).

[0096] To develop practical targeted therapies for future cancer treatment, it is necessary to understand PD-1-mediated functions and A more thorough and detailed understanding of the PD-1 and PD-C pathways is needed.

[0097] Pdcd1 and PD-1 sequences Exemplary mouse, human and humanized Pdcd1 and PD-1 sequences are set forth in FIG. Exemplary human nucleic acid sequences for humanizing non-human Pdcd1 genes are also set forth in FIG. There are.

[0098] Humanized Pdcd1 non-human animals An endogenous locus in a non-human animal that encodes the PD-1 protein (e.g., the Pdcd1 gene) Humanized PD-1 protein on the cell surface of non-human animals resulting from genetic modification of the PD-1 gene locus Suitable examples of animals described herein include rodents, particularly mammals that express the . includes the mouse.

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

[0100] In some embodiments, the endogenous Pdcd1 gene is deleted. The endogenous Pdcd1 has been altered, and a portion of the endogenous Pdcd1 gene has been replaced with a heterologous sequence ( In some embodiments, the PDCD1 sequence is replaced with a nucleotide sequence (e.g., all or part of a human PDCD1 sequence). In some embodiments, all or substantially all of the endogenous Pdcd1 gene is expressed in a heterologous vector (e.g., In some embodiments, a portion of a heterologous Pdcd1 gene is replaced with a heterologous Pdcd1 gene. A portion of the endogenous non-human Pdcd1 gene is inserted into the endogenous Pdcd1 locus. In embodiments, the heterologous gene is a human gene. In some embodiments, the heterologous gene is a modified or humanized This was performed on one of the two copies of the endogenous Pdcd1 gene, and the humanized Pdcd1 gene In another embodiment, the humanized Pdcd gene is a heterozygous non-human animal. Non-human animals homozygous for one gene are provided.

[0101] In various embodiments, the non-human animal contains an endogenous non-human PDCD1 gene that expresses all or a portion of the human PDCD1 gene. The human Pdcd1 locus is contained therein. Thus, such non-human animals can be used to express a heterologous Pdcd1 gene. These can be described as having substitutions, insertions, or modifications at the endogenous Pdcd1 locus. or a modified Pdcd1 gene or a protein expressed from such a gene. Quality can be measured by, for example, PCR, Western blot, Southern blot, restriction fragment length polymorphism (R Using a variety of methods, including FLP, or allele gain or loss assays, In some embodiments, the non-human animal can be detected by cloning the humanized Pdcd1 gene. It is heterozygous.

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

[0103] In various embodiments, the humanized Pdcd1 gene according to the present invention is the human PDCD1 gene of FIG. P, which has a second exon with a sequence that is virtually identical to the second exon found in gene 1 Contains the dcd1 gene.

[0104] In various embodiments, the humanized Pdcd1 gene according to the present invention is the human PDCD1 gene of FIG. Pdcd1 has a second exon with sequence identical to that found in gene 1. Contains genes.

[0105] In various embodiments, the humanized Pdcd1 gene according to the present invention is the humanized Pdc The third exon found in the d1 mRNA sequence is at least 50% (e.g., 50%, 5 5%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 9 3%, 94%, 95%, 96%, 97%, 98%, 99% or more) identical sequences The Pdcd1 gene contains a third exon carrying the

[0106] In various embodiments, the humanized Pdcd1 gene according to the present invention is the humanized Pdc A third exon with a sequence virtually identical to that found in the d1 mRNA sequence. It contains the Pdcd1 gene, which has

[0107] In various embodiments, the humanized Pdcd1 gene according to the present invention is the humanized Pdc Contains a third exon with sequence identical to that found in the d1 mRNA sequence Contains the Pdcd1 gene.

[0108] In various embodiments, the humanized Pdcd1 gene according to the present invention is SEQ ID NO: 21 or has at least 50% (e.g., 50%, 55%, 60%, 65%, 70%, %, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96 %, 97%, 98%, 99% or more) identical sequence to the Pdcd1 gene nothing.

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

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

[0111] In various embodiments, the humanized Pdcd1 gene according to the present invention is the human PDCD1 gene of FIG. Part of the second and third exons found in one gene and at least 50% ( For example, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more (above) have parts of the second and third exons that each have the same sequence Contains the Pdcd1 gene.

[0112] In various embodiments, the humanized Pdcd1 gene of the present invention is a humanized Pdcd1 gene of the mouse Pdc The first, fourth, and fifth exons found in the d1 gene and at least 50% (e.g., 5 0%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 9 2%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) identical The Pdcd1 gene contains the first, fourth, and fifth exons, each of which has the sequence .

[0113] In various embodiments, the humanized Pdcd1 gene of the present invention is a humanized Pdcd1 gene of the mouse Pdc The first, part of the third, fourth and fifth exons found in the d1 gene and at least 50 % (e.g. 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90 %, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or (and above) the first, a portion of the third, the fourth and the fifth exons, each of which has an identical sequence It contains the Pdcd1 gene, which has

[0114] In various embodiments, the humanized Pdcd1 gene of the present invention is a humanized Pdcd1 gene of the mouse Pdc The 5' untranslated region and 3' untranslated region found in the d1 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 higher) The Pdcd1 gene has 5' and 3' untranslated regions with the same sequence, respectively. include.

[0115] In various embodiments, the humanized Pdcd1 gene according to the present invention is the humanized Pdc The nucleotide coding sequence found in the d1 nucleotide coding sequence is at least 50% ( For example, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more (above) Pdcd1 gene having the same nucleotide coding sequence (e.g., cDNA sequence) Includes.

[0116] In various embodiments, the humanized Pdcd1 mRNA sequence according to the present invention is The humanized mRNA 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 in sequence.

[0117] In various embodiments, the humanized Pdcd1 gene of the present invention is a PD-1 polypeptide of FIG. The amino acid sequence found in the peptide sequence is at least 50% (e.g., 50%, 55%, 6 0%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 9 4%, 95%, 96%, 97%, 98%, 99% or more) identical amino acid sequence The present invention encodes a PD-1 polypeptide having the

[0118] In various embodiments, the humanized PD-1 protein produced by the non-human animals of the invention is The protein is at least 50% identical (e.g., 100% PD-1 protein) to the extracellular portion of the human PD-1 protein seen in Figure 8. For example, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91% %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher ) have an extracellular portion with an identical amino acid sequence.

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

[0134] In various embodiments, the humanized PD-1 protein produced by the non-human animals of the invention is The protein is the N-terminal immunoglobulin V of human or humanized PD-1 protein, as shown in Figure 8. area and 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 amino acid sequence to the N-terminal immunoglobulin V region Have.

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

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

[0137] In various embodiments, the humanized PD-1 protein produced by the non-human animals of the invention is The protein shares at least 50% (e.g., For example, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 9 1%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher Top) They have transmembrane domains with identical sequences.

[0138] In various embodiments, the humanized PD-1 protein produced by the non-human animals of the invention is The protein shared at least 50% (e.g., 100% of the intracellular tail of mouse PD-1 protein, as seen in Figure 8). For example, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 9 1%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher Top) They have intracellular tails with identical sequences.

[0139] In various embodiments, the humanized PD-1 protein produced by the non-human animals of the invention is The protein is located at amino acid residues 27 to 169 (or 27 to 29) of the human PD-1 protein (Figure 8). 6-169) and 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 amino acid sequence.

[0140] In various embodiments, the humanized PD-1 protein produced by the non-human animals of the invention is The protein is located at amino acid residues 27 to 169 (or 27 to 29) of the human PD-1 protein (Figure 8). 6-169) has substantially the same amino acid sequence as

[0141] In various embodiments, the humanized PD-1 protein produced by the non-human animals of the invention is The protein is located at amino acid residues 27 to 169 (or 27 to 29) of the human PD-1 protein (Figure 8). 6-169) has the same amino acid sequence.

[0142] In various embodiments, the humanized PD-1 protein produced by the non-human animals of the invention is The protein has at least a 50% ( For example, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more (above) have the same amino acid sequence.

