Non-human animals with humanized cluster of differentiation 47 gene

Abstract

The present invention provides non-human animals and methods and compositions for making and using the same, wherein the non-human animals comprise humanized endogenous cluster of differentiation (CD) genes, in particular humanized CD47 genes. In some embodiments, the non-human animals can be described as having genetic modifications of the endogenous CD47 gene such that the non-human animals express a CD47 polypeptide comprising a human portion and a non-human portion (e.g., a murine portion).

Description

[0001] This application is a divisional application of the Chinese invention patent application with application number 201580066047.0. The application date of the original application is November 25, 2015, the priority date is December 5, 2014, and the name of the invention is "Non-human animals with humanized differentiation cluster 47 genes".

[0002] Cross-references to related patent applications

[0003] This application claims priority to U.S. Patent Application Serial No. 62 / 087,992, filed December 5, 2014, which is hereby incorporated by reference in its entirety.

[0004] Sequence Listing Incorporated by Reference

[0005] The Sequence Listing is an ASCII text file named 32584_10108WO01_SequenceListing.txt, 96.0 KB in size, created on November 23, 2015, submitted to the U.S. Patent and Trademark Office via EFS-Web, and is incorporated herein by reference. Background Art

[0006] Cancer treatments can be divided into four major categories: chemotherapy / radiotherapy, hormone therapy, targeted therapy, and immunotherapy. The focus of medical research and development has been on targeted therapy and significant improvements have been achieved, but cancer remains a major challenge for patients and the global healthcare industry. This major challenge is partly due to the ability of cancer cells to evade the surveillance mechanisms of the innate and adaptive immune systems, which is in part the result of inhibition of phagocytic clearance. Currently, there is no such in vivo system that optionally determines the therapeutic potential of new cancer therapies designed to activate phagocytic clearance of cancer cells, as well as determining the molecular level of how cancer cells provide inhibitory signals to macrophages and phagocytes. Such systems provide a source for measuring phagocytosis and macrophage function in vivo, as well as the identification of new cancer therapies that aim to provide an anti-tumor environment by initiating pro-phagocytic signals to the immune system. Summary of the invention

[0007] The present invention encompasses the recognition that it is desirable to engineer non-human animals to allow for improved systems for identifying and developing new cancer therapeutics. The present invention also encompasses the recognition that it is desirable to engineer non-human animals to allow for improved engraftment of human hematopoietic stem cells. Additionally, the present invention also encompasses the recognition that non-human animals having a humanized CD47 gene and / or otherwise expressing, containing or producing a human or humanized CD47 polypeptide are desirable, for example, for use in identifying and developing cancer therapeutics that overcome systemic toxicity associated with CD47 blockade and overcome CD47-mediated inhibition of tumor cell phagocytosis, and provide a more effective in vivo system for engrafting human hematopoietic stem cells that provide for increased homeostasis of a wide range of human cell types.

[0008] In some embodiments, the present invention provides a non-human animal having a genome comprising a CD47 gene, wherein the CD47 gene comprises genetic material from two different species (e.g., human and non-human). In some embodiments, the CD47 gene of a non-human animal as described herein encodes a CD47 polypeptide comprising a human and a non-human portion, wherein the human and non-human portions are linked together and form a functional CD47 polypeptide.

[0009] In some embodiments, the non-human animals of the invention comprise a CD47 gene comprising an endogenous portion and a human portion, wherein the endogenous and human portions are operably linked to an endogenous promoter.

[0010] In some embodiments, the endogenous portion comprises exons downstream of exon 1 and exon 7 of the endogenous CD47 gene. In certain embodiments, the exons downstream of exon 1 and exon 7 of the endogenous CD47 gene are at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% identical to the corresponding exons downstream of exon 1 and exon 7 of the mouse CD47 gene presented in Table 3. In some embodiments, the exons downstream of exon 1 and exon 7 of the endogenous CD47 gene are identical to the corresponding exons downstream of exon 1 and exon 7 of the mouse CD47 gene presented in Table 3.

[0011] In some embodiments, the human portion encodes amino acids 16-292 of a human CD47 polypeptide. In some embodiments, the human portion encodes amino acids 19-292 of a human CD47 polypeptide. In some embodiments, the human portion encodes amino acids 19-141 of a human CD47 polypeptide. In some embodiments, the human portion encodes amino acids 19-127 of a human CD47 polypeptide. In some embodiments, the human portion comprises exons 2-7 of a human CD47 gene.

[0012] In some embodiments, exons 2-7 of the human CD47 gene are at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% identical to the corresponding exons 2-7 of the human CD47 gene presented in Table 3. In some embodiments, exons 2-7 of the human CD47 gene are identical to the corresponding exons 2-7 of the human CD47 gene presented in Table 3.

[0013] In some embodiments, the non-human animal of the present invention comprises a CD47 polypeptide comprising an extracellular portion of a human CD47 polypeptide and an intracellular portion of an endogenous CD47 polypeptide. In some embodiments, the CD47 polypeptide comprises a transmembrane portion of a human CD47 polypeptide. In other embodiments, the CD47 polypeptide comprises a transmembrane portion of a non-human CD47 polypeptide. In some embodiments, the CD47 polypeptide is translated by a non-human signal peptide in the cells of the non-human animal. In certain embodiments, the non-human signal peptide is a rodent (e.g., mouse or rat) signal peptide.

[0014] In some embodiments, the CD47 polypeptides of the invention are expressed from an endogenous non-human CD47 gene.

[0015] In some embodiments, the intracellular portion of the endogenous CD47 polypeptide comprises an intracytoplasmic tail having an amino acid sequence that is at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% identical to the intracytoplasmic tail of a mouse CD47 polypeptide presented in Table 3. In some embodiments, the intracellular portion of the endogenous CD47 polypeptide comprises an intracytoplasmic tail having an amino acid sequence that is identical to the intracytoplasmic tail of a mouse CD47 polypeptide presented in Table 3.

[0016] In some embodiments, the extracellular portion of the human CD47 polypeptide comprises amino acids corresponding to residues 19-141 of the human CD47 polypeptide. In some embodiments, the extracellular portion of the human CD47 polypeptide comprises amino acids corresponding to residues 19-127 of the human CD47 polypeptide. In some embodiments, the extracellular portion of the human CD47 polypeptide comprises an amino acid sequence that is at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% identical to the corresponding amino acid sequence of the extracellular portion of the human CD47 polypeptide presented in Table 3. In some embodiments, the extracellular portion of the human CD47 polypeptide comprises an amino acid sequence that is identical to the corresponding amino acid sequence of the extracellular portion of the human CD47 polypeptide presented in Table 3.

[0017] In some embodiments, the present invention provides a CD47 polypeptide encoded by a CD47 gene of a non-human animal as described herein. In certain embodiments, the encoded CD47 polypeptide comprises an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% or at least 98% identity to SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19 or SEQ ID NO: 20. In certain embodiments, the encoded CD47 polypeptide comprises an amino acid sequence identical to SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19 or SEQ ID NO: 20.

[0018] In some embodiments, the present invention provides a humanized CD47 gene, comprising one or more exons of a non-human CD47 gene operably linked to one or more exons of a human CD47 gene. In certain embodiments, the humanized CD47 gene of the present invention comprises non-human exons encoding the intracellular portion of a CD47 polypeptide and human exons encoding the extracellular portion of a human CD47 polypeptide. In some embodiments, the humanized CD47 gene further comprises human exons encoding the transmembrane portion of a human CD47 polypeptide. In certain embodiments, the humanized CD47 gene of the present invention comprises non-human exons encoding all or part of the signal peptide and the intracellular portion of a CD47 polypeptide, and human exons encoding the extracellular portion of a CD47 polypeptide and an optional transmembrane portion.

[0019] In some embodiments, the present invention provides cells or tissues separated from non-human animals as described herein. In some embodiments, the present invention provides cells or tissues separated comprising CD47 genes as described herein. In some embodiments, cells are selected from dendritic cells, lymphocytes (e.g., B cells or T cells), macrophages and monocytes. In some embodiments, tissues are selected from fat, bladder, brain, breast, bone marrow, eyes, heart, intestines, kidneys, liver, lungs, lymph nodes, muscles, pancreas, plasma, serum, skin, spleen, stomach, thymus, testicles, eggs and combinations thereof.

[0020] In some embodiments, the present invention provides non-human embryonic stem cells, whose genome comprises the CD47 gene as described herein. In some embodiments, the non-human embryonic stem cells are mouse embryonic stem cells and are from 129 strains, C57BL / 6 strains or BALB / c strains. In some embodiments, the non-human embryonic stem cells are mouse embryonic stem cells and are from a mixture of 129 and C57BL / 6 strains.

[0021] In some embodiments, the present invention provides the use of non-human embryonic stem cells as described herein for preparing non-human animals. In certain embodiments, the non-human embryonic stem cells are mouse embryonic stem cells and are used to prepare mice comprising the CD47 gene as described herein. In certain embodiments, the non-human embryonic stem cells are rat embryonic stem cells and are used to prepare rats comprising the CD47 gene as described herein.

[0022] In some embodiments, the present invention provides non-human embryos comprising, prepared by, obtained by, or produced by non-human embryonic stem cells containing a CD47 gene as described herein. In certain embodiments, the non-human embryo is a rodent embryo. In some embodiments, the rodent embryo is a mouse embryo. In some embodiments, the rodent embryo is a rat embryo.

[0023] In some embodiments, the present invention provides a method for preparing a non-human animal expressing a CD47 polypeptide from an endogenous CD47 gene, wherein the CD47 polypeptide comprises a human sequence, the method comprising inserting a genomic fragment into an endogenous CD47 gene in a non-human embryonic stem cell, the genomic fragment comprising a nucleotide sequence encoding all or part of a human CD47 polypeptide; obtaining a non-human embryonic stem cell comprising an endogenous CD47 gene, wherein the endogenous CD47 gene comprises a nucleotide sequence encoding all or part of a human CD47 polypeptide; and forming a non-human animal using the non-human embryonic stem cell comprising the nucleotide sequence encoding all or part of a human CD47 polypeptide.

[0024] In some embodiments, the human sequence comprises amino acids corresponding to residues 19-141 (or 19-292) of a human CD47 polypeptide. In some embodiments, the human sequence comprises amino acids corresponding to residues 19-127 of a human CD47 polypeptide.

[0025] In some embodiments, the nucleotide sequence comprises exons 2-7 of the human CD47 gene. In some embodiments, the nucleotide sequence comprises one or more selection markers. In some embodiments, the nucleotide sequence comprises one or more site-specific recombination sites.

[0026] In some embodiments, the method further comprises the step of inserting a genomic fragment into an endogenous SIRPα gene of a non-human embryonic stem cell, the genomic fragment comprising a nucleotide sequence encoding all or part of a human SIRPα polypeptide (e.g., encoding the extracellular portion of a human SIRPα polypeptide). In certain embodiments, a genomic fragment comprising a nucleotide sequence encoding all or part of a human SIRPα polypeptide (e.g., encoding the extracellular portion of a human SIRPα polypeptide) is inserted into an endogenous SIRPα gene of a non-human embryonic stem cell prior to inserting the endogenous CD47 gene.

[0027] In some embodiments, the method further comprises mating a non-human animal comprising an endogenous CD47 gene comprising a nucleotide sequence encoding all or part of a human CD47 polypeptide with a second non-human animal having a genome comprising a SIRPα gene encoding a SIRPα polypeptide comprising an extracellular portion of a human SIRPα polypeptide (e.g., amino acids corresponding to residues 28-362 of a human SIRPα polypeptide) and an intracellular portion of an endogenous SIRPα polypeptide.

[0028] In some embodiments, the present invention provides a method of providing a non-human animal, the genome of which comprises a CD47 gene, the CD47 gene encoding an extracellular portion of a human CD47 polypeptide linked to an intracellular portion of an endogenous CD47 polypeptide, the method comprising modifying the genome of the non-human animal so that it comprises a CD47 gene encoding an extracellular portion of a human CD47 polypeptide linked to an intracellular portion of an endogenous CD47 polypeptide, thereby providing the non-human animal. In some embodiments, the CD47 gene encodes a CD47 polypeptide comprising an extracellular portion and a transmembrane portion of a human CD47 polypeptide linked to an intracellular portion of an endogenous non-human CD47 polypeptide. In other embodiments, the CD47 gene encodes a CD47 polypeptide comprising an extracellular portion of a human CD47 polypeptide linked to a transmembrane portion and an intracellular portion of an endogenous non-human CD47 polypeptide.

[0029] In some embodiments, modifying the genome of a non-human animal is performed in a non-human embryonic stem cell. In certain embodiments, the non-human embryonic stem cell is a rodent embryonic stem cell; in some embodiments, a mouse embryonic stem cell; in some embodiments, a rat embryonic stem cell.

[0030] In some embodiments, the method further comprises modifying the genome of a non-human animal so that it comprises a SIRPα gene, which encodes an extracellular portion of a human SIRPα polypeptide linked to an intracellular portion of an endogenous SIRPα polypeptide (e.g., amino acids corresponding to residues 28-362 of a human SIRPα polypeptide). In certain embodiments, the genome of a non-human animal is modified so that it comprises a SIRPα gene, which encodes an extracellular portion of a human SIRPα polypeptide linked to an intracellular portion of an endogenous SIRPα polypeptide (e.g., amino acids corresponding to residues 28-362 of a human SIRPα polypeptide), and the modification of the genome of the non-human animal is performed before modifying the genome of the non-human animal so that it comprises a CD47 gene (the CD47 gene encodes an extracellular portion of a human CD47 polypeptide linked to an intracellular portion of an endogenous CD47 polypeptide and an optional transmembrane portion).

[0031] In some embodiments, the method further comprises mating the non-human animal with a second non-human animal, wherein the genome of the non-human animal comprises a CD47 gene, wherein the CD47 gene encodes an extracellular portion of a human CD47 polypeptide linked to an intracellular portion of an endogenous CD47 polypeptide, and the second non-human animal has a genome comprising a SIRPα gene encoding a SIRPα polypeptide, wherein the SIRPα polypeptide comprises an extracellular portion of a human SIRPα polypeptide (e.g., amino acids corresponding to residues 28-362 of the human SIRPα polypeptide) and an intracellular portion of an endogenous SIRPα polypeptide.

[0032] In some embodiments, the invention provides a non-human animal obtainable by a method as described herein.

[0033] In some embodiments, the present invention provides a method for implanting human cells into a non-human animal, the method comprising the steps of providing a non-human animal, the genome of the non-human animal comprising a CD47 gene encoding an extracellular portion of a human CD47 polypeptide linked to an intracellular portion of an endogenous CD47 polypeptide; and transplanting one or more human cells into the non-human animal. In certain embodiments, the method further comprises the step of determining the implantation of one or more human cells in the non-human animal. In certain embodiments, the step of determining comprises comparing the implantation of the one or more human cells with implantation in one or more wild-type non-human animals or in one or more non-human animals, the genome of which does not comprise a CD47 gene encoding an extracellular portion of a human CD47 polypeptide linked to an intracellular portion of an endogenous CD47 polypeptide.

[0034] In some embodiments, the human cells are hematopoietic stem cells. In some embodiments, the human cells are transplanted intravenously. In some embodiments, the human cells are transplanted intraperitoneally. In some embodiments, the human cells are transplanted subcutaneously.

[0035] In some embodiments, the present invention provides a method for evaluating the therapeutic efficacy of a drug targeted to human cells, the method comprising providing a non-human animal whose genome comprises a CD47 gene encoding an extracellular portion of a human CD47 polypeptide linked to an intracellular portion of an endogenous CD47 polypeptide; transplanting one or more human cells into the non-human animal; administering a candidate drug to the non-human animal; and monitoring the human cells in the non-human animal to determine the therapeutic efficacy of the candidate drug.

[0036] In some embodiments, the human cell is a cancer cell and the drug candidate is an anti-cancer drug candidate. In certain embodiments, the drug candidate is an antibody.

[0037] In some embodiments, the non-human animal further comprises a human immune cell.In certain embodiments, the drug candidate is a bispecific antibody that binds to human CD47 and an antigen on a transplanted human cancer cell.

[0038] In some embodiments, the invention provides a method comprising providing one or more cells whose genome comprises a CD47 gene encoding an extracellular portion of a human CD47 polypeptide linked to an intracellular portion of an endogenous CD47 polypeptide; incubating the one or more cells with a labeled substrate; and measuring phagocytosis of the labeled substrate by the one or more cells. In some embodiments, the substrate is fluorescently labeled. In some embodiments, the substrate is labeled with an antibody. In some embodiments, the substrate is one or more red blood cells. In some embodiments, the substrate is one or more bacterial cells. In some embodiments, the substrate is one or more tumor cells.

[0039] In some embodiments, the present invention provides a method comprising providing a non-human animal whose genome comprises a CD47 gene encoding an extracellular portion of a human CD47 polypeptide linked to an intracellular portion of an endogenous CD47 polypeptide; exposing the non-human animal to an antigen; and measuring phagocytosis of the antigen by one or more cells of the non-human animal. In some embodiments, the step of exposing comprises exposing the non-human animal to a fluorescently labeled antigen. In some embodiments, the step of exposing comprises exposing the non-human animal to one or more cells comprising the antigen. In some embodiments, the step of exposing comprises exposing the non-human animal to one or more human cells comprising the antigen or to one or more bacterial cells comprising the antigen. In some embodiments, the step of exposing comprises exposing the non-human animal to one or more cells transformed with the antigen such that the antigen is expressed on the surface of the one or more transformed cells. In some embodiments, the step of exposing comprises exposing the non-human animal to one or more tumor cells comprising the antigen.

[0040] In some embodiments, the present invention provides a method for identifying or validating a drug or vaccine, the method comprising the steps of delivering the drug or vaccine to a non-human animal whose genome comprises a CD47 gene encoding an extracellular portion of a human CD47 polypeptide linked to an intracellular portion of an endogenous CD47 polypeptide, and monitoring one or more of an immune response to the drug or vaccine, a safety profile of the drug or vaccine, or an effect on a disease or condition. In some embodiments, monitoring safety profiles comprises determining whether the non-human animal exhibits side effects or adverse reactions as a result of the delivery of the drug or vaccine. In some embodiments, the side effects or adverse reactions are selected from morbidity, mortality, changes in body weight, changes in the levels of one or more enzymes (e.g., liver), changes in the weight of one or more organs, loss of function (e.g., sensation, movement, organs, etc.), increased susceptibility to one or more diseases, changes in the genome of the non-human animal, an increase or decrease in food consumption, and complications of one or more diseases.

[0041] In some embodiments, the invention provides for use of a non-human animal as described herein in developing a drug or vaccine for use in medicine, such as for use as a medicament.

[0042] In some embodiments, the present invention provides use of a non-human animal as described herein in the manufacture of a medicament for treating cancer or a tumor.

[0043] In some embodiments, the present invention provides the use of non-human animals as described herein for evaluating the efficacy of therapeutic drugs targeting human cells. In multiple embodiments, the non-human animals of the present invention are transplanted with human cells, and the candidate drugs targeting the human cells are administered to the animals. In some embodiments, the efficacy of the drug is determined by monitoring the human cells in the non-human animal after administration of the drug.

[0044] In some embodiments, the invention provides non-human animals or cells as described herein for use in developing and / or identifying drugs (eg, antibodies) for treatment or diagnosis.

[0045] In some embodiments, the present invention provides non-human animals or cells as described herein for use in developing and / or identifying drugs (eg, antibodies) for treating, preventing, or ameliorating cancer or tumors.

[0046] In some embodiments, the present invention provides a method for evaluating the pharmacokinetics of a drug targeting human CD47, the method comprising the steps of administering the drug to a non-human animal as described herein, and performing an assay to determine one or more pharmacokinetic properties of the drug targeting human CD47.

[0047] In some embodiments, the present invention provides a method for evaluating on-target toxicity of a drug targeting human CD47, the method comprising the steps of administering the drug to a non-human animal as described herein, and measuring one or more parameters associated with the on-target toxicity of the drug.

[0048] In some embodiments, the present invention provides a method for evaluating off-target toxicity of a drug targeting human CD47, the method comprising the steps of administering the drug to a non-human animal as described herein, and measuring one or more parameters associated with the off-target toxicity of the drug.

[0049] In various embodiments, the non-human animal as described herein is a rodent whose genome includes a CD47 gene encoding an extracellular portion of a human CD47 polypeptide linked to an intracellular portion of an endogenous CD47 polypeptide; in some embodiments, the rodent is a mouse; in some embodiments, the rodent is a rat.

[0050] In some embodiments, the drug targeting human CD47 is a CD47 antagonist. In certain embodiments, the CD47 antagonist is an anti-CD47 antibody. In some embodiments, the drug targeting human CD47 is a CD47 agonist.

[0051] In various embodiments, the CD47 gene of the invention comprises a CD47 gene as described herein. In various embodiments, the CD47 polypeptide of the invention comprises a CD47 polypeptide as described herein.

[0052] In various embodiments, the non-human animals of the invention do not detectably express full-length endogenous non-human CD47 polypeptides. In various embodiments, the non-human animals of the invention do not detectably express extracellular portions of endogenous CD47 polypeptides. In various embodiments, the non-human animals of the invention do not detectably express extracellular portions of both endogenous CD47 polypeptides and endogenous SIRPα polypeptides.

[0053] In various embodiments, the extracellular portion of the human CD47 polypeptide comprises amino acids corresponding to residues 19-141 of the human CD47 polypeptide as described herein.

[0054] In various embodiments, the N-terminal immunoglobulin V domain of the human CD47 polypeptide comprises amino acids corresponding to residues 19-127 of the human CD47 polypeptide as described herein.

[0055] In various embodiments, the non-human animals, cells, tissues, embryonic stem cells and / or embryos of the present invention have a genome that also includes a SIRPα gene encoding a SIRPα polypeptide, wherein the SIRPα polypeptide includes an extracellular portion of a human SIRPα polypeptide (e.g., amino acids corresponding to residues 28-362 of the human SIRPα polypeptide) and an intracellular portion of an endogenous SIRPα polypeptide.

[0056] In various embodiments, the non-human animals of the invention are rodents; in some embodiments, mice; in some embodiments, rats.

[0057] As used in this application, the terms "about" and "approximately" are used equivalently. Any numerals used in this application with or without about / approximately are intended to encompass any normal fluctuations understood by a person of ordinary skill in the relevant art.

