RU2800428C2 - NON-HUMAN ANIMALS CONTAINING THE HUMANIZED TrkB LOCUS - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: invention relates to a non-human animal for the expression of a chimeric tropomyosin receptor kinase B (TRKB) protein containing a genetically modified endogenous TrkB locus encoding the TRKB protein, its cell, as well as to a method of obtaining the above animal.

EFFECT: invention is effective for evaluating the activity of a human TRKB-targeted reagent in vivo.

22 cl, 17 dwg, 23 tbl, 9 ex

Description

Link to related applications

[0001] The present application claims the benefit of U.S. Patent Application No. 62/592,905, filed November 30, 2017, and U.S. Patent Application No. 62/661,373, filed April 23, 2018, each of which is fully incorporated herein by reference for all purposes.

Link to a sequence listing provided as a text file via the EFS file system

[0002] The sequence listing provided in file 523380SEQLIST.txt is 154 KB in size and was created on November 30, 2018 and incorporated herein by reference.

Background of the Invention

[0003] Tropomyosin receptor kinase B (TRKB) is a promising target for neuroprotection in neurodegenerative diseases such as glaucoma. TRKB is one of the most widely distributed neurotrophic receptors (NTRs) in the brain and is highly represented in the neocortex, hippocampus, striatum, and brainstem. Binding of brain-derived neurotrophic factor (BDNF) to the TRKB receptor triggers its dimerization through conformational changes and autophosphorylation of tyrosine residues in the intracellular domain, leading to activation of signaling pathways involving mitogen-activated protein kinase (MAPK), phosphatidylinositol-3-kinase (PI3K), and phospholipases C-γ (PLC-γ).

[0004] TRKB is important for neuronal survival, differentiation, and function, and TRKB agonists may have therapeutic potential in numerous neurological, psychiatric, and metabolic disorders. However, there continues to be a need for suitable non-human animals that provide a true human target or near true human target for reagents targeting human TRKB, allowing testing of the efficacy and mode of action of such agents in living animals, as well as pharmacokinetic and pharmacodynamic studies.

Brief summary of the present invention

[0005] Non-human animals containing a humanized TRKB locus are provided, as well as methods for using such non-human animals. In addition, non-human animal genomes or cells containing a humanized TRKB locus are provided.

[0006] In one aspect, non-human animal genomes, non-human animal cells, or non-human animal genomes comprising a humanized TRKB locus are provided. Such non-human animal genomes, non-human animal cells, or non-human animals may contain a genetically modified endogenous TrkB locus encoding a TRKB protein, wherein the TRKB protein comprises a cytoplasmic domain, a transmembrane domain, and an extracellular domain, and all or part of the extracellular domain is encoded by the segment an endogenous TrkB locus that has been deleted and replaced with an orthologous human TRKB sequence.

[0007] In one aspect, non-human animals are provided that contain a humanized TrkB locus. Such non-human animals may contain a genetically modified endogenous TrkB locus encoding a TRKB protein, wherein the TRKB protein contains a cytoplasmic domain, a transmembrane domain, and an extracellular domain, and all or part of the extracellular domain is encoded by a segment of the endogenous TrkB locus that has been deleted and replaced with an orthologous sequence TRKB human.

[0008] According to another aspect, non-human animal cells are provided comprising in their genome a genetically modified endogenous TrkB locus encoding a TRKB protein, wherein the TRKB protein comprises a cytoplasmic domain, a transmembrane domain, and an extracellular domain, and all or part of the extracellular domain is encoded by a segment of the endogenous locus TrkB that has been deleted and replaced with an orthologous human TRKB sequence.

[0009] In another aspect, non-human animal genomes are provided comprising a genetically modified endogenous TrkB locus encoding a TRKB protein, wherein the TRKB protein comprises a cytoplasmic domain, a transmembrane domain, and an extracellular domain, and all or part of the extracellular domain is encoded by a segment of the endogenous TrkB locus that was deleted and replaced with an orthologous human TRKB sequence.

[0010] In some such non-human animal genomes, non-human animal cells, or non-human animal cells, the TRKB protein contains the human TRKB extracellular domain. Optionally, the extracellular domain contains the sequence shown in SEQ ID NO: 60. Optionally, the entire extracellular domain is encoded by a segment of the endogenous TrkB locus that has been deleted and replaced with an orthologous human TRKB sequence, optionally the coding sequence for the extracellular domain contains the sequence shown in SEQ ID NO: 72.

[0011] In some such non-human animal genomes, non-human animal cells, or non-human animal cells, the TRKB protein contains an endogenous signal peptide. Optionally, the signal peptide contains the sequence shown in SEQ ID NO: 51 or 55. Optionally, the entire signal peptide is encoded by the endogenous TrkB sequence, optionally, the coding sequence for the signal peptide contains the sequence shown in SEQ ID NO: 63 or 67.

[0012] In some such non-human animal genomes, non-human animal cells, or non-human animal cells, the TRKB protein contains an endogenous TRKB transmembrane domain. Optionally, the transmembrane domain contains the sequence shown in SEQ ID NO: 53 or 57. Optionally, the entire transmembrane domain is encoded by the endogenous TrkB sequence, optionally, the coding sequence for the transmembrane domain contains the sequence shown in SEQ III NO: 65 or 69.

[0013] In some such non-human animal genomes, non-human animal cells, or non-human animal cells, the TRKB protein contains an endogenous TRKB cytoplasmic domain. Optionally, the cytoplasmic domain contains the sequence shown in SEQ ID NO: 54 or 58. Optionally, the entire cytoplasmic domain is encoded by the endogenous TrkB sequence, optionally, the coding sequence for the cytoplasmic domain contains the sequence shown in SEQ ID NO: 66 or 70.

[0014] In some such non-human animal genomes, non-human animal cells, or non-human animal cells, the TRKB protein comprises an endogenous TRKB signal peptide, an endogenous TRKB transmembrane domain, and an endogenous TRKB cytoplasmic domain. Optionally, the signal peptide contains the sequence shown in SEQ ID NO: 51, the transmembrane domain contains the sequence shown in SEQ ID NO: 53, and the cytoplasmic domain contains the sequence shown in SEQ ID NO: 54. Optionally, the signal peptide contains the sequence shown in SEQ ID NO: 55, the transmembrane domain contains the sequence shown in SEQ ID NO: 57, and the cytoplasmic domain contains the sequence shown in SEQ ID NO: 58. Optionally, the entire signal peptide, the entire trans membrane domain, and the entire cytoplasmic domain are encoded by the endogenous TrkB sequence. Optionally, the coding sequence for the signal peptide contains the sequence shown in SEQ ID NO: 63, the coding sequence for the transmembrane domain contains the sequence shown in SEQ ID NO: 65, and the coding sequence for the cytoplasmic domain contains the sequence shown in SEQ ID NO: 66. Optionally, the coding sequence for the signal peptide contains the sequence shown in SEQ ID NO: 67, the coding sequence for the transmembrane domain contains the sequence shown in SEQ ID NO: 69, and the coding sequence for the cytoplasmic domain contains the sequence shown in SEQ ID NO: 70.

[0015] In some such non-human animal genomes, non-human animal cells, or non-human animal cells, the TRKB protein is a chimeric non-human animal/human TRKB protein. Optionally, the extracellular domain is the extracellular domain of human TRKB, the transmembrane domain is the transmembrane domain of the endogenous TRKB protein, and the cytoplasmic domain is the cytoplasmic domain of the endogenous TRKB protein. Optionally, the TRKB protein contains the sequence shown in SEQ ID NO: 4 or 5. Optionally, the coding sequence of the genetically modified TrkB locus encoding the TRKB protein contains the sequence shown in SEQ ID NO: 12 or 13.

[0016] Some such non-human animal genomes, non-human animal cells, or non-human animal genomes are heterozygous for a genetically modified endogenous TrkB locus. Some such non-human animal genomes, non-human animal cells, or non-human animal genomes are homozygous for the genetically modified endogenous TrkB locus.

[0017] Some such non-human animals are mammals. Optionally, the non-human animal is a rodent. Optionally, the rodent is a rat or mouse.

[0018] Some such non-human animals are rats. Optionally, the TRKB protein contains the sequence shown in SEQ ID NO: 5. Optionally, the coding sequence of the genetically modified TrkB locus encoding the TRKB protein contains the sequence shown in SEQ ID NO: 13.

[0019] Some such non-human animals are mice. Optionally, the TRKB protein contains the sequence shown in SEQ ID NO: 4. Optionally, the coding sequence of the genetically modified TrkB locus encoding the TRKB protein contains the sequence shown in SEQ ID NO: 12.

[0020] According to another aspect, methods are provided for evaluating the activity of a human TRKB-targeted reagent in vivo using the non-human animals described above. Some such methods include: (a) administering a human TRKB-targeted reagent to a non-human animal; and (b) evaluating the activity of a human TRKB-targeted reagent in a non-human animal.

[0021] In some such methods, the activity assessed is neuroprotective activity.

[0022] In some such methods, step (a) involves injecting a human TRKB-targeted reagent into a non-human animal.

[0023] In some such methods, step (b) involves assessing changes in one or more or all of the following: body weight, body composition, metabolism, and locomotion, compared to a control non-human animal. Optionally, the evaluation of changes in body tissue composition involves the evaluation of lean tissue mass and/or adipose tissue mass compared to a control non-human animal. Optionally, the assessment of changes in metabolism involves the assessment of changes in food intake and/or water intake.

[0024] In some such methods, step (b) involves assessing TRKB phosphorylation and/or activation of the MAPK/ERK and PI3K/Akt pathways compared to a control non-human animal.

[0025] In some such methods, step (b) involves assessing neuroprotective activity. In some such methods, step (b) involves assessing neuroprotective activity, and the non-human animal is a rat. In some such methods, step (b) involves assessing the viability of retinal ganglion cells. Optionally, the assessment of retinal ganglion cell viability includes an assessment of retinal ganglion cell density. Optionally, retinal ganglion cell density is measured in excised retinas stained for retinal ganglion cells. Optionally, retinal ganglion cell viability is assessed in a complete optic nerve transection model after optic nerve injury. Optionally, retinal ganglion cell viability is assessed in an optic nerve crush model.

[0026] In some such methods, the human TRKB-targeting reagent is an antigen-binding protein. Optionally, the antigen binding protein is a human TRKB agonist antibody. In some such methods, the human TRKB-targeting reagent is a small molecule. Optionally, the small molecule is a human TRKB agonist.

[0027] In another aspect, targeting vectors are provided to generate a genetically modified endogenous TrkB locus encoding a TRKB protein, wherein the TRKB protein comprises a cytoplasmic domain, a transmembrane domain, and an extracellular domain, and all or part of the extracellular domain is encoded by a segment of the endogenous TrkB locus that has been deleted and replaced by an orthologous human TRKB sequence, and wherein the targeting vector contains a nucleic acid insert containing the orthologous human TRKB sequence flanked by a 5' homologous arm targeting a 5' target sequence at the endogenous TrkB locus and a 3' homologous arm targeting 3' target sequence at the endogenous TrkB locus.

[0028] According to another aspect, methods are provided for obtaining any of the non-human animals described above. Some such methods may include the following: (a) introducing into a pluripotent cell of a non-human animal that is not a single cell embryo the following:

(i) an exogenous repair template containing a nucleic acid insert flanked by a 5' homologous arm that hybridizes to the 5' target sequence at the endogenous TrkB locus and a 3' homologous arm that hybridizes to the 3' target sequence at the endogenous TrkB locus, wherein nucleic acid - the insert contains an orthologous human TRKB sequence; And

(ii) a nuclease agent that targets a target sequence within the endogenous TrkB locus, wherein the genome is modified to include the genetically modified endogenous TrkB locus; (b) introducing the modified pluripotent non-human animal cell into the host embryo; and (c) implanting a host embryo into a surrogate mother to produce a genetically modified non-human animal of the F0 generation containing the genetically modified endogenous TrkB locus. Optionally, the pluripotent cell is an embryonic stem (ES) cell. Optionally, the nuclease agent is a Cas9 protein and a guide RNA that targets the target sequence for the guide RNA within the endogenous TrkB locus. Optionally, step (a) further comprises introducing into the non-human animal pluripotent cell a second guide RNA that targets a second guide RNA target sequence within the endogenous TrkB locus. Optionally, the exogenous repair template is a large targeting vector that is at least 10 kb in length, or where the exogenous repair template is a large targeting vector where the sum of the 5' homologous arm and the 3' homologous arm is at least 10 kb in length.

[0029] Some such methods include: (a) introducing the following into a single cell embryo of a non-human animal: a sequence at the endogenous TrkB locus, and a 3' homologous arm that hybridizes to a 3' target sequence at the endogenous TrkB locus, wherein the nucleic acid insert contains an orthologous human TRKB sequence; and (ii) a nuclease agent targeting a target sequence within the endogenous TrkB locus, wherein the genome is modified to include the genetically modified endogenous TrkB locus; and (b) implanting a modified single cell embryo of a non-human animal into a surrogate mother to produce a genetically modified F0 generation non-human animal containing the genetically modified endogenous TrkB locus. Optionally, the nuclease agent is a Cas9 protein and a guide RNA that targets the target sequence for the guide RNA within the endogenous TrkB locus. Optionally, step (a) further comprises introducing into the non-human embryo at the single cell stage a second guide RNA that targets a second guide RNA target sequence within the endogenous TrkB locus.

Brief description of the figures

[0030] Figure 1 (not to scale) shows a schematic representation of a targeting scheme for humanization of the mouse (Ntrk2 mouse) TrkB locus region encoding the TRKB extracellular domain. The upper part of the figure shows the endogenous mouse TrkB locus (mouse Ntrk2) and the lower part of the figure shows a large targeting vector.

[0031] Figure 2 (not to scale) shows a schematic representation of TAQMAN® assays for humanization screening of the mouse TrkB locus (mouse Ntrk2). Allele acquisition assays (GOA) include 7138hU and 7138hD. Allele loss assays (LOA) include 7138U and 7138D.

[0032] Figure 3 shows Western blots assessing total TRKB levels and phospho-TRKB levels in TRKB homozygous humanized mice at 1 hour, 4 hours, and 18 hours after direct hippocampal injection of H4H9816P2 TRKB agonist antibody or isotype control antibody.

[0033] Figure 4 (not to scale) shows a schematic representation of a targeting scheme for humanizing the rat TrkB (rat Ntrk2) locus region encoding the extracellular domain of TRKB. The top of the figure shows the endogenous rat TrkB (rat Ntrk2) locus and the bottom of the figure shows the large targeting vector.

[0034] Figure 5 (not to scale) shows a schematic representation of TAQMAN® assays for humanization screening of the rat TrkB locus (rat Ntrk2) and guide RNA position (target sequences for guide RNA as set out in SEQ ID NOs: 41-44) for targeting the rat TrkB locus (rat Ntrk2). Allele acquisition (GOA) assays include 7138hU and 7138hD. Allele loss assays (LOA) include rnoTU, rnoTM and rnoTD. CRISPR assays designed to cover the region that is disrupted by CRISPR/Cas9 targeting include rnoTGU and rnoTGD. Retention assays include rnoTAU2 and rnoTAD2.

[0035] Figure 6 shows the alignment of mouse, rat and human TRKB (NTRK2) proteins.

[0036] Figure 7 shows Western blots of phospho-TrkB, total TrkB, phospho-Akt, total AKT, phospho-ERK, and total ERK at 15 minutes and 2 hours post-treatment of primary cortical neurons isolated from homozygous day one pups. mice humanized against TRKB with various TrkB or BDNF agonist antibodies.

[0037] Figure 8 shows the pharmacokinetic profiles of the anti-TRKB antibody H4H9816P2 in homozygous TrkBhu/hu mice and wild-type mice.

[0038] Figure 9 shows cell survival in differentiated human neuroblastoma SH-SY5Y cells treated with various doses of TrkB or BDNF agonist antibodies. mAb1 to TrkB is H4H9816P2; mAb2 to TrkB is a control TrkB agonist antibody with affinity for human TrkB, rat TrkB and mouse TrkB. A human isotype control antibody was used as a negative control. Data were normalized to serum-free medium without antibodies.

[0039] Figure 10 shows cell survival in primary mouse retinal ganglion cells treated with various doses of TrkB agonist antibody or BDNF. mAb2 to TrkB is a control TrkB agonist antibody with affinity for human TrkB, rat TrkB and mouse TrkB. Data were normalized to serum-free medium without antibodies.

[0040] Figures 11A and 11B show the density of retinal ganglion cells in retinas excised and stained for retinal ganglion cells in wild-type rats and mice, respectively, after optic nerve transection and treatment with BDNF, TrkB agonist antibody, isotype antibody control or control-carrier. Rats were injected with BDNF (5 μg), TrkB agonist antibody (18 μg), isotype control antibody (18 μg) or vehicle control intravitreally 3 days and 10 days after optic nerve transection. Mice were injected with BDNF (2.5 μg), TrkB agonist antibody (10 μg), isotype control antibody (10 μg) or vehicle control intravitreally 3 days and 10 days after optic nerve transection. mAb2 to TrkB is a control TrkB agonist antibody with affinity for human TrkB, rat TrkB and mouse TrkB.

[0041] Figures 12A and 12B show the density of retinal ganglion cells in retinas excised and stained for retinal ganglion cells in wild-type mice and rats, respectively, after optic nerve transection or optic nerve crush and treatment with various doses of BDNF. Figure 12A shows the dose response of BDNF in an optic nerve crush (ONC) model in WT mice. Figure 12B shows the dose response of BDNF in the optic nerve transection model in WT rats from 0.13 μg to 30 μg.

[0042] Figure 13A shows the density of retinal ganglion cells in retinas excised and stained for retinal ganglion cells in homozygous, heterozygous, or wild-type TrkB rats treated with either a TrkB agonist antibody or an isotype control antibody intravitreally at 3 and 10 days after optic nerve transection (**** = p < 0.0001; *** p < 0.001; two-way analysis of variance). The retinas were excised 14 days after transection. mAb1 to TrkB is H4H9816P2.

[0043] Figure 13B shows the density of retinal ganglion cells in intact eyes excised from homozygous, heterozygous, or wild-type TrkB rats.

[0044] Figure 13C shows the body weight of human TRKB homozygous mice treated with a TrkB agonist antibody (H4H9816P2; TrkB) or an isotype control antibody (REGN1945; control).

[0045] Figure 14 shows the density of retinal ganglion cells in retinas excised and stained for retinal ganglion cells in human TRKB homozygous rats treated with either a TrkB agonist antibody (hTrkB; H4H9816P2) or an isotype control antibody (REGN1945) intravitreal 3 and 10 days after the intersection of the optic nerve. The retinas were excised 14 days after transection.

[0046] Figures 15A and 15B show the density of retinal ganglion cells in retinas excised and stained for retinal ganglion cells in human TRKB homozygous rats treated with various TrkB agonist antibodies (H4H9816P2-L9, H4H9814P-L9, H4H9780P- L5 or a combination of all three) or isotype control antibody (REGN1945) intravitreally 3 and 10 days after optic nerve transection (**p<0.01; Kruskal-Wallis test versus isotype control antibody). The retinas were excised 14 days after transection. Figure 15A includes an untreated control (intact contralateral eye) while Figure 15B does not.

[0047] Figure 16 shows the density of retinal ganglion cells in retinas excised and stained for retinal ganglion cells in wild-type rats treated with various TrkB agonist antibodies (H4H9780P and H4H9814P) or an isotype control antibody (REGN1945) intravitreally at 3 and 10 days after the intersection of the optic nerve. The retinas were excised 14 days after transection.

[0048] Figures 17A and 17B show the density of retinal ganglion cells in retinas excised and stained for retinal ganglion cells in human TRKB homozygous mice treated with a TrkB agonist antibody (H4H9780P) or an isotype control antibody (REGN1945) intravitreally via 3 and 10 days after the intersection of the optic nerve. The retinas were excised 14 days after transection. Figure 17A includes a normal control (intact contralateral eye), while Figure 17B does not.

[0049] Figure 17C shows body weights of human TRKB homozygous mice treated with a TrkB agonist antibody (H4H9780P) or an isotype control antibody (REGN1945).

Definitions

[0050] As used interchangeably herein, the terms "protein", "polypeptide", and "peptide" include polymeric forms of amino acids of any length, including encoded and non-encoded amino acids and chemically or biochemically modified or derivatized amino acids. The terms also include polymers that have been modified, such as polypeptides characterized by modified peptide backbones. The term "domain" refers to any portion of a protein or polypeptide that has a particular function or structure.

[0051] Proteins are considered to have an "N-terminus" and a "C-terminus". The term "N-terminus" refers to the beginning of a protein or polypeptide ending in an amino acid with a free amino group (-NH2). The term "C-terminus" refers to the end of an amino acid chain (of a protein or polypeptide) ending in a free carboxyl group (-COOH).

[0052] Used interchangeably herein, the terms "nucleic acid" and "polynucleotide" include polymeric forms of nucleotides of any length, including ribonucleotides, deoxyribonucleotides, or analogs or modified versions thereof. They include single-, double-, and multi-stranded DNA or RNA, genomic DNA, cDNA, DNA-RNA hybrids, and polymers containing purine bases, pyrimidine bases, or other natural, chemically modified, biochemically modified, non-natural, or derivatized nucleotide bases.

[0053] Nucleic acids are thought to have "5' ends" and "3' ends" because mononucleotides react to form oligonucleotides in such a way that the 5'-phosphate of one mononucleotide-pentose ring is attached to the 3'-oxygen of its neighbor in one through the phosphodiester bond. The end of an oligonucleotide is called the "5' end" if its 5' phosphate is not bonded to the 3' oxygen of the mononucleotide pentose ring. The end of an oligonucleotide is called the "3' end" if its 3' oxygen is not bonded to the 5' phosphate of another mononucleotide pentose ring. It can be said that the nucleic acid sequence, even if it is inside a larger oligonucleotide, is characterized by the presence of 5' and 3' ends. In a linear or circular DNA molecule, discrete elements are referred to as "upstream" or 5' from "downstream" or 3' elements.

[0054] The term "genomically integrated" refers to a nucleic acid that has been introduced into a cell such that the nucleotide sequence is integrated into the cell's genome and is capable of being inherited by its offspring. Any protocol can be used to stably incorporate a nucleic acid into a cell's genome.

[0055] The term "targeting vector" refers to a recombinant nucleic acid that can be introduced by homologous recombination, non-homologous end joining mediated ligation, or any other method of recombination with respect to a target position in the cell's genome.

[0056] The term "viral vector" refers to a recombinant nucleic acid that includes at least one element of viral origin and includes elements sufficient to or permit packaging into a viral vector particle. The vector and/or particle can be used to transfer DNA, RNA or other nucleic acids into cells, either ex vivo or in vivo. Numerous forms of viral vectors are known.

[0057] The term "wild type" includes entities characterized by structure and/or activity found in a normal (as opposed to mutated, diseased, altered, etc.) state or context. Wild-type genes and polypeptides often exist in several different forms (eg, alleles).

[0058] The term "endogenous" refers to a nucleic acid sequence that occurs naturally within a non-human cell or animal. For example, an endogenous TrkB sequence in a non-human animal refers to a native TrkB sequence that occurs naturally at the TrkB locus in a non-human animal.

[0059] "Exogenous" molecules or sequences include molecules or sequences that are not normally present in the cell in that form. Normal presence includes being present at a particular developmental stage and under particular environmental conditions of a cell. For example, an exogenous molecule or sequence may include a mutant version of the corresponding endogenous sequence in the cell, such as a humanized version of the endogenous sequence, or may include a sequence corresponding to the endogenous sequence in the cell, but in a different form (i.e., not within chromosomes). In contrast, endogenous molecules or sequences include molecules or sequences that are normally present in such form in a particular cell at a particular stage of development under certain environmental conditions.

[0060] The term "heterologous" when used in the context of a nucleic acid or protein indicates that the nucleic acid or protein contains at least two parts that do not naturally occur together in the same molecule. For example, the term "heterologous" when used in relation to parts of a nucleic acid or parts of a protein indicates that the nucleic acid or protein contains two or more subsequences that are not in the same relationship to each other (eg combined) in nature. As one example, a "heterologous" region of a nucleic acid vector is a nucleic acid segment within or attached to another nucleic acid molecule that is not found in association with another molecule in nature. For example, the heterologous region of a nucleic acid vector may include a coding sequence flanked by sequences that are not naturally found in association with the coding sequence. Similarly, a "heterologous" region of a protein is a segment of amino acids within or attached to another peptide molecule that is not naturally found in association with another peptide molecule (eg, a fusion or tagged protein). Similarly, the nucleic acid or protein may contain a heterologous tag or a heterologous secretion sequence or a heterologous localization sequence.

[0061] "Codon optimization" takes advantage of the degeneracy of codons, which manifests itself in the multiplicity of combinations of codons with three base pairs that define an amino acid, and typically involves the process of modifying the nucleic acid sequence for increased expression in specific cells - hosts by replacing at least one codon of the native sequence with a codon that is more or more frequently used in the genes of the host cell while maintaining the native amino acid sequence. For example, a nucleic acid encoding a Cas9 protein can be modified to replace codons having a higher frequency of use in a given prokaryotic or eukaryotic cell, including a bacterial cell, a yeast cell, a human cell, a non-human cell, a mammalian cell, a cell rodent, mouse cell, rat cell, hamster cell or any other host cell compared to a naturally occurring nucleic acid sequence. Codon usage tables are readily available, for example, in the "Codon Usage Database". These tables can be adapted in several ways. See Nakamura et al. (2000) Nucleic Acids Research 28:292, incorporated herein by reference in its entirety for all purposes. Computer algorithms are also available to optimize codons of a particular sequence for expression in a particular host (see, for example, Gene Forge).

[0062] The term "locus" refers to a specific location of a gene (or sequence of interest), a DNA sequence encoding a polypeptide sequence, or a position on a chromosome of an organism's genome. For example, a "TrkB locus" may refer to a specific location of a TrkB gene, a TrkB DNA sequence, a TrkB encoding sequence, or a position of TrkB on a chromosome of an organism's genome that has been identified relative to where such a sequence is located. A "TrkB locus" may comprise a regulatory element of the TrkB gene, including, for example, an enhancer, a promoter, a 5' and/or 3' untranslated region (UTR), or a combination thereof.

[0063] The term "gene" refers to a DNA sequence on a chromosome that encodes a product (e.g., an RNA product and/or a polypeptide product) and includes a coding region interrupted by non-coding introns and a sequence adjacent to the coding region by 5' and 3' ends so that the gene corresponds to the full length mRNA (including the 5' and 3' untranslated sequences). The term "gene" also includes other non-coding sequences, including regulatory sequences (e.g., promoters, enhancers, and transcription factor binding sites), polyadenylation signals, internal ribosome entry sites, silencers, an isolating sequence, and template attachment sites. These sequences may be close to the coding region of the gene (eg, within 10 kb), or may be located at remote sites, and they affect the level or rate of transcription and translation of the gene.

[0064] The term "allele" refers to a variant form of a gene. Some genes have many different forms that are located in the same position or genetic locus on the chromosome. A diploid organism contains two alleles at each genetic locus. Each pair of alleles represents the genotype of a specific genetic locus. Genotypes are described as homozygous if there are two identical alleles at a particular locus, and as heterozygous if the two alleles are different.

[0065] A "promoter" is a DNA regulatory region, typically containing a TATA box, capable of directing RNA polymerase II to initiate RNA synthesis at the appropriate transcription initiation site for a particular polynucleotide sequence. The promoter may further contain other regions that affect the rate of transcription initiation. The promoter sequences disclosed herein modulate the transcription of an operably linked polynucleotide. A promoter may be active in one or more of the cell types disclosed herein (e.g., eukaryotic cell, non-human mammalian cell, human cell, rodent cell, pluripotent cell, single cell embryo, differentiated cell, or a combination thereof). A promoter can be, for example, a constitutively active promoter, a conditional promoter, an inducible promoter, a time-limited promoter (eg, a developmentally regulated promoter), or a spatially-limited promoter (eg, a cell-specific or tissue-specific promoter). Examples of promoters can be found, for example, in International Patent Publication WO 2013/176772, incorporated herein by reference in its entirety for all purposes.

[0066] The term "operably linked" or "operably linked" includes the contiguous position of two or more components (e.g., a promoter and another element of the sequence) such that both components function normally and provide the possibility that at least one of the components may mediate a function that is acted upon by at least one of the other components. For example, a promoter may be operably linked to a coding sequence if the promoter controls the level of transcription of the coding sequence in response to the presence or absence of one or more transcriptional regulatory factors. The operative linkage may include sequences that are adjacent to each other or act in trans (eg, a regulatory sequence may act at a distance to direct transcription of a coding sequence).

[0067] The term "variant" refers to a nucleotide sequence different from the sequence most common in the population (eg, by one nucleotide), or a protein sequence different from the sequence most common in the population (eg, by one amino acid).

[0068] The term "fragment" in relation to a protein means a protein that is shorter or contains fewer amino acids than a full-length protein. The term "fragment" in relation to a nucleic acid means a nucleic acid that is shorter or contains fewer nucleotides than a full-length nucleic acid. A fragment may be, for example, an N-terminal fragment (ie, removal of part of the C-terminal end of the protein), a C-terminal fragment (ie, removal of part of the N-terminal end of the protein), or an internal fragment.

[0069] The term "sequence identity" or "identity" in the context of two polynucleotide or polypeptide sequences refers to residues in two sequences that are the same when aligned for maximum match in a specified comparison window. When percent sequence identity is used with proteins, positions of residues that are not identical are often distinguished by conservative amino acid substitutions, where amino acid residues are replaced by other amino acid residues with similar chemical properties (e.g., charge or hydrophobicity) and therefore do not change the functional properties of the molecule. . When sequences differ in conservative substitutions, the percent sequence identity can be increased to correct for the conservative nature of the substitution. Sequences that differ by such conservative substitutions are said to be characterized by "sequence similarity" or "similarity". Means for such adjustment are well known. Typically, this includes scoring a conservative substitution as a partial rather than a complete mismatch, which increases the percent sequence identity. Thus, for example, when an identical amino acid scores 1 and a non-conservative substitution scores 0, a conservative substitution scores 0 to 1. Conservative substitution scores are calculated, for example, as implemented in the PC/GENE program (Intelligenetics, Mountain View, California USA).

[0070] "Percent sequence identity" includes a value determined by comparing two optimally aligned sequences (highest number of perfectly matched residues) in the comparison window, wherein a portion of the polynucleotide sequence in the comparison window may contain additions or deletions (i.e., gaps ) compared to a reference sequence (which contains no addition or deletion) for optimal alignment of the two sequences. The percentage is calculated by determining the number of positions at which an identical nucleic acid base or amino acid residue occurs in both sequences to obtain the number of matching positions, dividing the number of matching positions by the total number of positions in the comparison window, and multiplying the result by 100 to obtain the percent sequence identity. . Unless otherwise noted (eg, the shorter sequence includes an associated heterologous sequence), the comparison window is the full length of the shorter of the two compared sequences.

[0071] Unless otherwise indicated, sequence identity/similarity values include the value obtained using GAP version 10 using the following parameters: % identity and % similarity for the nucleotide sequence using a GAP gap penalty of 50, and gap lengthening penalty of 3 and replacement matrix nwsgapdna.cmp; % identity and % similarity for amino acid sequence using GAP gap penalty of 8 and gap extension penalty of 2 and substitution matrix BLOSUM62; or any equivalent program. An "equivalent program" includes any sequence comparison program that, for any two sequences under consideration, generates an alignment characterized by identical nucleotide or amino acid residue matches and an identical percentage of sequence identity compared to the corresponding alignment generated by GAP version 10.

[0072] The term "conservative amino acid substitution" refers to the substitution of an amino acid that is normally present in a sequence with another amino acid of similar size, charge, or polarity. Examples of conservative substitutions include replacing a non-polar (hydrophobic) moiety such as isoleucine, valine, or leucine with another non-polar moiety. Similarly, examples of conservative substitutions include the substitution of one polar (hydrophilic) residue for another, such as a substitution between arginine and lysine, between glutamine and asparagine, or between glycine and serine. In addition, substitution of a basic residue such as lysine, arginine or histidine with another, or substitution of one acidic residue such as aspartic acid or glutamic acid with another acidic residue are further examples of conservative substitutions. Examples of non-conservative substitutions include replacing a non-polar (hydrophobic) amino acid residue such as isoleucine, valine, leucine, alanine, or methionine with a polar (hydrophilic) residue such as cysteine, glutamine, glutamic acid, or lysine, and/or a polar residue with non-polar residue. Typical classifications of amino acids are shown in the table below.

[0073] A "homologous" sequence (e.g., a nucleic acid sequence) includes a sequence that is either identical to or substantially similar to a known reference sequence such that it is, for example, 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%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the known reference sequence. Homologous sequences may include, for example, an orthologous sequence and paralogous sequences. Homologous genes, for example, typically derive from a common ancestral DNA sequence, either through a speciation event (orthologous genes) or a genetic duplication event (paralogous genes). "Orthologous" genes include genes in different species that have evolved from a common ancestral gene through speciation. Orthologs tend to retain the same function through evolution. "Paralogous" genes include genes associated with a duplication in the genome. Paralogs can develop new features in the course of evolution.

[0074] The term "in vitro" includes artificial environments and refers to processes or reactions that occur in an artificial environment (eg, in a test tube). The term "in vivo" includes natural environments (eg, cell, organism or body) and refers to processes or reactions that occur in a natural environment. The term "ex vivo" includes cells that have been removed from the human body and also refers to processes or reactions that take place in such cells.

[0075] The term "reporter gene" refers to a nucleic acid with a sequence encoding a gene product (typically an enzyme) that is readily and quantitatively analyzed when the construct containing the reporter gene sequence is operably linked to an endogenous or heterologous promoter and/or enhancer element introduced into cells containing (or can be made to contain) the factors necessary for the activation of promoter and/or enhancer elements. Examples of reporter genes include, but are not limited to, genes encoding beta-galactosidase (lacZ), genes for bacterial chloramphenicol acetyltransferase (feline), firefly luciferase genes, genes encoding beta-glucuronidase (GUS), and genes encoding fluorescent proteins. "Reporter protein" refers to a protein encoded by a reporter gene.

[0076] As used herein, the term "fluorescent reporter protein" means a reporter protein that can be detected based on fluorescence, where the fluorescence can be either directly from the reporter protein, the activity of the reporter protein on a fluorogenic substrate, or a protein with an affinity for binding to a fluorescent labeled compound. Examples of fluorescent proteins include green fluorescent proteins (e.g., GFP, GFP-2, tagGFP, turboGFP, eGFP, emerald green, Azami Green, monomeric Azami Green, CopGFP, AceGFP, and ZsGreenl), yellow fluorescent proteins (e.g., YFP, eYFP, citrine, Venus, YPet, PhiYFP, and ZsYellowl), blue fluorescent proteins (e.g., BFP, eBFP, eBFP2, azurite, mKalamal, GFPuv, sapphire, and T-sapphire), blue fluorescent proteins (e.g., CFP, eCFP, sky blue, CyPet, AmCyanl, and Midoriishi-Cyan), red fluorescent proteins (e.g., RFP, mKate, mKate2, mPlum, DsRed-Monomer, mCherry, mRFPl, DsRed-Express, DsRed2, DsRed-Monomer, HcRed-Tandem, HcRedl, AsRed2, eqFP611 , mRaspberry, mStrawberry and Jred), orange fluorescent proteins (e.g. mOrange, mKO, Kusabira-Orange, monomeric Kusabira-Orange, mTangerine and tdTomato) and any other suitable fluorescent protein whose presence in cells can be detected using flow cytometry methods.

[0077] The term "recombination" includes any process of exchange of genetic information between two polynucleotides and can occur by any mechanism. Recombination in response to double-strand breaks (DSBs) occurs primarily through two conservative DNA repair pathways: nonhomologous end joining (NHEJ) and homologous recombination (HR). See Kasparek & Humphrey (2011) Seminars in Cell & Dev. Biol. 22: 886-897, incorporated herein by reference in its entirety for all purposes. Similarly, repair of a target nucleic acid mediated by an exogenous donor nucleic acid may include any process of exchanging genetic information between two polynucleotides.

[0078] NHEJ involves repairing double-strand breaks in a nucleic acid by directly ligating the ends of the breaks to each other or to an exogenous sequence without the need for a homologous template. Ligation of non-contiguous sequences with NHEJ can often result in deletions, insertions, or translocations near the double-strand break site. For example, NHEJ can also result in targeted integration of the exogenous donor nucleic acid by direct ligation of the break ends to the ends of the exogenous donor nucleic acid (ie, NHEJ-based capture). Such NHEJ-mediated targeted integration may be preferred for exogenous donor nucleic acid insertion when homologous repair (HDR) pathways are not easily exploited (eg, in non-dividing cells, primary cells, and cells that perform poorly on homology-based DNA repair). In addition, in contrast to homologous repair, knowledge of the relatively large regions of sequence identity flanking the cleavage site is not required, which may be advantageous when attempting targeted insertion into organisms having genomes for which knowledge of the genomic sequence is limited. Integration can occur by blunt-end ligation between the exogenous donor nucleic acid and the cleaved genomic sequence, or by ligation of sticky ends (i.e., characterized by 5' or 3' hanging ends) using an exogenous donor nucleic acid that is flanked by hanging ends that are compatible with those created by the nuclease agent in the cleaved genomic sequence. See, for example, US 2011/020722, WO 2014/033644, WO 2014/089290 and Maresca et al. (2013) Genome Res. 23(3):539-546, each of which is incorporated herein by reference in its entirety for all purposes. If the blunt ends are ligated, it may be necessary to excise the target and/or donor to create regions of microhomology necessary for joining the fragments, which may lead to undesirable changes in the target sequence.

[0079] Recombination can occur via homologous repair (HDR) or homologous recombination (HR). HDR or HR involve a form of nucleic acid repair that may require nucleotide sequence homology, uses a "donor" molecule as a template to repair a "target" molecule (i.e. one that has experienced a double-strand break), and results in the transfer of genetic information from donor to target. While not wishing to be bound by any particular theory, such transfer may include correcting the heteroduplex DNA mismatch that forms between the damaged target and the donor, and/or strand synthesis dependent annealing, in which the donor is used to resynthesize the genetic information that will become part of the target and/or associated processes. In some cases, a donor polynucleotide, a portion of a donor polynucleotide, a copy of a donor polynucleotide, or a portion of a copy of a donor polynucleotide is integrated into the target DNA. See Wang et al. (2013) Cell 153:910-918; Mandalos et al. (2012) PLOS ONE7:e45768:l-9; and Wang et al. (2013) Nat Biotechnol. 31:530-532, each of which is hereby incorporated by reference in its entirety for all purposes.

[0080] The term "antigen binding protein" includes any protein that binds to an antigen. Examples of antigen-binding proteins include an antibody, an antigen-binding fragment of an antibody, a multispecific antibody (e.g., a bispecific antibody), scFV, bi-scFV, diabody, triabody, tetrabody, V-NAR, VHH, VL, F(ab), F(ab) ₂ , DVD (antigen-binding protein with dual variable domain), SVD (antigen-binding protein with a single variable domain), T-cell-attracting bispecific activator (BiTE), or Davisbody (U.S. Patent No. 8,586,713, incorporated herein by reference in its entirety for all purposes) .

[0081] As used herein, the term “anti-TRKB antibody” includes both monovalent antibodies with single specificity, as well as bispecific antibodies containing a first arm that binds to TRKB and a second arm that binds to a second (target) antigen, wherein the anti-TRKB arm contains, for example, any of the HCVR/LCVR or CDR sequences as set forth in Table 22 herein. The term "anti-TRKB antibody" also includes antibody-drug conjugates (ADCs) containing an anti-TRKB antibody, or an antigen-binding portion thereof, conjugated to a drug or toxin (ie, a cytotoxic agent). The term "anti-TRKB antibody" also includes antibody-radionuclide conjugates (ARCs) containing an anti-TRKB antibody, or an antigen-binding portion thereof, conjugated to a radionuclide.

[0082] As used herein, the term "anti-TRKB antibody" means any antigen-binding molecule or molecular complex containing at least one complementarity determining region (CDR) that specifically binds or interacts with TRKB or a portion of TRKB. The term "antibody" includes immunoglobulin molecules containing four polypeptide chains, two heavy (H) chains and two light (L) chains connected by disulfide bonds, as well as their multimers (eg, IgM). Each heavy chain contains a heavy chain variable region (abbreviated herein as HCVR or V _H ) and a heavy chain constant region. The heavy chain constant region contains three domains, C _H 1, C _H 2 and C _H 3. Each light chain contains a light chain variable region (abbreviated herein as "LCVR or "V _L ") and a light chain constant region (C _L ) . The V _H and V _L regions can be further subdivided into regions of hypervariability, called complementarity determining regions (CDRs), interspersed with regions that are more conserved, called framework regions (FRs). Each V _H and V _L consists of three CDRs and four FRs, arranged from amino to carboxy in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. In various embodiments, the FR of an antibody to TRKB (or an antigen-binding portion thereof) may be identical to human germline sequences, or may be naturally or artificially modified. The amino acid consensus sequence can be determined based on a parallel comparison of two or more CDRs.

[0083] As used herein, the term "antibody" also includes antigen-binding fragments of full length antibody molecules. As used herein, the terms "antigen-binding portion" of an antibody, "antigen-binding fragment" of an antibody, and the like include any enzymatically, synthetically, or genetically engineered polypeptide or glycoprotein that specifically binds to an antigen to form a complex. Antigen-binding fragments of an antibody can be obtained, for example, from complete antibody molecules using any suitable standard techniques such as proteolytic cleavage or recombinant genetic engineering techniques involving manipulation of DNA and expression of DNA encoding variable and optionally constant domains. Such DNA is known and/or readily available, for example from commercial sources, DNA libraries (including, for example, antibody phage libraries), or can be synthesized. DNA can be sequenced and manipulated chemically or using molecular biology techniques, for example, to arrange one or more variable and/or constant domains into a suitable configuration, or to introduce codons, create cysteine residues, modify, add or remove amino acids, etc. .

[0084] Non-limiting examples of antigen-binding fragments include the following: (i) Fab fragments; (ii) F(ab')2 fragments; (iii) Fd fragments; (iv) Fv fragments; (v) single chain Fv molecules (scFv); (vi) dAb fragments; and (vii) minimal recognition units consisting of amino acid residues that mimic an antibody hypervariable region (e.g., an isolated complementarity determining region (CDR) such as a CDR3 peptide) or a FR3-CDR3-FR4 conformationally restricted peptide. Other engineered molecules such as domain specific antibodies, single domain antibodies, domain deleted antibodies, chimeric antibodies, CDR grafted antibodies, diabodies, tribodies, tetrabodies, minibodies, nanobodies (e.g. monovalent nanobodies, bivalent nanobodies, etc.), small modular protein (SMIP) immunopharmaceuticals and shark IgNAR variable domains are also contemplated by the expression "antigen binding fragment" as used herein.

[0085] An antigen binding fragment of an antibody will typically contain at least one variable domain. The variable domain may be of any size or amino acid composition and will typically contain at least one CDR that is contiguous or in the same reading frame with one or more framework sequences. In antigen-binding fragments containing a V _H domain associated with a V _L domain, the V _H and V _L domains may be located relative to each other in any suitable arrangement. For example, the variable region may be dimeric and may contain V _H -V _H , V _H -V _L or V _L -V _L dimers. Alternatively, the antigen-binding fragment of an antibody may comprise a V _H or V _L monomeric domain.