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

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

[0145] Specific polymorphic forms, allelic variants (e.g., single amino acid differences) or alternatively We have developed a non-human animal that expresses humanized PD-1 protein, including spliced ​​isoforms. Compositions and methods for making such proteins are provided, including the preparation of human proteins. US201002326333A1 - Compositions and methods for making non-human animals that express human promoters and human regulatory sequences - Google Patents In some embodiments, such proteins are expressed by endogenous promoters and endogenous regulators. Compositions and methods for making non-human animals expressing the segmental sequences are also provided. In certain embodiments, the endogenous promoter and endogenous regulatory sequences are endogenous rodent promoters. The method comprises the steps of: generating a PD-1 gene encoding a human PD-1 protein; The genetic material encoding a portion of the genome of a non-human animal corresponding to the endogenous Pdcd1 gene is Inserting it into a precise location, thereby producing a fully or partially human PD-1 protein In some embodiments, the method includes generating a humanized Pdcd1 gene that expresses the Genomic sequences corresponding to all or part of exon 2 and exon 3 of the human PDCD1 gene DNA is inserted into the endogenous Pdcd1 gene of a non-human animal, thereby The present invention relates to a method for the production of a PD-1 protein comprising administering to a patient a human portion comprising the steps of: The method includes the step of generating a humanized gene that

[0146] Where appropriate, encoding all or part of the human (or humanized) PD-1 protein The coding region or polynucleotide sequence of the genetic material is encoded by a nucleic acid sequence encoding the polypeptide for expression from the cells of the non-human animal. The nucleic acid sequence may be modified to include codons optimized for (See US Pat. Nos. 70,356 and 5,874,304.) Codon-optimized sequences are synthetic sequences. and the same polypeptide encoded by the non-codon-optimized parent polynucleotide (also The biological activity of the full-length polypeptide is substantially the same as that of the full-length polypeptide. In some embodiments, it is preferred to encode a human (or humanized) The coding region of the genetic material that encodes all or part of the PD-1 protein is expressed in certain cells. Sequences modified to optimize codon usage for the cell type (e.g., rodent cells) For example, the endogenous Pdcd1 gene of a non-human animal (e.g., a rodent) A portion of exon 2 and exon 3 of the human PDCD1 gene (e.g., The codons in the genomic DNA corresponding to the 71 bp fragment are optimized for expression in non-human animal cells. Such sequences may be described as codon-optimized sequences. .

[0147] The humanized Pdcd1 gene approach utilizes relatively minimal modifications of the endogenous gene, The non-human animal provides natural PD-1 mediated signaling, which may be used in various embodiments. The genomic sequence of Pdcd1 sequence was altered in a single fragment, thus eliminating the need for further manipulation. The inclusion of the node sequence maintains normal function. So, Pdcd1 gene modification can affect other surrounding genes or other endogenous Pdcd1 genes. It does not affect interacting genes (e.g., PD-L1, PD-L2, etc.). In various embodiments, the modification affects the assembly of a functional PD-1 transmembrane protein on the cell membrane. through the cytoplasmic portion of the protein, which is unaffected by binding and modification Subsequent signal transduction maintains normal effector function.

[0148] Schematic representation of the genomic structure of the endogenous mouse Pdcd1 gene and the human PDCD1 gene (accurate A schematic representation (not to scale) of exon 2 and exon 3 of the human PDCD1 gene is provided in Figure 1. A genomic fragment containing a portion of chthon 3 was used to clone the endogenous mouse Pdcd1 gene. An exemplary method for humanizing human PDC is provided in Figure 2. As shown, Contains exon 2 and part of exon 3 (e.g., the first 71 bp) of the D1 gene An 883 bp genomic DNA fragment was cloned into the endogenous mouse Pdcd gene by the targeting construct. Inserted at 900 bp of sequence at 1 locus. 883 bp human DNA fragment. The nucleotides are cloned directly from human DNA or from the source sequence (e.g., Genb ank accession number NM_005018.2) may be synthesized from this genomic DNA. The extracellular portion of the human PD-1 protein involved in ligand binding (e.g., amino acid sequence The portion of the gene that encodes substantially all of residues 27-169 or 26-169 nothing.

[0149] A non-human animal (e.g., mouse) carrying a humanized Pdcd1 gene at the endogenous Pdcd1 locus. The stencils can be made by any method known in the art. For example, a selectable A targeting vector for introducing all or part of the human Pdcd1 gene with a marker gene Figure 2 shows the DNA fragments of exons 2 and 3 of the human PDCD1 gene. The mouse genome contains an insert of an 883 bp human DNA fragment containing the first 71 bp. 1 shows a targeting vector that contains the endogenous Pdcd1 locus of the mouse. As shown, the targeting construct contains sequences upstream of exon 2 of the endogenous mouse Pdcd1 gene (~61.7 Kb). A drug selection cassette followed by 5' homology arms (e.g., flanked by loxP sequences) The adjacent neomycin resistance gene (~5 Kb), exon 2 and and a genomic DNA fragment (883 bp) containing the first 71 bp of exon 3, and the remaining sequence of endogenous mouse exon 3 (i.e., the transmembrane domain of the PD-1 protein). 3, which contains exons 4 and 5 of endogenous mouse Pdcd1 'Contains homology arms (~84 Kb). The targeting construct is an auto-deletion drug selection cassette. (e.g., a neomycin resistance gene flanked by loxP sequences, U.S. Pat. No. 8,633,666; See Nos. 97,851, 8,518,392 and 8,354,389. all of which are incorporated herein by reference. Electroporating Embryonic Stem Cells and 900 bp of the endogenous wild-type Pdcd1 gene to 883 bp of the human PDCD1 gene. p (i.e., exon 2 and the first 71 bp of exon 3) The Pdcd1 gene is engineered and contained in a targeting vector. A child was created and cloned into the mouse model, which contains an 883 bp human DNA fragment (i.e., the human PDCD1 gene). Human genomic DNA containing the amino acids encoded by exon 2 and 71 bp of exon 3 of The result is a cell or non-human animal that expresses a modified PD-1 protein. The set is eliminated in a development-dependent manner, i.e., the germline cells are infected with the humanized Pd Offspring derived from mice containing the cd1 gene are selectable from differentiated cells during development (See the bottom of Figure 2.)

[0150] Humanized Pdcd1 gene from mouse (i.e., human protein portion and mouse portion) An embodiment using a mouse having a Pdcd1 gene encoding a PD-1 protein containing are broadly contemplated herein, but other non-human Animals are also provided. In some embodiments, such non-human animals include rodent Pdcd The humanized Pdcd1 gene is operably linked to the . In embodiments, such non-human animals are operably linked to the endogenous Pdcd1 promoter. In some embodiments, the humanized Pdcd1 gene is linked to an endogenous rodent Pdcd In some embodiments, such non-human animals are The humanized PD-1 protein is expressed from Protein amino acid residues 21-170 (or 26-169, or 27-169, 27- Such non-human animals include those disclosed herein. Animals genetically modified to express the PD-1 protein, e.g. , mice, rats, rabbits, pigs, cattle (e.g., cows, bulls, buffaloes) ), deer, sheep, goats, chickens, cats, dogs, ferrets, primates (e.g. marmoset For example, suitable genetically modifiable ES cells For non-human animals for which no genetic modification is readily available, Other methods may be employed to transfect non-ES cell genomes (e.g., fibroblasts or induced pluripotent cells) and somatic cell nuclear transfer (SCNT) ) to introduce the genetically modified genome into a suitable cell, e.g., an enucleated oocyte. and subjecting the modified cell (e.g., modified oocyte) to conditions suitable for the formation of an embryo. For example, the embryo may be gestation in a non-human animal.

[0151] Modification of non-human animal genomes (e.g., genomes of pigs, cows, rodents, chickens, etc.) Methods include, for example, using zinc finger nucleases (ZFNs) or TAL effectors Nuclease (transcription activator-like effect Using TALEN (talk-altering enzyme), the genome was modified to create humanized Pdcd1. Methods for including genes are included.

[0152] In some embodiments, the non-human animal of the invention is a mammal. Non-human animals of the invention are small mammals, for example of the superfamily Jerboidea or Murine superfamily. In some embodiments, the genetically modified animal of the present invention is a rodent. The rodent of the invention is selected from a mouse, a rat, and a hamster. The rodent is selected from the superfamily Murinoidea. In some embodiments, the genetically modified animal of the present invention The family Mycorrhizae (e.g. mouse-like hamsters), Cricetidae (e.g. hamsters), Tarra, New World Rats and Mice, Bank Voles, Muridae (Pure Mouse and rats, gerbils, spiny mice, maned mice, and mouse species (kino Boli mouse, rock mouse, white-tailed rat, Madagascar rat and mouse), Togeya Family Manicidae (e.g., the spiny dormouse), and Family Bucconidae (e.g., the mole rat, the bamboo rat, In some embodiments, the mammal is from a family selected from the group consisting of the rat, the house mouse, the plateau mole rat, and the plateau mole rat. The genetically modified rodents of the present invention are pure breeds of mice or rats (Muridae), In some embodiments, the mouse of the present invention is selected from the group consisting of mouse, spiny mouse, and maned mouse. The genetically modified mouse is from a member of the Muridae family. The non-human animals of the invention are rodents. In some embodiments, rodents of the invention include mice and rats. In some embodiments, the non-human animal of the invention is a mouse.