[0058] Other features, purposes and advantages of the present invention are apparent in the following detailed description. However, it should be understood that the detailed description, although indicating embodiments of the present invention, is provided by way of example only, and is not restrictive. According to the detailed description, various changes and modifications within the scope of the present invention will become apparent to those skilled in the art. Sequence Listing <110> Razerne Pharmaceuticals <120> Non-human animals with humanized cluster of differentiation 47 gene <130> IIC205133 <140> <141> <150> 62 / 087,992 <151> 2014-12-05 <160> 36 <170> PatentIn Version 3.5 <210> 1 <211> 5591 <212> DNA <213> House mouse <400> 1 gcctacaccg ggagagcagg gaggaggagt tggactgagg ttgggcggct ccgaggtcca 60 gggcgagctt ggccagaggg agtagagagc agcggggctg cgcagggacg cgtgccgtga 120 gttccggtga gcgtgtgtgt cccatgctcc cgtctttcag gccggcccag gacacgaagc 180 cggaagagag ctgggctggag ggacggggc cgtgagcaga gagtgcaacc cgcgcagccc 240 cggggacagg ctgattcttg gcgctctccg ccggagcctg cccagggctg ggtgtgaggc 300 tggcgtcacg tcaacgagca gaggcggcca ggcggggcgg agtgcgcgtg cgcggggcgg 360 cgagcacgcg cgcgcgcgca cccccgggca gcctgggcgg ccgctcctgc ctgtcactgc 420 tgcggcgctg ctggtcggtc gtttcccttg aaggcagcag cggaggcggc ggctgctcca 480 gacacctgcg gcggcgaccc cccggcggcg cggagatgtg gcccttggcg gcggcgctgt 540 tgctgggctc ctgctgctgc ggttcagctc aactactgtt tagtaacgtc aactccatag 600 agttcacttc atgcaatgaa 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tgttttctga tcggaggcat gacaaagcaa gactgaaatc 2400 tgaactgtgt ctcctgcatg gcaacacgtg tctccgtcag gccctcgcaa ggcccgggga 2460 gggggttcta cgcctcttgt ctctttgttg catgctgaac actcatcgcc ttcctactgt 2520 atcctgcctc ctgcagcctc cctcttcctc ctcctcttcc tcttcctcct cttcctcctc 2580 ctcctcctct tcctccaagt ttgaaaggtc aaacaaaact accacattcc ctacccagtt 2640 agaagaaaac caccgtcctg acagttgtga tcgcatggag tacttttaga ttattagcac 2700 ctgtttttac ctcgtttgtg ggcgtgtttg tatgtgcaca tgtatgaagt cggcacatgc 2760 accttctgta tgggcagagg cgtggcatct acagaagagc agatgccaac tttgtgcttt 2820 tagtgaatac attaaaaaaa aaaaaccaac ggtccttatt gagtggaatt ctatttgatg 2880 caaatatttg agctctttaa gactttaaaa ctagataatg tgccaagctt ttaggactgc 2940 tcaccagtgc cctctgaaga aacaccagta ctttttcctg tttgtgtaat aaaggcatat 3000 ttgtatttgt gtttgcatca ctaatggtta tttcttctta gtccactgaa tgtttccatg 3060 tgcctctcgt atgccaaact ttttgtcatc tttcatgtgg ggaccaaatg gtttgtctgt 3120 ggcaaaccta aacctatgac ctgctgaggc ctctcagaaa actgaccaca gtaccaagat 3180 agtacttcgc aaagaaaagt aggttccctc cctggttttg tagctgtcgc caatattagc 3240 gtaattccaa ggagctgaac gcctttatat aaatctgatg gcacctgatg cttttagttc 3300 tgaaaatatt tacactcgga tcatgttgtt gatgacttaa acaaagtttt gatgaagaga 3360 gcaaaaaaaa agcaggtgga tttggaacag tttcagggtt ttttttgttt tttgtttttt 3420 gtttttgttt tttttttttt atttttgttt ttctgttctc tgttagaaaa gtcaggtgtt 3480 ctctgtcagg ctatctttat agtcaatttt ttttacgaac taaagtagta ccttttaata 3540 tgtagtcaac gcccctctgc tcggggttca gttttgggtc ttaaccagct gtcatgttct 3600 ctatgctgcc tgccacttga ggcactgagt gccctagaca gtcccatcgg tggtagccag 3660 ggaaacgaaa gacgaactca actcttgctc ctaataatca actctctgta tgaaggatgg 3720 cagcattaag agtcctcctg cctgggcatt attgggccag ttcaccctct ttaaatcaaa 3780 cccgcagtgg ctcccagttc tcgtcccatc agatttaaat tgctaacagt atggggggca 3840 ccacgcatct gttttgtccc acaatgcgct tttctctccc aaatcccgat ttctgctgtc 3900 atagcctcta ttcaattttt atttatgtc tgccctccac ttatacaatc gtagagagca 3960 atgccatttg tcactttctg caacagtttt ttgagccttt atggctgaat cccatttttc 4020 ttctctttca aactgtttgc tccattgctc ctcccgcacg gctgtccgta cagtcatccc 4080 atccatctgg gggcctcttt catctctcac ccttcctggt gcttcgtgga tctctgctta 4140 cctctgtggg tttttttttt tttttttgac ttatcttct cactggactt taagattact 4200 tccacagcga aagtgctgcc tcccttttct gcccgcagtg ttctgcgtac tttagatact 4260 actcagtgct gacatttgat ggcaaaagtt gcctgcactt aaatttctct ttttaatagg 4320 gtgaactaga gttggagttt ttttctcttt tttctctttt ctctctctct ctctctctct 4380 ctctctctct ctctctctct ctccctccct ccctccctcc ctccctccct ccctctctct 4440 ctctttttct ttctttcttt cttctttct ttctttcttt cttctttct ttctttcttt 4500 ttttgacaaa tctcacaggc ttgagaatt ataaaaggtg acagttcacc tgaaaatcac 4560 aggtctggtc tgtttaaatt gttgagaaat atccgattaa aagtcttgtg gctgtgtcct 4620 aataggctct ctttcaggac gttgtagtca atagagtggc tgaaccatac ttgagttat 4680 aaagctcaaa aactgatgca cccactctgc tattatcgtg ttagtaagag ttcagctgta 4740 tatcattgtc taggtttatc ttgtcctaca gtgggtattc aaatatggcc accagaggat 4800 atgtgtaaat ataagcacct gtatttgcct gttgttgaga actggaggga aaacaaaaaa 4860 tgtctggcaa ccctttgcct ttttaaccgt attaattga cagtttattt agagataaga 4920 gttttcaaaa atctcttaac tgccacaacc cacagagggt cttgttttgc catcttcagt 4980 ggctcacaga tatgatccaa gttaacttga aagagatgag cagtacccag gaaattgtcc 5040 tgcctttaac tctggctgtc cttaattatg actgtttaat gctgaatttt ccatccgtct 5100 agtgtttgag ggtaaagaaa agcctttttt aaataagtat ttctgtaaaa cggcatcggt 5160 gggatcttct gtgttgctat cacgggtgaa agagggaaac atttcttatt tttattaagc 5220 agagcattat ttacagaaag ccattgttga gaattagttc ccacatcata taaatatcca 5280 ttaaccattc taaattgtaa gagaactcca gtgttgctat gcacaaggaa ctctcctggg 5340 ggcctttttt tgcatagcaa ttaaaggtat gctatttgtc agtagccatt ttttgcagtg 5400 atttaaagac caaagttgtt ttacagctgt gttaccctta aaggtttttt ttttatgtat 5460 taaatcaatt tatcactgtt tgaagctttg aatacctgca atctttgcca agatactttt 5520 ttatttaaaa aaataactgt gtaaatatta ccctgtaata ttatatatac ttaataaaac 5580 attttaagct a 5591 <210> 2 <211> 312 <212> PRT <213> Mus musculus <400> 2 Met Trp Pro Leu Ala Ala Ala Leu Leu Leu Gly Ser Cys Cys Cys Gly 1 5 10 15 Ser Ala Gln Leu Leu Phe Ser Asn Val Asn Ser Ile Glu Phe Thr Ser 20 25 30 Cys Asn Glu Thr Val Val Ile Pro Cys Ile Val Arg Asn Val Glu Ala 35 40 45 Gln Ser Thr Glu Glu Met Phe Val Lys Trp Lys Leu Asn Lys Ser Tyr 50 55 60 Ile Phe Ile Tyr Asp Gly Asn Lys Asn Ser Thr Thr Thr Asp Gln Asn 65 70 75 80 Phe Thr Ser Ala Lys Ile Ser Val Ser Asp Leu Ile Asn Gly Ile Ala 85 90 95 Ser Leu Lys Met Asp Lys Arg Asp Ala Met Val Gly Asn Tyr Thr Cys 100 105 110 Glu Val Thr Glu Leu Ser Arg Glu Gly Lys Thr Val Ile Glu Leu Lys 115 120 125 Asn Arg Thr Ala Phe Asn Thr Asp Gln Gly Ser Ala Cys Ser Tyr Glu 130 135 140 Glu Glu Lys Gly Gly Cys Lys Leu Val Ser Trp Phe Ser Pro Asn Glu 145 150 155 160 Lys Ile Leu Ile Val Ile Phe Pro Ile Leu Ala Ile Leu Leu Phe Trp 165 170 175 Gly Lys Phe Gly Ile Leu Thr Leu Lys Tyr Lys Ser Ser His Thr Asn 180 185 190 Lys Arg Ile Ile Leu Leu Leu Val Ala Gly Leu Val Leu Thr Val Ile 195 200 205 Val Val Val Gly Ala Ile Leu Leu Ile Pro Gly Glu Lys Pro Val Lys 210 215 220 Asn Ala Ser Gly Leu Gly Leu Ile Val Ile Ser Thr Gly Ile Leu Ile 225 230 235 240 Leu Leu Gln Tyr Asn Val Phe Met Thr Ala Phe Gly Met Thr Ser Phe 245 250 255 Thr Ile Ala Ile Leu Ile Thr Gln Val Leu Gly Tyr Val Leu Ala Leu 260 265 270 Val Gly Leu Cys Leu Cys Ile Met Ala Cys Glu Pro Val His Gly Pro 275 280 285 Leu Leu Ile Ser Gly Leu Gly Ile Ile Ala Leu Ala Glu Leu Leu Gly 290 295 300 Leu Val Tyr Met Lys Phe Val Glu 305 310 <210> 3 <211> 5560 <212> DNA <213> Mus musculus <400> 3 gcctacaccg ggagagcagg gaggaggagt tggactgagg ttgggcggct ccgaggtcca 60 gggcgagctt ggccagaggg agtagagagc agcggggctg cgcagggacg cgtgccgtga 120 gttccggtga gcgtgtgtgt cccatgctcc cgtctttcag gccggcccag gacacgaagc 180 cggaagagag ctgggctggag ggacggggc cgtgagcaga gagtgcaacc cgcgcagccc 240 cggggacagg ctgattcttg gcgctctccg ccggagcctg cccagggctg ggtgtgaggc 300 tggcgtcacg tcaacgagca gaggcggcca ggcggggcgg agtgcgcgtg cgcggggcgg 360 cgagcacgcg cgcgcgcgca cccccgggca gcctgggcgg ccgctcctgc ctgtcactgc 420 tgcggcgctg ctggtcggtc gtttcccttg aaggcagcag cggaggcggc ggctgctcca 480 gacacctgcg gcggcgaccc cccggcggcg cggagatgtg gcccttggcg gcggcgctgt 540 tgctgggctc ctgctgctgc ggttcagctc aactactgtt tagtaacgtc aactccatag 600 agttcacttc atgcaatgaa actgtggtca tcccttgcat cgtccgtaat gtggaggcgc 660 720 atggaataa aaatagcact actacagatc aaaactttac cagtgcaaaa atctcagtct 780 cagacttaat caatggcatt gcctctttga aaatggataa gcgcgatgcc atggtgggaa 840 actacacttg cgaagtgaca gagttatcca gagaaggcaaacagttata gagctgaaaa 900 accgcacggt ttcgtggttt tctccaatg aaagatcct cattgttatt ttcccaattt 960 tggctatact cctgttctgg ggaagtttg gtattttaac acccaaatat aaatccagcc 1020 atacgaataa gagaatcatt ctgctgctcg tgccgggct ggtgctcaca gtcatcgtgg 1080 ttgttggagc catccttctc atcccaggag aaagccccgt gaagaatgct tctggacttg 1140 gcctcattgt aatctctacg gggatattaa tactactca gtacaatgtg ttatgacag 1200 cttttggaat gaccctttc accattgcca tattgatcac tcaagtgctg ggctacgtcc 1260 ttgctttgt cgggctgtgt ctctgcatca tggcatgtga gccagtgcac ggcccctt 1320 tgatttcagg ttgggatc tagctctag cagaactact tggattt tatgagt 1380 ttgtcgcttc caccagagg actatccac ctcctaggaa taggtgaagg gaagtgacgg 1440 actgtaactt ggtcacga aatggaaa tacagttgtc tagcaccag gtcttcacga 1500 ctcacagctg gaaggaacag aacagtaa ctgacttcca tccaggaaaa catgtcacat 1560 aaatgattac taagtttata ttcaaagcag ctgtacttta cataataaaa aaaatatgat 1620 gtgctgtgta accaattgga atcccatttt tctattgttt ctactcaact aggggcaaac 1680 gtttcagggg caacttccaa gaatgatgct tgttagatcc tagagtctct gaacactgag 1740 tttaaattga ttccgagtga gactcgccaa gcactaacct gagggttagt tacccagaga 1800 tacctatgaa aaacagtggt atccagcaag ccttagtaaa ctcaggttgc cagcagcttt 1860 gccacttccg ctgctagctg aataacaaga ctgccacttc tgggtcatag tgatagac 1920 tgaagtagaa aaacgaatgt ggttgggcaa atcccgtgtg gcccctctgt gtgctatgat 1980 attgatggca ctggtgtctt cattcttggg ggttgccatc attcacacac acccctttga 2040 catacagtgc accccagttt tgaatacatt ttttttgcac cctgtcccgt tctgctactt 2100 tgattgcgt tatgatatat atatatatat ataatacctt ttctcctctt taaacatggt 2160 cctgtgacac aatagtcagt tgcagaaagg agccagactt attcgcaaag cactgtgctc 2220 aaactcttca gaaaaaaagg aaaaaaaaa aaagctatag ttgtaacata tgtattccag 2280 acctctggtt taaaggcaaa agaaaaaaaa tctacagtgt ttcttctcat gttttctgat 2340 cggaggcatg acaaagcaag actgaaatct gaactgtgtc tcctgcatgg caacacgtgt 2400 ctccgtcagg ccctcgcaag gccggggag ggggttctac gcctcttgtc tctttgttgc 2460 atgctgaaca ctcatcgcct tcctactgta tcctgcctc tgcagcctcc ctcttcctcc 2520 tctcttcct cttcctcctc tcctcctct cctccaagtt tgaaaggtca 2580 aacaaaacta ccacattccc tacccagtta gaagaaaacc accgtcctga cagttgtgat 2640 cgcatggagt acttttagat tattagcacc tgtttttacc tcgtttgtgg gcgtgtttgt 2700 atgtgcacat gtatgaagtc ggcacatgca ccttctgtat gggcagaggc gtggcatcta 2760 cagaagagca gatgccaact ttgtgctttt agtgaataca ttaaaaaaaa aaaaccaacg 2820 gtccttattg agtggaattc tatttgatgc aaatatttga gctctttaag actttaaaac 2880 tagataatgt gccaagcttt taggactgct caccagtgcc ctctgaagaa acaccagtac 2940 ttttcctgt ttgtgtaata aaggcatatt tgtatttgtg tttgcatcac taatggttat 3000 ttcttcttag tccactgaat gtttccatgt gcctctcgta tgccaaactt tttgtcatct 3060 ttcatgtggg gaccaaatgg tttgtctgtg gcaaacctaa acctatgacc tgctgaggcc 3120 tctcagaaaa ctgaccacag taccaagata gtacttcgca aagaaaagta ggttccctcc 3180 ctggttttgt agctgtcgcc aattatgcg taattccaag gagctgaacg cctttatata 3240 aatctgatgg cacctgatgc ttttagttct gaaaatatt acactcggat catgttgttg 3300 atgacttaaa caaagttttg atgaagagag caaaaaaaaa gcaggtggat ttggaacagt 3360 ttcagggttt ttttgtttt ttgttttttg ttttgtttt tttttttta ttttgtttt 3420 tctgttctct gttagaaaag tcaggtgttc tctgtcaggc tatctttata gtcaattttt 3480 tttacgaact aaagtagtac cttttaatat gtagtcaacg cccctctgct cggggttcag 3540 ttttgggtct taaccagctg tcatgttctc tatgctgcct gccacttgag gcactgagtg 3600 ccctagacag tcccatcggt ggtagccagg gaaacgaaag acgaactcaa ctcttgctcc 3660 tataatcaa ctctctgtat gaaggatggc agcattaaga gtcctcctgc ctgggcatta 3720 ttgggccagt tcaccctctt taaatcaaac ccgcagtggc tcccagttct cgtcccatca 3780 gatttaaatt gctaacagta tggggggcac cacgcatctg ttttgtccca caatgcgctt 3840 ttctctccca aatcccgatt tctgctgtca tagcctctat tcaattttta tttattgtct 3900 gccctccact tatacaatcg tagagagcaa tgccatttgt cactttctgc aacagttttt 3960 tgagccttta tggctgaatc ccatttttct tctctttcaa actgtttgct ccattgctcc 4020 tcccgcacgg ctgtccgtac agtcatccca tccatctggg ggcctctttc atctctcacc 4080 cttcctggtg cttcgtggat ctctgcttac ctctgtgggt tttttttttt ttttttgact 4140 tattcttctc actggacttt aagattactt ccacagcgaa agtgctgcct cccttttctg 4200 cccgcagtgt tctgcgtact ttagatacta ctcagtgctg acatttgatg gcaaaagttg 4260 cctgcactta aatttctctt tttaataggg tgaactagag ttggagtttt tttctctttt 4320 ttctcttttc tctctctctc tctctctctc tctctctctc tctctctctc tccctccctc 4380 cctccctccc tccctccctc cctctctctc tctttttctt tctttctttc tttctttctt 4440 tctttctttc tttctttctt tctttctttt tttgacaaat ctcacaggct ttgagaatta 4500 taaaaggtga cagttcacct gaaaatcaca ggtctggtct gtttaaattg ttgagaaata 4560 tccgattaaa agtcttgtgg ctgtgtccta ataggctctc tttcaggacg ttgtagtcaa 4620 tagagtggct gaaccatact tgagtttata aagctcaaaa actgatgcac ccactctgct 4680 attatcgtgt tagtaagagt tcagctgtat atcattgtct aggtttatct tgtcctacag 4740 tgggtattca aatatggcca ccagaggata tgtgtaaata taagcacctg tatttgcctg 4800 ttgttgagaa ctggagggaa aacaaaaaat gtctggcaac cctttgcctt tttaaccgta 4860 attaattgac agtttattta gagataagag ttttcaaaaa tctcttaact gccacaaccc 4920 acagagggtc ttgttttgcc atcttcagtg gctcacagat atgatccaag ttaacttgaa 4980 agagatgagc agtacccagg aaattgtcct gcctttaact ctggctgtcc ttaattatga 5040 ctgtttaatg ctgaattttc catccgtcta gtgtttgagg gtaaagaaaa gcctttttta 5100 aataagtatt tctgtaaaac ggcatcggtg ggatcttctg tgttgctatc acgggtgaaa 5160 gagggaaaca tttcttattt ttattaagca gagcattatt tacagaaagc cattgttgag 5220 aattagttcc cacatcatat aaatatccat taaccattct aaattgtaag agaactccag 5280 tgttgctatg cacaaggaac tctcctgggg gccttttttt gcatagcaat taaaggtatg 5340 ctatttgtca gtagccattt tttgcagtga tttaaagacc aaagttgttt tacagctgtg 5400 ttacccttaa aggttttttt tttatgtatt aaatcaattt atcactgttt gaagctttga 5460 atacctgcaa tctttgccaa gatacttttt tatttaaaaa aataactgtg taaatattac 5520 cctgtaatat tatatatact taataaaaca ttttaagcta 5560 <210> 4 <211> 303 <212> PRT <213> Mus musculus <400> 4 Met Trp Pro Leu Ala Ala Ala Leu Leu Leu Gly Ser Cys Cys Cys Gly 1 5 10 15 Ser Ala Gln Leu Leu Phe Ser Asn Val Asn Ser Ile Glu Phe Thr Ser 20 25 30 Cys Asn Glu Thr Val Val Ile Pro Cys Ile Val Arg Asn Val Glu Ala 35 40 45 Gln Ser Thr Glu Glu Met Phe Val Lys Trp Lys Leu Asn Lys Ser Tyr 50 55 60 Ile Phe Ile Tyr Asp Gly Asn Lys Asn Ser Thr Thr Thr Asp Gln Asn 65 70 75 80 Phe Thr Ser Ala Lys Ile Ser Val Ser Asp Leu Ile Asn Gly Ile Ala 85 90 95 Ser Leu Lys Met Asp Lys Arg Asp Ala Met Val Gly Asn Tyr Thr Cys 100 105 110 Glu Val Thr Glu Leu Ser Arg Glu Gly Lys Thr Val Ile Glu Leu Lys 115 120 125 Asn Arg Thr Val Ser Trp Phe Ser Pro Asn Glu Lys Ile Leu Ile Val 130 135 140 Ile Phe Pro Ile Leu Ala Ile Leu Leu Phe Trp Gly Lys Phe Gly Ile 145 150 155 160 Leu Thr Leu Lys Tyr Lys Ser Ser His Thr Asn Lys Arg Ile Ile Leu 165 170 175 Leu Leu Val Ala Gly Leu Val Leu Thr Val Ile Val Val Val Gly Ala 180 185 190 Ile Leu Leu Ile Pro Gly Glu Lys Pro Val Lys Asn Ala Ser Gly Leu 195 200 205 Gly Leu Ile Val Ile Ser Thr Gly Ile Leu Ile Leu Leu Gln Tyr Asn 210 215 220 Val Phe Met Thr Ala Phe Gly Met Thr Ser Phe Thr Ile Ala Ile Leu 225 230 235 240 Ile Thr Gln Val Leu Gly Tyr Val Leu Ala Leu Val Gly Leu Cys Leu 245 250 255 Cys Ile Met Ala Cys Glu Pro Val His Gly Pro Leu Leu Ile Ser Gly 260 265 270 Leu Gly Ile Ile Ala Leu Ala Glu Leu Leu Gly Leu Val Tyr Met Lys 275 280 285 Phe Val Ala Ser Asn Gln Arg Thr Ile Gln Pro Pro Arg Asn Arg 290 295 300 <210> 5 <211> 5648 <212> DNA <213> Mus musculus <400> 5 gcctacaccg ggagagcagg gaggaggagt tggactgagg ttgggcggct ccgaggtcca 60 gggcgagctt ggccagaggg agtagagagc agcggggctg cgcagggacg cgtgccgtga 120 gttccggtga gcgtgtgtgt cccatgctcc cgtctttcag gccggcccag gacacgaagc 180 cggaagagag ctgggctggag ggacggggc cgtgagcaga gagtgcaacc cgcgcagccc 240 cggggacagg ctgattcttg gcgctctccg ccggagcctg cccagggctg ggtgtgaggc 300 tggcgtcacg tcaacgagca gaggcggcca ggcggggcgg agtgcgcgtg cgcggggcgg 360 cgagcacgcg cgcgcgcgca cccccgggca gcctgggcgg ccgctcctgc ctgtcactgc 420 tgcggcgctg ctggtcggtc gtttcccttg aaggcagcag cggaggcggc ggctgctcca 480 gacacctgcg gcggcgaccc cccggcggcg cggagatgtg gcccttggcg gcggcgctgt 540 tgctgggctc ctgctgctgc ggttcagctc aactactgtt tagtaacgtc aactccatag 600 agttcacttc atgcaatgaa actgtggtca tcccttgcat cgtccgtaat gtggaggcgc 660 720 atggaataa aaatagcact actacagatc aaaactttac cagtgcaaaa atctcagtct 780 cagacttaat caatggcatt gcctctttga aaatggataa gcgcgatgcc atggtgggaa 840 actacacttg cgaagtgaca gagttatcca gaggaggcaa aacagttata gagctgaaaa 900 accgcacggc cttcaacact gaccaaggat cagcctgttc ttacgaggag gagaaaggag 960 gttgcaaatt agtttcgtgg ttttctccaa atgaaaagat cctcattgtt attttcccaa 1020 ttttggctat actcctgttc tggggaaagt ttggtatttt aacactcaaa tataaatcca 1080 gccatacgaa taagagaatc attctgctgc tcgttgccgg gctggtgctc acagtcatcg 1140 tggttgttgg agccatcctt ctcatcccag gagaaaagcc cgtgaagaat gcttctggac 1200 ttggcctcat tgtaatctct acggggatat taatactact tcagtacaat gtgtttatga 1260 cagcttttgg aatgacctct ttcaccattg ccatattgat cactcaagtg ctgggctacg 1320 tccttgcttt ggtcgggctg tgtctctgca tcatggcatg tgagccagtg cacggccccc 1380 ttttgatttc aggtttgggg atcatagctc tagcagaact acttggatta gtttatatga 1440 agtttgtcgc ttccaaccag aggactatcc aacctcctag gaaagctgta gaggaacccc 1500 ttaacgaata ggtgaaggga agtgacggac tgtaacttgg aagtcagaaa tggaagaata 1560 cagttgtcta agcaccaggt cttcacgact cacagctgga aggaacagac aacagtaact 1620 gacttccatc caggaaaaca tgtcacataa atgattacta agtttatatt caaagcagct 1680 gtactttaca tataaaaaa aatatgatgt gctgtgtaac caattggaat cccatttttc 1740 tattgtttct actcaactag gggcaaacgt ttcaggggca acttccaaga atgatgcttg 1800 ttagatccta gagtctctga acactgagtt taaattgatt ccgagtgaga ctcgccaagc 1860 actaacctga gggttagtta cccagagata cctatgaaaa acagtggtat ccagcaagcc 1920 ttagtaaact caggttgcca gcagctttgc cacttccgct gctagctgaa taacaagact 1980 gccacttctg ggtcatagtg atagagactg aagtagaaaa acgaatgtgg ttgggcaaat 2040 cccgtgtggc ccctctgtgt gctatgatat tgatggcact ggtgtcttca ttcttggggg 2100 ttgccatcat tcacacacac ccctttgaca tacagtgcac cccagttttg aatacattttt 2160 ttttgcaccc tgtcccgttc tgctactttg atttgcgtta tgatatatat atatatatat 2220 aatacctttt ctcctcttta aacatggtcc tgtgacacaa tagtcagttg cagaaaggag 2280 ccagacttat tcgcaaagca ctgtgctcaa actcttcaga aaaaaaggaa aaaaaaaaa 2340 agctatagtt gtaacatatg tattccagac ctctggttta aaggcaaaag aaaaaaaatc 2400 tacagtgttt cttctcatgt tttctgatcg gaggcatgac aaagcaagac tgaaatctga 2460 actgtgtctc ctgcatggca acacgtgtct ccgtcaggcc ctcgcaaggc ccggggaggg 2520 ggttctacgc ctcttgtctc tttgttgcat gctgaacact catcgccttc ctactgtatc 2580 ctgcctcctg cagcctccct cttcctcctc ctcttcctct tcctcctctt cctcctcctc 2640 ctcctcttcc tccaagtttg aaaggtcaaa caaaactacc acattcccta cccagttaga 2700 agaaaaccac cgtcctgaca gttgtgatcg catggagtac ttttagatta ttagcacctg 2760 tttttacctc gtttgtgggc gtgtttgtat gtgcacatgt atgaagtcgg cacatgcacc 2820 ttctgtatgg gcagaggcgt ggcatctaca gaagagcaga tgccaacttt gtgcttttag 2880 tgaatacatt aaaaaaaaaa aaccaacggt ccttattgag tggaattcta tttgatgcaa 2940 atatttgagc tctttaagac tttaaaacta gataatgtgc caagctttta ggactgctca 3000 ccagtgccct ctgaagaaac accagtactt tttcctgttt gtgtaataaa ggcatatttg 3060 3120. tatttgtgtt tgcatcacta atggttattt cttcttagtc cactgaatgt ttccatgtgc ctctcgtatg ccaaactttt tgtcatcttt catgtgggga ccaaatggtt tgtctgtggc 3180. aaacctaaac ctatgacctg ctgaggcctc tcagaaaact gaccacagta ccaagatagt acttcgcaaa gaaaagtagg ttccctccct ggttttgtag ctgtcgccaa tattagcgta attccaagga gctgaacgcc tttatataaa tctgatggca cctgatgctt ttagttctga aaatatttac actcggatca tgttgttgat gacttaaaca aagttttgat gaagagagca aaaaaaaagc aggtggattt ggaacagtttt cagggtttttt tttgtttttt gttttttgtt 3480 tttgtttttt tttttttatt tttgtttttc tgttctctgt tagaaagtc aggtgttctc 3540 tgtcaggcta tctttatagt caattttttt tacgaactaa agtagtacct tttaatatgt agtcaacgcc cctctgctcg gggttcagtt ttgggtctta accagctgtc atgttctcta 3660 tgctgcctgc cacttgaggc actgagtgcc ctagacagtc ccatcggtgg tagccaggga 3720 aacgaaagac gaactcaact cttgctccta father ctctgtatga aggatggcag cattaagagt cctcctgcct gggcattatt gggccagttc accctcttta aatcaaaccc 3840 gcagtggctc ccagttctcg tcccatcaga tttaaattgc taacagtatg gggggcacca 3900 cgcatctgtt ttgtcccaca atgcgctttt ctctcccaaa tcccgatttc tgctgtcata 3960 gcctctattc aatttttatt tattgtctgc cctccactta tacaatcgta gagagcaatg 4020 ccatttgtca ctttctgcaa cagttttttg agcctttatg gctgaatccc atttttcttc 4080 tctttcaaac tgtttgctcc attgctcctc ccgcacggct gtccgtacag tcatcccatc 4140 catctggggg cctctttcat ctctcaccct tcctggtgct tcgtggatct ctgcttacct 4200 ctgtgggttt tttttttttt ttttgactta ttcttctcac tggactttaa gattacttcc 4260 acagcgaaag tgctgcctcc cttttctgcc cgcagtgttc tgcgtacttt agatactact 4320 cagtgctgac atttgatggc aaaagttgcc tgcacttaaa tttctctttt taatagggtg 4380 aactagagtt ggagtttttt tctctttttt ctcttttctc tctctctctc tctctctctc 4440 tctctctctc tctctctctc cctccctccc tccctccctc cctccctccc tctctctctc 4500 ttttctttc ttctttcttt tctttctttc ttctttcttt tctttctttc ttcttttttt 4560 tgacaaatct cacaggcttt gagaattata aaaggtgaca gttcacctga aaatcacagg 4620 tctggtctgt ttaaattgtt gagaaatatc cgattaaaag tcttgtggct gtgtcctaat 4680 aggctctct tcaggacgtt gtagtcaata gagtggctga accatacttg agtttataaa 4740 gctcaaaaac tgatgcaccc actctgctat tatcgtgtta gtaagagttc agctgtatat 4800 cattgtctag gtttatcttg tcctacagtg ggtattcaaa tatggccacc agaggatatg 4860 tgtaaatata agcacctgta tttgcctgtt gttgagaact ggagggaaaa caaaaaatgt 4920 ctggcaaccc tttgcctttt taaccgtaat taattgacag tttatttaga gataagagtt 4980 ttcaaaaatc tcttaactgc cacaacccac agagggtctt gttttgccat cttcagtggc 5040 tcacagatat gatccaagtt aacttgaaag agatgagcag tacccaggaa attgtcctgc 5100 ctttaactct ggctgtcctt aattatgact gtttaatgct gaattttcca tccgtctagt 5160 gtttgaggt aaagaaaagc cttttttaaa taagtatttc tgtaaaacgg catcggtggg 5220 atcttctgtg ttgctatcac gggtgaaaga gggaaacatt tcttattttt attaagcaga 5280 gcattattta cagaaagcca ttgttgagaa ttagttccca catcatataa atatccatta 5340 accattctaa attgtaagag aactccagtg ttgctatgca caaggaactc tcctgggggc 5400 ctttttttgc atagcaatta aaggtatgct atttgtcagt agccattttt tgcagtgatt 5460 taaagaccaa agttgtttta cagctgtgtt acccttaaag gttttttttt tatgtattaa 5520 atcaatttat cactgtttga agctttgaat acctgcaatc tttgccaaga tactttttta 5580 tttaaaaaaa taactgtgta aatattaccc tgtaatatta tatatactta ataaaacatt 5640 ttaagcta 5648 <210> 6 <211> 331 <212> PRT <213> Mus musculus <400> 6 Met Trp Pro Leu Ala Ala Ala Leu Leu Leu Gly Ser Cys Cys Cys Gly 1 5 10 15 Ser Ala Gln Leu Leu Phe Ser Asn Val Asn Ser Ile Glu Phe Thr Ser 20 25 30 Cys Asn Glu Thr Val Val Ile Pro Cys Ile Val Arg Asn Val Glu Ala 35 40 45 Gln Ser Thr Glu Glu Met Phe Val Lys Trp Lys Leu Asn Lys Ser Tyr 50 55 60 Ile Phe Ile Tyr Asp Gly Asn Lys Asn Ser Thr Thr Thr Asp Gln Asn 65 70 75 80 Phe Thr Ser Ala Lys Ile Ser Val Ser Asp Leu Ile Asn Gly Ile Ala 85 90 95 Ser Leu Lys Met Asp Lys Arg Asp Ala Met Val Gly Asn Tyr Thr Cys 100 105 110 Glu Val Thr Glu Leu Ser Arg Glu Gly Lys Thr Val Ile Glu Leu Lys 115 120 125 Asn Arg Thr Ala Phe Asn Thr Asp Gln Gly Ser Ala Cys Ser Tyr Glu 130 135 140 Glu Glu Lys Gly Gly Cys Lys Leu Val Ser Trp Phe Ser Pro Asn Glu 145 150 155 160 Lys Ile Leu Ile Val Ile Phe Pro Ile Leu Ala Ile Leu Leu Phe Trp 165 170 175 Gly Lys Phe Gly Ile Leu Thr Leu Lys Tyr Lys Ser Ser His Thr Asn 180 185 190 Lys Arg Ile Ile Leu Leu Leu Val Ala Gly Leu Val Leu Thr Val Ile 195 200 205 Val Val Val Gly Ala Ile Leu Leu Ile Pro Gly Glu Lys Pro Val Lys 210 215 220 Asn Ala Ser Gly Leu Gly Leu Ile Val Ile Ser Thr Gly Ile Leu Ile 225 230 235 240 Leu Leu Gln Tyr Asn Val Phe Met Thr Ala Phe Gly Met Thr Ser Phe 245 250 255 Thr Ile Ala Ile Leu Ile Thr Gln Val Leu Gly Tyr Val Leu Ala Leu 260 265 270 Val Gly Leu Cys Leu Cys Ile Met Ala Cys Glu Pro Val His Gly Pro 275 280 285 Leu Leu Ile Ser Gly Leu Gly Ile Ile Ala Leu Ala Glu Leu Leu Gly 290 295 300 Leu Val Tyr Met Lys Phe Val Ala Ser Asn Gln Arg Thr Ile Gln Pro 305 310 315 320 Pro Arg Lys Ala Val Glu Glu Pro Leu Asn Glu 325 330 <210> 7 <211> 5618 <212> DNA <213> Mus musculus <400> 7 gcctacaccg ggagagcagg gaggaggagt tggactgagg ttgggcggct ccgaggtcca 60 gggcgagctt ggccagaggg agtaggagc agcggggctg cgcagggacg cgtgccgtga 120 gttccggtga gcgtgtgtgt cccatgctcc cgtctttcag gccggcccag gacacgaagc 180 cggaagagag ctggctggag ggacggggggc cgtgagcaga gagtgcaacc cgcgcagccc 240 cggggacagg ctgattcttg gcgctctccg ccggagcctg cccagggctg ggtgtgaggc 300 tggcgtcacg tcaacgagca gaggcggcca ggcggggcgg agtgcgcgtg cgcggggcgg 360 cgagcacgcg cgcgcgcgca cccccgggca gcctgggcgg ccgctcctgc ctgtcactgc 420 tgcggcgctg ctggtcggtc gtttcccttg aaggcagcag cggaggcggc ggctgctcca 480 gacacctgcg gcggcgaccc cccggcggcg cggagatgtg gcccttggcg gcggcgctgt 540 tgctgggctc ctgctgctgc ggttcagctc aactactgtt tagtaacgtc aactccatag agttcacttc atgcaatgaa actgtggtca tcccttgcat cgtccgtaat gtggaggcgc 660 aaagcaccga agaaatgttt gtgaagtgga agttgaacaa atcgtatatt ttcatctatg atggaaata aaatagcact actacagatc aaaactttac cagtgcaaaa