[0086] In certain embodiments, an antigen-binding fragment of an antibody may comprise at least one variable domain covalently linked to at least one constant domain. Non-limiting exemplary variable and constant domain configurations that can be found within an antigen-binding fragment of an antibody of the present invention include the following: (i) V _{H -CH} 1; (ii) V _H -C _H 2; (iii) V _H -C _H 3; (iv) V _H -C _H 1 -C _H 2; (v) V _H -C _H 1 -C _H 2 -C _H 3; (vi) V _H -C _H 2 -C _H 3; (vii) V _H -C _L ; (viii) V _L -C _H 1; (ix) V _{L -CH} 2; (x) V _L -C _H 3; (xi) V _L -C _H 1 -C _H 2; (xii) V _L -C _H 1 -C _H 2 -C _H 3; (xiii) V _L -C _H 2 -C _H 3; and (xiv) V _L -C _L . In any configuration of the variable and constant domains, including any of the exemplary configurations listed above, the variable and constant domains may either be directly linked to each other, or may be linked through a complete or partial hinge or linker region. The hinge region may be at least 2 (e.g., 5, 10, 15, 20, 40, 60 or more) amino acids, resulting in a flexible or semi-flexible connection between adjacent variable and/or constant domains in a single polypeptide molecule. Moreover, an antigen-binding fragment of an antibody of the present invention may comprise a homodimer or heterodimer (or other multimer) of any of the variable and constant domain configurations listed above in non-covalent association with each other and/or with one or more V _H or V _L monomeric domains. (for example, via disulfide(s) bond(s)).

[0087] As with full antibody molecules, antigen-binding fragments can be monospecific or multispecific (eg, bispecific). A multispecific antigen-binding fragment of an antibody will typically contain at least two different variable domains, each variable domain being capable of specifically binding to a separate antigen or to a different epitope on the same antigen. Any multispecific antibody format, including the exemplary bispecific antibody formats disclosed herein, can be adapted for use in the context of an antigen-binding fragment of an antibody of the present invention using standard techniques.

[0088] The term "epitope" refers to an antigenic determinant that interacts with a specific antigen binding site in the variable region of an antibody molecule known as a paratope. A single antigen may contain more than one epitope. Thus, different antibodies may bind to different regions on an antigen and may have different biological effects. Epitopes may be either conformational or linear. A conformational epitope is formed by spatially adjacent amino acids from different segments of a straight chain polypeptide chain. A linear epitope is formed by adjacent amino acid residues in a polypeptide chain. In certain circumstances, an epitope may include fragments of saccharides, phosphoryl groups, or sulfonyl groups on an antigen.

[0089] The term "specifically binds" or "specifically binds to" or the like means that an antibody or antigen-binding fragment forms a complex with an antigen that is relatively stable under physiological conditions. Specific binding may have an equilibrium dissociation constant of at least about 1×10 ^-6 M or less (eg, a smaller K _D value means tighter binding). Methods for determining whether two molecules are specifically binding are well known in the art and include, for example, equilibrium dialysis, surface plasmon resonance, and the like. As described herein, using surface plasmon resonance, for example, BIACORE™, antibodies have been identified that bind specifically to TRKB. Moreover, multispecific antibodies that bind to the TRKB protein and one or more additional antigens, or bispecific antibodies that bind to two different TRKB regions, are still considered to be antibodies that "bind specifically" as used herein.

[0090] Anti-TRKB antibodies disclosed herein may contain one or more amino acid substitutions, insertions and/or deletions in the framework regions and/or CDR regions of the heavy and light chain variable domains. Such mutations can be readily identified by comparing the amino acid sequences disclosed herein with sequences available, for example, from public antibody sequence databases. Once generated, antibodies and antigen-binding fragments that contain one or more mutations can be readily tested for one or more of the desired properties, such as improved binding specificity, increased binding affinity, improved or enhanced antagonistic or agonistic biological properties (depending on the specific situation) , reduced immunogenicity, etc. Antibodies and antigen-binding fragments obtained by this general method are provided by the present invention.

[0091] Also provided are anti-TRKB antibodies comprising variants of any of the amino acid sequences of HCVR, LCVR, and/or CDR disclosed herein with one or more conservative substitutions. For example, the present invention provides anti-TRKB antibodies with the amino acid sequences HCVR, LCVR, and/or CDR, e.g., 10 or less, 8 or less, 6 or less, 4 or less, etc. conservative amino acid substitutions with respect to any of the HCVR, LCVR and/or CDR amino acid sequences disclosed in Table 22.

[0092] The term "substantial identity" or "substantially identical" when referring to a nucleic acid or fragment thereof in the context of anti-TRKB antibodies indicates that when optimally aligned with the appropriate nucleotide insertions or deletions with another nucleic acid (or its complementary strand) there is a nucleotide sequence identity of at least about 90%, and more preferably at least about 95%, 96%, 97%, 98%, or 99% of the nucleotide bases, as measured by any well-known identity measurement algorithm. sequences such as FASTA, BLAST or GAP as discussed below. A nucleic acid molecule with significant identity to a reference nucleic acid molecule may, in some instances, encode a polypeptide having the same or substantially similar amino acid sequence as the polypeptide encoded by the reference nucleic acid molecule.

[0093] When applied to polypeptides in the context of anti-TRKB antibodies, the term "substantial similarity" or "substantially similar" means that two peptide sequences, when optimally aligned, for example, using the GAP or BESTFIT programs using default gap penalties, have at least 95% sequence identity, even more preferably at least 98% or 99% sequence identity. Preferably, positions of residues that are not identical are distinguished by conservative amino acid substitutions. A "conservative amino acid substitution" in relation to polypeptides in the context of anti-TRKB antibodies is one in which an amino acid residue is replaced with another amino acid residue containing a side chain (R group) with similar chemical properties (eg, charge or hydrophobicity). In general, a conservative amino acid substitution will not significantly alter the functional properties of a protein. In cases where two or more amino acid sequences differ from each other by conservative substitutions, the percentage or degree of similarity may be adjusted upwards to adjust for the conservative nature of the substitution. Means for effecting this adjustment are well known to those skilled in the art. See, for example, Pearson (1994) Methods Mol. Biol. 24: 307-331, which is incorporated herein by reference. Examples of groups of amino acids that contain side chains with similar chemical properties include the following: 1) aliphatic side chains: glycine, alanine, valine, leucine and isoleucine; 2) aliphatic-hydroxyl side chains: series and threonine; 3) amide-containing side chains: asparagine and glutamine; 4) aromatic side chains: phenylalanine, tyrosine and tryptophan; 5) main side chains: lysine, arginine and histidine; 6) acidic side chains: aspartate and glutamate; and 7) sulfur-containing side chains: cysteine and methionine. Preferred groups of conservative amino acid substitutions are: valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine, alanine-valine, glutamate-aspartate, and asparagine-glutamine. Alternatively, a conservative substitution is any change characterized by a positive value in the PAM250 log-likelihood matrix disclosed in Gonnet et al. (1992) Science 256: 1443 45, incorporated herein by reference. A "moderately conservative" substitution is any change that has a non-negative value in the PAM250 log-likelihood matrix.

[0094] Compositions or methods "providing" or "comprising" one or more of the listed elements may include other elements that are not specifically indicated. For example, a composition that "comprises" or "comprises" a protein may contain the protein alone or in combination with other ingredients. The transitional phrase "consisting essentially of" means that the scope of the claims is to be interpreted as covering the specified elements listed in the claims and elements that do not significantly affect the essential(s) and new feature(s) of the claimed invention. . Thus, it is not intended that the term "consisting essentially of" when used in the claims should be interpreted as equivalent to "comprising".

[0095] "Optional" or "optional" means that the subsequently described event or circumstance may or may not occur, and that the description includes cases in which the event or circumstance occurs and cases in which it does not occur.

[0096] The designation of a range of values includes all integers within the range or defining a range and all subranges defined by integers within the range.

[0097] Unless otherwise clear from the context, the term "approximately" encompasses values within a standard error margin of measurement (eg, SEM) of a specified value.

[0098] The term "and/or" refers to and encompasses any possible combinations of one or more of the related listed items, as well as the absence of combinations when interpreted in the alternative ("or").

[0099] The term "or" refers to any one member of a particular list, and also includes any combination of members of that list.

[00100] Singular forms include plural forms unless the context clearly dictates otherwise. For example, the term "protein" or "at least one protein" can include a variety of proteins, including mixtures thereof.

[00101] Statistically significant means p ≤ 0.05.

Detailed disclosure of the present invention

I. Overview

[00102] Disclosed herein are the genomes of a non-human

animal, non-human animal and non-human animal cells containing the humanized TRKB locus, and methods of using such non-human animal and non-human animal cells. Non-human or non-human animal cells containing a humanized TRKB locus express a human TRKB protein or a chimeric TRKB protein containing one or more human TRKB protein fragments (eg, all or part of the human TRKB extracellular domain).

[00103] A humanized TRKB allele (e.g., resulting from a one-to-one replacement of all or part of the genomic DNA of a non-human animal with orthologous human genomic DNA) will provide a true human target or a close approximation to the true human target of human TRKB-targeting reagents (e.g. , agonist antibodies or small molecule agonists designed to target human TRKB), which allows testing the efficacy and mode of action of such agents in live animals, as well as pharmacokinetic and pharmacodynamic studies. For example, as shown in the working examples disclosed herein, intravitreal administration of anti-TRKB agonists has a significant neuroprotective effect following optic nerve injury in humanized TrkB rats.

II. Non-human animals containing the humanized TRKB locus

[00104] Non-human animal genomes, non-human animal cells, and non-human animal genomes disclosed herein contain the humanized TRKB locus. Cells or non-human animals containing a humanized TRKB locus express a human TRKB protein or a partially humanized, chimeric TRKB protein in which one or more fragments of the native TRKB protein have been replaced with corresponding fragments from human TRKB (e.g., all or part of the extracellular domain).

A.TRKB

[00105] Cells and non-human animals described herein

document contain the humanized TRKB locus. TRKB (also known as growth factor receptor BDNF-NT-3, GP145-TrkB, Trk-B, TrkB, neurotrophic tyrosine kinase type 2 receptor, tyrosine kinase TrkB, tropomyosin-related kinase B, tropomyosin receptor kinase B, neurotrophic receptor tyrosine kinase 2 and NTRK2) is encoded by the TRKB gene (also known as NTRK2, OBHD, TRK-B and GP145-TRKB). TRKB is a receptor tyrosine kinase involved in the development and maturation of the central and peripheral nervous systems through the regulation of survival, proliferation, migration, neuronal differentiation, and synapse formation and plasticity. TRKB is the receptor for BDNF/brain-derived neurotrophic factor and NTF4/neurotrophin-4. Alternatively, TRKB can also bind NTF/neurotrophin-3, which is less efficient in activating the receptor but regulates neuronal survival through TRKB. After ligand binding, TRKB undergoes homodimerization, autophosphorylation, and activation. The canonical TRKB isoform is expressed in the central and peripheral nervous systems. In the central nervous system (CNS), expression is observed in the cerebral cortex, hippocampus, thalamus, choroid plexus, cerebellar granular layer, brainstem, and spinal cord. In the peripheral nervous system, it is expressed in many cranial ganglia, the optic nerve, the vestibular system, multiple facial structures, the submandibular glands, and the dorsal root ganglia.

[00106] Human TRKB corresponds to human 9q21.33 on chromosome 9 (gene accession number according to RefSeq NCBI 4915; assembly GRCh38.p7; NC position 000009.12 (84668368..85027070)). The gene has been reported to be characterized by 23 exons. The wild-type human TRKB protein has been assigned the accession number UniProt Q16620. At least seven isoforms are known (Q16620-1 - Q16620-7). The sequence for one isoform, Q16620-4 (identical to NCBI accession number NP 006171.2), is shown in SEQ ID NO: 3. The mRNA (cDNA) encoding the canonical isoform has been assigned NCBI accession number AF410899.1 and is shown in SEQ ID NO: 8 Another example of mRNA (cDNA) encoding the human TRKB isoform has been assigned RefSeq mRNA accession number NM 006180.4. An exemplary coding sequence (CDS) is shown in SEQ ID NO: 11. The full-length human TRKB protein shown in SEQ TD NO: 3 contains 838 amino acids, including a signal peptide (amino acids 1-31), an extracellular domain (amino acids 32-430 ), a transmembrane domain (amino acids 431-454) and a cytoplasmic domain (amino acids 455-838). The demarcations between these domains are as outlined in UniProt. Reference to human TRKB includes canonical forms (wild type) as well as all allelic forms and isoforms. Any other forms of human TRKB contain amino acids numbered for maximum alignment with the wild-type form, with the aligned amino acids designated by the same number. An example of another isoform of human TRKB is Q16620-1 (identical to NCBI accession number NP 001018074.1) shown in SEQ ID NO: 75. The mRNA (cDNA) encoding this isoform has been assigned NCBI accession number NM 001018064.2 and is shown in SEQ ID NO: 76. An exemplary coding sequence (CDS) for this isoform (CCDS GO CCDS35050.1) is shown in SEQ ID NO: 77.

[00107] Rat TrkB corresponds to rat 17p14 on chromosome 17 (gene registration number according to RefSeq NCBI 25054; Rnor assembly 6.0; NC position 005116.4 (5934651..6245778, complementary strand)). The gene has been reported to be characterized by 23 exons. The wild-type rat TRKB protein was assigned UniProt accession number Q63604. At least three isoforms are known (Q63604-1 - Q63604-3). The sequence for the canonical isoform, Q63604-1 (identical to NCBI accession number NP 036863.1), is shown in SEQ ID NO: 2. The mRNA (cDNA) encoding the canonical isoform has been assigned NCBI accession number NM 012731.2 and is shown in SEQ ID NO: 7. Another An example mRNA (cDNA) encoding the rat TRKB isoform is assigned RefSeq mRNA M55291. An exemplary coding sequence (CDS) is shown in SEQ ID NO: 10. The canonical full-length rat TRKB protein shown in SEQ ID NO: 2 contains 821 amino acids, including a signal peptide (amino acids 1-31), an extracellular domain (amino acids 32- 429), a transmembrane domain (amino acids 430-453) and a cytoplasmic domain (amino acids 454-821). The demarcations between these domains are as outlined in UniProt. Reference to rat TRKB includes canonical forms (wild type) as well as all allelic forms and isoforms. Any other forms of rat TRKB contain amino acids numbered for maximum alignment with the wild-type form, with the aligned amino acids designated by the same number.

[00108] Mouse TrkB corresponds to 13 B1; 13 31.2 cM mice on chromosome 12 (gene registration number according to RefSeq NCBI 18212; assembly GRCm38.p4 (GCF 000001635.24); NC position 000079.6 (58806569..59133970)). The gene has been reported to be characterized by 23 exons. The wild-type mouse TRKB protein has been assigned the accession number UniProt PI 5209. At least four isoforms are known (P15209-1 to P15209-4). The sequence for the canonical isoform, P15209-1 (identical to NCBI accession numbers NP 001020245.1 and NP 001269890.1), is shown in SEQ ID NO: 1. An exemplary mRNA (cDNA) isoform encoding the canonical isoform has been assigned NCBI accession number NM 001025074.2 and is shown in SEQ ID NO: 6. An exemplary coding sequence (CDS) (CCDS ID CCDS26 573.1) is shown in SEQ ID NO: 9. The canonical full-length mouse TRKB protein shown in SEQ ID NO: 1 contains 821 amino acids, including the signal peptide (amino acids 1-31), extracellular domain (amino acids 32-429), transmembrane domain (amino acids 430-453), and cytoplasmic domain (amino acids 454-821). The demarcations between these domains are as indicated by UniProt. Reference to mouse TRKB includes canonical forms (wild-type) as well as all allelic forms and isoforms. Any other forms of mouse TRKB contain amino acids numbered for maximum alignment with the wild-type form, with the aligned amino acids designated by the same number.

B. Humanized TRKB loci

[00109] A humanized TRKB locus can be a TrkB locus in which the entire TrkB gene is replaced by a corresponding orthologous human TRKB sequence, or a TrkB locus in which only a portion of the TrkB gene is replaced by a corresponding orthologous human TRKB sequence (i.e., humanized). Optionally, the corresponding orthologous human TRKB sequence is modified to optimize codons based on the frequency of codon usage in the non-human animal. Replaced (ie, humanized) regions may include coding regions such as an exon, non-coding regions such as an intron, an untranslated region, or a regulatory region (eg, a promoter, enhancer, or transcriptional repressor binding element), or any combination thereof. As one example, exons corresponding to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22 or all 23 exons of the human TRKB gene may be humanized. For example, exons corresponding to exons 3-10 of the human TRKB gene can be humanized, including a segment of exon 2 (coding exon 1) from the codon encoding amino acid 33, starting immediately after the signal peptide. Alternatively, a region of TrkB encoding an epitope recognized by an antigen-binding protein to human TRKB, or a region targeted by a human TRKB-targeting reagent (eg, a small molecule), may be humanized. Similarly, nitrons corresponding to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 or all 22 introns human TRKB gene may be humanized or may remain endogenous. For example, introns corresponding to introns between exons 2 and 10 (i.e., introns 2-9, between coding exon 1 and exon 10) of the human TRKB gene may be humanized, optionally including a portion of the intron after exon 10 (i.e., .intron 10). The flanking non-translated regions, including regulatory sequences, may also be humanized or remain endogenous. For example, the 5' untranslated region (UTR), 3'UTR, or both the 5'UTR and 3'UTR may be humanized, or the 5'UTR, 3'UTR, or both the 5'UTR and 3'UTR may remain endogenous. . In a specific example, both the 5'UTR and the 3'UTR remain endogenous. Depending on the degree of replacement with orthologous sequences, regulatory sequences such as a promoter may be endogenous or provided with a replacement human orthologous sequence. For example, a humanized TRKB locus may include the endogenous TrkB promoter of a non-human animal.

[00110] One or more or all of the signal peptide, cytoplasmic domain, transmembrane domain, or extracellular coding regions may be humanized, or one or more such regions may remain endogenous. Exemplary coding sequences for the signal peptide, extracellular domain, transmembrane domain, and mouse TRKB cytoplasmic domain are shown in SEQ ID NOs: 63-66, respectively. Exemplary coding sequences for the signal peptide, extracellular domain, transmembrane domain and cytoplasmic domain of rat TRKB are shown in SEQ ID NOs: 67-70, respectively. Exemplary coding sequences for the signal peptide, extracellular domain, transmembrane domain, and cytoplasmic domain of human TRKB are shown in SEQ ID NOs: 71-74, respectively.

[00111] For example, all or part of the TrkB locus region encoding the signal peptide may be humanized, and/or all or part of the TrkB locus region encoding the extracellular domain may be humanized, and/or all or part of the TrkB locus region encoding the transmembrane domain may be humanized, and/or all or part of the region of the TrkB locus encoding the cytoplasmic domain may be humanized. In one example, all or part of the extracellular domain encoding region of the TrkB locus is humanized. Optionally, the human TRKB extracellular domain CDS contains, consists essentially of, or consists of a sequence that is at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical with respect to SEQ ID NO: 72 (or its degenerate sequences). The TRKB protein may retain the activity of native TRKB (eg, retain the ability to be phosphorylated, retain the ability to activate downstream signaling pathways such as the PI3K/AKT and MAPK/ERK pathways, or retain the ability to regulate survival, proliferation, migration, neuronal differentiation, or synapse formation and plasticity) or produce any of the phenotypes disclosed elsewhere herein). For example, the region of the TrkB locus encoding the extracellular domain can be humanized such that the chimeric TRKB protein is produced with an endogenous signal peptide, an endogenous cytoplasmic domain, an endogenous transmembrane domain, and a humanized extracellular domain.

[00112] One or more of the signal peptide, cytoplasmic domain, transmembrane domain, or extracellular coding regions may remain endogenous. For example, the region encoding the signal peptide and/or the cytoplasmic domain and/or the transmembrane domain may remain endogenous. Optionally, the CDS of the endogenous signal peptide TRKB contains, consists essentially of, or consists of a sequence that is at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO : 63 or 67 (or their degenerate sequences). Optionally, the CDS of the endogenous transmembrane domain TRKB contains, consists essentially of, or consists of a sequence that is at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO : 65 or 69 (or their degenerate sequences). Optionally, the CDS of the endogenous cytoplasmic domain TRKB contains, consists essentially of, or consists of a sequence that is at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO : 66 or 70 (or their degenerate sequences). In each case, the TRKB protein can retain the activity of native TRKB.

[00113] The TRKB protein encoded by the humanized TRKB locus may contain one or more domains that are derived from the human TRKB protein and/or one or more domains that are derived from the endogenous (ie, native) TRKB protein. Exemplary amino acid sequences of the signal peptide, extracellular domain, transmembrane domain, and mouse TRKB cytoplasmic domain are shown in SEQ ID NOs: 51-54, respectively. Exemplary amino acid sequences of the signal peptide, extracellular domain, transmembrane domain, and cytoplasmic domain of rat TRKB are shown in SEQ ID NOs: 55-58, respectively. Exemplary amino acid sequences of the signal peptide, extracellular domain, transmembrane domain, and cytoplasmic domain of human TRKB are shown in SEQ ID NOs: 59-62, respectively.

[00114] The TRKB protein may comprise one or more or all of the following: human TRKB signal peptide, human TRKB extracellular domain, human TRKB transmembrane domain, and human TRKB cytoplasmic domain. As one example, a TRKB protein may comprise the human TRKB extracellular domain.

[00115] The TRKB protein encoded by the humanized TRKB locus may also contain one or more domains that are derived from the endogenous (ie, native) TRKB protein of a non-human animal. As one example, a TRKB protein encoded by a humanized TRKB locus may comprise a signal peptide from an endogenous (i.e., native) non-human TRKB protein and/or a cytoplasmic domain from an endogenous (i.e., native) non-human TRKB protein an animal and/or a transmembrane domain from an endogenous (ie, native) TRKB protein of a non-human animal.

[00116] Domains in the chimeric TRKB protein that are derived from the human TRKB protein may be encoded by a fully humanized sequence (i.e., the entire sequence encoding that domain is replaced by an orthologous human TRKB sequence) or may be encoded by a partially humanized sequence (i.e., some of the sequence encoding this domain is replaced by an orthologous human TRKB sequence, and the rest of the endogenous (i.e., native) sequence encoding this domain encodes the same amino acids as the orthologous human TRKB sequence, so that the encoded domain is identical to that domain in the human TRKB protein). Similarly, domains in a chimeric protein that are derived from the endogenous TRKB protein may be encoded by an entirely endogenous sequence (i.e., the entire sequence encoding that domain is an endogenous TrkB sequence) or may be encoded by a partially humanized sequence (i.e., some of the sequence encoding this domain is replaced by an orthologous human TRKB sequence, but the orthologous human TRKB sequence encodes the same amino acids as the replaced endogenous TrkB sequence, so that the encoded domain is identical to that of the endogenous TRKB protein). For example, a portion of the region of the TrkB locus encoding a transmembrane domain (e.g., encoding the N-terminal region of the transmembrane domain) can be replaced with an orthologous human TRKB sequence, wherein the amino acid sequence of the transmembrane domain region encoded by the orthologous human TRKB sequence is identical to the corresponding endogenous amino acid sequence.

[00117] As one example, a TRKB protein encoded by a humanized TRKB locus may contain a human TRKB extracellular domain. Optionally, the human TRKB extracellular domain contains, consists essentially of, or consists of a sequence that is at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 60. The TRKB protein retains the activity of native TRKB (for example, retains the ability to be phosphorylated, retains the ability to activate downstream signaling pathways such as the PI3K/AKT and MAPK/ERK pathways, or retains the ability to regulate survival, proliferation, migration, differentiation of neurons, or the formation of synapses and their plasticity or produce any of the phenotypes disclosed elsewhere herein). As another example, a TRKB protein encoded by a humanized TRKB locus may comprise an endogenous TRKB cytoplasmic domain of a non-human animal (eg, mouse TRKB cytoplasmic domain or rat TRKB cytoplasmic domain). Optionally, the TRKB cytoplasmic domain of the non-human animal contains, consists essentially of, or consists of a sequence that is at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 54 or 58. As another example, the TRKB protein encoded by the humanized TRKB locus may comprise an endogenous non-human TRKB transmembrane domain (eg, mouse TRKB transmembrane domain or rat TRKB transmembrane domain). Optionally, the non-human TRKB transmembrane domain contains, consists essentially of, or consists of a sequence that is at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 53 or 57. As another example, the TRKB protein encoded by the humanized TRKB locus may comprise an endogenous TRKB signal peptide from a non-human animal (eg, mouse TRKB signal peptide or rat TRKB signal peptide). Optionally, the non-human animal TRKB signal peptide comprises, consists essentially of, or consists of a sequence that is at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 51 or 55. In each case, the TRKB protein can retain the activity of native TRKB. For example, a TRKB protein encoded by a humanized TRKB locus may contain, consist essentially of, or consist of a sequence that is at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100 % identical to SEQ ID NO: 4 or 5. Optionally, the TRKB CDS encoded by the humanized TRKB locus may contain, consist essentially of, or consist of a sequence that is at least 85%, 90%, 95%, 96%, 97 %, 98%, 99% or 100% identical to SEQ ID NO: 12 or 13 (or their degenerate sequences). In each case, the TRKB protein can retain the activity of native TRKB.

[00118] Optionally, the humanized TRKB locus may contain other elements. Examples of such elements may include selection cassettes, reporter genes, recombinase recognition sites, or other elements. Alternatively, other elements may be missing from the humanized TRKB locus (eg, the selection marker or selection cassette may be missing). Examples of suitable reporter genes and reporter proteins are disclosed elsewhere in this document. Examples of suitable selection markers include neomycin phosphotransferase (peog), hygromycin B phosphotransferase (hygr), puromycin N-acetyltransferase (puro _r ), blasticidin S-deaminase (bsr _r ), xanthine/guanine phosphoribosyl transferase (gpt), and herpes simplex virus thymidine kinase. (HSV-k). Examples of recombinases include the Cre, Flp and Dre recombinases. One example of a Cre recombinase gene is Crei, in which two exons encoding Cre recombinase are separated by an intron to prevent its expression in a prokaryotic cell. Such recombinases may additionally contain a nuclear localization signal to facilitate nuclear localization (eg, NLS-Crei). Recombinase recognition sites include nucleotide sequences that are recognized by a site-specific recombinase and can serve as a substrate for a recombination event. Examples of recombinase recognition sites include FRT, FRT11, FRT71, attp, att, rox and lox sites such as loxP, lox511, lox2272, lox66, lox71, loxM2 and lox5171.

[00119] Other elements, such as reporter genes or selection cassettes, may be self-removing cassettes flanked by recombinase recognition sites. See, for example, US 8697851 and US 2013/0312129, each of which is incorporated herein by reference in its entirety for all purposes. As an example, a self-removing cassette may contain the Crei gene (contains two exons encoding Cre recombinase separated by an intron) operably linked to the mouse Prml promoter and a neomycin resistance gene operably linked to the human ubiquitin promoter. By using the Prml promoter, the self-removing cassette can be specifically removed in the male germ cells of F0 animals. The polynucleotide encoding the selection marker may be operably linked to a promoter active in the target cell. Examples of promoters are presented elsewhere in this document. As another specific example, a self-removing selection cassette may comprise a hygromycin resistance gene coding sequence operably linked to one or more promoters (e.g., both human ubiquitin and EM7 promoters) followed by a polyadenylation signal followed by a Crei coding sequence operably linked with one or more promoters (eg, the mPrml promoter) followed by another polyadenylation signal, with the entire cassette flanked by loxP sites.

[00120] The humanized TRKB locus can also be a conditional allele. For example, a conditional allele may be a multifunctional allele as described in US 2011/0104799, incorporated herein by reference in its entirety for all purposes. For example, a conditional allele may contain the following: (a) a starter sequence in sense orientation relative to the transcription of the target gene; (b) a drug susceptibility selection (DSC) cassette in sense or antisense orientation; (c) the nucleotide sequence of interest (NSI) in antisense orientation; and (d) a conditional inversion module (COIN, which uses an exon-cleaving intron and a gene trap-like reversible module) in reverse orientation. See, for example, US 2011/0104799. The conditional allele may further comprise recombinant units that recombine when exposed to the first recombinase to form a conditional allele that (i) does not contain the trigger sequence and DSC; and (ii) contains NSI in sense orientation and COIN in antisense orientation. See, for example, US 2011/0104799.

[00121] One exemplary humanized TRKB locus (e.g., humanized mouse TrkB locus or humanized rat TrkB locus) is a locus in which the region in exon 2/coding exon 1 from the codon encoding amino acid 33, starting immediately after the signal peptide (or codon corresponding to the codon encoding amino acid 33 in mouse TrkB, rat TrkB, or human TRKB when optimally aligned with CDS mouse TrkB, rat TrkB, or human TRKB, respectively) to exon 10 (or exon corresponding to exon 10 of mouse TrkB, rat TrkB, or human TRKB TRKB when optimally aligned with CDS mouse TrkB, rat TrkB or human TRKB, respectively), optionally including a portion of Nitron 10, is replaced with the corresponding human sequence. The replaced region encodes the TRKB extracellular domain. See figures 1 and 4 and SEQ ID NOs: 4 and 5.

C. Non-human animal genomes, non-human animal cells, and non-human animal genomes containing the humanized TRKB locus

[00122] Non-human animal genomes, non-human animal cells, and non-human animal genomes containing a humanized TRKB locus are provided, as described elsewhere herein. Genomes, cells, or non-human animals may be male or female. Genomes, cells, or non-human animals may be heterozygous or homozygous for the humanized TRKB locus. A diploid organism has two alleles at each genetic locus. Each pair of alleles represents the genotype of a particular genetic locus. Genotypes are described as homozygous if two identical alleles are present at a particular locus, and as heterozygous if the two alleles are different.

[00123] The genomes or cells provided herein are not

a human animal can be, for example, any non-human animal genome or cell containing the TrkB locus or a genomic locus homologous or orthologous to the human TRKB locus. The genomes may be derived from the following, or the cells may be, for example, the following: eukaryotic cells, which include, for example, fungal (eg, yeast) cells, plant cells, animal cells, mammalian cells, non-human mammalian cells, and human cells. The term "animal" includes any member of the animal kingdom, including, for example, mammals, fish, reptiles, amphibians, birds, and worms. The mammalian cell may be, for example, a non-human mammalian cell, a rodent cell, a rat cell, a mouse cell, or a hamster cell. Other non-human mammals include, for example, non-human primates, apes, great apes, orangutans, cats, dogs, rabbits, horses, bulls, deer, bison, livestock (for example, cattle species, such as cows, bulls, etc., ovine species such as sheep, goats, etc., and swine species such as pigs and wild boars). Birds include, for example, chickens, turkeys, ostriches, geese, ducks, and so on. Pets and farm animals are also provided. The term "non-human" excludes humans.

[00124] Cells can also be in an undifferentiated or differentiated state of any type. For example, the cell may be a totipotent cell, a pluripotent cell (eg, a human pluripotent cell or a non-human pluripotent cell such as a mouse embryonic stem (ES) cell or a rat ES cell), or a non-pluripotent cell. Totipotent cells include undifferentiated cells that can give rise to any type of cell, and pluripotent cells include undifferentiated cells that have the ability to develop into more than one type of differentiated cell. Such pluripotent and/or totipotent cells may be, for example, ES cells or ES-like cells such as induced pluripotent stem (iPS) cells. ES cells include embryonic-derived totipotent or pluripotent cells that are capable of contributing to any tissue in the developing embryo when introduced into the embryo. ES cells can be derived from the inner cell mass of the blastocyst and are capable of differentiating into cells from any of the three vertebrate germ layers (endoderm, ectoderm, and mesoderm).

[00125] The cells provided herein can also be germ cells (eg, spermatozoa or oocytes). The cells may be mitotically competent cells or mitotically inactive cells, meiotically competent cells or meiotically inactive cells. Similarly, the cells may also be primary somatic cells or cells that are not primary somatic cells. Somatic cells include any cell that is not a gamete, germ cell, gametocyte, or undifferentiated stem cell. For example, the cells may be neurons, such as hippocampal neurons or cortical neurons.

[00126] Suitable cells provided herein also include primary cells. Primary cells include cells or cell cultures that have been isolated directly from an organism, organ, or tissue. Primary cells include cells that are neither transformed nor immortal. They include any cell derived from an organism, organ, or tissue that has not previously been transferred to tissue culture, or has previously been transferred to tissue culture, but is not capable of being transferred to tissue culture indefinitely. Such cells can be isolated using conventional techniques and include, for example, hippocampal neurons or cortical neurons.

[00127] Other suitable cells provided herein include immortalized cells. Immortalized cells include cells of a multicellular organism that do not normally reproduce indefinitely, but due to mutation or change have escaped normal cellular senescence and may instead continue to undergo division. Such mutations or changes may occur naturally or be intentionally induced. A specific example of an immortalized cell line is a neuroblastoma cell line such as N18TG2 or T48, or a cell line such as the NIH-3T3 cell line. Numerous types of immortalized cells are well known. Immortalized or primary cells include cells that are typically used for culture or for the expression of recombinant genes or proteins.

[00128] The cells provided herein also include embryos at the single cell stage (i.e., fertilized oocytes or zygotes). Such single cell embryos may be from any genetic background (eg, BALB/c, C57BL/6, 129, or combinations thereof in mice), may be fresh or frozen, and may be derived from natural breeding or in vitro fertilization.

[00129] The cells provided herein may be normal, healthy cells, or may be pathological or mutated cells.

[00130] Non-human animals containing a humanized TRKB locus as described herein can be obtained using the methods described elsewhere herein. The term "animal" includes any member of the animal kingdom, including, for example, mammals, fish, reptiles, amphibians, birds, and worms. In a specific example, the non-human animal is a non-human mammal. Non-human mammals include, for example, non-human primates, apes, great apes, orangutans, cats, dogs, horses, bulls, deer, bison, sheep, rabbits, rodents (e.g., mice, rats, hamsters, and marine gilts) and livestock (e.g., bovine species such as cows and bulls; ovine species such as sheep and goats; and porcine species such as pigs and boars). Birds include, for example, chickens, turkeys, ostriches, geese and ducks. Pets and farm animals are also provided. The term "non-human animal" excludes humans. Preferred non-human animals include, for example, rodents such as mice and rats.

[00131] Non-human animals can be of any genetic origin. For example, suitable mice may be the 129 line, the C57BL/6 line, a mixture of 129 and C57BL/6, the BALB/c line, or the Swiss Webster line. Examples of lines 129 include 129P1, 129P2, 129P3, 129X1, 129S1 (e.g., 129S1/SV, 129S1/Svlm), 129S2, 129S4, 129S5, 129S9/SvEvH, 129S6 (129/SvEvTac), 129S7, 129S8, 129T1 and 129T2. See, for example, Festing et al. (1999) Mammalian Genome 10:836, incorporated herein by reference in its entirety for all purposes. Examples of C57BL lineages include C57BL/A, C57BL/An, C57BL/GrFa, C57BL/Kal wN, C57BL/6, C57BL/6J, C57BL/6ByJ, C57BL/6NJ, C57BL/10, C57BL/10ScSn, C57BL/10Cr and C57BL/Ola. Suitable mice can also be from a mixture of the aforementioned 129 line and the aforementioned C57BL/6 line (eg 50% 129 and 50% C57BL/6). Likewise, suitable mice can be from a mixture of the aforementioned 129 lines or a mixture of the aforementioned BL/6 lines (eg 129S6 (129/SvEvTac) line).

[00132] Similarly, the rats can be from any strain of rats, including, for example, the ACI rat strain, the Dark Agouti (DA) rat strain, the Wistar rat strain, the LEA rat strain, the Sprague Dawley (SD) rat strain, or the Fischer like Fisher F344 or Fisher F6. Rats can also be obtained from a line derived from a mixture of two or more of the lines above. For example, a suitable rat may be from the DA strain or the ACI strain. The ACI rat strain is characterized by having a black agouti coat color, white belly and paws, and the RT1 ^av1 haplotype. Such lines are available from various sources, including Harlan Laboratories. The Dark Agouti (DA) rat strain is characterized by having an agouti coat color and the RT1 ^av1 haplotype. Such rats are available from a variety of sources including Charles River and Harlan Laboratories. In some cases, suitable rats may be inbred rats. See, for example, US 2014/0235933, which is hereby incorporated by reference in its entirety for all purposes.

III. Methods of using non-human animals containing a humanized TRKB locus to evaluate the efficacy of human TRKB-targeting reagents in vivo or ex vivo

[00133] Various methods are contemplated for using non-human animals containing a humanized TRKB locus, as described elsewhere herein, to evaluate or optimize the delivery or efficacy of human TRKB-targeting reagents (e.g., therapeutic agonist molecules) in vivo or ex vivo. Because non-human animals contain a humanized TRKB locus, non-human animals will more accurately reflect the efficacy of a human TRKB-targeted reagent.

A. Methods for testing the efficacy of human TRKB-targeting reagents in vivo or ex vivo

[00134] Various methods are provided to evaluate the delivery or efficacy of human TRKB-targeted reagents in vivo using non-human animals containing a humanized TRKB locus, as described elsewhere herein. Such methods may include: (a) administering to a non-human animal a human TRKB-targeting reagent; and (b) evaluating the activity of a human TRKB-targeted reagent.

[00135] The human TRKB-targeted reagent can be a human TRKB-targeted antibody or antigen-binding protein, or any other large molecule or small molecule that targets human TRKB. Alternatively, the human TRKB targeting reagent can be any biological or chemical agent that targets the human TRKB locus (human TRKB gene), human TRKB mRNA, or human TRKB protein. Examples of human TRKB-targeting reagents are disclosed elsewhere in this document.

[00136] Such human TRKB-targeting reagents can be administered to a cell or non-human animal via any delivery method (e.g., AAV, LNP, or HDD) as disclosed elsewhere herein, and by any route of administration. Means of delivery of therapeutic molecules and routes of administration are disclosed in more detail elsewhere in this document. According to specific methods, the reagents are delivered by injection (eg, direct hippocampal injection, subcutaneous injection, or intravitreal injection).

[00137] Methods for assessing the activity of a human TRKB-targeted reagent are well known and are provided elsewhere herein. In some methods, evaluating the activity of a human TRKB-targeting reagent (e.g., agonistic activity or inhibitory activity) involves evaluating TRKB activity (e.g., TRKB phosphorylation, TRKB-mediated activation of downstream signaling pathways, or TRKB-induced phenotypes), as disclosed elsewhere herein. document. The activity assessment can be performed in any cell type, any tissue type, or any type of organ, as disclosed elsewhere herein. In some methods, activity is assessed in brain tissue (eg, hippocampus or striatum) or neurons (eg, retinal ganglion cells, hippocampal neurons, or cortical neurons).

[00138] If the TRKB-targeting reagent is a genome editing reagent (eg, a nuclease agent), such methods may involve assessing modification of the humanized TRKB locus. For example, the evaluation may involve sequencing the humanized TRKB locus in one or more cells isolated from a non-human animal (eg, next generation sequencing). The assessment may include isolating the target organ (eg, brain) or tissue from a non-human animal and assessing the modification of the humanized TRKB locus in the target organ or tissue. The evaluation may also include evaluation of the modification of the humanized TRKB locus in two or more different cell types within the target organ or tissue. Similarly, the assessment may include isolating a non-target organ or tissue (eg, two or more non-target organs or tissues) from a non-human animal and assessing the modification of the humanized TRKB locus in the non-target organ or tissue.

[00139] Such methods may also comprise measuring expression levels of mRNA produced by the humanized TRKB locus or measuring expression levels of the protein encoded by the humanized TRKB locus. For example, protein levels can be measured in a particular type of cell, tissue, or organ (eg, brain) or secreted levels can be measured in serum. Methods for assessing the expression of TRKB mRNA or a protein expressed from a humanized TRKB locus are provided elsewhere herein and are well known.

[00140] The various methods provided above for evaluating in vivo activity can also be used to evaluate the activity of human TRKB-targeted reagents ex vivo, as described elsewhere herein.

B. Methods for optimizing the delivery or efficacy of a human TRKB-targeted reagent in vivo or ex vivo

[00141] Various methods are contemplated for optimizing delivery of human TRKB-targeted reagents to a cell or non-human animal, or optimizing the activity or delivery efficiency of human TRKB-targeted reagents in vivo. Such methods may include, for example, the following: (a) performing a method for testing the efficacy of a human TRKB-targeted reagent as described above for the first time in a first non-human animal or in a first cell; (b) changing the variable and performing the method a second time in a second non-human animal (ie the same species) or in a second cage with the changed variable; and (c) comparing the activity of the human TRKB-targeting reagent in step (a) with that of the human TRKB-targeting reagent in step (b), and selecting the method that results in greater efficacy or activity.

[00142] Methods for measuring the delivery, potency, or potency of human TRKB-targeted reagents are disclosed elsewhere herein. Higher potency can mean different things depending on the desired effect in the non-human animal body or cell. For example, higher efficiency may mean higher activity and/or higher specificity. Higher activity may be, for example, TRKB activation activity or TRKB inhibition activity. This may refer to a higher percentage of cells to be targeted in a particular target cell type (eg, neurons such as retinal ganglion cells) or in a particular target tissue or organ (eg, brain). Higher specificity may refer to higher specificity for TRKB compared to off-target effects, higher specificity for a target cell type, or higher specificity for a target tissue or organ type.

[00143] The variable that changes can be any parameter. As one example, the altered variable may be a packaging or delivery method by which a human TRKB-targeted reagent or reagents are administered to a cell or non-human animal. Examples of delivery methods are disclosed elsewhere herein. As another example, the altered variable may be the route of administration for introducing a human TRKB-targeted reagent or reagents into a cell or non-human animal. Examples of routes of administration are disclosed elsewhere in this document.

[00144] As another example, the altered variable may be the concentration or amount of administered human TRKB-targeting reagent or reagents. As another example, the altered variable may be the time of administration of the human TRKB-targeted reagent or reagents relative to the time the activity or efficacy of the reagents was assessed. As another example, the variable variable may be the number of times or frequency with which the human TRKB-targeted reagent or reagents are administered. As another example, the altered variable may be the human TRKB-targeting reagent or reagents that are administered (eg, comparing one reagent with another reagent).

C. Human TRKB Targeted Reagents

[00145] The human TRKB targeting reagent can be any reagent that targets the human TRKB protein, human TRKB gene, or human TRKB mRNA. The human TRKB-targeting reagent may be, for example, an agonist (ie, a molecule that indirectly or directly activates human TRKB) or may be an antagonist (ie, an inhibitor or inhibitory reagent that blocks human TRKB activity). In a specific example, the human TRKB targeting reagent is a TRKB agonist. The human TRKB-targeting reagents in the methods disclosed herein may be known human TRKB-targeting reagents, may be putative human TRKB-targeting reagents (e.g., candidate reagents designed to target human TRKB), or may be screened for human TRKB-targeted activity.