[0153] In some embodiments, the non-human animals of the invention are C57BL / A, C57BL / An, C 57BL / GrFa, C57BL / KaLwN, C57BL / 6, C57BL / 6J, C 57BL / 6ByJ, C57BL / 6NJ, C57BL / 10, C57BL / 10ScS C57BL lineage mice selected from C57BL / n, C57BL / 10Cr, and C57BL / Ola In some embodiments, the mouse of the present invention is a rodent that is a 129P1, 129 P2, 129P3, 129X1, 129S1 (e.g., 129S1 / SV, 129S1 / SvIm), 129S2, 129S4, 129S5, 129S9 / SvEvH, 129 / SvJae, 129S6 (129 / SvEvTac), 129S7, 129S8, 129 129T1, 129T2, or a 129 line selected from the group consisting of lines that are Fest ing et al., 1999, Mammalian Genome 10:83 6; Auerbach, W. et al., 2000, Biotechniq (see E. 29(5):1024-1028, 1030, 1032). In an embodiment, the genetically modified mouse of the present invention is a mouse of the 129 strain and the C57BL / 6 strain. In some embodiments, the genetically modified mouse of the present invention is a mixture of the aforementioned 129 strains. or a mixture of the aforementioned BL / 6 lineages. The mixed 129 lineage is the 129S6 (129 / SvEvTac) lineage. In some embodiments, the mouse of the present invention is of the BALB strain (e.g., the BALB / c strain). The mice of the present invention are a mix of the BALB strain and another of the aforementioned strains.

[0154] In some embodiments, the non-human animal of the invention is a rat. The rats used in this study were Wistar rats, LEA strains, Sprague Dawley strains, and Fischer rats. The sirah series, F344, F6 and Dark Agouti are selected from the group consisting of In particular, the rat strains described herein are Wistar, LEA, Sprague Dawley and Wley, Fisher, F344, F6, and Dark Agouti It is a mixture of two or more systems selected from the above.

[0155] Methods using non-human animals carrying a humanized Pdcd1 gene Investigating PD-1 function using various Pdcd1 mutants and transgenic non-human The use of animals was employed (e.g., Nishimura, H. et al. (199 8) Intern. Immunol. 10(10):1563-1572; Shimura, H. et al. (1999) Immunity 11:14 1-151; Nishimura, H. et al. (2001) Scien ce 291:319-322; Iwai, Y. et al. (2004) I ntern. Immunol. 17(2):133-144; Keir, M. E. et al. (2005) J. Immunol. 175:7372-73 79; Keir, ME et al. (2007) J. Immunol 179:5064-5070; Carter, LL et al. (20 07) J. Neuroimmunol. 182:124-134; Chen, L. et al. (2007) Europ. Soc. Organ Trans plant. 21:21-29; Okazaki, T. et al. (201 1) J. Exp. Med. 208(2):395-407; U.S. Pat. No. 7,411,633; See, for example, US Pat. No. 4,171 and European Patent No. 1 334 659 B1, which are incorporated herein by reference. (The disclosure of which is incorporated herein by reference in its entirety). Such mutants and transgenic animals are Useful in determining the molecular aspects of PD-1 expression, function and regulation of various cellular processes However, they have limitations. For example, PD-1-deficient mice were generated by knocking in human PD-1 cDNA into 1. , expressing human PD-1 even after stimulation with PMA (Carter, LL et al., supra). Furthermore, there were considerable differences in phenotypes between PD-1 mutant animals of different genetic backgrounds. In particular, studies have attempted to assign various functions and / or regulatory activities to PD-1. However, other transgenic animals that overexpress PD-1 have been Such animals have been generated using transgenic mice (Chen, L. et al., supra). The results show a distinct expression pattern that can reasonably be attributed to the construct design. Because the same source genetic material (i.e., mice) was used, PD-1 overexpression was observed in Due to potential position effects of the gene, endogenous PD-1, but not transgenic PD-1, was not detected. PD-1 transgenic mice may have been resistant to some PD- 1 Although it has been shown to be useful in clarifying vector functions, the results obtained The mice show variability in their DNA, which is at least in part due to the different DNA fragments used to generate the mice. Therefore, current in vivo studies exploiting PD-1 mediated biology are The molecular aspects of PD-1-mediated functions and signaling pathways are being investigated. Transgenic mice have not yet been utilized to their full potential.

[0156] The non-human animals of the invention express human (or humanized) PD-1, which is useful in a variety of assays. To provide an improved in vivo system and source of biological material (e.g., cells) for expressing In various embodiments, the non-human animals of the invention are provided with a PD-1-targeting and / or or modulating PD-1 signaling (e.g., PD-L1 and / or PD-L2 In various embodiments, the present invention is used to develop therapeutic agents that prevent the interaction of The non-human animals of the invention can be used to identify and screen candidate therapeutic agents (e.g., antibodies) that bind human PD-1. In various embodiments, the present invention is used to clean and / or develop Clear non-human animals have demonstrated the interaction of human PD-1 with human PD-L1 and / or human PD-L2. Used to screen and develop candidate therapeutic agents (e.g., antibodies) that block the interaction. In various embodiments, the non-human animals of the invention are adapted to use the non-human Binding profiles of humanized PD-1 antagonists and / or agonists on the surface of mouse cells In some embodiments, the non-human animals of the invention are used to determine the level of human PD- To determine the epitope or epitopes of one or more candidate therapeutic antibodies that bind to 1. It is used to

[0157] In various embodiments, the non-human animals of the invention are used to characterize the pharmacokinetic profile of anti-PD-1 antibodies. In various embodiments, one or more non-human animals of the present invention are used to determine and one or more control or reference non-human animals each containing one or more candidate therapeutic anti-PD The 1-1 antibody was administered at various doses (e.g., 0.1 mg / kg, 0.2 mg / kg, 0.3 mg / kg, 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 / k The candidate treatments were exposed to 100 mg / kg or 40 mg / kg or more. The therapeutic antibody may be administered by any desired route of administration, including parenteral and non-injectable routes of administration. Parenteral routes include, for example, intravenous, intraarterial, intraportal, intramuscular, subcutaneous, and intraperitoneal. , intrathecal, intrathecal, intraventricular, intracranial, intrapleural or other routes of injection. Non-injection routes include, for example, oral, nasal, transdermal, pulmonary, rectal, buccal, vaginal, and ocular. Continuous injection, local administration, sustained release from implants (gels, membranes, etc.), and / or intravenous Blood was drawn from non-human animals (humanized and control) at various time points ( For example, 0 hours, 6 hours, 1st, 2nd, 3rd, 4th, 5th, 6th, 7th, 8th, 9th, 1st The cells are isolated at 0, 11, or up to 30 or more days. The present disclosure includes, but is not limited to, total IgG, anti-treatment antibody responses, agglutination, etc. Using samples obtained from the non-human animals described in This may be performed to determine the pharmacokinetic profile.

[0158] In various embodiments, the non-human animals of the invention are characterized by the ability to inhibit or modulate PD-1 signaling. used to measure the effect of treatment on gene expression as a result of cellular changes In various embodiments, the non-human animals of the invention or cells isolated therefrom are Humanized PD-1 protein (or the human portion of the PD-1 protein) on the cell surface of a human animal After a period of exposure to a candidate therapeutic agent that binds to the cells (minutes), e.g., adhesion, apoptosis, These include inflammation, proliferation, self-tolerance, and viral infection (or response). The effects of these drugs on PD-1-dependent processes were analyzed.

[0159] The non-human animals of the invention express humanized PD-1 proteins, and thus the cells, cell lines, Cell cultures are generated to, for example, specifically detect antagonists or agonists expressing human PD-1 sequences or or alternatively, associated with PD-L1 and / or PD-L2. PD-1 antagonists or For use in binding and functional assays to assay agonist binding or function In various embodiments, the humanized PD-1 can serve as a source of Using cells isolated from the non-human animal of the invention, P bound by a candidate therapeutic antibody can be detected. In various embodiments, the D-1 epitope can be determined using the methods described herein. The humanized PD-1 protein expressed by the non-human animal has a variant amino acid sequence Variant human PD-1 associated with autoimmune and infectious diseases Protein (e.g., polymorphisms) have been reported (e.g., Lee, YH et al. l. (2014) Z. Rheumatol. PMID: 24942602; Mansur, A. et al. (2014) J. Investig. Me d. 62(3):638-643; Nasi, M. et al. (2013) Intern. J. Infect. Dis. 17:e845-e850; P iskin, IE et al. (2013) Neuropediatric s 44(4):187-190; Carter, LL et al. (20 07) J. Neuroimmunol. 182(1-2):124-134; W an, B. et al. (2006) J. Immunol. 177(12) Exemplary human PD-1 variants are listed in SN P Includes those listed on the GeneView webpage and summarized in Table 3 In various embodiments, the non-human animals of the invention express a humanized PD-1 protein variant. In various embodiments, the variants are expressed at amino acid positions associated with ligand binding. In various embodiments, the non-human animals of the invention are characterized by the polymorphism of human PD-1. It is used to determine the effect of ligand binding through interaction with the ant. In embodiments, the non-human animals of the invention comprise a human PD-1 protein variant found in Table 3. is expressed. [Table 3-1] [Table 3-2] [Table 3-3]

[0160] The cells from the non-human animals of the invention can be isolated and used ad hoc or can be cultured for multiple generations. In various embodiments, the non-human animals of the present invention can be maintained in culture for up to 10 days. Cells from the vector can be immortalized (e.g., by the use of a virus) and grown in culture (e.g., continuously in culture) indefinitely.

