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Title:  Isolated DNA molecules encoding humanized calcitonin gene-related peptide receptor, related non-human transgenic animals and assay methods
United States Patent: 
7,193,070
Issued: 
March 20, 2007

Inventors: 
Kane; Stefanie A. (Schwenksville, PA), Salvatore; Christopher A. (Philadelphia, PA), Mallee; John J. (Collegeville, PA), Koblan; Kenneth S. (Carversville, PA), Oliver; Kevin R. (Huntingdon, GB)
Assignee: 
Merck & Co., Inc. (Rahway, NJ)
Appl. No.: 
10/490,594
Filed: 
September 26, 2002
PCT Filed: 
September 26, 2002
PCT No.: 
PCT/US02/30501
371(c)(1),(2),(4) Date: 
March 23, 2004
PCT Pub. No.: 
WO03/027252
PCT Pub. Date: 
April 03, 2003


 

Pharm Bus Intell & Healthcare Studies


Abstract

Disclosed herein are isolated nucleic acid molecules encoding a humanized version of a calcitonin gene-related peptide (CGRP) receptor, which comprises the G-protein coupled receptor calcitonin-receptor-like receptor (CRLR) and the receptor-activity-modifying protein 1 (RAMP1). The humanized CGRP receptors of the present invention attain pharmacological profiles similar to the wild type human receptor via modifications to the respective mammalian RAMP1 nucleotide sequence, specifically at amino acid 74. Also described are related recombinant vectors, recombinant hosts and associated methods for generating such humanized CGRP receptors. Also presented are non-human transgenic animals which express humanized RAMP1. Such animals have been engineered to provide for a CGRP pharmacological profile similar to human CGRP.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to an isolated or purified nucleic acid molecule (polynucleotide) which encodes a humanized version of a calcitonin gene-related peptide (CGRP) receptor, which comprises the G-protein coupled receptor calcitonin-receptor-like receptor (CRLR) and the receptor-activity-modifying protein-1 (RAMP1). More specifically, the present invention relates to isolated or purified vertebrate, and preferably mammalian, nucleic acid molecules which encode derivative, humanized versions of the CGRP receptor, namely via DNA molecules which encode chimeric, hybrid or mutant derivatives of a mammalian RAMP1 sequence, which are shown herein to be responsible for the "humanization" of the CGRP receptor upon association with a vertebrate (and again, preferably a mammalian) CRLR receptor protein. The CRLR and RAMP1 DNA molecules disclosed herein may be co-transfected into a host cell of choice wherein the recombinant host cell provides a source for substantial levels of an expressed functional, humanized version of a CGRP receptor. Therefore, these recombinantly expressed humanized CGRP receptor proteins form a receptor complex in which small molecule CGRP receptor antagonists display potency similar to that for a "wild type" human CGRP receptor. Such mutant receptors will be useful in cell based assays, receptor binding assays and/or radioligand binding assays, and, as noted below, in the generation of transgenic animals which provide for this humanized CGRP receptor activity.

To this end, a particularly preferred aspect of the present invention which is afforded only in view of this specification is the generation of non-human animal cells, non-human transgenic animals, such as founders and littermates, especially transgenic "knock-in" animals, wherein the endogenous gene encoding RAMP1 has been engineered (i.e., "humanized") to provide for a CGRP receptor pharmacological profile similar to human CGRP receptor. Such non-human transgenic animals will preferably provide for an altered genotype (endogenous CRLR and "humanized" RAMP1), which will provide for a phenotype whereby the pharmacological profile of the non-human transgenic animal in regard to modulators of CGRP will mimic the human form of CGRP receptor. Various non-human transgenic animals may be contemplated in view of the finding disclosed herein that alteration of a single amino acid residue in a non-human RAMP1 sequence (such as rat, mouse and pig, as shown herein, as well as additional species, such as cyno and canine) results in a "humanized" version of RAMP1 when complexed with a mammalian version of CRLR. In other words, the species-specific pharmacology of known antagonists is shown herein to be localized to the region at or around amino acid residue 74 of human RAMP1 (a tryptophan residue), such that non-human RAMP1 forms may be generated and used to generate transgenic animals which express the humanized version along with or instead of the endogenous RAMP1 protein.