[00146] For example, a human TRKB-targeting reagent can be an antigen-binding protein (eg, an agonist antibody) targeted to an epitope of the human TRKB protein. An example of such a reagent is the TRKB agonist antibody, H4H9816P2. Other anti-TRKB antibodies are disclosed elsewhere herein. In some instances, anti-TRKB antibodies bind to human TRKB with a Kd value of less than about 200 nM as measured by surface plasmon resonance at 25°C or 37°C. In other instances, anti-TRKB antibodies bind to human TRKB with a Kd value of less than about 600 pM, less than about 300 pM, less than about 200 pM, less than about 150 pM, less than about 100 pM, less than about 80 pM less than about 50 pM, less than about 40 pM, less than about 30 pM, less than about 20 pM, less than about 10 pM, less than about 5 pM, less than about 3 pM, or less than about 1 pM. In some instances, anti-TRKB antibodies bind to human TRKB with a dissociative half-life (t½) of greater than about 10 minutes, as measured by surface plasmon resonance at 25°C or 37°C. In other instances, anti-TRKB antibodies bind to human TRKB with a t½ of greater than about 20 minutes, greater than about 50 minutes, greater than about 100 minutes, greater than about 120 minutes, greater than about 150 minutes, greater than about 300 minutes more than about 350 minutes, more than about 400 minutes, more than about 450 minutes, more than about 500 minutes, more than about 550 minutes, more than about 600 minutes, more than about 700 minutes, more than about 800 minutes, more than about 900 minutes, more than about 1000 minutes, more than about 1100 minutes, or more than about 1200 minutes. As a specific example, an anti-TRKB antibody may comprise a set of six CDRs (HCDR1-HCDR2-HCDR3-LCDR1-LCDR2-LCDR3) selected from the group shown in Table 22 or substantially similar sequences characterized by at least 90% at least 95%, at least 98%, or at least 99% sequence identity with respect to them.

[00147] Other human TRKB targeting reagents include small molecules (eg, agonists) targeting the human TRKB protein. Examples of small molecule TRKB agonists include 7,8-dihydroxyflavone (7,8-DHF), deoxygedunin, LM22A-4 (N,N',N'',-tris(2-hydroxyethyl)-1,3,5 -benzenetricarboxamide) and LM22B-10 (2-[[4-[[4-[bis-(2-hydroxyethyl)amino]phenyl]-(4-chlorophenyl)methyl]phenyl]-(2-hydroxyethyl)amino]ethanol) . See, for example, Liu et al. (2015) Translational Neurodegeneration 5:2; Massa et al. (2010) j. Clin. Invest. 120(5):1774-1785; and Yang et al. (2016) Neuropharmacology 110:343-361, each of which is hereby incorporated by reference in its entirety for all purposes. An example of a TRKB-targeting reagent that is an inhibitor is K252a. See, for example, Yang et al. (2016) Neuropharmacology 110:343-361, incorporated herein by reference in its entirety for all purposes.

[00148] Other reagents targeting human TRKB include peptides or peptide mimetics (eg, agonists) targeting the human TRKB protein. Examples of peptide mimetics that serve as human TRKB agonists are disclosed, for example, in O'Leary et al. (2003) J. Biol. Chem. 278(28):25738-25744, incorporated herein by reference in its entirety for all purposes.

[00149] Other reagents targeting human TRKB may include genome editing reagents such as a nuclease agent (e.g., CRISPR/CRISPR-associated protein nuclease, zinc finger nuclease (ZFN ) and an effector nuclease like transcription activator (TALEN)), which cleaves a recognition site within the human TRKB gene. Similarly, the human TRKB-targeting reagent can be an exogenous donor nucleic acid (eg, a targeting vector or a single-stranded oligodeoxynucleotide (ssODN)) designed to recombine with the human TRKB gene).

[00150] Other reagents targeting human TRKB may include antisense oligonucleotides (eg, siRNAs (small interfering RNAs) or siRNAs (small hairpin RNAs)) targeting human TRKB mRNA. Antisense oligonucleotides (ASOs) or antisense RNAs are short synthetic nucleotide sequences designed to prevent the expression of a target protein by selectively binding to an RNA that encodes the target protein and thereby preventing translation. These compounds bind to RNA with high affinity and selectivity through a well-characterized Watson-Crick pairing (hybridization). RNA interference (RNAi) is an endogenous cellular mechanism for the control of gene expression, in which small interfering RNAs (siRNAs), which are associated with an RNA-induced silencing complex (RISC), mediate cleavage of a target messenger RNA (mRNA).

[00151] The activity of any other known or suspected human TRKB-targeting reagent can also be assessed using the non-human animals disclosed herein. Similarly, any other molecule can be screened for human TRKB-targeted activity using the non-human animals disclosed herein.

D. Administration of Human TRKB Targeting Reagents to Non-Human Animals or Cells

[00152] The methods disclosed herein may involve administering to a non-human animal body or cell various molecules (e.g., human TRKB-targeting reagents such as antibodies or small molecules), including nucleic acids, proteins, nucleic acid complexes, and protein, peptide mimetics, antigen-binding proteins or small molecules. "Introducing" includes presenting a molecule (e.g., nucleic acid or protein or small molecule) to a cell or non-human animal in such a way that it gains access to the interior of the cell or interior of the cells in the body of the non-human animal. The introduction can be done by any means. If multiple components are administered, they may be administered simultaneously or sequentially in any combination. In addition, two or more components can be introduced into a cell or body of a non-human animal using the same delivery method or using different delivery methods. Similarly, two or more components can be administered to a non-human animal by the same route of administration or by different routes of administration.

[00153] Molecules introduced into the body or cell of a non-human animal can be provided in compositions containing a carrier that increases the stability of the introduced molecules (for example, prolonging the period under given storage conditions (for example, -20 ° C, 4 ° C or temperature environment) for which degradation products remain below a threshold, such as below 0.5% by weight of the parent nucleic acid or protein, or an increase in in vivo stability). Non-limiting examples of such carriers include poly(lactic acid) (PLA) microspheres, microspheres, D,L-lactic-glycolic acid (PLGA) copolymer microspheres, liposomes, micelles, reverse micelles, lipid cochleates, and lipid microtubules.

[00154] Various methods and compositions are contemplated herein that allow the administration of a human TRKB-targeted reagent into a non-human cell or animal. Methods for introducing nucleic acids into various cell types are known in the art and include, for example, stable transfection methods, transient transfection methods, and virus-mediated methods.

[00155] Protocols for transfection, as well as protocols for introducing nucleic acid sequences into cells, may vary. Non-limiting methods of transfection include chemical methods of transfection; methods using liposomes; nanoparticles; calcium phosphate (Graham et al. (1973) Virology 52 (2): 456-67, Bacchetti et al. (1977) Proc. Natl. Acad. Sci. USA 74 (4): 1590-4, and Kriegler, M ( 1991. Transfer and Expression: A Laboratory Manual, New York: W. H. Freeman and Company, pp. 96-97); dendrimers or cationic polymers such as DEAE-dextran or polyethyleneimine. Non-chemical methods include electroporation, sonoporation and optical transfection. Particle-based transfection includes the use of a gene gun or magnetic transfection (Bertram (2006) Current Pharmaceutical Biotechnology 7, 277-28). Viral techniques can also be used for transfection.

[00156] The introduction of human TRKB-targeting reagents into a cell can also be mediated by electroporation, intracytoplasmic injection, viral infection, adenovirus, adeno-associated virus, lentivirus, retrovirus, transfection, lipid-mediated transfection, or nucleofection. Nucleofection is an advanced electroporation technology that delivers nucleic acid substrates not only into the cytoplasm, but also across the nuclear membrane and into the nucleus. In addition, the use of nucleofection in the methods disclosed herein typically requires much fewer cells than conventional electroporation (eg, only about 2 million compared to 7 million with conventional electroporation). In one example, nucleofection is performed using the LONZA® NUCLEOFECTOR™ system.

[00157] Introducing human TRKB-targeted reagents into a cell (eg, zygote) can also be accomplished by microinjection. In zygotes (ie, single cell stage embryos), microinjection can be performed into the mother's and/or father's pronucleus or into the cytoplasm. If microinjection occurs in only one pronucleus, the paternal pronucleus is preferred due to its larger size. Microinjection of mRNA is preferably carried out in the cytoplasm (for example, to deliver mRNA directly to the translation apparatus), while microinjection of a protein or polynucleotide encoding a protein or encoding RNA is preferably carried out in the nucleus/pronucleus. Alternatively, microinjection can be done by injection into both the nucleus/pronucleus and the cytoplasm: first, the needle can be inserted into the nucleus/pronucleus and the first amount can be injected, when the needle is removed from the embryo at the single cell stage, the second amount can be injected into the cytoplasm. Methods for conducting microinjection are well known. See, for example, Nagy et al. (Nagy A, Gertsenstein M, Vintersten K, Behringer R., 2003, Manipulating the Mouse Embryo. Cold Spring Harbor, New York: Cold Spring Harbor Laboratory Press); see also Meyer et al. (2010) Proc. Natl. Acad. sci. USA 107:15022-15026 and Meyer et al. (2012) Proc. Natl. Acad. sci. USA 109:9354-9359.

[00158] Other methods of introducing human TRKB-targeting reagents into a cell or non-human animal may include, for example, vector delivery, particle-mediated delivery, exosome-mediated delivery, lipid nanoparticle-mediated delivery, cell-penetrating peptide-mediated delivery, or implantable device-mediated delivery. As specific examples, the nucleic acid or protein can be administered to a non-human cell or animal in a carrier such as a poly(lactic acid) (PLA) microsphere, a D,L-lactic-glycolic acid copolymer (PLGA) microsphere, liposome, micelle, reverse micelle, lipid cochleate, or lipid microtubule. Some specific examples of delivery to a non-human animal include hydrodynamic delivery, virus-mediated delivery (eg, adeno-associated virus (AAV) mediated delivery), and lipid nanoparticle-mediated delivery.

[00159] The introduction of human TRKB-targeted reagents into non-human cells or organisms can be accomplished using hydrodynamic delivery (HDD). Hydrodynamic delivery has emerged as a method for intracellular delivery of DNA in vivo. To deliver genes to parenchymal cells through a chosen blood vessel, only the necessary DNA sequences need to be introduced, eliminating the safety concerns associated with current viral and synthetic vectors. When injected into the bloodstream, DNA is able to reach cells in various tissues accessible to the blood. Hydrodynamic delivery uses the force generated by rapidly injecting a large volume of solution into incompressible blood in the circulation to overcome the physical barriers of the endothelium and cell membranes that prevent large, membrane-impermeable compounds from entering parenchymal cells. In addition to DNA delivery, this method is applicable for efficient intracellular delivery of RNA, proteins and other small compounds in vivo. See, for example, Bonamassa et al. (2011) Pharm. Res. 28(4):694-701, incorporated herein by reference in its entirety for all purposes.

[00160] Administration of human TRKB-targeting reagents can also be accomplished by virus-mediated delivery, such as AAV-mediated delivery or lentivirus-mediated delivery. Other exemplary viruses/viral vectors include retroviruses, adenoviruses, vaccinia viruses, poxviruses, and herpes simplex viruses. Viruses can infect dividing cells, non-dividing cells, or both dividing and non-dividing cells. Viruses may integrate into the host genome or, alternatively, may not integrate into the host genome. Such viruses can also be designed to give reduced immunity. Viruses may be capable of replication or may be replication defective (eg, defective in one or more genes required for additional rounds of replication and/or virion packaging). Viruses can cause transient expression, long-term expression (eg, for at least 1 week, 2 weeks, 1 month, 2 months, or 3 months), or persistent expression. Exemplary virus titers (eg, AAV titers) include 10 ¹² , 10 ¹³ , 10 ¹⁴ , 10 ¹⁵ , and 10 ¹⁶ vector genomes/ml.

[00161] The AAV ssDNA genome consists of two open reading frames, Rep and Cap, flanked by two inverted terminal repeats that allow the synthesis of a complementary DNA strand. When constructing an AAV transfer plasmid, the transgene is placed between two ITRs and Rep and Cap can be delivered in trans. In addition to Rep and Cap, AAV may require a helper plasmid containing adenovirus genes. These genes (E4, E2a and VA) mediate AAV replication. For example, a transfer plasmid, Rep/Cap, and a helper plasmid can be transfected into HEK293 cells containing the adenovirus E1+ gene to generate infectious AAV particles. Alternatively, Rep, Cap, and adenovirus helper genes can be combined into a single plasmid. Similar packaging cells and methods can be used for other viruses such as retroviruses.

[00162] Numerous AAV serotypes have been identified. These serotypes differ in the cell types they infect (ie, their tropism), which allows for preferential transduction of certain cell types. CNS tissue serotypes include AAV1, AAV2, AAV4, AAV5, AAV8 and AAV9. Serotypes for heart tissue include AAV1, AAV8 and AAV9. Renal tissue serotypes include AAV2. Lung tissue serotypes include AAV4, AAV5, AAV6 and AAV9. Pancreatic tissue serotypes include AAV8. Serotypes for photoreceptor cells include AAV2, AAV5 and AAV8. Retinal pigment epithelial tissue serotypes include AAV1, AAV2, AAV4, AAV5 and AAV8. Skeletal muscle tissue serotypes include AAV1, AAV6, AAV7, AAV8 and AAV9. Liver tissue serotypes include AAV7, AAV8 and AAV9 and in particular AAV8.

[00163] Tropism can be further refined using pseudotyping, which is the mixing of capsid and genome from different viral serotypes. For example, AAV2/5 indicates a virus containing a serotype 2 genome packaged in a capsid from serotype 5. The use of pseudotyped viruses can increase transduction efficiency as well as alter tropism. Hybrid capsids derived from different serotypes can also be used to modify viral tropism. For example, AAV-DJ contains a hybrid capsid of eight serotypes and exhibits high infectivity across a wide range of cell types in vivo. AAV-DJ8 is another example that exhibits the properties of AAV-DJ, but with improved brain uptake. AAV serotypes can also be modified through mutations. Examples of AAV2 mutations include Y444F, Y500F, Y730F and S662V. Examples of AAV3 mutations include Y705F, Y731F and T492V. Examples of AAV6 mutational modifications include S663V and T492V. Other pseudotyped/modified AAV variants include AAV2/1, AAV2/6, AAV2/7, AAV2/8, AAV2/9, AAV2.5, AAV8.2, and AAV/SASTG.

[00164] Self-complementary AAV variants (scAAV) can be used to accelerate transgene expression. Because AAV depends on the cell's DNA replication machinery to synthesize the complementary strand of the AAV single-stranded DNA genome, transgene expression can be delayed. To eliminate this delay, scAAVs containing complementary sequences that are capable of spontaneous annealing upon infection can be used, eliminating the need for host cell DNA synthesis.

[00165] To increase packaging capacity, longer transgenes can be split between two AAV transfer plasmids, the first with a 3' splice donor site and the second with a 5' splice acceptor site. When cells are co-infected, these viruses form concatemers, splice together, and a full-length transgene can be expressed. While this allows for increased expression of the transgene, expression is less efficient. Homologous recombination is used in such capacity increasing methods. For example, a transgene can be split between two transfer plasmids, but with significant sequence overlap such that co-expression induces homologous recombination and full-length transgene expression.

[00166] Administration of human TRKB-targeted reagents can also be accomplished by lipid nanoparticle (LNP) mediated delivery. Lipid formulations can protect biological molecules from degradation while improving their cellular uptake. Lipid nanoparticles are particles containing many lipid molecules physically connected to each other by intermolecular forces. These include microspheres (including single-layer and multilayer vesicles such as liposomes), dispersed phase in emulsion, micelles, or internal phase in suspension. Such lipid nanoparticles can be used to encapsulate one or more nucleic acids or proteins for delivery. Compositions that contain cationic lipids are useful for delivering polyanions such as nucleic acids. Other lipids that can be included are neutral lipids (i.e., uncharged or zwitterionic lipids), anionic lipids, accessory lipids that enhance transfection, and stealth lipids that increase the length of time the nanoparticles can exist in vivo. . Examples of suitable cationic lipids, neutral lipids, anionic lipids, accessory lipids and invisible lipids can be found in International Patent Publication WO 2016/010840 A1, incorporated herein by reference in its entirety for all purposes. An exemplary lipid nanoparticle may contain a cationic lipid and one or more other components. In one example, the other component may contain an auxiliary lipid such as cholesterol. In another example, other components may contain an auxiliary lipid such as cholesterol and a neutral lipid such as DSPC. In another example, other components may contain an accessory lipid such as cholesterol, an optional neutral lipid such as DSPC, and a stealth lipid such as S010, S024, S027, S031, or S033.

[00167] The delivery method can be chosen to reduce immunogenicity. For example, if there are several components, they can be delivered in different ways (for example, bimodal delivery). These different methods can confer different pharmacodynamic or pharmacokinetic properties on the target molecule to be delivered. For example, different methods may result in different tissue distributions, different half-lives, or different time distributions. Some delivery methods (eg, delivery of a nucleic acid vector that is maintained in the cell through autonomous replication or genomic integration) result in more permanent expression and presence of the molecule, while other delivery methods are transient and less permanent (eg, RNA or protein delivery).

[00168] In vivo administration can be by any suitable route, including, for example, parenteral, intravenous, oral, subcutaneous, intra-arterial, intracranial, intrathecal, intraperitoneal, topical, intranasal, or intramuscular. Methods of systemic administration include, for example, oral and parenteral routes. Examples of parenteral routes include intravenous, intraarterial, intraosseous, intramuscular, intradermal, subcutaneous, intranasal, and intraperitoneal routes. A specific example is intravenous infusion. Nasal instillation and intravitreal injection are other specific examples. Methods of topical administration include, for example, intrathecal, intracerebroventricular, intraparenchymal (eg, localized intraparenchymal delivery to the following: striatum (eg, caudate nucleus or putamen), cerebral cortex, precentral gyrus, hippocampus (eg, dentate gyrus or area CA3), temporal cortex, amygdala, frontal cortex, thalamus, cerebellum, medulla oblongata, hypothalamus, midbrain roof, midbrain tegmentum, or substantia nigra), intraocular, intraorbital, subconjunctival, intravitreal, subretinal, and transscleral pathways. Significantly lower amounts of components (compared to systemic approaches) may be effective when administered topically (eg, intraparenchymal or intravitreal) compared to systemic administration (eg, intravenously). Topical routes of administration may also reduce or eliminate the occurrence of potentially toxic side effects that may occur when therapeutically effective amounts of a component are administered systemically. In a specific example, human TRKB-targeting reagents are administered by direct hippocampal injection, subcutaneous injection, or intravitreal injection.

[00169] Compositions containing human TRKB-targeting reagents can be formulated using one or more physiologically and pharmaceutically acceptable carriers, diluents, excipients, or additives. The composition may depend on the chosen route of administration. The term "pharmaceutically acceptable" means that the carrier, diluent, excipient or additive is compatible with the other ingredients of the formulation and is not substantially harmful to its recipient.

[00170] The frequency of administration and the number of dosages may depend on the half-life of human TRKB-targeted reagents and the route of administration, among other factors. Administration of human TRKB-targeted reagents to a cell or non-human animal can be performed once or multiple times over a period of time. For example, the administration can be performed at least twice during a period of time, at least three times during a period of time, at least four times during a period of time, at least five times during a period of time, at least six times over a period of time, at least seven times over a period of time, at least eight times over a period of time, at least nine times over a period of time, at least ten times over a period of time, at least eleven times, at least twelve times in a period of time, at least thirteen times in a period of time, at least fourteen times in a period of time, at least fifteen times in a period of time, at least sixteen times in a period of time time, at least seventeen times during a period of time, at least eighteen times during a period of time, at least nineteen times during a period of time, or at least twenty times during a period of time.

E. Measurement of in vivo or ex vivo delivery, potency, or efficacy of human TRKB-targeted reagents

[00171] The methods disclosed herein may further comprise detecting or measuring the activity of reagents targeted to human TRKB. Measurement of the activity of such reagents (eg, agonist activity or inhibitor activity) may include measurement of TRKB activity. TRKB activity can be measured by any known means. For example, TRKB phosphorylation can be assessed (eg, in the brain or neurons), activation of downstream pathways such as PI3K/AKT and MAPK/ERK by TRKB can be assessed (eg, in the brain or neurons such as primary cortical neurons), or cell survival can be assessed (eg, neuronal cell survival, such as retinal ganglion cell survival). For example, phosphorylation or activation of downstream signaling pathways can be assessed at 15 minutes, 30 minutes, 1 hour, 2 hours, 4 hours, or 18 hours post-dose. An increase in TRKB phosphorylation, activation of downstream signaling pathways, or cell survival may be indicative of TRKB activation, while a decrease may indicate TRKB inhibition.

[00172] In non-human animals, the assessment may include an assessment of one or more or all of the following: body weight, body composition, metabolism, and locomotion, compared with a control non-human animal (for example, after 12 hours, 24 hours, 48 hours, 72 hours, 96 hours, or 120 hours after dosing). See, for example, Lin et al. (2008) PLoS ONE 3(4):e1900; Rios et al. (2013) Trends in Neurosciences 36(2):83-90; and Zomer et al. (2003) Biol. Psychiatry 54:972-982, each of which is hereby incorporated by reference in its entirety for all purposes. Assessment of changes in body tissue composition may include, for example, assessment of lean tissue mass and/or adipose tissue mass. Assessing changes in metabolism may include, for example, assessing changes in food intake and/or water intake. A decrease in body weight, adipose tissue mass, lean mass, food intake and water intake may be a sign of TRKB activation, while a decrease in these indicators may be a sign of TRKB inhibition. An increase in locomotion may be a sign of TRKB activation, while a decrease in this indicator may be a sign of TRKB inhibition.

[00173] The assessment may include an assessment of neuroprotective activity. As one example, cell survival can be assessed in non-human animals. For example, rodent retinal ganglion cells (RGCs) are often used to study neurodegenerative processes associated with axonal injury, as well as to evaluate neuroprotective therapy. See, for example, (2009) Invest. Ophthalmol. Vis. Sic. 50(8):3860-3868, incorporated herein by reference in its entirety for all purposes. Survival/viability of retinal ganglion cells can be assessed (eg, in a complete optic nerve transection model after optic nerve injury) after treatment with a human TRKB-targeted reagent compared to a non-human control animal. For example, survival/viability of retinal ganglion cells can be assessed in a complete optic nerve transection model after optic nerve injury. See, for example, (2009) Invest. Ophthalmol. Vis. Sic. 50(8):3860-3868, incorporated herein by reference in its entirety for all purposes. As another example, retinal ganglion cell survival/viability can be assessed in an optic nerve crush model. In this model, crush injury to the optic nerve leads to gradual apoptosis of retinal ganglion cells. See, for example, Tang et al. (2011) J. Vis. Exp. 50:2685, incorporated herein by reference in its entirety for all purposes. Survival/viability of retinal ganglion cells can be assessed, for example, by measuring the density of retinal ganglion cells (eg, in retinas excised and stained for retinal ganglion cells). Increased survival/viability may be indicative of TRKB activation, while decreased survival/viability may be indicative of TRKB inhibition.

[00174] If the human TRKB-targeting reagent is a genome editing reagent, the measurement may comprise an assessment of the humanized TRKB locus for modifications. Various methods can be used to identify cells with a targeted genetic modification. Screening may include a quantitative analysis to assess the modification of the allele (MOA) of the parent chromosome. For example, quantitative analysis can be performed using quantitative PCR, such as real-time PCR (qPCR). Real-time PNR may use a first primer set that recognizes a target locus and a second primer set that recognizes a non-target reference locus. The primer set may contain a fluorescent probe that recognizes the amplified sequence. Other examples of suitable assays include fluorescence-mediated in situ hybridization (FISH), comparative genomic hybridization, isothermal DNA amplification, quantitative hybridization with immobilized probe(s), ^INVADER® probes, ^TAQMAN® Molecular Beacon probes, or probe technology. ECLIPSE™ (see, for example, US 2005/0144655, incorporated herein by reference in its entirety for all purposes). Next generation sequencing (NGS) can also be used for screening. Next generation sequencing may also be referred to as "NGS" or "massive parallel sequencing" or "high throughput sequencing". NGS can be used as a screening tool in addition to MOA assays to determine the exact nature of the targeted genetic modification and whether it is consistent across cell types or tissue types or organ types.

[00175] An assessment in a non-human animal can be performed in any cell type from any tissue or organ. For example, the assessment can be performed on multiple cell types from the same tissue or organ (eg, brain) or on cells from multiple locations in a tissue or organ (eg, hippocampus and striatum). This can provide information about which cell types in the target tissue or organ are modified or which tissue or organ sites are reached by a human TRKB-targeted reagent. As another example, the evaluation can be performed in several types of tissues or in several organs. In methods in which a particular tissue or organ is targeted, this can provide information on how effective the targeted effect is on that tissue or organ and whether there are off-target effects in other tissues or organs.

[00176] If the reagent is to inactivate the humanized TRKB locus, affect the expression of the humanized TRKB locus, or prevent translation of the humanized TRKB mRNA, the measurement may include assessing the expression of the humanized TRKB mRNA or protein. This measurement can be done in the brain or specific cell types (eg, neurons such as retinal ganglion cells).

IV. Methods for producing non-human animals containing a humanized TRKB locus

[00177] Various methods are provided for obtaining the genome of a non-human animal, a cell of a non-human animal, or a non-human animal containing a humanized TRKB locus, as disclosed elsewhere herein. Any conventional method or protocol for producing a genetically modified organism is suitable for producing such a genetically modified non-human animal. See, for example, Cho et al. (2009) Current Protocols in Cell Biology 42:19.11:19.11.1-19.11.22 and Gama Sosa et al. (2010) Brain Struct. fund. 214(2-3):91-109, each of which is incorporated herein by reference in its entirety for all purposes. Such genetically modified non-human animals can be obtained, for example, by knocking a gene at the target TrkB locus.

[00178] For example, a method for obtaining a non-human animal containing a humanized TRKB locus may include the following: (1) modifying the genome of a pluripotent cell to include a humanized TRKB locus; (2) identifying or selecting a genetically modified pluripotent cell containing the humanized TRKB locus; (3) introducing the genetically modified pluripotent cell into a host embryo of a non-human animal; and (4) implantation and gestation of the host embryo in the body of a surrogate mother. For example, a method for obtaining a non-human animal containing a humanized TRKB locus may include the following: (1) modifying the genome of a pluripotent cell to include a humanized TRKB locus; (2) identifying or selecting a genetically modified pluripotent cell containing the humanized TRKB locus; (3) introducing the genetically modified pluripotent cell into a host embryo of a non-human animal; and (4) carrying the host embryo in the body of a surrogate mother. Optionally, a host embryo containing a modified pluripotent cell (eg, a non-human ES cell) may be incubated to the blastocyst stage prior to implantation and gestation in a surrogate mother to produce a non-human F0 animal. The surrogate mother can then produce a non-human F0 generation animal containing the humanized TRKB locus.

[00179] The methods may further comprise identifying a cell or animal with a modified target genomic locus. Various methods can be used to identify cells and animals with targeted genetic modification.

[00180] The genome modification step can use, for example, exogenous repair templates (eg, targeting vectors) to modify the TrkB locus to contain the humanized TRKB locus disclosed herein. As one example, a targeting vector can be provided to produce a humanized TRKB gene at an endogenous TrkB locus (e.g., an endogenous TrkB locus of a non-human animal), wherein the targeting vector contains a 5' homologous arm targeting a 5' target sequence at the endogenous TrkB locus, and a 3' homologous arm targeting the 3' target sequence at the endogenous TrkB locus. Exogenous repair templates can also contain nucleic acids - inserts, including DNA segments to be integrated into the TrkB locus. Integration of an insert nucleic acid into the TrkB locus may result in the addition of the nucleic acid sequence of interest to the TrkB locus, deletion of the nucleic acid sequence of interest to the TrkB locus, or substitution of the nucleic acid sequence of interest to the TrkB locus (i.e., deletion and insertion). Homologous arms can flank a nucleic acid insert containing a human TRKB sequence to create a humanized TRKB locus (eg, to delete a segment of the endogenous TrkB locus and replace with an orthologous human TRKB sequence).

[00181] Exogenous repair templates can be provided for non-homologous end joining (NHEJ) mediated insertion or homologous repair. Exogenous repair templates may contain deoxyribonucleic acid (DNA) or ribonucleic acid (RNA), they may be single or double stranded, and they may be in linear or circular form. For example, the repair template may be a single stranded oligodeoxynucleotide (ssODN).

[00182] Exogenous repair templates may also contain a heterologous sequence that is not present at the untargeted endogenous TrkB locus. For example, the exogenous repair matrix may contain a selection cassette, such as a selection cassette, flanked by recombinase recognition sites.

[00183] Some exogenous repair matrices contain homologous arms. If the exogenous repair template also contains an insert nucleic acid, homologous arms may flank the insert nucleic acid. For ease of reference, homologous arms are referred to herein as 5' and 3' (ie, upstream and downstream) homologous arms. This terminology refers to the relative position of the homologous arms with respect to the nucleic acid insert within the exogenous repair matrix. The 5' and 3' homologous arms correspond to regions within the TrkB locus, which are referred to herein as "5' target sequence" and "3' target sequence", respectively.

[00184] A homologous arm and a target sequence "match" or are "matched" to each other when the two regions have a sufficient level of sequence identity relative to each other to act as substrates for the homologous recombination reaction. The term "homology" includes DNA sequences that are either identical or have sequence identity with respect to a corresponding sequence. The sequence identity between a given target sequence and the corresponding homologous arm occurring in the exogenous repair template may be any degree of sequence identity that allows homologous recombination to occur. For example, the amount of sequence identity shared by the homologous arm of the exogenous repair template (or fragment thereof) and the target sequence (or fragment thereof) may be at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96% , 97%, 98%, 99%, or 100% sequence identity such that the sequences undergo homologous recombination. Moreover, the corresponding region of homology between the homologous arm and the corresponding target sequence may be of any length that is sufficient to promote homologous recombination. In some targeting vectors, the intended mutation at the endogenous TrkB locus is incorporated into a nucleic acid insert flanked by homologous arms.

[00185] In cells other than single cell stage embryos, the exogenous repair template may be a "large targeting vector" or "LTVEC", which includes targeting vectors that contain homologous arms that match and are derived from nucleic acid sequences , greater than those typically used in other approaches envisaged for carrying out homologous recombination in cells. LTVECs also include targeting vectors containing nucleic acids - inserts characterized by nucleic acid sequences larger than those typically used in other approaches envisaged for conducting homologous recombination in cells. For example, LTVECs allow the modification of large loci that cannot fit into conventional plasmid-based targeting vectors due to their size limitations. For example, the target locus may be (i.e., the 5' and 3' homologous arms may correspond to the locus) a cell locus that cannot be targeted using a conventional method, or that can only be targeted incorrectly or only with significantly low efficiency in the absence of a single- or double-strand break caused by a nuclease agent (for example, the Cas protein). LTVECs can be of any length and are typically at least 10 kb in length. The total sum of the 5' homologous arm and the 3' homologous arm in LTVEC is typically at least 10 kb.

[00186] The screening step may include, for example, a quantitative analysis to assess the allele modification (MOA) of the parent chromosome. For example, quantitative analysis can be performed using a quantitative PNR such as real-time PCR (qPCR). Real-time PNR can use a first primer set that recognizes a target locus and a second primer set that recognizes a non-target reference locus. The primer set may contain a fluorescent probe that recognizes the amplified sequence

[00187] Other examples of suitable quantitative assays include fluorescence-mediated in situ hybridization (FISH), comparative genomic hybridization, isothermal DNA amplification, quantitative hybridization with immobilized probe(s), ^INVADER® probes, ^TAQMAN® Molecular Beacon probes or ECLIPSE™ probe technology (see, for example, US 2005/0144655, incorporated herein by reference in its entirety for all purposes).

[00188] An example of a suitable pluripotent cell is an embryonic stem (ES) cell (eg, a mouse ES cell or a rat ES cell). A modified pluripotent cell can be generated, for example, by: (a) introducing into the cell one or more exogenous donor nucleic acids (eg, targeting vectors) containing an insert nucleic acid flanked by, for example, 5' and 3' homologous arms corresponding to 5' and 3' target sites, wherein the nucleic acid insert contains a human TRKB sequence to create a humanized TRKB locus; and (b) identifying at least one cell containing in its genome an insert nucleic acid integrated into an endogenous TrkB locus (ie, identifying at least one cell containing a humanized TRKB locus). A modified pluripotent cell can be created, for example, by recombination by the following: (a) introducing into the cell one or more targeting vectors containing a nucleic acid insert flanked by 5' and 3' homologous arms corresponding to the 5' and 3' target sites, wherein nucleic acid - the insert contains a humanized TRKB locus; and (b) identifying at least one cell containing in its genome an insert nucleic acid integrated at a target genomic locus.

[00189] Alternatively, a modified pluripotent cell can be prepared as follows: (a) introducing the following into the cell: (i) a nuclease agent, wherein the nuclease agent induces a single-strand break or double-strand break at a target site within the endogenous TrkB locus; and (ii) one or more exogenous donor nucleic acids (eg, targeting vectors) containing an insert nucleic acid flanked by, for example, 5' and 3' homologous arms corresponding to 5' and 3' target sites located in sufficient proximity to a nuclease target site, wherein the nucleic acid insert contains a human TRKB sequence to create a humanized TRKB locus; and (c) identifying at least one cell containing in its genome an insert nucleic acid integrated at an endogenous TrkB locus (ie, identifying at least one cell containing a humanized TRKB locus). Alternatively, a modified pluripotent cell can be prepared as follows: (a) introducing the following into the cell: (i) a nuclease agent, wherein the nuclease agent induces a single-strand break or double-strand break at a target site within the target genomic locus; and (ii) one or more targeting vectors containing a nucleic acid insert flanked by 5' and 3' homologous arms corresponding to 5' and 3' target sites located in sufficient proximity to the recognition site, wherein the nucleic acid insert contains a humanized locus TRKB; and (c) identifying at least one cell containing the modification (eg, nucleic acid integration - insert) at the target genomic locus. Any nuclease agent that causes a single strand or double strand break at the desired recognition site can be used. Examples of suitable nucleases include a transcription activator-like effector nuclease (TALEN), a zinc finger domain nuclease (ZFN), a meganuclease, and clustered regularly interspersed short palindromic repeat (CRISPRyCRISPR-associated proteins (Cas)) systems or components of such a system (e.g., CRISPR/Cas9) See, for example, US 2013/0309670 and US 2015/0159175, each of which is incorporated herein by reference in its entirety for all purposes.

[00190] The donor cell can be introduced into the host embryo at any stage, such as the blastocyst stage or premorula stage (ie, 4 cell stage or 8 cell stage). Offspring are created that can pass on the genetic modification through the germline. See, for example, US Pat. No. 7,294,754, incorporated herein by reference in its entirety for all purposes.

[00191] Alternatively, a method for producing non-human animals described elsewhere herein may include the following: (1) modifying the single-cell stage embryonic genome to include a humanized TRKB locus using the methods described above to modify pluripotent cells; (2) selection of genetically modified embryos; and (3) implantation and gestation of a genetically modified embryo in the body of a surrogate mother. Alternatively, a method for producing non-human animals described elsewhere herein may include the following: (1) modifying the single cell stage embryonic genome to include a humanized TRKB locus using the methods described above to modify pluripotent cells; (2) selection of genetically modified embryos; and (3) carrying a genetically modified embryo in the body of a surrogate mother. Offspring are created that can pass on the genetic modification through the germline.

[00192] Nuclear transfer techniques can also be used to produce non-human mammals. Briefly, nuclear transfer methods may include the following steps: (1) enucleating an oocyte or obtaining an enucleated oocyte; (2) isolating or obtaining a donor cell or nucleus to combine with the enucleated oocyte; (3) inserting a cell or nucleus into an enucleated oocyte to form a remodeled cell; (4) implanting the reconstructed cell into the uterus of the animal to form an embryo; and (5) ensuring the development of the embryo. In such methods, oocytes are typically recovered from deceased animals, although they may also be isolated from the oviducts and/or ovaries of living animals. Oocytes can mature in a variety of well known media prior to enucleation. Enucleation of the oocyte can be accomplished by a number of well known methods. Insertion of a donor cell or nucleus into an enucleated oocyte to form a remodeled cell can be accomplished by microinjection of the donor cell under the vitellin layer prior to fusion. Fusion can be induced by applying a direct current electrical pulse across the contact/fusion plane (electrofusion), by exposing the cells to fusion-promoting chemicals such as polyethylene glycol, or by an inactivated virus such as Sendai virus. The reconstructed cell can be activated by electrical and/or non-electrical means before, during and/or after the fusion of the donor-nucleus and recipient-oocyte. Activation methods include electrical impulses, chemically induced shock, penetration by spermatozoa, increase in the content of divalent cations in the oocyte, and decrease in phosphorylation of cellular proteins (as with kinase inhibitors) in the oocyte. Activated remodeled cells or embryos can be cultured in well known media and then transferred into the uterus of an animal. See, for example, US 2008/0092249, WO 1999/005266, US 2004/0177390, WO 2008/017234, and US Pat. No. 7,612,250, each of which is incorporated herein by reference in its entirety for all purposes.

[00193] Various methods provided herein allow the creation of a genetically modified non-human F0 animal, wherein the cells of the genetically modified F0 animal contain the humanized TRKB locus. It is recognized that depending on the method used to obtain the F0 animal, the number of cells in the F0 animal that contain the humanized TRKB locus will vary. The introduction of donor ES cells into a premorula embryo from an appropriate organism (e.g., an 8-cell stage mouse embryo), for example, using the ^{VELOCIMOUSE®} method, allows a greater percentage of the cell population of an F0 animal to be obtained that contains cells containing a nucleotide sequence of interest containing targeted genetic modification. For example, at least 50%, 60%, 65%, 70%, 75%, 85%, 86%, 87%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% of the cellular contribution of the non-human F0 animal may contain a cell population containing the target modification.

[00194] The cells of the genetically modified F0 animal may be heterozygous for the humanized TRKB locus or may be homozygous for the humanized TRKB locus.

[00195] All patent applications, websites, other publications, serial numbers, and the like referred to above or below are incorporated by reference in their entirety for all purposes to the same extent as if it were specifically and individually indicated that each individual document incorporated herein by reference. If different versions of the sequence are associated with the registration number at different times, the version associated with the registration number on the filing date of this application is assumed. Effective filing date means the earlier of the actual filing date or the filing date of the priority application with reference to the registration number, as appropriate. Likewise, if different versions of a publication, website, or the like are published at different times, the latest version published on the actual date of submission is assumed, unless otherwise indicated. Any feature, step, element, embodiment, or aspect of the present invention may be used in conjunction with any other, unless specifically noted otherwise. Although the present invention has been described in more detail by way of illustration and example for clarity and understanding, it will be apparent that certain changes and modifications can be made within the scope of the appended claims.

Brief description of the sequences

[00196] The nucleotide and amino acid sequences listed in the accompanying sequence listing are shown using standard letter abbreviations for nucleotide bases and a three-letter code for amino acids. Nucleotide sequences are shown according to standard convention, starting at the 5' end of the sequence and moving forward (ie, left to right on each line) to the 3' end. Only one strand of each nucleotide sequence is shown, but a complementary strand is considered to be included in any reference to the displayed strand. When a nucleotide sequence is presented encoding an amino acid sequence, it is understood that codon-degenerate variants thereof are also presented which encode the same amino acid sequence. Amino acid sequences are shown according to standard convention, starting at the amino end of the sequence and moving forward (ie, left to right on each line) towards the carboxy end.

[00197]

EXAMPLES

Example 1 Generation of mice containing the humanized TRKB locus

[00198] A large targeting vector (LTVEC) containing a 5' homologous arm containing 41.6 kb, the mouse TrkB locus and a 3' homologous arm containing 62.4 kb. mouse TrkB locus was designed to replace the 65.7 kb region from the mouse TrkB gene encoding the mouse TRKB extracellular domain by 74.4 kb. the corresponding human TRKB sequence. Information on mouse and human TRKBs is presented in Table 2. Description of the generation of large targeting vector is presented in Table 3. Generation and use of large targeting vectors (LTVECs) derived from bacterial artificial chromosome (BAC) DNA via Bacterial Homologous Recombination Recombination (BHR) reactions using ^VELOCIGENE® genetic engineering technologies are described, for example, in US Pat. No. 6,586,251 and Valenzuela et al. (2003) Nat. Biotechnol. 21(6):652-659, each of which is incorporated herein by reference in its entirety for all purposes. The creation of LTVECs by in vitro assembly methods is described, for example, in US 2015/0376628 and WO 2015/200334, each of which is incorporated herein by reference in its entirety for all purposes.

[00199]

[00200]

[00201] Specifically, the region from exon 2 (coding for exon 1; from amino acid 32, conserving the signal peptide) to exon 10, including the first 137 bp of intron 10 and all introns between exons 2 and 10 (i.e. between coding exon 1 and exon 10) were deleted from the mouse TrkB locus (retaining the mouse transmembrane domain encoded by exons 10 and 11). Region including exon 2/coding exon 1 (from amino acid 32 after the signal peptide) to exon 10, including the first 177 bp of intron 10 and all introns between exons 2 and 10 (i.e. between the coding exon 1 and exon 10) were inserted in place of the deleted rat region (retaining the rat transmembrane domain encoded by exons 10 and 11).

[00202] The sequences for the signal peptide, extracellular domain, transmembrane domain, and mouse TRKB cytoplasmic domain are shown in SEQ ID NOs: 51-54, respectively, with the corresponding coding sequence shown in SEQ ID NOs: 63-66, respectively. The sequences for the signal peptide, extracellular domain, transmembrane domain and cytoplasmic domain of human TRKB are shown in SEQ ID NOs: 59-62, respectively, with the corresponding coding sequence shown in SEQ ID NOs: 71-74, respectively. The putative encoded TRKB chimeric protein contains the mouse TRKB transmembrane and intracellular domains, the mouse TRKB signal peptide, and the extracellular human TRKB domain. See fig. 1. Alignment of mouse and human TRKB proteins in FIG. 6. The mouse and human TrkB/TRKB coding sequences are shown in SEQ ID NOs: 9 and 11, respectively. Mouse and human TRKB protein sequences are shown in SEQ ID NOs: 1 and 3, respectively. The sequences for the putative mouse/human TRKB chimeric coding sequence and the putative mouse/human TRKB chimeric protein are shown in SEQ ID NOS: 12 and 4, respectively.

[00203] To create a mutant allele, a large targeting vector was introduced into mouse F1H4 embryonic stem cells. After antibiotic selection, colonies were selected, expanded and screened with ^TAQMAN® . See fig. 2. Allele loss assays were performed to detect loss of the endogenous rat allele, and allele gain assays were performed to detect humanized allele acquisition using the primers and probes shown in Table 4.

[00204]

[00205] Allele modification assays (MOAs), including allele loss (LOA) and allele gain (GOA) assays, are described, for example, in US 2014/0178879; US 2016/0145646; WO2016/081923; and Frendewey et al. (2010) Methods Enzymol. 476:295-307, each of which is hereby incorporated by reference in its entirety for all purposes. The allele loss assay (LOA) reverses the traditional screening logic and quantifies the copy number in a genomic DNA sample of the native locus targeted by the mutation. In a properly targeted clone of heterozygous cells, LOA analysis detects one of two native alleles (for genes not on the X or Y chromosome), with the other allele destroyed by the targeted modification. The same principle can be applied in reverse as the allele amplification assay (GOA) to quantify the copy number of an inserted targeting vector in a genomic DNA sample.

[00206] F0 mice were created using the ^{VELOCFMOUSE®} method. See, for example, US Pat. Nos. 7,576,259; 7659442; 7294754; documents US 2008/0078000; and Poueymirou et al. (2007) Nat. Biotechnol. 25(1):91-99, each of which is incorporated herein by reference in its entirety for all purposes. In the ^{VELOCIMOUSE®} method, targeted mouse embryonic stem (ES) cells are injected by laser injection into pre-morula embryos, eg eight cell stage embryos, which effectively give rise to F0 generation mice that are entirely derived from ES cells. All experiments performed on mice humanized for TRKB, as described below, were performed on mice in which the self-removing selection cassette was self-removed.