atctcagtct cagacttaat caatggcatt gcctctttga aaatggataa gcgcgatgcc atggtgggaa 840 actacacttg cgaagtgaca gagttatcca gagaaggcaaacagttata gagctgaaaa 900 accgcacggt ttcgtggttt tctccaatg aaagatcct cattgttatt ttcccaattt 960 tggctatact cctgttctgg ggaagtttg gtattttaac acccaaatat aaatccagcc 1020 atacgaataa gagaatcatt ctgctgctcg tgccgggct ggtgctcaca gtcatcgtgg 1080 ttgttggagc catccttctc atcccaggag aaagccccgt gaagaatgct tctggacttg 1140 gcctcattgt aatctctacg gggatattaa tactactca gtacaatgtg ttatgacag 1200 cttttggaat gaccctttc accattgcca tattgatcac tcaagtgctg ggctacgtcc 1260 ttgctttgt cgggctgtgt ctctgcatca tggcatgtga gccagtgcac ggcccctt 1320 tgatttcagg ttgggatc tagctctag cagaactact tggattt tatgagt 1380 ttgtcgcttc caccagagg actatccaac ctcctaggaa agctgtagag gaacccctta 1440 acgcatttaa agtcaaaa ggcattga atgacgaat ggtgaagga agtgacggac 1500 tgtaacttgg aagtcagaaa tggaagaata cagttgcta agcaccaggt cttcacgact 1560 cacagctgga aggaacagac aacagtaact gacttccatc caggaaaaca tgtcacataa 1620 atgattacta agtttatatt caaagcagct gtactttaca tataaaaaa aatatgatgt 1680 gctgtgtaac caattggaat cccatttttc tattgtttct actcaactag gggcaaacgt 1740 ttcaggggca acttccaaga atgatgcttg ttagatccta gagtctctga acactgagtt 1800 taaattgatt ccgagtgaga ctcgccaagc actaacctga gggttagtta cccagagata 1860 cctatgaaaa acagtggtat ccagcaagcc ttagtaaact caggttgcca gcagctttgc 1920 cacttccgct gctagctgaa taacaagact gccacttctg ggtcatagtg atagagactg 1980 aagtagaaaa acgaatgtgg ttgggcaaat cccgtgtggc ccctctgtgt gctatgatat 2040 tgatggcact ggtgtcttca ttcttggggg ttgccatcat tcacacacac ccctttgaca 2100 tacagtgcac cccagttttg aatacattt ttttgcaccc tgtcccgtc tgctactttg 2160 atttgcgtta tgatatatat atatatatat aatacctttt ctcctcttta aacatggtcc 2220 tgtgacacaa tagtcagttg cagaaaggag ccagacttat tcgcaaagca ctgtgctcaa 2280 actcttcaga aaaaaaggaa aaaaaaaaaa agctatagtt gtaacatatg tattccagac 2340 ctctggttta aaggcaaaag aaaaaaaatc tacagtgttt cttctcatgt tttctgatcg 2400 gaggcatgac aaagcaagac tgaaatctga actgtgtctc ctgcatggca acacgtgtct 2460 ccgtcaggcc ctcgcaaggc ccggggaggg ggttctacgc ctcttgtctc tttgttgcat 2520 gctgaacact catcgccttc ctactgtatc ctgcctcctg cagcctccct cttcctcctc 2580 ctcttcctct tcctcctctt cctcctcctc ctcctcttcc tccaagtttg aaaggtcaaa 2640 caaaactacc acattcccta cccagttaga agaaaaccac cgtcctgaca gttgtgatcg 2700 catggagtac ttttagatta ttagcacctg tttttacctc gtttgtgggc gtgtttgtat 2760 gtgcacatgt atgaagtcgg cacatgcacc ttctgtatgg gcagaggcgt ggcatctaca 2820 gaagagcaga tgccaacttt gtgcttttag tgaatacatt aaaaaaaaaa aaccaacggt 2880 ccttattgag tggaattcta tttgatgcaa atatttgagc tctttaagac tttaaaacta 2940 gataatgtgc caagctttta ggactgctca ccagtgccct ctgaagaaac accagtactt 3000 tttcctgttt gtgtaataaa ggcatatttg tatttgtgtt tgcatcacta atggttattt 3060 cttcttagtc cactgaatgt ttccatgtgc ctctcgtatg ccaaactttt tgtcatcttt 3120 catgtgggga ccaaatggtt tgtctgtggc aaacctaaac ctatgacctg ctgaggcctc 3180 tcagaaaact gaccacagta ccaagatagt acttcgcaaa gaaaagtagg ttccctccct 3240 ggttttgtag ctgtcgccaa tattagcgta attccaagga gctgaacgcc tttatataaa 3300 tctgatggca cctgatgctt ttagttctga aaatatttac actcggatca tgttgttgat 3360 gacttaaaca aagttttgat gaagagagca aaaaaaaagc aggtggattt ggaacagttt 3420 cagggttttt tttgtttttt gttttttgtt tttgtttttt tttttttatt tttgtttttc 3480 tgttctctgt tagaaaagtc aggtgttctc tgtcaggcta tctttatagt caattttttt 3540 tacgaactaa agtagtacct tttaatatgt agtcaacgcc cctctgctcg gggttcagtt 3600 ttgggtctta accagctgtc atgttctcta tgctgcctgc cacttgaggc actgagtgcc 3660 ctagacagtc ccatcggtgg tagccaggga aacgaaagac gaactcaact cttgctccta 3720 ataatcaact ctctgtatga aggatggcag cattaagagt cctcctgcct gggcattatt 3780 gggccagttc accctcttta aatcaaaccc gcagtggctc ccagttctcg tcccatcaga 3840 tttaaattgc taacagtatg gggggcacca cgcatctgtt ttgtcccaca atgcgctttt 3900 ctctcccaaa tcccgatttc tgctgtcata gcctctattc aatttttatt tattgtctgc 3960 cctccactta tacaatcgta gagagcaatg ccatttgtca ctttctgcaa cagttttttg 4020 agcctttatg gctgaatccc atttttcttc tctttcaaac tgtttgctcc attgctcctc 4080 ccgcacggct gtccgtacag tcatcccatc catctggggg cctctttcat ctctcaccct 4140 tcctggtgct tcgtggatct ctgcttacct ctgtgggttt tttttttttt ttttgactta 4200 ttcttctcac tggactttaa gattacttcc acagcgaaag tgctgcctcc cttttctgcc 4260 cgcagtgttc tgcgtacttt agatactact cagtgctgac atttgatggc aaaagttgcc 4320 tgcacttaaa tttctctttt taatagggtg aactagagtt ggagtttttt tctctttttt 4380 ctcttttctc tctctctctc tctctctctc tctctctctc tctctctctc cctccctccc 4440 tccctccctc cctccctccc tctctctctc tttttctttc tttctttctt tctttctttc 4500 tttctttctt tctttctttc tttctttttt tgacaaatct cacaggcttt gagaattata 4560 aaaggtgaca gttcacctga aaatcacagg tctggtctgt ttaaattgtt gagaaatatc 4620 cgattaaaag tcttgtggct gtgtcctaat aggctctctt tcaggacgtt gtagtcaata 4680 gagtggctga accatacttg agtttataaa gctcaaaaac tgatgcaccc actctgctat 4740 tatcgtgtta gtaagagttc agctgtatat cattgtctag gtttatcttg tcctacagtg 4800 ggtattcaaa tatggccacc agaggatatg tgtaaatata agcacctgta tttgcctgtt 4860 gttgagaact ggagggaaaa caaaaaatgt ctggcaaccc tttgcctttt taaccgtaat 4920 taattgacag tttatttaga gataagagtt ttcaaaaatc tcttaactgc cacaacccac 4980 agagggtctt gttttgccat cttcagtggc tcacagatat gatccaagtt aacttgaaag 5040 agatgagcag tacccaggaa attgtcctgc ctttaactct ggctgtcctt aattatgact 5100 gtttaatgct gaattttcca tccgtctagt gtttgagggt aaagaaaagc cttttttaaa 5160 taagtatttc tgtaaaacgg catcggtggg atcttctgtg ttgctatcac gggtgaaaga 5220 gggaaacatt tcttattttt attaagcaga gcattattta cagaaagcca ttgttgagaa 5280 ttagttccca catcatataa atatccatta accattctaa attgtaagag aactccagtg 5340 ttgctatgca caaggaactc tcctgggggc ctttttttgc atagcaatta aaggtatgct 5400 atttgtcagt agccattttt tgcagtgatt taaagaccaa agttgtttta cagctgtgtt 5460 acccttaaag gttttttttt tatgtattaa atcaatttat cactgtttga agctttgaat 5520 acctgcaatc tttgccaaga tactttttta tttaaaaaaa taactgtgta aatattaccc 5580 tgtaatatta tatatactta ataaaacatt ttaagcta 5618 <210> 8 <211> 321 <212> PRT <213> Mus musculus <400> 8 Met Trp Pro Leu Ala Ala Ala Leu Leu Leu Gly Ser Cys Cys Cys Gly 1 5 10 15 Ser Ala Gln Leu Leu Phe Ser Asn Val Asn Ser Ile Glu Phe Thr Ser 20 25 30 Cys Asn Glu Thr Val Val Ile Pro Cys Ile Val Arg Asn Val Glu Ala 35 40 45 Gln Ser Thr Glu Glu Met Phe Val Lys Trp Lys Leu Asn Lys Ser Tyr 50 55 60 Ile Phe Ile Tyr Asp Gly Asn Lys Asn Ser Thr Thr Thr Asp Gln Asn 65 70 75 80 Phe Thr Ser Ala Lys Ile Ser Val Ser Asp Leu Ile Asn Gly Ile Ala 85 90 95 Ser Leu Lys Met Asp Lys Arg Asp Ala Met Val Gly Asn Tyr Thr Cys 100 105 110 Glu Val Thr Glu Leu Ser Arg Glu Gly Lys Thr Val Ile Glu Leu Lys 115 120 125 Asn Arg Thr Val Ser Trp Phe Ser Pro Asn Glu Lys Ile Leu Ile Val 130 135 140 Ile Phe Pro Ile Leu Ala Ile Leu Leu Phe Trp Gly Lys Phe Gly Ile 145 150 155 160 Leu Thr Leu Lys Tyr Lys Ser Ser His Thr Asn Lys Arg Ile Ile Leu 165 170 175 Leu Leu Val Ala Gly Leu Val Leu Thr Val Ile Val Val Val Gly Ala 180 185 190 Ile Leu Leu Ile Pro Gly Glu Lys Pro Val Lys Asn Ala Ser Gly Leu 195 200 205 Gly Leu Ile Val Ile Ser Thr Gly Ile Leu Ile Leu Leu Gln Tyr Asn 210 215 220 Val Phe Met Thr Ala Phe Gly Met Thr Ser Phe Thr Ile Ala Ile Leu 225 230 235 240 Ile Thr Gln Val Leu Gly Tyr Val Leu Ala Leu Val Gly Leu Cys Leu 245 250 255 Cys Ile Met Ala Cys Glu Pro Val His Gly Pro Leu Leu Ile Ser Gly 260 265 270 Leu Gly Ile Ile Ala Leu Ala Glu Leu Leu Gly Leu Val Tyr Met Lys 275 280 285 Phe Val Ala Ser Asn Gln Arg Thr Ile Gln Pro Pro Arg Lys Ala Val 290 295 300 Glu Glu Pro Leu Asn Ala Phe Lys Glu Ser Lys Gly Met Met Asn Asp 305 310 315 320 Glu <210> 9 <211> 5021 <212> DNA <213> Homo sapiens <400> 9 agtgggagcg cgcgtgcgcg cggccgtgca gcctgggcg tgggctcgc ctgtgacgcg 60 cggcggcggt cggtcctgcc tgtaacggcg gcggcggctg ctgctccgga cacctgcggc 120 ggcggcggcg accccgcggc gggcgcggag atgtggcccc tggtagcggc gctgttgctg 180 ggctcggcgt gctgcggatc agctcagcta ctatttaata aaaaaaatc tgtagaattc 240 acgttttgta atgacactgt cgtcattcca tgctttgtta ctaatatgga ggcacaaaac 300 actactgaag tatacgtaaa gtggaaattt aaaggaagag atattacac ctttgatgga 360 gctctaaaca agtccactgt ccccactgac tttagtagtg caaaaattga agtctcacaa 420 ttactaaaag gagatgcctc tttgaagatg gataagagtg atgctgtctc acacacagga 480 aactacactt gtgaagtaac agaattaacc agagaaggtg aaacgatcat cgagctaaaa 540 tatcgtgttg tttcatggtt ttctccaaat gaaaatattc ttattgttat ttcccaatt 600 tttgctatac tcctgttctg gggacagttt ggtattaaaa cacttaaata tagatccggt 660 ggtatggatg agaaaacaat tgctttactt gttgctggac tagtgatcac tgtcattgtc 720 attgttggag ccattctttt cgtcccaggt gaatattcat taaagaatgc tactggcctt 780 ggtttaattg tgactttac agggatatta atattacttc actactatgt gtttagtaca 840 gcgattggat taacctcctt cgtcattgcc atattggtta ttcaggtgat agcctatatc 900 ctcgctgtgg ttggactgag tctctgtatt gcggcgtgta taccaatgca tggccctctt 960 ctgatttcag gtttgagtat cttagctcta gcacaattac ttggactagt ttatatgaaa 1020 tttgtggaat aactgaagtg aagtgatgga ctccgatttg gagagtagta agacgtgaaa 1080 ggaatacact tgtgtttaag caccatggcc ttgatgattc actgttgggg agaagaaaca 1140 agaaaagtaa ctggttgtca cctatgagac ccttacgtga ttgttagtta agtttttatt 1200 caaagcagct gtaatttagt taataaaata attatgatct atgttgtttg cccaattgag 1260 atccagtttt ttgttgttat tttaatcaa ttaggggcaa tagtagaatg gacaatttcc 1320 aagaatgatg cctttcaggt cctagggcct ctggcctcta ggtaaccagt ttaaattggt 1380 tcagggtgat aactacttag cactgccctg gtgattaccc agagatatct atgaaaacca 1440 gtggcttcca tcaaaccttt gccaactcag gttcacagca gctttgggca gttatggcag 1500 tatggcatta gctgagaggt gtctgccact tctgggtcaa tggaataata attaagtac 1560 aggcaggaat ttggttggga gcatcttgta tgatctccgt atgatgtgat attgatggag 1620 atagtggtcc tcattcttgg gggttgccat tcccacattc ccccttcaac aaacagtgta 1680 acaggtcctt cccagattta gggtactttt attgatggat atgttttcct tttattcaca 1740 taaccccttg aaaccctgtc ttgtcctcct gttacttgct tctgctgtac aagatgtagc 1800 accttttctc ctctttgaac atggtctagt gacacggtag caccagttgc aggaaggagc 1860 cagacttgtt ctcagagcac tgtgttcaca cttttcagca aaatagcta tggttgtaac 1920 atatgtattc ccttcctctg atttgaaggc aaaaatctac agtgtttctt cacttcttt 1980 ctgatctggg gcatgaaaaa agcaagattg aaatttgaac tatgagtctc ctgcatggca 2040 acaaaatgtg tgtcaccatc aggccaacag gccagccctt gaatggggat ttattactgt 2100 tgtatctatg ttgcatgata aacattcatc accttcctcc tgtagtcctg cctcgtactc 2160 cccttcccct atgattgaaa agtaaacaa accacattt cctatcctgg ttagaagaaa 2220 attaatgttc tgacagttgt gatcgcctgg agtacttta gactttagc attcgtttt 2280 tacctgtttg tggatgtgtg tttgtatgtg catacgtatg agataggcac atgcatcttc 2340 tgtatggaca aaggtggggt acctacagga gagcaaggt taatttgtg cttttagtaa 2400 aaacatttaa atacaaagtt ctttattggg tggaattata tttgatgcaa atatttgatc 2460 acttaaaact tttaaaactt ctagtaatt tgccacgctt tttgactgct caccaatacc 2520 ctgtaaaaat acgtaattct tcctgtttgt gtaataagat attcatattt gtagttgcat 2580 taataatagt tattcttag tccatcagat gttcccgtgt gcctcttta tgccaaattg 2640 attgtcatat ttcatgttgg gaccaagtag ttgcccatg gcaacctaa atttatgacc 2700 tgctgaggcc tctcagaaaa ctgagcatac tagcagaca gctctcttg aaaaaaaaaa 2760 tatgtataca caatatata cgtatatcta tatatacgta tgtatataca cacatgtata 2820 ttctccttg attgtgtagc tgtccaaaat aaacatat atagaggag ctgtattcct 2880 ttatacaaat ctgatggctc ctgcagcact ttttccttct gaaaatattt acattttgct 2940 aacctagttt gttactttaa aaatcagttt tgatgaaagg agggaaaagc agatggactt 3000 gaaaaagatc caagctccta ttagaaaagg tatgaaaatc tttatagtaa aattttttat 3060 aaactaaagt tgtacctttt aatatgtagt aaactctcat ttatttgggg ttcgctcttg 3120 gatctcatcc atccattgtg ttctctttaa tgctgcctgc cttttgaggc attcactgcc 3180 ctagacaatg ccaccagaga tagtggggga aatgccagat gaaaccaact cttgctctca 3240 ctagttgtca gcttctctgg ataagtgacc acagaagcag gagtcctcct gcttgggcat 3300 cattgggcca gttccttctc tttaaatcag atttgtaatg gctcccaaat tccatcacat 3360 cacatttaaa ttgcagacag tgttttgcac atcatgtatc tgttttgtcc cataatatgc 3420 tttttactcc ctgatcccag tttctgctgt tgactcttcc attcagtttt atttattgtg 3480 tgttctcaca gtgacaccat ttgtcctttt ctgcaacaac ctttccagct acttttgcca 3540 aattctattt gtcttctcct tcaaaacatt ctcctttgca gttcctcttc atctgtgtag 3600 ctgctcttt gtctcttaac ttaccattcc tatagtactt tatgcatctc tgcttagttc 3660 tattagtttt ttggccttgc tcttctcctt gattttaaaa ttccttctat agctagagct 3720 tttctttctt tcattctctc ttcctgcagt gttttgcata catcagaagc taggtacata 3780 agttaaatga ttgagagttg gctgtattta gatttatcac ttttaatag ggtgagcttg 3840 agagttttct ttctttctgt tttttttttt tttttttt tttttttt tttttttttt 3900 ttttgactaa tttcacatgc tctaaaaacc ttcaaaggtg attatttttc tcctggaaac 3960 tccaggtcca ttctgtttaa atccctaaga atgtcagaat taaaataaca gggctatccc 4020 gtaattggaa atatttcttt tttcaggatg ctatagtcaa tttagtaagt gaccaccaaa 4080 ttgttatttg cactaacaaa gctcaaaaca cgataagtttt actcctccat ctcagtaata 4140 aaaattaagc tgtaatcaac cttctaggtt tctcttgtct taaaatgggt attcaaaaat 4200 ggggatctgt ggtgtatgta tggaaacaca tactccttaa tttacctgtt gttggaaact 4260 ggagaaatga ttgtcgggca accgtttatt ttttattgta ttttattgg ttgagggatt 4320 tttttataaa cagttttact tgtgtcatat tttaaaatta ctaactgcca tcacctgctg 4380 gggtccttg ttaggtcatt ttcagtgact aatagggata atccaggtaa ctttgaagag 4440 atgagcagtg agtgaccagg cagtttttct gcctttagct ttgacagttc ttaattaaga 4500 tcattgaaga ccagctttct cataaatttc tctttttgaa aaaaagaaag catttgtact 4560 aagctcctct gtaagacaac atcttaaatc ttaaaagtgt tgttatcatg actggtgaga 4620 gaagaaaaca ttttgttttt attaaatgga gcattattta caaaaagcca ttgttgagaa 4680 ttagatccca catcgtataa atatctatta accattctaa ataaagagaa ctccagtgtt 4740 gctatgtgca agatcctctc ttggagcttt tttgcatagc aattaaaggt gtgctatttg 4800 tcagtagcca ttttttgca gtgatttgaa gaccaaagtt gttttacagc tgtgttaccg 4860 ttaaaggttt tttttttat atgtattaaa tcaatttatc actgtttaaa gctttgaata 4920 tctgcaatct ttgccaaggt actttttat ttaaaaaaaa acataacttt gtaaatatta 4980 ccctgtaata ttatatac ttaataaaac attttaagct a 5021 <210> 10 <211> 293 <212> PRT <213> Homo sapiens <400> 10 Met Trp Pro Leu Val Ala Ala Leu Leu Leu Gly Ser Ala Cys Cys Gly 1 5 10 15 Ser Ala Gln Leu Leu Phe Asn Lys Thr Lys Ser Val Glu Phe Thr Phe 20 25 30 Cys Asn Asp Thr Val Val Ile Pro Cys Phe Val Thr Asn Met Glu Ala 35 40 45 Gln Asn Thr Thr Glu Val Tyr Val Lys Trp Lys Phe Lys Gly Arg Asp 50 55 60 Ile Tyr Thr Phe Asp Gly Ala Leu Asn Lys Ser Thr Val Pro Thr Asp 65 70 75 80 Phe Ser Ser Ala Lys Ile Glu Val Ser Gln Leu Leu Lys Gly Asp Ala 85 90 95 Ser Leu Lys Met Asp Lys Ser Asp Ala Val Ser His Thr Gly Asn Tyr 100 105 110 Thr Cys Glu Val Thr Glu Leu Thr Arg Glu Gly Glu Thr Ile Ile Glu 115 120 125 Leu Lys Tyr Arg Val Val Ser Trp Phe Ser Pro Asn Glu Asn Ile Leu 130 135 140 Ile Val Ile Phe Pro Ile Phe Ala Ile Leu Leu Phe Trp Gly Gln Phe 145 150 155 160 Gly Ile Lys Thr Leu Lys Tyr Arg Ser Gly Gly Met Asp Glu Lys Thr 165 170 175 Ile Ala Leu Leu Val Ala Gly Leu Val Ile Thr Val Ile Val Ile Val 180 185 190 Gly Ala Ile Leu Phe Val Pro Gly Glu Tyr Ser Leu Lys Asn Ala Thr 195 200 205 Gly Leu Gly Leu Ile Val Thr Ser Thr Gly Ile Leu Ile Leu Leu His 210 215 220 Tyr Tyr Val Phe Ser Thr Ala Ile Gly Leu Thr Ser Phe Val Ile Ala 225 230 235 240 Ile Leu Val Ile Gln Val Ile Ala Tyr Ile Leu Ala Val Val Gly Leu 245 250 255 Ser Leu Cys Ile Ala Ala Cys Ile Pro Met His Gly Pro Leu Leu Ile 260 265 270 Ser Gly Leu Ser Ile Leu Ala Leu Ala Gln Leu Leu Gly Leu Val Tyr 275 280 285 Met Lys Phe Val Glu 290 <210> 11 <211> 5288 <212> DNA <213> Homo sapiens <400> 11 ggggagcagg cgggggagcg ggcgggaagc agtgggagcg cgcgtgcgcg cggccgtgca 60 gcctgggcag tgggtcctgc ctgtgacgcg cggcggcggt cggtcctgcc tgtaacggcg 120 gcggcggctg ctgctccaga cacctgcggc ggcggcggcg accccgcggc gggcgcggag 180 atgtggcccc tggtagcggc gctgttgctg ggctcggcgt gctgcggatc agctcagcta 240 ctatttaata aaacaaaatc tgtagaattc acgttttgta atgacactgt cgtcattcca 300 tgctttgtta ctaatatgga ggcacaaaac actactgaag tatacgtaaa gtggaaattt 360 aaaggaagag atatttacac ctttgatgga gctctaaaca agtccactgt ccccactgac 420 tttagtagtg caaaaattga agtctcacaa ttactaaaag gagatgcctc tttgaagatg 480 gataagagtg atgctgtctc acacacagga aactacactt gtgaagtaac agaattaacc 540 agagaaggtg aaacgatcat cgagctaaaa tatcgtgttg tttcatggtt ttctccaaat 600 gaaaatattc ttattgttat tttcccaatt tttgctatac tcctgttctg gggacagttt 660 ggtattaaaa cacttaaata tagatccggt ggtatggatg agaaaacaat tgctttactt 720 gttgctggac tagtgatcac tgtcattgtc attgttggag ccattctttt cgtcccaggt 780 gaatattcat taaagaatgc tactggcctt ggtttaattg tgacttctac agggatatta 840 atattacttc actactatgt gtttagtaca gcgattggat taacctcctt cgtcattgcc 900 atattggtta ttcaggtgat agcctatatc ctcgctgtgg ttggactgag tctctgtatt 960 gcggcgtgta taccaatgca tggccctctt ctgatttcag gtttgagtat cttagctcta 1020 gcacaattac ttggactagt ttatatgaaa tttgtggctt ccaatcagaa gactatacaa 1080 cctcctagga ataactgaag tgaagtgatg gactccgatt tggagagtag taagacgtga 1140 aaggaataca cttgtgttta agcaccatgg ccttgatgat tcactgttgg ggaagaaa 1200 caagaaaagt aactggttgt cacctatgag acccttacgt gattgttagt taagttttta 1260 ttcaaagcag ctgtaattta gttaataaaa taattatgat ctatgttgtt tgcccaattg 1320 agatccagtt ttttgttgtt atttttaatc aattaggggc aattagtagaa tggacaattt 1380 ccaagaatga tgcctttcag gtcctagggc ctctggcctc taggtaacca gtttaaattg 1440 gttcagggtg ataactactt agcactgccc tggtgattac ccagagatat ctatgaaaac 1500 cagtggcttc catcaaacct ttgccaactc aggttcacag cagctttggg cagttatggc 1560 agtatggcat tagctgagag gtgtctgcca cttctgggtc aatggaataa taaattaagt 1620 acaggcagga atttggttgg gagcatcttg tatgatctcc gtatgatgtg atattgatgg 1680 agatagtggt cctcattctt gggggttgcc attcccacat tcccccttca acaaacagtg 1740 taacaggtcc ttcccagatt tagggtactt ttattgatgg atatgttttc cttttattca 1800 cataacccct tgaaaccctg tcttgtcctc ctgttacttg cttctgctgt acaagatgta 1860 gcaccttttc tcctctttga acatggtcta gtgacacggt agcaccagtt gcaggaagga 1920 gccagacttg ttctcagagc actgtgttca cacttttcag caaaaatagc tatggttgta 1980 acatatgtat tcccttcctc tgatttgaag gcaaaaatct acagtgtttc ttcacttctt 2040 ttctgatctg gggcatgaaa aaagcaagat tgaaatttga actatgagtc tcctgcatgg 2100 caacaaaatg tgtgtcacca tcaggccaac aggccagccc ttgaatgggg atttattact 2160 gttgtatcta tgttgcatga taaacattca tcaccttcct cctgtagtcc tgcctcgtac 2220 tccccttccc ctatgattga aaagtaaaca aaacccacat ttcctatcct ggttagaaga 2280 aaattaatgt tctgacagtt gtgatcgcct ggagtacttt tagactttta gcattcgttt 2340 tttacctgtt tgtggatgtg tgtttgtatg tgcatacgta tgagataggc acatgcatct 2400 tctgtatgga caaaggtggg gtacctacag gagagcaaag gttaattttg tgcttttagt 2460 aaaaacattt aaatacaaag ttctttattg ggtggaatta tatttgatgc aaatatttga 2520 tcacttaaaa cttttaaaac ttctaggtaa tttgccacgc tttttgactg ctcaccaata 2580 ccctgtaaaa atacgtaatt cttcctgttt gtgtaataag atattcatat ttgtagttgc 2640 attaataata gttatttctt agtccatcag atgttcccgt gtgcctcttt tatgccaaat 2700 tgattgtcat atttcatgtt gggaccaagt agtttgccca tggcaaacct aaatttatga 2760 cctgctgagg cctctcagaa aactgagcat actagcaaga cagctcttct tgaaaaaaaa 2820 atatgtata cacaata tacgtatatc tatatatacg tatgtata cacacatgta 2880 tattctctcct tgattgtgta gctgtccaaa ataacaat atagaggg agctgtattc 2940 ctttatacaa atctgatggc tcctgcagca ctttttcctt ctgaaaatat ttacattttg 3000 ctaacctagt ttgttacttt aaaaatcagt ttgatgaaa ggagggaaa gcagatggac 3060 ttgaaaaaga tccaagctcc tattagaaaa ggtatgaaaa tctttagt aaaatttttt 3120 aaactaaa gttgtacctt ttaatatgta gtaaactctc atttattgg ggttcgctct 3180 tggatctcat gcattcattg tgttcttt aatgctgcct gcctttgag gcattcactg 3240 ccctagacaa tgccaccaga gatagtgggg gaatgccag atgaaccaa ctcttgctct 3300 cactagttgt cagcttctct ggataagtga ccacagaagc aggagtccctc ctgcttgggc 3360 atcattgggc cagttccttc tctttaatc agatttgtaa tggctccca attccatcac 3420 atcacattta aattgcagac agtgttttgc acatcatgta tctgttttgt cccatatatat 3480 gctttttact ccctgatccc agttctgct gttgactctt ccattcagtt ttatttg 3540 tgtgttctca cagtgacacc atttgtcctt ttctgcaaca acctttccag ctacttttgc 3600 caaattctat ttgtcttctc cttcaaaaca ttctcctttg cagttcctct tcatctgtgt 3660 agctgctctt ttgtctctta acttaccatt cctatagtac tttatgcatc tctgcttagt 3720 tctattagtt ttttggcctt gctcttctcc ttgattttaa aattccttct atagctagag 3780 cttttctttc tttcattctc tcttcctgca gtgttttgca tacatcagaa gctaggtaca 3840 taagttaaat gattgagagt tggctgtatt tagatttatc actttttaat agggtgagct 3900 tgagagttt ctttctttct gttttttttt tttgtttttt tttttttttt ttttttttt 3960 ttttttgact aatttcacat gctctaaaaa ccttcaaagg tgattatttt tctcctggaa 4020 actccaggtc cattctgttt aaatccctaa gaatgtcaga attaaaataa cagggctatc 4080 ccgtaattgg aaatatttct tttttcagga tgctatagtc aatttagtaa gtgaccacca 4140 aattgttatt tgcactaaca aagctcaaaa cacgataagt ttactcctcc atctcagtaa 4200 taaaaattaa gctgtaatca accttctagg tttctcttgt cttaaaatgg gtattcaaaa 4260 atggggatct gtggtgtatg tatggaaaca catactcctt aatttacctg ttgttggaaa 4320 ctggagaaat gattgtcggg caaccgttta ttttttattg tattttattt ggttgaggga 4380 tttttttata aacagtttta cttgtgtcat attttaaaat tactaactgc catcacctgc 4440 tggggtcctt tgttaggtca ttttcagtga ctaataggga taatccaggt aactttgaag 4500 agatgagcag tgagtgacca ggcagttttt ctgcctttag ctttgacagt tcttaattaa 4560 gatcattgaa gaccagcttt ctcataaatt tctctttttg aaaaaaagaa agcatttgta 4620 ctaagctcct ctgtaagaca acatcttaaa tcttaaaagt gttgttatca tgactggtga 4680 gagaagaaaa cattttgttt ttattaaatg gagcattatt tacaaaaagc cattgttgag 4740 aattagatcc cacatcgtat aaatatctat taaccattct aaataaagag aactccagtg 4800 ttgctatgtg caagatcctc tcttggagct tttttgcata gcaattaaag gtgtgctatt 4860 tgtcagtagc catttttttg cagtgatttg aagaccaaag ttgttttaca gctgtgttac 4920 cgttaaaggt tttttttttt atatgtatta aatcaattta tcactgttta aagctttgaa 4980 tatctgcaat ctttgccaag gtactttttt atttaaaaaa aaacataact ttgtaaatat 5040 taccctgtaa tattatatat acttaataaa acattttaag ctattttgtt gggctatttc 5100 tattgctgct acagcagacc acaagcacat ttctgaaaaa tttaatttat taatgtattt 5160 ttaagttgct tatattctag gtaacaatgt aaagaatgat ttaaaatatt aattatgaat 5220 tttttgagta taatacccaa taagctttta attagagcag agttttaatt aaaagtttta 5280 aatcagtc 5288 <210> 12 <211> 305 <212> PRT <213> Homo sapiens <400> 12 Met Trp Pro Leu Val Ala Ala Leu Leu Leu Gly Ser Ala Cys Cys Gly 1 5 10 15 Ser Ala Gln Leu Leu Phe Asn Lys Thr Lys Ser Val Glu Phe Thr Phe 20 25 30 Cys Asn Asp Thr Val Val Ile Pro Cys Phe Val Thr Asn Met Glu Ala 35 40 45 Gln Asn Thr Thr Glu Val Tyr Val Lys Trp Lys Phe Lys Gly Arg Asp 50 55 60 Ile Tyr Thr Phe Asp Gly Ala Leu Asn Lys Ser Thr Val Pro Thr Asp 65 70 75 80 Phe Ser Ser Ala Lys Ile Glu Val Ser Gln Leu Leu Lys Gly Asp Ala 85 90 95 Ser Leu Lys Met Asp Lys Ser Asp Ala Val Ser His Thr Gly Asn Tyr 100 105 110 Thr Cys Glu Val Thr Glu Leu Thr Arg Glu Gly Glu Thr Ile Ile Glu 115 120 125 Leu Lys Tyr Arg Val Val Ser Trp Phe Ser Pro Asn Glu Asn Ile Leu 130 135 140 Ile Val Ile Phe Pro Ile Phe Ala Ile Leu Leu Phe Trp Gly Gln Phe 145 150 155 160 Gly Ile Lys Thr Leu Lys Tyr Arg Ser Gly Gly Met Asp Glu Lys Thr 165 170 175 Ile Ala Leu Leu Val Ala Gly Leu Val Ile Thr Val Ile Val Ile Val 180 185 190 Gly Ala Ile Leu Phe Val Pro Gly Glu Tyr Ser Leu Lys Asn Ala Thr 195 200 205 Gly Leu Gly Leu Ile Val Thr Ser Thr Gly Ile Leu Ile Leu Leu His 210 215 220 Tyr Tyr Val Phe Ser Thr Ala Ile Gly Leu Thr Ser Phe Val Ile Ala 225 230 235 240 Ile Leu Val Ile Gln Val Ile Ala Tyr Ile Leu Ala Val Val Gly Leu 245 250 255 Ser Leu Cys Ile Ala Ala Cys Ile Pro Met His Gly Pro Leu Leu Ile 260 265 270 Ser Gly Leu Ser Ile Leu Ala Leu Ala Gln Leu Leu Gly Leu Val Tyr 275 280 285 Met Lys Phe Val Ala Ser Asn Gln Lys Thr Ile Gln Pro Pro Arg Asn 290 295 300 Asn 305 <210> 13 <211> 5078 <212> DNA <213> Homo sapiens <400> 13 agtgggagcg cgcgtgcgcg cggccgtgca gcctgggcag tgggtcctgc ctgtgacgcg 60 cggcggcggt cggtcctgcc tgtaacggcg gcggcggctg ctgctccgga cacctgcggc 120 ggcggcggcg accccgcggc gggcgcggag atgtggcccc tggtagcggc gctgttgctg 180 ggctcggcgt gctgcggatc agctcagcta ctatttaata aaacaaaatc tgtagaattc 240 acgttttgta atgacactgt cgtcattcca tgctttgtta ctaatatgga ggcacaaaac 300 actactgaag tatacgtaaa gtggaaattt aaaggaagag atattacac ctttgatgga 360 gctctaaaca agtccactgt ccccactgac tttagtagtg caaaaattga agtctcacaa 420 ttactaaaag gagatgcctc tttgaagatg gataagagtg atgctgtctc acacacagga 480 aactacactt gtgaagtaac agaattaacc agagaaggtg aaacgatcat cgagctaaaa 540 tatcgtgttg tttcatggtt ttctccaaat gaaaatattc ttattgttat ttcccaatt 600 tttgctatac tcctgttctg gggacagttt ggtattaaaa cacttaaata tagatccggt 660 ggtatggatg agaaaacaat tgctttactt gttgctggac tagtgatcac tgtcattgtc 720 attgttggag ccattctttt cgtcccaggt gaatattcat taaagaatgc tactggcctt 780 ggttaattg tgacttctac agggatatta atattacttc actactatgt gtttagtaca 840 gcgattggat taacctcctt cgtcattgcc atattggtta ttcaggtgat agcctatatc 900 ctcgctgtgg ttggactgag tctctgtatt gcggcgtgta taccaatgca tggccctctt 960 ctgatttcag gtttgagtat cttagctcta gcacaattac ttggactagt ttatatgaaa 1020 ttgtggctt ccaatcagaa gactatacaa cctcctagga aagctgtaga ggaacccctt 1080 aatgaatac tgaagtgaag tgatggactc cgatttggag agtagtaga cgtgaaagga 1140 attacktgt gtttaagcac catggccttg atgattcact gttggggaga agaaacaaga 1200 aaagtaactg gttgtcacct atgagaccct tacgtgattg ttagttagt ttttattca 1260 agcagctgta atttagttaa taaaatatt atgatctatg ttgtttgccc aattgagatc 1320 cagttttg ttgttattt