[0161] In various embodiments, the cells and / or non-human animals of the invention are immunized against an antigen. It will be used in various immunization regimens to determine the PD-1 mediated function of the response. In some embodiments, the antibody binds to human (or humanized) PD-1 or one or more Candidate therapeutic agents that inhibit function are characterized in the non-human animals of the invention. Suitable assays include , various cell assays, proliferation assays, serum immunoglobulin analysis (e.g., antibody titers) , cytotoxicity assays, and characterization of ligand-receptor interactions (e.g., immunoprecipitation assays) In some embodiments, the non-human animals of the invention include a P In some embodiments, the antigen is used to characterize D-1 mediated functional regulation. 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., an ovoa In some embodiments, the antigen is an antigen that is a cellular component of the human body that is in need of treatment. The target is associated with a disease or condition suffered by the human patient.

[0162] In various embodiments, the non-human animals of the invention are capable of expressing candidate therapeutic agents that target human PD-1. Serum assays were performed to determine the titer of autoantibody production to examine the pharmacological and toxicological aspects of In some embodiments, autoantibody production in the non-human animals of the invention is used in It results from one or more autoimmune diseases, disorders or conditions induced in the animal.

[0163] In various embodiments, the non-human animals of the invention are capable of expressing specific T cell-dependent T cells against an antigen. and PD-1-dependent regulation of immune responses, including but not limited to B cell-dependent responses. The present invention provides a method for determining the therapeutic potential of a compound or biological agent that modulates one or more of the antigens. It is used for exposure to

[0164] In various embodiments, the cells and / or non-human animals of the invention express human PD-1 signaling. To screen and develop candidate therapeutic agents that modulate survival and / or or proliferation assays (e.g., with B or T cells). Loss or activation of PD-1 plays an important role in regulating T cell responses and may contribute to the establishment of PD-1-mediated self-tolerance. Regulation may result from activation of specific epitopes on the extracellular domain of PD-1. Thus, the non-human animal cells of the invention and / or the non-human animals described herein are used In response, candidate PD-1 modulators (e.g., antagonists or agonists) are identified, characterized, and developed. In some embodiments, the cells and / or non-human animals of the invention may In addition, the presence and absence of PD-1 may be involved in the proliferation or apoptosis of certain cells (e.g., cancer cells). The antibody may be used in a survival or mortality assay to determine the effect on the cis site.

[0165] In various embodiments, the cells and / or non-human animals of the invention are transplanted human cells. To determine PD-1-mediated functions in physiological (e.g., immune) responses to cells or tissues In some embodiments, human PD Candidate therapeutic agents that bind to or inhibit one or more functions of .DELTA.-1 are used in the non-human animal models of the present invention. Suitable assays include a variety of cell assays, proliferation assays, and serum Immunoglobulin analysis (e.g., antibody titers), cytotoxicity assays, and ligand-receptor phase In some embodiments, the non-human IgG antibodies of the present invention can be used to characterize the interactions between the antibodies and the human IgG antibodies (immunoprecipitation assays). These animals will be used to characterize PD-1-mediated functional regulation of immune responses to antigens. In some embodiments, the antigen is associated with a neoplasm. In some embodiments, the antigen is autologous. In some embodiments, the antigen is associated with an immune disease, disorder or condition. In some embodiments, the antigen is associated with a particular These targets are associated with a disease or condition suffered by a human patient.

[0166] In various embodiments, the non-human animals of the present invention are provided with novel lymphocytes and their immune functions. To determine the therapeutic potential of compounds or biological agents that modulate PD-1-dependent regulation, or adoptive transfer experiments. In various embodiments, the non-human animals of the invention are human T In some embodiments, the subject is transplanted with naive T cells, and in some embodiments, activated T cells. It will be transplanted.

[0167] In various embodiments, the cells of the non-human animals of the invention are capable of mediating T cell dependent responses and functions. To determine the therapeutic potential of compounds or biological agents that modulate PD-1-dependent regulation, Exemplary T cell assays include ELISpot, intracellular cytokine assays, and Insulin staining, major histocompatibility complex (MHC) restriction, viral inhibition assay, cytotoxicity These assays include, but are not limited to, T cell proliferation assays and regulatory T cell suppression assays. stomach.

[0168] In various embodiments, the cells of the non-human animals of the invention are used to identify candidate therapeutics that modulate human PD-1. It is used in cell migration assays to screen and develop therapeutic agents. The migration assay involves the movement of cells across the endothelium, and migration assays have been used to study the endothelial involvement of leukocytes or tumor cells. This allows the measurement of the interaction with the epithelium and transendothelial migration.

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

[0170] In various embodiments, the cells of the non-human animals of the present invention are capable of expressing cytokines released from T cells. To determine the therapeutic potential of compounds or biological agents that modulate PD-1-dependent regulation of In some embodiments, the non-human animals of the present invention are used in cytokine production assays. The cells are transfected with humanized PD-1 and human PD-1 or a PD-1 ligand (e.g., PD-L1 Detection of intracellular cytokine release resulting from interaction of targeted drugs with PD-L2 (and PD-L2) and / or measurement.

[0171] In various embodiments, the PD-1 inhibitor is a potent inhibitor of one or more functions of an autoimmune disease, disorder, or condition. In vivo assays to determine the therapeutic potential of compounds or biological agents that modulate I-dependent regulation In order to provide a system, an autoimmune disease, disorder or condition is treated with one or more non-human Exemplary autoimmune diseases induced in one or more non-human animals of the present invention The disorders or conditions include diabetes, experimental autoimmune encephalomyelitis (e.g., multiple sclerosis), del), rheumatoid arthritis, and systemic lupus erythematosus.

[0172] The one or more non-human animals of the present invention may be used as an input for drug or vaccine analysis and testing. In various embodiments, a candidate drug or vaccine is provided according to the present invention. and then monitoring the non-human animals to determine whether the drug or vaccine is effective. the immune response to, the safety profile of, or the severity of, a disease or condition In some embodiments, the vaccine may determine one or more of the effects on, e.g., Targeting viruses such as human immunodeficiency virus or hepatitis virus (e.g., HCV) Exemplary methods used to determine the safety profile include toxicity, optimal dose, These include measurements of concentrations, efficacy of drugs or vaccines, and potential risk factors. Drugs or vaccines may be improved and / or developed in such non-human animals. .

[0173] The non-human animals of the present invention are useful as an indicator for evaluating the pharmacokinetic properties of drugs targeting human PD-1. In various embodiments, the human PD-1 targeting agent is a Delivered or administered to one or more non-human animals and then isolated from the non-human animals or perform one or more assays to assess the efficacy and safety of the product in a non-human animal. Pharmacokinetic properties describe how an animal uses a drug. to various metabolites (or the presence or absence of one or more drug metabolites, including toxic metabolites) detection), drug half-life, circulating levels of drug after administration (e.g., serum concentration of drug), antidrug reaction response (e.g., anti-drug antibodies), drug absorption and distribution, route of administration, drug excretion and / or In some embodiments, the drug (e.g., The pharmacokinetic and pharmacodynamic properties of the PD-1 modulators (PD-1 modulators) can be determined in or on the non-human animals of the invention. This is monitored through the use of

[0174] The non-human animals of the present invention are useful in assessing the on-target toxicity of drugs targeting human PD-1 in vivo. In various embodiments, the human PD-1 targeting agent is one of the methods of the present invention. or the like, and then delivered or administered to the non-human animal (or isolated therefrom). and performing one or more assays to assess the efficacy of the drug in a non-human animal. This may determine the target toxic effect of the agent. Generally, an agent modulates one or more functions of a target. In one example, a PD-1 modulator may target the expression of one or more cells. By interacting in some way with PD-1 molecules on the surface of the In some embodiments, the present invention is directed to modulating PD-1 signaling. Such modulators may have adverse effects that are an exaggeration of the desired pharmacological action of the modulator. Such effects are called target effects. Exemplary target effects include too high a dose, chronic activity, In some embodiments, the present invention provides a method for the activation / inactivation of a polypeptide of the present invention. The targeted effects of PD-1-targeting agents identified in or through the use of non-human animals include: It is used to determine previously unknown functions of PD-1.

[0175] The non-human animals of the present invention are useful in in vitro studies to assess off-target toxicity of drugs targeting human PD-1. In various embodiments, the human PD-1 targeting agent is one of the methods of the present invention. or more non-human animals and then delivered or administered to the non-human animals (or isolated therefrom) or perform one or more assays to assess the efficacy and safety of the antibody in a non-human animal. These may determine the off-target toxic effects of a drug. Off-target effects occur when a drug interacts with an unintended target. This may occur when antibodies interact with each other (e.g., cross-react to a common epitope). Such interactions can occur in intended or unintended tissues. In addition, drug enantiomers may have off-target toxic effects. In turn, drugs may inappropriately interact with different receptor subtypes, activating them unintentionally. Exemplary off-target effects include mistargeting of tissues that is not related to the tissue of the wrong target. In some embodiments, the human PD-1 targeted agent may be The off-target effect is the effect of administering the agent to a non-human animal of the invention and to one or more reference non-human animals. The effect is determined by comparing the dose.