Example 2 In vivo comparison of the effect of H4H9816 and REGN1945 isotype control antibodies on body weight and metabolism in TrkB ^hu/hu mice (MAID7139)

Experimental procedure

[00207] To determine the effect of the TRKB agonist antibody, H4H9816P2, on body weight and body composition, a metabolic study of mice homozygous for expression of the human TRKB receptor instead of the mouse TRKB receptor (TrkB ^hu/hu mice) was performed after a single subcutaneous injection of the antibody. These studies were undertaken in part on the basis of previous studies on TrkB agonists and TrkB knockout mice. See, for example, Lin et al. (2008) PLoS ONE 3(4):e1900; Rios et al. (2013) Trends in Neurosciences 36(2):83-90; and Zorner et al. (2003) Biol. Psychiatry 54:972-982, each of which is hereby incorporated by reference in its entirety for all purposes. TrkB ^hu/hu mice (male, 20 weeks old) were first transferred from a group cage to an individual cage, where they were housed for two weeks for acclimatization. After this period, mice were transferred to metabolic cages (CLAMS, Columbus Instruments) to evaluate changes in food and water intake, locomotion, energy expenditure, and respiration following antibody administration. Conventional powdered food was stored in a floor chamber on a spring pressure balance (Mettler Toledo, PL602E) to measure food intake by changing the total weight of the food. Water was available through a drinker located at the top of the cage and consumption was measured by monitoring changes in pump line volume ( ^Oxymax® /CLAMS Liquid Unit). Metabolic CLAMS cells measured each of these parameters at continuous 16-18 minute intervals throughout the study. Metabolic data were analyzed in single measurements and summarized in 24-hour intervals containing one complete dark and light cycle using ^OXYMAX® /CLAMS software (Columbus instruments, v5.35). After acclimating to the cages for two weeks, TrkB ^hu/hu mice received a single 50 mg/kg subcutaneous dose of either the TRKB agonist antibody, H4H9816P2, or the IgG4 isotype control antibody in PBS at pH 7.2. A group of untreated TrkB ^hu/hu control mice did not receive an injection. Mice were weighed immediately before dosing and 24, 48, 72, 96 and 120 hours after dosing. To measure the body composition of each mouse, an EchoMRI™-500 analyzer (EchoMRI LLC) was used to perform nuclear magnetic resonance relaxometry, also referred to as quantitative magnetic resonance. Prior to dosing, mice were placed in a clear plastic holder and inserted into an NMR-MRI device to measure lean tissue mass, adipose tissue mass, and hydration status of each subject. Measurements were taken within 0.5-3.2 minutes per mouse and taken again approximately 120 hours post-dose.

Results and conclusions

[00208] Daily body weight monitoring was performed to determine if a single subcutaneous injection of H4H9816P2 caused weight loss in TrkB ^hu/hu mice. Before dose, there were no significant differences in mean body weight between the three treatment groups, as each had a mean pre-dose body weight of 28.39-29.85 g (Table 5). However, 48 hours post-dose, TrkB ^hu/hu mice treated with H4H9816P2 lost an average of 1.70 g, or 5.96% of pre-dose body weight. At the same time, untreated and TrkB ^hu/hu mice treated with isotype control antibodies gained 1.79 to 2.37% of their pre-dose body weight. TrkB ^hu/hu mice treated with H4H9816P2 continued to lose body weight throughout the course of the study, and at 72 and 96 hours post-dose, these mice lost an average of 8.42% and 11.80% of pre-dose body weight. doses, respectively. At 120 hours post-dose, TrkB ^hu/hu mice treated with H4H9816P2 lost an average of 12.67% of pre-dose body weight. Conversely, untreated and TrkB ^hu/hu mice treated with isotype control antibodies showed no pre-dose weight loss throughout the study. Since the body weight of TrkB ^hu/hu mice treated with H4H9816P2 was significantly reduced compared to both untreated and isotype controls at 48, 72, 96 and 120 hours post-dose, it was found that the antibody - TRKB agonist H4H9816P2 causes significant weight loss in TrkB ^hu/hu mice.

[00209]

[00210] The effect of an injection of the H4H9816P2 TRKB agonist antibody on body tissue composition was also measured by performing pre- and post-dose NMR-MRI on each subject. Prior to dosing, the three groups of TrkB ^hu/hu mice did not show any significant difference in adipose tissue mass or lean tissue mass, with each group having an average of 4.19-4.75 g of adipose tissue mass and 21.32- 21.70 g lean tissue mass (Table 6). After administration of antibodies, however, TrkB ^hu/hu mice dosed with H4H9816P2 lost an average of 48.90% of their total body fat mass during the entire study (Table 6). Untreated and treated with the TrkB ^hu/hu isotype control antibody, mice lost an average of 8.49% and 9.48% of their pre-dose body fat mass, respectively, which was significantly less than those treated with H4H9816P2 subjects (table 6). Moreover, H4H9816P2 TrkB ^hu/hu mice treated with H4H9816P2 lost an average of 7.84% of their lean mass during the entire study, which was significantly greater than the 2.41% and 1.75% mean loss of lean mass before dosing in untreated and treated with isotype control antibodies, respectively (Table 6). Thus, the described body weight loss can be explained by a significant loss of adipose tissue mass and a moderate loss of non-adipose tissue mass after injection of the TRKB agonist antibody H4H9816P2 in TrkB ^hu/hu mice.

[00211]

[00212] In addition to evaluating the effects of injection of the TRKB agonist antibody H4H9816P2 on body weight and body composition in TrkB ^hu/hu mice, food intake, drinking activity, and locomotor activity were continuously measured with the metabolic cell. Before dosing with TrkB ^hu/hu mice consumed an average of 3.49 to 3.73 g of food per day. However, 24 hours post-dose, H4H9816P2 TrkB-treated ^hu/hu mice significantly reduced their food intake to 2.20 g per day. Mean food intake in H4H9816P2 TrkB ^hu/hu treated mice did not exceed 2.49 g of food per day for the remainder of the study, while untreated and treated with TrkB ^hu/hu isotype antibody continuously consumed an average of 3.62-4.07 g of feed per day (Table 7).

[00213] Similarly, there were no significant differences in daily water intake between the pre-dose treatment groups. TrkB ^hu/hu mice consumed an average of 4.67-5.55 ml of water per day in each treatment group (Table 8). After dosing, H4H9816P2 TrkB-treated ^hu/hu mice reduced their water intake to 2.05-3.24 ml of water per day. This was significantly lower than in untreated and treated with TrkB ^hu/hu isotype control antibody mice, which consistently consumed 4.50-5.77 ml of water per day throughout the study (Table 8). Thus, injection of the TRKB agonist antibody, H4H9816P2, appears to result in a significant reduction in food and water intake in TrkB ^hu/hu mice compared to both untreated mice and isotype controls.

[00214]

[00215]

[00216] To determine the effects of antibody treatment on activity, locomotion was analyzed using the ^OXYMAX® /CLAMS software (Columbus instruments, v5.35), which continuously measured the total x-plane locomotion of each mouse. One mouse showed pre-dose hyperactivity and was excluded from post-dose statistical analysis. Whereas untreated and isotype antibody treated subjects consistently averaged 11,000-15,000 movements per day throughout the study, H4H9816P2 TrkB ^hu/hu treated mice recorded 28,260 movements during 24-48 hours post-dose and recorded 21193 and 27028 movements from 48-72 to 72-96 hours post-dose, respectively (Table 9). H4H9816P2 TrkB ^hu/hu mice treated with H4H9816P2 TrkB hu/hu mice showed a greater total number of movements at each time point after antibody administration, indicating hyperactivity as additional activity upon H4H9816P2 injection. Together, these effects suggest that a single subcutaneous injection of the TRKB agonist antibody, H4H9816P2, caused significant changes in body weight, body composition, metabolism, and locomotion in TrkB ^hu/hu mice.

[00219]

Example 3 In vivo comparison of the effect of the TRKB agonist antibody H4H9816P2 and the IgG4 isotype control REGN1945 on TRKB phosphorylation in the brain after stereotaxic injection in TrkB ^hu/hu mice (MAID7139)

Experimental procedure

[00218] Tyrosine receptor kinase B (TRKB) is activated by binding to its brain-derived neurotrophic factor (BDNF) ligand on the extracellular domain of the receptor, which induces dimerization and autophosphorylation of tyrosine residues in the intracellular domain of the receptor and subsequent activation of cytoplasmic signaling pathways. See, for example, Haniu et al. (1997) J. Biol. Chem. 272(40):25296-25303 and Rogalski et al. (2000) J. Biol. Chem. 275(33):25082-25088, each of which is incorporated herein by reference in its entirety for all purposes. To determine the effect of the TRKB agonist antibody, H4H9816P2, on the kinetics of TRKB activation, a study was made of the temporal dynamics of TRKB phosphorylation after direct hippocampal injection in mice homozygous for the murine/human TRKB chimeric receptor in which the extracellular domain has been humanized (MAID7139) (referred to as TrkB ^hu/hu mice). TrkB ^hu/hu mice (N=48) received bilateral stereotaxic injections with either 2 µl of either vehicle (PBS) or REGN1945 designated IgG4 isotype control antibody (final concentration 27.5 mg/ml) or TRKB agonist antibody H4H9816P2 (final concentration 27.5 mg/ml) in the hippocampus, -2 mm posterior and +1.5 mm lateral to the bregma. To minimize tissue damage, needle injection and removal were performed gradually over 5-minute intervals. TrkB ^hu/hu mice were then sacrificed by CO ₂ euthanasia at approximately 30 minutes, 1 hour, 4 hours, or 18 hours after injection. Terminal bleeding was performed by cardiac puncture to collect blood, and the mice were then transcardially perfused with cold heparinized saline. The brain was carefully removed from the skull and a 2 mm ³ tissue section surrounding the injection site was dissected, collected in an Eppendorf tube and stored on ice. The brain section was then lysed in 300 μl of RIPA lysis buffer (ThermoFisher Scientific, cat. no. 89901) containing 2x protease and phosphatase inhibitors (ThermoFisher Scientific, cat. no. 78444) and stored on ice. The lysed tissue was then homogenized for further processing, aliquoted and stored at -80°C.

[00219] To evaluate TRKB phosphorylation in brain tissue, immunoprecipitation and Western blotting were performed. The anti-human TRKB antibody H4H10108N, which does not compete for binding to H4H9816P2, was bound to NHS-activated Sepharose beads (prepared using the manufacturer's protocol; GE Healthcare, Cat # 17-0906) and washed three times with DPBS to remove residual preservative solution. Homogenized brain lysates were thawed on ice and diluted to a concentration of 1 mg/ml (brain weight to buffer volume) in buffer consisting of 1% NP-40, 0.1% Tween-20, protease inhibitors and phosphatase in TBST. The protein concentration in the homogenized brain lysate was quantified by performing a standard BCA assay according to the manufacturer's instructions (Thermo Scientific Pierce, Cat # 23225). For every 100 µg of protein, 15 µl of NHS-activated sepharose beads with anti-human TRKB antibody (H4H10108N) were added to the brain lysate solution and the mixture was incubated overnight at 4° C. with gentle shaking at 20 rpm (Thermo shaker). The next day, the samples were centrifuged at 1000 x g for one minute, and then the supernatant was carefully removed. The beads were then washed twice with 400 µl of Tris buffered saline (Bio-Rad, Cat # 1706435) with 1% Tween-20 (Sigma Aldrich, Cat # P9416) (TBST). After carefully aspirating the wash buffer, 60 μl of 0.1% trifluoroacetic acid (TFA; Sigma-Aldrich, T62200) in water at pH 3.0 was added to each sample. The solution was stirred and left for two minutes, then collected and transferred to a separate tube. This process was repeated with another 60 μl of 0.1% TFA at pH 3.0. The two 0.1% TFA solutions for each sample were then combined and 2 µl of 1M Tris-HCl (ThermoFisher Scientific, Cat # 15567-027) was added at pH 8.5.

[00220] The solution was dried using a high speed vacuum and then resuspended and reconstituted with a mixture of 20 µl of 1x Laemmli's buffer (Bio-Rad, cat # 1610737) along with 355 nM 2-mercaptoethanol (BME; Gibco, # 21985-023). Samples were boiled at 95° C. for 10 minutes and loaded onto a 10-well 4-15% Tris-Glycine Mini-Protean gel (Bio-Rad, Cat # 4561086). After electrophoresis, protein samples were transferred from a Tris-Glycine gel to a PVDF membrane (Bio-Rad, cat. no. 170-4156) via the Trans-Blot Turbo Transfer System (Bio-Rad, cat. no. 1704156) for 30 minutes with constant speed 1.3A and 25V. After transfer, the membrane was blocked with 2.5% milk (Bio-Rad, Cat # 170-6406) in TBST for one hour at room temperature and then assayed overnight with any of the following: anti-phospho-TRKB antibody (Novus, cat no. NB100-92656) diluted 1:1000 in 2.5% BSA or primary anti-TRKB antibody (Cell Signaling cat no. 4603) diluted 1:1000 in 2.5% milk TBST at 4 °C on a shaker at 30 rpm. The next day, blots were washed with TBST and incubated with horseradish peroxidase-conjugated anti-rabbit IgG (Jackson cat# 111-035-144) at a 1:1000 dilution in 1% milk in TBST for 1 hour at room temperature. The blots were then washed again, developed in ECL solution (PerkinElmer, Inc. Cat # RPN2106), and subsequent images were taken every 30 seconds.

Results and conclusions

[00221] Immunoprecipitation and subsequent Western blot analysis of protein derived from ^hu/hu TrkB mouse brain lysates demonstrated that hippocampal TRKB phosphorylation was detectable in mice injected with the TRKB agonist antibody, H4H9816P2, but not in vehicle-treated mice. or an isotype control antibody as shown in Figure 3. Among the time points assessed, TRKB phosphorylation peaked 4 hours after stereotaxic injection in mice dosed with H4H9816P2. Phosphorylation of TRKB was also detected by Western blotting 18 hours post-dose in some but not all mice. Conversely, injection of vehicle and IgG4 isotype control antibody did not cause TRKB phosphorylation at any time point. Western blotting also showed that total TRKB receptor levels were reduced in some but not all H4H9816P2 dosed TrkB ^hu/hu mice relative to vehicle and isotype control dosed mice. Total TRKB levels appear to be slightly reduced in subjects treated with H4H9816P2 18 hours post-dose. Thus, these results indicate that direct injection of the TRKB agonist antibody, H4H9816P2, induces phosphorylation of hippocampal TRKB receptors in TrkB ^hu/hu mice.

Example 4 Activation of downstream signaling pathways by TrkB agonist antibodies in primary cortical neurons from TrkB ^hu/hu mice on the first day after birth

Experimental procedure

[00222] All procedures were performed in accordance with the ARVO Statement on the Use of Animals in Ophthalmology and Vision Research and IACUC (Institutional Committee for the Care and Use of Animals) of Regeneron Pharmaceutical Inc. Mouse primary cortical neurons were isolated and cultured from TrkB humanized mice (MAS 7139). See, for example, Beaudoin et al. (2012) Nat. Protocol. 7(9):1741-1754, incorporated herein by reference in its entirety for all purposes. Western blotting was performed to determine the effects of TrkB agonist antibodies on the downstream Akt and Erk pathways (p-Akt, p-Erk1/2). Primary cortical neurons from TrkB humanized baby mice on the first day after birth (P1) were cultured for 4 days (DIV-4) in NeuralQ basal medium (Global Stem, cat. no. GSM-9420) supplemented with GS21 neuronal supplement ( Global Stem, cat. no. GSM-3100), Glutamax (Invitrogen, cat. no. 35050-061) and penicillin/streptomycin. Cells were treated with TrkB agonist antibody H4H9816P-L1 (10 µg/ml), TrkB agonist antibody H4H9780P-L1 (10 µg/ml), TrkB agonist antibody H4H9814P-L1 (10 µg/ml), IgG4 REGN1945 isotype control (10 µg/ml), control antibody H1M8037C-L1 (10 µg/ml) or BDNF (1 µg/ml) for 15 minutes or 2 hours. Western blot was performed to determine if the agonists have a difference in the maintenance and activity of downstream signals. Treated cells were washed and purified in cold PBS containing 1% protease and phosphatase inhibitors (Sigma). Protein concentration was determined by Bradford protein analysis (Pierce). Samples (50 μg) were separated by SDS-PAGE on 3-8% tris-acetate reconstituted gels (Novex) and transferred to a nitrocellulose membrane (Bio-Rad).

[00223] The membrane was incubated for 1 hour in a blocking solution containing 5% milk and 0.1% Tween-20, pH 7.6. This was followed by an overnight incubation at 4° C. in blocking buffer containing 5% BSA, 0.1% Tween-20 and rabbit anti-phospho-Trk antibody (Cell Signaling cat. #9141, 1:500), rabbit antibody to phospho-Akt (Cell Signaling, cat. No. 9271, 1:1000) or rabbit antibody to phospho-ERK1/2 (Sigma, cat. No. E7028, 1:5000). The labeled proteins were then visualized by incubation with horseradish peroxidase (HRP) conjugated anti-goat, mouse or rabbit IgG followed by development with a chemiluminescent substrate for HRP (Pierce). To determine the amount of total TrkB, MAPK, or Akt present in each band, nitrocellulose membranes were stripped of antibodies in Stripping Buffer (Pierce) for 20 minutes and incubated with rabbit anti-TrkB antibody (Cell Signaling cat #4603, 1 :1000), rabbit anti-Erk1/2 antibody (Cell Signaling cat. no. 06-182, 1:1000) or rabbit anti-Akt antibody (Cell Signaling cat. no. 9272, 1:1000) and then visualized as described above . Beta-actin (Sigma, cat. no. A5316, 1:20000 and GAPDH (Sigma, cat. no. G9295) were tested as a sample loading control.

materials

[00224]

[00225]

[00226]

[00227]

[00228]

Results and conclusions

[00229] As shown in Figure 7, although all TrkB agonist antibodies showed activation of the MAPK/ERK and PI3K/Akt pathways 15 minutes after incubation, only BDNF and H4H9814P showed TrkB phosphorylation. 2 hours after incubation, all TrkB agonist antibodies showed TrkB activation.

Example 5 Pharmacokinetic Evaluation of Anti-TRKB Antibody in TrkB Humanized and Wild Type Mice

Experimental procedure

[00230] An evaluation of the pharmacokinetics of an anti-TrkB antibody, H4H9816P2 (series H4H9816P2-L7), was performed in TrkB humanized (mice homozygous for the expression of chimeric mouse/human TrkB, TrkB ^hu/hu ) (MAID7139) mice and wild-type (WT ). The cohorts contained 5 mice per mouse strain. All mice received a single subcutaneous (SC) dose of 10 mg/kg. Blood samples were collected at 6 hours and 1, 2, 3, 6, 9, 16, 21 and 30 days post-dose. Blood was processed to serum and frozen at -80°C until analysis.

[00231] Circulating antibody concentrations were determined by assaying total human IgG4/hIgG1 antibody using GyroLab xPlore™ (Gyros, Uppsala, Sweden). Briefly, a biotinylated mouse monoclonal antibody specific for human IgG4/IgG1 (REGN2567; series RSCH15088) diluted to 100 μg/ml in antibody dilution buffer (0.05% Tween-20 + PBS) was captured on a CD Gyrolab Bioaffy 200 that contained affinity columns preloaded with streptavidin coated beads (Dynospheres™). The standard used for calibration in this assay was H4H9816P at concentrations from 0.488 to 2000 ng/ml in dilution buffer (0.5% BSA+PBS) containing 0.1% normal mouse serum (NMS). Serum samples were diluted 1:100 in antibody dilution buffer. Human IgG captured on anti-REGN2567 antibody-coated affinity columns on CD was examined at room temperature, detected by addition of 0.5 μg/mL Alexa-647 conjugated mouse anti-human kappa monoclonal antibody (REGN654; RSCH13067 series), diluted in detection buffer (Rexxip F buffer); and the resulting fluorescent signal was recorded in response units (RU) using the GyroLab xPlore instrument. Sample concentrations were determined by interpolation from a standard curve fitted using a 5-parameter logistic curve fitting using Gyrolab Evaluator software. Average concentrations from two replicate experiments were used for subsequent PK analysis.

[00232] PK parameters were determined by non-compartmental analysis (NCA) using ^{Phoenix® WinNonlin®} version 6.3 software (Certara, LP, Princeton, NJ) and an extravascular dosing model. Using the appropriate average concentration values for each antibody, all PK parameters, including the observed maximum serum concentration (Six), the estimated observed half-life (t _1/2 ) and the area under the concentration versus time curve up to the last measured concentration (AUC _last ) was determined using the linear trapezoidal rule with linear interpolation and uniform weighting.

Results and conclusions

[00233] After 10 mg/kg subcutaneous administration of the anti-TrkB antibody, H4H9816P2, similar maximum concentrations (C _max ) of the antibody were observed by day 1 or 2 in both TrkB ^hu/hu mice and WT mice (135 and 131 μg/ml respectively). By day 9, H4H9816P2 showed greater drug clearance in TrkB ^hu/hu mice than in WT mice, indicating a target-mediated effect. At day 30, antibody concentrations were approximately 35 times lower in TrkB ^hu/hu mice. Antibody exposure (AUC _last ) for H4H9816P2 in WT mice was ~1.7 times higher than in TrkB ^hu/hu mice (1730 and 1020 d*µg/mL, respectively). WT mice also showed an approximately 3-fold increase in half-life (T _1/2 ) compared to TrkB ^hu/hu mice (8.4 and 2.9 days, respectively).

[00234] A summary of data for total anti-TrkB antibody concentrations is presented in Table 15, mean PK parameters are described in Table 16, and mean total antibody concentrations versus time are shown in Figure 8. In Figure 8, mice were administered a single subcutaneous dose of 10 mg /kg on day 0. Serum concentrations of total H4H9816P2 were measured using the Gyros immunoassay. Post-dose data points at 6 hours, 1, 2, 3, 6, 9, 16, 21, and 30 days indicate the mean antibody concentration. Total H4H9816P2 antibody concentrations are represented by solid circles for TrkB ^hu/hu mice and solid squares for wild-type mice. Data are presented as mean±SD.

[00235]

[00236]

Example 6 Production of rats containing the humanized TRKB locus

[00237] A large targeting vector containing a 5' homologous arm containing 7 kb. rat TrkB locus, and a 3' homologous arm containing 47 kb. the rat TrkB locus was designed to replace the 68.5 kb region. from the rat TrkB gene encoding the rat TRKB extracellular domain at 74.4 kb. the corresponding human TRKB sequence. The construction and use of large targeting vectors (LTVECs) derived from bacterial artificial chromosome (BAC) DNA via bacterial homologous recombination (BHR) reactions using ^VELOCIGENE® genetic engineering technology are described, for example, in US Pat. No. 6,586,251 and Valenzuela et al. (2003) Nat. Biotechnol. 21(6):652-659, each of which is incorporated herein by reference in its entirety for all purposes. The creation of LTVECs by in vitro assembly methods is described, for example, in US 2015/0376628 and WO 2015/200334, each of which is incorporated herein by reference in its entirety for all purposes. Information on rat and human TRKB is shown in Table 17. Description of the large targeting vector generation is shown in Table 18.

[00238]

[00239]

[00240] In particular, the region from exon 2 (coding for exon 1; from amino acid 32, retaining the signal peptide) to exon 10, including the first 50 base pairs of intron 10 and all introns between exons 2 and 10 (i.e. between coding exon 1 and exon 10) were deleted from the rat TrkB locus (retaining the rat transmembrane domain encoded by exons 10 and 11). Region including exon 2/coding exon 1 (from amino acid 32 after the signal peptide) to exon 10, including the first 66 base pairs of intron 10 and all introns between exons 2 and 10 (i.e. between the coding exon 1 and exon 10) were inserted in place of the deleted rat region (retaining the rat transmembrane domain encoded by exons 10 and 11).

[00241] The sequences for signal peptide, extracellular domain, transmembrane domain and cytoplasmic domain of rat TRKB are shown in SEQ ID NOs: 55-58, respectively, with the corresponding coding sequence shown in SEQ ID NOs: 67-70, respectively. The sequences for the signal peptide, extracellular domain, transmembrane domain and cytoplasmic domain of human TRKB are shown in SEQ ID NOs: 59-62, respectively, with the corresponding coding sequence shown in SEQ ID NOs: 71-74, respectively. The putative encoded TRKB chimeric protein contains the rat TRKB transmembrane and intracellular domains, the rat TRKB signal peptide, and the extracellular human TRKB domain. See fig. 4. Alignment of rat and human TRKB proteins in Figure 6. The rat and human TrkB/TRKB coding sequences are shown in SEQ ID NOS: 10 and 11, respectively. The rat and human TRKB protein sequences are shown in SEQ ID NOs: 2 and 3, respectively. The sequences for the putative rat/human TRKB chimeric coding sequence and the putative rat/human TRKB chimeric protein are shown in SEQ ID NOS: 13 and 5, respectively.

[00242] To create a mutant allele, CRISPR/Cas9 components, including four guide RNAs (target sequences for guide RNAs given in SEQ ID NOs: 41-44), were introduced into rat embryonic stem cells along with a large targeting vector. Specifically, 4×10 ⁶ rat ES cells (Dark Agouti DA2B line) were electroporated with the following: 2 mg TrkB LTVEC; 5 mg Cas9 expressing plasmid; and 5 mg each of gRNA: gU, gU2, gD and gD2. Electroporation conditions: voltage 400 V; capacitance 100 mF; and resistance 0 W. Selection with antibiotics was performed using G418 at a concentration of 75 mg/ml. Colonies were selected, propagated and screened with TAQMAN ^® . See fig. 5. Allele loss assays were performed to detect loss of the endogenous rat allele, and allele acquisition assays were performed to detect acquisition of the humanized allele, and CRISPR assays and retention assays were performed using the primers and probes shown in Table 19.

[00243]

[00244] Allele modification assays (MOAs), including allele loss (LOA) and allele gain (GOA) assays, are described, for example, in US 2014/0178879; US 2016/0145646; WO2016/081923; and Frendewey et al. (2010) Methods Enzymol. 476:295-307, each of which is hereby incorporated by reference in its entirety for all purposes. The allele loss assay (LOA) reverses the traditional screening logic and quantifies the copy number in a genomic DNA sample of the native locus targeted by the mutation. In a properly targeted clone of heterozygous cells, LOA analysis detects one of two native alleles (for genes not on the X or Y chromosome), with the other allele destroyed by the targeted modification. The same principle can be applied in reverse as the allele amplification assay (GOA) to quantify the copy number of an inserted targeting vector in a genomic DNA sample.

[00245] Retention assays are described in US 2016/0145646 and WO 2016/081923, each of which is incorporated herein by reference in its entirety for all purposes. In retention assays, a distinction is made between correct targeted nucleic acid insertions - inserts at a target genomic locus and random transgenic nucleic acid insertions - insertions at genomic locations outside the target genomic locus by evaluating the number of copies of DNA templates from the 5' and 3' target sequences corresponding to 5 ' and 3' homologous arms of the targeting vector, respectively. In particular, retention assays determine the number of copies in a genomic DNA sample of the 5' target sequence DNA template intended to be retained at the modified target genomic locus and/or the target sequence 3' DNA template intended to be retained at the modified target genomic locus. In diploid cells, properly targeted clones will retain a copy number of two. A copy number greater than two generally indicates transgenic integration of the targeting vector at random outside the target genomic locus rather than at the target genomic locus. A copy number less than usual indicates large deletions outside the area targeted for the deletion.

[00246] CRISPR assays are ^TAQMAN® assays designed to cover the region that is disrupted by CRISPR gRNA. When a CRISPR gRNA cuts and creates an indel (insert or deletion), the ^TAQMAN® assay will fail to amplify and thus report CRISPR cleavage.

[00247] The positive clone CB1 was thawed, expanded and re-confirmed with TAQMAN ^® . CB1 was also confirmed by successful PCR from the 5' end of the human substitution sequence to the flanking rat genomic sequence beyond the 5' end of the homologous arm. The PCR amplicon was confirmed as correct by end sequencing.

[00248] F0 and F1 rats were generated using the methods described in US 2014/0235933, US 2014/0310828, WO 2014/130706 and WO 2014/172489, each of which is incorporated herein by reference in its entirety for all purposes. In such methods, validated target rat ES cell clones (e.g., Dark Agouti ES cell clones) are microinjected into blastocysts (e.g., Sprague Dawley (SD) blastocysts), which are then transferred to pseudopregnant female recipients (e.g., female SD recipients) for gestation using standard techniques. Chimeras are identified (eg, by coat color) and F0 male chimeras are bred to wild-type female rats of the same strain (eg, SD females). The germline (eg, Agouti) F1 pups are then genotyped for the target allele. All experiments performed on TRKB humanized rats as described below were performed on rats in which the self-removing selection cassette self-removed.

Example 7 In vivo comparison of the effect of the TRKB agonist antibody H4H9816P2 and the IgG4 isotype control REGN1945 on the survival of retinal ganglion cells (RGC) in TrkB ^hu/hu rats

Experimental procedure

[00249] All procedures were performed in accordance with the ARVO Statement on the Use of Animals in Ophthalmology and Vision Research and IACUC (Institutional Committee for the Care and Use of Animals) of Regeneron Pharmaceutical Inc. Adult TrkB humanized rats (MAID100010) aged 8-10 weeks, each weighing 200-250 g were used. All surgical interventions on rats were performed under general anesthesia using an intraperitoneal injection of ketamine (63 mg/kg) and xylazine (6.0 mg/kg). An eye ointment containing erythromycin (0.5%, Bausch & Lomb) was used to protect the cornea.

[00250] Intraorbital optic nerve axotomy and intravitreal injection. The left optic nerve (ON) was affected intraorbitally, its dura was opened. ON was transected approximately 1.5 mm behind the eyeball. Care was taken not to damage the blood supply to the retina. Intravitreal injections were performed directly behind the flat part of the ciliary body using an extended glass pipette connected to a 50 µl Hamilton syringe. Care was taken not to damage the lens. Rats with any significant postoperative complications (eg, retinal ischemia, cataracts) were excluded from further analysis. Animals were divided into different experimental groups. One control group received intravitreal injections of 3 μl of REGN1945 isotype control (46.6 μg/μl); the other group received an injection of 3 μl of anti-human TRKB antibody H4H9816P2 (45.7 μg/μl) 3 and 10 days after ON-axotomy.

[00251] Immunohistochemical staining and counting of viable retinal ganglion cells (RGCs). BRN3A (brain-specific homeobox/ROA 3A domain protein) has been used as a marker for surviving retinal ganglion cells (RGCs) as it has been shown to be an efficient and reliable way to selectively label viable RGCs in retinal whole preparations after optic nerve injury. See, for example, (2009) Invest. Ophthalmol. Vis. Sic. 50(8):3860-3868, incorporated herein by reference in its entirety for all purposes. For BRN3A immunostaining, retinas were blocked in 10% normal donkey serum and 0.5% Triton X-100 for 1 hour, then incubated in the same medium with anti-BRN3A antibody (1:400; cat no.: sc-31984, Santa Cruz) 2 h at room temperature. After further washing, retinas were incubated with Alexa594-conjugated donkey anti-goat secondary antibody (1:400; Cat #: A-11058, mvitrogen) overnight at 4°C.

Results and conclusions

[00252] To evaluate the effect of a TRKB agonist antibody on RGC in vivo survival, we used a complete optic nerve transection model. TRKB agonist antibody (H4H9816P2) or isotype control antibody was used 3 and 10 days after surgery. Animals were sacrificed 14 days after axotomy. RGC density in the intact contralateral eye is similar for the three TRKB genotypes (homozygous humanized, heterozygous humanized and wild type), averaging about 1600 per mm ² as shown in Table 20. Density of surviving RGCs was assessed in total retinal preparations using BRN3A staining. The present inventors found that in homozygous TrkB ^hu/hu humanized rats, the TRKB agonist antibody (H4H9816P2) significantly (p<0.01, Mann-Whitney test) increased RGC survival compared to controls (685±106 versus 255± 66 RGC per ^mm2 ). Heterozygous TrkB ^hu/+ humanized rats also showed a significant (p<0.05, Mann-Whitney test) effect on survival from the TrkB agonist antibody (444±90 versus 208±50 RGC/mm ² ). In wild-type rats, a small but non-significant increase in RGC was observed in rats treated with the TRKB agonist antibody compared to isotype controls (Table 21). In conclusion, the TRKB agonist antibody (H4H9816P2) significantly increased RGC survival in humanized TrkB ^hu/hu rats.

[00253]

[00254]

Example 8. Amino acid sequences of the variable region of the heavy and light chains of anti-TRKB antibodies used in the examples.

[00255] Several fully human anti-TRKB antibodies (i.e., antibodies having human variable domains and human constant domains) were tested in the examples, including those designated as H4H9780P, H4H9814P, and H4H9816P2. Table 22 lists the amino acid sequence identifiers for the heavy and light chain variable regions and CDRs of selected anti-TRKB antibodies used in the examples. Table 23 lists the heavy and light chain variable region nucleic acid sequence identifiers and CDRs of selected anti-TRKB antibodies used in the examples. These antibodies are described in more detail in US Patent Application No. 16/202881, filed Nov. 28, 2018, which is incorporated herein by reference in its entirety for all purposes.

[00256]

[00257]

[00258] Generally, antibodies are referred to herein according to the following nomenclature: Fc prefix (e.g., "H4H") followed by a numeric identifier (e.g., "9780", "9816", etc., as shown in Table 22) followed by the suffix "P" or "P2". The H4H prefixes in antibody designations used herein indicate the specific isotype of the Fc region of an antibody. Thus, in accordance with this nomenclature, an antibody may be referred to herein, for example, "H4H9780P", etc., which indicates a human IgG4 Fc. The variable regions are fully human, as indicated by the first "H" in the designation of the antibody. One of ordinary skill in the art will appreciate that an antibody with a particular Fc isotype can be converted into an antibody with a different Fc isotype (e.g., a mouse IgG1 Fc antibody can be converted into a human IgG4 antibody, etc.), but in any case, the variable domains (including CDRs) which are indicated by the numeric identifiers shown in Table 22 will remain the same and antigen binding properties are expected to be identical or substantially similar regardless of the nature of the Fc domain.

Example 9. Neuroprotective effect of anti-human TrkB agonists in humanized TrkB rats

[00259] The following experiments were undertaken to evaluate the neuroprotective effects of endogenous TRKB agonist, brain-derived neurotrophic factor (BDNF) and TRKB agonist monoclonal antibody (mAb) in wild type (WT) mice and rats and in TrkB humanized mice and rats.

[00260] The in vitro effects of BDNF and anti-TRKB antibody (Ab) were quantified by cell survival assays using the differentiated human neuroblastoma cell line SH-SY5Y. In vitro, BDNF or Ab to TRKB significantly increased cell survival in retinoic acid-differentiated SH-SY5Y cells. The effects showed bell-shaped dose-response curves with the optimal dose being 1 μg/ml for BDNF or 10 μg/ml for Ab to TRKB. The SH-SY5Y neuroblastoma cell line was cultured in differentiation medium containing 10 μM all-trans retinoic acid for 4 days. The culture was changed to serum-free differentiation medium containing different doses of antibodies (0.01-100 μg/ml). Two days later, the CCK8 reagent was added, the plates were incubated for 3-4 hours and OD450 was measured to determine the percentage of surviving cells. Data were normalized to serum-free medium without antibodies. As shown in Figure 9, an anti-TRKB mAb (anti-TrkB mAb1 is H4H9816P2; anti-TrkB mAb2 is a control TrkB agonist antibody) increased the survival of SH-SY5Y cells in a dose dependent manner. Human isotype control did not affect the survival of SH-SY5Y cells. Serum-free medium without antibodies resulted in 100% survival.

[00261] Retinas from P2 C57BL/6J mice were then excised and dissociated. Retinal ganglion cells were purified by immuno-panning and cultured in a 96-well plate with or without treatment. After 24 hours, MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) was added to each well to calculate cell viability for each group. As shown in Figure 10, BDNF had a bell-shaped response curve and an optimal dose at 1 μg/ml. anti-TrkB mAb2 (a control TrkB agonist antibody with affinity for human TrkB, mouse TrkB, and rat TrkB) can also exhibit a bell curve at higher doses, but exhibits a neuroprotective effect.

[00262] WT and TrkB humanized mice and rats were used to test the neuroprotective effect in vivo. Animals received intravitreal (IVT) injections of BDNF or anti-TRKB mAb on days 3 and 10 after optic nerve transection (ONT). Retinal ganglion cell (RGC) counts were quantified using HALO software (Indica Labs) 1 week in mouse or 2 weeks in rat after optic nerve transection with Brn3a IHC on flat retinal holders.

[00263] RGC death in TrkB ^hu/hu mice was similar to that in WT mice 1 or 2 weeks after optic nerve transection. BDNF or Ab to TRKB had little or no significant neuroprotective effect in WT or TrkB ^hu/hu mice. In contrast, TrkB ^hu/hu rats showed significant neuroprotection of RGC by IVT Ab to TRKB. Weight loss was observed in TrkB ^hu/hu mice, but not in rats after treatment with IVT Ab to TRKB. BDNF did not affect body weight in either mice or rats.

[00264] Figures 11A and 11B show the results of a neuroprotection experiment in an optic nerve transection model in WT mice and rats. 11A, Dark Agouti rats aged 8-9 weeks were injected with BDNF (5 μg), anti-TrkB mAb2 (18 μg), isotype control antibody (18 μg) or vehicle control intravitreally 3 days and 10 days after crossing. mAb2 to TrkB is a control TrkB agonist antibody with affinity for human TrkB, mouse TrkB and rat TrkB. Retinas were dissected and stained for retinal ganglion cells 14 days after transection. BDNF and anti-TRKB mAbs showed significant neuroprotection as measured by retinal ganglion cell (RGC) density. In Figure 11B, 8-week-old C57BL/6J WT mice were injected with BDNF (2.5 μg), anti-TrkB mAb2 (10 μg), isotype control antibody (10 μg) or vehicle control intravitreally 3 days and 10 days post-crossing. mAb2 to TrkB is a control TrkB agonist antibody with affinity for human TrkB, mouse TrkB and rat TrkB. No significant neuroprotection was observed. Thus, treatment with BDNF and anti-TRKB mAb resulted in a significant increase in RGC density in excised retinas in wild-type rats after optic nerve transection, while no significant effect on RGC density was observed in the same model in wild-type mice.

[00265] Figures 12A and 12B show the dose response of BDNF in WT mice and rats. In Figure 12A, the BDNF dose response in the WT mouse optic nerve crush (ONC) model shows a small window of neuroprotection. Figure 12B shows the dose response of BDNF in the optic nerve transection model in WT rats from 0.13 μg to 30 μg. There is a bell-shaped response similar to in vitro data, with an optimal dose of 0.8 µg. Retinas were dissected and stained for retinal ganglion cells 14 days after transection. Thus, treatment with BDNF resulted in a much stronger dose-response curve as measured by RGC density in excised retinas of wild-type rats after optic nerve transection compared to a much less pronounced dose-response curve of BDNF as measured by RGC density in excised retinas of wild-type mice after crushing the optic nerve.

[00266] The neuroprotective effect of Ab against TRKB was then tested in humanized TrkB rats. The results in Figures 13A and 13B show that intravitreal injection of anti-TRKB mAb in TrkB humanized optic nerve transected rats demonstrates significant neuroprotection of retinal ganglion cells. Human TRKB homozygous, human TRKB heterozygous, or wild-type TrkB rats were injected with either anti-TrkB mAb1 or an isotype control antibody intravitreally (3 μl) 3 and 10 days after optic nerve transection. Fourteen days after transection, the retinas were excised and stained for RGC. The rats were females aged 17-19 weeks. As shown in Figure 13A, rats treated with anti-TrkB mAb1 (H4H9816P2) showed neuroprotection across all three genotypes compared to corresponding isotype control antibody-treated rats. Human TRKB homozygous and heterozygous rats given isotype control had a higher RGC density than wild-type rats given isotype control. Figure 13B shows no difference in the number of RGCs in untreated eyes between genotypes. Figure 13C shows body weights of human TRKB homozygous mice treated with either the TrkB agonist antibody (H4H9816P2) or the isotype control antibody (REGN1945) 14 days post-crossing.

[00267] Rat retinal whole preparation RGC isodensity maps were then generated showing Brn3a labeled cells of intact and treated injured eyes in three genotypes (data not shown). The reconstruction of the total preparation was obtained using a motorized stage on a fluorescence microscope (Nikon Eclipse Ti). RGC was counted using image analysis software (HALO®; mdica Labs, Corrales, NM, USA). Isodensity maps were obtained using Matlab. Higher RGC density was observed in TrkB humanized rats treated with anti-TrkB mAb1 (H4H9816P2) compared to isotype control treated rats (data not shown).

[00268] Taken together, the data shown in Figures 11A, 11B, 12A, 12B, and 13A-13C demonstrate that intravitreal administration of a TRKB agonist mAb has a significant neuroprotective effect following optic nerve injury in TrkB humanized rats, in contrast to little or no significant neuroprotective effect observed after optic nerve injury in humanized TrkB mice.

[00269] To further evaluate the effect of TRKB agonist antibodies on RGC survival in rats in an optic nerve transection (ONT) model, a dose-response study was undertaken. Human TRKB homozygous rats (MAGO100010; 75% SD, 25% DA) aged 1-9 months were used. Six rats were used in each group. Human TRKB homozygous rats were injected with various doses of either anti-TrkB mAb1 or isotype control antibody (REGN1945) intravitreally (3 µl) 3 and 10 days after optic nerve transection. Fourteen days after transection, the retinas were excised and stained for RGC. As shown in Figure 14, anti-TrkB mAb1 dose-dependently increased RGC survival in TrkB humanized rats.

[00270] The neuroprotective effects of various TrkB agonist antibodies were then compared in human TRKB homozygous rats in an optic nerve transection (ONT) model. TrkB humanized rats (MAID100010; 75% SD, 25% DA) aged 8-10 weeks were used. Five to six rats were used in each group. Human TRKB homozygous rats were injected with either H4H9816P2-L9 (10 µg), H4H9814P-L9 (10 µg), H4H9780P-L5 (10 µg), a combination of all three (3.3 µg each), or an isotype control antibody (REGN1945; 10 ) μg) intravitreal (3 μl) 3 and 10 days after the intersection of the optic nerve. Fourteen days after transection, the retinas were excised and stained for RGC. The results are shown in figures 15A and 15B. Each TrkB agonist antibody has a neuroprotective effect compared to the isotype control antibody. Body weight in each group was the same (data not shown).

[00271] In contrast, the TrkB agonist antibodies, H4H9780P and H4H9814P, had no neuroprotective effect in wild-type rats. The neuroprotective effect was assessed in wild-type rats using the optic nerve transection (ONT) model. Wild-type female rats aged 8-10 weeks were used. Five to six rats were used in each group. Wild-type rats were injected intravitreally (3 μl) with H4H9780P (120 μg), H4H9814P (120 μg) or an isotype control antibody (REGN1945; 120 μg) 3 and 10 days after optic nerve transection. Fourteen days after transection, the retinas were excised and stained for RGC. As shown in Figure 16, none of the TrkB agonist antibodies had a significant neuroprotective effect in wild-type rats.

[00272] In addition, the TrkB agonist antibody (H4H9780P) had no neuroprotective effect in homozygous human TRKB mice. TRKB homozygous male mice (MAID7139; 75% C57BL/6, 25% 129) were used and were 5 months old. Five to six mice were used in each group. Human TRKB homozygous mice were injected with either H4H9780P (40 μg/eye) or an isotype control antibody (REGN1945; 40 μg/eye) intravitreally (1 μl) 3 and 10 days after optic nerve transection. Fourteen days after transection, the retinas were excised for the RGC as well. As shown in Figures 17A and 17B, the TrkB agonist antibody had no neuroprotective effect in human TRKB homozygous mice, in contrast to the neuroprotective effect observed in human TRKB homozygous rats. Figure 17C shows body weights of human TRKB homozygous mice treated with either H4H9780P or an isotype control antibody 14 days post-crossing.