taatcatta ggggcaatag tagaatggac aatttccaag 1380 aatgatgcct ttcaggctcct agggctctg gcctctaggt aaccagttta aattggttca 1440 gggtgataac tacttagcac tgccctggtg attacccaga gatatcttg aaaaccagtg 1500 gcttccatca aaccttgcc aactcaggtt cacagcagct tggcagtt atggcagtt 1560 ggcattagct gagaggtgtc tgccacttct gggtcaatgg ataataat tagtacagg 1620 caggaatttg gttgggagca tcttgtatga tctccgtatg atgtgatatt gatggagata 1680 gtggtcctca ttctggggg ttgccattcc cacattcccc cttcacaa cagtgtaaca 1740 ggtccttcc agatttaggg tacttttatt gatggatatg ttttccttt attcacataa 1800 ccccttgaaa ccctgtcttg tcctcctgtt acttgcttct gctgtacaag atgtagcacc 1860 ttttctcctc tttgaacatg gtctagtgac acggtagcac cagttgcagg aaggagccag 1920 acttgttctc agagcactgt gttcacactt ttcagcaaaa atagctatgg ttgtaacata 1980 tgtattccct tcctctgatt tgaaggcaaa aatctacagt gtttcttcac ttcttttctg 2040 atctggggca tgaaaaaagc aagattgaaa tttgaactat gagtctcctg catggcaaca 2100 aaatgtgtgt caccatcagg ccaacaggcc agcccttgaa tggggattta ttactgttgt 2160 atctatgttg catgataaac attcatcacc ttcctcctgt agtcctgcct cgtactcccc 2220 ttcccctatg attgaaaagt aaacaaaacc cacatttcct atcctggtta gaaaaatt 2280 aatgttctga cagttgtgat cgcctggagt acttttagac ttttagcatt cgttttttac 2340 ctgtttgtgg atgtgtgttt gtatgtgcat acgtatgaga taggcacatg catcttctgt 2400 atggacaaag gtggggtacc tacaggagag caaaggttaa ttttgtgctt ttagtaaaaa 2460 catttaaata caaagttctt tattgggtgg aattatattt gatgcaaata tttgatcact 2520 taaaactttt aaaacttcta ggtaatttgc cacgcttttt gactgctcac caataccctg 2580 taaaaatacg taattcttcc tgtttgtgta ataagatatt catatttgta gttgcattaa 2640 taatagttat ttcttagtcc atcagatgtt cccgtgtgcc tcttttatgc caaattgatt 2700 gtcatatttc atgttgggac caagtagttt gcccatggca aacctaaatt tatgacctgc 2760 tgaggcctct cagaaaactg agcatactag caagacagct cttcttgaaa aaaaaaatat 2820 gtatacacaa atatatacgt atatctatat atacgtatgt atatacacac atgtatattc 2880 ttccttgatt gtgtagctgt ccaaaataat aacatatata gagggagctg tattccttta 2940 tacaaatctg atggctcctg cagcactttt tccttctgaa aatatttaca ttttgctaac 3000 ctagtttgtt actttaaaaa tcagttttga tgaaaggagg gaaaagcaga tggacttgaa 3060 aaagatccaa gctcctatta gaaaaggtat gaaaatcttt atagtaaaat tttttataaa 3120 ctaaagttgt acctttaat atgtagtaaa ctctcattta tttggggttc gctcttggat 3180 ctcatccatc cattgtgttc tctttaatgc tgcctgcctt ttgaggcatt cactgcccta 3240 gacaatgcca ccagagatag tgggggaaat gccagatgaa accaactctt gctctcacta 3300 gttgtcagct tctctggata agtgaccaca gaagcaggag tcctcctgct tgggcatcat 3360 tgggccagtt ccttctcttt aaatcagatt tgtaatggct cccaaattcc atcacatcac 3420 atttaaattg cagacagtgt tttgcacatc atgtatctgt tttgtcccat aatatgcttt 3480 ttactccctg atcccagttt ctgctgttga ctcttccatt cagttttatt tattgtgtgt 3540 tctcacagtg acaccatttg tccttttctg caacaacctt tccagctact tttgccaaat 3600 tctatttgtc ttctccttca aaacattctc ctttgcagtt cctcttcatc tgtgtagctg 3660 ctcttttgtc tcttaactta ccattcctat agtactttat gcatctctgc ttagttctat 3720 tagttttttg gccttgctct tctccttgat tttaaaattc cttctatagc tagagctttt 3780 ctttctttca ttctctcttc ctgcagtgtt ttgcatacat cagaagctag gtacataagt 3840 taaatgattg agagttggct gtatttagat ttatcacttt ttaatagggt gagcttgaga 3900 gttttctttc tttctgtttt ttttttttgt tttttttttt tttttttttt ttttttttt 3960 tgactaattt cacatgctct aaaaaccttc aaaggtgatt attttctcc tggaaactcc 4020 aggtccattc tgtttaaatc cctaagaatg tcagaattaa aataacaggg ctatcccgta 4080 attggaaata tttctttttt caggatgcta tagtcaattt agtaagtgac caccaaattg 4140 ttatttgcac taacaaagct caaaacacga taagtttact cctccatctc agtaataaaa 4200 attaagctgt aatcaacctt ctaggtttct cttgtcttaa aatgggtatt caaaaatggg 4260 gatctgtggt gtatgtatgg aaacacatac tccttaattt acctgttgtt ggaactgga 4320 gaaatgattg tcgggcaacc gtttattttt tattgtattt tatttggttg agggatttt 4380 ttaaaacag ttttacttgt gtcatatttt aaaattacta actgccatca cctgctgggg 4440 tcctttgtta ggtcattttc agtgactaat agggataatc caggtaactt tgaagagatg 4500 agcagtgagt gaccaggcag tttttctgcc tttagctttg acagttctta attaagatca 4560 ttgaagacca gctttctcat aaatttctct ttttgaaaaa aagaaagcat ttgtactaag 4620 ctcctctgta agacaacatc ttaaatctta aaagtgttgt tatcatgact ggtgagagaa 4680 gaaaacattt tgtttttatt aaatggagca ttatttacaa aaagccattg ttgagaatta 4740 gatcccacat cgtataaata tctattaacc attctaaata aagagaactc cagtgttgct 4800 atgtgcaaga tcctctcttg gagctttttt gcatagcaat taaaggtgtg ctatttgtca 4860 gtagccattt ttttgcagtg atttgaagac caaagttgtt ttacagctgt gttaccgtta 4920 aaggtttttt tttttatatg tattaaatca atttatcact gtttaaagct ttgaatatct 4980 gcaatctttg ccaaggtact tttttattta aaaaaaaaca taactttgta aatattaccc 5040 tgtaatatta tatatactta ataaaacatt ttaagcta 5078 <210> 14 <211> 312 <212> PRT <213> Homo sapiens <400> 14 Met Trp Pro Leu Val Ala Ala Leu Leu Leu Gly Ser Ala Cys Cys Gly 1 5 10 15 Ser Ala Gln Leu Leu Phe Asn Lys Thr Lys Ser Val Glu Phe Thr Phe 20 25 30 Cys Asn Asp Thr Val Val Ile Pro Cys Phe Val Thr Asn Met Glu Ala 35 40 45 Gln Asn Thr Thr Glu Val Tyr Val Lys Trp Lys Phe Lys Gly Arg Asp 50 55 60 Ile Tyr Thr Phe Asp Gly Ala Leu Asn Lys Ser Thr Val Pro Thr Asp 65 70 75 80 Phe Ser Ser Ala Lys Ile Glu Val Ser Gln Leu Leu Lys Gly Asp Ala 85 90 95 Ser Leu Lys Met Asp Lys Ser Asp Ala Val Ser His Thr Gly Asn Tyr 100 105 110 Thr Cys Glu Val Thr Glu Leu Thr Arg Glu Gly Glu Thr Ile Ile Glu 115 120 125 Leu Lys Tyr Arg Val Val Ser Trp Phe Ser Pro Asn Glu Asn Ile Leu 130 135 140 Ile Val Ile Phe Pro Ile Phe Ala Ile Leu Leu Phe Trp Gly Gln Phe 145 150 155 160 Gly Ile Lys Thr Leu Lys Tyr Arg Ser Gly Gly Met Asp Glu Lys Thr 165 170 175 Ile Ala Leu Leu Val Ala Gly Leu Val Ile Thr Val Ile Val Ile Val 180 185 190 Gly Ala Ile Leu Phe Val Pro Gly Glu Tyr Ser Leu Lys Asn Ala Thr 195 200 205 Gly Leu Gly Leu Ile Val Thr Ser Thr Gly Ile Leu Ile Leu Leu His 210 215 220 Tyr Tyr Val Phe Ser Thr Ala Ile Gly Leu Thr Ser Phe Val Ile Ala 225 230 235 240 Ile Leu Val Ile Gln Val Ile Ala Tyr Ile Leu Ala Val Val Gly Leu 245 250 255 Ser Leu Cys Ile Ala Ala Cys Ile Pro Met His Gly Pro Leu Leu Ile 260 265 270 Ser Gly Leu Ser Ile Leu Ala Leu Ala Gln Leu Leu Gly Leu Val Tyr 275 280 285 Met Lys Phe Val Ala Ser Asn Gln Lys Thr Ile Gln Pro Pro Arg Lys 290 295 300 Ala Val Glu Glu Pro Leu Asn Glu 305 310 <210> 15 <211> 5346 <212> DNA <213> Homo sapiens <400> 15 ggggagcagg cgggggagcg ggcgggaagc agtgggagcg cgcgtgcgcg cggccgtgca 60 gcctgggcag tgggtcctgc ctgtgacgcg cggcggcggt cggtcctgcc tgtaacggcg 120 gcggcggctg ctgctccaga cacctgcggc ggcggcggcg acccgcggc gggcgcggag 180 atgtggcccc tggtagcggc gctgttgctg ggctcggcgt gctgcggatc agctcagcta 240 ctatttaata aaacaaaatc tgtagaattc acgttttgta atgacactgt cgtcattcca 300 tgctttgtta ctaatatgga ggcacaaaac actactgaag tatacgtaaa gtggaaattt 360 aaaggaagag atattacac ctttgatgga gctctaaaca agtccactgt ccccactgac 420 tttagtagtg caaaaattga agtctcacaa ttactaaaag gagatgcctc tttgaagatg 480 gataagagtg atgctgtctc acacacagga aactacactt gtgaagtaac agaattaacc 540 agagaaggtg aaacgatcat cgagctaaaa tatcgtgttg tttcatggtt ttctccaaat 600 gaaaatattc ttattgttat tttcccaatt tttgctatac tcctgttctg gggacagttt 660 ggtattaaaa cacttaaata tagatccggt ggtatggatg agaaaacaat tgctttactt 720 gttgctggac tagtgatcac tgtcattgtc attgttggag ccattctttt cgtcccaggt 780 gaatattcat taaagaatgc tactggcctt ggtttaattg tgactttac agggatatta 840 atattacttc actactatgt gtttagtaca gcgattggat taacctcctt cgtcattgcc 900 atattggtta ttcaggtgat agcctatatc ctcgctgtgg ttggactgag tctctgtatt 960 gcggcgtgta taccaatgca tggccctctt ctgatttcag gtttgagtat cttagctcta 1020 gcacaattac ttggactagt ttatatgaaa tttgtggctt ccaatcagaa gactatacaa 1080 cctcctagga aagctgtaga ggaacccctt aatgcattca aagaatcaaa aggaatgatg 1140 aatgatgaat aactgaagtg aagtgatgga ctccgatttg gagagtagta agacgtgaaa 1200 ggaatacact tgtgtttaag caccatggcc ttgatgattc actgttgggg agaagaaaca 1260 agaaaagtaa ctggttgtca cctatgagac ccttacgtga ttgttagtta agtttttatt 1320 caaagcagct gtaatttagt taataaaata attatgatct atgttgtttg cccaattgag 1380 atccagtttt ttgttgttat tttaatcaa ttaggggcaa tagtagaatg gacaatttcc 1440 aagaatgatg cctttcaggt cctagggcct ctggcctcta ggtaaccagt ttaaattggt 1500 tcagggtgat aactacttag cactgccctg gtgattaccc agagatatct atgaaaacca 1560 gtggcttcca tcaaaccttt gccaactcag gttcacagca gctttgggca gttatggcag 1620 tatggcatta gctgagaggt gtctgccact tctgggtcaa tggaataata attaagtac 1680 aggcaggaat ttggttggga gcatcttgta tgatctccgt atgatgtgat attgatggag 1740 atagtggtcc tcattcttgg gggttgccat tcccacattc ccccttcaac aaacagtgta 1800 acaggtcctt cccagattta gggtactttt attgatggat atgttttcct tttattcaca 1860 taaccccttg aaaccctgtc ttgtcctcct gttacttgct tctgctgtac aagatgtagc 1920 accttttctc ctctttgaac atggtctagt gacacggtag caccagttgc aggaaggagc 1980 cagacttgtt ctcagagcac tgtgttcaca cttttcagca aaatagcta tggttgtaac 2040 atatgtattc ccttcctctg atttgaaggc aaaaatctac agtgtttctt cacttctttt 2100 ctgatctggg gcatgaaaaa agcaagattg aaatttgaac tatgagtctc ctgcatggca 2160 acaaaatgtg tgtcaccatc aggccaacag gccagccctt gaatggggat ttattactgt 2220 tgtatctatg ttgcatgata aacattcatc accttcctcc tgtagtcctg cctcgtactc 2280 cccttcccct atgattgaa agtaaacaa accacattt cctatcctgg ttagagaaa 2340 attaatgttc tgacagttgt gatcgcctgg agtacttta gactttagc attcgttttt 2400 tacctgtttg tggatgtgtg tttgtatgtg catacgtatg agataggcac atgcatcttc 2460 tgtatggaca aaggtggggt acctacagga gagcaaggt taatttgtg cttttagtaa 2520 aaacatttaa atacaaagtt ctttattggg tggaattata tttgatgcaa atatttgatc 2580 acttaaaact tttaaaactt ctagtaatt tgccacgctt tttgactgct caccaatacc 2640 ctgtaaaaat acgtaattct tcctgtttgt gtaataagat attcatattt gtagttgcat 2700 taataatagt tattcttag tccatcagat gttcccgtgt gcctcttta tgccaaattg 2760 attgtcatat ttcatgttgg gaccaagtag ttgcccatg gcaacctaa atttatgacc 2820 tgctgaggcc tctcagaaaa ctgagcatac tagcagaca gctctcttg aaaaaaaaaa 2880 tatgtataca caatatata cgtatatcta tatatacgta tgtataca cacatgtata 2940 ttcttccttg attgtgtagc tgtccaaaat aataacatat atagagggag ctgtattcct 3000 ttatacaaat ctgatggctc ctgcagcact ttttccttct gaaaatattt acattttgct 3060 aacctagttt gttactttaa aaatcagttt tgatgaaagg agggaaaagc agatggactt 3120 gaaaaagatc caagctccta ttagaaaagg tatgaaaatc tttatagtaa aattttttat 3180 aaactaaagt tgtacctttt aatatgtagt aaactctcat ttatttgggg ttcgctcttg 3240 gatctcatcc atccattgtg ttctctttaa tgctgcctgc cttttgaggc attcactgcc 3300 ctagacaatg ccaccagaga tagtggggga aatgccagat gaaaccaact cttgctctca 3360 ctagttgtca gcttctctgg ataagtgacc acagaagcag gagtcctcct gcttgggcat 3420 cattgggcca gttccttctc tttaaatcag atttgtaatg gctcccaaat tccatcacat 3480 cacatttaaa ttgcagacag tgttttgcac atcatgtatc tgttttgtcc cataatatgc 3540 tttttactcc ctgatcccag tttctgctgt tgactcttcc attcagtttt atttattgtg 3600 tgttctcaca gtgacaccat ttgtcctttt ctgcaacaac ctttccagct acttttgcca 3660 aattctattt gtcttctcct tcaaaacatt ctcctttgca gttcctcttc atctgtgtag 3720 ctgctcttt gtctcttaac ttaccattcc tatagtactt tatgcatctc tgcttagttc 3780 tattagtttt ttggccttgc tcttctcctt gattttaaaa ttccttctat agctagagct 3840 tttctttctt tcattctctc ttcctgcagt gttttgcata catcagaagc taggtacata 3900 agttaaatga ttgagagttg gctgtattta gatttatcac ttttaatag ggtgagcttg 3960 agagttttct ttctttctgt tttttttttt tttttttt tttttttt tttttttttt 4020 ttttgactaa tttcacatgc tctaaaaacc ttcaaaggtg attatttttc tcctggaaac 4080 tccaggtcca ttctgtttaa atccctaaga atgtcagaat taaaataaca gggctatccc 4140 gtaattggaa atatttcttt tttcaggatg ctatagtcaa tttagtaagt gaccaccaaa 4200 ttgttatttg cactaacaaa gctcaaaaca cgataagtttt actcctccat ctcagtaata 4260 aaaattaagc tgtaatcaac cttctaggtt tctcttgtct taaaatgggt attcaaaaat 4320 ggggatctgt ggtgtatgta tggaaacaca tactccttaa tttacctgtt gttggaaact 4380 ggagaaatga ttgtcgggca accgtttatt ttttattgta ttttattgg ttgagggatt 4440 tttttataaa cagttttact tgtgtcatat tttaaaatta ctaactgcca tcacctgctg 4500 gggtccttg ttaggtcatt ttcagtgact aatagggata atccaggtaa ctttgaagag 4560 atgagcagtg agtgaccagg cagtttttct gcctttagct ttgacagttc ttaattaaga 4620 tcattgaaga ccagctttct cataaatttc tctttttgaa aaaaagaaag catttgtact 4680 aagctcctct gtaagacaac atcttaaatc ttaaaagtgt tgttatcatg actggtgaga 4740 gaagaaaaca ttttgttttt attaaatgga gcattattta caaaaagcca ttgttgagaa 4800 ttagatccca catcgtataa atatctatta accattctaa ataaagagaa ctccagtgtt 4860 gctatgtgca agatcctctc ttggagcttt tttgcatagc aattaaaggt gtgctatttg 4920 tcagtagcca ttttttgca gtgatttgaa gaccaaagtt gttttacagc tgtgttaccg 4980 ttaaaggttt tttttttat atgtattaaa tcaatttatc actgtttaaa gctttgaata 5040 tctgcaatct ttgccaaggt actttttat ttaaaaaaaa acataacttt gtaaatatta 5100 ccctgtaata ttatatatac ttaataaaac attttaagct attttgttgg gctatttcta 5160 ttgctgctac agcagaccac aagcacattt ctgaaaaatt taatttatta atgtattttt 5220 aagttgctta tattctaggt aacaatgtaa agaatgattt aaaatattaa ttatgaattt 5280 tttgagtata atacccaata agcttttaat tagagcagag ttttaattaa aagttttaaa 5340 tcagtc 5346 <210> 16 <211> 323 <212> PRT <213> Homo sapiens <400> 16 Met Trp Pro Leu Val Ala Ala Leu Leu Leu Gly Ser Ala Cys Cys Gly 1 5 10 15 Ser Ala Gln Leu Leu Phe Asn Lys Thr Lys Ser Val Glu Phe Thr Phe 20 25 30 Cys Asn Asp Thr Val Val Ile Pro Cys Phe Val Thr Asn Met Glu Ala 35 40 45 Gln Asn Thr Thr Glu Val Tyr Val Lys Trp Lys Phe Lys Gly Arg Asp 50 55 60 Ile Tyr Thr Phe Asp Gly Ala Leu Asn Lys Ser Thr Val Pro Thr Asp 65 70 75 80 Phe Ser Ser Ala Lys Ile Glu Val Ser Gln Leu Leu Lys Gly Asp Ala 85 90 95 Ser Leu Lys Met Asp Lys Ser Asp Ala Val Ser His Thr Gly Asn Tyr 100 105 110 Thr Cys Glu Val Thr Glu Leu Thr Arg Glu Gly Glu Thr Ile Ile Glu 115 120 125 Leu Lys Tyr Arg Val Val Ser Trp Phe Ser Pro Asn Glu Asn Ile Leu 130 135 140 Ile Val Ile Phe Pro Ile Phe Ala Ile Leu Leu Phe Trp Gly Gln Phe 145 150 155 160 Gly Ile Lys Thr Leu Lys Tyr Arg Ser Gly Gly Met Asp Glu Lys Thr 165 170 175 Ile Ala Leu Leu Val Ala Gly Leu Val Ile Thr Val Ile Val Ile Val 180 185 190 Gly Ala Ile Leu Phe Val Pro Gly Glu Tyr Ser Leu Lys Asn Ala Thr 195 200 205 Gly Leu Gly Leu Ile Val Thr Ser Thr Gly Ile Leu Ile Leu Leu His 210 215 220 Tyr Tyr Val Phe Ser Thr Ala Ile Gly Leu Thr Ser Phe Val Ile Ala 225 230 235 240 Ile Leu Val Ile Gln Val Ile Ala Tyr Ile Leu Ala Val Val Gly Leu 245 250 255 Ser Leu Cys Ile Ala Ala Cys Ile Pro Met His Gly Pro Leu Leu Ile 260 265 270 Ser Gly Leu Ser Ile Leu Ala Leu Ala Gln Leu Leu Gly Leu Val Tyr 275 280 285 Met Lys Phe Val Ala Ser Asn Gln Lys Thr Ile Gln Pro Pro Arg Lys 290 295 300 Ala Val Glu Glu Pro Leu Asn Ala Phe Lys Glu Ser Lys Gly Met Met 305 310 315 320 Asn Asp Glu <210> 17 <211> 293 <212> PRT <213> Artificial sequence <220> <221> source <223> / Note="Artificial sequence description: Synthetic Peptide" <400> 17 Met Trp Pro Leu Ala Ala Ala Leu Leu Leu Gly Ser Cys Cys Cys Gly 1 5 10 15 Ser Ala Gln Leu Leu Phe Asn Lys Thr Lys Ser Val Glu Phe Thr Phe 20 25 30 Cys Asn Asp Thr Val Val Ile Pro Cys Phe Val Thr Asn Met Glu Ala 35 40 45 Gln Asn Thr Thr Glu Val Tyr Val Lys Trp Lys Phe Lys Gly Arg Asp 50 55 60 Ile Tyr Thr Phe Asp Gly Ala Leu Asn Lys Ser Thr Val Pro Thr Asp 65 70 75 80 Phe Ser Ser Ala Lys Ile Glu Val Ser Gln Leu Leu Lys Gly Asp Ala 85 90 95 Ser Leu Lys Met Asp Lys Ser Asp Ala Val Ser His Thr Gly Asn Tyr 100 105 110 Thr Cys Glu Val Thr Glu Leu Thr Arg Glu Gly Glu Thr Ile Ile Glu 115 120 125 Leu Lys Tyr Arg Val Val Ser Trp Phe Ser Pro Asn Glu Asn Ile Leu 130 135 140 Ile Val Ile Phe Pro Ile Phe Ala Ile Leu Leu Phe Trp Gly Gln Phe 145 150 155 160 Gly Ile Lys Thr Leu Lys Tyr Arg Ser Gly Gly Met Asp Glu Lys Thr 165 170 175 Ile Ala Leu Leu Val Ala Gly Leu Val Ile Thr Val Ile Val Ile Val 180 185 190 Gly Ala Ile Leu Phe Val Pro Gly Glu Tyr Ser Leu Lys Asn Ala Thr 195 200 205 Gly Leu Gly Leu Ile Val Thr Ser Thr Gly Ile Leu Ile Leu Leu His 210 215 220 Tyr Tyr Val Phe Ser Thr Ala Ile Gly Leu Thr Ser Phe Val Ile Ala 225 230 235 240 Ile Leu Val Ile Gln Val Ile Ala Tyr Ile Leu Ala Val Val Gly Leu 245 250 255 Ser Leu Cys Ile Ala Ala Cys Ile Pro Met His Gly Pro Leu Leu Ile 260 265 270 Ser Gly Leu Ser Ile Leu Ala Leu Ala Gln Leu Leu Gly Leu Val Tyr 275 280 285 Met Lys Phe Val Glu 290 <210> 18 <211> 305 <212> PRT <213> Artificial sequence <220> <221> source <223> / Note="Artificial sequence description: Synthetic Peptide" <400> 18 Met Trp Pro Leu Ala Ala Ala Leu Leu Leu Gly Ser Cys Cys Cys Gly 1 5 10 15 Ser Ala Gln Leu Leu Phe Asn Lys Thr Lys Ser Val Glu Phe Thr Phe 20 25 30 Cys Asn Asp Thr Val Val Ile Pro Cys Phe Val Thr Asn Met Glu Ala 35 40 45 Gln Asn Thr Thr Glu Val Tyr Val Lys Trp Lys Phe Lys Gly Arg Asp 50 55 60 Ile Tyr Thr Phe Asp Gly Ala Leu Asn Lys Ser Thr Val Pro Thr Asp 65 70 75 80 Phe Ser Ser Ala Lys Ile Glu Val Ser Gln Leu Leu Lys Gly Asp Ala 85 90 95 Ser Leu Lys Met Asp Lys Ser Asp Ala Val Ser His Thr Gly Asn Tyr 100 105 110 Thr Cys Glu Val Thr Glu Leu Thr Arg Glu Gly Glu Thr Ile Ile Glu 115 120 125 Leu Lys Tyr Arg Val Val Ser Trp Phe Ser Pro Asn Glu Asn Ile Leu 130 135 140 Ile Val Ile Phe Pro Ile Phe Ala Ile Leu Leu Phe Trp Gly Gln Phe 145 150 155 160 Gly Ile Lys Thr Leu Lys Tyr Arg Ser Gly Gly Met Asp Glu Lys Thr 165 170 175 Ile Ala Leu Leu Val Ala Gly Leu Val Ile Thr Val Ile Val Ile Val 180 185 190 Gly Ala Ile Leu Phe Val Pro Gly Glu Tyr Ser Leu Lys Asn Ala Thr 195 200 205 Gly Leu Gly Leu Ile Val Thr Ser Thr Gly Ile Leu Ile Leu Leu His 210 215 220 Tyr Tyr Val Phe Ser Thr Ala Ile Gly Leu Thr Ser Phe Val Ile Ala 225 230 235 240 Ile Leu Val Ile Gln Val Ile Ala Tyr Ile Leu Ala Val Val Gly Leu 245 250 255 Ser Leu Cys Ile Ala Ala Cys Ile Pro Met His Gly Pro Leu Leu Ile 260 265 270 Ser Gly Leu Ser Ile Leu Ala Leu Ala Gln Leu Leu Gly Leu Val Tyr 275 280 285 Met Lys Phe Val Ala Ser Asn Gln Arg Thr Ile Gln Pro Pro Arg Asn 290 295 300 Arg 305 <210> 19 <211> 312 <212> PRT <213> Artificial sequence <220> <221> source <223> / Note="Artificial sequence description: Synthetic Peptide" <400> 19 Met Trp Pro Leu Ala Ala Ala Leu Leu Leu Gly Ser Cys Cys Cys Gly 1 5 10 15 Ser Ala Gln Leu Leu Phe Asn Lys Thr Lys Ser Val Glu Phe Thr Phe 20 25 30 Cys Asn Asp Thr Val Val Ile Pro Cys Phe Val Thr Asn Met Glu Ala 35 40 45 Gln Asn Thr Thr Glu Val Tyr Val Lys Trp Lys Phe Lys Gly Arg Asp 50 55 60 Ile Tyr Thr Phe Asp Gly Ala Leu Asn Lys Ser Thr Val Pro Thr Asp 65 70 75 80 Phe Ser Ser Ala Lys Ile Glu Val Ser Gln Leu Leu Lys Gly Asp Ala 85 90 95 Ser Leu Lys Met Asp Lys Ser Asp Ala Val Ser His Thr Gly Asn Tyr 100 105 110 Thr Cys Glu Val Thr Glu Leu Thr Arg Glu Gly Glu Thr Ile Ile Glu 115 120 125 Leu Lys Tyr Arg Val Val Ser Trp Phe Ser Pro Asn Glu Asn Ile Leu 130 135 140 Ile Val Ile Phe Pro Ile Phe Ala Ile Leu Leu Phe Trp Gly Gln Phe 145 150 155 160 Gly Ile Lys Thr Leu Lys Tyr Arg Ser Gly Gly Met Asp Glu Lys Thr 165 170 175 Ile Ala Leu Leu Val Ala Gly Leu Val Ile Thr Val Ile Val Ile Val 180 185 190 Gly Ala Ile Leu Phe Val Pro Gly Glu Tyr Ser Leu Lys Asn Ala Thr 195 200 205 Gly Leu Gly Leu Ile Val Thr Ser Thr Gly Ile Leu Ile Leu Leu His 210 215 220 Tyr Tyr Val Phe Ser Thr Ala Ile Gly Leu Thr Ser Phe Val Ile Ala 225 230 235 240 Ile Leu Val Ile Gln Val Ile Ala Tyr Ile Leu Ala Val Val Gly Leu 245 250 255 Ser Leu Cys Ile Ala Ala Cys Ile Pro Met His Gly Pro Leu Leu Ile 260 265 270 Ser Gly Leu Ser Ile Leu Ala Leu Ala Gln Leu Leu Gly Leu Val Tyr 275 280 285 Met Lys Phe Val Ala Ser Asn Gln Arg Thr Ile Gln Pro Pro Arg Lys 290 295 300 Ala Val Glu Glu Pro Leu Asn Glu 305 310 <210> 20 <211> 323 <212> PRT <213> artificial sequence <220> <221> source <223> / Note="Artificial sequence description: Synthetic Peptide" <400> 20 Met Trp Pro Leu Ala Ala Ala Leu Leu Leu Gly Ser Cys Cys Cys Gly 1 5 10 15 Ser Ala Gln Leu Leu Phe Asn Lys Thr Lys Ser Val Glu Phe Thr Phe 20 25 30 Cys Asn Asp Thr Val Val Ile Pro Cys Phe Val Thr Asn Met Glu Ala 35 40 45 Gln Asn Thr Thr Glu Val Tyr Val Lys Trp Lys Phe Lys Gly Arg Asp 50 55 60 Ile Tyr Thr Phe Asp Gly Ala Leu Asn Lys Ser Thr Val Pro Thr Asp 65 70 75 80 Phe Ser Ser Ala Lys Ile Glu Val Ser Gln Leu Leu Lys Gly Asp Ala 85 90 95 Ser Leu Lys Met Asp Lys Ser Asp Ala Val Ser His Thr Gly Asn Tyr 100 105 110 Thr Cys Glu Val Thr Glu Leu Thr Arg Glu Gly Glu Thr Ile Ile Glu 115 120 125 Leu Lys Tyr Arg Val Val Ser Trp Phe Ser Pro Asn Glu Asn Ile Leu 130 135 140 Ile Val Ile Phe Pro Ile Phe Ala Ile Leu Leu Phe Trp Gly Gln Phe 145 150 155 160 Gly Ile Lys Thr Leu Lys Tyr Arg Ser Gly Gly Met Asp Glu Lys Thr 165 170 175 Ile Ala Leu Leu Val Ala Gly Leu Val Ile Thr Val Ile Val Ile Val 180 185 190 Gly Ala Ile Leu Phe Val Pro Gly Glu Tyr Ser Leu Lys Asn Ala Thr 195 200 205 Gly Leu Gly Leu Ile Val Thr Ser Thr Gly Ile Leu Ile Leu Leu His 210 215 220 Tyr Tyr Val Phe Ser Thr Ala Ile Gly Leu Thr Ser Phe Val Ile Ala 225 230 235 240 Ile Leu Val Ile Gln Val Ile Ala Tyr Ile Leu Ala Val Val Gly Leu 245 250 255 Ser Leu Cys Ile Ala Ala Cys Ile Pro Met His Gly Pro Leu Leu Ile 260 265 270 Ser Gly Leu Ser Ile Leu Ala Leu Ala Gln Leu Leu Gly Leu Val Tyr 275 280 285 Met Lys Phe Val Ala Ser Asn Gln Arg Thr Ile Gln Pro Pro Arg Lys 290 295 300 Ala Val Glu Glu Pro Leu Asn Ala Phe Lys Glu Ser Lys Gly Met Met 305 310 315 320 Asn Asp Glu <210> twenty one <211> twenty two <212> DNA <213> Artificial sequence <220> <221> source <223> / Note="Artificial sequence description: Synthetic Primer" <400> twenty one tgcagaagtc actaggagga at 22 <210> twenty two <211> 27 <212> DNA <213> Artificial sequence <220> <221> source <223> / Note="Artificial sequence description: Synthetic Probe" <400> twenty two tcagtcaact tcttctgggt tgtttcc 27 <210> twenty three <211> twenty three <212> DNA <213> Artificial sequence <220> <221> source <223> / Note="Artificial sequence description: Synthetic Primer" <400> twenty three gtgccagact cactttctat cca 23 <210> twenty four <211> twenty two <212> DNA <213> Artificial sequence <220> <221> source <223> / Note="Artificial sequence description: Synthetic Primer" <400> twenty four tgctgccaat atacggcttc tg 22 <210> 25 <211> 26 <212> DNA <213> Artificial sequence <220> <221> source <223> / Note="Artificial sequence description: Synthetic Probe" <400> 25 cagctctcat agccaactat ggtgcc 26 <210> 26 <211> twenty two <212> DNA <213> Artificial sequence <220> <221> source <223> / Note="Artificial sequence description: Synthetic Primer" <400> 26 tcaagcagag cctggttatc tg 22 <210> 27 <211> twenty three <212> DNA <213> Artificial sequence <220> <221> source <223> / Note="Artificial sequence description: Synthetic Primer" <400> 27 gtcgtcattc catgctttgt tac 23 <210> 28 <211> 30 <212> DNA <213> Artificial sequence <220> <221> source <223> / Note="Artificial sequence description: Synthetic Probe" <400> 28 tggaggcaca aaacactact gaagtatacg 30 <210> 29 <211> twenty two <212> DNA <213> Artificial sequence <220> <221> source <223> / Note="Artificial sequence description: Synthetic Primer" <400> 29 ggacagtgga cttgtttaga gc 22 <210> 30 <211> twenty one <212> DNA <213> Artificial sequence <220> <221> source <223> / Note="Artificial sequence description: Synthetic Primer" <400> 30 ggcttggtgg ctgattgttc t 21 <210> 31 <211> 28 <212> DNA <213> Artificial sequence <220> <221> source <223> / Note="Artificial sequence description: Synthetic Probe" <400> 31 agcacccaaa ctgatatgcc tgtatttg 28 <210> 32 <211> twenty three <212> DNA <213> Artificial sequence <220> <221> source <223> / Note="Artificial sequence description: Synthetic Primer" <400> 32 tgggaactgg tgtttcaagt cta 23 <210> 33 <211> 158 <212> DNA <213> Artificial sequence <220> <221> source <223> / Note="Artificial sequence description: Synthetic Polynucleotide" <400> 33 gcagacatga ttacttcaga gctttcaaag ctagatactg taccttgcat attccaacac 60 gcgatcgcat tttaagattt tccatcctag tggaaagata tgatttgatt catcctattt 120 actttgtata ttaaagtaca gtagaacctg ccactttt 158 <210> 34 <211> 160 <212> DNA <213> Artificial sequence <220> <221> source <223> / Note="Artificial sequence description: Synthetic Polynucleotide" <400> 34 ggatccattt taagtaatag aataggattt ttaattgttc cagtgtttct gtgatagagc 60 tgtcctgcac agacctgttt ctcgagataa cttcgtataa tgtatgctat acgaagttat 120 atgcatggcc tccgcgccgg gttttggcgc ctcccgcggg 160 <210> 35 <211> 160 <212> DNA <213> Artificial sequence <220> <221> source <223> / Note="Artificial sequence description: Synthetic Polynucleotide" <400> 35 catgtctgga ataacttcgt ataatgtatg ctatacgaag ttatgctagt aactataacg 60 gtcctaaggt agcgactagc attagtatgg aaggtccgtc cactgtccag gttcctcttg 120 cggagctctt tgtctctctg gactctgtat acactgcttg 160 <210> 36 <211> 239 <212> DNA <213> Artificial sequence <220> <221> source <223> / Note="Artificial sequence description: Synthetic Polynucleotide" <400> 36 ggatccattt taagtaatag aataggattt ttaattgttc cagtgtttct gtgatagagc 60 tgtcctgcac agacctgttt ctcgagataa cttcgtataa tgtatgctat acgaagttat 120 gctagtaact ataacggtcc taaggtagcg actagcatta gtatggaagg tccgtccact 180 gtccaggttc ctcttgcgga gctctttgtc tctctggact ctgtatacac tgcttgcat 239 BRIEF DESCRIPTION OF THE DRAWINGS