The present invention therefore relates to isolated or purified nucleic acid molecules which encode a chimeric, hybrid and/or mutant version of a RAMP1 protein where such a protein is functional (i.e., when co-expressed with CRLR will exhibit predicted pharmacological properties), and furthermore wherein such a protein is humanized by virtue of altering the amino acid that corresponds to human amino acid residue 74 to a tryptophan residue. Such a nucleic acid molecule is part of the present invention whether it encodes a chimeric, hybrid or various mutant protein, so long as amino acid 74 has been altered from its native residue to the human residue, namely tryptophan.

The present invention further relates to isolated or purified nucleic acid molecules which encode a chimeric, hybrid and/or mutant version of a RAMP1 protein, wherein such a derivative RAMP1 protein comprises the respective amino acid sequence at least from about amino acid 1 to amino acid 65 and from about amino acid 113 to about amino acid 148, wherein the region corresponding from about amino acid 66 to amino acid 112 is at least partially derived from the human RAMP1 coding region. Such DNA molecules will encode "humanized" RAMP1 proteins which, when co-expressed with a CRLR gene, or functional derivative thereof, will result in a CGRP receptor which mimics human CGRP receptor pharmacological properties.

The present invention further relates to isolated or purified nucleic acid molecules which encode a chimeric, hybrid and/or mutant version of a RAMP1 protein, wherein such a derivative RAMP1 protein at least comprises a nucleotide change which results in an alteration of amino acid residue 74 to a tryptophan residue, which results in a humanized form of RAMP1. To this end, a specific embodiment of the present invention relates to an isolated or purified nucleic acid molecule from rat wherein the codon for amino acid residue 74 is altered from a lysine residue to a tryptophan residue. Another specific embodiment of the present invention relates to an isolated or purified nucleic acid molecule from mouse wherein the codon for amino acid residue 74 is altered from a lysine residue to a tryptophan residue. Yet another specific embodiment of the present invention relates to an isolated or purified nucleic acid molecule from cynomolgous wherein the codon for the amino acid residue corresponding to human residue 74 is altered from a cysteine residue to a tryptophan residue (i.e., a "C74W" mutant). Still another specific embodiment of the present invention relates to an isolated or purified nucleic acid molecule from porcine (pig) wherein the codon for the amino acid residue corresponding to human residue 74 is altered from a arginine residue to a tryptophan residue (i.e., a "R74W" mutant). Therefore, the present invention further relates to an isolated nucleic acid molecule (polynucleotide) which encodes mRNA which expresses a humanized RAMP1 protein, this DNA molecule comprising the nucleotide sequence disclosed herein in Table 1 (see Original Patent) and listed as SEQ ID NO:1 (rat), SEQ ID NO:3 (mouse), SEQ ID NO:5 (a partial sequence from cyno) and SEQ ID NO:7 (a partial sequence from porcine (pig)). Table 1 (see Original Patent) discloses the nucleotide and predicted amino acid sequences of these various mammalian RAMP1 sequences which, when expressed as a full length RAMP1 protein, correspond to a "humanized" form of RAMP1.

The present invention also relates to biologically active fragments or mutants of SEQ ID NOs:1, 3, 5 and 7 which encode mRNA expressing a humanized RAMP1 protein. Any such biologically active fragment and/or mutant will encode either a protein or protein fragment which at least substantially mimics the pharmacological properties of human RAMP1, including but not limited to the humanized RAMP1 proteins as set forth in SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, and SEQ ID NO:8, with SIDs 6 and 8 representing partial sequences which span the region manipulated for humanization of the respective RAMP1 protein. Any such polynucleotide includes but is not necessarily limited to chimeric constructs (including but not limited to the exemplified chimeric constructs described herein), hybrid constructs, nucleotide substitutions, deletions, additions, amino-terminal truncations and carboxy-terminal truncations such that these mutations encode mRNA which may co-express a functional humanized RAMP1 with a mammalian CRLR protein in a eukaryotic cell so as to be useful for screening for agonists and/or antagonists of CGRP activity. To this end, preferred aspects of this portion of the present invention are disclosed in Table 1 as SEQ ID NOs:1; 3 and 5, all of which encode a humanized version of RAMP1.

The isolated nucleic acid molecules of the present invention may include a deoxyribonucleic acid molecule (DNA), such as genomic DNA and complementary DNA (cDNA), which may be single (coding or noncoding strand) or double stranded, as well as synthetic DNA, such as a synthesized, single stranded polynucleotide. The isolated nucleic acid molecule of the present invention may also include a ribonucleic acid molecule (RNA).