--->

SEQUENCE LIST

<110> Regeneron Pharmaceuticals, Inc.

<120> NON-HUMAN ANIMALS CONTAINING HUMANIZED

LOCUS TrkB

<130> 57766-523380

<150> US 62/592905

<151> 2017-11-30

<150> US 62/661373

<151> 2018-04-23

<160> 125

<170> PatentIn version 3.5

<210> 1

<211> 821

<212> PRT

<213> Mus musculus

<220>

<221> MISC_FEATURE

<222> (1)..(31)

<223> Signal peptide

<220>

<221> MISC_FEATURE

<222> (32)..(429)

<223> Extracellular domain

<220>

<221> MISC_FEATURE

<222> (430)..(453)

<223> Transmembrane domain

<220>

<221> MISC_FEATURE

<222> (454)..(821)

<223> Cytoplasmic domain

<400> 1

Met Ser Pro Trp Leu Lys Trp His Gly Pro Ala Met Ala Arg Leu Trp

1 5 10 15

Gly Leu Cys Leu Leu Val Leu Gly Phe Trp Arg Ala Ser Leu Ala Cys

20 25 30

Pro Thr Ser Cys Lys Cys Ser Ser Ala Arg Ile Trp Cys Thr Glu Pro

35 40 45

Ser Pro Gly Ile Val Ala Phe Pro Arg Leu Glu Pro Asn Ser Val Asp

50 55 60

Pro Glu Asn Ile Thr Glu Ile Leu Ile Ala Asn Gln Lys Arg Leu Glu

65 70 75 80

Ile Ile Asn Glu Asp Asp Val Glu Ala Tyr Val Gly Leu Arg Asn Leu

85 90 95

Thr Ile Val Asp Ser Gly Leu Lys Phe Val Ala Tyr Lys Ala Phe Leu

100 105 110

Lys Asn Ser Asn Leu Arg His Ile Asn Phe Thr Arg Asn Lys Leu Thr

115 120 125

Ser Leu Ser Arg Arg His Phe Arg His Leu Asp Leu Ser Asp Leu Ile

130 135 140

Leu Thr Gly Asn Pro Phe Thr Cys Ser Cys Asp Ile Met Trp Leu Lys

145 150 155 160

Thr Leu Gln Glu Thr Lys Ser Ser Pro Asp Thr Gln Asp Leu Tyr Cys

165 170 175

Leu Asn Glu Ser Ser Lys Asn Met Pro Leu Ala Asn Leu Gln Ile Pro

180 185 190

Asn Cys Gly Leu Pro Ser Ala Arg Leu Ala Ala Pro Asn Leu Thr Val

195 200 205

Glu Glu Gly Lys Ser Val Thr Leu Ser Cys Ser Val Gly Gly Asp Pro

210 215 220

Leu Pro Thr Leu Tyr Trp Asp Val Gly Asn Leu Val Ser Lys His Met

225 230 235 240

Asn Glu Thr Ser His Thr Gln Gly Ser Leu Arg Ile Thr Asn Ile Ser

245 250 255

Ser Asp Asp Ser Gly Lys Gln Ile Ser Cys Val Ala Glu Asn Leu Val

260 265 270

Gly Glu Asp Gln Asp Ser Val Asn Leu Thr Val His Phe Ala Pro Thr

275 280 285

Ile Thr Phe Leu Glu Ser Pro Thr Ser Asp His His Trp Cys Ile Pro

290 295 300

Phe Thr Val Arg Gly Asn Pro Lys Pro Ala Leu Gln Trp Phe Tyr Asn

305 310 315 320

Gly Ala Ile Leu Asn Glu Ser Lys Tyr Ile Cys Thr Lys Ile His Val

325 330 335

Thr Asn His Thr Glu Tyr His Gly Cys Leu Gln Leu Asp Asn Pro Thr

340 345 350

His Met Asn Asn Gly Asp Tyr Thr Leu Met Ala Lys Asn Glu Tyr Gly

355 360 365

Lys Asp Glu Arg Gln Ile Ser Ala His Phe Met Gly Arg Pro Gly Val

370 375 380

Asp Tyr Glu Thr Asn Pro Asn Tyr Pro Glu Val Leu Tyr Glu Asp Trp

385 390 395 400

Thr Thr Pro Thr Asp Ile Gly Asp Thr Thr Asn Lys Ser Asn Glu Ile

405 410 415

Pro Ser Thr Asp Val Ala Asp Gln Ser Asn Arg Glu His Leu Ser Val

420 425 430

Tyr Ala Val Val Val Ile Ala Ser Val Val Gly Phe Cys Leu Leu Val

435 440 445

Met Leu Leu Leu Leu Lys Leu Ala Arg His Ser Lys Phe Gly Met Lys

450 455 460

Gly Pro Ala Ser Val Ile Ser Asn Asp Asp Asp Ser Ala Ser Pro Leu

465 470 475 480

His His Ile Ser Asn Gly Ser Asn Thr Pro Ser Ser Ser Glu Gly Gly

485 490 495

Pro Asp Ala Val Ile Ile Gly Met Thr Lys Ile Pro Val Ile Glu Asn

500 505 510

Pro Gln Tyr Phe Gly Ile Thr Asn Ser Gln Leu Lys Pro Asp Thr Phe

515 520 525

Val Gln His Ile Lys Arg His Asn Ile Val Leu Lys Arg Glu Leu Gly

530 535 540

Glu Gly Ala Phe Gly Lys Val Phe Leu Ala Glu Cys Tyr Asn Leu Cys

545 550 555 560

Pro Glu Gln Asp Lys Ile Leu Val Ala Val Lys Thr Leu Lys Asp Ala

565 570 575

Ser Asp Asn Ala Arg Lys Asp Phe His Arg Glu Ala Glu Leu Leu Thr

580 585 590

Asn Leu Gln His Glu His Ile Val Lys Phe Tyr Gly Val Cys Val Glu

595 600 605

Gly Asp Pro Leu Ile Met Val Phe Glu Tyr Met Lys His Gly Asp Leu

610 615 620

Asn Lys Phe Leu Arg Ala His Gly Pro Asp Ala Val Leu Met Ala Glu

625 630 635 640

Gly Asn Pro Pro Thr Glu Leu Thr Gln Ser Gln Met Leu His Ile Ala

645 650 655

Gln Gln Ile Ala Ala Gly Met Val Tyr Leu Ala Ser Gln His Phe Val

660 665 670

His Arg Asp Leu Ala Thr Arg Asn Cys Leu Val Gly Glu Asn Leu Leu

675 680 685

Val Lys Ile Gly Asp Phe Gly Met Ser Arg Asp Val Tyr Ser Thr Asp

690 695 700

Tyr Tyr Arg Val Gly Gly His Thr Met Leu Pro Ile Arg Trp Met Pro

705 710 715 720

Pro Glu Ser Ile Met Tyr Arg Lys Phe Thr Thr Glu Ser Asp Val Trp

725 730 735

Ser Leu Gly Val Val Leu Trp Glu Ile Phe Thr Tyr Gly Lys Gln Pro

740 745 750

Trp Tyr Gln Leu Ser Asn Asn Glu Val Ile Glu Cys Ile Thr Gln Gly

755 760 765

Arg Val Leu Gln Arg Pro Arg Thr Cys Pro Gln Glu Val Tyr Glu Leu

770 775 780

Met Leu Gly Cys Trp Gln Arg Glu Pro His Thr Arg Lys Asn Ile Lys

785 790 795 800

Ser Ile His Thr Leu Leu Gln Asn Leu Ala Lys Ala Ser Pro Val Tyr

805 810 815

Leu Asp Ile Leu Gly

820

<210> 2

<211> 821

<212> PRT

<213> Rattus norvegicus

<220>

<221> MISC_FEATURE

<222> (1)..(31)

<223> Signal peptide

<220>

<221> MISC_FEATURE

<222> (32)..(429)

<223> Extracellular domain

<220>

<221> MISC_FEATURE

<222> (430)..(453)

<223> Transmembrane domain

<220>

<221> MISC_FEATURE

<222> (454)..(821)

<223> Cytoplasmic domain

<400> 2

Met Ser Pro Trp Pro Arg Trp His Gly Pro Ala Met Ala Arg Leu Trp

1 5 10 15

Gly Leu Cys Leu Leu Val Leu Gly Phe Trp Arg Ala Ser Leu Ala Cys

20 25 30

Pro Met Ser Cys Lys Cys Ser Thr Thr Arg Ile Trp Cys Thr Glu Pro

35 40 45

Ser Pro Gly Ile Val Ala Phe Pro Arg Leu Glu Pro Asn Ser Ile Asp

50 55 60

Pro Glu Asn Ile Thr Glu Ile Leu Ile Ala Asn Gln Lys Arg Leu Glu

65 70 75 80

Ile Ile Asn Glu Asp Asp Val Glu Ala Tyr Val Gly Leu Lys Asn Leu

85 90 95

Thr Ile Val Asp Ser Gly Leu Lys Phe Val Ala Tyr Lys Ala Phe Leu

100 105 110

Lys Asn Gly Asn Leu Arg His Ile Asn Phe Thr Arg Asn Lys Leu Thr

115 120 125

Ser Leu Ser Arg Arg His Phe Arg His Leu Asp Leu Ser Asp Leu Ile

130 135 140

Leu Thr Gly Asn Pro Phe Thr Cys Ser Cys Asp Ile Met Trp Leu Lys

145 150 155 160

Thr Leu Gln Glu Thr Lys Ser Ser Pro Asp Thr Gln Asp Leu Tyr Cys

165 170 175

Leu Asn Glu Ser Ser Lys Asn Thr Pro Leu Ala Asn Leu Gln Ile Pro

180 185 190

Asn Cys Gly Leu Pro Ser Ala Arg Leu Ala Ala Pro Asn Leu Thr Val

195 200 205

Glu Glu Gly Lys Ser Val Thr Ile Ser Cys Ser Val Gly Gly Asp Pro

210 215 220

Leu Pro Thr Leu Tyr Trp Asp Val Gly Asn Leu Val Ser Lys His Met

225 230 235 240

Asn Glu Thr Ser His Thr Gln Gly Ser Leu Arg Ile Thr Asn Ile Ser

245 250 255

Ser Asp Asp Ser Gly Lys Gln Ile Ser Cys Val Ala Glu Asn Leu Val

260 265 270

Gly Glu Asp Gln Asp Ser Val Asn Leu Thr Val His Phe Ala Pro Thr

275 280 285

Ile Thr Phe Leu Glu Ser Pro Thr Ser Asp His His Trp Cys Ile Pro

290 295 300

Phe Thr Val Arg Gly Asn Pro Lys Pro Ala Leu Gln Trp Phe Tyr Asn

305 310 315 320

Gly Ala Ile Leu Asn Glu Ser Lys Tyr Ile Cys Thr Lys Ile His Val

325 330 335

Thr Asn His Thr Glu Tyr His Gly Cys Leu Gln Leu Asp Asn Pro Thr

340 345 350

His Met Asn Asn Gly Asp Tyr Thr Leu Met Ala Lys Asn Glu Tyr Gly

355 360 365

Lys Asp Glu Arg Gln Ile Ser Ala His Phe Met Gly Arg Pro Gly Val

370 375 380

Asp Tyr Glu Thr Asn Pro Asn Tyr Pro Glu Val Leu Tyr Glu Asp Trp

385 390 395 400

Thr Thr Pro Thr Asp Ile Gly Asp Thr Thr Asn Lys Ser Asn Glu Ile

405 410 415

Pro Ser Thr Asp Val Ala Asp Gln Thr Asn Arg Glu His Leu Ser Val

420 425 430

Tyr Ala Val Val Val Ile Ala Ser Val Val Gly Phe Cys Leu Leu Val

435 440 445

Met Leu Leu Leu Leu Lys Leu Ala Arg His Ser Lys Phe Gly Met Lys

450 455 460

Gly Pro Ala Ser Val Ile Ser Asn Asp Asp Asp Ser Ala Ser Pro Leu

465 470 475 480

His His Ile Ser Asn Gly Ser Asn Thr Pro Ser Ser Ser Glu Gly Gly

485 490 495

Pro Asp Ala Val Ile Ile Gly Met Thr Lys Ile Pro Val Ile Glu Asn

500 505 510

Pro Gln Tyr Phe Gly Ile Thr Asn Ser Gln Leu Lys Pro Asp Thr Phe

515 520 525

Val Gln His Ile Lys Arg His Asn Ile Val Leu Lys Arg Glu Leu Gly

530 535 540

Glu Gly Ala Phe Gly Lys Val Phe Leu Ala Glu Cys Tyr Asn Leu Cys

545 550 555 560

Pro Glu Gln Asp Lys Ile Leu Val Ala Val Lys Thr Leu Lys Asp Ala

565 570 575

Ser Asp Asn Ala Arg Lys Asp Phe His Arg Glu Ala Glu Leu Leu Thr

580 585 590

Asn Leu Gln His Glu His Ile Val Lys Phe Tyr Gly Val Cys Val Glu

595 600 605

Gly Asp Pro Leu Ile Met Val Phe Glu Tyr Met Lys His Gly Asp Leu

610 615 620

Asn Lys Phe Leu Arg Ala His Gly Pro Asp Ala Val Leu Met Ala Glu

625 630 635 640

Gly Asn Pro Pro Thr Glu Leu Thr Gln Ser Gln Met Leu His Ile Ala

645 650 655

Gln Gln Ile Ala Ala Gly Met Val Tyr Leu Ala Ser Gln His Phe Val

660 665 670

His Arg Asp Leu Ala Thr Arg Asn Cys Leu Val Gly Glu Asn Leu Leu

675 680 685

Val Lys Ile Gly Asp Phe Gly Met Ser Arg Asp Val Tyr Ser Thr Asp

690 695 700

Tyr Tyr Arg Val Gly Gly His Thr Met Leu Pro Ile Arg Trp Met Pro

705 710 715 720

Pro Glu Ser Ile Met Tyr Arg Lys Phe Thr Thr Glu Ser Asp Val Trp

725 730 735

Ser Leu Gly Val Val Leu Trp Glu Ile Phe Thr Tyr Gly Lys Gln Pro

740 745 750

Trp Tyr Gln Leu Ser Asn Asn Glu Val Ile Glu Cys Ile Thr Gln Gly

755 760 765

Arg Val Leu Gln Arg Pro Arg Thr Cys Pro Gln Glu Val Tyr Glu Leu

770 775 780

Met Leu Gly Cys Trp Gln Arg Glu Pro His Thr Arg Lys Asn Ile Lys

785 790 795 800

Asn Ile His Thr Leu Leu Gln Asn Leu Ala Lys Ala Ser Pro Val Tyr

805 810 815

Leu Asp Ile Leu Gly

820

<210> 3

<211> 838

<212> PRT

<213> Homo sapiens

<220>

<221> MISC_FEATURE

<222> (1)..(31)

<223> Signal peptide

<220>

<221> MISC_FEATURE

<222> (32)..(430)

<223> Extracellular domain

<220>

<221> MISC_FEATURE

<222> (431)..(454)

<223> Transmembrane domain

<220>

<221> MISC_FEATURE

<222> (455)..(838)

<223> Cytoplasmic domain

<400> 3

Met Ser Ser Trp Ile Arg Trp His Gly Pro Ala Met Ala Arg Leu Trp

1 5 10 15

Gly Phe Cys Trp Leu Val Val Gly Phe Trp Arg Ala Ala Phe Ala Cys

20 25 30

Pro Thr Ser Cys Lys Cys Ser Ala Ser Arg Ile Trp Cys Ser Asp Pro

35 40 45

Ser Pro Gly Ile Val Ala Phe Pro Arg Leu Glu Pro Asn Ser Val Asp

50 55 60

Pro Glu Asn Ile Thr Glu Ile Phe Ile Ala Asn Gln Lys Arg Leu Glu

65 70 75 80

Ile Ile Asn Glu Asp Asp Val Glu Ala Tyr Val Gly Leu Arg Asn Leu

85 90 95

Thr Ile Val Asp Ser Gly Leu Lys Phe Val Ala His Lys Ala Phe Leu

100 105 110

Lys Asn Ser Asn Leu Gln His Ile Asn Phe Thr Arg Asn Lys Leu Thr

115 120 125

Ser Leu Ser Arg Lys His Phe Arg His Leu Asp Leu Ser Glu Leu Ile

130 135 140

Leu Val Gly Asn Pro Phe Thr Cys Ser Cys Asp Ile Met Trp Ile Lys

145 150 155 160

Thr Leu Gln Glu Ala Lys Ser Ser Pro Asp Thr Gln Asp Leu Tyr Cys

165 170 175

Leu Asn Glu Ser Ser Lys Asn Ile Pro Leu Ala Asn Leu Gln Ile Pro

180 185 190

Asn Cys Gly Leu Pro Ser Ala Asn Leu Ala Ala Pro Asn Leu Thr Val

195 200 205

Glu Glu Gly Lys Ser Ile Thr Leu Ser Cys Ser Val Ala Gly Asp Pro

210 215 220

Val Pro Asn Met Tyr Trp Asp Val Gly Asn Leu Val Ser Lys His Met

225 230 235 240

Asn Glu Thr Ser His Thr Gln Gly Ser Leu Arg Ile Thr Asn Ile Ser

245 250 255

Ser Asp Asp Ser Gly Lys Gln Ile Ser Cys Val Ala Glu Asn Leu Val

260 265 270

Gly Glu Asp Gln Asp Ser Val Asn Leu Thr Val His Phe Ala Pro Thr

275 280 285

Ile Thr Phe Leu Glu Ser Pro Thr Ser Asp His His Trp Cys Ile Pro

290 295 300

Phe Thr Val Lys Gly Asn Pro Lys Pro Ala Leu Gln Trp Phe Tyr Asn

305 310 315 320

Gly Ala Ile Leu Asn Glu Ser Lys Tyr Ile Cys Thr Lys Ile His Val

325 330 335

Thr Asn His Thr Glu Tyr His Gly Cys Leu Gln Leu Asp Asn Pro Thr

340 345 350

His Met Asn Asn Gly Asp Tyr Thr Leu Ile Ala Lys Asn Glu Tyr Gly

355 360 365

Lys Asp Glu Lys Gln Ile Ser Ala His Phe Met Gly Trp Pro Gly Ile

370 375 380

Asp Asp Gly Ala Asn Pro Asn Tyr Pro Asp Val Ile Tyr Glu Asp Tyr

385 390 395 400

Gly Thr Ala Ala Asn Asp Ile Gly Asp Thr Thr Asn Arg Ser Asn Glu

405 410 415

Ile Pro Ser Thr Asp Val Thr Asp Lys Thr Gly Arg Glu His Leu Ser

420 425 430

Val Tyr Ala Val Val Val Ile Ala Ser Val Val Gly Phe Cys Leu Leu

435 440 445

Val Met Leu Phe Leu Leu Lys Leu Ala Arg His Ser Lys Phe Gly Met

450 455 460

Lys Asp Phe Ser Trp Phe Gly Phe Gly Lys Val Lys Ser Arg Gln Gly

465 470 475 480

Val Gly Pro Ala Ser Val Ile Ser Asn Asp Asp Asp Ser Ala Ser Pro

485 490 495

Leu His His Ile Ser Asn Gly Ser Asn Thr Pro Ser Ser Ser Glu Gly

500 505 510

Gly Pro Asp Ala Val Ile Ile Gly Met Thr Lys Ile Pro Val Ile Glu

515 520 525

Asn Pro Gln Tyr Phe Gly Ile Thr Asn Ser Gln Leu Lys Pro Asp Thr

530 535 540

Phe Val Gln His Ile Lys Arg His Asn Ile Val Leu Lys Arg Glu Leu

545 550 555 560

Gly Glu Gly Ala Phe Gly Lys Val Phe Leu Ala Glu Cys Tyr Asn Leu

565 570 575

Cys Pro Glu Gln Asp Lys Ile Leu Val Ala Val Lys Thr Leu Lys Asp

580 585 590

Ala Ser Asp Asn Ala Arg Lys Asp Phe His Arg Glu Ala Glu Leu Leu

595 600 605

Thr Asn Leu Gln His Glu His Ile Val Lys Phe Tyr Gly Val Cys Val

610 615 620

Glu Gly Asp Pro Leu Ile Met Val Phe Glu Tyr Met Lys His Gly Asp

625 630 635 640

Leu Asn Lys Phe Leu Arg Ala His Gly Pro Asp Ala Val Leu Met Ala

645 650 655

Glu Gly Asn Pro Thr Glu Leu Thr Gln Ser Gln Met Leu His Ile

660 665 670

Ala Gln Gln Ile Ala Ala Gly Met Val Tyr Leu Ala Ser Gln His Phe

675 680 685

Val His Arg Asp Leu Ala Thr Arg Asn Cys Leu Val Gly Glu Asn Leu

690 695 700

Leu Val Lys Ile Gly Asp Phe Gly Met Ser Arg Asp Val Tyr Ser Thr

705 710 715 720

Asp Tyr Tyr Arg Val Gly Gly His Thr Met Leu Pro Ile Arg Trp Met

725 730 735

Pro Pro Glu Ser Ile Met Tyr Arg Lys Phe Thr Thr Glu Ser Asp Val

740 745 750

Trp Ser Leu Gly Val Val Leu Trp Glu Ile Phe Thr Tyr Gly Lys Gln

755 760 765

Pro Trp Tyr Gln Leu Ser Asn Asn Glu Val Ile Glu Cys Ile Thr Gln

770 775 780

Gly Arg Val Leu Gln Arg Pro Arg Thr Cys Pro Gln Glu Val Tyr Glu

785 790 795 800

Leu Met Leu Gly Cys Trp Gln Arg Glu Pro His Met Arg Lys Asn Ile

805 810 815

Lys Gly Ile His Thr Leu Leu Gln Asn Leu Ala Lys Ala Ser Pro Val

820 825 830

Tyr Leu Asp Ile Leu Gly

835

<210> 4

<211> 822

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic

<220>

<221> MISC_FEATURE

<222> (1)..(31)

<223> Signal peptide

<220>

<221> MISC_FEATURE

<222> (1)..(31)

<223> Mouse sequence

<220>

<221> MISC_FEATURE

<222> (32)..(430)

<223> Extracellular domain

<220>

<221> MISC_FEATURE

<222> (32)..(432)

<223> Human Sequence

<220>

<221> MISC_FEATURE

<222> (431)..(454)

<223> Transmembrane domain

<220>

<221> MISC_FEATURE

<222> (433)..(822)

<223> Mouse sequence

<220>

<221> MISC_FEATURE

<222> (455)..(822)

<223> Cytoplasmic domain

<400> 4

Met Ser Pro Trp Leu Lys Trp His Gly Pro Ala Met Ala Arg Leu Trp

1 5 10 15

Gly Leu Cys Leu Leu Val Leu Gly Phe Trp Arg Ala Ser Leu Ala Cys

20 25 30

Pro Thr Ser Cys Lys Cys Ser Ala Ser Arg Ile Trp Cys Ser Asp Pro

35 40 45

Ser Pro Gly Ile Val Ala Phe Pro Arg Leu Glu Pro Asn Ser Val Asp

50 55 60

Pro Glu Asn Ile Thr Glu Ile Phe Ile Ala Asn Gln Lys Arg Leu Glu

65 70 75 80

Ile Ile Asn Glu Asp Asp Val Glu Ala Tyr Val Gly Leu Arg Asn Leu

85 90 95

Thr Ile Val Asp Ser Gly Leu Lys Phe Val Ala His Lys Ala Phe Leu

100 105 110

Lys Asn Ser Asn Leu Gln His Ile Asn Phe Thr Arg Asn Lys Leu Thr

115 120 125

Ser Leu Ser Arg Lys His Phe Arg His Leu Asp Leu Ser Glu Leu Ile

130 135 140

Leu Val Gly Asn Pro Phe Thr Cys Ser Cys Asp Ile Met Trp Ile Lys

145 150 155 160

Thr Leu Gln Glu Ala Lys Ser Ser Pro Asp Thr Gln Asp Leu Tyr Cys

165 170 175

Leu Asn Glu Ser Ser Lys Asn Ile Pro Leu Ala Asn Leu Gln Ile Pro

180 185 190

Asn Cys Gly Leu Pro Ser Ala Asn Leu Ala Ala Pro Asn Leu Thr Val

195 200 205

Glu Glu Gly Lys Ser Ile Thr Leu Ser Cys Ser Val Ala Gly Asp Pro

210 215 220

Val Pro Asn Met Tyr Trp Asp Val Gly Asn Leu Val Ser Lys His Met

225 230 235 240

Asn Glu Thr Ser His Thr Gln Gly Ser Leu Arg Ile Thr Asn Ile Ser

245 250 255

Ser Asp Asp Ser Gly Lys Gln Ile Ser Cys Val Ala Glu Asn Leu Val

260 265 270

Gly Glu Asp Gln Asp Ser Val Asn Leu Thr Val His Phe Ala Pro Thr

275 280 285

Ile Thr Phe Leu Glu Ser Pro Thr Ser Asp His His Trp Cys Ile Pro

290 295 300

Phe Thr Val Lys Gly Asn Pro Lys Pro Ala Leu Gln Trp Phe Tyr Asn

305 310 315 320

Gly Ala Ile Leu Asn Glu Ser Lys Tyr Ile Cys Thr Lys Ile His Val

325 330 335

Thr Asn His Thr Glu Tyr His Gly Cys Leu Gln Leu Asp Asn Pro Thr

340 345 350

His Met Asn Asn Gly Asp Tyr Thr Leu Ile Ala Lys Asn Glu Tyr Gly

355 360 365

Lys Asp Glu Lys Gln Ile Ser Ala His Phe Met Gly Trp Pro Gly Ile

370 375 380

Asp Asp Gly Ala Asn Pro Asn Tyr Pro Asp Val Ile Tyr Glu Asp Tyr

385 390 395 400

Gly Thr Ala Ala Asn Asp Ile Gly Asp Thr Thr Asn Arg Ser Asn Glu

405 410 415

Ile Pro Ser Thr Asp Val Thr Asp Lys Thr Gly Arg Glu His Leu Ser

420 425 430

Val Tyr Ala Val Val Val Ile Ala Ser Val Val Gly Phe Cys Leu Leu

435 440 445

Val Met Leu Leu Leu Leu Lys Leu Ala Arg His Ser Lys Phe Gly Met

450 455 460

Lys Gly Pro Ala Ser Val Ile Ser Asn Asp Asp Asp Ser Ala Ser Pro

465 470 475 480

Leu His His Ile Ser Asn Gly Ser Asn Thr Pro Ser Ser Ser Glu Gly

485 490 495

Gly Pro Asp Ala Val Ile Ile Gly Met Thr Lys Ile Pro Val Ile Glu

500 505 510

Asn Pro Gln Tyr Phe Gly Ile Thr Asn Ser Gln Leu Lys Pro Asp Thr

515 520 525

Phe Val Gln His Ile Lys Arg His Asn Ile Val Leu Lys Arg Glu Leu

530 535 540

Gly Glu Gly Ala Phe Gly Lys Val Phe Leu Ala Glu Cys Tyr Asn Leu

545 550 555 560

Cys Pro Glu Gln Asp Lys Ile Leu Val Ala Val Lys Thr Leu Lys Asp

565 570 575

Ala Ser Asp Asn Ala Arg Lys Asp Phe His Arg Glu Ala Glu Leu Leu

580 585 590

Thr Asn Leu Gln His Glu His Ile Val Lys Phe Tyr Gly Val Cys Val

595 600 605

Glu Gly Asp Pro Leu Ile Met Val Phe Glu Tyr Met Lys His Gly Asp

610 615 620

Leu Asn Lys Phe Leu Arg Ala His Gly Pro Asp Ala Val Leu Met Ala

625 630 635 640

Glu Gly Asn Pro Thr Glu Leu Thr Gln Ser Gln Met Leu His Ile

645 650 655

Ala Gln Gln Ile Ala Ala Gly Met Val Tyr Leu Ala Ser Gln His Phe

660 665 670

Val His Arg Asp Leu Ala Thr Arg Asn Cys Leu Val Gly Glu Asn Leu

675 680 685

Leu Val Lys Ile Gly Asp Phe Gly Met Ser Arg Asp Val Tyr Ser Thr

690 695 700

Asp Tyr Tyr Arg Val Gly Gly His Thr Met Leu Pro Ile Arg Trp Met

705 710 715 720

Pro Pro Glu Ser Ile Met Tyr Arg Lys Phe Thr Thr Glu Ser Asp Val

725 730 735

Trp Ser Leu Gly Val Val Leu Trp Glu Ile Phe Thr Tyr Gly Lys Gln

740 745 750

Pro Trp Tyr Gln Leu Ser Asn Asn Glu Val Ile Glu Cys Ile Thr Gln

755 760 765

Gly Arg Val Leu Gln Arg Pro Arg Thr Cys Pro Gln Glu Val Tyr Glu

770 775 780

Leu Met Leu Gly Cys Trp Gln Arg Glu Pro His Thr Arg Lys Asn Ile

785 790 795 800

Lys Ser Ile His Thr Leu Leu Gln Asn Leu Ala Lys Ala Ser Pro Val

805 810 815

Tyr Leu Asp Ile Leu Gly

820

<210> 5

<211> 822

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic

<220>

<221> MISC_FEATURE

<222> (1)..(31)

<223> Signal peptide

<220>

<221> MISC_FEATURE

<222> (1)..(31)

<223> Rat sequence

<220>

<221> MISC_FEATURE

<222> (32)..(430)

<223> Extracellular domain

<220>

<221> MISC_FEATURE

<222> (32)..(432)

<223> Human Sequence

<220>

<221> MISC_FEATURE

<222> (431)..(454)

<223> Transmembrane domain

<220>

<221> MISC_FEATURE

<222> (433)..(822)

<223> Rat sequence

<220>

<221> MISC_FEATURE

<222> (455)..(822)