[0059] This patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee.

[0060] The drawings consisting of the following figures included herein are for illustration purposes only and are not intended to be limiting.

[0061] Figure 1Schematic diagram not to scale showing the genomic organization of the non-human (eg, mouse) and human cluster of differentiation 47 (CD47) genes. The numbering of the exons is below each exon.

[0062] Figure 2 A schematic diagram not to scale shows an exemplary method for humanizing a non-human cluster of differentiation 47 (CD47) gene.

[0063] Figure 3 Schematic diagram not to scale showing the genomic organization of the mouse and human cluster of differentiation 47 (CD47) genes. The locations of the probes used in the assays described in Example 1 are indicated.

[0064] Figure 4 An exemplary histogram of CD47 expression in erythrocytes from humanized CD47 mice detected by anti-CD47 antibodies is shown. Ab A, Ab B, Ab C, Ab D, and Ab E: anti-CD47 antibodies; hIgG4s: human IgG4 with a modified Fc region with reduced effector function and irrelevant specificity; hIgG4: human IgG4 antibody with irrelevant specificity.

[0065] Figure 5 Exemplary hemagglutination of mouse erythrocytes from wild-type (n=2) and humanized CD47 (n=2) mice is shown by anti-CD47 antibodies. WT: wild-type; HuCD47: humanized CD47; Ab A, Ab B, Ab C, Ab D, and Ab E: anti-CD47 antibodies; hIgG4s: human IgG4 with a modified Fc region with reduced effector function and with irrelevant specificity; hIgG4: human IgG4 antibody with irrelevant specificity.

[0066] Figure 6 Exemplary pharmacokinetic profiles of anti-CD47 antibodies in humanized CD47 mice are shown as antibody concentration (in μg / mL, y-axis) over time (in days, x-axis); Ab F, Ab G, Ab H, and Ab I: anti-CD47 antibodies; hIgG4s: human IgG4 antibodies with a modified Fc region with reduced effector function and with an irrelevant specificity.

[0067] Figure 7 The results show the antibody concentration (in mcg / mL, y-axis) over time (in days, x-axis) in humanized CD47 / SIRPα mice (CD47 hu / hu SIRPα hu / hu ) in the exemplary pharmacokinetic profile of the anti-CD47 antibody. Ab J, Ab F, AbG and Ab I: anti-CD47 antibodies; hIgG4s: human IgG4 antibodies with a modified Fc region with reduced effector function and with irrelevant specificity.

[0068] Figure 8 The results show the antibody concentration (in mcg / mL, y-axis) over time (in days, x-axis) in humanized CD47 / SIRPα mice (CD47 hu / hu SIRPα hu / hu ) in the exemplary pharmacokinetic profile of the anti-CD47 antibody. Ab J, Ab F: anti-CD47 antibodies; Ab Fs: Ab F with a modified Fc region with reduced effector function; Ab Fmono: a monovalent version of Ab F; hIgG4s: a human IgG4 antibody with a modified Fc region with reduced effector function and with an unrelated specificity.

[0069] Fig. 9 An exemplary pharmacokinetic profile of an anti-CD47 antibody in wild-type mice is shown as antibody concentration (in mcg / mL, y-axis) over time (in days, x-axis). Mice exhibiting mouse anti-human antibody response (MAHA) were excluded. Ab J, Ab F, Ab I, and Ab G: anti-CD47 antibodies; hIgG4s: human IgG4 antibodies with modified Fc regions with reduced effector function and irrelevant specificity (asterisk: all points excluded from the hIgG4s treatment group after 15 days due to MAHA); AbJ F(ab')2: F(ab')2 fragment of Ab J.

[0070] definition

[0071] The present invention is not limited to the specific methods and experimental conditions described herein, as such methods and conditions may vary. It should also be understood that the terminology used herein is for the purpose of describing specific embodiments only, and is not intended to be limiting, as the scope of the present invention is limited only by the claims.

[0072] Unless otherwise defined, all terms and phrases used herein include the meanings that the terms and phrases have acquired in the art, unless expressly indicated to the contrary or clearly contrary from the context in which the term or phrase is used. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, specific methods and materials are now described. All publications mentioned are hereby incorporated by reference.

[0073] The term "approximately" as used herein for one or more target values ​​refers to a value similar to the reference value. In certain embodiments, unless otherwise indicated or obviously different according to context, the term "approximately" or "about" refers to 25%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1% or a smaller range of values ​​(except that such a number will exceed 100% of a possible value) falling on the reference value in either direction (greater than or less than).

[0074] As used herein, the term "biological activity" refers to the property of any agent that is active in a biological system, either in vitro or in vivo (e.g., in an organism). For example, an agent that has a biological effect in vivo when present in an organism is considered to have biological activity. In specific embodiments, when a protein or polypeptide has biological activity, a portion of the protein or polypeptide that shares at least one biological activity of the protein or polypeptide is generally referred to as a "biologically active" portion.

[0075] As used herein, the term "comparable" refers to two or more agents, entities, conditions, sets of conditions, etc., which may be different from each other but are sufficiently similar to allow comparisons to be made between them so that conclusions can be reasonably drawn based on the observed differences or similarities. One of ordinary skill in the art will understand in context what degree of identity is required for two or more such agents, entities, conditions, sets of conditions, etc. to be considered comparable in any given case.

[0076] The term "conservative" as used herein to describe conservative amino acid substitutions refers to the substitution of an amino acid residue by another amino acid residue having a side chain R group with similar chemical properties (e.g., charge or hydrophobicity). In general, conservative amino acid substitutions do not substantially change the target functional properties of a protein, for example, the ability of a receptor to bind a ligand. Examples of groups of amino acids with side chains with similar chemical properties include: aliphatic side chains such as glycine, alanine, valine, leucine, and isoleucine; aliphatic-hydroxy side chains such as serine and threonine; amide-containing side chains such as asparagine and glutamine; aromatic side chains such as phenylalanine, tyrosine, and tryptophan; basic side chains such as lysine, arginine, and histidine; acidic side chains such as aspartic acid and glutamic acid; and sulfur-containing side chains such as cysteine ​​and methionine. Conservative amino acid substitution groups include, for example, valine / leucine / isoleucine, phenylalanine / tyrosine, lysine / arginine, alanine / valine, glutamic acid / aspartic acid, and asparagine / glutamine. In some embodiments, conservative amino acid substitutions may be substitutions of alanine to any native residue in a protein, such as used in, for example, alanine scanning mutagenesis. In some embodiments, conservative substitutions are substitutions with positive values ​​in the PAM250 log-likelihood matrix disclosed in Gonnet et al. (1992) Exhaustive Matching of the Entire Protein Sequence Database, Science 256: 1443-45 (incorporated by reference). In some embodiments, a substitution is considered to be "moderately conservative" if it has a non-negative value in the PAM250 log-likelihood matrix.

[0077] As used herein, the term "control" refers to the meaning of "control" as understood in the art, as a standard for result comparison. Typically, the control is used to enhance experimental integrity by separating variables to draw conclusions about such variables. In some embodiments, the control is a reaction or determination carried out simultaneously with a test reaction or determination to provide a contrast. As used herein, "control" may refer to a "control animal". A "control animal" may have a modification as described herein, a modification different from the modification described herein, or no modification (i.e., a wild-type animal). In an experiment, a "test" (i.e., a variable tested) is applied. In a second experiment "control", the variable tested is not applied. In some embodiments, the control is a historical control (i.e., a test or determination previously performed, or an amount or result previously known). In some embodiments, the control is or includes a record printed or otherwise preserved. The control may be a positive control or a negative control.

[0078] As used herein, the term "disruption" refers to the result of homologous recombination with a DNA molecule (e.g., with an endogenous homologous sequence such as a gene or locus). In some embodiments, disruption can achieve or represent insertion, deletion, substitution, replacement, missense mutation, or DNA sequence frameshift or a combination thereof. Insertion can include insertion of an entire gene or gene fragment, such as an exon, which can be derived from other sources (e.g., heterologous sequences) other than an endogenous sequence. In some embodiments, disruption can increase the expression and / or activity of a gene or gene product (e.g., a protein encoded by a gene). In some embodiments, disruption can reduce the expression and / or activity of a gene or gene product. In some embodiments, disruption can change the sequence of a gene or an encoded gene product (e.g., an encoded protein). In some embodiments, disruption can truncate a gene or an encoded gene product (e.g., an encoded protein) or fragment it. In some embodiments, disruption can extend a gene or an encoded gene product; in some such embodiments, disruption can achieve the assembly of a fusion protein. In some embodiments, disruption can affect the level of a gene or a gene product but not its activity. In some embodiments, disruption can affect the activity of a gene or a gene product but not its level. In some embodiments, the destruction may have no significant effect on the level of a gene or a gene product. In some embodiments, the destruction may have no significant effect on the activity of a gene or a gene product. In some embodiments, the destruction may have no significant effect on the level or activity of a gene or a gene product.

[0079] The terms "determine," "measure," "evaluate," "assess," "assay," and "analyze" are used interchangeably herein to refer to any form of measurement, and include determining whether an element is present or not. These terms include quantitative and / or qualitative determinations. Determinations may be relative or absolute. "Determining the presence of" includes determining the amount of something present and / or determining whether it is present or not.

[0080] As used herein, the term "endogenous locus" or "endogenous gene" refers to a genetic locus that is found in a parent or reference organism prior to the introduction of a break, deletion, substitution, alteration, or modification as described herein. In some embodiments, the endogenous locus has a naturally occurring sequence. In some embodiments, the endogenous locus is a wild-type locus. In some embodiments, the reference organism is a wild-type organism. In some embodiments, the reference organism is an engineered organism. In some embodiments, the reference organism is a laboratory-grown organism (whether wild-type or engineered).

[0081] The term "endogenous promoter" refers to a promoter naturally associated with an endogenous gene, such as in a wild-type organism.

[0082] As used herein, the term "heterologous" refers to an agent or entity from a different source. For example, when a reference polypeptide, gene, or gene product is used or present in a particular cell or organism, the term illustrates that the relevant polypeptide, gene, or gene product: 1) is artificially engineered; 2) is artificially (e.g., via genetic engineering) introduced into a cell or organism (or a precursor thereof); and / or 3) is non-naturally produced by a related cell or organism (e.g., a related cell type or organism type) or is non-naturally present in the cell or organism.

[0083] As used herein, the term "host cell" refers to a cell into which a heterologous (e.g., exogenous) nucleic acid or protein has been introduced. Those skilled in the art will appreciate that such terms refer not only to a specific subject cell, but also to the progeny of the cell after reading this disclosure. Because some modifications may occur in subsequent generations due to mutations or environmental influences, such progeny may not in fact be equivalent to the parent cell, but are still included in the scope of the term "host cell" as used herein. In some embodiments, the host cell is a prokaryotic or eukaryotic cell or comprises a prokaryotic or eukaryotic cell. Generally speaking, a host cell is any cell suitable for receiving and / or producing a heterologous nucleic acid or protein, regardless of the kingdom of life to which the cell is designated. Exemplary cells include cells of prokaryotes and eukaryotes (single or multicellular), bacterial cells (e.g., strains of E. coli, Bacillus spp., Streptomyces spp., etc.), mycobacterial cells, fungal cells, yeast cells (e.g., S. cerevisiae, S. pombe, P. pastoris, P. methanolica, etc.), plant cells, insect cells (e.g., SF-9, SF-21, baculovirus-infected insect cells, Trichoplusia ni, etc.), non-human animal cells, human cells, or cell fusions such as hybridomas or quadromas. In some embodiments, the cells are human, monkey, ape, hamster, rat, or mouse cells. In some embodiments, the cell is a eukaryotic cell and is selected from the following cells: CHO (e.g., CHO K1, DXB-11 CHO, Veggie-CHO), COS (e.g., COS-7), retinal cells, Vero, CV1, kidney (e.g., HEK293, 293EBNA, MSR 293, MDCK, HaK, BHK), HeLa, HepG2, WI38, MRC 5, Colo205, HB 8065, HL-60, (e.g., BHK21), Jurkat, Daudi, A431 (epidermal), CV-1, U937, 3T3, L cells, C127 cells, SP2 / 0, NS-0, MMT 060562, Sertoli cells, BRL 3A cells, HT1080 cells, myeloma cells, tumor cells, and cell lines derived from the foregoing cells. In some embodiments, the cell comprises one or more viral genes, such as retinal cells expressing viral genes (e.g., PER.C6TM cells). In some embodiments, the host cell is an isolated cell or comprises an isolated cell. In some embodiments, the host cell is a part of a tissue. In some embodiments, the host cell is a part of an organism.

[0084] As used herein, the term "humanization" refers to a nucleic acid or protein according to its field-understood meaning: the structure of the nucleic acid or protein (i.e., nucleotide or amino acid sequence) includes a portion that substantially or completely corresponds to the structure of a specific gene or protein naturally present in a non-human animal, and also includes a portion that is different from the portion present in the relevant specific non-human gene or protein but more closely corresponds to the comparable structure present in the corresponding human gene or protein. In some embodiments, a "humanized" gene is a gene encoding a polypeptide having an amino acid sequence substantially identical to the amino acid sequence of a human polypeptide (e.g., a human protein or a portion thereof-e.g., a characteristic portion thereof). To give just one example, in the case of a membrane receptor, a "humanized" gene may encode a polypeptide having an extracellular portion, the amino acid sequence of the extracellular portion being identical to the amino acid sequence of the human extracellular portion, and the remaining sequence being identical to the sequence of a non-human (e.g., mouse) polypeptide. In some embodiments, a humanized gene comprises at least a portion of the DNA sequence of a human gene. In some embodiments, a humanized gene comprises the complete DNA sequence of a human gene. In some embodiments, a humanized protein comprises a sequence of a portion that appears in a human protein. In some embodiments, a humanized protein comprises the entire sequence of a human protein and is expressed from an endogenous locus of a non-human animal corresponding to a homolog or ortholog of the human gene.

[0085] The term "identity" as used herein in conjunction with sequence comparison refers to the identity as determined by any of the many different algorithms known in the art that can be used to measure nucleotide and / or amino acid sequence identity. In some embodiments, ClustalW v.1.83 (slow) alignment (using an open gap penalty of 10.0, an extension gap penalty of 0.1) and using a Gonnet similarity matrix (MACVECTORTM10.0.2, MacVector Inc., 2008) are used to determine the identity described herein.

[0086] As used herein, the term "isolated" refers to substances and / or entities that (1) have been separated from at least some of the components with which they were associated when originally produced (whether naturally or in an experimental setting), and / or (2) have been artificially designed, produced, prepared and / or manufactured. An isolated substance and / or entity may be separated from about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or more than about 99% of other components with which they were originally associated. In some embodiments, the isolated agent is about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99% or more than about 99% pure. As used herein, a substance is "pure" if it is substantially free of other components. In some embodiments, as will be understood by those skilled in the art, a substance may still be considered "isolated" or even "pure" after being combined with certain other components, such as one or more carriers or excipients (e.g., buffers, solvents, water, etc.); in such embodiments, the percentage of separation or purity of the substance is calculated without including such carriers or excipients. To give just one example, in some embodiments, a naturally occurring biopolymer such as a polypeptide or polynucleotide is considered "isolated" a) when, by virtue of the origin or source from which it is derived, it is not associated with some or all of the components that naturally accompany it in its native state; b) when it is substantially free of other polypeptides or nucleic acids of the same species as the species in which it is naturally produced; c) when expressed by or otherwise associated with components from a cell or other expression system of a species other than the species in which it is naturally produced. Thus, for example, in some embodiments, a polypeptide that is chemically synthesized or synthesized in a cellular system different from the cellular system in which it is naturally produced is considered an "isolated" polypeptide. Alternatively or additionally, in some embodiments, a polypeptide that has been subjected to one or more purification techniques may be considered an "isolated" polypeptide to the extent that it has been separated from other components with which it is a) associated in nature; and / or b) associated when originally produced.

[0087] As used herein, the term "non-human animal" refers to any non-human vertebrate organism. In some embodiments, the non-human animal is a cyclostomum vertebrate, a bony fish, a cartilaginous fish (e.g., a shark or ray), an amphibian, a reptile, a mammal, or a bird. In some embodiments, the non-human mammal is a primate, a goat, a sheep, a pig, a dog, a cow, or a rodent. In some embodiments, the non-human animal is a rodent such as a rat or a mouse.

[0088] As used herein, the term "nucleic acid" refers to any compound and / or substance that is or can be incorporated into an oligonucleotide chain in its broadest sense. In some embodiments, "nucleic acid" is a compound and / or substance that is or can be incorporated into an oligonucleotide chain through a phosphodiester linkage. As can be seen from the context, in some embodiments, "nucleic acid" refers to a single nucleic acid residue (e.g., nucleotide and / or nucleoside); in some embodiments, "nucleic acid" refers to an oligonucleotide chain containing a single nucleic acid residue. In some embodiments, "nucleic acid" is RNA or contains RNA; in some embodiments, "nucleic acid" is DNA or contains DNA. In some embodiments, "nucleic acid" is one or more natural nucleic acid residues, contains one or more natural nucleic acid residues, or consists of one or more natural nucleic acid residues. In some embodiments, "nucleic acid" is one or more nucleic acid analogs, contains one or more nucleic acid analogs, or consists of one or more nucleic acid analogs. In some embodiments, the difference between nucleic acid analogs and "nucleic acids" is that they do not use a phosphodiester backbone. For example, in some embodiments, the "nucleic acid" is, comprises, or consists of one or more "peptide nucleic acids", which are known in the art and have peptide bonds in the backbone rather than phosphodiester bonds, which are considered to be within the scope of the present invention. Alternatively or additionally, in some embodiments, the "nucleic acid" has one or more phosphorothioate and / or 5'-N-phosphoramidite linkages rather than phosphodiester bonds. In some embodiments, the "nucleic acid" is, comprises, or consists of one or more natural nucleosides (e.g., adenosine, thymidine, guanosine, cytidine, uridine, deoxyadenosine, deoxythymidine, deoxyguanosine, and deoxycytidine). In some embodiments, a "nucleic acid" is one or more nucleoside analogs (e.g., 2-aminoadenosine, 2-thiothymidine, inosine, pyrrolopyrimidine, 3-methyladenosine, 5-methylcytidine, C-5 propynyl-cytidine, C-5 propynyl-uridine, 2-aminoadenosine, C5-bromouridine, C5-fluorouridine, C5-iodouridine, C5-propynyl-uridine, C5-propynyl-cytidine, C5-methylcytidine, 2-aminoadenosine, 7-deazaadenosine, 7-deazaguanosine, 8-oxoadenosine, 8-oxoguanosine, O(6)-methylguanine, 2-thiocytidine, methylated bases, intercalated bases, and combinations thereof), comprises one or more nucleoside analogs, or consists of one or more nucleoside analogs. In some embodiments, a "nucleic acid" comprises one or more modified sugars (e.g., 2'-fluororibose, ribose, 2'-deoxyribose, arabinose, and hexose) compared to sugars in natural nucleic acids. In some embodiments, a "nucleic acid" has a nucleotide sequence encoding a functional gene product such as an RNA or a protein. In some embodiments, a "nucleic acid" includes one or more introns.In some embodiments, a "nucleic acid" is prepared by one or more of the following: isolation from a natural source, enzymatic synthesis by complementary template-based polymerization (in vivo or in vitro), replication in a recombinant cell or system, chemical synthesis, and combinations thereof. In some embodiments, a "nucleic acid" is at least 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 20, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, 500, 600, 700, 800, 900, 1000, 1500, 2000, 2500, 3000, 3500, 4000, 4500, 5000, or more residues in length. In some embodiments, the "nucleic acid" is single-stranded; in some embodiments, the "nucleic acid" is double-stranded. In some embodiments, the "nucleic acid" has a nucleotide sequence comprising at least one element that encodes a polypeptide or is complementary to a sequence encoding a polypeptide. In some embodiments, the "nucleic acid" has enzymatic activity.

[0089] As used herein, the term "operably linked" refers to a juxtaposition in which the components described are in a relationship that allows them to function in the intended manner. A control sequence that is "operably linked" to a coding sequence is linked in such a way that expression of the coding sequence is obtained under conditions compatible with the control sequence. "Operably linked" sequences include expression control sequences adjacent to a target gene and expression control sequences that act in a trans manner or act remotely to control a target gene. As used herein, the term "expression control sequence" refers to a polynucleotide sequence that is necessary to achieve expression and processing of a coding sequence to which it is linked. "Expression control sequences" include suitable transcription initiation, termination, promoter, and enhancer sequences; effective RNA processing signals such as splicing and polyadenylation signals; sequences that stabilize cytoplasmic mRNA; sequences that enhance translation efficiency (i.e., Kozak consensus sequences); sequences that enhance protein stability and qualitative properties; and sequences that enhance protein secretion when necessary. The nature of such control sequences varies with the host organism. For example, in prokaryotes, such control sequences generally include promoters, ribosome binding sites, and transcription termination sequences, while in eukaryotes, such expression control sequences typically include promoters and transcription termination sequences. The term "control sequence" is intended to include components whose presence is essential for expression and processing, and may also include additional components whose presence is advantageous, for example, leader sequences and fusion partner sequences.

[0090] As used herein, the term "polypeptide" refers to any amino acid polymer chain. In some embodiments, the polypeptide has a naturally occurring amino acid sequence. In some embodiments, the polypeptide has a non-naturally occurring amino acid sequence. In some embodiments, the polypeptide has an amino acid sequence that includes naturally occurring parts (i.e., from two or more different organisms, e.g., human and non-human parts) that are separate from each other. In some embodiments, the polypeptide has an engineered amino acid sequence that is designed and / or produced by artificial behavior.

[0091] As used herein, the term "recombinant" is intended to refer to polypeptides (e.g., CD47 polypeptides described herein) that are designed, engineered, prepared, expressed, produced, or isolated by recombinant means, such as polypeptides expressed using recombinant expression vectors transfected into host cells, polypeptides isolated from a recombinant combinatorial human polypeptide library (Hoogenboom HR, (1997) TIB Tech. 15:62-70; Azzazy H. and Highsmith WE, (2002) Clin. Biochem. 35:425-445; Gavilondo JV and Larrick JW (2002) BioTechniques 29:128-145; Hoogenboom H. and Chames P. (2000) Immunology Today 21:371-378), antibodies isolated from animals (e.g., mice) transgenic for human immunoglobulin genes (see, e.g., Taylor, LD et al. (1992) Nucl. Acids Res.20:6287-6295; Kellermann SA. and Green LL (2002) Current Opinion in Biotechnology 13:593-597; Little M. et al. (2000) Immunology Today 21:364-370; Murphy, AJ et al. (2014) Proc. Natl. Acad. Sci. USA 111 (14):5153-5158) or by any other means involving splicing selected sequence elements to each other to prepare, express, produce or isolate polypeptides. In some embodiments, one or more of such selected sequence elements are naturally occurring. In some embodiments, one or more of such selected sequence elements are computer designed (in silico). In some embodiments, one or more such selected sequence elements are generated by mutagenesis (e.g., in vivo or in vitro) of known sequence elements, such as from natural sources or synthetic sources. For example, in some embodiments, the recombinant polypeptide is composed of a sequence present in the genome of a target source organism (e.g., human, mouse, etc.). In some embodiments, the recombinant polypeptide has an amino acid sequence produced by mutagenesis (e.g., in vitro or in vivo, such as in a non-human animal) so that the amino acid sequence of the recombinant polypeptide is a sequence that, while derived from and related to the polypeptide sequence, may not naturally exist in the genome of the non-human animal.

[0092] As used herein, the term "replacement" refers to a process by which a "replaced" nucleic acid sequence (e.g., a gene) present in a host locus (e.g., in a genome) is removed from the locus and a different "replacement" nucleic acid is placed in the position. In some embodiments, the replaced nucleic acid sequence is comparable to the replacement nucleic acid sequence because, for example, they are homologous to each other and / or contain corresponding elements (e.g., protein coding elements, regulatory elements, etc.). In some embodiments, the replaced nucleic acid sequence comprises one or more of a promoter, an enhancer, a splice donor site, a splice acceptor site, an intron, an exon, and an untranslated region (UTR); in some embodiments, the replacement nucleic acid sequence comprises one or more coding sequences. In some embodiments, the replacement nucleic acid sequence is a homolog of the replaced nucleic acid sequence. In some embodiments, the replacement nucleic acid sequence is a direct homolog of the replaced sequence. In some embodiments, the replacement nucleic acid sequence is a human nucleic acid sequence or comprises a human nucleic acid sequence. In some embodiments, including in the case where the replacement nucleic acid sequence is a human nucleic acid sequence or comprises a human nucleic acid sequence, the replaced nucleic acid sequence is a rodent sequence or comprises a rodent sequence (e.g., a mouse or rat sequence). The nucleic acid sequence so placed may include one or more regulatory sequences (e.g., promoters, enhancers, 5'- or 3'-untranslated regions, etc.) that are part of the source nucleic acid sequence used to obtain the sequence so placed. For example, in various embodiments, the replacement is the replacement of an endogenous sequence by a heterologous sequence, the replacement resulting in the production of a gene product from the nucleic acid sequence so placed (comprising the heterologous sequence), but without the expression of the endogenous sequence; the replacement is the replacement of an endogenous genomic sequence (e.g., the endogenous genomic sequence encodes a CD47 protein, and the DNA fragment encodes one or more human CD47 proteins) with a nucleic acid sequence encoding a protein having a similar function to that encoded by the endogenous sequence. In various embodiments, the endogenous gene or fragment thereof is replaced by a corresponding human gene or fragment thereof. The corresponding human gene or fragment thereof is a human gene or fragment that is an ortholog of the replaced endogenous gene or fragment thereof, or a human gene or fragment that is substantially similar or identical in structure and / or function to the replaced endogenous gene or fragment thereof.

[0093] As used herein, the term "cluster of differentiation 47 protein" or "CD47 protein" refers to a multi-transmembrane protein belonging to the immunoglobulin superfamily and having an extracellular amino-terminal immunoglobulin V domain, five transmembrane domains and a short carboxyl-terminal intracellular tail. CD47 is expressed on the cell surface and is involved in the interaction between membrane surface proteins such as integrins, SIRPα and thrombospondin-1 (TSP-1). CD47 is expressed in normal tissues and is upregulated in many human cancers. CD47 has been shown to be involved in multiple cellular processes such as apoptosis, proliferation, adhesion and migration. Several alternatively spliced ​​CD47 isoforms have been identified between mice and humans. By way of example only, the nucleotide and amino acid sequences of mouse and human CD47 genes are provided in Table 3. Those skilled in the art will recognize after reading the present disclosure that one or more endogenous CD47 genes (or all) in the genome can be replaced by one or more heterologous CD47 genes (e.g., polymorphic variants, subtypes or mutants, genes from another species, humanized forms, etc.).

[0094] As used herein, "CD47 expressing cells" refers to cells expressing CD47 transmembrane proteins. In some embodiments, CD47 expressing cells express CD47 transmembrane proteins on their surface. In some embodiments, CD47 proteins are expressed on the surface of cells in sufficient amounts to mediate cell-to-cell interactions through CD47 transmembrane proteins expressed on the surface of cells. Exemplary CD47 expressing cells include neurons, immune cells, keratinocytes, and circulating cells. CD47 expressing cells regulate the interaction of immune cells and circulating cells to regulate various cellular processes, such as adhesion, cell proliferation and / or apoptosis, angiogenesis, and inflammation. In some embodiments, the non-human animals of the present invention exhibit regulation of multiple cellular processes (as described herein) via humanized CD47 proteins expressed on the surface of one or more cells of the non-human animals.

[0095] As used herein, the term "reference" describes a standard or control reagent, queue, individual, colony, sample, sequence or value compared to a target reagent, animal, queue, individual, colony, sample, sequence or value. In some embodiments, the test or determination of a reference reagent, queue, individual, colony, sample, sequence or value is substantially performed simultaneously with the test or determination of a target reagent, queue, individual, colony, sample, sequence or value. In some embodiments, the reference reagent, queue, individual, colony, sample, sequence or value is a historical reference, which is optionally embodied in a tangible medium. In some embodiments, reference may refer to control. As used herein, "reference" may refer to "reference animals". "Reference animals" may have modifications as described herein, modifications different from the modifications described herein or no modifications (i.e., wild-type animals). Generally, those skilled in the art will understand that the reference reagent, animal, queue, individual, colony, sample, sequence or value are measured or characterized under certain conditions, and the conditions are similar to the conditions used to measure or characterize the target reagent, animal (e.g., mammal), queue, individual, colony, sample, sequence or value.

[0096] As used herein, the term "substantially" refers to a qualitative condition that exhibits all or nearly all of the extent or degree of a target characteristic or property. One of ordinary skill in the biological field will appreciate that biological and chemical phenomena are rarely, if ever, carried to completion and / or carried to completeness, or achieve or avoid absolute results. Thus the term "substantially" is used to capture the potential lack of completeness inherent in many biological and chemical phenomena.

[0097] As used herein, the term "substantially homologous" refers to a comparison between amino acid or nucleic acid sequences. As will be appreciated by one of ordinary skill in the art, two sequences are generally considered to be "substantially homologous" if they contain homologous residues at corresponding positions. Homologous residues may be identical residues. Alternatively, homologous residues may be non-identical residues having appropriately similar structural and / or functional properties. For example, as is well known to one of ordinary skill in the art, certain amino acids are generally classified as "hydrophobic" or "hydrophilic" amino acids, and / or as having "polar" or "non-polar" side chains. The substitution of one amino acid for another amino acid of the same type may generally be considered a "homologous" substitution. Typical amino acid classes are summarized in Tables 1 and 2.

[0098] Table 1

[0099]

[0100]

[0101] Table 2

[0102]

[0103] As is well known in the art, amino acid or nucleic acid sequences can be compared using any of a variety of algorithms, including those available in commercial computer programs such as BLASTN for nucleotide sequences and BLASTP, Gapped BLAST, and PSI-BLAST for amino acid sequences. Exemplary such programs are described in Altschul et al. (1990) Basic local alignment search tool, J. Mol. Biol., 215(3): 403-410; Altschul et al. (1997) Methods in Enzymology; Altschul et al., "Gapped BLAST and PSI-BLAST: a new generation of protein database search programs", Nucleic Acids Res. 25: 3389-3402; Baxevanis et al. (1998) Bioinformatics: A Practical Guide to the Analysis of Genes and Proteins, Wiley; and Misener et al. (eds.) (1999) Bioinformatics Methods and Protocols (Methods in Molecular Biology, Vol. 132), Humana Press. In addition to identifying homologous sequences, the above-mentioned programs generally provide an indication of the degree of homology. In some embodiments, if at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more of the corresponding residues of the two sequences are homologous on the relevant residue sequence stretch, then the two sequences are considered to be substantially homologous. In some embodiments, the relevant sequence stretch is a complete sequence. In some embodiments, the relevant sequence stretch is at least 9, 10, 11, 12, 13, 14, 15, 16, 17 or more residues. In some embodiments, the relevant sequence stretch includes continuous residues along the complete sequence. In some embodiments, the relevant sequence stretch includes discontinuous residues along the complete sequence. In some embodiments, the coherent stretch is at least 10, 15, 20, 25, 30, 35, 40, 45, 50 or more residues.