The present invention also relates to recombinant vectors and recombinant host cells, both prokaryotic and eukaryotic, which contain the nucleic acid molecules disclosed throughout this specification and which encode a humanized version of a CGRP receptor and associated fragment thereof, substantially purified forms of associated humanized version of a CGRP receptor, recombinant membrane fractions comprising these proteins (e.g., active CGRP receptors comprising CRLR and humanized RAMP1 proteins), associated mutant proteins, and methods associated with identifying compounds which specifically modulated human CGRP receptor utilizing the humanized version of RAMP1 in various assays.

The present invention also relates to a substantially purified form of a humanized RAMP1 protein, which comprises the amino acid sequence disclosed in Table 1 (e.g., SEQ ID NOs:2, 4, 6 and 8). The invention further relates to a humanized RAMP1 protein which consists of the amino acid sequence disclosed in Table 1 (e.g., SEQ ID NOs:2, 4, 6 and 8). As noted herein, while vertebrate sequences are within the scope of the invention, mammalian sequences, including but not limited to those exemplified herein, are preferred.

Another preferred aspect of the present invention relates to a substantially purified, fully processed (including proteolytic processing, glycosylation and/or phosphorylation), mature humanized RAMP1 protein obtained from a recombinant host cell containing a DNA expression vector comprising nucleotide sequence as set forth in SEQ ID NOs: 1, 3, 5 and 7 which express the respective humanized RAMP1 protein. It is especially preferred that the recombinant host cell be a eukaryotic host cell, such as a mammalian cell line.

Another aspect of the present invention relates to a substantially purified membrane preparation, partially purified membrane preparation, or cell lysate which has been obtained from a recombinant host cell transformed or transfected with a DNA expression vector which comprises and appropriately expresses a complete open reading frame as set forth, for example, in SEQ ID NOs: 1, 3, 5 and 7, which results in a functional form of the respective humanized RAMP1 protein. These recombinant membranes will comprise humanized RAMP1 proteins such as those disclosed in Table 1 (i.e., SEQ ID NOs: 2, 4, 6 and 8), or additional equivalents which results in a humanized form of RAMP1, namely mammalian RAMP1 proteins wherein the amino acid residue corresponding to human amino acid residue 74 has been altered to code for a tryptophan residue.

A preferred aspect of this portion of the present invention relates to a substantially purified membrane preparation, partially purified membrane preparation, or cell lysate which has been obtained from a recombinant host cell transformed or transfected with a DNA expression vector which comprises and appropriately expresses a humanized RAMP1 protein as described throughout this specification, in conjunction with a DNA expression vector which comprises and appropriately expresses a mammalian CRLR GPCR protein. Examples of mammalian nucleotide sequences which may be utilized for such a purpose included but are not limited to the human, rat and mouse nucleic acid molecules disclosed in Table 2 (see Original Patent) and set forth as SEQ ID NOs: 7, 9, and 11, which results in a functional form of a mammalian CRLR GPCR which, when co-expressed with a humanized RAMP1 protein, will be useful to screen for modulators which effect the human CGRP receptor. The subcellular membrane fractions and/or membrane-containing cell lysates from the recombinant host cells (both prokaryotic and eukaryotic as well as both stably and transiently transformed cells) contain the functional and processed proteins encoded by the nucleic acid molecules disclosed herein. This recombinant-based membrane preparation will comprise a mammalian CRLR protein and a humanized RAMP1 protein which is essentially free from contaminating proteins. These subcellular membrane fractions will comprise "humanized" CGRP receptors which function efficiently for the screening of modulators (e.g., agonists and especially antagonists) of the human CGRP receptor at levels which are at least similar to or possibly substantially above endogenous levels. Any such "humanized" CGRP receptor-containing membrane preparation will be useful in various assays to select for modulators of the respective CGRP receptor. A preferred eukaryotic host cell of choice to express the CGRP receptor of the present invention is a mammalian cell line.

The present invention also relates to biologically active fragments and/or mutants of a humanized RAMP1 protein, comprising the amino acid sequence as set forth in SEQ ID NOs: 2, 4, 6 or 8, including but not necessarily limited to amino acid substitutions, deletions, additions, amino terminal truncations and carboxy-terminal truncations such that these mutations provide for proteins or protein fragments of diagnostic, therapeutic or prophylactic use and would be useful for screening for selective modulators, including but not limited to agonists and/or antagonists for human CGRP receptor pharmacology.