<223> Cytoplasmic domain

<400> 5

Met Ser Pro Trp Pro Arg Trp His Gly Pro Ala Met Ala Arg Leu Trp

1 5 10 15

Gly Leu Cys Leu Leu Val Leu Gly Phe Trp Arg Ala Ser Leu Ala Cys

20 25 30

Pro Thr Ser Cys Lys Cys Ser Ala Ser Arg Ile Trp Cys Ser Asp Pro

35 40 45

Ser Pro Gly Ile Val Ala Phe Pro Arg Leu Glu Pro Asn Ser Val Asp

50 55 60

Pro Glu Asn Ile Thr Glu Ile Phe Ile Ala Asn Gln Lys Arg Leu Glu

65 70 75 80

Ile Ile Asn Glu Asp Asp Val Glu Ala Tyr Val Gly Leu Arg Asn Leu

85 90 95

Thr Ile Val Asp Ser Gly Leu Lys Phe Val Ala His Lys Ala Phe Leu

100 105 110

Lys Asn Ser Asn Leu Gln His Ile Asn Phe Thr Arg Asn Lys Leu Thr

115 120 125

Ser Leu Ser Arg Lys His Phe Arg His Leu Asp Leu Ser Glu Leu Ile

130 135 140

Leu Val Gly Asn Pro Phe Thr Cys Ser Cys Asp Ile Met Trp Ile Lys

145 150 155 160

Thr Leu Gln Glu Ala Lys Ser Ser Pro Asp Thr Gln Asp Leu Tyr Cys

165 170 175

Leu Asn Glu Ser Ser Lys Asn Ile Pro Leu Ala Asn Leu Gln Ile Pro

180 185 190

Asn Cys Gly Leu Pro Ser Ala Asn Leu Ala Ala Pro Asn Leu Thr Val

195 200 205

Glu Glu Gly Lys Ser Ile Thr Leu Ser Cys Ser Val Ala Gly Asp Pro

210 215 220

Val Pro Asn Met Tyr Trp Asp Val Gly Asn Leu Val Ser Lys His Met

225 230 235 240

Asn Glu Thr Ser His Thr Gln Gly Ser Leu Arg Ile Thr Asn Ile Ser

245 250 255

Ser Asp Asp Ser Gly Lys Gln Ile Ser Cys Val Ala Glu Asn Leu Val

260 265 270

Gly Glu Asp Gln Asp Ser Val Asn Leu Thr Val His Phe Ala Pro Thr

275 280 285

Ile Thr Phe Leu Glu Ser Pro Thr Ser Asp His His Trp Cys Ile Pro

290 295 300

Phe Thr Val Lys Gly Asn Pro Lys Pro Ala Leu Gln Trp Phe Tyr Asn

305 310 315 320

Gly Ala Ile Leu Asn Glu Ser Lys Tyr Ile Cys Thr Lys Ile His Val

325 330 335

Thr Asn His Thr Glu Tyr His Gly Cys Leu Gln Leu Asp Asn Pro Thr

340 345 350

His Met Asn Asn Gly Asp Tyr Thr Leu Ile Ala Lys Asn Glu Tyr Gly

355 360 365

Lys Asp Glu Lys Gln Ile Ser Ala His Phe Met Gly Trp Pro Gly Ile

370 375 380

Asp Asp Gly Ala Asn Pro Asn Tyr Pro Asp Val Ile Tyr Glu Asp Tyr

385 390 395 400

Gly Thr Ala Ala Asn Asp Ile Gly Asp Thr Thr Asn Arg Ser Asn Glu

405 410 415

Ile Pro Ser Thr Asp Val Thr Asp Lys Thr Gly Arg Glu His Leu Ser

420 425 430

Val Tyr Ala Val Val Val Ile Ala Ser Val Val Gly Phe Cys Leu Leu

435 440 445

Val Met Leu Leu Leu Leu Lys Leu Ala Arg His Ser Lys Phe Gly Met

450 455 460

Lys Gly Pro Ala Ser Val Ile Ser Asn Asp Asp Asp Ser Ala Ser Pro

465 470 475 480

Leu His His Ile Ser Asn Gly Ser Asn Thr Pro Ser Ser Ser Glu Gly

485 490 495

Gly Pro Asp Ala Val Ile Ile Gly Met Thr Lys Ile Pro Val Ile Glu

500 505 510

Asn Pro Gln Tyr Phe Gly Ile Thr Asn Ser Gln Leu Lys Pro Asp Thr

515 520 525

Phe Val Gln His Ile Lys Arg His Asn Ile Val Leu Lys Arg Glu Leu

530 535 540

Gly Glu Gly Ala Phe Gly Lys Val Phe Leu Ala Glu Cys Tyr Asn Leu

545 550 555 560

Cys Pro Glu Gln Asp Lys Ile Leu Val Ala Val Lys Thr Leu Lys Asp

565 570 575

Ala Ser Asp Asn Ala Arg Lys Asp Phe His Arg Glu Ala Glu Leu Leu

580 585 590

Thr Asn Leu Gln His Glu His Ile Val Lys Phe Tyr Gly Val Cys Val

595 600 605

Glu Gly Asp Pro Leu Ile Met Val Phe Glu Tyr Met Lys His Gly Asp

610 615 620

Leu Asn Lys Phe Leu Arg Ala His Gly Pro Asp Ala Val Leu Met Ala

625 630 635 640

Glu Gly Asn Pro Thr Glu Leu Thr Gln Ser Gln Met Leu His Ile

645 650 655

Ala Gln Gln Ile Ala Ala Gly Met Val Tyr Leu Ala Ser Gln His Phe

660 665 670

Val His Arg Asp Leu Ala Thr Arg Asn Cys Leu Val Gly Glu Asn Leu

675 680 685

Leu Val Lys Ile Gly Asp Phe Gly Met Ser Arg Asp Val Tyr Ser Thr

690 695 700

Asp Tyr Tyr Arg Val Gly Gly His Thr Met Leu Pro Ile Arg Trp Met

705 710 715 720

Pro Pro Glu Ser Ile Met Tyr Arg Lys Phe Thr Thr Glu Ser Asp Val

725 730 735

Trp Ser Leu Gly Val Val Leu Trp Glu Ile Phe Thr Tyr Gly Lys Gln

740 745 750

Pro Trp Tyr Gln Leu Ser Asn Asn Glu Val Ile Glu Cys Ile Thr Gln

755 760 765

Gly Arg Val Leu Gln Arg Pro Arg Thr Cys Pro Gln Glu Val Tyr Glu

770 775 780

Leu Met Leu Gly Cys Trp Gln Arg Glu Pro His Thr Arg Lys Asn Ile

785 790 795 800

Lys Asn Ile His Thr Leu Leu Gln Asn Leu Ala Lys Ala Ser Pro Val

805 810 815

Tyr Leu Asp Ile Leu Gly

820

<210> 6

<211> 8744

<212> DNA

<213> Mus musculus

<400> 6

gtctggaggg tgctatgcta tgcgtgtgtg cgtgtgtgtg 60

tgtgtgtttt tggatttcat actaattttc tggagtttct gcccctgctc tgcgtcagcc 120

ctcacgtcac ttcgccagca gtagcagagg cggcggcggc ggcggccgcc ggttagagcc 180

cagtcgctgc ttcagctgct gttgctgctt ctgcagcgct ctgctccctg cgcttgctac 240

gggaggccgg ggaagccgcg cggacagtcc tcggtggcct gggccggcac tgtcctgcta 300

ccgcagttgc tccccagccc tgaggtgcgc accgatatcg atattcgtgc cggtttagcg 360

gttctgcgac ccaaagagtc cagggagatc caccgagtgg tgcctggcgt ataggactat 420

gcagccgcct tgtggctcgg agcagcggcc cgcgatgtcc cagccactgt gaaccatttg 480

gtcagcgcca acctgctcag ccccagcacc gacaggctca gcctctggta cgctccactc 540

cgcgggaggc caccagcacc aagcagcaag agggcgcagg gaaggcctcc cccctccggc 600

gggggacgcc tggctcagcg tagggacacg cactccgact gactggcact ggcagctcgg 660

gatgtcgccc tggctgaagt ggcatggacc cgccatggcg cggctctggg gcttatgcct 720

gctggtcttg ggcttctgga gggcctctct cgcctgcccg acgtcctgca aatgcagttc 780

cgctaggatt tggtgtactg agccttctcc aggcatcgtg gcattcccga ggttggaacc 840

taacagcgtt gacccggaga acatcacgga aattctcatt gcaaaccaga aaaggctaga 900

aatcatcaat gaagatgacg ttgaagctta cgtggggctg agaaacctta caattgtgga 960

ttccggctta aagtttgtgg cttacaaagc gtttctgaaa aacagcaacc tgcggcacat 1020

aaatttcaca cgaaacaagc tgacgagttt gtccaggaga catttccgcc accttgactt 1080

gtctgacctg atcctgacgg gtaatccgtt cacgtgctcc tgcgacatca tgtggctcaa 1140

gactctccag gagactaaat ccagccccga cactcaggat ttgtactgcc tcaatgagag 1200

cagcaagaac atgcccctgg cgaacctgca gatacccaat tgtggtctgc catctgcacg 1260

tctggctgct cctaacctca ccgtggagga aggaaagtct gtgacccttt cctgcagtgt 1320

ggggggtgac ccactcccca ccttgtactg ggacgttggg aatttggttt ccaagcacat 1380

gaatgaaaca agccacacac agggctcctt aaggataacg aacatttcat ctgatgacag 1440

tggaaagcaa atctcttgtg tggcagaaaa ccttgtagga gaagatcaag attctgtgaa 1500

cctcactgtg cattttgcgc caactatcac gtttctcgag tctccaacct cagatcacca 1560

ctggtgcatt ccattcactg tgagaggcaa ccccaagcct gcgcttcagt ggttctacaa 1620

tggggccata ctgaatgagt ccaagtacat ctgtactaag atccacgtca ccaatcacac 1680

ggagtaccat ggctgcctcc agctggataa ccccactcat atgaataacg gagactacac 1740

cctgatggcc aagaacgagt atgggaagga tgagagacag atctccgctc acttcatggg 1800

ccggcctgga gtcgactacg agacaaaccc aaattaccct gaagtcctct atgaagactg 1860

gaccacgcca actgacattg gggatactac gaacaaaagt aatgaaatcc cctccacgga 1920

tgttgctgac caaagcaatc gggagcatct ctcggtctat gccgtggtgg tgattgcatc 1980

tgtggtggga ttctgcctgc tggtgatgtt gctcctgctc aagttggcga gacattccaa 2040

gtttggcatg aaaggcccag cttcggtcat cagcaacgac gatgactctg ccagccccct 2100

ccaccacatc tccaatggga gtaacactcc atcttcttcg gagggcggtc ccgacgctgt 2160

cattattgga atgaccaaga ttcctgttat tgaaaacccc cagtactttg gcatcaccaa 2220

cagtcagctc aagccagaca catttgttca gcatatcaag agacacaaca tcgttctgaa 2280

gagggaactt ggggaaggag ccttcgggaa agttttcctt gccgagtgct acaacctctg 2340

ccgagcag gataagatcc tggtggctgt gaagacgctg aaggacgcca gcgacaatgc 2400

2460

caagttctac ggtgtctgtg tggagggcga cccactcatc atggtctttg agtacatgaa 2520

gcacggggac ctcaacaagt tccttagggc acacgggccc gacgcagtgc tgatggcaga 2580

gggtaacccg cccacagagc tgacgcagtc gcagatgctg cacatcgctc agcaaatcgc 2640

agcaggtatg gtctacctgg cgtcccaaca ctttgtgcac cgtgacctgg ccacccggaa 2700

ctgcctggtg ggagagaacc tgctggtgaa aattggggac tttgggatgt cccgagatgt 2760

gtacagcacc gactactatc gggtcggtgg ccacacaatg ttgcccatcc gatggatgcc 2820

2880

tgtgttgtgg gagatcttca cctacggcaa gcagccctgg tatcagctat cgaacaatga 2940

ggtgatagag tgcatcaccc agggaagagt ccttcagcgg cctcgaacgt gtccccagga 3000

ggtgtatgag ctcatgcttg gatgctggca gcgggaacca cacacccggga agaacatcaa 3060

gagcatccac accctccttc agaacttggc caaggcatct cccgtctacc tggatatcct 3120

aggctagggt cctccttctg cccagaccgt ccttcccaag gccctcctca gactggccta 3180

cacgacgaac ctcttgactg ccgctgacgt catgaccttg ctgtccttcg ctctgacagt 3240

gttgacagga ccaggagcgg ctctttgggg gaggcagtgt gtgcttctcc atccacagac 3300

agtattaact cgcttctggc attgtctctt tctctccctt gggtttgttt ctttcttttg 3360

ccccttcccc ttttatcatt atttattcat ttatttattt tctggtcttc accgcttcac 3420

ggccctcagt ctctccttga ccaatctggc ttctgcattc ctattaactg tacatagaca 3480

3540

aacaatgcct tgttgtattc ctgcctttga cgtggatgaa aaaaagggaa aaaaaaatca 3600

aacatctgac ttaagctgtc acttccgatg tacagacgtg gggcgtttct atggattcac 3660

3720

3780

atgaaaacga gagcgagcaa gcgagcaaga gagggagaga gagagagaag cgttaccata 3840

aaccacggca tgagcgagac agagacaagc catgggatca gtcgggagtc cgttgtgctt 3900

aggaaaaccc agcagccatt agctggggga gcatgttcgg ctctgtcccc caagcacctt 3960

tctgaggagg acacaggatg ttgaactctg cttcacgggc agagcttcta atgacagata 4020

ctggcttgca ctggaaagac agttccccg ggacctggac ggacaacaca tcctacattc 4080

agacattgtg gtcgggcacg gtgacagagt tgatccgttt ctcaagtgtt atctaccaag 4140

cttttgtgaa gttccatcga aggaggtaga ttcttgctca gatataattt caggaaaacc 4200

4260

actttttcag acagcaactc cgccagccc ccatcttcca ctctcacctg tcttgtaact 4320

gtgcaaacaa aagtgtgcat ggtctttgtc aattgatacc tatgtgcacg tgtgcagaaa 4380

ctgttgttcc agctggggtg tctgattagg agggcagatc cataaaaggt ctaacctagg 4440

caacttcggg aaaggagacc agatcagtag ctggaggcac tctccagtag gcggtgaggg 4500

gtttactgag taggcatgct gaagccggga tattcaccca tctcaaaccc ccggggctgc 4560

aggacaggca caggccatcc ctgaggagaa ggggagccct tttgggatac cacctgaggt 4620

tatgttcagt gtgctctggt caagtccctt gctcggggct ctgtttgggg agagtggttt 4680

cattccaagg tactcattat tagtatgctg ttttgttaac tatactccat taaaaagtta 4740

aaaaaaaaaa gaattaagcc ttgacactgt atggctgaca ggaggctgtg cccagactga 4800

gcctggagat ttgcgcccgc acatggtcat tggttttccg aaaagagagg gtaaatttat 4860

atagaaattt acaggtattt gggtagtcat ttagaccgag ggagaccagt gtttccattt 4920

ctctgcgccc cctctgtgag gggaagttcg atacacttga cacctttata aacggagcca 4980

5040

gtgcaagagc tctgtgtagg tttcatttgt gcccgagcat ttctggagca ctgtgttttc 5100

tagcagaaac tccgagagcc agttctcaca atgaaacttt aaaacctgtg taaattgaca 5160

gagagcagaa ggcgatccaa gaggcccact caagtgagtg gtggcacgag gcacatgcgg 5220

tggccctttc tgttgtgctg gcccatgaga gatgggagct attttgtcct cttcgtccat 5280

taaaacaaac ccctccagaa tacctatagt aatataatga aagccatatc tctgtgatct 5340

ataccgtcag gtatagatca ttaaaggacc catggtactg tcaggcactg tggaaccgtg 5400

aggcagcgga aaggcaaggg cacatttgta catgttcctc tagcttccgc cagccgtgac 5460

ctatgaactc acataggccg gttgctccta gtctgacggg ctgccctggg aggaaaagct 5520

gcaagatgct cagcagagag caaagggag gatagtcttg gaaaaggcca aggttgggga 5580

tgccatacag gattacacca aagggctgta ctgagaatgt gggagcattc cattccaggt 5640

ttgtattttc ctcagcaaga aaaaagagca gatagaaggg cagaggagga tagagaggaa 5700

gagaaaggag agggagaaga gagcagagaa gagagagaga gagagagaga gagagaatct 5760

ttatactttt tggcaagtcc tcgaaggtct caaaatgaaa gtgtctatgc aagtgcaaat 5820

5880

ttgtctcaag aatatgtgca gatttcagag cattaaagag tatttggtttt cttttaaagt 5940

agttgggtga gccaaggaca ccttaaaata attgtcactg ttcatctgct atcgcctttc 6000

6060

agatgacaaa aaaaaatggt gttagcaaac cgaaacctcc cccgattcct gtctgacatc 6120

6180

6240

accgaagtct gtctgccaga tacaaactaa catacaaaca agacagccgg gaaggaaatg 6300

tgctggtagc gtgtctgtgg agcagggtgg gggttagaac agaaggagac ctgggcagtg 6360

cacgcagaag agatgttaca tcatgtgctt ctgctcctgg cttctgctga ctcccaactt 6420

tgcgctcagc ctcatttaga aaacgcttca aacactgctc ttcaggggttc tgtcagaact 6480

tatgtacttc tatataaata 6540

tatacataat atatgttata aacacttgat ttatacatat acaaatgcac acatgtagtg 6600

6660

tgcaatatga ataatttgct taatatttgc taagtaacca aaactttcca acgtcatgtg 6720

6780

gtgcagccct tggatggaag atgtgttgaa aaaaaaatca catcttccct gaaacaactg 6840

actgaaagcc atcctctaca ttgaatctgt tctctggctc tttgcaaagg agaaatgcag 6900

acaaatggtc tccttgtgtc ccagtcctgg gaaggagaag tgggttttct cttcttgcac 6960

atttgggccg tgctcacctt tcctggctct atgcccttta cactgggctc agagatgaag 7020

cagtctagac ccaagctgca gggctgtagg gaggcaaatg tcttcgaggc tgatagcaaa 7080

gcatccaaaa atctatagca agatgcgcag cttgaagtca ctcctcatcc acacggcaag 7140

caagagccag ggcatgcagt gctgtccatc tctaggactg ggcagtgggg tccccggtca 7200

agggcagtct ttccctaagc ctctcctttt gcaaatggca ctgccagcag tacctggcat 7260

taagtcacga aaaatttcta aaatggcagc acagacccgt gtgccctcat tgctctgccc 7320

ccacatgcct catcattaga gggctccagt tttcaaactt gactttccat ttggccctgt 7380

atcgtggtta aaggaaatct caacagaggg atggccaatt gacctaaatc cccagatgtc 7440

caagtttgca gtgaaagcca gttcttccca aatgtataac tggaatgtag ggactcaggt 7500

tgtgatactt catacaggaa tctcagatgt tattgtaaag aactgggttg ggagggattc 7560

atcactaagc caacaataat gcaggagggc gtggctgatc gttaagccaa tagtagtgca 7620

ggagggcgtg gctgctgtga gggctggaat ccatctctgc tgctaaagtt cagggttctc 7680

aactcttttc agctgttgca gcccttcact tctttccctt cggcgttatt tatttgtgta 7740

tttatttatt tatttattta tttatctatc tatctatcaa cttttggtcc attcatcacc 7800

aacataaagc aaaaacaatt ttttacatat atgtaaatgt gttgcaagtg aaacactgtg 7860

aatctcacaa ccacctccta gcaactatgc tgccatcttg acgtgcgtct caggagtcga 7920

gacgggaaaa gacgaggacg tcattttgca gcctacgcat tctgggccag gtccgtgttc 7980

gttttatttc tttagtctgt gagttaagaa aacaatccga gtggagggtg acggatgttc 8040

ggcaagttcg gtgaagcagc acgagctctt tgcagctata ggttcgagca agaagaaaaa 8100

ccatggggaga tggagaaggg aaatttgatg gaggggtggg gtggggtgggg ggctctgtct 8160

tacagctcgt gtagcttact gttgcttaaa aatgtataca acagctggaa atgttttcaa 8220

tgtgaatctt aaatatgtac tcccttaagg aatgaacata 8280

ttatggtgaa atattgctcc cccgcgtccc ctacaaatct gcctagagat gtggatacct 8340

acatttgctt aagcatcaga ccagtgtttg tatatacccg ggccatacac actcatctcg 8400

acgtctctct gcggatgtat ttccccttca ctggtgacct ggtatttaga actccgtctt 8460

ttcagttggt ttatttccct ttaatgtgat gtctctgtgc cgattattac cggttcttac 8520

ttgtttttgc aatccgtttt gaggtccagt gttttactga gactcattgc atcttggctg 8580

atttcaaagt gacacccgaa tacagtgttt aaaaaaaaag tttgtttgta aatcatgtga 8640

ccagcttctc tcaccctgac atggaatgtc tcttgtacta cagtgtattt aataataaaa 8700

gaaaaaaatg atgtcttaca ataaataaca tcttccacaa gagg 8744

<210> 7

<211> 4750

<212> DNA

<213> Rattus norvegicus

<400> 7

60 tgtgcgtgcg tgcgtgtgtg tgtgtgtgtg tgtgtgtgtg tgtgtgtgtg

120

tcagccctca cgtcacttcg ccagcagtag cagaggcggc ggcggccgcc ggttagagcc 180

cagtcgctgc ttcagctgct gttgctgctt ctgcggcgct ctgctccctg cgctggctac 240

gggaggccgg gggagccgcg ccgacagtcc tctgtggcca gggccggcac tgtcctgcta 300

ccgcagttgc tccccagccc tgaggtgcgc accgatatcg atatccgtgc cggtttagcg 360

gttctgcgac ccaaagagtc cagggaggc caccgagtgg cgcctggcgt ataggaccat 420

gcagccgcct tgtggcttgg agcagcggcc cgtgatgttc cagccactgt gaaccatttg 480

gtcagcgcca acctgctcag ccccagcacc gacaggctca gcctctggta cgctcctctc 540

ggcgggaggc catcagcacc aagcagcaag agggctcagg gaaggcctcc cccctccggc 600

gggggacgcc tggctcagcg tagggacacg cactctgact gactggcact ggcagctcgg 660

gatgtcgccc tggccgaggt ggcatggacc cgccatggcg cggctctggg gcttatgctt 720

gctggtcttg ggcttctgga gggcttctct tgcctgcccc atgtcctgca aatgcagcac 780

cactaggatt tggtgtaccg agccttctcc tggcatcgtg gcatttccga ggttggaacc 840

taacagcatt gacccagaga acatcaccga aattctcatt gcaaaccaga aaaggttaga 900

aatcatcaat gaagatgatg tcgaagctta cgtggggctg aaaaacctta caattgtgga 960

ttccggctta aagtttgtgg cttacaaggc gtttctgaag aacggcaacc tgcggcacat 1020

caatttcact cgaaacaagc tgacgagttt gtccaggaga catttccgcc accttgactt 1080

gtctgacctg atcctgacgg gtaatccgtt cacgtgttcc tgtgacatca tgtggctcaa 1140

gactctccag gagacgaaat ccagccccga cactcaggat ttgtattgcc tcaatgagag 1200

cagcaagaat acccctctgg cgaacctgca gattcccaat tgtggtctgc cgtctgcacg 1260

tctggccgct cctaacctca cggtggagga agggaagtct gtgaccattt cctgcagcgt 1320

cgggggtgac ccgctcccca ccttgtactg ggacgttggg aatttggttt ccaaacacat 1380

gaatgaaaca agccacacac agggctcctt aaggataaca aacatttcat cggatgacag 1440

tgggaaacaa atctcttgtg tggcagaaaa cctcgttgga gaagatcaag actctgtgaa 1500

cctcactgtg cattttgcac caaccatcac atttctcgaa tctccaacct cagaccacca 1560

ctggtgcatc ccattcactg tgagaggcaa ccccaagcca gcacttcagt ggttctacaa 1620

cggagccata ctgaatgaat ccaagtacat ctgtaccaaa atacacgtca ccaatcacac 1680

ggagtaccac ggctgcctcc agctggataa ccccactcat atgaataatg gagactacac 1740

cctaatggcc aagaatgaat atgggaagga cgagagacag atttctgctc acttcatggg 1800

ccggcctgga gttgactatg agacaaaccc aaattaccct gaagtcctct atgaagactg 1860

gaccacgcca actgacatcg gggatactac aaacaaaagt aatgagatcc cctccacgga 1920

tgttgctgac caaaccaatc gggagcatct ctcggtctat gccgtggtgg tgattgcctc 1980

tgtggtagga ttctgcctgc tggtgatgct gcttctgctc aagttggcga gacattccaa 2040

gtttggcatg aaaggcccag cttccgtcat cagcaacgac gatgactctg ccagccctct 2100

ccaccacatc tccaacggga gcaacactcc gtcttcttcg gagggcgggc ccgatgctgt 2160

catcattggg atgaccaaga tccctgtcat tgaaaacccc cagtacttcg gtatcaccaa 2220

cagccagctc aagccggaca catttgttca gcacatcaag agacacaaca tcgttctgaa 2280

gagggagctt ggagaaggag cctttgggaa agttttccta gcggagtgct ataacctctg 2340

ccccgagcag gataagatcc tggtggccgt gaagacgctg aaggacgcca gcgacaatgc 2400

2460

caagttctac ggtgtctgtg tggagggcga cccactcatc atggtctttg agtacatgaa 2520

gcacggggac ctcaacaagt tccttagggc acacggggcca gatgcagtgc tgatggcaga 2580

gggtaacccg cccaccgagc tgacgcagtc gcagatgctg cacatcgctc agcaaatcgc 2640

agcaggcatg gtctacctgg catcccaaca cttcgtgcac cgagacctgg ccacccggaa 2700

ctgcttggta ggagagaacc tgctggtgaa aattggggac ttcgggatgt cccgggatgt 2760

atacagcacc gactactacc gggttggtgg ccacacaatg ttgcccatcc gatggatgcc 2820

2880

tgtgttgtgg gagatcttca cctacggcaa gcagccctgg tatcagctat caaacaacga 2940

ggtgatagaa tgcatcaccc agggcagagt ccttcagcgg cctcgcacgt gtccccagga 3000

ggtgtacgag ctgatgctgg gatgctggca gcgggaacca cacacaagga agaacatcaa 3060

gaacatccac acactccttc agaacttggc gaaggcgtcg cccgtctacc tggacatcct 3120

aggctagact ccctcttctc ccagacggcc cttcccaagg cacccctcag acctcttaac 3180

tgccgctgat gtcaccacct tgctgtcctt cgctctgaca gtgttaacaa gacaaggagc 3240

ggctctccgg ggtgaggcag tgcgcacttc cccatccaca gacagtatcg actcgcttct 3300

3360

3420

tatttattta tttattggtc ttcactgctt catggtcctc ggcctctctc cttgaccgat 3480

3540

atatcagcag acactccagt ttgcccacca caactaacaa tgccttgttg tattcctgcc 3600

tttgatgtgg atgaaaaaaa gggaaaaaaa aataatcaaa catctgactt aaaccgtcac 3660

3720

ttatctattt atttatttct cttctttgtt gttttccggt ggttttagcc tgtgtatgag 3780

aagggaaagt catgtacagt ctgggaaaac tttatctgtg ggaaatggaa accagaaagg 3840

gaaagaagct ttaccataaa gcacagcagg agtgagacac agaaaagcca ttggatcagc 3900

cagagtccgt cctgcatagg aaaacccagc agccatcagg ctggaggatc atgttcggca 3960

ctgacccccg aggacctttc tgaggaggac acagaatgtt aaactctgca tcatggacac 4020

agtttccgat cacagatact ggccttcaat ggaaaaaaaa aaaaacccag atagttcttg 4080

tgagacctgg acagcacgtc caacatccag acattgtggt cgggcacagt gacagagttg 4140

atgcatttct cacgggttat tctacagagc ttttgtcaag tccaatggaa ggaggtagat 4200

tcttgttcag atatgatttc gggaaaaacc gagtccttga caaagacagg agacaccctc 4260

agttgggagg caagtttctc ttaccttggga ctttctcaca cagcaattct cacccccacc 4320

ccctccactc tcacctgtct tgtaactgtg caaacaaaag tgtgcatggt ctttgtcagt 4380

tgataccttt gtgcacctct gtgcagaaac tgctgtctgt cccggctgtg gtacccgatc 4440

agtggggtag atccacgaaa ggtctcattt taggccgctt tgggaaggta accagatcgg 4500

tagctggaag cactctccag taggtggcga agggtgagtg ggtctgctga agcctgcata 4560

tcttcaccca cctcaaaccc accgggctgc acaggggaca ggcacaggcc acccctgagg 4620

gacagggaag ctctcttggg ataccacctg agtttacatt cagtgtgctc aggtcaagtc 4680

tctcgctcgg ggctctgttt cggggagaat ggtttcattc caacgcactc attatcagga 4740

ttctgttttc 4750

<210> 8

<211> 4057

<212> DNA

<213> Homo sapiens

<400> 8

gggagcagga gcctcgctgg ctgcttcgct cgcgctctac gcgctcagtc cccggcggta 60

gcaggagcct ggacccaggc gccggcggcg ggcgtgaggc gccggagccc ggcctcgagg 120

tgcataccgg acccccattc gcatctaaca aggaatctgc gccccagaga gtcccggacg 180

ccgccggtcg gtgcccggcg cgccggggcca tgcagcgacg gccgccgcgg agctccgagc 240

agcggtagcg cccccctgta aagcggttcg ctatgccggg accactgtga accctgccgc 300

ctgccggaac actcttcgct ccggaccagc tcagcctctg ataagctgga ctcggcacgc 360

ccgcaacaag caccgaggag ttaagagagc cgcaagcgca gggaaggcct ccccgcacgg 420

gtgggggaaa gcggccggtg cagcgcgggg acaggcactc gggctggcac tggctgctag 480

ggatgtcgtc ctggataagg tggcatggac ccgccatggc gcggctctgg ggcttctgct 540

ggctggttgt gggcttctgg agggccgctt tcgcctgtcc cacgtcctgc aaatgcagtg 600

cctctcggat ctggtgcagc gacccttctc ctggcatcgt ggcatttccg agattggagc 660

ctaacagtgt agatcctgag aacatcaccg aaattttcat cgcaaaccag aaaaggttag 720

aaatcatcaa cgaagatgat gttgaagctt atgtgggact gagaaatctg acaattgtgg 780

attctggatt aaaatttgtg gctcataaag catttctgaa aaacagcaac ctgcagcaca 840

tcaattttac ccgaaacaaa ctgacgagtt tgtctaggaa acatttccgt caccttgact 900

tgtctgaact gatcctggtg ggcaatccat ttacatgctc ctgtgacatt atgtggatca 960

agactctcca agaggctaaa tccagtccag acactcagga tttgtactgc ctgaatgaaa 1020

gcagcaagaa tattcccctg gcaaacctgc agatacccaa ttgtggtttg ccatctgcaa 1080

atctggccgc acctaacctc actgtggagg aaggaaagtc tatcacatta tcctgtagtg 1140

tggcaggtga tccggttcct aatatgtatt gggatgttgg taacctggtt tccaaacata 1200

tgaatgaaac aagccacaca cagggctcct taaggataac taacatttca tccgatgaca 1260

gtgggaagca gatctcttgt gtggcggaaa atcttgtagg agaagatcaa gattctgtca 1320

acctcactgt gcattttgca ccaactatca catttctcga atctccaacc tcagaccacc 1380

actggtgcat tccattcact gtgaaaggca accccaaacc agcgcttcag tggttctata 1440

acggggcaat attgaatgag tccaaataca tctgtactaa aatacatgtt accaatcaca 1500

cggagtacca cggctgcctc cagctggata atcccactca catgaacaat ggggactaca 1560

ctctaatagc caagaatgag tatgggaagg atgagaaaca gatttctgct cacttcatgg 1620

gctggcctgg aattgacgat ggtgcaaacc caaattatcc tgatgtaatt tatgaagatt 1680

atggaactgc agcgaatgac atcggggaca ccacgaacag aagtaatgaa atcccttcca 1740

cagacgtcac tgataaaacc ggtcgggaac atctctcggt ctatgctgtg gtggtgattg 1800

1860

ccaagtttgg catgaaagat ttctcatggt ttggatttgg gaaagtaaaa tcaagacaag 1920

gtgttggccc agcctccgtt atcagcaatg atgatgactc tgccagccca ctccatcaca 1980

tctccaatgg gagtaacact ccatcttctt cggaaggtgg cccagatgct gtcattattg 2040

gaatgaccaa gatccctgtc attgaaaatc cccagtactt tggcatcacc aacagtcagc 2100

tcaagccaga cacatttgtt cagcacatca agcgacataa cattgttctg aaaagggagc 2160

taggcgaagg agccttttgga aaagtgttcc tagctgaatg ctataacctc tgtcctgagc 2220

aggacaagat cttggtggca gtgaagaccc tgaaggatgc cagtgacaat gcacgcaagg 2280

acttccaccg tgaggccgag ctcctgacca acctccagca tgagcacatc gtcaagttct 2340

atggcgtctg cgtggagggc gaccccctca tcatggtctt tgagtacatg aagcatgggg 2400

acctcaacaa gttcctcagg gcacacggcc ctgatgccgt gctgatggct gagggcaacc 2460

cgcccacgga actgacgcag tcgcagatgc tgcatatagc ccagcagatc gccgcgggca 2520

tggtctacct ggcgtcccag cacttcgtgc accgcgattt ggccaccagg aactgcctgg 2580

tcggggagaa cttgctggtg aaaatcgggg actttgggat gtcccgggac gtgtacagca 2640

ctgactacta cagggtcggt ggccacacaa tgctgcccat tcgctggatg cctccagaga 2700

gcatcatgta caggaaattc acgacggaaa gcgacgtctg gagcctgggg gtcgtgttgt 2760

gggagatttt cacctatggc aaacagccct ggtaccagct gtcaaacaat gaggtgatag 2820

agtgtatcac tcagggccga gtcctgcagc gaccccgcac gtgcccccag gaggtgtatg 2880

agctgatgct ggggtgctgg cagcgagagc cccacatgag gaagaacatc aagggcatcc 2940

ataccctcct tcagaacttg gccaaggcat ctccggtcta cctggacatt ctaggctagg 3000

3060 gcccttttcc ccagaccgat ccttcccaac

atcttttaac tgccgctgga ggccaccaag ctgctctcct tcactctgac agtattaaca 3120

tcaaagactc cgagaagctc tcgagggaag cagtgtgtac ttcttcatcc atagacacag 3180

3240

ttcttttttt aaatttttctt ttttcttcttt tttttcgtct tccctgcttc acgattctta 3300

ccctttcttt tgaatcaatc tggcttctgc attactatta actctgcata gacaaaggcc 3360

3420

3480

aaactttgtc acttctgctg tacagatatc gagagtttct atggattcac ttctatttat 3540

ttattattat tactgttctt attgtttttg gatggcttaa gcctgtgtat aaaaaagaaa 3600

3660

tttattatga accgcaatat gggaggaaca aagacaacca ctgggatcag ctggtgtcag 3720

tccctactta ggaaatactc agcaactgtt agctgggaag aatgtattcg gcaccttccc 3780

3840 ctgaggacct ttctgaggag taaaaagact

cccatcacca gaaatgatag cgtgcagtag agagcaaaga tggcttccgt gagacacaag 3900

atggcgcata gtgtgctcgg acacagtttt gtcttcgtag gttgtgatga tagcactggt 3960

ttgtttctca agcgctatcc acagaacctt tgtcaacttc agttgaaaag aggtggattc 4020

atgtccagag ctcatttcgg ggtcaggtgg gaaagcc 4057

<210> 9

<211> 2466

<212> DNA

<213> Mus musculus

<220>

<221> misc_feature

<222> (1)..(93)

<223> Signal peptide

<220>

<221> misc_feature

<222> (94)..(1287)

<223> Extracellular domain

<220>

<221> misc_feature

<222> (1288)..(1359)

<223> Transmembrane domain

<220>

<221> misc_feature

<222> (1360)..(2463)

<223> Cytoplasmic domain

<400> 9

atgtcgccct ggctgaagtg gcatggaccc gccatggcgc ggctctgggg cttatgcctg 60

ctggtcttgg gcttctggag ggcctctctc gcctgcccga cgtcctgcaa atgcagttcc 120

gctaggattt ggtgtactga gccttctcca ggcatcgtgg cattcccgag gttggaacct 180

aacagcgttg acccggagaa catcacggaa attctcattg caaaccagaa aaggctagaa 240

atcatcaatg aagatgacgt tgaagcttac gtggggctga gaaaccttac aattgtggat 300

360

aatttcacac gaaacaagct gacgagtttg tccaggagac atttccgcca ccttgacttg 420

tctgacctga tcctgacggg taatccgttc acgtgctcct gcgacatcat gtggctcaag 480

actctccagg agactaaatc cagccccgac actcaggatt tgtactgcct caatgagagc 540

agcaagaaca tgcccctggc gaacctgcag atacccaatt gtggtctgcc atctgcacgt 600

ctggctgctc ctaacctcac cgtggaggaa ggaaagtctg tgaccctttc ctgcagtgtg 660

gggggtgacc cactccccac cttgtactgg gacgttggga atttggtttc caagcacatg 720

aatgaaacaa gccacacaca gggctcctta aggataacga acatttcatc tgatgacagt 780

ggaaagcaaa tctcttgtgt ggcagaaaac cttgtaggag aagatcaaga ttctgtgaac 840

ctcactgtgc attttgcgcc aactatcacg tttctcgagt ctccaacctc agatcaccac 900

tggtgcattc cattcactgt gagaggcaac cccaagcctg cgcttcagtg gttctacaat 960

ggggccatac tgaatgagtc caagtacatc tgtactaaga tccacgtcac caatcacacg 1020

gagtaccatg gctgcctcca gctggataac cccactcata tgaataacgg agactacacc 1080

ctgatggcca agaacgagta tgggaaggat gagagacaga tctccgctca cttcatgggc 1140

cggcctggag tcgactacga gacaaaccca aattaccctg aagtcctcta tgaagactgg 1200

accacgccaa ctgacattgg ggatactacg aacaaaagta atgaaatccc ctccacggat 1260

gttgctgacc aaagcaatcg ggagcatctc tcggtctatg ccgtggtggt gattgcatct 1320

gtggtgggat tctgcctgct ggtgatgttg ctcctgctca agttggcgag acattccaag 1380

tttggcatga aaggcccagc ttcggtcatc agcaacgacg atgactctgc cagccccctc 1440

caccacatct ccaatgggag taacactcca tcttcttcgg agggcggtcc cgacgctgtc 1500

attattggaa tgaccaagat tcctgttatt gaaaaccccc agtactttgg catcaccaac 1560

agtcagctca agccagacac atttgttcag catatcaaga gacacaacat cgttctgaag 1620

agggaacttg gggaaggagc cttcgggaaa gttttccttg ccgagtgcta caacctctgc 1680

ccagagcagg ataagatcct ggtggctgtg aagacgctga aggacgccag cgacaatgca 1740

cgcaaggact ttcatcggga agctgagctg ctgaccaacc tccagcacga gcacattgtc 1800

aagttctacg gtgtctgtgt ggagggcgac ccactcatca tggtctttga gtacatgaag 1860

cacggggacc tcaacaagtt ccttagggca cacgggcccg acgcagtgct gatggcagag 1920

ggtaacccgc ccacagagct gacgcagtcg cagatgctgc acatcgctca gcaaatcgca 1980

gcaggtatgg tctacctggc gtcccaacac tttgtgcacc gtgacctggc cacccggaac 2040

tgcctggtgg gagagaacct gctggtgaaa attggggact ttgggatgtc ccgagatgtg 2100

tacagcaccg actactatcg ggtcggtggc cacacaatgt tgcccatccg atggatgcct 2160

ccagagagca tcatgtacag gaaattcacc accgagagcg acgtctggag cctgggcgtt 2220

gtgttgtggg agatcttcac ctacggcaag cagccctggt atcagctatc gaacaatgag 2280

gtgatagagt gcatcaccca gggaagagtc cttcagcggc ctcgaacgtg tccccaggag 2340

gtgtatgagc tcatgcttgg atgctggcag cgggaaccac acacccggaa gaacatcaag 2400

agcatccaca ccctccttca gaacttggcc aaggcatctc ccgtctacct ggatatccta 2460

ggctag 2466

<210> 10

<211> 2466

<212> DNA

<213> Rattus norvegicus

<220>

<221> misc_feature

<222> (1)..(93)

<223> Signal peptide

<220>

<221> misc_feature

<222> (94)..(1287)

<223> Extracellular domain

<220>

<221> misc_feature

<222> (1288)..(1359)

<223> Transmembrane domain

<220>

<221> misc_feature

<222> (1360)..(2463)

<223> Cytoplasmic domain

<400> 10

atgtcgccct ggccgaggtg gcatggaccc gccatggcgc ggctctgggg cttatgcttg 60

ctggtcttgg gcttctggag ggcttctctt gcctgcccca tgtcctgcaa atgcagcacc 120

actaggattt ggtgtaccga gccttctcct ggcatcgtgg catttccgag gttggaacct 180

aacagcattg acccagagaa catcaccgaa attctcattg caaaccagaa aaggttagaa 240

atcatcaatg aagatgatgt cgaagcttac gtggggctga aaaaccttac aattgtggat 300

tccggcttaa agtttgtggc ttacaaggcg tttctgaaga acggcaacct gcggcacatc 360

aatttcactc gaaacaagct gacgagtttg tccaggagac atttccgcca ccttgacttg 420

tctgacctga tcctgacggg taatccgttc acgtgttcct gtgacatcat gtggctcaag 480

actctccagg agacgaaatc cagccccgac actcaggatt tgtattgcct caatgagagc 540

agcaagaata cccctctggc gaacctgcag attcccaatt gtggtctgcc gtctgcacgt 600

ctggccgctc ctaacctcac ggtggaggaa gggaagtctg tgaccatttc ctgcagcgtc 660

gggggtgacc cgctccccac cttgtactgg gacgttggga atttggtttc caaacacatg 720

aatgaaacaa gccacacaca gggctcctta aggataacaa acatttcatc ggatgacagt 780

gggaaacaaa tctcttgtgt ggcagaaaac ctcgttggag aagatcaaga ctctgtgaac 840

ctcactgtgc attttgcacc aaccatcaca tttctcgaat ctccaacctc agaccaccac 900

tggtgcatcc cattcactgt gagaggcaac cccaagccag cacttcagtg gttctacaac 960

ggagccatac tgaatgaatc caagtacatc tgtaccaaaa tacacgtcac caatcacacg 1020

gagtaccacg gctgcctcca gctggataac cccactcata tgaataatgg agactacacc 1080

ctaatggcca agaatgaata tgggaaggac gagagacaga tttctgctca cttcatgggc 1140

cggcctggag ttgactatga gacaaaccca aattaccctg aagtcctcta tgaagactgg 1200

accacgccaa ctgacatcgg ggatactaca aacaaaagta atgagatccc ctccacggat 1260

gttgctgacc aaaccaatcg ggagcatctc tcggtctatg ccgtggtggt gattgcctct 1320

gtggtaggat tctgcctgct ggtgatgctg cttctgctca agttggcgag acattccaag 1380

tttggcatga aaggcccagc ttccgtcatc agcaacgacg atgactctgc cagccctctc 1440

caccacatct ccaacgggag caacactccg tcttcttcgg agggcgggcc cgatgctgtc 1500

atcattggga tgaccaagat ccctgtcatt gaaaaccccc agtacttcgg tatcaccaac 1560

agccagctca agccggacac atttgttcag cacatcaaga gacacaacat cgttctgaag 1620

agggagcttg gagaaggagc ctttgggaaa gttttcctag cggagtgcta taacctctgc 1680

cccgagcagg ataagatcct ggtggccgtg aagacgctga aggacgccag cgacaatgct 1740

cgcaaggact ttcatcgcga agccgagctg ctgaccaacc tccagcacga gcacattgtc 1800

aagttctacg gtgtctgtgt ggagggcgac ccactcatca tggtctttga gtacatgaag 1860

cacggggacc tcaacaagtt ccttagggca cacgggccag atgcagtgct gatggcagag 1920

ggtaacccgc ccaccgagct gacgcagtcg cagatgctgc acatcgctca gcaaatcgca 1980

gcaggcatgg tctacctggc atcccaacac ttcgtgcacc gagacctggc cacccggaac 2040

tgcttggtag gagagaacct gctggtgaaa attggggact tcgggatgtc ccgggatgta 2100

tacagcaccg actactaccg ggttggtggc cacacaatgt tgcccatccg atggatgcct 2160

ccagagagca tcatgtacag gaaattcacc accgagagtg acgtctggag cctgggagtt 2220

gtgttgtggg agatcttcac ctacggcaag cagccctggt atcagctatc aaacaacgag 2280

gtgatagaat gcatcaccca gggcagagtc cttcagcggc ctcgcacgtg tccccaggag 2340

gtgtacgagc tgatgctggg atgctggcag cgggaaccac acacaaggaa gaacatcaag 2400

aacatccaca cactccttca gaacttggcg aaggcgtcgc ccgtctacct ggacatccta 2460

ggctag 2466

<210> 11

<211> 2517

<212> DNA

<213> Homo sapiens

<220>

<221> misc_feature

<222> (1)..(93)

<223> Signal peptide

<220>

<221> misc_feature

<222> (94)..(1290)

<223> Extracellular domain

<220>

<221> misc_feature

<222> (1291)..(1362)

<223> Transmembrane domain

<220>

<221> misc_feature

<222> (1363)..(2514)

<223> Cytoplasmic domain

<400> 11

atgtcgtcct ggataaggtg gcatggaccc gccatggcgc ggctctgggg cttctgctgg 60

ctggttgtgg gcttctggag ggccgctttc gcctgtccca cgtcctgcaa atgcagtgcc 120

tctcggatct ggtgcagcga cccttctcct ggcatcgtgg catttccgag attggagcct 180

aacagtgtag atcctgagaa catcaccgaa attttcatcg caaaccagaa aaggttagaa 240

atcatcaacg aagatgatgt tgaagcttat gtgggactga gaaatctgac aattgtggat 300

tctggattaa aatttgtggc tcataaagca tttctgaaaa acagcaacct gcagcacatc 360

aattttaccc gaaacaaact gacgagtttg tctaggaaac atttccgtca ccttgacttg 420

tctgaactga tcctggtggg caatccattt acatgctcct gtgacattat gtggatcaag 480

actctccaag aggctaaatc cagtccagac actcaggatt tgtactgcct gaatgaaagc 540

agcaagaata ttcccctggc aaacctgcag atacccaatt gtggtttgcc atctgcaaat 600

ctggccgcac ctaacctcac tgtggaggaa ggaaagtcta tcacattatc ctgtagtgtg 660

gcaggtgatc cggttcctaa tatgtattgg gatgttggta acctggtttc caaacatatg 720

aatgaaacaa gccacacaca gggctcctta aggataacta acatttcatc cgatgacagt 780

gggaagcaga tctcttgtgt ggcggaaaat cttgtaggag aagatcaaga ttctgtcaac 840

ctcactgtgc attttgcacc aactatcaca tttctcgaat ctccaacctc agaccaccac 900

tggtgcattc cattcactgt gaaaggcaac cccaaaccag cgcttcagtg gttctataac 960

ggggcaatat tgaatgagtc caaatacatc tgtactaaaa tacatgttac caatcacacg 1020

gagtaccacg gctgcctcca gctggataat cccactcaca tgaacaatgg ggactacact 1080

ctaatagcca agaatgagta tgggaaggat gagaaacaga tttctgctca cttcatgggc 1140

tggcctggaa ttgacgatgg tgcaaaccca aattatcctg atgtaattta tgaagattat 1200

ggaactgcag cgaatgacat cggggacacc acgaacagaa gtaatgaaat cccttccaca 1260

gacgtcactg ataaaaccgg tcgggaacat ctctcggtct atgctgtggt ggtgattgcg 1320

1380

1440

gttggcccag cctccgttat cagcaatgat gatgactctg ccagcccact ccatcacatc 1500

tccaatggga gtaacactcc atcttcttcg gaaggtggcc cagatgctgt cattattgga 1560

atgaccaaga tccctgtcat tgaaaatccc cagtactttg gcatcaccaa cagtcagctc 1620

aagccagaca catttgttca gcacatcaag cgacataaca ttgttctgaa aagggagcta 1680

ggcgaaggag cctttggaaa agtgttccta gctgaatgct ataacctctg tcctgagcag 1740

gacaagatct tggtggcagt gaagaccctg aaggatgcca gtgacaatgc acgcaaggac 1800

ttccaccgtg aggccgagct cctgaccaac ctccagcatg agcacatcgt caagttctat 1860

ggcgtctgcg tggagggcga ccccctcatc atggtctttg agtacatgaa gcatggggac 1920

ctcaacaagt tcctcagggc acacggccct gatgccgtgc tgatggctga gggcaacccg 1980

cccacggaac tgacgcagtc gcagatgctg catatagccc agcagatcgc cgcgggcatg 2040

gtctacctgg cgtcccagca cttcgtgcac cgcgatttgg ccaccaggaa ctgcctggtc 2100

ggggagaact tgctggtgaa aatcggggac tttgggatgt cccgggacgt gtacagcact 2160

gactactaca gggtcggtgg ccacacaatg ctgcccattc gctggatgcc tccagagagc 2220

atcatgtaca ggaaattcac gacggaaagc gacgtctgga gcctgggggt cgtgttgtgg 2280

2340

2400

ctgatgctgg ggtgctggca gcgagagccc cacatgagga agaacatcaa gggcatccat 2460

accctccttc agaacttggc caaggcatct ccggtctacc tggacattct aggctag 2517

<210> 12

<211> 2469

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic

<220>

<221> misc_feature

<222> (1)..(93)

<223> Signal peptide

<220>

<221> misc_feature

<222> (1)..(93)

<223> Mouse sequence

<220>

<221> misc_feature

<222> (94)..(1290)

<223> Extracellular domain

<220>

<221> misc_feature

<222> (94)..(1296)

<223> Human Sequence

<220>

<221> misc_feature

<222> (1291)..(1362)

<223> Transmembrane domain

<220>

<221> misc_feature

<222> (1297)..(2466)

<223> Mouse sequence

<220>

<221> misc_feature

<222> (1363)..(2466)

<223> Cytoplasmic domain

<400> 12

atgtcgccct ggctgaagtg gcatggaccc gccatggcgc ggctctgggg cttatgcctg 60

ctggtcttgg gcttctggag ggcctctctc gcctgtccca cgtcctgcaa atgcagtgcc 120

tctcggatct ggtgcagcga cccttctcct ggcatcgtgg catttccgag attggagcct 180

aacagtgtag atcctgagaa catcaccgaa attttcatcg caaaccagaa aaggttagaa 240

atcatcaacg aagatgatgt tgaagcttat gtgggactga gaaatctgac aattgtggat 300

tctggattaa aatttgtggc tcataaagca tttctgaaaa acagcaacct gcagcacatc 360

aattttaccc gaaacaaact gacgagtttg tctaggaaac atttccgtca ccttgacttg 420

tctgaactga tcctggtggg caatccattt acatgctcct gtgacattat gtggatcaag 480

actctccaag aggctaaatc cagtccagac actcaggatt tgtactgcct gaatgaaagc 540

agcaagaata ttcccctggc aaacctgcag atacccaatt gtggtttgcc atctgcaaat 600

ctggccgcac ctaacctcac tgtggaggaa ggaaagtcta tcacattatc ctgtagtgtg 660

gcaggtgatc cggttcctaa tatgtattgg gatgttggta acctggtttc caaacatatg 720

aatgaaacaa gccacacaca gggctcctta aggataacta acatttcatc cgatgacagt 780

gggaagcaga tctcttgtgt ggcggaaaat cttgtaggag aagatcaaga ttctgtcaac 840

ctcactgtgc attttgcacc aactatcaca tttctcgaat ctccaacctc agaccaccac 900

tggtgcattc cattcactgt gaaaggcaac cccaaaccag cgcttcagtg gttctataac 960

ggggcaatat tgaatgagtc caaatacatc tgtactaaaa tacatgttac caatcacacg 1020

gagtaccacg gctgcctcca gctggataat cccactcaca tgaacaatgg ggactacact 1080

ctaatagcca agaatgagta tgggaaggat gagaaacaga tttctgctca cttcatgggc 1140

tggcctggaa ttgacgatgg tgcaaaccca aattatcctg atgtaattta tgaagattat 1200

ggaactgcag cgaatgacat cggggacacc acgaacagaa gtaatgaaat cccttccaca 1260

gacgtcactg ataaaaccgg tcgggaacat ctctcggtct atgccgtggt ggtgattgca 1320

tctgtggtgg gattctgcct gctggtgatg ttgctcctgc tcaagttggc gagacattcc 1380

aagtttggca tgaaaggccc agcttcggtc atcagcaacg acgatgactc tgccagcccc 1440

ctccaccaca tctccaatgg gagtaacact ccatcttctt cggagggcgg tcccgacgct 1500

gtcattattg gaatgaccaa gattcctgtt attgaaaacc cccagtactt tggcatcacc 1560

aacagtcagc tcaagccaga cacatttgtt cagcatatca agagacacaa catcgttctg 1620

aagagggaac ttggggaagg agccttcggg aaagttttcc ttgccgagtg ctacaacctc 1680

tgccgagc aggataagat cctggtggct gtgaagacgc tgaaggacgc cagcgacaat 1740

gcacgcaagg actttcatcg ggaagctgag ctgctgacca acctccagca cgagcacatt 1800

gtcaagttct acggtgtctg tgtggagggc gacccactca tcatggtctt tgagtacatg 1860

aagcacgggg acctcaacaa gttccttagg gcacacgggc ccgacgcagt gctgatggca 1920

gagggtaacc cgcccacaga gctgacgcag tcgcagatgc tgcacatcgc tcagcaaatc 1980

gcagcaggta tggtctacct ggcgtcccaa cactttgtgc accgtgacct ggccacccgg 2040

aactgcctgg tgggagagaa cctgctggtg aaaattgggg actttgggat gtcccgagat 2100

gtgtacagca ccgactacta tcgggtcggt ggccacacaa tgttgcccat ccgatggatg 2160

cctccagaga gcatcatgta caggaaattc accaccgaga gcgacgtctg gagcctgggc 2220

gttgtgttgt gggagatctt cacctacggc aagcagccct ggtatcagct atcgaacaat 2280

gaggtgatag agtgcatcac ccagggaaga gtccttcagc ggcctcgaac gtgtccccag 2340

gaggtgtatg agctcatgct tggatgctgg cagcgggaac cacacacccg gaagaacatc 2400

aagagcatcc acaccctcct tcagaacttg gccaaggcat ctcccgtcta cctggatatc 2460

ctaggctag 2469

<210> 13

<211> 2469

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic

<220>

<221> misc_feature

<222> (1)..(93)

<223> Signal peptide

<220>

<221> misc_feature

<222> (1)..(93)

<223> Rat sequence

<220>

<221> misc_feature

<222> (94)..(1290)

<223> Extracellular domain

<220>

<221> misc_feature

<222> (94)..(1296)

<223> Human Sequence

<220>

<221> misc_feature

<222> (1291)..(1362)

<223> Transmembrane domain

<220>

<221> misc_feature

<222> (1297)..(2466)

<223> Rat sequence

<220>

<221> misc_feature

<222> (1363)..(2466)