[0176] In some embodiments, performing the assay includes determining the phenotype of the non-human animal to which the agent is administered. and / or determining the effect on genotype. This includes determining lot-to-lot variability of PD-1 modulators (e.g., antagonists or agonists). In some embodiments, performing the assay includes determining whether the non-human animal of the invention To determine the difference between the efficacy of PD-1-targeted drugs administered to mice and their efficacy in reference non-human animals. In various embodiments, the reference non-human animal comprises a modification as described herein, Alterations other than those described in the specification (e.g., disruption, deletion or deletion of the Pdcd1 gene) otherwise have a non-functional Pdcd1 gene) or no alteration (all i.e., wild-type non-human animals).

[0177] Evaluate the pharmacokinetic properties, target toxicity, and / or off-target toxicity of human PD-1-targeting agents in non-human animals (or in and / or using cells isolated therefrom) to Exemplary parameters that can be measured (using a cellular marker) include aggregation, autophagy, cell division, cell Death, complement-mediated hemolysis, DNA integrity, drug-specific antibody titers, drug metabolism, gene expression arrays, Metabolic activity, mitochondrial activity, oxidative stress, phagocytosis, protein biosynthesis, protein breakdown Solution, protein secretion, stress response, target tissue drug concentration, non-target tissue drug concentration, transcriptional activity In various embodiments, the non-human animals of the present invention include, but are not limited to, , are used to determine a pharma- ceutical effective amount of a PD-1 modulator.

[0178] The non-human animals of the present invention are useful as modified models for the development and characterization of candidate therapeutic agents for use in cancer. In various embodiments, the non-human animals of the present invention are provided with improved in vivo systems. The tumor may be implanted, followed by administration of one or more candidate therapeutic agents. So, candidate therapeutics include multispecific antibodies (e.g., bispecific antibodies) or antibody cocktails. In some embodiments, the candidate therapeutic agent may be administered, for example, concomitantly or simultaneously. The tumor may be one or more tumors in a non-human animal, including combination therapy, such as administration of a monospecific antibody to This may provide sufficient time for tumor cells to establish themselves in the affected area. These may be measured both before and after administration of the candidate therapeutic agent. , which may be measured in non-human animals if desired.

[0179] The non-human animals of the present invention may be used to effectively treat a disease, disorder or condition that affects humans. , candidate therapeutic agents and / or treatment regimens (e.g., monotherapy, combination therapy, dose ranging studies) may be used to develop one or more disease models to evaluate or assess Various disease states may be established in the non-human animals of the invention, and then one or more of the disease states may be One or more candidate molecules can be assayed to determine the efficacy of a candidate molecule (e.g., a PD-1-targeting drug). In some embodiments, disease models include autoimmune, inflammatory, and / or neoplastic diseases, disorders or conditions.

[0180] In one example, the non-human animal of the invention can be used to prevent and / or treat tumors or tumor cells. In various embodiments, the present invention provides an improved animal model for therapeutic treatment. A human animal is implanted with one or more tumor cells and then administered one or more candidate therapeutic agents, e.g. In some embodiments, the administration of one or more candidate therapeutic agents may include administering one or more Implantation of one or more tumor cells and one or more candidate therapeutic agents in the non-human animal of the invention. Evaluate efficacy in preventing solid tumor establishment and / or tumor cell growth in rats The test is conducted after the test has been approved (e.g., minutes or hours later, but typically the same day). In embodiments, administration of one or more candidate therapeutic agents is performed after implantation of one or more tumor cells (e.g., In some specific embodiments, one or more implanted tumor cells are This is performed after sufficient time for the animals to reach a desired size (e.g., volume). One or more candidate therapeutic agents are evaluated for effectiveness in treating one or more established tumors. Human animals will allow determination of the optimal dose or dose range that correlates with effective treatment of established tumors. Patients may be placed into different treatment arms based on their dose, such as:

[0181] Candidate molecules can be administered to non-human animals using any method of administration, including parenteral and non-injectable routes of administration. Parenteral routes include, for example, intravenous, intraarterial, portal vein, and the like. Intramuscular, subcutaneous, intraperitoneal, intrathecal, intraarachnoid, intraventricular, intracranial, intrathoracic or intravenous Non-injection routes include, for example, oral, nasal, transdermal, pulmonary, rectal, buccal, Administration may be by continuous injection, topical administration, or sustained administration from an implant (gel, membrane, etc.). The combination therapy may be evaluated in the non-human animals of the invention. When evaluating the efficacy and safety of the candidate molecules, the candidate molecules can be administered by the same or different routes of administration. When a therapeutic regimen is evaluated in a non-human animal of the invention, the candidate molecule is identified as being capable of functioning in one or more disease models. To determine the dosing regimen that will provide the desired therapeutic or prophylactic effect in the non-human animal for which the study is being established. every two months, every month, every three weeks, every two weeks, every week, every day, at variable intervals, and / or It may be administered in gradually increasing concentrations.

[0182] The non-human animals of the invention may be used as therapeutic agents for the development and characterization of candidate therapeutic agents for use in infectious diseases. In various embodiments, the non-human animals of the present invention provide improved in vivo systems. is a virus (e.g., MHV, HIV, HCV, etc.) or pathogen (e.g., bacteria) The infected individuals may be injected with one or more potential therapeutic agents and then administered one or more potential therapeutic agents. In embodiments, the candidate therapeutics include multispecific antibodies (e.g., bispecific antibodies) or antibody complexes. In some embodiments, the candidate therapeutic agent may include, for example, a concomitant or simultaneous In some particular embodiments, therapies include combination therapies such as administration of a monospecific antibody administered in combination with a The complementary medicine may include a vaccine. one or more symptoms associated with infection with the non-human animal, such that the non-human animal develops one or more symptoms associated with infection with the In some cases, the T cells may be given enough time to colonize the upper sites or cells. In addition, survival, blood flow, and growth may be measured both before and after administration of the candidate therapeutic agent. Serum and / or intracellular cytokine analysis, liver and / or spleen histopathology will be performed. In some embodiments, the present invention may be measured in non-human animals infected with a pathogen or pathogens. The non-human animals of the invention are used to determine the extent of organ damage associated with viral infection. In some embodiments, the non-human animals of the present invention are non-human animals infected with a particular virus. Used to determine cytokine expression profiles in various organs.

[0183] The non-human animals of the invention can be used to evaluate human cell-targeted therapeutic agents. In various embodiments, the non-human animals of the invention are implanted with human cells, targeting human cells, etc. A targeted drug candidate is administered to such a non-human animal. The therapeutic efficacy of the drug is then evaluated by The amount of the human cell in the non-human animal is determined by monitoring the human cells in the non-human animal after administration of The drugs that can be tested include small molecule compounds, i.e., 1500 kD, 1200 kD, 100 Compounds with a molecular weight of less than 0 kD or 800 Daltons, and large molecules (proteins, etc.) These include both proteins, e.g., antibodies, which target (e.g., bind to) human cells. and / or affecting the treatment of human diseases and conditions. It has an effect.

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

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

[0186] The following examples will demonstrate to one of ordinary skill in the art how to make and use the methods and compositions of the present invention. It is offered for illustrative purposes and is 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 atmospheric or is nearby.

[0187] Example 1: Humanization of the endogenous programmed cell death 1 (Pdcd1) gene An example of this is programmed cell death 1 (P1) in non-human mammals such as rodents (e.g., mice). 1 shows an exemplary method for humanizing the endogenous Pdcd1 gene encoding the Pdcd1 polypeptide. The method described in the example can be used to select any human sequence, or a desired human sequence (or sequence flag A combination of these markers was used to humanize the endogenous Pdcd1 gene in non-human animals. In this example, GenBank accession number NM_005018.2 Exon 2, intron 2, and A ~883 bp human DNA fragment containing the first 71 bp of exon 3 was identified. It has been used to humanize the endogenous Pdcd1 gene in mice. The targeting vector for humanization of the genetic material encoding the extracellular N-terminal IgV region of the child is Made using VELOCIGENE® technology (U.S. Patent No. 6,586, No. 251 and Valenzuela et al., 2003, Nature B iotech. 21(6):652-659, which are incorporated herein by reference. (included).