[0104] As used herein, the term "substantially identical" refers to a comparison between amino acid or nucleic acid sequences. As will be appreciated by one of ordinary skill in the art, two sequences are generally considered to be "substantially identical" if they contain identical residues at corresponding positions. As is well known in the art, amino acid or nucleic acid sequences can be compared using any of a variety of algorithms, including those available in commercial computer programs such as BLASTN for nucleotide sequences and BLASTP, GAP BLAST, and PSI-BLAST for amino acid sequences. Exemplary such programs are described in Altschul et al. (1990) Basic local alignment search tool, J. Mol. Biol., 215 (3): 403-410; Altschul et al., Methods in Enzymology; Altschul et al. (1997) Nucleic Acids Res. 25: 3389-3402; Baxevanis et al. (1998) Bioinformatics: A Practical Guide to the Analysis of Genes and Proteins, Wiley; and Misener et al., (eds.) (1999) Bioinformatics Methods and Protocols (Methods in Molecular Biology, Vol. 132), Humana Press. In addition to identifying identical sequences, the above-mentioned programs generally provide an indication of the degree of identity. In some embodiments, two sequences are considered to be substantially identical if at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more of the corresponding residues of the two sequences are identical over the relevant residue sequence segment. In some embodiments, the relevant sequence segment is a complete sequence. In some embodiments, the relevant segment is at least 10, 15, 20, 25, 30, 35, 40, 45, 50 or more residues.

[0105] As used herein, the term "targeting vector" or "targeting construct" refers to a polynucleotide molecule comprising a targeting region. The targeting region comprises a sequence identical or substantially identical to a sequence in a target cell, tissue, or animal and provides a targeting construct for integration via homologous recombination into a position in the genome of the cell, tissue, or animal. Also included is a targeting region for targeting using a site-specific recombinase recognition site (e.g., loxP or Frt site). In some embodiments, the targeting construct of the present invention also comprises a specific target nucleic acid sequence or gene, a selectable marker, a control and / or regulatory sequence, and other nucleic acid sequences that allow for the reorganization mediated by the exogenous addition of a protein that helps or promotes the reorganization of such sequences. In some embodiments, the targeting construct of the present invention also comprises all or part of a target gene, wherein the target gene is a heterologous gene encoding all or part of a protein having a function similar to that of a protein encoded by an endogenous sequence. In some embodiments, the targeting construct of the present invention also comprises all or part of a target humanized gene, wherein the target humanized gene encodes all or part of a protein having a function similar to that of a protein encoded by an endogenous sequence.

[0106] As used herein, the term "variant" refers to an entity that exhibits significant structural identity to a reference entity but is structurally different from the reference entity in the presence or level of one or more chemical moieties compared to the reference entity. In many embodiments, a "variant" is also functionally different from its reference entity. Generally speaking, whether a particular entity is properly considered to be a "variant" of a reference entity is based on the degree of structural identity between it and the reference entity. As will be appreciated by those skilled in the art, any biological or chemical reference entity has certain characteristic structural elements. By definition, a "variant" is a different chemical entity that shares one or more such characteristic structural elements. Just to give some examples, a small molecule may have a characteristic core structural element (e.g., a macrocyclic core) and / or one or more characteristic side chain moieties, such that variants of a small molecule are molecules that share the core structural element and the characteristic side chain moieties, but differ in other side chain moieties and / or in the type of bonds present in the core (single bonds versus double bonds, E versus Z, etc.), a polypeptide may have a characteristic sequence element comprising a plurality of amino acids that have specified positions relative to each other in linear or three-dimensional space and / or contribute to a specific biological function, a nucleic acid may have a characteristic sequence element comprising a plurality of nucleotide residues that have specified positions relative to each other in linear or three-dimensional space. For example, a "variant polypeptide" may differ from a reference polypeptide due to one or more differences in the amino acid sequence and / or one or more differences in the chemical moieties (e.g., carbohydrates, lipids, etc.) covalently attached to the polypeptide backbone. In some embodiments, a "variant polypeptide" shows an overall sequence identity of at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97% or 99% with a reference polypeptide. Alternatively or additionally, in some embodiments, a "variant polypeptide" does not share at least one characteristic sequence element with a reference polypeptide. In some embodiments, a reference polypeptide has one or more biological activities. In some embodiments, a "variant polypeptide" shares one or more biological activities of a reference polypeptide. In some embodiments, a "variant polypeptide" lacks one or more biological activities of a reference polypeptide. In some embodiments, a "variant polypeptide" shows one or more biological activities that are reduced in level compared to a reference polypeptide. In many embodiments, if a target polypeptide has an amino acid sequence that is identical to the amino acid sequence of a parent but has an amino acid sequence with a few sequence changes at a specific position, the target polypeptide is considered to be a "variant" of a parent or reference polypeptide. Typically, less than 20%, 15%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2% of the residues in the variant are replaced compared to the parent. In some embodiments, a "variant" variant has 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 replaced residues compared to the parent.Typically, a "variant" has a very small number (e.g., less than 5, 4, 3, 2, or 1) of substituted functional residues (i.e., residues involved in a particular biological activity). In addition, a "variant" typically has no more than 5, 4, 3, 2, or 1 additions or deletions, and typically has no additions or deletions, compared to the parent. In addition, any additions or deletions are typically less than about 25, about 20, about 19, about 18, about 17, about 16, about 15, about 14, about 13, about 10, about 9, about 8, about 7, about 6 residues, and typically less than about 5, about 4, about 3, or about 2 residues. In some embodiments, the parent or reference polypeptide is a naturally found polypeptide. As will be appreciated by one of ordinary skill in the art, multiple variants of a particular target polypeptide may typically occur in nature, particularly when the target polypeptide is an infectious agent polypeptide.

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

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

[0109] DETAILED DESCRIPTION OF CERTAIN EMBODIMENTS

[0110] The present invention provides, inter alia, improved and / or engineered non-human animals having humanized genetic material encoding a cluster of differentiation 47 (CD47) gene, which are used to determine the therapeutic efficacy of CD47 antagonists (e.g., anti-CD47 antibodies) for the treatment of cancer and for determination of transplant implants, activation and phagocytosis, and signal transduction. It is conceivable that such non-human animals provide improvements in determining the therapeutic efficacy of CD47 antagonists and their potential for CD47 blockade. It is also conceivable that such non-human animals provide improvements in transplant implantation of human cells. Therefore, the present invention is particularly useful for developing anti-CD47 therapies for the treatment of a variety of cancers, as well as for maintaining human hematopoietic cells in non-human animals. Specifically, the present invention includes humanization of the murine CD47 gene, which results in expression of a humanized CD47 protein on the surface of non-human animal cells. Such humanized CD47 proteins are capable of providing human CD47 for determining the efficacy of anti-CD47 therapeutic agents in activating phagocytosis of tumor cells. +The source of cells. In addition, such humanized CD47 proteins are able to recognize implanted human cells by engaging other cell surface proteins and ligands (e.g., SIRPα) present on the surface of implanted human cells. In some embodiments, the non-human animals of the present invention are able to activate phagocytosis by blocking CD47 signaling through humanized CD47 proteins expressed on the surface of non-human animal cells. In some embodiments, the non-human animals of the present invention are able to accept transplanted human hematopoietic cells; in some embodiments, such non-human mammals develop and / or have an immune system comprising human cells. In some embodiments, the humanized CD47 protein has a sequence corresponding to the N-terminal immunoglobulin V domain of the human CD47 protein. In some embodiments, the humanized CD47 protein has a sequence corresponding to the N-terminal portion of the human CD47 protein, and the human CD47 protein comprises an extracellular portion and a transmembrane portion of the human CD47 protein, wherein the extracellular portion includes the N-terminal immunoglobulin V domain of the human CD47 protein, and the transmembrane portion includes five transmembrane domains of the human CD47 protein. In some embodiments, the humanized CD47 protein has a sequence corresponding to the intracytoplasmic tail of a non-human (e.g., murine) CD47 protein. In some embodiments, the humanized CD47 protein has a sequence corresponding to amino acid residues 19-292 (or 19-141 or 19-127) of a human CD47 protein. In some embodiments, the non-human animal of the present invention comprises an endogenous CD47 gene containing genetic material from a non-human animal and a heterologous species (e.g., human). In some embodiments, the non-human animal of the present invention comprises a humanized CD47 gene, wherein the humanized CD47 gene comprises an exon of a human CD47 gene encoding an extracellular portion, the extracellular portion comprising an N-terminal immunoglobulin V domain of a human CD47 gene. In some embodiments, the humanized CD47 gene comprises human CD47 exons, such as exons 2-7 encoding the N-terminal portion of the human CD47 protein, the human CD47 protein comprising the extracellular portion and the transmembrane portion of the human CD47 protein, wherein the extracellular portion includes the N-terminal immunoglobulin V domain of the human CD47 protein, and the transmembrane portion includes five transmembrane domains of the human CD47 protein. In some embodiments, the humanized CD47 gene comprises a non-human CD47 exon encoding all or part of the signal peptide and the intracytoplasmic tail of the non-human CD47 protein. In some embodiments, the humanized CD47 gene comprises non-human CD47 exons encoding the intracytoplasmic tail and 3'UTR and exons downstream of exon 7. Depending on the isotype, one or more exons downstream of exon 7 with a stop codon and 3'UTR may be present in the last exon of all isotypes. For example, isoform 2 of both mouse and human CD47 shown in Table 3 has two exons downstream of exon 7, referred to as exons 8 and 9.

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

[0112] Cluster of differentiation 47 (CD47) gene

[0113] CD47, formerly known as integrin-associated protein (IAP) due to its role in signal transduction from integrins on immune cells, is a transmembrane protein that includes an N-terminal immunoglobulin V (IgV) domain, five transmembrane domains, and a short C-terminal intracytoplasmic tail. The length of the intracytoplasmic tail varies according to the four alternatively spliced ​​isoforms that have been identified. CD47 (or IAP) was originally described as being expressed on all tissues (isoform 2), neurons (isoform 4), and keratinocytes and macrophages (isoform 1; see Reinhold et al. (1995) J. Cell Sci. 108: 3419-3425). Little is known about isoform 3, although this form has the second longest intracytoplasmic tail of the four isoforms. In addition to integrins, CD47 is known to interact with many other cell surface proteins such as thrombospondin and members of the SIRP family. Most notably, CD47 interacts with SIRPα and causes bidirectional signaling that regulates many intercellular responses, such as inhibition of phagocytosis and T cell activation. In fact, the CD47-SIRPα interaction has attracted much attention in recent years due to its role in enabling tumor cells to evade immune surveillance. The binding of CD47 to SIRPα usually provides protection through anti-phagocytic signals for normal cells ("don't eat me"). However, it has been found that tumors also express anti-phagocytic signals, including CD47, to avoid destruction by phagocytosis. Interestingly, CD47 is known to be upregulated in many blood cancers and contributes to tumor growth and metastasis (Chao et al. (2012) Curr Opin Immunol. 24 (2): 225–232).

[0114] As a new treatment for cancer, the full effects of targeting CD47 and the CD47-SIRPα pathway are not yet understood, and some possible toxicities have been investigated. A more complete and detailed understanding of CD47 signaling and the CD47-SIRPα pathway is needed to develop better targeted therapies for future cancer treatment.

[0115] CD47 sequence

[0116] Exemplary CD47 sequences for mice and humans are shown in Table 3. For mRNA sequences, bold indicates coding sequences, and when consecutive exons are indicated, they are separated by alternating underlined text. For mouse and human protein sequences, the signal peptide is underlined, the extracellular sequence is bold, and the intracytoplasmic sequence is italic. For humanized protein sequences, non-human sequences are represented in regular font, human sequences are represented in bold, and the signal peptide is underlined. As shown in the figure, the number of exons of isoforms is different. For example, isoforms 1-4 of the human CD47 gene have a total of 8, 9, 10, and 11 exons, respectively, and exons 2-7 of each isoform encode an extracellular domain and five transmembrane domains.

[0117] Table 3

[0118]

[0119]

[0120]

[0121]

[0122]

[0123]

[0124]

[0125]

[0126]

[0127]

[0128]

[0129]

[0130]

[0131]

[0132]

[0133]

[0134]

[0135]

[0136]

[0137]

[0138]

[0139]

[0140]

[0141]

[0142]

[0143]

[0144]

[0145]

[0146]

[0147]

[0148]

[0149]

[0150]

[0151]

[0152]

[0153] Humanized CD47 non-human animals

[0154] A non-human animal is provided, which expresses a humanized CD47 protein on the cell surface of the non-human animal, wherein the humanized CD47 protein is produced by genetically modifying the endogenous gene locus encoding the CD47 protein of the non-human animal. Suitable examples described herein include rodents, in particular, mice.

[0155] In some embodiments, the humanized CD47 gene comprises genetic material from a heterologous species (e.g., a human), wherein the humanized CD47 gene encodes a CD47 protein, and the CD47 protein comprises an encoded portion of genetic material from a heterologous species. In some embodiments, the humanized CD47 gene of the present invention comprises genomic DNA from a heterologous species, and the genomic DNA encodes the extracellular portion of the CD47 protein expressed on the surface of the cytoplasmic membrane. In some embodiments, the humanized CD47 gene of the present invention comprises genomic DNA from a heterologous species, and the genomic DNA encodes the extracellular portion and transmembrane portion of the CD47 protein expressed on the surface of the cytoplasmic membrane. Non-human animals, embryos, cells, and targeting constructs for preparing non-human animals, non-human embryos, and cells comprising the humanized CD47 gene are provided.

[0156] In some embodiments, the endogenous CD47 gene is deleted. In some embodiments, the endogenous CD47 gene is changed, wherein a portion of the endogenous CD47 gene is replaced by a heterologous sequence (e.g., all or part of the human CD47 sequence). In some embodiments, all or substantially all of the endogenous CD47 genes are replaced by heterologous genes (e.g., human CD47 genes). In some embodiments, a portion of the heterologous CD47 gene is inserted into the endogenous CD47 locus of the endogenous non-human CD47 gene. In some embodiments, the heterologous gene is a human gene. In some embodiments, one of the two copies of the endogenous CD47 gene is modified or humanized, thereby producing a non-human animal heterozygous for the humanized CD47 gene. In other embodiments, a non-human animal heterozygous for the humanized CD47 gene is provided.

[0157] The non-human animals of the present invention contain all or part of the human CD47 gene on the endogenous non-human CD47 locus. Therefore, such non-human animals can be described as having a heterologous CD47 gene. The replaced, inserted, modified or altered CD47 gene at the endogenous CD47 locus can be detected using a variety of methods (including, for example, PCR, Western blot, Southern blot, restriction fragment length polymorphism (RFLP) or allele gain or loss assay). In some embodiments, the non-human animal is heterozygous for the humanized CD47 gene. In other embodiments, a non-human animal homozygous for the humanized CD47 gene is provided.

[0158] In various embodiments, the humanized CD47 gene according to the present invention includes a CD47 gene having a second exon, a third exon, a fourth exon, a fifth exon, a sixth exon and a seventh exon, wherein the second exon, the third exon, the fourth exon, the fifth exon, the sixth exon and the seventh exon each have a sequence that is at least 50% (e.g., 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) identical to the second exon, the third exon, the fourth exon, the fifth exon, the sixth exon and the seventh exon presented in the human CD47 gene of Table 3.

[0159] In various embodiments, the humanized CD47 gene according to the present invention includes a CD47 gene having a first exon and an exon downstream of exon 7 (e.g., the eighth exon and the ninth exon of isoform 2), wherein the first exon and the exon downstream of exon 7 each have a sequence that is at least 50% (e.g., 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) identical to the respective exons presented in the mouse CD47 gene of Table 3.

[0160] In various embodiments, the humanized CD47 gene according to the present invention includes a CD47 gene having a 5' untranslated region and a 3' untranslated region, each of which has a sequence that is at least 50% (e.g., 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) identical to the 5' untranslated region and 3' untranslated region presented in the mouse CD47 gene of Table 3.

[0161] In various embodiments, the humanized CD47 gene according to the present invention includes a CD47 gene having a nucleotide coding sequence (e.g., a cDNA sequence) that is at least 50% (e.g., 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) identical to the nucleotide coding sequence presented in Table 3.

[0162] In various embodiments, the humanized CD47 protein produced by the non-human animal of the invention has an extracellular portion having an amino acid sequence that is at least 50% (e.g., 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) identical to the extracellular portion of the human CD47 protein presented in Table 3.

[0163] In various embodiments, the humanized CD47 protein produced by the non-human animal of the invention has an extracellular portion having an amino acid sequence identical to amino acid residues 19-141 presented in the human CD47 protein of Table 3.

[0164] In various embodiments, the humanized CD47 protein produced by the non-human animal of the invention has an N-terminal immunoglobulin V domain having an amino acid sequence that is at least 50% (e.g., 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) identical to the N-terminal immunoglobulin V domain of the human CD47 protein presented in Table 3.

[0165] In various embodiments, the humanized CD47 protein produced by the non-human animal of the invention has an N-terminal immunoglobulin V domain having an amino acid sequence identical to amino acid residues 19-127 presented in the human CD47 protein of Table 3.

[0166] In various embodiments, the humanized CD47 protein produced by the non-human animal of the invention has an N-terminal immunoglobulin V domain and five transmembrane domains, each of which has a sequence that is at least 50% (e.g., 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) identical to the N-terminal immunoglobulin V domain and five transmembrane domains of the human CD47 protein presented in Table 3.

[0167] In various embodiments, the humanized CD47 protein produced by the non-human animal of the invention has an intracytoplasmic tail having a sequence that is at least 50% (e.g., 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) identical to the intracytoplasmic tail of the mouse CD47 protein presented in Table 3.

[0168] In various embodiments, the humanized CD47 protein produced by the non-human animal of the invention has an amino acid sequence that is at least 50% (e.g., 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) identical to amino acid residues 16-292 presented in the human CD47 protein of Table 3.

[0169] In various embodiments, the humanized CD47 protein produced by the non-human animal of the invention has an amino acid sequence that is at least 50% (e.g., 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) identical to amino acid residues 19-292 presented in the human CD47 protein of Table 3.

[0170] In various embodiments, the humanized CD47 protein produced by the non-human animal of the invention has an amino acid sequence identical to amino acid residues 19-292 (or 16-292) presented in the human CD47 protein of Table 3.

[0171] In various embodiments, the humanized CD47 protein produced by the non-human animal of the invention has an amino acid sequence that is at least 50% (e.g., 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) identical to the amino acid sequence of the humanized CD47 protein presented in Table 3.

[0172] In various embodiments, the humanized CD47 protein produced by the non-human animal of the invention has an amino acid sequence identical to the amino acid sequence of the humanized CD47 protein presented in Table 3.

[0173] Compositions and methods for preparing non-human animals are provided, the non-human animals expressing humanized CD47 proteins, including specific polymorphic forms, allelic variants (e.g., single amino acid differences), or alternatively spliced ​​isoforms, including compositions and methods for preparing non-human animals expressing such proteins from human promoters and human regulatory sequences. In some embodiments, compositions and methods for preparing non-human animals expressing such proteins from endogenous promoters and endogenous regulatory sequences are also provided. The method includes inserting genetic material encoding all or part of a human CD47 protein into the genome of a non-human animal at a precise location corresponding to an endogenous CD47 gene, thereby producing a humanized CD47 gene expressing all or part of a human CD47 protein. In some embodiments, the method includes inserting genomic DNA corresponding to exons 2-7 of a human CD47 gene into an endogenous CD47 gene of a non-human animal, thereby producing a humanized gene encoding a CD47 protein, the CD47 protein comprising a human portion containing amino acids encoded by the inserted exons.

[0174] Where appropriate, the coding region of the genetic material or polynucleotide sequence encoding all or part of the human CD47 protein may be modified to include codons that are optimized for expression in non-human animals (e.g., see U.S. Pat. Nos. 5,670,356 and 5,874,304). A codon-optimized sequence is a synthetic sequence and preferably encodes the same polypeptide as the polypeptide encoded by the non-codon-optimized parent polynucleotide (or a biologically active fragment of a full-length polypeptide having substantially the same activity as the full-length polypeptide). In some embodiments, the coding region of the genetic material encoding all or part of the human CD47 protein may include a sequence that is altered to optimize codon usage for a specific cell type (e.g., a rodent cell). For example, the codons of the genomic DNA corresponding to exons 2-7 of the human CD47 gene to be inserted into the endogenous CD47 gene of a non-human animal (e.g., a rodent) may be optimized for expression in the cells of the non-human animal. This sequence may be described as a codon-optimized sequence.

[0175] The humanized CD47 gene method uses relatively minimal endogenous gene modification and produces natural CD47-mediated signal transduction in non-human animals, in multiple embodiments, because the genomic sequence of the CD47 sequence is modified in a single fragment and thus maintains normal functionality by including necessary regulatory sequences. Therefore, in such embodiments, the CD47 gene modification will not affect other surrounding genes or other endogenous CD47 interaction genes (e.g., thrombospondin, SIRP, integrin, etc.). In addition, in multiple embodiments, the modification will not affect the assembly of the functional CD47 transmembrane protein on the plasma membrane, and maintain normal effector function via binding and subsequent signal transduction through the cytoplasmic portion of the protein that is not affected by the modification.

[0176] A schematic diagram (not drawn to scale) of the genomic organization of the endogenous murine CD47 gene and the human CD47 gene is provided at Figure 1 An exemplary method for humanizing the endogenous murine CD47 gene using a genomic fragment comprising exons 2-7 of the human CD47 gene is provided in Figure 2 As shown in the figure, genomic DNA containing exons 2-7 of the human CD47 gene is inserted into the locus of the endogenous murine CD47 gene by a targeting construct. This genomic DNA includes the gene portion encoding the extracellular portion and transmembrane domain (e.g., amino acid residues 16-292) of the human CD47 protein responsible for ligand binding.

[0177] A non-human animal (eg, mouse) having a humanized CD47 gene at the endogenous CD47 locus can be prepared by any method known in the art. For example, a targeting vector introducing all or part of a human CD47 gene with a selectable marker gene can be prepared. Figure 2 The endogenous CD47 locus of the mouse genome containing exon 2-7 insertion of the human CD47 gene is shown. As shown, the targeting construct comprises a 5' homology arm (~39Kb) containing a sequence upstream of exon 2 of the endogenous murine CD47 gene, followed by a genomic DNA fragment (~23.9Kb) containing exon 2-7 of the human CD47 gene, a drug selection box (e.g., a neomycin resistance gene flanked by loxP sequences on both sides; ~5Kb) and a 3' homology arm (~99Kb) containing a sequence downstream of exon 7 of the endogenous murine CD47 gene. The targeting construct comprises a self-deleted drug selection box (e.g., a neomycin resistance gene flanked by loxP sequences; see U.S. Patent Nos. 8,697,851, 8,518,392, and 8,354,389, all of which are incorporated herein by reference). During homologous recombination, exons 2-7 of the endogenous murine CD47 gene are replaced by sequences contained in the targeting vector (i.e., exons 2-7 of the human CD47 gene). The formation of a humanized CD47 gene produces cells or non-human animals expressing a humanized CD47 protein comprising amino acids encoded by exons 2-7 of the human CD47 gene. The drug selection cassette is removed in a development-dependent manner, i.e., offspring derived from mice whose germline cells contain the humanized CD47 gene will shed the selectable marker from differentiated cells during development.

[0178] The non-human animals of the present invention may be prepared as described above or using methods known in the art to include additional human or humanized genes, generally depending on the intended use of the non-human animals. The genetic material of such additional human or humanized genes may be introduced by further changing the genome of cells (e.g., embryonic stem cells) having the above genetic modifications, or by breeding techniques known in the art using other desired genetically modified strains. In some embodiments, the non-human animals of the present invention are prepared to further include one or more human or humanized genes selected from SIRPα (CD172a), IL-3, M-CSF, GM-CSF, and TPO. In some embodiments, the non-human animals of the present invention may be prepared by introducing a targeting vector as described herein into cells from a modified strain. To give just one example, the above targeting vector may be introduced into Rag2-deficient and IL-2Rγ-deficient mice, and includes four human cytokines (Rag2 - / - IL2Rγc - / - 、M-CSF Hu , IL-3 / GM-CSF Hu , hSIRPα tg 、TPO Hu ). In some embodiments, the non-human animals of the present invention are prepared to further comprise a human or humanized signal regulatory protein alpha (SIRPα) gene. In some embodiments, the non-human animals of the present invention comprise a humanized CD47 gene as described herein and genetic material from a heterologous species (e.g., human), wherein the genetic material encodes in whole or in part one or more heterologous proteins selected from SIRPα (CD172a), IL-3, M-CSF, GM-CSF, and TPO. In certain embodiments, the non-human animals of the present invention comprise a humanized CD47 gene as described herein and genetic material from a heterologous species (e.g., human), wherein the genetic material encodes in whole or in part a heterologous (e.g., human) SIRPα (CD172a) protein. In certain embodiments, the non-human animal of the present invention further comprises a SIRPα gene, wherein the SIRPα comprises an endogenous portion and a human portion (e.g., exons 2-4 of the human SIRPα gene), wherein the human portion encodes the extracellular domain of the SIRPα protein (e.g., amino acids corresponding to residues 28-362 of the human SIRPα protein), and the endogenous portion encodes the intracellular domain of the endogenous SIRPα protein; in some embodiments, the human portion and the endogenous portion are operably linked to an endogenous SIRPα promoter.

[0179] For example, as described herein, non-human animals comprising a humanized CD47 gene may also comprise (e.g., by hybridization or multi-gene targeting strategies) PCT / US2010 / 051339 filed on October 4, 2010, PCT / US2013 / 058448 filed on September 6, 2013, PCT / US2013 / 045788 filed on June 14, 2013, PCT / US2014 / 056910 filed on September 23, 2014, PCT / US2014 / 056929 filed on October 23, 2014, One or more modifications described in PCT / US2014 / 060568, filed on February 15, 2014, PCT / US2012 / 025040, filed on February 14, 2014, PCT / US2012 / 062379, filed on October 29, 2012, PCT / US2014 / 064806, filed on November 10, 2014, and PCT / US2014 / 064810, filed on November 10, 2014; the entireties of these patent applications are incorporated herein by reference. In certain embodiments, a rodent comprising a humanized CD47 gene (i.e., exons 2-7 of a human CD47 gene are operably linked to exons 1 and exon 8 (and thus any downstream exons) of an endogenous rodent CD47 gene, such that the humanized CD47 gene encodes a CD47 polypeptide having an extracellular portion from a human CD47 protein and an intracellular portion from a rodent CD47 protein) is hybridized with a rodent comprising a humanized SIRPα gene (e.g., exons 2-4 of a human SIRPα gene are operably linked to exons 1 and 5-8 of an endogenous rodent SIRPα gene, such that the humanized SIRPα gene encodes a SIRPα polypeptide having an extracellular portion from a human SIRPα protein (e.g., amino acids corresponding to residues 28-362) and an intracellular portion from a rodent SIRPα protein; see, e.g., PCT / US2014 / 056910, filed September 23, 2014, which is incorporated herein by reference).

[0180] Although the humanized CD47 gene is widely discussed in the text for application to mice (i.e., mice having a CD47 gene encoding a CD47 protein, the CD47 protein comprising a human portion and a mouse portion), other non-human animals comprising a humanized CD47 gene are also provided. In some embodiments, such non-human animals comprise a humanized CD47 gene operably linked to an endogenous CD47 promoter. In some embodiments, such non-human animals express a humanized CD47 protein from an endogenous locus, wherein the humanized CD47 protein comprises amino acid residues 16-292 (or 19-141 or 19-127) of a human CD47 protein. Such non-human animals include any of those animals that can be genetically modified to express a CD47 protein as described herein, including, for example, mammals, such as mice, rats, rabbits, pigs, cattle (e.g., cows, bulls, buffaloes), deer, sheep, goats, chickens, cats, dogs, minks, primates (e.g., marmosets, macaques), etc. For example, for non-human animals for which suitable ES cells that can be genetically modified are not readily available, other methods are used to prepare non-human animals comprising genetic modifications. Such methods include, for example, modifying a non-ES cell genome (e.g., a fibroblast or an induced pluripotent cell) and using somatic cell nuclear transfer (SCNT) to transfer the genetically modified genome to a suitable cell, such as an enucleated oocyte, and gestating the modified cell (e.g., a modified oocyte) in a non-human animal under conditions suitable for forming an embryo.

[0181] Methods for modifying non-human animal genomes (e.g., pig, cow, rodent, chicken, etc. genomes) include, for example, using zinc finger nucleases (ZFNs) or transcription activator-like effector nucleases (TALENs) to modify the genome to include a humanized CD47 gene.

[0182] In some embodiments, the non-human animal of the present invention is a mammal. In some embodiments, the non-human animal of the present invention is a small mammal, such as a mammal of the superfamily Jerboidea or Muroidea. In some embodiments, the genetically modified animal of the present invention is a rodent. In some embodiments, the rodent of the present invention is selected from mice, rats and hamsters. In some embodiments, the rodent of the present invention is selected from the superfamily Muroidea. In some embodiments, the genetically modified animal of the present invention is from a family selected from the following: Cricetidae (e.g., hamsters of mouse-like hamsters), Cricetidae (e.g., hamsters, New World rats and mice, voles), Muridae (true mice and rats, gerbils, spiny mice, crown mice), Isla Muscidae (climbing mice, rock mice, rats with tails, Madagascar rats and mice), Spiny Mountain Muridae (e.g., spiny rod dormice) and Mole-shaped Muridae (e.g., moles, bamboo rats and zokors). In certain embodiments, the genetically modified rodent of the present invention is selected from true mice or rats (Muridae), gerbils, spiny mice and crown mice. In certain embodiments, the genetically modified mouse of the present invention is a member from the family Muridae. In some embodiments, the non-human animal of the present invention is a rodent. In certain embodiments, the rodent of the present invention is selected from mice and rats. In some embodiments, the non-human animal of the present invention is a mouse.

[0183] In some embodiments, the non-human animal of the present invention is a rodent, which is a mouse of the following C57BL strains: C57BL / A, C57BL / An, C57BL / GrFa, C57BL / KaLwN, C57BL / 6, C57BL / 6J, C57BL / 6ByJ, C57BL / 6NJ, C57BL / 10, C57BL / 10ScSn, C57BL / 10Cr and C57BL / Ola. In certain embodiments, the present invention The mouse is a 129 strain selected from the following strains: 129P1, 129P2, 129P3, 129X1, 129S1 (e.g., 129S1 / SV, 129S1 / SvIm), 129S2, 129S4, 129S5, 129S9 / SvEvH, 129 / SvJae, 129S6 (129 / SvEvTac), 129S7, 129S8, 129T1, 129T2 (see, e.g., Festing et al., 1999, Mammalian Genome 10:836; Auerbach, W. et al., 2000, Biotechniques 29(5):1024-1028, 1030, 1032). In certain embodiments, the genetically modified mouse of the present invention is a mixture of the above-mentioned 129 strain and the aforementioned C57BL / 6 strain. In certain embodiments, the mouse of the present invention is a mixture of the above-mentioned 129 strains, or a mixture of the aforementioned BL / 6 strains. In certain embodiments, the 129 strain of the mixture as described herein is a 129S6 (129 / SvEvTac) strain. In some embodiments, the mouse of the present invention is a BALB strain, such as a BALB / c strain. In some embodiments, the mouse of the present invention is a mixture of a BALB strain and another of the above-mentioned strains.

[0184] In some embodiments, the non-human animal of the present invention is a rat. In certain embodiments, the rat of the present invention is selected from Wistar rats, LEA strains, Sprague Dawley strains, Fischer strains, F344, F6 and Dark Agouti. In certain embodiments, the rat strain described herein is a mixture of two or more strains selected from Wistar, LEA, Sprague Dawley, Fischer, F344, F6 and Dark Agouti.