<223> Cytoplasmic domain

<400> 13

atgtcgccct ggccgaggtg gcatggaccc gccatggcgc ggctctgggg cttatgcttg 60

ctggtcttgg gcttctggag ggcttctctt gcctgtccca cgtcctgcaa atgcagtgcc 120

tctcggatct ggtgcagcga cccttctcct ggcatcgtgg catttccgag attggagcct 180

aacagtgtag atcctgagaa catcaccgaa attttcatcg caaaccagaa aaggttagaa 240

atcatcaacg aagatgatgt tgaagcttat gtgggactga gaaatctgac aattgtggat 300

tctggattaa aatttgtggc tcataaagca tttctgaaaa acagcaacct gcagcacatc 360

aattttaccc gaaacaaact gacgagtttg tctaggaaac atttccgtca ccttgacttg 420

tctgaactga tcctggtggg caatccattt acatgctcct gtgacattat gtggatcaag 480

actctccaag aggctaaatc cagtccagac actcaggatt tgtactgcct gaatgaaagc 540

agcaagaata ttcccctggc aaacctgcag atacccaatt gtggtttgcc atctgcaaat 600

ctggccgcac ctaacctcac tgtggaggaa ggaaagtcta tcacattatc ctgtagtgtg 660

gcaggtgatc cggttcctaa tatgtattgg gatgttggta acctggtttc caaacatatg 720

aatgaaacaa gccacacaca gggctcctta aggataacta acatttcatc cgatgacagt 780

gggaagcaga tctcttgtgt ggcggaaaat cttgtaggag aagatcaaga ttctgtcaac 840

ctcactgtgc attttgcacc aactatcaca tttctcgaat ctccaacctc agaccaccac 900

tggtgcattc cattcactgt gaaaggcaac cccaaaccag cgcttcagtg gttctataac 960

ggggcaatat tgaatgagtc caaatacatc tgtactaaaa tacatgttac caatcacacg 1020

gagtaccacg gctgcctcca gctggataat cccactcaca tgaacaatgg ggactacact 1080

ctaatagcca agaatgagta tgggaaggat gagaaacaga tttctgctca cttcatgggc 1140

tggcctggaa ttgacgatgg tgcaaaccca aattatcctg atgtaattta tgaagattat 1200

ggaactgcag cgaatgacat cggggacacc acgaacagaa gtaatgaaat cccttccaca 1260

gacgtcactg ataaaaccgg tcgggaacat ctctcggtct atgccgtggt ggtgattgcc 1320

tctgtggtag gattctgcct gctggtgatg ctgcttctgc tcaagttggc gagacattcc 1380

aagtttggca tgaaaggccc agcttccgtc atcagcaacg acgatgactc tgccagccct 1440

ctccaccaca tctccaacgg gagcaacact ccgtcttctt cggagggcgg gcccgatgct 1500

gtcatcattg ggatgaccaa gatccctgtc attgaaaacc cccagtactt cggtatcacc 1560

aacagccagc tcaagccgga cacatttgtt cagcacatca agagacacaa catcgttctg 1620

aagagggagc ttggagaagg agcctttggg aaagttttcc tagcggagtg ctataacctc 1680

tgccccgagc aggataagat cctggtggcc gtgaagacgc tgaaggacgc cagcgacaat 1740

gctcgcaagg actttcatcg cgaagccgag ctgctgacca acctccagca cgagcacatt 1800

gtcaagttct acggtgtctg tgtggagggc gacccactca tcatggtctt tgagtacatg 1860

aagcacgggg acctcaacaa gttccttagg gcacacggggc cagatgcagt gctgatggca 1920

gagggtaacc cgcccaccga gctgacgcag tcgcagatgc tgcacatcgc tcagcaaatc 1980

gcagcaggca tggtctacct ggcatcccaa cacttcgtgc accgagacct ggccacccgg 2040

aactgcttgg taggagagaa cctgctggtg aaaattgggg acttcgggat gtcccgggat 2100

gtatacagca ccgactacta ccgggttggt ggccacacaa tgttgcccat ccgatggatg 2160

cctccagaga gcatcatgta caggaaattc accaccgaga gtgacgtctg gagcctggga 2220

gttgtgttgt gggagatctt cacctacggc aagcagccct ggtatcagct atcaaacaac 2280

gaggtgatag aatgcatcac ccagggcaga gtccttcagc ggcctcgcac gtgtccccag 2340

gaggtgtacg agctgatgct gggatgctgg cagcgggaac cacacacaag gaagaacatc 2400

aagaacatcc acacactcct tcagaacttg gcgaaggcgt cgcccgtcta cctggacatc 2460

ctaggctag 2469

<210> 14

<211> 22

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic

<400> 14

aggtgggtag gtcctggaag tg 22

<210> 15

<211> 20

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic

<400> 15

aatgctgtcc caagagtggg 20

<210> 16

<211> 21

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic

<400> 16

gtcctgcatc ccttgtcttt g 21

<210> 17

<211> 21

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic

<400> 17

atgtgggcgt tgtgcagtct c 21

<210> 18

<211> 24

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic

<400> 18

cgctgcagtg cattgaactc agca 24

<210> 19

<211> 20

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic

<400> 19

ctgtggaggg acgtgaccag 20

<210> 20

<211> 22

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic

<400> 20

tccgctagga tttggtgtac tg 22

<210> 21

<211> 24

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic

<400> 21

agccttctcc aggcatcgtg gcat 24

<210> 22

<211> 19

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic

<400> 22

tccgggtcaa cgctgttag 19

<210> 23

<211> 18

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic

<400> 23

tcctgcgagg gttctgac 18

<210> 24

<211> 29

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic

<400> 24

tgggtgctca tatgccagag aaattgtca 29

<210> 25

<211> 22

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic

<400> 25

cgatctgtga tggcctgctt ac 22

<210> 26

<211> 22

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic

<400> 26

gggctcaggc aggtatatgt tg 22

<210> 27

<211> 27

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic

<400> 27

acagatgctg tcccaaacat agcaaga 27

<210> 28

<211> 23

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic

<400> 28

ccaaccctaa gccagtgaaa cag 23

<210> 29

<211> 20

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic

<400> 29

gtgctggaga ccaggagact 20

<210> 30

<211> 28

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic

<400> 30

tgccatactc agtttatacg gtgctgac 28

<210> 31

<211> 20

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic

<400> 31

gcctggtggc tcagtcaatg 20

<210> 32

<211> 19

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic

<400> 32

gcagacactg gatgggtca 19

<210> 33

<211> 26

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic

<400> 33

ccattcgcga gttatgagaa gctgca 26

<210> 34

<211> 22

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic

<400> 34

acagggttag ctggtgaatg ga 22

<210> 35

<211> 19

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic

<400> 35

tcggagcaca ggactacag 19

<210> 36

<211> 25

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic

<400> 36

caagaggaac tgtgtccagg aaagc 25

<210> 37

<211> 22

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic

<400> 37

agcgtgcctc acctaacctc ta 22

<210> 38

<211> 19

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic

<400> 38

gcacagcact gtaaaggca 19

<210> 39

<211> 29

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic

<400> 39

acggaactcg aaggaattgg tattgttgt 29

<210> 40

<211> 23

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic

<400> 40

acacagctat gggagaaaga ctg 23

<210> 41

<211> 23

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic

<400> 41

ccggttggcg agtgcgcatg cac 23

<210> 42

<211> 23

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic

<400> 42

ccttccggtt ggcgagtgcg cat 23

<210> 43

<211> 23

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic

<400> 43

ccaagggtgc gttgatggat cta 23

<210> 44

<211> 23

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic

<400> 44

gaatgccaag ggtgcgttga tgg 23

<210> 45

<211> 23

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic

<400> 45

ctgggtgatt gggactgaga aag 23

<210> 46

<211> 24

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic

<400> 46

cagccttgaa agtatggctt gggc 24

<210> 47

<211> 18

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic

<400> 47

gcactcgcca accggaag 18

<210> 48

<211> 23

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic

<400> 48

gaccagctca cccttactta tgg 23

<210> 49

<211> 23

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic

<400> 49

actgaatgcc aagggtgcgt tga 23

<210> 50

<211> 23

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic

<400> 50

tcttggaaat ccgctgaaga gtt 23

<210> 51

<211> 31

<212> PRT

<213> Mus musculus

<400> 51

Met Ser Pro Trp Leu Lys Trp His Gly Pro Ala Met Ala Arg Leu Trp

1 5 10 15

Gly Leu Cys Leu Leu Val Leu Gly Phe Trp Arg Ala Ser Leu Ala

20 25 30

<210> 52

<211> 398

<212> PRT

<213> Mus musculus

<400> 52

Cys Pro Thr Ser Cys Lys Cys Ser Ser Ala Arg Ile Trp Cys Thr Glu

1 5 10 15

Pro Ser Pro Gly Ile Val Ala Phe Pro Arg Leu Glu Pro Asn Ser Val

20 25 30

Asp Pro Glu Asn Ile Thr Glu Ile Leu Ile Ala Asn Gln Lys Arg Leu

35 40 45

Glu Ile Ile Asn Glu Asp Asp Val Glu Ala Tyr Val Gly Leu Arg Asn

50 55 60

Leu Thr Ile Val Asp Ser Gly Leu Lys Phe Val Ala Tyr Lys Ala Phe

65 70 75 80

Leu Lys Asn Ser Asn Leu Arg His Ile Asn Phe Thr Arg Asn Lys Leu

85 90 95

Thr Ser Leu Ser Arg Arg His Phe Arg His Leu Asp Leu Ser Asp Leu

100 105 110

Ile Leu Thr Gly Asn Pro Phe Thr Cys Ser Cys Asp Ile Met Trp Leu

115 120 125

Lys Thr Leu Gln Glu Thr Lys Ser Ser Pro Asp Thr Gln Asp Leu Tyr

130 135 140

Cys Leu Asn Glu Ser Ser Lys Asn Met Pro Leu Ala Asn Leu Gln Ile

145 150 155 160

Pro Asn Cys Gly Leu Pro Ser Ala Arg Leu Ala Ala Pro Asn Leu Thr

165 170 175

Val Glu Glu Gly Lys Ser Val Thr Leu Ser Cys Ser Val Gly Gly Asp

180 185 190

Pro Leu Pro Thr Leu Tyr Trp Asp Val Gly Asn Leu Val Ser Lys His

195 200 205

Met Asn Glu Thr Ser His Thr Gln Gly Ser Leu Arg Ile Thr Asn Ile

210 215 220

Ser Ser Asp Asp Ser Gly Lys Gln Ile Ser Cys Val Ala Glu Asn Leu

225 230 235 240

Val Gly Glu Asp Gln Asp Ser Val Asn Leu Thr Val His Phe Ala Pro

245 250 255

Thr Ile Thr Phe Leu Glu Ser Pro Thr Ser Asp His His Trp Cys Ile

260 265 270

Pro Phe Thr Val Arg Gly Asn Pro Lys Pro Ala Leu Gln Trp Phe Tyr

275 280 285

Asn Gly Ala Ile Leu Asn Glu Ser Lys Tyr Ile Cys Thr Lys Ile His

290 295 300

Val Thr Asn His Thr Glu Tyr His Gly Cys Leu Gln Leu Asp Asn Pro

305 310 315 320

Thr His Met Asn Asn Gly Asp Tyr Thr Leu Met Ala Lys Asn Glu Tyr

325 330 335

Gly Lys Asp Glu Arg Gln Ile Ser Ala His Phe Met Gly Arg Pro Gly

340 345 350

Val Asp Tyr Glu Thr Asn Pro Asn Tyr Pro Glu Val Leu Tyr Glu Asp

355 360 365

Trp Thr Thr Pro Thr Asp Ile Gly Asp Thr Thr Asn Lys Ser Asn Glu

370 375 380

Ile Pro Ser Thr Asp Val Ala Asp Gln Ser Asn Arg Glu His

385 390 395

<210> 53

<211> 24

<212> PRT

<213> Mus musculus

<400> 53

Leu Ser Val Tyr Ala Val Val Val Ile Ala Ser Val Val Gly Phe Cys

1 5 10 15

Leu Leu Val Met Leu Leu Leu Leu

20

<210> 54

<211> 368

<212> PRT

<213> Mus musculus

<400> 54

Lys Leu Ala Arg His Ser Lys Phe Gly Met Lys Gly Pro Ala Ser Val

1 5 10 15

Ile Ser Asn Asp Asp Asp Ser Ala Ser Pro Leu His His Ile Ser Asn

20 25 30

Gly Ser Asn Thr Pro Ser Ser Ser Glu Gly Gly Pro Asp Ala Val Ile

35 40 45

Ile Gly Met Thr Lys Ile Pro Val Ile Glu Asn Pro Gln Tyr Phe Gly

50 55 60

Ile Thr Asn Ser Gln Leu Lys Pro Asp Thr Phe Val Gln His Ile Lys

65 70 75 80

Arg His Asn Ile Val Leu Lys Arg Glu Leu Gly Glu Gly Ala Phe Gly

85 90 95

Lys Val Phe Leu Ala Glu Cys Tyr Asn Leu Cys Pro Glu Gln Asp Lys

100 105 110

Ile Leu Val Ala Val Lys Thr Leu Lys Asp Ala Ser Asp Asn Ala Arg

115 120 125

Lys Asp Phe His Arg Glu Ala Glu Leu Leu Thr Asn Leu Gln His Glu

130 135 140

His Ile Val Lys Phe Tyr Gly Val Cys Val Glu Gly Asp Pro Leu Ile

145 150 155 160

Met Val Phe Glu Tyr Met Lys His Gly Asp Leu Asn Lys Phe Leu Arg

165 170 175

Ala His Gly Pro Asp Ala Val Leu Met Ala Glu Gly Asn Pro Pro Thr

180 185 190

Glu Leu Thr Gln Ser Gln Met Leu His Ile Ala Gln Gln Ile Ala Ala

195 200 205

Gly Met Val Tyr Leu Ala Ser Gln His Phe Val His Arg Asp Leu Ala

210 215 220

Thr Arg Asn Cys Leu Val Gly Glu Asn Leu Leu Val Lys Ile Gly Asp

225 230 235 240

Phe Gly Met Ser Arg Asp Val Tyr Ser Thr Asp Tyr Tyr Arg Val Gly

245 250 255

Gly His Thr Met Leu Pro Ile Arg Trp Met Pro Pro Glu Ser Ile Met

260 265 270

Tyr Arg Lys Phe Thr Thr Glu Ser Asp Val Trp Ser Leu Gly Val Val

275 280 285

Leu Trp Glu Ile Phe Thr Tyr Gly Lys Gln Pro Trp Tyr Gln Leu Ser

290 295 300

Asn Asn Glu Val Ile Glu Cys Ile Thr Gln Gly Arg Val Leu Gln Arg

305 310 315 320

Pro Arg Thr Cys Pro Gln Glu Val Tyr Glu Leu Met Leu Gly Cys Trp

325 330 335

Gln Arg Glu Pro His Thr Arg Lys Asn Ile Lys Ser Ile His Thr Leu

340 345 350

Leu Gln Asn Leu Ala Lys Ala Ser Pro Val Tyr Leu Asp Ile Leu Gly

355 360 365

<210> 55

<211> 31

<212> PRT

<213> Rattus norvegicus

<400> 55

Met Ser Pro Trp Pro Arg Trp His Gly Pro Ala Met Ala Arg Leu Trp

1 5 10 15

Gly Leu Cys Leu Leu Val Leu Gly Phe Trp Arg Ala Ser Leu Ala

20 25 30

<210> 56

<211> 398

<212> PRT

<213> Rattus norvegicus

<400> 56

Cys Pro Met Ser Cys Lys Cys Ser Thr Thr Arg Ile Trp Cys Thr Glu

1 5 10 15

Pro Ser Pro Gly Ile Val Ala Phe Pro Arg Leu Glu Pro Asn Ser Ile

20 25 30

Asp Pro Glu Asn Ile Thr Glu Ile Leu Ile Ala Asn Gln Lys Arg Leu

35 40 45

Glu Ile Ile Asn Glu Asp Asp Val Glu Ala Tyr Val Gly Leu Lys Asn

50 55 60

Leu Thr Ile Val Asp Ser Gly Leu Lys Phe Val Ala Tyr Lys Ala Phe

65 70 75 80

Leu Lys Asn Gly Asn Leu Arg His Ile Asn Phe Thr Arg Asn Lys Leu

85 90 95

Thr Ser Leu Ser Arg Arg His Phe Arg His Leu Asp Leu Ser Asp Leu

100 105 110

Ile Leu Thr Gly Asn Pro Phe Thr Cys Ser Cys Asp Ile Met Trp Leu

115 120 125

Lys Thr Leu Gln Glu Thr Lys Ser Ser Pro Asp Thr Gln Asp Leu Tyr

130 135 140

Cys Leu Asn Glu Ser Ser Lys Asn Thr Pro Leu Ala Asn Leu Gln Ile

145 150 155 160

Pro Asn Cys Gly Leu Pro Ser Ala Arg Leu Ala Ala Pro Asn Leu Thr

165 170 175

Val Glu Glu Gly Lys Ser Val Thr Ile Ser Cys Ser Val Gly Gly Asp

180 185 190

Pro Leu Pro Thr Leu Tyr Trp Asp Val Gly Asn Leu Val Ser Lys His

195 200 205

Met Asn Glu Thr Ser His Thr Gln Gly Ser Leu Arg Ile Thr Asn Ile

210 215 220

Ser Ser Asp Asp Ser Gly Lys Gln Ile Ser Cys Val Ala Glu Asn Leu

225 230 235 240

Val Gly Glu Asp Gln Asp Ser Val Asn Leu Thr Val His Phe Ala Pro

245 250 255

Thr Ile Thr Phe Leu Glu Ser Pro Thr Ser Asp His His Trp Cys Ile

260 265 270

Pro Phe Thr Val Arg Gly Asn Pro Lys Pro Ala Leu Gln Trp Phe Tyr

275 280 285

Asn Gly Ala Ile Leu Asn Glu Ser Lys Tyr Ile Cys Thr Lys Ile His

290 295 300

Val Thr Asn His Thr Glu Tyr His Gly Cys Leu Gln Leu Asp Asn Pro

305 310 315 320

Thr His Met Asn Asn Gly Asp Tyr Thr Leu Met Ala Lys Asn Glu Tyr

325 330 335

Gly Lys Asp Glu Arg Gln Ile Ser Ala His Phe Met Gly Arg Pro Gly

340 345 350

Val Asp Tyr Glu Thr Asn Pro Asn Tyr Pro Glu Val Leu Tyr Glu Asp

355 360 365

Trp Thr Thr Pro Thr Asp Ile Gly Asp Thr Thr Asn Lys Ser Asn Glu

370 375 380

Ile Pro Ser Thr Asp Val Ala Asp Gln Thr Asn Arg Glu His

385 390 395

<210> 57

<211> 24

<212> PRT

<213> Rattus norvegicus

<400> 57

Leu Ser Val Tyr Ala Val Val Val Ile Ala Ser Val Val Gly Phe Cys

1 5 10 15

Leu Leu Val Met Leu Leu Leu Leu

20

<210> 58

<211> 368

<212> PRT

<213> Rattus norvegicus

<400> 58

Lys Leu Ala Arg His Ser Lys Phe Gly Met Lys Gly Pro Ala Ser Val

1 5 10 15

Ile Ser Asn Asp Asp Asp Ser Ala Ser Pro Leu His His Ile Ser Asn

20 25 30

Gly Ser Asn Thr Pro Ser Ser Ser Glu Gly Gly Pro Asp Ala Val Ile

35 40 45

Ile Gly Met Thr Lys Ile Pro Val Ile Glu Asn Pro Gln Tyr Phe Gly

50 55 60

Ile Thr Asn Ser Gln Leu Lys Pro Asp Thr Phe Val Gln His Ile Lys

65 70 75 80

Arg His Asn Ile Val Leu Lys Arg Glu Leu Gly Glu Gly Ala Phe Gly

85 90 95

Lys Val Phe Leu Ala Glu Cys Tyr Asn Leu Cys Pro Glu Gln Asp Lys

100 105 110

Ile Leu Val Ala Val Lys Thr Leu Lys Asp Ala Ser Asp Asn Ala Arg

115 120 125

Lys Asp Phe His Arg Glu Ala Glu Leu Leu Thr Asn Leu Gln His Glu

130 135 140

His Ile Val Lys Phe Tyr Gly Val Cys Val Glu Gly Asp Pro Leu Ile

145 150 155 160

Met Val Phe Glu Tyr Met Lys His Gly Asp Leu Asn Lys Phe Leu Arg

165 170 175

Ala His Gly Pro Asp Ala Val Leu Met Ala Glu Gly Asn Pro Pro Thr

180 185 190

Glu Leu Thr Gln Ser Gln Met Leu His Ile Ala Gln Gln Ile Ala Ala

195 200 205

Gly Met Val Tyr Leu Ala Ser Gln His Phe Val His Arg Asp Leu Ala

210 215 220

Thr Arg Asn Cys Leu Val Gly Glu Asn Leu Leu Val Lys Ile Gly Asp

225 230 235 240

Phe Gly Met Ser Arg Asp Val Tyr Ser Thr Asp Tyr Tyr Arg Val Gly

245 250 255

Gly His Thr Met Leu Pro Ile Arg Trp Met Pro Pro Glu Ser Ile Met

260 265 270

Tyr Arg Lys Phe Thr Thr Glu Ser Asp Val Trp Ser Leu Gly Val Val

275 280 285

Leu Trp Glu Ile Phe Thr Tyr Gly Lys Gln Pro Trp Tyr Gln Leu Ser

290 295 300

Asn Asn Glu Val Ile Glu Cys Ile Thr Gln Gly Arg Val Leu Gln Arg

305 310 315 320

Pro Arg Thr Cys Pro Gln Glu Val Tyr Glu Leu Met Leu Gly Cys Trp

325 330 335

Gln Arg Glu Pro His Thr Arg Lys Asn Ile Lys Asn Ile His Thr Leu

340 345 350

Leu Gln Asn Leu Ala Lys Ala Ser Pro Val Tyr Leu Asp Ile Leu Gly

355 360 365

<210> 59

<211> 31

<212> PRT

<213> Homo sapiens

<400> 59

Met Ser Ser Trp Ile Arg Trp His Gly Pro Ala Met Ala Arg Leu Trp

1 5 10 15

Gly Phe Cys Trp Leu Val Val Gly Phe Trp Arg Ala Ala Phe Ala

20 25 30

<210> 60

<211> 399

<212> PRT

<213> Homo sapiens

<400> 60

Cys Pro Thr Ser Cys Lys Cys Ser Ala Ser Arg Ile Trp Cys Ser Asp

1 5 10 15

Pro Ser Pro Gly Ile Val Ala Phe Pro Arg Leu Glu Pro Asn Ser Val

20 25 30

Asp Pro Glu Asn Ile Thr Glu Ile Phe Ile Ala Asn Gln Lys Arg Leu

35 40 45

Glu Ile Ile Asn Glu Asp Asp Val Glu Ala Tyr Val Gly Leu Arg Asn

50 55 60

Leu Thr Ile Val Asp Ser Gly Leu Lys Phe Val Ala His Lys Ala Phe

65 70 75 80

Leu Lys Asn Ser Asn Leu Gln His Ile Asn Phe Thr Arg Asn Lys Leu

85 90 95

Thr Ser Leu Ser Arg Lys His Phe Arg His Leu Asp Leu Ser Glu Leu

100 105 110

Ile Leu Val Gly Asn Pro Phe Thr Cys Ser Cys Asp Ile Met Trp Ile

115 120 125

Lys Thr Leu Gln Glu Ala Lys Ser Ser Pro Asp Thr Gln Asp Leu Tyr

130 135 140

Cys Leu Asn Glu Ser Ser Lys Asn Ile Pro Leu Ala Asn Leu Gln Ile

145 150 155 160

Pro Asn Cys Gly Leu Pro Ser Ala Asn Leu Ala Ala Pro Asn Leu Thr

165 170 175

Val Glu Glu Gly Lys Ser Ile Thr Leu Ser Cys Ser Val Ala Gly Asp

180 185 190

Pro Val Pro Asn Met Tyr Trp Asp Val Gly Asn Leu Val Ser Lys His

195 200 205

Met Asn Glu Thr Ser His Thr Gln Gly Ser Leu Arg Ile Thr Asn Ile

210 215 220

Ser Ser Asp Asp Ser Gly Lys Gln Ile Ser Cys Val Ala Glu Asn Leu

225 230 235 240

Val Gly Glu Asp Gln Asp Ser Val Asn Leu Thr Val His Phe Ala Pro

245 250 255

Thr Ile Thr Phe Leu Glu Ser Pro Thr Ser Asp His His Trp Cys Ile

260 265 270

Pro Phe Thr Val Lys Gly Asn Pro Lys Pro Ala Leu Gln Trp Phe Tyr

275 280 285

Asn Gly Ala Ile Leu Asn Glu Ser Lys Tyr Ile Cys Thr Lys Ile His

290 295 300

Val Thr Asn His Thr Glu Tyr His Gly Cys Leu Gln Leu Asp Asn Pro

305 310 315 320

Thr His Met Asn Asn Gly Asp Tyr Thr Leu Ile Ala Lys Asn Glu Tyr

325 330 335

Gly Lys Asp Glu Lys Gln Ile Ser Ala His Phe Met Gly Trp Pro Gly

340 345 350

Ile Asp Asp Gly Ala Asn Pro Asn Tyr Pro Asp Val Ile Tyr Glu Asp

355 360 365

Tyr Gly Thr Ala Ala Asn Asp Ile Gly Asp Thr Thr Asn Arg Ser Asn

370 375 380

Glu Ile Pro Ser Thr Asp Val Thr Asp Lys Thr Gly Arg Glu His

385 390 395

<210> 61

<211> 24

<212> PRT

<213> Homo sapiens

<400> 61

Leu Ser Val Tyr Ala Val Val Val Ile Ala Ser Val Val Gly Phe Cys

1 5 10 15

Leu Leu Val Met Leu Phe Leu Leu

20

<210> 62

<211> 384

<212> PRT

<213> Homo sapiens

<400> 62

Lys Leu Ala Arg His Ser Lys Phe Gly Met Lys Asp Phe Ser Trp Phe

1 5 10 15

Gly Phe Gly Lys Val Lys Ser Arg Gln Gly Val Gly Pro Ala Ser Val

20 25 30

Ile Ser Asn Asp Asp Asp Ser Ala Ser Pro Leu His His Ile Ser Asn

35 40 45

Gly Ser Asn Thr Pro Ser Ser Ser Glu Gly Gly Pro Asp Ala Val Ile

50 55 60

Ile Gly Met Thr Lys Ile Pro Val Ile Glu Asn Pro Gln Tyr Phe Gly

65 70 75 80

Ile Thr Asn Ser Gln Leu Lys Pro Asp Thr Phe Val Gln His Ile Lys

85 90 95

Arg His Asn Ile Val Leu Lys Arg Glu Leu Gly Glu Gly Ala Phe Gly

100 105 110

Lys Val Phe Leu Ala Glu Cys Tyr Asn Leu Cys Pro Glu Gln Asp Lys

115 120 125

Ile Leu Val Ala Val Lys Thr Leu Lys Asp Ala Ser Asp Asn Ala Arg

130 135 140

Lys Asp Phe His Arg Glu Ala Glu Leu Leu Thr Asn Leu Gln His Glu

145 150 155 160

His Ile Val Lys Phe Tyr Gly Val Cys Val Glu Gly Asp Pro Leu Ile

165 170 175

Met Val Phe Glu Tyr Met Lys His Gly Asp Leu Asn Lys Phe Leu Arg

180 185 190

Ala His Gly Pro Asp Ala Val Leu Met Ala Glu Gly Asn Pro Pro Thr

195 200 205

Glu Leu Thr Gln Ser Gln Met Leu His Ile Ala Gln Gln Ile Ala Ala

210 215 220

Gly Met Val Tyr Leu Ala Ser Gln His Phe Val His Arg Asp Leu Ala

225 230 235 240

Thr Arg Asn Cys Leu Val Gly Glu Asn Leu Leu Val Lys Ile Gly Asp

245 250 255

Phe Gly Met Ser Arg Asp Val Tyr Ser Thr Asp Tyr Tyr Arg Val Gly

260 265 270

Gly His Thr Met Leu Pro Ile Arg Trp Met Pro Pro Glu Ser Ile Met

275 280 285

Tyr Arg Lys Phe Thr Thr Glu Ser Asp Val Trp Ser Leu Gly Val Val

290 295 300

Leu Trp Glu Ile Phe Thr Tyr Gly Lys Gln Pro Trp Tyr Gln Leu Ser

305 310 315 320

Asn Asn Glu Val Ile Glu Cys Ile Thr Gln Gly Arg Val Leu Gln Arg

325 330 335

Pro Arg Thr Cys Pro Gln Glu Val Tyr Glu Leu Met Leu Gly Cys Trp

340 345 350

Gln Arg Glu Pro His Met Arg Lys Asn Ile Lys Gly Ile His Thr Leu

355 360 365

Leu Gln Asn Leu Ala Lys Ala Ser Pro Val Tyr Leu Asp Ile Leu Gly

370 375 380

<210> 63

<211> 93

<212> DNA

<213> Mus musculus

<400> 63

atgtcgccct ggctgaagtg gcatggaccc gccatggcgc ggctctgggg cttatgcctg 60

ctggtcttgg gcttctggag ggcctctctc gcc 93

<210> 64

<211> 1194

<212> DNA

<213> Mus musculus

<400> 64

tgcccgacgt cctgcaaatg cagttccgct aggatttggt gtactgagcc ttctccaggc 60

atcgtggcat tcccgaggtt ggaacctaac agcgttgacc cggagaacat cacggaaatt 120

ctcattgcaa accagaaaag gctagaaatc atcaatgaag atgacgttga agcttacgtg 180

gggctgagaa accttacaat tgtggattcc ggcttaaagt ttgtggctta caaagcgttt 240

ctgaaaaaca gcaacctgcg gcacataaat ttcacacgaa acaagctgac gagtttgtcc 300

aggagacatt tccgccacct tgacttgtct gacctgatcc tgacgggtaa tccgttcacg 360

tgctcctgcg acatcatgtg gctcaagact ctccaggaga ctaaatccag ccccgacact 420

caggatttgt actgcctcaa tgagagcagc aagaacatgc ccctggcgaa cctgcagata 480

cccaattgtg gtctgccatc tgcacgtctg gctgctccta acctcaccgt ggaggaagga 540

aagtctgtga ccctttcctg cagtgtgggg ggtgacccac tccccacctt gtactgggac 600

gttgggaatt tggtttccaa gcacatgaat gaaacaagcc acacacaggg ctccttaagg 660

ataacgaaca tttcatctga tgacagtgga aagcaaatct cttgtgtggc agaaaacctt 720

gtaggagaag atcaagattc tgtgaacctc actgtgcatt ttgcgccaac tatcacgttt 780

ctcgagtctc caacctcaga tcaccactgg tgcattccat tcactgtgag aggcaacccc 840

aagcctgcgc ttcagtggtt ctacaatggg gccatactga atgagtccaa gtacatctgt 900

actaagatcc acgtcaccaa tcacacggag taccatggct gcctccagct ggataacccc 960

actcatatga ataacggaga ctacaccctg atggccaaga acgagtatgg gaaggatgag 1020

agacagatct ccgctcactt catgggccgg cctggagtcg actacgagac aaacccaaat 1080

taccctgaag tcctctatga agactggacc acgccaactg acattgggga tactacgaac 1140

aaaagtaatg aaatcccctc cacggatgtt gctgaccaaa gcaatcggga gcat 1194

<210> 65

<211> 72

<212> DNA

<213> Mus musculus

<400> 65

ctctcggtct atgccgtggt ggtgattgca tctgtggtgg gattctgcct gctggtgatg 60

ttgctcctgc tc 72

<210> 66

<211> 1104

<212> DNA

<213> Mus musculus

<400> 66

aagttggcga gacattccaa gtttggcatg aaaggcccag cttcggtcat cagcaacgac 60

gatgactctg ccagccccct ccaccacatc tccaatggga gtaacactcc atcttcttcg 120

gagggcggtc ccgacgctgt cattattgga atgaccaaga ttcctgttat tgaaaacccc 180

cagtactttg gcatcaccaa cagtcagctc aagccagaca catttgttca gcatatcaag 240

agacacaaca tcgttctgaa gagggaactt ggggaaggag ccttcgggaa agttttcctt 300

gccgagtgct acaacctctg ccgagcag gataagatcc tggtggctgt gaagacgctg 360

aaggacgcca gcgacaatgc acgcaaggac tttcatcggg aagctgagct gctgaccaac 420

ctccagcacg agcacattgt caagttctac ggtgtctgtg tggagggcga cccactcatc 480

atggtctttg agtacatgaa gcacggggac ctcaacaagt tccttagggc acacgggccc 540

gacgcagtgc tgatggcaga gggtaacccg cccacagagc tgacgcagtc gcagatgctg 600

cacatcgctc agcaaatcgc agcaggtatg gtctacctgg cgtcccaaca ctttgtgcac 660

cgtgacctgg ccacccggaa ctgcctggtg ggagagaacc tgctggtgaa aattggggac 720

tttgggatgt cccgagatgt gtacagcacc gactactatc gggtcggtgg ccacacaatg 780

ttgcccatcc gatggatgcc tccagagagc atcatgtaca ggaaattcac caccgagagc 840

gacgtctgga gcctgggcgt tgtgttgtgg gagatcttca cctacggcaa gcagccctgg 900

tatcagctat cgaacaatga ggtgatagag tgcatcaccc agggaagagt ccttcagcgg 960

cctcgaacgt gtccccagga ggtgtatgag ctcatgcttg gatgctggca gcgggaacca 1020

cacacccggga agaacatcaa gagcatccac accctccttc agaacttggc caaggcatct 1080

cccgtctacc tggatatcct aggc 1104

<210> 67

<211> 93

<212> DNA

<213> Rattus norvegicus

<400> 67

atgtcgccct ggccgaggtg gcatggaccc gccatggcgc ggctctgggg cttatgcttg 60

ctggtcttgg gcttctggag ggcttctctt gcc 93

<210> 68

<211> 1194

<212> DNA

<213> Rattus norvegicus

<400> 68

tgccccatgt cctgcaaatg cagcaccact aggatttggt gtaccgagcc ttctcctggc 60

atcgtggcat ttccgaggtt ggaacctaac agcattgacc cagagaacat caccgaaatt 120

ctcattgcaa accagaaaag gttagaaatc atcaatgaag atgatgtcga agcttacgtg 180

gggctgaaaa accttacaat tgtggattcc ggcttaaagt ttgtggctta caaggcgttt 240

ctgaagaacg gcaacctgcg gcacatcaat ttcactcgaa acaagctgac gagtttgtcc 300

aggagacatt tccgccacct tgacttgtct gacctgatcc tgacgggtaa tccgttcacg 360

tgttcctgtg acatcatgtg gctcaagact ctccaggaga cgaaatccag ccccgacact 420

caggatttgt attgcctcaa tgagagcagc aagaataccc ctctggcgaa cctgcagatt 480

cccaattgtg gtctgccgtc tgcacgtctg gccgctccta acctcacggt ggaggaaggg 540

aagtctgtga ccatttcctg cagcgtcggg ggtgacccgc tccccacctt gtactgggac 600

660

ataacaaaca tttcatcgga tgacagtggg aaacaaatct cttgtgtggc agaaaacctc 720

gttggagaag atcaagactc tgtgaacctc actgtgcatt ttgcaccaac catcacattt 780

ctcgaatctc caacctcaga ccaccactgg tgcatcccat tcactgtgag aggcaacccc 840

aagccagcac ttcagtggtt ctacaacgga gccatactga atgaatccaa gtacatctgt 900

accaaaatac acgtcaccaa tcacacggag taccacggct gcctccagct ggataacccc 960

actcatatga ataatggaga ctacacccta atggccaaga atgaatatgg gaaggacgag 1020

agacagattt ctgctcactt catgggccgg cctggagttg actatgagac aaacccaaat 1080

taccctgaag tcctctatga agactggacc acgccaactg acatcgggga tactacaaac 1140

aaaagtaatg agatcccctc cacggatgtt gctgaccaaa ccaatcggga gcat 1194

<210> 69

<211> 72

<212> DNA

<213> Rattus norvegicus

<400> 69

ctctcggtct atgccgtggt ggtgattgcc tctgtggtag gattctgcct gctggtgatg 60

ctgcttctgc tc 72

<210> 70

<211> 1104

<212> DNA

<213> Rattus norvegicus

<400> 70

aagttggcga gacattccaa gtttggcatg aaaggcccag cttccgtcat cagcaacgac 60

gatgactctg ccagccctct ccaccacatc tccaacggga gcaacactcc gtcttcttcg 120

gagggcgggc ccgatgctgt catcattggg atgaccaaga tccctgtcat tgaaaacccc 180

cagtacttcg gtatcaccaa cagccagctc aagccggaca catttgttca gcacatcaag 240

agacacaaca tcgttctgaa gagggagctt ggagaaggag cctttgggaa agttttccta 300

gcggagtgct ataacctctg ccccgagcag gataagatcc tggtggccgt gaagacgctg 360

aaggacgcca gcgacaatgc tcgcaaggac tttcatcgcg aagccgagct gctgaccaac 420

ctccagcacg agcacattgt caagttctac ggtgtctgtg tggagggcga cccactcatc 480

atggtctttg agtacatgaa gcacggggac ctcaacaagt tccttagggc acacggggcca 540

gatgcagtgc tgatggcaga gggtaacccg cccaccgagc tgacgcagtc gcagatgctg 600

cacatcgctc agcaaatcgc agcaggcatg gtctacctgg catcccaaca cttcgtgcac 660

cgagacctgg ccacccggaa ctgcttggta ggagagaacc tgctggtgaa aattggggac 720

ttcgggatgt cccgggatgt atacagcacc gactactacc gggttggtgg ccacacaatg 780

ttgcccatcc gatggatgcc tccagagagc atcatgtaca ggaaattcac caccgagagt 840

gacgtctgga gcctgggagt tgtgttgtgg gagatcttca cctacggcaa gcagccctgg 900

tatcagctat caaacaacga ggtgatagaa tgcatcaccc agggcagagt ccttcagcgg 960

cctcgcacgt gtccccagga ggtgtacgag ctgatgctgg gatgctggca gcgggaacca 1020

cacacaagga agaacatcaa gaacatccac acactccttc agaacttggc gaaggcgtcg 1080

cccgtctacc tggacatcct aggc 1104

<210> 71

<211> 93

<212> DNA

<213> Homo sapiens

<400> 71

atgtcgtcct ggataaggtg gcatggaccc gccatggcgc ggctctgggg cttctgctgg 60

ctggttgtgg gcttctggag ggccgctttc gcc 93

<210> 72

<211> 1197

<212> DNA

<213> Homo sapiens

<400> 72

tgtccccgt cctgcaaatg cagtgcctct cggatctggt gcagcgaccc ttctcctggc 60

atcgtggcat ttccgagatt ggagcctaac agtgtagatc ctgagaacat caccgaaatt 120

ttcatcgcaa accagaaaag gttagaaatc atcaacgaag atgatgttga agcttatgtg 180

ggactgagaa atctgacaat tgtggattct ggattaaaat ttgtggctca taaagcattt 240

ctgaaaaaca gcaacctgca gcacatcaat tttacccgaa acaaactgac gagtttgtct 300

aggaaacatt tccgtcacct tgacttgtct gaactgatcc tggtgggcaa tccatttaca 360

tgctcctgtg acattatgtg gatcaagact ctccaagagg ctaaatccag tccagacact 420

caggatttgt actgcctgaa tgaaagcagc aagaatattc ccctggcaaa cctgcagata 480

cccaattgtg gtttgccatc tgcaaatctg gccgcaccta acctcactgt ggaggaagga 540

aagtctatca cattatcctg tagtgtggca ggtgatccgg ttcctaatat gtattgggat 600

gttggtaacc tggtttccaa acatatgaat gaaacaagcc acacacaggg ctccttaagg 660

ataactaaca tttcatccga tgacagtggg aagcagatct cttgtgtggc ggaaaatctt 720

gtaggagaag atcaagattc tgtcaacctc actgtgcatt ttgcaccaac tatcacattt 780

ctcgaatctc caacctcaga ccaccactgg tgcattccat tcactgtgaa aggcaacccc 840

aaaccagcgc ttcagtggtt ctataacggg gcaatattga atgagtccaa atacatctgt 900

actaaaatac atgttaccaa tcacacggag taccacggct gcctccagct ggataatccc 960

actcacatga acaatgggga ctacactcta atagccaaga atgagtatgg gaaggatgag 1020

aaacagattt ctgctcactt catgggctgg cctggaattg acgatggtgc aaacccaaat 1080

tatcctgatg taatttatga agattatgga actgcagcga atgacatcgg ggacaccacg 1140

aacagaagta atgaaatccc ttccacagac gtcactgata aaaccggtcg ggaacat 1197

<210> 73

<211> 72

<212> DNA

<213> Homo sapiens

<400> 73

ctctcggtct atgctgtggt ggtgattgcg tctgtggtgg gattttgcct tttggtaatg 60

ctgtttctgc tt 72

<210> 74

<211> 1152

<212> DNA

<213> Homo sapiens

<400> 74

aagttggcaa gacactccaa gtttggcatg aaagatttct catggtttgg atttgggaaa 60

gtaaaatcaa gacaaggtgt tggcccagcc tccgttatca gcaatgatga tgactctgcc 120

agccactcc atcacatctc caatgggagt aacactccat cttcttcgga aggtggccca 180

gatgctgtca ttattggaat gaccaagatc cctgtcattg aaaatcccca gtactttggc 240

atcaccaaca gtcagctcaa gccagacaca tttgttcagc acatcaagcg acataacatt 300

360

aacctctgtc ctgagcagga caagatcttg gtggcagtga agaccctgaa ggatgccagt 420

gacaatgcac gcaaggactt ccaccgtgag gccgagctcc tgaccaacct ccagcatgag 480

cacatcgtca agttctatgg cgtctgcgtg gagggcgacc ccctcatcat ggtctttgag 540

tacatgaagc atggggacct caacaagttc ctcagggcac acggccctga tgccgtgctg 600

atggctgagg gcaacccgcc cacggaactg acgcagtcgc agatgctgca tatagcccag 660

cagatcgccg cgggcatggt ctacctggcg tcccagcact tcgtgcaccg cgatttggcc 720

accaggaact gcctggtcgg ggagaacttg ctggtgaaaa tcggggactt tgggatgtcc 780

cgggacgtgt acagcactga ctactacagg gtcggtggcc acacaatgct gcccattcgc 840

tggatgcctc cagagagcat catgtacagg aaattcacga cggaaagcga cgtctggagc 900

ctgggggtcg tgttgtggga gattttcacc tatggcaaac agccctggta ccagctgtca 960

aacaatgagg tgatagagtg tatcactcag ggccgagtcc tgcagcgacc ccgcacgtgc 1020

ccccaggagg tgtatgagct gatgctgggg tgctggcagc gagagcccca catgaggaag 1080

aacatcaagg gcatccatac cctccttcag aacttggcca aggcatctcc ggtctacctg 1140

gacattctag gc 1152

<210> 75

<211> 822

<212> PRT

<213> Homo sapiens

<220>

<221> MISC_FEATURE

<222> (1)..(31)

<223> Signal peptide

<220>

<221> MISC_FEATURE

<222> (32)..(430)

<223> Extracellular domain

<220>

<221> MISC_FEATURE

<222> (431)..(454)

<223> Transmembrane domain

<220>

<221> MISC_FEATURE

<222> (455)..(822)