[0188] Briefly, mouse bacterial artificial chromosome (BAC) clone RP23-93N20 (I (Invitrogen) encodes amino acids 26 to 169 of the human PD-1 polypeptide. A ~883 bp human DNA fragment was used to clone the endogenous Pdcd1 gene. A deletion of intron 2, intron 2, and part of exon 3 was performed to identify the human PDCD1 gene. It was modified to insert exon 2, intron 2 and part of exon 3 of the gene. Exon 1, part of exon 3 (i.e., encoding the transmembrane domain), 4, and 5 The endogenous DNA, including the 5' and 3' untranslated regions (UTRs), was retained. Sequence analysis of a 1 bp human DNA fragment identified all human PDCD1 exons (i.e. , 71 bp in exon 2 and exon 3) and splicing signals were identified. Sequence analysis confirmed that the sequence was identical to the reference genome and the PDCD1 transcript NM_005018.2. It turns out that this was the case.

[0189] More specifically, first, a small bacterial homologous recombination donor is prepared by integrating a synthetic DNA fragment containing: A fragment was generated from: [(HindIII)-(mouse upstream 78 bp)-(X hoI / NheI restriction enzyme site)-(human PDCD1 883bp)-(mouse downstream 7 5bp)- (HindIII)]. This fragment was obtained from Genescript Inc. (Piscataway, NJ) and ampicillin resistant. The recombinase was cloned into a plasmid vector using the XhoI-NheI sites. The ligation ligates a ~4,996 bp auto-deleting neomycin cassette flanked by ribozyme recognition sites. (loxP-hUb1-em7-Neo-pA-mPrm1-Crei-loxP, U.S. Patent Nos. 8,697,851, 8,518,392 and 8,354,389 (See, for example, US Pat. No. 6,333,345, which are incorporated herein by reference). Subsequent selections include neomycin. The flanking HindIII sites were used to clone the mouse BAC clone RP23-93 Prior to N20 homologous recombination, the targeting vector was linearized. The junction between the 883bp fragment and the mouse downstream 75bp opens exon 3 The reading frame was maintained (Figure 2). The resulting targeting vector contained a 5' to 3' Up to 61.7 kb of mouse genomic DNA from BAC clone RP23-93N20 5' homology arms containing b, a self-deleting neomycin cassette flanked by loxP sites , an 883 bp human genomic DNA fragment (from exon 2 of the human Pdcd1 gene (including the first 71 bp of exon 3) and from BAC clone RP23-93N20 It contained ∼84 kb of mouse genomic DNA.

[0190] The modified RP23-93N20 BAC clone described above was used to induce the expression of mouse embryonic stem (ES) cells. The gene was humanized from exon 2 to part of exon 3 (i.e., endogenous Pd (a 900 bp deletion in the cd1 gene and an 883 bp insertion of human sequences) endogenous Pdc The resulting clone was used to generate engineered ES cells containing the humanized Pdcd1 gene. ES cells targeted to human PDCD1 sequences (e.g., exon 2 and exon 3) (part of) The mouse Pdcd1 sequence (e.g., exon 2 and part of exon 3, and / or Table 4 shows the loss and / or retention of endogenous Pd The primers and probes used to confirm the humanization of the cd1 gene are shown (Figure 1). 3). The nucleotide sequence spanning the upstream insertion point includes the following, which contains a loxP site (boldface): ) adjacent to the endogenous 5' end of the neomycin cassette. Mouse sequence (included in brackets below, XhoI restriction site in italics) and deletion points The cassette sequence is: (TCAAAGGACA GAATAGTAGC CTCC AGACCC TAGGTTCAGT TATGCTGAAG GAAGAGCCCT CTCGAG)ATAACTTCGT ATAATGTATG CTATACGAAG TTATATGCAT GGCCTCCGCG CCGGGTTTTG GCGCCTC CCG CGGGCGCCCC CCTCCTCACG (SEQ ID NO: 19). Automatic deletion The nucleotide sequence spanning the downstream insertion point at the 3' end of the omycin cassette includes: This cassette sequence (summarized below) is adjacent to the human Pdcd1 genomic sequence downstream of the insertion point. The following sequence is shown: (CTGGAATAAC TTCGTATAAT GTATGCTATA CGAAGT TATG CTAGTAACTA TAACGGTCCT AAGGTAGCGA GC TAGC) AAGAGGCTCT GCAGTGGAGG CCAGTGCCCA T CCCCGGGTG GCAGAGGCCC CAGCAGAGAC TTCTCAAT GA CATTCCAGCT GGGGTGGCCC TTCCAGAGCC CTTG CTGCCC GAGGGATGTG AGCAGGTGGC CGGGGAGGCT TTGTGGGGCC ACCCAGCCCC (SEQ ID NO: 20). Human PDCD1 genome The nucleotide sequence spanning the downstream insertion point at the 3' end of the mouse sequence includes the following: The human PDCD1 sequences adjacent to the Pdcd1 genomic sequence (included in brackets below) Shown:CCCTTCCAGA GAGAAGGGCA GAAGTGCCCA CAGC CCACCC CAGCCCCTCA CCCAGGCCAG CCGGCCAGTT CCAAACCCTG (GTCATTGGTA TCATGAGTGC CCTAGT GGGT ATCCCTGTAT TGCTGCTGCT GGCCTGGGCC CT AGCTGTCT TCTGCTCAAC) (SEQ ID NO: 21). Neomycin cassette After deletion of the remaining 77 bp, the nucleotide sequence spanning the upstream insertion point contains The remaining cassette sequence is the loxP sequence (included in brackets below, and restricted by XhoI and NheI) The restriction sites are in italics, and the loxP sequences are in bold. Showing the column:TCAAAGGACA GAATAGTAGC CTCCAGACCC TA GGTTCAGT TATGCTGAAG GAAGAGCCCT (CTCGAG A TAACTTCGT ATAATGTATG CTATACGAAG TTATGCTA GT AACTATAACG GTCCTAAGGT AGCGA GCTAGC) A AGAG GCTCTGCAGT GGAGGCCAGT GCCCATCCCC GG GTGGCAGA GGCCCCAGCA GAGACTTCTC AATGACATT C CAGCTGGGGT GGCCCTTCCA (sequence number: 22).

[0191] The positive ES cell clones are then used to culture the VELOCIMOUSE® method (e.g. For example, U.S. Patent No. 7,294,754 and Poueymirou et al., 2 007, Nature Biotech. 25(1):91-99) The mouse strain was then transplanted with human PDCD1 exon 2 and a portion of human PDCD1 exon 3. The researchers then produced a litter containing an insertion into the endogenous Pdcd1 gene of the mouse. Part of exons 2 and 3 of the human Mice carrying the mutation were analyzed by an allele assay (Val) that detected the presence of the human PDCD1 gene sequence. Genetic analysis of DNA isolated from tail fragments using a modified version of the method described by Enzuela et al., supra. The offspring was genotyped and identified again using humanized Pdcd1 genes. A cohort of animals heterozygous for the offspring construct was selected for characterization. [Table 4]

[0192] Example 2: Expression of humanized PD-1 on activated T cells This example shows a non-human animal that has been modified to contain a humanized Pdcd1 gene according to Example 1. The subject (e.g., rodent) expresses humanized PD-1 protein on the surface of activated lymphocytes. In this example, we demonstrate that stimulatory T cells isolated from wild-type and humanized mice To determine the expression of PD-1, human PD-1 clones were cloned from the endogenous Pdcd1 gene as described in Example 1. Activated T cells from mice heterozygous for PD-1 activation were stained with anti-PD-1 antibody.

[0193] Briefly, wild-type mice and those expressing the endogenous Pdcd1 gene as described in Example 1 were Spleens were harvested from mice heterozygous for humanization and cultured in single-cell suspensions by mechanical dissociation. The cells were washed in culture medium (RPMI supplemented with 10% FBS) and diluted in 1× 0 6 The cells were resuspended at 100 / mL and 200 μL (200,000 cells) was placed on a 96-well plate. The cells in the selected wells were incubated with anti-CD3 and anti-CD28 antibodies (both at 100 ng / ml). Cells were stimulated with 10 μg / mL of 100 mM erythrocyte / mL sucrose for 72 h. Cells were then cultured for FACS according to the manufacturer's specifications. CD4, CD8, CD19 and human (clone MIH4, BD Biosciences) ces) or mouse (clone J43, eBioscience) PD1-recognizing antibodies The stained cells were run on an LSRII flow cytometer and analyzed by Flo Data were analyzed using the wjo software. + T cells are human and mouse Gated for expression of mouse PD1 (CD19 - CD8 + ) An illustrative result is shown in Figure 4. As shown.

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

[0195] Example 3: In vivo efficacy of PD-1 modulators An example of this would be screening for PD-1 modulators (e.g., anti-PD-1 antibodies), e.g. , to determine various characteristics such as inhibition of tumor growth and / or tumor cell killing. A non-human animal (e.g., This example demonstrates that a novel marker for tumor growth inhibition, known as cytotoxicity, can be used in in vivo assays. Determine the optimal antibody dose to inhibit and the extent to which anti-PD-1 antibodies mediate tumor cell killing To this end, a mouse model was prepared that was homozygous for the humanization of the endogenous Pdcd1 gene described in Example 1. Several anti-PD-1 antibodies are being screened in mice.