[0185] Methods using non-human animals with a humanized CD47 gene

[0186] CD47 mutants and transgenic non-human animals (e.g., minipigs) and cells have been reported (Koshimizu H. et al. (2014) PLoS One, 9(2): e89584; Lavender, KJ et al. (2014) J. Immunol. Methods, 407: 127-134; Tena, A. et al. (2014) Am. J. Transplant. doi: 10.1111 / ajt.12918; Lavender K.J. et al. (2013) Blood, 122(25): 4013-4020; Tena, A. et al. (2012) Transplantation 94(10S): 776; Wang, C. et al. (2011) Cell Transplant.20(11-12):1915-1920; Johansen, ML and Brown, EJ (2007) J.Biol.Chem.282:24219-24230; Wang, H. et al. (2007) Proc.Nat.Acad.Sci.USA104:13744-13749; Tulasne D. et al. (2001) Blood,98(12):3346-52; Oldenborg, P. et al. (2000) Science 288:2051-2054; Verdrengh, M. et al. (1999) Microbes Infect.1(10):745-751; Chang, HP et al. (1999) Learn Mem.6(5):448-457; Wang, XQ et al. (1999) J.Cell. Biol. 147(2):389-400; Lindberg, FP et al. (1996) Science 274(5288):795-798). Such animals have been used in multiple assays to determine, for example, the molecular aspects of CD47 expression, function, and regulation. Important species differences have been found. In fact, non-obese diabetic / severe combined immunodeficiency (NOD / SCID) mice express SIRPα protein, which is able to interact with human CD47 and is therefore widely used in the development of mouse models containing components of the human immune system (see, for example, Takenaka, K. et al. (2007) Nat. Immunol. 8(120:1313-1323). The SIRPα alleles present in these mice are not representative of the SIRPα alleles present in other mouse strains, and in general, there is little cross-reactivity between CD47 and SIRPα between species.In addition, it has been reported that CD47 on mouse cells has almost complete mobility, while CD47 on human cells only shows about 30-40% (Bruce, L. et al. (2003) Blood 101: 4180-4188; Mouro-Chanteloup, L. et al. (2000) Vox Sanguinis 78: P030; Mouro-Chanteloup, L. et al. (2003) Blood 101: 338-344). Therefore, NOD / SCID mice are not unlimited. For example, although in some genetic backgrounds can support multi-lineage human hematopoiesis (e.g., BALB / c Rag2). - / - IL-2Rγc - / - ), but homeostasis of other cell types remains inefficient (e.g., T and NK cells; see, e.g., Gimeno, R. et al. (2004) Blood 104:3886-3893; Traggiai, E. et al. (2004) Science 304:104-107; Legrand, N. et al. (2006) Blood 108:238-245). In addition, CD47 is also known to interact with other cell surface proteins and provide bidirectional signaling. Therefore, for the elucidation of CD47-dependent functions in various biological processes such as implantation and phagocytosis, existing mice represent an inefficient in vivo system. In addition, for the development of CD47-targeted therapies, existing mice represent a suboptimal in vivo system.

[0187] The non-human animals of the present invention provide improved in vivo systems and sources of biological materials (e.g., cells) expressing human CD47, which are useful for a variety of assays. In multiple embodiments, the non-human animals of the present invention are used to develop therapeutic agents that target CD47 and / or regulate CD47-SIRPα signaling. In multiple embodiments, the mice of the present invention are used to screen and develop candidate therapeutic agents (e.g., antibodies) that bind to human CD47. In multiple embodiments, the non-human animals of the present invention are used to screen and develop therapeutic agents (e.g., antibodies) that block the interaction between human CD47 and human SIRPα. In multiple embodiments, the non-human animals of the present invention are used to determine the binding curves of antagonists and / or agonists of humanized CD47 on the cell surface of non-human animals as described herein. In some embodiments, the non-human animals of the present invention are used to determine one or more epitopes of one or more candidate therapeutic antibodies that bind to human CD47.

[0188] In various embodiments, non-human animals of the invention are used to determine the pharmacokinetic profile of anti-CD47 antibodies. In various embodiments, one or more non-human animals of the invention and one or more control or reference non-human animals are exposed to multiple doses (e.g., 0.1 mg / kg, 0.2 mg / kg, 0.3 mg / kg, 0.4 mg / kg, 0.5 mg / kg, 1 mg / kg, 2 mg / kg, 3 mg / kg, 4 mg / kg, 5 mg / mg, 7.5 mg / kg, 10 mg / kg, 15 mg / kg, 20 mg / kg, 25 mg / kg, 30 mg / kg, 40 mg / kg or 50 mg / kg or more) of one or more candidate therapeutic anti-CD47 antibodies. Candidate therapeutic antibodies can be administered via any desired route of administration (e.g., subcutaneous, intravenous, intramuscular, intraperitoneal, etc.). Blood is isolated from non-human animals (humanized and control groups) at various time points (e.g., 0 hr, 6 hr, 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, or up to 30 or more days). Various assays can be performed using samples obtained from non-human animals as described herein to determine the pharmacokinetic properties of the administered candidate therapeutic antibodies, including, but not limited to, total IgG, anti-therapeutic antibody responses, agglutination, etc.

[0189] In various embodiments, the non-human animals of the invention are used to measure the therapeutic effects of blocking or modulating CD47 signaling and the effects of cellular changes on gene expression. In various embodiments, the non-human animals of the invention or cells isolated therefrom are exposed to candidate therapeutic agents that bind to humanized CD47 proteins (or human portions of CD47 proteins) on the surface of non-human animal cells, and then, after a period of time, the effects on CD47-dependent processes of tumors (or tumor cells), such as adhesion, angiogenesis, apoptosis, inflammation, migration, phagocytosis, proliferation, and clearance, are analyzed.

[0190] The non-human animals of the present invention express humanized CD47 proteins, and thus cells, cell lines, and cell cultures can be generated to serve as sources of humanized CD47 for binding and functional assays, such as binding and functional assays for CD47 antagonists or agonists, particularly where the antagonists or agonists are specific for human SIRPα sequences or epitopes. In various embodiments, the CD47 epitope bound by a candidate therapeutic antibody can be determined using cells isolated from the non-human animals of the present invention.

[0191] Cells from non-human animals of the present invention can be isolated and used at any time, or can be maintained in culture for many generations. In various embodiments, cells from non-human animals of the present invention are immortalized and maintained in culture indefinitely (e.g., in continuous culture).

[0192] In multiple embodiments, the cells and / or non-human animals of the present invention are used for survival and / or proliferation assays (e.g., using B or T cells) to screen and develop candidate therapeutic agents that modulate human CD47 signaling. The activation or loss of CD47 can play an important role in regulating cell proliferation, and CD47-induced apoptosis can be caused by activation of specific epitopes of the extracellular domain of CD47, therefore, the cells of the non-human animals of the present invention and / or the non-human animals as described herein can be used to identify, characterize and develop candidate CD47 regulators (e.g., antagonists or agonists). In some embodiments, the cells and / or non-human animals of the present invention are used in survival or death assays to determine the effects of proliferation and apoptosis on specific cells (e.g., cancer cells) in the presence or absence of CD47.

[0193] In various embodiments, the cells and / or non-human animals of the present invention are used for xenotransplantation of allogeneic (e.g., human) cells to determine the CD47-mediated functions in the physiological (e.g., immune) response to transplanted human cells. In some embodiments, candidate therapeutic agents that bind or block one or more functions of human CD47 are characterized in non-human animals of the present invention. Suitable measurements include various cell assays, proliferation assays, serum immunoglobulin analysis (e.g., antibody titers), cytotoxicity assays, and characterization of ligand-receptor interactions (immunoprecipitation assays). In some embodiments, non-human animals of the present invention are used to characterize CD47-mediated functions that regulate immune responses to antigens. In some embodiments, the antigen is associated with a tumor. In some embodiments, the antigen is associated with an autoimmune disease or condition. In some embodiments, the antigen is a target associated with a disease or condition suffered by one or more patients in need of treatment.

[0194] In various embodiments, the non-human animals of the invention are used in transplantation or adoptive transfer experiments to determine the therapeutic potential of compounds or biologics for CD47-dependent regulation of neo-lymphocytes and their immune functions. In various embodiments, the non-human animals of the invention are transplanted with human B cells.

[0195] In various embodiments, cells of the non-human animals of the invention are used in cell migration or spreading assays to screen and develop candidate therapeutic agents that modulate human CD47. Such processes are essential for multiple cellular processes, including wound healing, differentiation, proliferation, and survival.

[0196] In various embodiments, cells of the non-human animals of the invention are used in phagocytosis assays to determine the therapeutic potential of compounds or biological agents that modulate CD47-dependent phagocytosis.

[0197] In various embodiments, cells of the non-human animals of the invention are used in tumor cell growth (or proliferation) assays to determine the therapeutic potential of compounds or biological agents to modulate CD47-dependent regulation and / or tumor cell apoptosis.

[0198] In various embodiments, an inflammatory disease or condition is induced in one or more non-human animals of the invention to provide an in vivo system to determine the therapeutic potential of a compound or biological agent to modulate CD47-dependent regulation of one or more functions of an inflammatory disease or condition. In some embodiments, the inflammatory disease or condition is associated with a tumor.

[0199] In various embodiments, an anti-angiogenic disorder is induced in one or more non-human animals of the invention to provide an in vivo system for determining the therapeutic potential of a compound or biological agent to modulate CD47-dependent regulation of one or more functions of an anti-angiogenic disorder. Exemplary functions assessed to determine therapeutic efficacy include chemokine expression, nitric oxide (NO) stimulation response, and blood flow restoration.

[0200] Non-human animals of the present invention provide systems in vivo for analyzing and testing drugs or vaccines. In a plurality of embodiments, candidate drugs or vaccines can be delivered to one or more non-human animals of the present invention, then the non-human animals are monitored to determine one or more immunne responses to the drugs or vaccines, the safety characteristics of the drugs or vaccines, or the effect of a disease or illness. Exemplary methods for determining safety characteristics include measuring toxicity, optimal dose concentration, the efficacy of the drugs or vaccines, and possible risk factors. Such drugs or vaccines can be improved and / or developed in such non-human animals.

[0201] The non-human animals of the present invention provide an in vivo system for evaluating the pharmacokinetic properties of drugs targeting CD47. In various embodiments, a drug targeting CD47 can be delivered or administered to one or more non-human animals of the present invention, followed by monitoring the non-human animals (or cells isolated therefrom) or performing one or more assays to determine the effect of the drug on the non-human animals. Pharmacokinetic properties include, but are not limited to, the manner in which the animal processes the drug into various metabolites (or one or more drug metabolites, including detection of the presence or absence of toxic metabolites), drug half-life, circulating levels of the drug after administration (e.g., serum concentration of the drug), anti-drug responses (e.g., anti-drug antibodies), drug absorption and distribution, route of administration, route of excretion, and / or drug clearance. In some embodiments, the pharmacokinetic and pharmacodynamic properties of a drug (e.g., a CD47 modulator) are monitored in or by using the non-human animals of the present invention.

[0202] The non-human animals of the present invention provide an in vivo system for evaluating the on-target toxicity of drugs targeting CD47. In multiple embodiments, drugs targeting CD47 can be delivered or administered to one or more non-human animals of the present invention, followed by monitoring the non-human animals (or cells isolated therefrom) or performing one or more assays to determine the on-target toxic effects of the drugs on non-human animals. In general, drugs are intended to modulate the functions of one or more of their targets. To give just one example, CD47 modulators are intended to modulate CD47-mediated functions (e.g., CD47-induced apoptosis) by interacting with CD47 molecules on the surface of one or more cells in some way. In some embodiments, such modulators may have the adverse effect of amplifying the desired pharmacological effects of the modulators. Such effects are referred to as on-target effects. Exemplary on-target effects include too high a dose, chronic activation / inactivation, and correct effects in incorrect tissues. In some embodiments, previously unknown CD47 functions are determined using the on-target effects of drugs targeting CD47 identified in the non-human animals of the present invention or by using the non-human animals of the present invention.

[0203] The non-human animals of the present invention provide an in vivo system for evaluating the off-target toxicity of drugs targeting CD47. In various embodiments, a drug targeting CD47 may be delivered or administered to one or more non-human animals of the present invention, followed by monitoring the non-human animals (or cells isolated therefrom) or performing one or more assays to determine the off-target toxic effects of the drug on the non-human animals. Off-target effects may occur when a drug interacts with an unintended target (e.g., cross-reactivity to a common epitope). Such interactions may occur in intended or unintended tissues. Just to give an example, mirror image isomers (enantiomers) of a drug may lead to off-target toxic effects. In addition, the drug may not be suitable for interacting with different receptor subtypes or is not intended to activate different receptor subtypes. Exemplary off-target effects include incorrect activation / inhibition of incorrect targets, regardless of the tissue in which the incorrect target is found. In some embodiments, the off-target effects of a drug targeting CD47 are determined by comparing the effects of the drug administered to the non-human animals of the present invention with the effects of the drug administered to one or more reference non-human animals.

[0204] In some embodiments, performing the assay comprises determining the effect on the phenotype (e.g., weight change) and / or genotype of the non-human animal to which the drug is administered. In some embodiments, performing the assay comprises determining the batch-to-batch variability of a CD47 modulator (e.g., antagonist or agonist). In some embodiments, performing the assay comprises determining the difference between the effects of a drug targeting CD47 administered to a non-human animal of the present invention and a reference non-human animal. In various embodiments, the reference non-human animal may have modifications as described herein, modifications different from those described herein (e.g., modifications with a disrupted, missing, or otherwise non-functional CD47 gene), or no modifications (i.e., a wild-type non-human animal).

[0205] Exemplary parameters that can be measured in non-human animals (or in and / or using cells isolated therefrom) to assess pharmacokinetic properties, on-target toxicity, and / or off-target toxicity of drugs targeting CD47 include, but are not limited to, aggregation, autophagy, cell division, cell death, complement-mediated hemolysis, DNA integrity, drug-specific antibody titers, drug metabolism, gene expression arrays, hematocrit levels, hematuria, metabolic activity, mitochondrial activity, oxidative stress, phagocytosis, protein biosynthesis, protein degradation, protein secretion, stress response, target tissue drug concentration, non-target tissue drug concentration, transcriptional activity, etc. In various embodiments, the non-human animals of the invention are used to determine a pharmaceutically effective dose of a CD47 modulator.

[0206] The non-human animal system of the present invention provides an in vivo system for developing and characterizing candidate therapeutic agents for cancer. In multiple embodiments, the non-human animal of the present invention can be implanted with a tumor, followed by administration of one or more candidate therapeutic agents. In some embodiments, the candidate therapeutic agent may include a multispecific antibody (e.g., a bispecific antibody) or an antibody mixture; in some embodiments, the candidate therapeutic agent system includes a combination therapy, such as the administration of a monospecific antibody administered sequentially or simultaneously. Allow the tumor enough time to establish in one or more locations in the non-human animal. Tumor cell proliferation, growth, etc. can be measured before and after administration of the candidate therapeutic agent. The cytotoxicity of the candidate therapeutic agent can also be measured in the non-human animal as needed.

[0207] The non-human animals of the present invention provide an improved in vivo system for elucidating the mechanisms of human intercellular interactions through adoptive transfer. In various embodiments, the non-human animals of the present invention can be transplanted with tumor xenografts, and then the tumor infiltrating lymphocytes can be transplanted into the non-human animals by adoptive transfer to determine the effectiveness of eradicating solid tumors or other malignancies. Such experiments can be performed with human cells due to the unique presence of human CD47, which does not compete with the endogenous CD47 of the non-human animal. Alternatively, such experiments may include the use of human cells from NOD / SCID or BRG (BALB / c Rag2 - / -IL-2Rγc - / - ) background. In addition, therapies and drugs for xenotransplantation can be improved and / or developed in such non-human animals.

[0208] The non-human animal of the present invention provides an improved in vivo system for maintaining and developing human hematopoietic stem cells by implantation. In multiple embodiments, the non-human animal of the present invention provides improved development and maintenance of human stem cells in non-human animals. In multiple embodiments, the increase of people's B and T cell colonies of differentiation is observed in the blood, bone marrow, spleen and thymus of non-human animals. In multiple embodiments, the best T and NK cell homeostasis is observed in the blood, bone marrow, spleen and thymus cells of non-human animals. In multiple embodiments, compared with one or more reference non-human animals, the non-human animal of the present invention shows that the level or amount of red blood cells (RBC) increases.

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

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

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

[0212] In some embodiments, the non-human animals of the present invention are implanted with human immune cells or cells differentiated into human immune cells. Human cancer cells are transplanted into such non-human animals implanted with human immune cells, and anti-cancer drugs, such as bispecific antibodies that bind to antigens on human cancer cells and antigens on human immune cells (e.g., T cells), are administered. The therapeutic efficacy of the bispecific antibodies can be evaluated based on their ability to inhibit the growth or metastasis of human cancer cells in non-human animals. In specific embodiments, the non-human animals of the present invention are implanted with human CD34 + Hematopoietic progenitor cells, which human CD34+ hematopoietic progenitor cells generate human immune cells (including T cells, B cells, NK cells, etc.). Human B cell lymphoma cells (e.g., Raji cells) are transplanted into such non-human animals implanted with human immune cells, and then a bispecific antibody that binds to a tumor antigen (e.g., an antigen on normal B cells and certain B cell malignancies such as CD20) and the CD3 subunit of the T cell receptor is administered to test the ability of the bispecific antibody to inhibit tumor growth in the non-human animal. Example

[0213] The following examples are provided to describe to those of ordinary skill in the art how to make and use the methods and compositions of the invention and are not intended to limit the scope of what the inventors regard as their invention. Unless otherwise indicated, temperatures are given in degrees Celsius and pressures are at or near atmospheric pressure.

[0214] Example 1. Humanization of the endogenous cluster of differentiation 47 (CD47) gene

[0215] This example illustrates an exemplary method for humanizing an endogenous gene encoding cluster of differentiation 47 (CD47) in a non-human mammal, such as a rodent (e.g., mouse). The method described in this example can be used to humanize an endogenous CD47 gene of a non-human animal using any human sequence or combination of human sequences (or sequence fragments) as described. In this example, the human CD47 gene present in the bacterial artificial chromosome (BAC) clone RP11-69A17 was used to humanize an endogenous CD47 gene of a mouse.

[0216] use Technology is used to construct a targeting vector for humanizing the genetic material encoding the extracellular N-terminal IgV domain and five transmembrane domains of the endogenous CD47 gene (see, for example, U.S. Patent No. 6,586,251 and Valenzuela et al. (2003) High-throughput engineering of the mouse genome coupled with high-resolution expression analysis, Nature Biotech. 21(6): 652-659; these documents are incorporated herein by reference).

[0217] Briefly, the mouse bacterial artificial chromosome (BAC) clone RP23-230L20 (Invitrogen) was modified to delete sequences containing exons 2-7 of the endogenous CD47 gene, and the human BAC clone RP11-69A17 (Invitrogen) was used to insert exons 2-7 of the human CD47 gene, which encodes amino acids 16-292 of the human CD47 polypeptide. Endogenous DNA containing genomic DNA corresponding to exons 1, 8 and 9 of isoform 2 and 5' and 3' untranslated regions (UTRs) was retained. Sequence analysis of the human CD47 sequence contained in the BAC clone RP11-69A17 confirmed all CD47 exons and splicing signals. Sequence analysis showed that the sequence matched the reference genome and CD47 transcripts NM_001777.3 and NM_198793.2. The genomic DNA corresponding to exons 2-7 of the endogenous CD47 gene (~30.8 kb) was used to replace the BAC clone RP23-230L20 by homologous recombination in bacterial cells to insert a DNA fragment containing ~23.9 kb of genomic human DNA corresponding to exons 2-7 of the human CD47 gene from the BAC clone RP11-69A17 and a ~4995 bp DNA fragment corresponding to a self-deleting neomycin cassette flanked by recombinase recognition sites (loxP-hUb1-em7-Neo-pA-mPrm1-Crei-loxP; see U.S. Patent Nos. 8,697,851, 8,518,392 and 8,354,389, which are incorporated herein by reference). The self-deleting neomycin cassette was added to the end of the ~23.9 kb human DNA fragment containing exons 2-7 of the human CD47 gene ( Figure 2). The targeting vector contains from 5' to 3' a 5' homology arm containing ~39 kb of mouse genomic DNA from BAC clone RP23-230L20, ~29.3 kb of human genomic DNA from BAC clone RP11-69A17 (comprising exons 2-7 of the human CD47 gene), a self-deleting neomycin cassette flanked by loxP sites, and ~98.8 kb of mouse genomic DNA from BAC clone RP23-230L20. After homologous recombination with the above-mentioned targeting vector in bacterial cells, a modified RP23-230L20 BAC clone is formed, generating a humanized CD47 gene comprising mouse 5'UTR, mouse exon 1, human exons 2-7, mouse exons 8-9, and mouse 3'UTR. The protein sequences of the four designed alternative splicing isoforms of humanized CD47 are provided in Table 3, which shows the mouse and human amino acids encoded by the resulting mouse and human DNA, respectively.

[0218] The modified BAC clones described above were used to electroporate F1H4 (50% 129 / S6 / SvEv / Tac, 50% C57BL / 6NTac; Auerbach, W. et al. (2000) Biotechniques 29(5):1024-8, 1030, 1032) mouse embryonic stem (ES) cells to form modified ES cells containing an endogenous CD47 gene humanized from exons 2-7. Forward-targeted ES cells containing the humanized CD47 gene were identified by detecting the presence of human CD47 sequences (e.g., exons 2-7) and confirming the loss and / or retention of mouse CD47 sequences (e.g., exons 1, 8 and 9 and / or exons 2-7) (Valenzuela et al., supra). Table 4 shows the primers and probes used to confirm the humanization of the endogenous CD47 gene as described above ( Figure 3). The nucleotide sequence spanning the upstream insertion point includes the following sequence, which shows the endogenous mouse sequence upstream of the insertion point (contained in parentheses below, with the AsiSI restriction site in italics) adjacent to the human CD47 sequence present at the insertion point: (GCAGACATGA TTACTTCAGA GCTTTCAAAG CTAGATACTG TACCTTGCAT ATTCCAACAC) GCGATCGCATTTTAAGAT TTTCCATCCT AGTGGAAAGA TATGATTTGA TTCATCCTAT TTACTTTGTA TATTAAAGTACAGTAGAACC TGCCACTTTT (SEQ ID NO: 33). The nucleotide sequence spanning the downstream insertion point located at the 5' end of the self-deleting neomycin cassette includes the following sequence, which shows that the human CD47 genomic sequence is adjacent to the cassette sequence downstream of the insertion point (contained in parentheses below, with loxP sequences in italics): GGATCCATTT TAAGTAATAG AATAGGATTTTTAATTGTTC CAGTGTTTCT GTGATAGAGC TGTCCTGCAC AGACCTGTTT(CTCGAGATAA CTTCGTATAATGTATGCTAT ACGAAGTTAT ATGCATGGCC TCCGCGCCGG GTTTTGGCGC CTCCCGCGGG) (SEQ ID NO:34). The nucleotide sequence spanning the downstream insertion point located at the 3' end of the neomycin cassette included the following sequence, which shows that the cassette sequence is adjacent to the mouse genomic sequence 3' of exon 7 of the endogenous CD47 gene (contained in parentheses below, with loxP sequences in italics): CATGTCTGGA ATAACTTCGT ATAATGTATG CTATACGAAG TTATGCTAGT AACTATAACGGTCCTAAGGT AGCGACTAGC (ATTAGTATGG AAGGTCCGTC CACTGTCCAG GTTCCTCTTG CGGAGCTCTTTGTCTCTCTG GACTCTGTAT ACACTGCTTG) (SEQ ID NO:35).The nucleotide sequence spanning the downstream insertion point after the neomycin cassette deletion (76 bp remaining) includes the following sequence, which shows the human and mouse genomic sequence juxtaposed with the remaining cassette sequence loxP sequence (contained in parentheses below, with the loxP sequence in italics): GGATCCATTT TAAGTAATAGAATAGGATTT TTAATTGTTC CAGTGTTTCT GTGATAGAGC TGTCCTGCAC AGACCTGTTT(CTCGAGATAACTTCGTATAA TGTATGCTAT ACGAAGTTAT GCTAGTAACT ATAACGGTCC TAAGGTAGCG ACTAGC)ATTAGTATGGAAG GTCCGTCCAC TGTCCAGGTT CCTCTTGCGG AGCTCTTTGT CTCTCTGGAC TCTGTATACACTGCTTGCAT (SEQ ID NO:36).

[0219] Then use Methods, using forward ES cell clones to transplant female mice (see, e.g., U.S. Pat. No. 7,294,754 and Poueymirou et al. F0 generation mice that are essentially fully derived from the donor gene-targeted ES cells allowing immediate phenotypic analyses, 2007, Nature Biotech. 25 (1): 91-99), to generate a litter of pups containing exons 2-7 of the human CD47 gene inserted into the mouse endogenous CD47 gene. Using allele assay remodeling (Valenzuela et al., supra) that detects the presence of human CD47 gene sequences, mice with exons 2-7 of the humanized endogenous CD47 gene are reconfirmed and identified by genotyping of DNA isolated from tail snips. The pups are genotyped and a cohort of animals heterozygous for the humanized CD47 gene construct is selected for characterization.

[0220] Table 4

[0221]

[0222]

[0223] Example 2. Expression of humanized CD47 polypeptide by mouse erythrocytes.

[0224] This example demonstrates that non-human animals (e.g., rodents) modified to contain a humanized CD47 gene according to Example 1 can be used to screen CD47 modulators (e.g., anti-CD47 antibodies) and determine various properties such as pharmacokinetics and safety properties. In this example, a plurality of anti-CD47 antibodies were screened on mouse red blood cells (RBCs) isolated from rodents prepared according to Example 1, which express a humanized CD47 polypeptide as described herein.

[0225] Briefly, 2 mL of whole blood from humanized CD47 mice (n=2) was transferred to a 15 mL tube and centrifuged at 200×g for 10 minutes at 4°C. The plasma and buffy coat were aspirated, and 15 mL of PBS was added to gently mix the cells. The mixture was centrifuged again at 200×g for five minutes at 4°C. The supernatant was aspirated and the cells were washed twice more. The precipitated RBCs were resuspended in a final volume of 10 mL PBS. The resuspended RBCs were centrifuged for the last time at 200×g for 10 minutes at 4°C. The volume of the packed RBCs was expected to be 0.5 mL and was diluted to a concentration of 0.5% with PBS (0.5 mL packed RBCs / 100 mL PBS). CellometerAuto T4 (1.5×10 7 / mL; Nexcelom Bioscience) to determine the actual concentration of RBCs.

[0226] Eighty (80) μL of 0.5% mouse RBCs were added to each well of a 96-well V-bottom plate. Anti-CD47 antibody (20 μL, 33 nM) was added to each well. The plate was tapped to mix and incubated on ice for 30 minutes. The plate was then washed twice with staining buffer (PBS containing 2% FBS). A secondary antibody Fab-488 (Alexa Fluor 488 conjugated AffiniPure mouse anti-human IgG, F(ab')2 fragment specific, Jackson Immuno Research) was added to each well at a concentration of 10 μg / mL. The plate was incubated again on ice for 30 minutes and then washed once with staining buffer. The cells in each well were resuspended in 200 μL of staining buffer and filtered through a 96-well filter plate. Filtered using a BD ACCURI TM The cells in the plates were analyzed using the C6 system (BD Biosciences). Exemplary results are shown in Figure 4 The mean fluorescence intensity (MFI) of each tested antibody above the isotype control is shown in Table 5.

[0227] Table 5

[0228]

[0229] hIgG4s: human IgG4 with a modified Fc region with reduced effector function

[0230] like Figure 4 As shown, all anti-CD47 antibodies bind to RBCs from humanized CD47 mice. In summary, this example demonstrates that (1) non-human animals (e.g., rodents) engineered to contain a humanized CD47 gene as described herein express humanized CD47 polypeptides on the surface of cells (e.g., RBCs) of the non-human animal, and (2) such cells are used to screen CD47 modulators (e.g., CD47 antibodies) and determine the pharmacokinetic profile of such modulators.

[0231] Example 3. Hemagglutination of mouse erythrocytes expressing humanized CD47 polypeptide.

[0232] This example also demonstrates that non-human animals (e.g., rodents) modified to contain a humanized CD47 gene according to Example 1 can be used in various assays (e.g., hemagglutination assays) to screen CD47 modulators (e.g., anti-CD47 antibodies) and determine various properties such as pharmacokinetics and safety properties. In this example, several anti-CD47 antibodies were screened on mouse red blood cells (RBCs) expressing a humanized CD47 polypeptide as described herein to determine the antibody concentration that promotes hemagglutination.

[0233] Briefly, RBCs from wild-type and humanized CD47 mice (n=2) were prepared as described in Example 2. Twenty (20) μL of anti-CD47 antibody (5-fold serial dilutions) were added to wells 1-12 of a 96-well V-bottom plate, and then 80 μL of 0.5% mouse RBCs were added to all wells of the plate. The plate was tapped to mix and incubated at room temperature (24-27°C) for 30 minutes. The agglutination endpoint (i.e., RBCs settled to the bottom of negative samples, while RBCs agglutinated in positive samples) was visually observed. Exemplary results are shown in Figure 5 Shown in , the boxes depict wells showing blood coagulation.

[0234] like Figure 5 As shown, only lectin caused agglutination in wild-type mice. However, in addition to lectin, two anti-CD47 antibodies (Ab E and Ab C) also caused agglutination in RBCs from two humanized CD47 rodents prepared according to Example 1. Both antibodies induced agglutination starting from a concentration of 11 nM. In summary, this example demonstrates that non-human animals (e.g., rodents) engineered to contain a humanized CD47 gene as described herein can be used to evaluate one or more properties (e.g., hemagglutination) of a CD47 modulator (e.g., a CD47 antibody).

[0235] Example 4. Pharmacokinetic clearance of CD47 modulators in humanized CD47 rodents.

[0236] This example demonstrates a method for evaluating the pharmacokinetic clearance of a CD47 modulator (e.g., an anti-CD47 antibody) in a non-human animal (e.g., a rodent) that has been modified to contain a humanized CD47 gene according to Example 1. In this example, anti-CD47 antibodies are administered to wild-type and humanized CD47 rodents (e.g., mice), and serum levels of the antibodies are determined using an ELISA assay.

[0237] Briefly, four anti-CD47 antibodies (Ab F, Ab G, Ab Hand Ab I) and IgG4s isotype control antibody (IgG4s) were administered to wild type (n=5) or mouse homozygotes of humanized CD47 (n=5; as described above). The genetic background of the mice was 75% CD57BL / 6 and 25% 129Sv. Each antibody was tested in five humanized CD47 rodents. All antibodies were administered subcutaneously at a dose of 50 mg / kg. Pre-bleeding was collected once a day before the administration of the antibody (day 0). Post-injection bleeding was collected at 6 hours, 1 day, 2 days, 3 days, 4 days, 7 days, 10 days and 14 days. The serum fraction from the bleed was separated and subjected to total human antibody analysis using an ELISA immunoassay.

[0238] Briefly, goat anti-human IgG polyclonal antibody (Jackson ImmunoResearch) was coated on a 96-well plate to capture the test human antibodies in the serum, and then horseradish peroxidase-conjugated goat anti-human IgG polyclonal antibody (Jackson ImmunoResearch) and TMB substrate (BD Pharmingen) were used to detect the antibodies bound to the plate. Serum samples were diluted in six doses and the reference standard of each antibody was diluted in 12 doses. The drug antibody concentration in the serum was calculated based on the reference standard curve generated using GraphpadPrism software. Exemplary results are shown in Figure 6 and are shown in Table 6.

[0239] The data show that antibodies administered to wild-type and humanized mice as described herein are well tolerated. In summary, this example demonstrates that the non-human animals of the present invention can be used to evaluate one or more pharmacokinetic properties of drugs targeting CD47 (e.g., anti-CD47 antibodies), such as circulating drug levels. In addition, the non-human animals described herein can be used to evaluate the toxicity of drugs targeting CD47 by determining adverse effects after administration.

[0240] Table 6

[0241]

[0242]

[0243] Example 5. Pharmacokinetic profiles of CD47 modulators in humanized CD47 / SIRPα rodents.

[0244] This example demonstrates a method for evaluating the pharmacokinetic clearance of a CD47 modulator (e.g., an anti-CD47 antibody) in a non-human animal (e.g., a rodent) that is modified to contain a humanized CD47 (according to Example 1) and a SIRPα gene. Specifically, the humanized CD47 rodent described herein is modified to also contain a humanized SIRPα gene containing an endogenous portion and a human portion, the human portion encoding the extracellular domain of the human SIRPα protein (e.g., amino acids 28-362 of the human SIRPα protein), the endogenous portion encoding the intracellular domain of the endogenous SIRPα protein (e.g., amino acids encoding the transmembrane and intracellular portions of the murine SIRPα protein), as described in PCT / US14 / 56910 filed on September 23, 2014, which is incorporated herein by reference. Double humanized CD47 / SIRPα mice are prepared by mating humanized SIRPα mice with humanized CD47 mice. In this example, various anti-CD47 antibodies were administered to double humanized CD47 / SIRPα rodents (eg, mice), and their corresponding pharmacokinetic profiles were determined.