<223> Cytoplasmic domain

<400> 75

Met Ser Ser Trp Ile Arg Trp His Gly Pro Ala Met Ala Arg Leu Trp

1 5 10 15

Gly Phe Cys Trp Leu Val Val Gly Phe Trp Arg Ala Ala Phe Ala Cys

20 25 30

Pro Thr Ser Cys Lys Cys Ser Ala Ser Arg Ile Trp Cys Ser Asp Pro

35 40 45

Ser Pro Gly Ile Val Ala Phe Pro Arg Leu Glu Pro Asn Ser Val Asp

50 55 60

Pro Glu Asn Ile Thr Glu Ile Phe Ile Ala Asn Gln Lys Arg Leu Glu

65 70 75 80

Ile Ile Asn Glu Asp Asp Val Glu Ala Tyr Val Gly Leu Arg Asn Leu

85 90 95

Thr Ile Val Asp Ser Gly Leu Lys Phe Val Ala His Lys Ala Phe Leu

100 105 110

Lys Asn Ser Asn Leu Gln His Ile Asn Phe Thr Arg Asn Lys Leu Thr

115 120 125

Ser Leu Ser Arg Lys His Phe Arg His Leu Asp Leu Ser Glu Leu Ile

130 135 140

Leu Val Gly Asn Pro Phe Thr Cys Ser Cys Asp Ile Met Trp Ile Lys

145 150 155 160

Thr Leu Gln Glu Ala Lys Ser Ser Pro Asp Thr Gln Asp Leu Tyr Cys

165 170 175

Leu Asn Glu Ser Ser Lys Asn Ile Pro Leu Ala Asn Leu Gln Ile Pro

180 185 190

Asn Cys Gly Leu Pro Ser Ala Asn Leu Ala Ala Pro Asn Leu Thr Val

195 200 205

Glu Glu Gly Lys Ser Ile Thr Leu Ser Cys Ser Val Ala Gly Asp Pro

210 215 220

Val Pro Asn Met Tyr Trp Asp Val Gly Asn Leu Val Ser Lys His Met

225 230 235 240

Asn Glu Thr Ser His Thr Gln Gly Ser Leu Arg Ile Thr Asn Ile Ser

245 250 255

Ser Asp Asp Ser Gly Lys Gln Ile Ser Cys Val Ala Glu Asn Leu Val

260 265 270

Gly Glu Asp Gln Asp Ser Val Asn Leu Thr Val His Phe Ala Pro Thr

275 280 285

Ile Thr Phe Leu Glu Ser Pro Thr Ser Asp His His Trp Cys Ile Pro

290 295 300

Phe Thr Val Lys Gly Asn Pro Lys Pro Ala Leu Gln Trp Phe Tyr Asn

305 310 315 320

Gly Ala Ile Leu Asn Glu Ser Lys Tyr Ile Cys Thr Lys Ile His Val

325 330 335

Thr Asn His Thr Glu Tyr His Gly Cys Leu Gln Leu Asp Asn Pro Thr

340 345 350

His Met Asn Asn Gly Asp Tyr Thr Leu Ile Ala Lys Asn Glu Tyr Gly

355 360 365

Lys Asp Glu Lys Gln Ile Ser Ala His Phe Met Gly Trp Pro Gly Ile

370 375 380

Asp Asp Gly Ala Asn Pro Asn Tyr Pro Asp Val Ile Tyr Glu Asp Tyr

385 390 395 400

Gly Thr Ala Ala Asn Asp Ile Gly Asp Thr Thr Asn Arg Ser Asn Glu

405 410 415

Ile Pro Ser Thr Asp Val Thr Asp Lys Thr Gly Arg Glu His Leu Ser

420 425 430

Val Tyr Ala Val Val Val Ile Ala Ser Val Val Gly Phe Cys Leu Leu

435 440 445

Val Met Leu Phe Leu Leu Lys Leu Ala Arg His Ser Lys Phe Gly Met

450 455 460

Lys Gly Pro Ala Ser Val Ile Ser Asn Asp Asp Asp Ser Ala Ser Pro

465 470 475 480

Leu His His Ile Ser Asn Gly Ser Asn Thr Pro Ser Ser Ser Glu Gly

485 490 495

Gly Pro Asp Ala Val Ile Ile Gly Met Thr Lys Ile Pro Val Ile Glu

500 505 510

Asn Pro Gln Tyr Phe Gly Ile Thr Asn Ser Gln Leu Lys Pro Asp Thr

515 520 525

Phe Val Gln His Ile Lys Arg His Asn Ile Val Leu Lys Arg Glu Leu

530 535 540

Gly Glu Gly Ala Phe Gly Lys Val Phe Leu Ala Glu Cys Tyr Asn Leu

545 550 555 560

Cys Pro Glu Gln Asp Lys Ile Leu Val Ala Val Lys Thr Leu Lys Asp

565 570 575

Ala Ser Asp Asn Ala Arg Lys Asp Phe His Arg Glu Ala Glu Leu Leu

580 585 590

Thr Asn Leu Gln His Glu His Ile Val Lys Phe Tyr Gly Val Cys Val

595 600 605

Glu Gly Asp Pro Leu Ile Met Val Phe Glu Tyr Met Lys His Gly Asp

610 615 620

Leu Asn Lys Phe Leu Arg Ala His Gly Pro Asp Ala Val Leu Met Ala

625 630 635 640

Glu Gly Asn Pro Thr Glu Leu Thr Gln Ser Gln Met Leu His Ile

645 650 655

Ala Gln Gln Ile Ala Ala Gly Met Val Tyr Leu Ala Ser Gln His Phe

660 665 670

Val His Arg Asp Leu Ala Thr Arg Asn Cys Leu Val Gly Glu Asn Leu

675 680 685

Leu Val Lys Ile Gly Asp Phe Gly Met Ser Arg Asp Val Tyr Ser Thr

690 695 700

Asp Tyr Tyr Arg Val Gly Gly His Thr Met Leu Pro Ile Arg Trp Met

705 710 715 720

Pro Pro Glu Ser Ile Met Tyr Arg Lys Phe Thr Thr Glu Ser Asp Val

725 730 735

Trp Ser Leu Gly Val Val Leu Trp Glu Ile Phe Thr Tyr Gly Lys Gln

740 745 750

Pro Trp Tyr Gln Leu Ser Asn Asn Glu Val Ile Glu Cys Ile Thr Gln

755 760 765

Gly Arg Val Leu Gln Arg Pro Arg Thr Cys Pro Gln Glu Val Tyr Glu

770 775 780

Leu Met Leu Gly Cys Trp Gln Arg Glu Pro His Met Arg Lys Asn Ile

785 790 795 800

Lys Gly Ile His Thr Leu Leu Gln Asn Leu Ala Lys Ala Ser Pro Val

805 810 815

Tyr Leu Asp Ile Leu Gly

820

<210> 76

<211> 8498

<212> DNA

<213> Homo sapiens

<400> 76

ccctcgaggtg cataccggac ccccattcgc atctaacaag gaatctgcgc cccagagagt 60

120

agctccgagc agcggtagcg cccccctgta aagcggttcg ctatgccggg gccactgtga 180

accctgccgc ctgccggaac actcttcgct ccggaccagc tcagcctctg ataagctgga 240

ctcggcacgc ccgcaacaag caccgaggag ttaagagagc cgcaagcgca gggaaggcct 300

ccccgcacgg gtgggggaaa gcggccggtg cagcgcgggg acaggcactc gggctggcac 360

tggctgctag ggatgtcgtc ctggataagg tggcatggac ccgccatggc gcggctctgg 420

ggcttctgct ggctggttgt gggcttctgg agggccgctt tcgcctgtcc cacgtcctgc 480

aaatgcagtg cctctcggat ctggtgcagc gacccttctc ctggcatcgt ggcatttccg 540

agattggagc ctaacagtgt agatcctgag aacatcaccg aaattttcat cgcaaaccag 600

aaaaggttag aaatcatcaa cgaagatgat gttgaagctt atgtgggact gagaaatctg 660

acaattgtgg attctggatt aaaatttgtg gctcataaag catttctgaa aaacagcaac 720

ctgcagcaca tcaattttac ccgaaacaaa ctgacgagtt tgtctaggaa acatttccgt 780

caccttgact tgtctgaact gatcctggtg ggcaatccat ttacatgctc ctgtgacatt 840

atgtggatca agactctcca agaggctaaa tccagtccag acactcagga tttgtactgc 900

ctgaatgaaa gcagcaagaa tattcccctg gcaaacctgc agatacccaa ttgtggtttg 960

ccatctgcaa atctggccgc acctaacctc actgtggagg aaggaaagtc tatcacatta 1020

tcctgtagtg tggcaggtga tccggttcct aatatgtatt gggatgttgg taacctggtt 1080

tccaaacata tgaatgaaac aagccacaca cagggctcct taaggataac taacatttca 1140

tccgatgaca gtgggaagca gatctcttgt gtggcggaaa atcttgtagg agaagatcaa 1200

gattctgtca acctcactgt gcattttgca ccaactatca catttctcga atctccaacc 1260

tcagaccacc actggtgcat tccattcact gtgaaaggca accccaaacc agcgcttcag 1320

tggttctata acggggcaat attgaatgag tccaaataca tctgtactaa aatacatgtt 1380

accaatcaca cggagtacca cggctgcctc cagctggata atcccactca catgaacaat 1440

ggggactaca ctctaatagc caagaatgag tatgggaagg atgagaaaca gatttctgct 1500

cacttcatgg gctggcctgg aattgacgat ggtgcaaacc caaattatcc tgatgtaatt 1560

1620

atcccttcca cagacgtcac tgataaaacc ggtcgggaac atctctcggt ctatgctgtg 1680

gtggtgattg cgtctgtggt gggattttgc cttttggtaa tgctgtttct gcttaagttg 1740

gcaagacact ccaagtttgg catgaaaggc ccagcctccg ttatcagcaa tgatgatgac 1800

tctgccagcc cactccatca catctccaat gggagtaaca ctccatcttc ttcggaaggt 1860

ggcccagatg ctgtcattat tggaatgacc aagatccctg tcattgaaaa tccccagtac 1920

tttggcatca ccaacagtca gctcaagcca gacacatttg ttcagcacat caagcgacat 1980

aacattgttc tgaaaaggga gctaggcgaa ggagcctttg gaaaagtgtt cctagctgaa 2040

tgctataacc tctgtcctga gcaggacaag atcttggtgg cagtgaagac cctgaaggat 2100

gccagtgaca atgcacgcaa ggacttccac cgtgaggccg agctcctgac caacctccag 2160

catgagcaca tcgtcaagtt ctatggcgtc tgcgtggagg gcgaccccct catcatggtc 2220

tttgagtaca tgaagcatgg ggacctcaac aagttcctca gggcacacgg ccctgatgcc 2280

gtgctgatgg ctgagggcaa cccgccccg gaactgacgc agtcgcagat gctgcatata 2340

gcccagcaga tcgccgcggg catggtctac ctggcgtccc agcacttcgt gcaccgcgat 2400

ttggccacca ggaactgcct ggtcggggag aacttgctgg tgaaaatcgg ggactttgggg 2460

atgtcccggg acgtgtacag cactgactac tacagggtcg gtggccacac aatgctgccc 2520

attcgctgga tgcctccaga gagcatcatg tacaggaaat tcacgacgga aagcgacgtc 2580

tggagcctgg gggtcgtgtt gtgggagatt ttcacctatg gcaaacagcc ctggtaccag 2640

ctgtcaaaca atgaggtgat agagtgtatc actcagggcc gagtcctgca gcgaccccgc 2700

acgtgccccc aggaggtgta tgagctgatg ctggggtgct ggcagcgaga gccccacatg 2760

aggaagaaca tcaagggcat ccataccctc cttcagaact tggccaaggc atctccggtc 2820

tacctggaca ttctaggcta gggccctttt ccccagaccg atccttccca acgtactcct 2880

cagacggggct gagaggatga acatctttta actgccgctg gaggccacca agctgctctc 2940

cttcactctg acagtattaa catcaaagac tccgagaagc tctcgaggga agcagtgtgt 3000

acttcttcat ccatagacac agtattgact tctttttggc attatctctt tctctctttc 3060

3120

ttccctgctt cacgattctt accctttctt ttgaatcaat ctggcttctg cattactatt 3180

aactctgcat agacaaaggc cttaacaaac gtaatttgtt atatcagcag acactccagt 3240

ttgcccacca caactaacaa tgccttgttg tattcctgcc tttgatgtgg atgaaaaaaa 3300

gggaaaacaa atatttcact taaactttgt cacttctgct gtacagatat cgagagtttc 3360

3420

3480

taaaaccaga gagaaagaag atttattatg aaccgcaata tgggaggaac aaagacaacc 3540

actgggatca gctggtgtca gtccctactt aggaaatact cagcaactgt tagctgggaa 3600

gaatgtattc ggcaccttcc cctgaggacc tttctgagga gtaaaaagac tactggcctc 3660

tgtgccatgg atgattcttt tcccatcacc agaaatgata gcgtgcagta gagagcaaag 3720

3780

ggttgtgatg atagcactgg tttgtttctc aagcgctatc cacagaacct ttgtcaactt 3840

cagttgaaaa gaggtggatt catgtccaga gctcatttcg gggtcaggtg ggaaagccaa 3900

3960

tttcagatct cacttccctc cgacccctaa cttccatgcc cacccgtcct tttaactgtg 4020

caagcaaaat tgtgcatggt cttcgtcgat taataccttg tgtgcagaca ctactgctcc 4080

agacgtcgtt tccctgatag gtagagcaga tccataaaaa ggtatgactt atacaattag 4140

gggaagctaa tggagtttat tagctgagta tcaatgtctc tgcgttgtac ggtggtgatg 4200

ggttttaatg aatatggacc ctgaagcctg gaaatcctca tccacgtcga acccacagga 4260

ctgtgggaag ggcagaatca atccctaagg gaaaggaaac ctcaccctga gggcatcaca 4320

tgcactcatg ttcagtgtac acaggtcaag tcccttgctc tgggctctag ttgggagagt 4380

ggtttcattc caagtgtact ccattgtcag tatgctgttt ttgtttcctt cactccattc 4440

aaaaagtcaa aatacaaaat ttggcacagc atgccaacgg gaggctgtgc ccagaccaag 4500

cactggaagt gtgcttctag gcatagtcat tggttttgca aaaagaggggc tcaaatttaa 4560

atagaaattt acagctattt gaatggtcag atataccaag aaagaaaaat atttctgttc 4620

ctcaagaaaa cttgctaccc tctgtgaggg gaattttgct aaacttgaca tctttataac 4680

atgagccaga ttgaaaggga gtgattttca ttcatcttag gtcatgttat ttcatatttg 4740

tttctgaagg tgcgatagct ctgttttagg ttttgcttgc gcctgttaat tactggaaca 4800

ccttattttt cattaaaggc tttgaaagcc aattctcaaa aattcaaaag tgcaaattaa 4860

cagaacaaaa ggaaatccag tagcaactgc agtcaagcga gggagttgac aagataaacc 4920

ttacgtccat tcaagttata tgctggccta tgagagatga gagttggggtc gtttgttctc 4980

tttgttgatg attttaaaaa aaccctctag aatacacata ataacataat gaaagccata 5040

tctccatgat atatatgtgc acatatatat acatacatgt gcatgtatgt atcatattaa 5100

ggacccatgg tactcttaaa acactgtaga actctgtgac gcagtaagga aggggcagat 5160

ttgtacaaaa acttttctag attccatcag caaaaaccaa cacaggtttg tcacgctgca 5220

tgtctggcca gctaatctcg ggggaaaagc tacaagttat ttattttatt ttaagagaat 5280

5340

atttgtttaa ggcaaatgag actttgggag catcccattc cagttttgtc ttttttttct 5400

ctgaaagaaa aaagcaaaaa ataaaataaa attccactta taccttctga caagtcccta 5460

aaggtcttga aataaaaggt tctatgcaag tgcaaagttt tatagttatt tttattgctg 5520

attatacta ttactatctc tgttgtctta agagtatgtg ctgatttcag agacatctca 5580

aattgaaaga atatcagatt gcttttaaag tagctgaacg agccacagaa tatctgaaat 5640

tattcattgt tgttcctcca ccaccccctt tctcatggtc tgatttttag aagagtggca 5700

tcctcgttct aaaatgtaat gatcaccaaa tacggccttc catcaaattt gtgaaaacta 5760

caacagtata acagtgacaa acctaattct ctagcccaaa cctggtctga caatcatttc 5820

catttagaag tcattgaata gttttccaaa cactttccat gtgtgttagc aaattattcc 5880

tattttgtgt agatgaggac gttgagactc agagacattc agaggcacgc tagaggtctc 5940

cagcctagct tccagcacca ttgggactga atccaagtac tctcactctg aacttcgtgg 6000

ttctgtccac tagagactct aatatgcaaa caagcagttc aggaaagaaa gcatgctaac 6060

acattcatga agcagtatat gaagttagaa gaacaaaagg aaatacagga gatgacaagc 6120

aactgagata ttgtgatata taatcatgct cttagcttca gctaaattca gctaaattct 6180

6240

aaagattata tcagaattca tattatacat gtgttcacat cagcgctacc tgtgatgttt 6300

tcatgtattt atgtatgtgt tataaatact tgatttatac atatacaaat gcacatacgt 6360

6420

taggatgcgg tatgaataat ttgcttaaaa tatgctaaat aaccaaaact gtttaacgtc 6480

atgttgctgt tagtgcttcc atactccacg tgggtaggac tacatcacac ttttcaactc 6540

tgtgcagtac tgcatgggtg gaagacatat ttaagataat gtgcttccca aaacaactga 6600

ataaaagcca tcccactaca ttgagtgctt tctctggctc cttgcaaaga aagatacttt 6660

6720

gctcaccttg tctctgtctc actgtgaccc ctttacactt gagttcagag ttcaagcatt 6780

ccaaatataa attggaatgt tggcagccca gtggcttgaa ggccaatgat gagcagtcca 6840

agaccccaca gcgagatgag caactcttag gaattcccac atcctagagt gaatgcacca 6900

actaacagta tagaatgctg tccttttcaa agcgtcctaa cagcaggatt acctggtcaa 6960

gtatggactt tctttgaatc tttcttttca caaattggac tgcctgtaat accaataaca 7020

ttgttgtatc taactaaata aatgactgca tatacacaca taccctcaat tctcttgctt 7080

ccccattttc ttttcatcc cctgtctcag gacttttatt ttcaatgttg acctttggtt 7140

tggccatata tcactgttat aggaaatctc atgagaggaa tggctagtga cccaactctc 7200

caaatgtcta agttagtagt tacagctgat tttttatgat gcataattgg aatgtggagc 7260

ctctgaggtt gtgatagctt gtacatgaat ttcaaatgtc attctaaaga atgaggggtg 7320

ggagggattt atagttagaa acgacagtgc aggaaggggt attttcttgt tgtcaggggct 7380

ggaatgaatc actgctgctc aagtcaaagg ttcttgaata tccttagttt ttgcatttcc 7440

cctccttttc ctttgacctt tatttattta attatgtatt tatttattta tttatatactact 7500

tttgctccat tcagcacaaa cacaaagcaa agcaaaaaaa aaaatatata tatatatatc 7560

tgtatatgtg ttgtaggcaa aacactgtga atttcacaac aaccaccacc aagcaactat 7620

tttgccatct taacatacat ctcaggagac gaaatgagaa aagatgggga tgtcattttt 7680

tagtctatgc gtttgaggcc aggtccatgt ttatttattt ctttagtcta tgcattaatg 7740

aaaatgatcc tgagtggagg ttagctgaac gttcaatgta ctggagcaag catcataaaa 7800

gctgctagta gccatgtgtt tgaacaggaa aaatattaca gaaaatgaaa tgtaaaggcc 7860

tatatcttgc agcttgtata tcttactatt gcttaaaaaa tgtataaagc agctggaaat 7920

gttttaaata caaggtcttt gaattaaatg tggattttaa atatgtaatc ccttgacaaa 7980

tgaccaaatt atggtgaact attgctccct gcgttctttg atcattacct atgacttaca 8040

aatctgcctg gagatgtgga cattctgcat ttgcttctgt atctggagag atgtttgtat 8100

atatccaggc cgtatacaca cacatttcca tatctctcta cagatatatt tccccttcaa 8160

tcgtgacctg gtatttggaa ctctcctttt catttggctt atcttccttt taatgtgatg 8220

tctctgtgct aatacttacc agttcttgtt ttgcaatctg ttttgaggtc cattgcttta 8280

ctaagaccca ctgcatcttg gctgatttca aagtgacacc tgaatacagt gtttaaaaaa 8340

aaaaaagttt tgtttgtaaa tcatgtgacc agcttctctc aacctgacat ggaaagtctc 8400

ttgtactaca gtgtatttaa taaaaatgat gtcttacaat aaataacata ctccaaaaga 8460

8498

<210> 77

<211> 2469

<212> DNA

<213> Homo sapiens

<220>

<221> misc_feature

<222> (1)..(93)

<223> Signal peptide

<220>

<221> misc_feature

<222> (94)..(1290)

<223> Extracellular domain

<220>

<221> misc_feature

<222> (1291)..(1362)

<223> Transmembrane domain

<220>

<221> misc_feature

<222> (1363)..(2466)

<223> Cytoplasmic domain

<400> 77

atgtcgtcct ggataaggtg gcatggaccc gccatggcgc ggctctgggg cttctgctgg 60

ctggttgtgg gcttctggag ggccgctttc gcctgtccca cgtcctgcaa atgcagtgcc 120

tctcggatct ggtgcagcga cccttctcct ggcatcgtgg catttccgag attggagcct 180

aacagtgtag atcctgagaa catcaccgaa attttcatcg caaaccagaa aaggttagaa 240

atcatcaacg aagatgatgt tgaagcttat gtgggactga gaaatctgac aattgtggat 300

tctggattaa aatttgtggc tcataaagca tttctgaaaa acagcaacct gcagcacatc 360

aattttaccc gaaacaaact gacgagtttg tctaggaaac atttccgtca ccttgacttg 420

tctgaactga tcctggtggg caatccattt acatgctcct gtgacattat gtggatcaag 480

actctccaag aggctaaatc cagtccagac actcaggatt tgtactgcct gaatgaaagc 540

agcaagaata ttcccctggc aaacctgcag atacccaatt gtggtttgcc atctgcaaat 600

ctggccgcac ctaacctcac tgtggaggaa ggaaagtcta tcacattatc ctgtagtgtg 660

gcaggtgatc cggttcctaa tatgtattgg gatgttggta acctggtttc caaacatatg 720

aatgaaacaa gccacacaca gggctcctta aggataacta acatttcatc cgatgacagt 780

gggaagcaga tctcttgtgt ggcggaaaat cttgtaggag aagatcaaga ttctgtcaac 840

ctcactgtgc attttgcacc aactatcaca tttctcgaat ctccaacctc agaccaccac 900

tggtgcattc cattcactgt gaaaggcaac cccaaaccag cgcttcagtg gttctataac 960

ggggcaatat tgaatgagtc caaatacatc tgtactaaaa tacatgttac caatcacacg 1020

gagtaccacg gctgcctcca gctggataat cccactcaca tgaacaatgg ggactacact 1080

ctaatagcca agaatgagta tgggaaggat gagaaacaga tttctgctca cttcatgggc 1140

tggcctggaa ttgacgatgg tgcaaaccca aattatcctg atgtaattta tgaagattat 1200

ggaactgcag cgaatgacat cggggacacc acgaacagaa gtaatgaaat cccttccaca 1260

gacgtcactg ataaaaccgg tcgggaacat ctctcggtct atgctgtggt ggtgattgcg 1320

1380

aagtttggca tgaaaggccc agcctccgtt atcagcaatg atgatgactc tgccagccca 1440

ctccatcaca tctccaatgg gagtaacact ccatcttctt cggaaggtgg cccagatgct 1500

gtcattattg gaatgaccaa gatccctgtc attgaaaatc cccagtactt tggcatcacc 1560

aacagtcagc tcaagccaga cacatttgtt cagcacatca agcgacataa cattgttctg 1620

1680

tgtcctgagc aggacaagat cttggtggca gtgaagaccc tgaaggatgc cagtgacaat 1740

gcacgcaagg acttccaccg tgaggccgag ctcctgacca acctccagca tgagcacatc 1800

gtcaagttct atggcgtctg cgtggagggc gaccccctca tcatggtctt tgagtacatg 1860

aagcatgggg acctcaacaa gttcctcagg gcacacggcc ctgatgccgt gctgatggct 1920

gagggcaacc cgcccacgga actgacgcag tcgcagatgc tgcatatagc ccagcagatc 1980

gccgcgggca tggtctacct ggcgtcccag cacttcgtgc accgcgattt ggccaccagg 2040

aactgcctgg tcggggagaa cttgctggtg aaaatcgggg actttgggat gtcccgggac 2100

gtgtacagca ctgactacta cagggtcggt ggccacacaa tgctgcccat tcgctggatg 2160

cctccagaga gcatcatgta caggaaattc acgacggaaa gcgacgtctg gagcctgggg 2220

gtcgtgttgt gggagatttt cacctatggc aaacagccct ggtaccagct gtcaaacaat 2280

gaggtgatag agtgtatcac tcagggccga gtcctgcagc gaccccgcac gtgcccccag 2340

gaggtgtatg agctgatgct ggggtgctgg cagcgagagc cccacatgag gaagaacatc 2400

aagggcatcc ataccctcct tcagaacttg gccaaggcat ctccggtcta cctggacatt 2460

ctaggctag 2469

<210> 78

<211> 354

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic

<400> 78

caggtgcagc tggtggagtc tgggggaggc gtggtccagc ctgggaggtc cctgagactc 60

tcctgtgcag cctctggatt cagcttcagt agctttggca tgcactgggt ccgccaggct 120

ccaggcaagg gactggagtg ggtgtcagtt atatcatatg atggaattaa tacatactat 180

acagactccg tgaagggccg attcaccatc tccagagaca attccaagaa cacgctgtat 240

ctgcaaatga acggcctgag agctgaggac acggctcttt attactgtgt gcaagggtca 300

attggaaccg tttttgaata ctggggccag ggaaccctgg tcaccgtctc ctca 354

<210> 79

<211> 118

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic

<400> 79

Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Ser Phe Ser Ser Phe

20 25 30

Gly Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ser Val Ile Ser Tyr Asp Gly Ile Asn Thr Tyr Tyr Thr Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr

65 70 75 80

Leu Gln Met Asn Gly Leu Arg Ala Glu Asp Thr Ala Leu Tyr Tyr Cys

85 90 95

Val Gln Gly Ser Ile Gly Thr Val Phe Glu Tyr Trp Gly Gln Gly Thr

100 105 110

Leu Val Thr Val Ser Ser

115

<210> 80

<211> 24

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic

<400> 80

ggattcagct tcagtagctt tggc 24

<210> 81

<211> 8

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic

<400> 81

Gly Phe Ser Phe Ser Ser Phe Gly

15

<210> 82

<211> 24

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic

<400> 82

atatcatatg atggaattaa taca 24

<210> 83

<211> 8

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic

<400> 83

Ile Ser Tyr Asp Gly Ile Asn Thr

15

<210> 84

<211> 33

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic

<400> 84

gtgcaagggt caattggaac cgtttttgaa tac 33

<210> 85

<211> 11

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic

<400> 85

Val Gln Gly Ser Ile Gly Thr Val Phe Glu Tyr

1 5 10

<210> 86

<211> 321

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic

<400> 86

gacatccaga tgacccagtc tccatcctcc ctgtctgcat ctgtaggaga cagagtcacc 60

atcacttgcc gggcgagtca gggcattagc aattatttag cctggtatca gcagaaacca 120

gggaaagttc ctaaactcct gatctatgct gcatccactt tacaatcagg ggtcccatct 180

cggttcggtg gcagtggatc tgggacagat ttcactctca ccatcagcag cctgcagcct 240

gaagatgttg caacttatta ctgtcaaaag tataccagtg ccccattcac tttcggccct 300

gggaccaaag tggatatcaa a 321

<210> 87

<211> 107

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic

<400> 87

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Gly Ile Ser Asn Tyr

20 25 30

Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Val Pro Lys Leu Leu Ile

35 40 45

Tyr Ala Ala Ser Thr Leu Gln Ser Gly Val Pro Ser Arg Phe Gly Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Val Ala Thr Tyr Tyr Cys Gln Lys Tyr Thr Ser Ala Pro Phe

85 90 95

Thr Phe Gly Pro Gly Thr Lys Val Asp Ile Lys

100 105

<210> 88

<211> 18

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic

<400> 88

cagggcatta gcaattat 18

<210> 89

<211> 6

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic

<400> 89

Gln Gly Ile Ser Asn Tyr

15

<210> 90

<211> 9

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic

<400> 90

gctgcatcc 9

<210> 91

<211> 3

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic

<400> 91

Ala Ala Ser

1

<210> 92

<211> 27

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic

<400> 92

caaaagtata ccagtgcccc attcact 27

<210> 93

<211> 9

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic

<400> 93

Gln Lys Tyr Thr Ser Ala Pro Phe Thr

15

<210> 94

<211> 366

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic

<400> 94

gaggtgcagc tggtggagtc tgggggaggc ttggtccagc ctggggggtc cctaaaactc 60

tcctgtacag cctctgggtt caccttcagt ggctctgtta ttcactgggt ccgccaggct 120

tccgggaaag ggctggagtg gattggccgt attagaaaca aggctaacag ttacgcgaca 180

gcatatggtg cgtcggtgac aggcaggttc accatctcca gagatgattc aaagaacacg 240

gcgtatctgc aaatgaacag cctgaaaacc gaggacacgg ccgtttatta ctgtactttc 300

ccgggtgtag tgggacgagg aggttttgac tactggggcc agggcaccct ggtcaccgtc 360

tcctca 366

<210> 95

<211> 122

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic

<400> 95

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Lys Leu Ser Cys Thr Ala Ser Gly Phe Thr Phe Ser Gly Ser

20 25 30

Val Ile His Trp Val Arg Gln Ala Ser Gly Lys Gly Leu Glu Trp Ile

35 40 45

Gly Arg Ile Arg Asn Lys Ala Asn Ser Tyr Ala Thr Ala Tyr Gly Ala

50 55 60

Ser Val Thr Gly Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Asn Thr

65 70 75 80

Ala Tyr Leu Gln Met Asn Ser Leu Lys Thr Glu Asp Thr Ala Val Tyr

85 90 95

Tyr Cys Thr Phe Pro Gly Val Val Gly Arg Gly Gly Phe Asp Tyr Trp

100 105 110

Gly Gln Gly Thr Leu Val Thr Val Ser Ser

115 120

<210> 96

<211> 24

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic

<400> 96

gggttcacct tcagtggctc tgtt 24

<210> 97

<211> 8

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic

<400> 97

Gly Phe Thr Phe Ser Gly Ser Val

15

<210> 98

<211> 30

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic

<400> 98

attagaaaca aggctaacag ttacgcgaca 30

<210> 99

<211> 10

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic

<400> 99

Ile Arg Asn Lys Ala Asn Ser Tyr Ala Thr

1 5 10

<210> 100

<211> 39

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic

<400> 100

actttcccgg gtgtagtggg acgaggaggt tttgactac 39

<210> 101

<211> 13

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic

<400> 101

Thr Phe Pro Gly Val Val Gly Arg Gly Gly Phe Asp Tyr

1 5 10

<210> 102

<211> 339

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic

<400> 102

gacatcgtga tgacccagtc tccagactcc ctggctgtgt ctctgggcga gggggccacc 60

atcaactgca tgtccagcca gagtgtttta ttcagctcca acaataagaa ctacttagct 120

tggtaccaac agaaaccagg acagcctcct aagttgctct tttactgggc atctacccgg 180

gaatccgggg tccctgaccg attcggtggc agcgggtctg ggacagattt ctctctcacc 240

atcaacagcc tgcagactga agatgtggca gtttattact gtctccaata ttatagtatt 300

ccgtggacgt tcggccaagg gaccaaggtg gaaatcaaa 339

<210> 103

<211> 113

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic

<400> 103

Asp Ile Val Met Thr Gln Ser Pro Asp Ser Leu Ala Val Ser Leu Gly

1 5 10 15

Glu Gly Ala Thr Ile Asn Cys Met Ser Ser Gln Ser Val Leu Phe Ser

20 25 30

Ser Asn Asn Lys Asn Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln

35 40 45

Pro Pro Lys Leu Leu Phe Tyr Trp Ala Ser Thr Arg Glu Ser Gly Val

50 55 60

Pro Asp Arg Phe Gly Gly Ser Gly Ser Gly Thr Asp Phe Ser Leu Thr

65 70 75 80

Ile Asn Ser Leu Gln Thr Glu Asp Val Ala Val Tyr Tyr Cys Leu Gln

85 90 95

Tyr Tyr Ser Ile Pro Trp Thr Phe Gly Gln Gly Thr Lys Val Glu Ile

100 105 110

Lys

<210> 104

<211> 36

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic

<400> 104

cagagtgttt tattcagctc caacaataag aactac 36

<210> 105

<211> 12

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic

<400> 105

Gln Ser Val Leu Phe Ser Ser Asn Asn Lys Asn Tyr

1 5 10

<210> 106

<211> 9

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic

<400> 106

tgggcatct 9

<210> 107

<211> 3

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic

<400> 107

Trp Ala Ser

1

<210> 108

<211> 27

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic

<400> 108

ctccaatatt atagtattcc gtggacg 27

<210> 109

<211> 9

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic

<400> 109

Leu Gln Tyr Tyr Ser Ile Pro Trp Thr

15

<210> 110

<211> 381

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic

<400> 110

gaggtgcagc tggtggagtc tgggggaggc ttggtacagc ctggagggtc cctgacactc 60

tcctgtgcag cctctggatt caatttccgt gattatgaaa tgatctgggt ccgccagact 120

ccagggaagg ggctggagtg gatttcatac attagtaata gtggttatac catatactac 180

gcagactctg tgaagggccg attcaccatc tccagagaca acgccaggaa ctcaatatat 240

ctgcaagtga acagcctgag agccgaggac acggctgttt attactgttc gagacgtact 300

actatgattc ggggcattag ggcgtactac tattacggtc tggacgtctg gggccaaggg 360

accacggtca ccgtctcctc a 381

<210> 111

<211> 127

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic

<400> 111

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Thr Leu Ser Cys Ala Ala Ser Gly Phe Asn Phe Arg Asp Tyr

20 25 30

Glu Met Ile Trp Val Arg Gln Thr Pro Gly Lys Gly Leu Glu Trp Ile

35 40 45

Ser Tyr Ile Ser Asn Ser Gly Tyr Thr Ile Tyr Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Arg Asn Ser Ile Tyr

65 70 75 80

Leu Gln Val Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ser Arg Arg Thr Thr Met Ile Arg Gly Ile Arg Ala Tyr Tyr Tyr Tyr

100 105 110

Gly Leu Asp Val Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser

115 120 125

<210> 112

<211> 24

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic

<400> 112

ggattcaatt tccgtgatta tgaa 24

<210> 113

<211> 8

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic

<400> 113

Gly Phe Asn Phe Arg Asp Tyr Glu

15

<210> 114

<211> 24

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic

<400> 114

attagtaata gtggttatac cata 24

<210> 115

<211> 8

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic

<400> 115

Ile Ser Asn Ser Gly Tyr Thr Ile

15

<210> 116

<211> 60

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic

<400> 116

tcgagacgta ctactatgat tcggggcatt agggcgtact actattacgg tctggacgtc 60

<210> 117

<211> 20

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic

<400> 117

Ser Arg Arg Thr Thr Met Ile Arg Gly Ile Arg Ala Tyr Tyr Tyr Tyr

1 5 10 15

Gly Leu Asp Val

20

<210> 118

<211> 324

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic

<400> 118

gacatccaga tgacccagtc tccatcctcc ctgtctgcat ctgtaggaga cagagtcacc 60

atcacttgcc gggcaagtca gagcattagc agctatttaa attggtatca gcagaaacca 120

gggaaagccc ctaagctcct gatctatgct gcatccagtt tgcaaagtgg ggtcccgtca 180

aggttcagtg gcagtggatc tgggacagat ttcactctca ccatcagcag tctgcaacct 240

gaagattttg caacttacta ctgtcaacag agttacagta cccctccgat caccttcggc 300

caagggacac gactggagat taaa 324

<210> 119

<211> 108

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic

<400> 119

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile Ser Ser Tyr

20 25 30

Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

Tyr Ala Ala Ser Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ser Tyr Ser Thr Pro Pro

85 90 95

Ile Thr Phe Gly Glyn Gly Thr Arg Leu Glu Ile Lys

100 105

<210> 120

<211> 18

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic

<400> 120

cagagcatta gcagctat 18

<210> 121

<211> 6

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic

<400> 121

Gln Ser Ile Ser Ser Tyr

15

<210> 122

<211> 9

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic

<400> 122

gctgcatcc 9

<210> 123

<211> 3

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic

<400> 123

Ala Ala Ser

1

<210> 124

<211> 30

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic

<400> 124

caacagagtt acagtacccc tccgatcacc 30

<210> 125

<211> 10

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic

<400> 125

Gln Gln Ser Tyr Ser Thr Pro Pro Ile Thr

1 5 10

<---

Claims (41)

1. A non-human animal for expressing a chimeric tropomyosin receptor kinase B (TRKB) protein containing one or more human TRKB protein fragments, wherein the non-human animal contains a genetically modified endogenous TrkB locus encoding the TRKB protein, wherein the non-human animal is is a mouse or a rat, wherein the TRKB protein contains an endogenous TRKB signal peptide, an endogenous TRKB cytoplasmic domain, an endogenous TRKB transmembrane domain, and a human TRKB extracellular domain, and the entire extracellular domain is encoded by a segment of the endogenous TrkB locus that has been deleted and replaced by an orthologous human TRKB sequence. 2. A non-human animal according to claim 1, wherein the extracellular domain contains the sequence shown in SEQ ID NO: 60, optionally the coding sequence for the extracellular domain contains the sequence shown in SEQ ID NO: 72. 3. A non-human animal according to claim 1 or 2, wherein: (I) the signal peptide contains the sequence shown in SEQ ID NO: 51 or 55; or (II) the entire signal peptide is encoded by the endogenous TrkB sequence, optionally the coding sequence for the signal peptide contains the sequence shown in SEQ ID NO: 63 or 67. 4. A non-human animal according to any one of the preceding paragraphs, wherein: (I) the transmembrane domain contains the sequence shown in SEQ ID NO: 53 or 57; or (II) the entire transmembrane domain is encoded by the endogenous TrkB sequence, optionally the coding sequence for the transmembrane domain contains the sequence shown in SEQ ID NO: 65 or 69. 5. A non-human animal according to any one of the preceding paragraphs, wherein: (I) moreover, the cytoplasmic domain contains the sequence presented in SEQ ID NO: 54 or 58; or (II) the entire cytoplasmic domain is encoded by the endogenous TrkB sequence, optionally the coding sequence for the cytoplasmic domain contains the sequence shown in SEQ ID NO: 66 or 70. 6. A non-human animal according to any one of the preceding claims, wherein the signal peptide contains the sequence shown in SEQ ID NO: 51, the transmembrane domain contains the sequence shown in SEQ ID NO: 53, and the cytoplasmic domain contains the sequence shown in SEQ ID NO: : 54, or moreover, the signal peptide contains the sequence shown in SEQ ID NO: 55, the transmembrane domain contains the sequence shown in SEQ ID NO: 57, and the cytoplasmic domain contains the sequence shown in SEQ ID NO: 58. 7. A non-human animal according to any of the preceding claims, wherein the entire signal peptide, the entire transmembrane domain, and the entire cytoplasmic domain are encoded by the endogenous TrkB sequence, optionally, the coding sequence for the signal peptide contains the sequence shown in SEQ ID NO: 63, the coding sequence for the transmembrane domain contains the sequence shown in SEQ ID NO: 65, and the coding sequence for the cytoplasmic domain contains the sequence shown in SEQ ID NO: 66, or optionally, the coding sequence for the signal peptide contains the sequence shown in SEQ ID NO: 67, the coding sequence for the transmembrane domain contains the sequence shown in SEQ ID NO: 69, and the coding sequence for the cytoplasmic domain contains the sequence shown in SEQ ID NO: 70. 8. A non-human animal according to any one of the preceding claims, wherein the TRKB protein contains the sequence shown in SEQ ID NO: 4 or 5. 9. A non-human animal according to claim 8, wherein the coding sequence of the genetically modified TrkB locus encoding the TRKB protein contains the sequence shown in SEQ ID NO: 12 or 13. 10. A non-human animal according to any one of the preceding claims, wherein the rat or mouse is homozygous for the genetically modified endogenous TrkB locus. 11. A non-human animal according to any one of the preceding claims, wherein the non-human animal is a rat, optionally the TRKB protein comprising the sequence shown in SEQ ID NO: 5 and optionally the coding sequence of the genetically modified TrkB locus encoding the TRKB protein comprises the sequence shown in SEQ ID NO: 13. 12. Non-human animal according to any one of paragraphs. 1-10, wherein the non-human animal is a mouse, optionally the TRKB protein contains the sequence shown in SEQ ID NO: 4 and optionally the coding sequence of the genetically modified TrkB locus encoding the TRKB protein contains the sequence shown in SEQ ID NO: 12. 13. A method for evaluating the activity of a human TRKB-targeted reagent in vivo, comprising the following: (a) administering a human TRKB-targeted reagent to a non-human animal according to any one of paragraphs. 1-12; And (b) assessing the activity of a human TRKB-targeted reagent in a non-human animal. 14. The method of claim 13, wherein step (a) comprises injecting the human TRKB-targeted reagent into a non-human animal. 15. The method of claim 13 or 14, wherein step (b) comprises assessing changes in one or more or all of the following: body weight, body composition, metabolism, and locomotion, compared to a control non-human animal, optionally assessing changes in body tissue composition involves assessing lean tissue mass and/or adipose tissue mass compared to a control non-human animal, and optionally assessing changes in metabolism involves assessing changes in food intake and/or water intake. 16. The method according to any one of paragraphs. 13-15, in which step (b) involves assessing TRKB phosphorylation and/or activation of the MAPK/ERK and PI3K/Akt pathways. 17. The method according to any one of paragraphs. 13-16, in which step (b) involves the evaluation of neuroprotective activity. 18. The method according to any one of paragraphs. 13-17, in which step (b) involves assessing retinal ganglion cell viability, optionally assessing retinal ganglion cell viability in a complete optic nerve transection after optic nerve injury model or in an optic nerve crush model. 19. The method according to any one of paragraphs. 13-18, wherein the human TRKB targeting reagent is an antigen-binding protein, optionally the antigen-binding protein is a human TRKB agonist antibody. 20. The method according to any one of paragraphs. 13-18, wherein the human TRKB targeting reagent is a small molecule, optionally the small molecule is a human TRKB agonist. 21. The method of obtaining a non-human animal according to any one of paragraphs. 1-12, providing the following: (I) (a) modifying the genome of a mouse or rat pluripotent cell to include a genetically modified endogenous TrkB locus encoding the TRKB protein, wherein the TRKB protein contains an endogenous TRKB signal peptide, an endogenous TRKB cytoplasmic domain, an endogenous TRKB transmembrane domain, and a human TRKB extracellular domain, and the entire extracellular domain is encoded by a segment of the endogenous TrkB locus that has been deleted and replaced with an orthologous human TRKB sequence; (b) identifying or selecting genetically modified mouse or rat pluripotent cells containing the genetically modified endogenous TrkB locus; (c) introducing a genetically modified mouse or rat pluripotent cell into a mouse or rat host embryo; And (d) implantation and incubation of a mouse or rat host embryo by a mouse or rat surrogate mother; or (II) (a) modifying the genome of a mouse or rat embryo at the single cell stage to include a genetically modified endogenous TrkB locus encoding the TRKB protein, wherein the TRKB protein contains an endogenous TRKB signal peptide, an endogenous TRKB cytoplasmic domain, an endogenous TRKB transmembrane domain, and an extracellular TRKB domain human, and the entire extracellular domain is encoded by a segment of the endogenous TrkB locus that has been deleted and replaced with an orthologous human TRKB sequence; (b) selecting a genetically modified mouse or rat embryo at the single cell stage containing the genetically modified endogenous TrkB locus; And (c) implantation and incubation of a genetically modified mouse or rat embryo at the single cell stage by a mouse or rat surrogate mother. 22. A non-human animal cell for expressing a tropomyosin receptor kinase B (TRKB) chimeric protein containing one or more human TRKB protein fragments, wherein the non-human animal contains a genetically modified endogenous TrkB locus encoding the TRKB protein, wherein the non-human animal is a mouse or rat, wherein the TRKB protein comprises an endogenous TRKB signal peptide, an endogenous TRKB cytoplasmic domain, an endogenous TRKB transmembrane domain, and a human TRKB extracellular domain, and the entire extracellular domain is encoded by a segment of the endogenous TrkB locus that has been deleted and replaced by the orthologous human TRKB sequence.