[0196] Briefly, mice were divided into 5 treatment or control groups (n=5) in Study 1 according to body weight. / group), eight treatment or control groups in study 2 (n=5 / group), and five treatment or control groups in study 3 (n=5 / group). On day 0, mice were treated with isoflurane The subjects were anesthetized by inhalation and then inoculated with MC38.ova cells suspended in 100 μL of DMEM on the right side of the subject. Injected subcutaneously into the abdomen (Test 1: 5 × 10 5 , Test 2 / 3: 1×10 6 ).MC38.ov a (mouse colon adenocarcinoma) cells were treated with chicken ovalbumin to increase tumor immunogenicity. In study 1, treatment groups received one of three anti-PD-1 antibodies: 200 μg of one or an isotype control antibody of irrelevant specificity was added to the third, seventh, and eighth of the experiment. On days 10, 14, and 17, a group of mice was injected intraperitoneally with 10-, 14-, and 17-mg IgG1. In Study 2, treatment groups received either 10 mg / kg or 5 mg / kg of anti-PD-1 -1 antibodies, one of three, anti-PD-1 antibodies at 10 mg / kg per dose (Ab B, IgG4) or 10 mg / kg of an isotype control antibody of irrelevant specificity In Study 3, the treatment groups were: one of two anti-PD-1 antibodies at a dose of 5 mg / kg or 2.5 mg / kg; or 5 mg / kg of a control antibody not specific for PD-1 (Control) on days 3, 7, 10, and 14 of the experiment. The mice were injected intraperitoneally on days 14 and 17. Table 5 shows the experimental doses and treatments for the groups of mice. The treatment protocol is shown.

[0197] For each study, the 14th or 17th day and the 23rd or 30th day of each study for each treatment group were The mean tumor volume, as determined by caliper measurement, and the percent survival at day 24 were reported. The number of tumor-free mice was also recorded at the end of the study (day 42 in study 1 and day 43 in study 2 and day 44 in study 3). The mean tumor volume (mm 3 ) (±SD), percent survival The number of tumor-free mice was calculated for each study (Tables 7-9). The growth curves are provided in FIG.

[0198] As shown in Table 6 for Study 1, mice treated with Ab A had Mice treated with Ab C did not develop detectable tumors in either study 17 or 2. At day 4, tumor volume was sustainedly reduced compared to controls, with 3 of 5 mice retaining tumors until the end of the study. In contrast, treatment with Ab B did not result in a significant reduction in tumor volume compared to controls in this study. By day 23 of the study, the group receiving Ab B had One of five mice died, compared with two of five mice in the isotype control antibody-treated group. In the untreated and isotype control groups, some mice showed spontaneous tumor regression ( (1 of 5 and 2 of 5 mice, respectively).

[0199] Mice treated with 10 mg / kg Ab A as shown in Table 7 for Study 2 did not develop detectable tumors during the course of the study. Groups of mice treated with either Ab D showed significantly higher mean ... The 10 mg / kg dose of either Ab C or Ab D significantly reduced tumor volume. Four out of five mice in each group treated with isotonic acid were tumor-free at day 31, whereas In the control treatment group, only 1 of 5 mice was tumor-free as a result of spontaneous tumor regression. Ab B tested at 100 mg / kg increased tumor volume compared to controls on days 17 and 24 of the study. Although the antibody showed a large reduction in PD-1 expression at the end of the study, this antibody was the least effective anti-PD-1 antibody. Only 2 of 5 mice survived.

[0200] A dose-dependent response of tumor inhibition was observed at the doses tested (5 mg / kg and 10 mg / kg). , observed in Ab A, Ab C and Ab D treatment groups. Ab A at 5 mg / kg Alternatively, Ab C therapy was less effective, with only 5 mice remaining tumor-free at the end of the study on day 31. In the 10 mg / kg dose group of Ab A, 5 out of 5 mice developed tumors, compared with 4 out of 4 mice in the 10 mg / kg dose group of Ab A. In the two-way ANOVA multiple comparison Dunnett's test, the 10 Groups treated with 10 mg / kg of isotype control antibody and 10 mg / kg of Ab A, A b The difference in tumor growth between groups treated with Ab C or Ab D was statistically significant with a p value < 0.005. As a reference, 10 mg / kg of isotype control antibody was used. The groups treated with Ab A, Ab C, or Ab D at 5 mg / kg were compared. The difference in tumor growth between the groups was also statistically significant with a p value of <0.05.

[0201] Treatment with 5 mg / kg Ab A or Ab C as shown in Table 8 for Study 3 Six of seven mice treated were tumor-free at the end of the study, compared with 1.1% in the isotype control group. None of the mice were tumor-free. One mouse with a tumor in the IgG4 control group died 17 days after implantation. Only 4 of 7 mice treated with 2.5 mg / kg Ab C died on day 1 of treatment. Patients remained tumor-free at the end of the study. Differences in tumor volume at day 21 between the groups were analyzed using one-way post-test Dunnett's multiple comparison test. Statistically significant, p<0.01, was determined by ANOVA. For all anti-PD-1 antibodies, the 5 mg / kg dose was better than the 2.5 mg / kg dose. was more effective.

[0202] As shown in FIG. 5, the anti-PD-1 antibody was synthesized using the PD-1 humanized antibody prepared according to Example 1. Significantly inhibited tumor growth in a prophylactic MC38.ova tumor growth model in mice. Anti-PD-1 Ab therapy at 100 mg / kg was administered to all mice (5 mice) throughout the course of the experiment. The treatment promoted tumor regression in 1 out of 5 mice (5 out of 5 mice), whereas in the control group, tumors spontaneously regressed in 1 out of 5 mice (1 out of 5 mice). Anti-PD-1 therapy at 5 mg / kg was only slightly more effective. At the end of the study, four out of five mice were tumor-free. One-way ANOVA with test comparisons showed p-values ​​< 0.05 (5 mg / kg) and and p-value <0.01 (10 mg / kg), there was a significant difference between anti-PD-1 treatment and control antibody treatment. It turns out there is a significant difference.

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

[0204] Briefly, PD-1 or control antibody-treated patients were randomly assigned to receive either the anti-PD-1 or control antibody treatment at the end of the study on day 21. Spleen cells were obtained from the -1 humanized mouse (75% C57BL / 6 / 25% 129). Total RNA was isolated and subjected to oligonucleotide and PCR using mouse CD8b (forward primer) ima: GCTCTGGCTG GTCTTCAGTA TG, SEQ ID NO: 24; Primer: TTGCCGTATG GTTGGTTTGA AC, SEQ ID NO:25; Probe: AGCAGCTCTG CCCTCAT, SEQ ID NO: 26), mouse CD3ζ (Mm00446171_m1, Applied Biosystems), mouse IF N-γ (Mm01168134_m1, Applied Biosystems), human PD-1 (forward primer: ACTTCCACAT GAGCGTGG, SEQ ID NO: :27; reverse primer: GGGCTGTGGG CACTTCTG, SEQ ID NO:2 8; probe: GCAGATCAAA GAGAGCCTGC, SEQ ID NO: 29) and Mouse PD-1 (Mm01285676_m1, Applied Biosystems ) was used to probe the reverse transcribed cDNA. Real-time PCR was performed. Samples were analyzed for mouse cyclophilin B expression. Normalized. Example results are provided in FIG.

[0205] As shown in FIG. 6, administration of anti-hPD-1 antibody (made according to Example 1) Splenic CD8 in humanized mice bearing C38.ova tumors + and CD3 + T cell production Furthermore, anti-hPD-1 antibody activity in tumor-bearing PD-1-humanized mice was increased by I Dependent on FNγ, which confirms proper signaling through humanized PD-1 on the cell surface Overall, there was an increase in T cells and IFNγ in both treatments compared to control-treated mice. was observed in groups.

[0206] Human PD-1 mRNA expression was engineered against the extracellular portion of the PD-1 protein. Proper expression of humanized PD-1 protein on the cell surface, as measured by a human-specific probe In addition, primers designed to detect the extracellular portion of mouse PD-1 were Measurement of mouse PD-1 mRNA expression in failed to produce a product.