[0245] Briefly, wild-type (n=5) and homozygous mouse groups (CD47 hu / hu SIRPα hu / hu ; n=5 per group) were administered with selected anti-CD47 antibodies and IgG4 isotype control antibodies (hIgG4s). The genetic background of the mice was 75% CD57BL / 6 and 25% 129Sv. All antibodies were administered as a single subcutaneous dose of 50 mg / kg. Pre-bleeding was collected once a day before administration of the antibody (day 0). Post-injection bleeds were collected at 6 hours, 1 day, 2 days, 3 days, 4 days, 7 days and 10 days. The serum fractions from the bleeds were separated and subjected to total human antibody analysis using an ELISA immunoassay (as described above). In addition, hematocrit levels were determined at 6 hours, 1 day, 2 days, 3 days, 4 days, 7 days and 10 days, and urine tests were performed as needed (at 6 hours and when the urine color deviated from yellow) to determine the red blood cell count. Exemplary results are presented in Figure 7-9 Shown in.

[0246] like Figure 7 and 8 As shown, all anti-CD47 antibodies showed CD47 hu / hu SIRPα hu / huTarget-mediated clearance in mice, specifically, many anti-CD47 antibodies showed similar pharmacokinetic profiles. In addition, a monovalent version of an anti-CD47 antibody (Ab F) showed higher bioavailability than its bivalent equivalent ( Figure 8 The inventors found that in humanized CD47 and double humanized animals (i.e., CD47 hu / hu SIRPα hu / hu In multiple experiments in mice, the antibodies had similar pharmacokinetic profiles.

[0247] Ab J had less effect on hematocrit levels than the other tested anti-CD47 antibodies (Ab F, Ab G, Ab I, etc.) and hu / hu SIRPα hu / hu In mice, hematocrit levels produced comparable changes compared to controls (hIgG4s). Hematocrit measurements on days 2-4 showed the greatest decrease from the normal range (~38.5-45.1%), which included groups administered AbF, G, and I. Specifically, the monovalent form of Ab F showed a delayed lowering effect on hematocrit compared to the other antibodies tested. The inventors speculate that the differences in hematocrit levels between the multiple treatment groups can be attributed to differences in the epitopes recognized by the various antibodies. In addition, mice administered with select anti-CD47 antibodies showed a positive urine dipstick test for hemoglobin at 6 hours. For example, the Ab J and Ab F treatment groups each had one mouse that was positive for hemoglobin on day 1, while all other time points were negative. No significant weight loss (>20%) was observed in any treatment group.

[0248] In summary, this example demonstrates that the non-human animals of the present invention provide an in vivo system for evaluating the pharmacokinetic properties and / or profiles of one or more drugs targeting CD47 (e.g., one or more anti-CD47 antibodies), such as circulating drug levels. In addition, non-human animals engineered to further contain other humanized genes (e.g., humanized SIRPα) as described herein can be used to evaluate target-mediated clearance of one or more drugs targeting CD47.

[0249] Equivalent

[0250] Having thus described several aspects of at least one embodiment of the present invention, it will be appreciated by those skilled in the art that various changes, modifications and improvements will be readily made by those skilled in the art. Such changes, modifications and improvements are intended to be a part of the present disclosure and are intended to be within the spirit and scope of the present invention. Therefore, the above description and accompanying drawings are intended only as examples, and the present invention is described in detail by the following claims.

[0251] The use of ordinal terms such as "first", "second", "third", etc. in the claims to modify claim elements does not in itself imply any priority, precedence or order of one claim element relative to another element or the temporal order in which method acts are performed, but is merely used as a mark to distinguish one claim element with a certain name from another element with the same name (but using ordinal terms) to distinguish the claim elements.

[0252] Unless expressly indicated to the contrary, the articles "a" and "an" used in the specification and claims herein should be understood to include plural referents in the specification and claims. Claims or descriptions that include "or" between one or more members of a group should be deemed to satisfy the following situation, that is, one, more than one, or all of the group members are present in, applied to, or otherwise related to a given product or method, unless otherwise indicated or clearly different from the context. The present invention includes such embodiments in which an exact member of the group is present in, applied to, or otherwise related to a given product or method. The present invention also includes such embodiments in which more than one group member or all group members are present in, applied to, or otherwise related to a given product or method. In addition, it should be understood that the present invention includes and covers all variations, combinations and permutations in which one or more limitations, elements, clauses, descriptive terms from one or more of the listed claims are introduced into another claim (or any other claim related thereto) that is subordinate to the same basic claim, unless otherwise indicated or unless it is obvious to a person of ordinary skill in the art that it would cause a contradiction or inconsistency. When elements are presented in the form of a list (e.g., in Markush groups or similar forms), it should be understood that each subgroup of these elements is also disclosed, and any element can be removed from the group. It should be understood that, in general, when the present invention or aspects of the present invention are referred to as comprising specific elements, features, etc., certain embodiments of the present invention or aspects of the present invention are composed of such elements, features, etc. or are essentially composed of them. For the purpose of simplification, these embodiments are not specifically described in each case with so many words stated herein. It should be understood that any embodiment or aspect of the present invention can be explicitly excluded from the claims, regardless of whether such specific exclusions are described in the specification.

[0253] Those skilled in the art will appreciate the typical standard deviation or error that may be attributed to values ​​obtained in the assays or other methods described herein.

[0254] Publications, websites, and other reference materials cited herein to describe the background of the invention and to provide additional details regarding its implementation are hereby incorporated by reference.

[0255] The present invention also includes the following embodiments:

[0256] 1. A rodent comprising a CD47 gene comprising an endogenous portion and a human portion, wherein the endogenous portion and the human portion are operably linked to an endogenous CD47 promoter.

[0257] 2. The rodent of embodiment 1, wherein the endogenous portion comprises exon 1 and exon 7 downstream of the endogenous CD47 gene.

[0258] 3. The rodent of embodiment 2, wherein the exons downstream of exon 1 and exon 7 of the endogenous CD47 gene are at least 90% or at least 95% identical to the corresponding exons downstream of exon 1 and exon 7 of the mouse CD47 gene presented in Table 3.

[0259] 4. The rodent of any one of embodiments 1-3, wherein the human portion encodes amino acids 16-292 of a human CD47 polypeptide.

[0260] 5. The rodent of any one of embodiments 1-3, wherein the human portion comprises exons 2-7 of the human CD47 gene.

[0261] 6. The rodent of embodiment 5, wherein the exons 2-7 of the human CD47 gene are at least 50% identical to the corresponding exons 2-7 of the human CD47 gene presented in Table 3.

[0262] 7. The rodent of embodiment 5, wherein the exons 2-7 of the human CD47 gene are at least 60% identical to the corresponding exons 2-7 of the human CD47 gene presented in Table 3.

[0263] 8. The rodent of embodiment 5, wherein the exons 2-7 of the human CD47 gene are at least 70% identical to the corresponding exons 2-7 of the human CD47 gene presented in Table 3.

[0264] 9. The rodent of embodiment 5, wherein the exons 2-7 of the human CD47 gene are at least 80% identical to the corresponding exons 2-7 of the human CD47 gene presented in Table 3.

[0265] 10. The rodent of embodiment 5, wherein the exons 2-7 of the human CD47 gene are at least 90% identical to the corresponding exons 2-7 of the human CD47 gene presented in Table 3.

[0266] 11. The rodent of embodiment 5, wherein the exons 2-7 of the human CD47 gene are at least 95% identical to the corresponding exons 2-7 of the human CD47 gene presented in Table 3.

[0267] 12. The rodent of embodiment 5, wherein the exons 2-7 of the human CD47 gene are identical to the corresponding exons 2-7 of the human CD47 gene presented in Table 3.

[0268] 13. A rodent expressing a CD47 polypeptide, wherein the CD47 polypeptide comprises an extracellular portion and optionally a transmembrane portion of a human CD47 polypeptide and an intracellular portion of an endogenous CD47 polypeptide.

[0269] 14. The rodent of embodiment 13, wherein the CD47 polypeptide is translated from a rodent signal peptide in cells of the rodent.

[0270] 15. The rodent of embodiment 13, wherein the intracellular portion of the endogenous CD47 polypeptide comprises an intracytoplasmic tail having an amino acid sequence that is at least 50% identical to the intracytoplasmic tail of the mouse CD47 polypeptide presented in Table 3.

[0271] 16. The rodent of embodiment 13, wherein the intracellular portion of the endogenous CD47 polypeptide comprises an intracytoplasmic tail having an amino acid sequence that is at least 60% identical to the intracytoplasmic tail of the mouse CD47 polypeptide presented in Table 3.

[0272] 17. The rodent of embodiment 13, wherein the intracellular portion of the endogenous CD47 polypeptide comprises an intracytoplasmic tail having an amino acid sequence that is at least 70% identical to the intracytoplasmic tail of the mouse CD47 polypeptide presented in Table 3.

[0273] 18. The rodent of embodiment 13, wherein the intracellular portion of the endogenous CD47 polypeptide comprises an intracytoplasmic tail having an amino acid sequence that is at least 80% identical to the intracytoplasmic tail of the mouse CD47 polypeptide presented in Table 3.

[0274] 19. The rodent of embodiment 13, wherein the intracellular portion of the endogenous CD47 polypeptide comprises an intracytoplasmic tail having an amino acid sequence that is at least 90% identical to the intracytoplasmic tail of the mouse CD47 polypeptide presented in Table 3.

[0275] 20. The rodent of embodiment 13, wherein the intracellular portion of the endogenous CD47 polypeptide comprises an intracytoplasmic tail having an amino acid sequence that is at least 95% identical to the intracytoplasmic tail of the mouse CD47 polypeptide presented in Table 3.

[0276] 21. The rodent of embodiment 13, wherein the intracellular portion of the endogenous CD47 polypeptide comprises an intracytoplasmic tail having amino acids that are identical to the intracytoplasmic tail of the mouse CD47 polypeptide presented in Table 3.

[0277] 22. The rodent of any one of embodiments 13-21, wherein the extracellular portion of the human CD47 polypeptide comprises amino acids corresponding to residues 19-141 of the human CD47 polypeptide.

[0278] 23. The rodent of any one of embodiments 13-21, wherein the extracellular portion of the human CD47 polypeptide comprises an amino acid sequence that is at least 50% identical to the corresponding amino acid sequence of the extracellular portion of the human CD47 polypeptide presented in Table 3.

[0279] 24. The rodent of any one of embodiments 13-21, wherein the extracellular portion of the human CD47 polypeptide comprises an amino acid sequence that is at least 60% identical to the corresponding amino acid sequence of the extracellular portion of the human CD47 polypeptide presented in Table 3.

[0280] 25. The rodent of any one of embodiments 13-21, wherein the extracellular portion of the human CD47 polypeptide comprises an amino acid sequence that is at least 70% identical to the corresponding amino acid sequence of the extracellular portion of the human CD47 polypeptide presented in Table 3.

[0281] 26. The rodent of any one of embodiments 13-21, wherein the extracellular portion of the human CD47 polypeptide comprises an amino acid sequence that is at least 80% identical to the corresponding amino acid sequence of the extracellular portion of the human CD47 polypeptide presented in Table 3.

[0282] 27. The rodent of any one of embodiments 13-21, wherein the extracellular portion of the human CD47 polypeptide comprises an amino acid sequence that is at least 90% identical to the corresponding amino acid sequence of the extracellular portion of the human CD47 polypeptide presented in Table 3.

[0283] 28. The rodent of any one of embodiments 13-21, wherein the extracellular portion of the human CD47 polypeptide comprises an amino acid sequence that is at least 95% identical to the corresponding amino acid sequence of the extracellular portion of the human CD47 polypeptide presented in Table 3.

[0284] 29. The rodent of any one of embodiments 13-21, wherein the extracellular portion of the human CD47 polypeptide comprises an amino acid sequence that is identical to the corresponding amino acid sequence of the extracellular portion of the human CD47 polypeptide presented in Table 3.

[0285] 30. A rodent according to any one of embodiments 1-29, wherein the rodent further comprises a SIRPα gene encoding a SIRPα polypeptide, wherein the SIRPα polypeptide comprises an extracellular portion of a human SIRPα polypeptide and an intracellular portion of an endogenous SIRPα polypeptide, wherein optionally the SIRPα gene comprises exon 1, exon 5, exon 6, exon 7 and exon 8 of the endogenous SIRPα gene and exons 2-4 of the human SIRPα gene.

[0286] 31. The rodent of any one of embodiments 1-30, wherein the rodent is a rat or a mouse.

[0287] 32. A CD47 polypeptide encoded by a gene of the rodent according to any one of embodiments 1-31.

[0288] 33. The CD47 polypeptide of embodiment 32, wherein the encoded polypeptide comprises an amino acid sequence that is at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or more identical to SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19 or SEQ ID NO: 20.

[0289] 34. An isolated rodent cell or tissue, the genome of which comprises a CD47 gene encoding the extracellular portion and, optionally, the transmembrane portion of a human CD47 polypeptide linked to an intracellular portion of an endogenous CD47 polypeptide.

[0290] 35. The isolated cell or tissue of embodiment 34, wherein the CD47 gene is operably linked to a rodent CD47 promoter.

[0291] 36. The isolated cell or tissue of embodiment 34 or embodiment 35, wherein the CD47 gene comprises exons 2-7 of the human CD47 gene.

[0292] 37. The isolated rodent cell or tissue of any one of embodiments 34-36, wherein the cell or tissue further comprises a SIRPα gene encoding a SIRPα polypeptide comprising an extracellular portion of a human SIRPα polypeptide and an intracellular portion of an endogenous SIRPα polypeptide.

[0293] 38. An isolated rodent cell or tissue according to any one of embodiments 34-37, wherein the rodent cell or tissue is a mouse cell or mouse tissue or a rat cell or rat tissue.

[0294] 39. A rodent embryonic stem cell, the genome of which comprises a CD47 gene encoding an extracellular portion and, optionally, a transmembrane portion of a human CD47 polypeptide linked to an intracellular portion of an endogenous CD47 polypeptide.

[0295] 40. The rodent embryonic stem cell of embodiment 39, wherein the CD47 gene comprises exons 2-7 of a human CD47 gene operably linked to an endogenous CD47 promoter.

[0296] 41. The rodent embryonic stem cell of embodiment 39 or embodiment 40, wherein the rodent embryonic stem cell is a mouse embryonic stem cell and is from the 129 strain, the C57BL strain, or a mixture thereof.

[0297] 42. The rodent embryonic stem cell of embodiment 41, wherein the rodent embryonic stem cell is a mouse embryonic stem cell and is a mixture of 129 and C57BL strains.

[0298] 43. A rodent embryonic stem cell according to any one of embodiments 39-42, wherein the genome of the rodent embryonic stem cell also comprises a SIRPα gene encoding a SIRPα polypeptide, the SIRPα polypeptide comprising an extracellular portion of a human SIRPα polypeptide and an intracellular portion of an endogenous SIRPα polypeptide, wherein optionally the SIRPα gene comprises exon 1, exon 5, exon 6, exon 7 and exon 8 of the endogenous SIRPα gene and exons 2-4 of the human SIRPα gene.

[0299] 44. The rodent embryonic stem cell of any one of Embodiment 39, Embodiment 40, or Embodiment 43, wherein the rodent embryonic stem cell is a mouse or rat embryonic stem cell.

[0300] 45. A rodent embryo produced from the embryonic stem cells of any one of embodiments 39-44.

[0301] 46. ​​A rodent embryo according to embodiment 45, wherein the embryo is a mouse or rat embryo.

[0302] 47. A method for producing a rodent that expresses a CD47 polypeptide from an endogenous CD47 locus, wherein the CD47 polypeptide comprises a human sequence, the method comprising:

[0303] (a) inserting a genomic fragment comprising a nucleotide sequence encoding all or part of a human CD47 polypeptide into an endogenous CD47 locus in a rodent embryonic stem cell, thereby forming a humanized CD47 gene;

[0304] (b) obtaining rodent embryonic stem cells comprising the humanized CD47 gene described in (a); and

[0305] (c) Generating a rodent using the rodent embryonic stem cells described in (b).

[0306] 48. A method according to embodiment 47, wherein the human sequence comprises amino acids corresponding to residues 19-127, residues 19-141, residues 19-292 or residues 16-292 of the human CD47 polypeptide.

[0307] 49. A method according to embodiment 47 or embodiment 48, wherein the nucleotide sequence comprises exons 2-7 of the human CD47 gene.

[0308] 50. The method of any one of embodiments 47-49, wherein the nucleotide sequence comprises one or more selection markers, optionally with one or more site-specific recombination sites.

[0309] 51. A method according to any one of embodiments 47-50, wherein the humanized CD47 gene comprises exon 1 and exons downstream of exon 7 of the rodent CD47 gene.

[0310] 52. A method according to any one of embodiments 47-51, wherein the method further comprises the step of inserting a genomic fragment into the endogenous SIRPα gene of the rodent embryonic stem cells described in (a), wherein the genomic fragment comprises a nucleotide sequence encoding all or part of the human SIRPα polypeptide, wherein optionally the genomic fragment comprises exons 2-4 of the human SIRPα gene.

[0311] 53. A method according to embodiment 52, wherein the genomic fragment comprising a nucleotide sequence encoding all or part of a human SIRPα polypeptide is inserted into the endogenous SIRPα gene of the rodent embryonic stem cell described in (a) before inserting the endogenous CD47 gene.

[0312] 54. A method according to any one of embodiments 47-51, wherein the method further comprises mating the rodent described in (c) with a second rodent, the second rodent having a genome comprising a SIRPα gene encoding a SIRPα polypeptide, the SIRPα polypeptide comprising an extracellular portion of a human SIRPα polypeptide and an intracellular portion of an endogenous SIRPα polypeptide, wherein optionally the SIRPα gene comprises exon 1, exon 5, exon 6, exon 7 and exon 8 of the endogenous SIRPα gene and exons 2-4 of the human SIRPα gene.

[0313] 55. The method of any one of embodiments 47-54, wherein the rodent is a mouse or a rat.

[0314] 56. A rodent obtainable by the method of any one of embodiments 47-55.

[0315] 57. The rodent of embodiment 56, wherein the rodent is a mouse or a rat.

[0316] 58. A method of providing a rodent, the genome of the rodent comprising a CD47 gene encoding an extracellular portion of a human CD47 polypeptide linked to an intracellular portion of an endogenous CD47 polypeptide, the method comprising

[0317] The genome of the rodent is modified so that it comprises a CD47 gene encoding the extracellular portion and optionally the transmembrane portion of a human CD47 polypeptide linked to an intracellular portion of the endogenous CD47 polypeptide, thereby providing the rodent.

[0318] 59. A method according to embodiment 58, wherein the CD47 gene includes exons 2-7 of the human CD47 gene.

[0319] 60. A method according to embodiment 58 or embodiment 59, wherein the CD47 gene comprises exon 1 of a rodent CD47 gene and an exon downstream of exon 7.

[0320] 61. The method of any one of embodiments 58-60, wherein modifying the genome is performed in rodent embryonic stem cells.

[0321] 62. A method according to any one of embodiments 58-61, wherein the extracellular portion of the human CD47 polypeptide corresponds to amino acids 19-141 of the human CD47 polypeptide.

[0322] 63. A method according to any one of embodiments 58-62, wherein the method further comprises modifying the genome of the rodent so that it comprises a SIRPα gene encoding the extracellular portion of the human SIRPα polypeptide linked to the intracellular portion of the endogenous SIRPα polypeptide, wherein optionally the SIRPα gene comprises exons 2-4 of the human SIRPα gene and exon 1 and exons 5-8 of the endogenous SIRPα gene.

[0323] 64. A method according to embodiment 63, wherein the modification of the genome of the rodent so that it contains the SIRPα gene encoding the extracellular portion of the human SIRPα polypeptide linked to the intracellular portion of the endogenous SIRPα polypeptide is performed before modifying the genome of the rodent so that it contains the CD47 gene encoding the extracellular portion of the human CD47 polypeptide linked to the intracellular portion of the endogenous CD47 polypeptide.

[0324] 65. A method according to any one of embodiments 58-62, wherein the method further comprises mating the rodent with a second rodent, the rodent's genome comprising a CD47 gene encoding an extracellular portion of a human CD47 polypeptide linked to an intracellular portion of an endogenous CD47 polypeptide, the second rodent having a genome comprising a SIRPα gene encoding a SIRPα polypeptide comprising an extracellular portion of a human SIRPα polypeptide and an intracellular portion of an endogenous SIRPα polypeptide, wherein optionally the SIRPα gene comprises exons 2-4 of the human SIRPα gene and exons 1 and exons 5-8 of the endogenous SIRPα gene.

[0325] 66. The method of any one of embodiments 58-65, wherein the rodent is a mouse or a rat.

[0326] 67. A rodent obtainable by the method of any one of embodiments 58-66.

[0327] 68. The rodent of embodiment 67, wherein the rodent is a mouse or a rat.

[0328] 69. A method of implanting human cells into a rodent, the method comprising the steps of:

[0329] (a) providing a rodent according to any one of embodiments 1-31; and

[0330] (b) transplanting one or more human cells into the rodent.

[0331] 70. The method according to embodiment 69, further comprising the steps of:

[0332] (c) assaying the rodent for engraftment of the one or more human cells.

[0333] 71. A method according to embodiment 70, wherein the determining step comprises comparing the implantation of the one or more human cells with implantation in the one or more wild-type or control rodents.

[0334] 72. A method according to any one of embodiments 69-71, wherein the human cells are hematopoietic stem cells.

[0335] 73. A method according to any one of embodiments 69-72, wherein the human cells are transplanted intravenously.

[0336] 74. A method according to any one of embodiments 69-72, wherein the human cells are transplanted intraperitoneally.

[0337] 75. A method according to any one of embodiments 69-72, wherein the human cells are transplanted subcutaneously.

[0338] 76. A method for evaluating the therapeutic efficacy of a drug targeting human cells, the method comprising:

[0339] Providing a rodent according to any one of embodiments 1-31;

[0340] transplanting one or more human cells into the rodent;

[0341] administering a candidate drug to the rodent; and

[0342] The human cells in the rodent are monitored to determine the therapeutic efficacy of the candidate drug.

[0343] 77. A method according to embodiment 76, wherein the human cell is a cancer cell and the candidate drug is an anti-cancer candidate drug.

[0344] 78. The method of embodiment 77, wherein the candidate drug is an antibody.

[0345] 79. A method according to embodiment 78, wherein the rodent also contains human immune cells.

[0346] 80. A method according to embodiment 79, wherein the candidate drug is a bispecific antibody that binds to human CD47 and an antigen on the transplanted human cancer cells.

[0347] 81. A method comprising:

[0348] (a) providing one or more rodent cells, the genome of which comprises a CD47 gene encoding an extracellular portion of a human CD47 polypeptide linked to an intracellular portion of an endogenous rodent CD47 polypeptide;

[0349] (b) incubating the one or more rodent cells of step (a) with a labeled substrate; and

[0350] (c) measuring phagocytosis of the labeled substrate by the one or more rodent cells of step (b).

[0351] 82. A method according to embodiment 81, wherein the CD47 gene is operably linked to an endogenous rodent CD47 promoter.

[0352] 83. A method according to embodiment 81 or embodiment 82, wherein the CD47 gene comprises exons 2-7 of the human CD47 gene.

[0353] 84. A method according to embodiment 83, wherein the CD47 gene comprises exons 1 and exons downstream of exon 7 of the endogenous rodent CD47 gene.

[0354] 85. The method of any one of embodiments 81-84, wherein the substrate is fluorescently labeled.

[0355] 86. A method according to any one of embodiments 81-84, wherein the substrate is labeled with an antibody.

[0356] 87. The method of any one of embodiments 81-84, wherein the substrate is one or more red blood cells.

[0357] 88. The method of any one of embodiments 81-84, wherein the substrate is one or more bacterial cells.

[0358] 89. The method of any one of embodiments 81-84, wherein the substrate is one or more tumor cells.

[0359] 90. A method according to any one of embodiments 81-84, wherein the genome of the one or more rodent cells described in step (a) also contains a SIRPα gene encoding a SIRPα polypeptide, and the SIRPα polypeptide contains an extracellular portion of a human SIRPα polypeptide and an intracellular portion of an endogenous SIRPα polypeptide.

[0360] 91. The method according to embodiment 90, wherein the SIRPα gene comprises exons 2-4 of the human SIRPα gene and exons 1 and exons 5-8 of the endogenous SIRPα gene.

[0361] 92. The method of any one of embodiments 81-91, wherein the one or more cells of step (a) are mouse or rat cells.

[0362] 93. A method comprising:

[0363] (a) providing a rodent according to any one of embodiments 1-31;

[0364] (b) exposing the rodent to an antigen; and

[0365] (c) measuring phagocytosis of the antigen by one or more cells of the rodent.

[0366] 94. A method according to embodiment 93, wherein the exposing step comprises exposing the rodent to a fluorescently labeled antigen.

[0367] 95. A method according to embodiment 93, wherein the exposing step comprises exposing the rodent to one or more cells containing the antigen.

[0368] 96. A method according to embodiment 95, wherein the exposing step comprises exposing the rodent to one or more human cells containing the antigen or to one or more bacterial cells containing the antigen.

[0369] 97. A method according to embodiment 95, wherein the exposing step comprises exposing the rodent to one or more cells transformed with the antigen, such that the antigen is expressed on the surface of the one or more transformed cells.

[0370] 98. A method according to embodiment 95, wherein the exposing step comprises exposing the rodent to one or more tumor cells containing the antigen.

[0371] 99. A method for evaluating the pharmacokinetics of a drug targeting human CD47, the method comprising the following steps:

[0372] administering the medicament to a rodent according to any one of embodiments 1-31; and

[0373] Assays are performed to determine one or more pharmacokinetic properties of the drug targeting human CD47.

[0374] 100. A method for evaluating the on-target toxicity of a drug targeting human CD47, the method comprising the following steps:

[0375] administering the medicament to a rodent according to any one of embodiments 1-31; and

[0376] Determination of one or more parameters related to on-target toxicity of the drug is performed.

[0377] 101. A method for evaluating off-target toxicity of a drug targeting human CD47, the method comprising the following steps:

[0378] administering the medicament to a rodent according to any one of embodiments 1-31; and

[0379] One or more parameters associated with off-target toxicity of the drug are measured.

[0380] 102. A method according to any one of embodiments 99-101, wherein the drug targeting human CD47 is a CD47 antagonist.

[0381] 103. A method according to embodiment 102, wherein the CD47 antagonist is an anti-CD47 antibody.

[0382] 104. A method according to any one of embodiments 99-101, wherein the drug targeting human CD47 is a CD47 agonist.

[0383] 105. The method of embodiment 99, wherein the drug is an anti-CD47 antibody, and wherein the assay determines the pharmacokinetic clearance of the antibody in the rodent.

[0384] 106. A method comprising:

[0385] (a) providing a rodent according to any one of embodiments 1-31;

[0386] (b) isolating red blood cells from the rodent;

[0387] (c) incubating the isolated red blood cells in the presence of a candidate modulator of human CD47 protein; and

[0388] (d) evaluating whether the candidate modulator induces agglutination of the erythrocytes.

[0389] 107. A method according to embodiment 106, wherein the candidate modulator is an antibody.

[0390] 108. A method according to embodiment 106 or embodiment 107, wherein the evaluation comprises determining the concentration of the candidate modulator at which it induces agglutination of the red blood cells.

Claims

1. An isolated rodent tissue, the genome of which comprises a humanized CD47 gene, wherein the humanized CD47 gene comprises exon 1 of an endogenous rodent CD47 gene, exons 2-7 of a human CD47 gene, and exons downstream of exon 7 of the endogenous rodent CD47 gene, wherein the humanized CD47 gene is operably linked to an endogenous rodent CD47 promoter, wherein the isolated rodent tissue does not have the ability to regenerate a complete rodent, and wherein the rodent is a mouse or a rat.

2. The isolated rodent tissue of claim 1, wherein the humanized CD47 gene is at an endogenous rodent CD47 locus.

3. The isolated rodent tissue according to claim 1, wherein the genome of the rodent further comprises a SIRPα gene encoding a SIRPα polypeptide, wherein the SIRPα polypeptide comprises an extracellular domain of a human SIRPα polypeptide and an intracellular domain of an endogenous rodent SIRPα polypeptide, wherein the SIRPα polypeptide interacts with a CD47 polypeptide. 4 . The isolated rodent tissue of claim 3 , wherein the SIRPα gene comprises exon 1, exon 5, exon 6, exon 7 and exon 8 of an endogenous rodent SIRPα gene and exons 2-4 of a human SIRPα gene.

5. An isolated cell of a rodent, the genome of the rodent comprising a humanized CD47 gene, wherein the humanized CD47 gene comprises exon 1 of an endogenous rodent CD47 gene, exons 2-7 of a human CD47 gene, and exons downstream of exon 7 of the endogenous rodent CD47 gene, wherein the humanized CD47 gene is operably linked to an endogenous rodent CD47 promoter, wherein the isolated cell of the rodent does not have the ability to regenerate a complete rodent, and wherein the rodent is a mouse or a rat.

6. The isolated cell of claim 5, wherein the humanized CD47 gene is at an endogenous rodent CD47 locus.

7. The isolated cell according to claim 5, wherein the genome of the rodent further comprises a SIRPα gene encoding a SIRPα polypeptide, wherein the SIRPα polypeptide comprises an extracellular domain of a human SIRPα polypeptide and an intracellular domain of an endogenous rodent SIRPα polypeptide, wherein the SIRPα polypeptide interacts with a CD47 polypeptide. 8 . The isolated cell according to claim 7 , wherein the SIRPα gene comprises exon 1, exon 5, exon 6, exon 7 and exon 8 of an endogenous rodent SIRPα gene and exons 2-4 of a human SIRPα gene.

9. A method for evaluating the pharmacokinetic properties of drugs targeting human CD47 in rodents, wherein the genome of the rodent comprises a humanized CD47 gene at an endogenous rodent CD47 locus, The humanized CD47 gene comprises exon 1 of the endogenous rodent CD47 gene, exons 2-7 of the human CD47 gene, and exons downstream of exon 7 of the endogenous rodent CD47 gene, wherein the humanized CD47 gene is operably linked to an endogenous rodent CD47 promoter at an endogenous rodent CD47 locus, and wherein the rodent is a mouse or a rat, The method comprises administering the drug to the rodent and performing an assay to determine one or more pharmacokinetic properties of the drug.

10. A method for evaluating on-target or off-target toxicity of a drug targeting human CD47 in rodents, wherein the genome of the rodent comprises a humanized CD47 gene at an endogenous rodent CD47 locus, The humanized CD47 gene comprises exon 1 of the endogenous rodent CD47 gene, exons 2-7 of the human CD47 gene, and exons downstream of exon 7 of the endogenous rodent CD47 gene, wherein the humanized CD47 gene is operably linked to an endogenous rodent CD47 promoter at an endogenous rodent CD47 locus, and wherein the rodent is a mouse or a rat, The method includes administering the drug to the rodent and performing an assay to determine on-target or off-target toxicity of the drug.

11. The method of claim 9 or 10, wherein the drug is an antibody.

12. The method according to claim 9 or 10, wherein the genome of the rodent further comprises a SIRPα gene encoding a SIRPα polypeptide, wherein the SIRPα polypeptide comprises an extracellular domain of a human SIRPα polypeptide and an intracellular domain of an endogenous rodent SIRPα polypeptide, wherein the SIRPα polypeptide interacts with a CD47 polypeptide. 13 . The method of claim 12 , wherein the SIRPα gene comprises exon 1, exon 5, exon 6, exon 7, and exon 8 of an endogenous rodent SIRPα gene and exons 2-4 of a human SIRPα gene.

14. A nucleic acid vector comprising a humanized CD47 gene encoding a humanized CD47 polypeptide, The humanized CD47 gene comprises exon 1 of the rodent CD47 gene, exons 2-7 of the human CD47 gene, and exons downstream of exon 7 of the rodent CD47 gene; wherein the humanized CD47 polypeptide comprises a portion of a human CD47 polypeptide and an intracellular portion of a rodent CD47 polypeptide; wherein the portion of the human CD47 polypeptide comprises the extracellular domain and the transmembrane domain of the human CD47 polypeptide, and is encoded by exons 2-7 of the human CD47 gene; wherein the intracellular portion of the rodent CD47 polypeptide is encoded by an exon downstream of exon 7 of the rodent CD47 gene; and The rodent is a mouse or a rat.

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