[0207] In summary, this example demonstrates that the non-human animals of the invention can be administered an agent that targets PD-1, e.g. Such animals can be used to assess the in vivo efficacy of anti-PD-1 antibodies. Further, the present invention has been shown to be useful in differentiating therapeutic effects in the body. Non-human animals have demonstrated that drugs targeting PD-1 inhibit tumor growth and / or inhibit tumor cells. The non-human animals of the present invention (e.g., mice) can be used to assess the extent to which the antibodies mediate cell killing. mouse) as evidenced by T cell and cytokine expression (e.g., IFN-γ). Humanized PD-1 mediates functional PD-1 signaling and proper PD-1-dependent immune responses. This demonstrates the response. [Table 5] [Table 6] [Table 7] [Table 8]

[0208] Example 4: Rodent Model of Anti-PD-1 Tumor Therapy This example illustrates a non-human animal modified to contain a humanized Pdcd1 gene according to Example 1. (e.g., rodents) to determine the optimal therapeutic dose of a PD-1 modulator (e.g., an anti-PD-1 antibody). In this example, we demonstrate that anti-PD-1 antibodies can be used in tumor models to determine To determine the optimal therapeutic dose for the treatment of established tumors, the intravenous injections were performed using the intravenous injections described in Example 1. The vaccine is administered to mice homozygous for the humanized Pdcd1 gene.

[0209] Briefly, mice containing a humanized Pdcd1 gene (as described in Example 1) were (above) 1×10 6 MC38.Ova cells were implanted subcutaneously, and the tumor volume then increased to 80 ~120 mm 3 Once this was reached, patients were randomized into six treatment arms (n=8–9 per arm). Mice were administered a stepwise escalation range of 0.3 to 25 mg / kg (i.e., 0. 3, 1, 3, 10 or 25 mg / kg) of anti-hPD-1 antibody or 25 mg / kg The antibodies were administered intraperitoneally on days 0, 3, 7, 10, and 13. Tumor volumes were monitored by caliper measurements twice weekly for the duration of the study (60 days). Exemplary tumor growth curves are provided in FIG.

[0210] As shown in Figure 7, mice that received the control antibody were tumor-free at the end of the experiment. In contrast, the dose range of 3–25 mg / kg of anti-hPD-1 antibodies , resulting in approximately 44–55% tumor-free mice in the different treatment groups. The present invention provides a method for the administration of drugs targeting PD-1 to effectively treat established tumors in non-human animals. as a rodent tumor model to determine the optimal dose and / or dose range of an agent (e.g., an antibody); It has been demonstrated that it can be used.

[0211] equivalent With any of these described aspects of at least one embodiment of the present invention, It will be appreciated that various modifications, alterations, and improvements will readily occur to those skilled in the art. Such modifications, variations, and improvements are intended to be part of this disclosure. and are intended to be within the spirit and scope of the present invention. The above aspects are by way of example only, and the invention is further described in the following claims.

[0212] The use of sequences such as "first," "second," and "third" to modify claims in the claims The use of a term does not, by itself, imply priority, precedence, or sequence of one claim element over another. A single claim with a specific title that does not imply a sequence or a chronological order in which the acts of the method are performed. Distinguishing claim elements from other elements of the same name (except for the use of ordinal terms) Used only as a distinguishing marker.

[0213] As used in this specification and claims, the articles "a" and "an" are intended to mean Plural references should be understood to include more than one of a group unless expressly indicated otherwise. Any claim or statement containing "or" between the above elements is understood to be construed as including "or" whether or not it is indicated otherwise. Unless otherwise clear from the context, one, more than one, or all of the elements of a group An element is present in, used in, or otherwise relevant to a given product or process. The invention is satisfied when exactly one member of the group is present in a given product or process. The present invention includes embodiments that are found in, employed in, or otherwise related to the One or more, or the entire group of members, may be present in, used in, or in a given product or process. Further, the present invention includes, unless otherwise specified, Unless it is obvious to a person skilled in the art that a contradiction or inconsistency would occur, and one or more limitations, elements, phrases, descriptive terms, etc. from one or more of the claims. leads to another claim (or any other related claim) that depends on the same basic claim It should be understood that this encompasses all permutations of the elements that may be included. If there are subgroups of elements (e.g., in the form of Markush groups or similar), each subgroup of elements is also It will be appreciated that in general, the present invention is not limited to the above embodiments, and any element may be removed from the group. Whenever an embodiment or aspect of the invention is referred to as including a particular element, feature, etc., the invention Particular embodiments or aspects of the invention may consist of or consist essentially of such elements, features, etc. For the sake of brevity, these embodiments are in all cases largely This is not specifically described herein in any of the terms of the present invention. Any embodiment or aspect may be used in any manner that satisfies the claims, regardless of whether a specific exclusion is recited in the specification. may be expressly excluded from the claim.

[0214] Those skilled in the art will appreciate that values ​​obtained in the assays or other processes described herein are The reader will understand the typical standard deviation or error that may result from the The publications, websites and other information contained herein provide additional details regarding the practice of Other references are incorporated herein by reference.

Claims

1. A humanized programmed cell death 1 (Pdcd1) gene for expressing a humanized PD-1 polypeptide in a rodent, the humanized Pdcd1 gene encodes the humanized PD-1 polypeptide and comprises exon 2 and a portion of exon 3 of the human Pdcd1 gene; the humanized PD-1 polypeptide comprises a human portion and a rodent portion; the human portion comprises the extracellular domain of a human PD-1 polypeptide and is encoded by exon 2 and a portion of exon 3 of the human Pdcd1 gene; the rodent portion comprises an intracellular portion of a rodent PD-1 polypeptide; the rodent is a mouse or a rat; A humanized Pdcd1 gene, said humanized Pdcd1 gene having 5' and 3' untranslated regions identical to those present in a mouse or rat Pdcd1 gene.

2. 2. The humanized Pdcd1 gene of claim 1, wherein the humanized PD-1 polypeptide comprises a transmembrane portion of the rodent PD-1 polypeptide.

3. 3. The humanized Pdcd1 gene of claim 1 or 2, wherein the human portion of the humanized PD-1 polypeptide comprises amino acids 26-169 of human PD-1 polypeptide.

4. The humanized Pdcd1 gene of any one of claims 1 to 3, wherein the humanized Pdcd1 gene comprises exons 1, 4 and 5 of a rodent Pdcd1 gene.

5. The humanized Pdcd1 gene of claim 4 , wherein the humanized Pdcd1 gene comprises a portion of exon 3 of the rodent Pdcd1 gene.

6. The humanized Pdcd1 gene of any one of claims 1 to 5, operably linked to a rodent Pdcd1 promoter.

7. A targeted nucleic acid vector comprising, 5' to 3': a 5' homology arm comprising a nucleotide sequence of the mouse Pdcd1 gene upstream of exon 2 of the mouse Pdcd1 gene; A genomic DNA fragment of the human Pdcd1 gene, comprising exon 2 and a portion of exon 3 of the human Pdcd1 gene, encoding the extracellular domain of the human PD-1 polypeptide encoded by the human Pdcd1 gene; A targeting nucleic acid vector comprising a nucleotide sequence of the mouse Pdcd1 gene including a portion of exon 3, exon 4 and exon 5 of the mouse Pdcd1 gene, the portion of exon 3 encoding amino acids of the transmembrane portion of the mouse PD-1 polypeptide encoded by the mouse Pdcd1 gene, the vector comprising a 3' homology arm.

8. 8. The targeted nucleic acid vector of claim 7, wherein the genomic DNA fragment of the human Pdcd1 gene encodes amino acids 26 to 169 of the human PD-1 polypeptide.

9. 1. A method for evaluating a property of an agent that targets human PD-1, the method comprising: administering said agent to a mouse whose genome comprises a humanized Pdcd1 gene at the endogenous mouse Pdcd1 locus, operably linked to the mouse Pdcd1 promoter; the humanized Pdcd1 gene encodes a humanized Pdcd1 polypeptide and comprises exon 2 and a portion of exon 3 of the human Pdcd1 gene; the humanized PD-1 polypeptide comprises a human portion and an endogenous portion; the human portion comprises the extracellular domain of a human PD-1 polypeptide and is encoded by exon 2 and a portion of exon 3 of the human Pdcd1 gene; the endogenous portion comprises the intracellular portion of an endogenous mouse PD-1 polypeptide; and monitoring said mouse to determine said characteristic of said agent.

10. The method of claim 9, wherein the agent that targets human PD-1 is a PD-1 antagonist.

11. The method of claim 9, wherein the agent targeting human PD-1 is an anti-human PD-1 antibody.

12. 10. The method of claim 9, wherein the mouse comprises a tumor, the property of the agent comprises an anti-tumor effect of the agent, and the monitoring comprises monitoring the mouse to determine the effect of the agent on the tumor.

13. 10. The method of claim 9, wherein the humanized PD-1 polypeptide is translated in a cell of the mouse with a mouse signal peptide.

14. 10. The method of claim 9, wherein the humanized PD-1 polypeptide comprises a transmembrane portion of the endogenous mouse PD-1 polypeptide.

15. 10. The method of claim 9, wherein the human portion of the humanized PD-1 polypeptide comprises amino acids 26-169 of the human PD-1 polypeptide.

16. 10. The method of claim 9, wherein the humanized Pdcd1 gene comprises exons 1, 4 and 5 of endogenous mouse Pdcd1.

17. The method of claim 9, wherein the humanized Pdcd1 gene comprises a portion of exon 3 of endogenous Pdcd1.

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