A preferred aspect of the present invention is disclosed in Table 1 as SEQ ID NOs:2, 4, 6 and 8, respective amino acid sequences which are mammalian RAMP1 proteins, or portions thereof, which have been "humanized" solely by altering amino acid residue 74 to a tryptophan ("Trp" or "W") residue. As noted above, co-expression of a humanized RAMP1 protein of the present invention along with a mammalian CRLR protein will be useful in screening for antagonists of the CGRP receptor.

The present invention also relates to polyclonal and monoclonal antibodies raised against forms of humanized RAMP1, a biologically active fragment of humanized RAMP1, or a CGRP receptor complex which comprises a humanized RAMP1.

The present invention also relates to isolated nucleic acid molecules which encode humanized RAMP1 fusion constructs (as well as the substantially purified protein expressed within and recovered from the respective host cell which houses the fusion construct, most likely in the form of a DNA expression vector), including but not limited to fusion constructs which express a portion of humanized RAMP1 to various markers, including but in no way limited to GFP (Green fluorescent protein), the MYC epitope, GST, Fc, Flag, HA, and His-tag. Any such fusion construct will comprise at least a portion of the RAMP1 open reading frame which encodes for the alteration at amino acid 74 to a tryptophan residue, such that the respective fusion protein will exhibit human-like pharmacological properties when complexed with a mammalian CRLR protein.

As noted above, the heterodimeric CGRP receptor requires co-expression of calcitonin receptor-like receptor (CRLR) and an accessory protein called receptor activity modifying protein 1, or RAMP1. Several small molecule CGRP receptor antagonists have been shown to exhibit marked species selectivity, with >100-fold higher affinities for the human CGRP receptor than for receptors from other species. It is shown herein that species selectivity of CGRP modulators is determined exclusively by RAMP1. By constructing hybrid human/rat CRLR/RAMP1 receptors, it is disclosed herein that co-expression of hCRLR with rRAMP1 produced rat receptor pharmacology, and vice versa (h=human, r=rat, m=mouse). Moreover, with rat/human RAMP1 chimeras and site-directed mutants, it is further disclosed herein that a single amino acid at position 74 of the RAMP1 protein modulates the affinity of small molecule antagonists for CRLR/RAMP1. Co-expression of rCRLR with rK74W RAMP1 and mCRLR with mK74W RAMP1 increased the affinities of these antagonists by >100-fold, resulting in IC.sub.50 values similar to those observed for the human receptor. Therefore, it is disclosed herein that the affinities of small molecule antagonists for the CGRP receptor are heavily influenced by the nature of amino acid 74 of RAMP1 and provide evidence that RAMP1 participates in the antagonist binding sites.

It is shown herein that amino acid position 74 of RAMP1 is responsible for the observed species selectivity of several known antagonists of the CGRP receptor, suggesting that that the affinity of small molecule antagonists can be affected by a single amino acid change and that these antagonists may interact directly with RAMP1. The identification of a single amino acid mutation that can convert the mouse CGRP receptor into one that displays human-like pharmacology shows that a humanized CGRP receptor mouse may be created by a "knock-in" strategy, wherein lysine-74 is replaced with tryptophan by various techniques well known in the art. Such a humanized non-human transgenic animal (e.g., a transgenic mouse), will have utility in drug discovery and development programs for in vivo pharmacological studies of CGRP receptor antagonists, as well as complementing marmoset as a suitable animal model for such studies.

To this end, the present invention relates to a transgenic non-human animal, such as a founder animal or subsequent littermate, wherein both alleles of the endogenous RAMP1 gene have been humanized, as well as heterozygous transgenic non-human animals wherein a single endogenous RAMP1 allele has been humanized and to non-human transgenic animal comprising wild type endogenous RAMP1 alleles in addition to at least one humanized RAMP1 allele stably integrated within the respective target genome. To this end, the present invention relates to animal cells, non-human transgenic embryos, non-human transgenic animals and non-human transgenic littermates which are homozygous for humanized RAMP1 and whereby endogenous RAMP1 has been disrupted, namely by replacement of the endogenous RAMP1 coding region, or portion thereof, by direct gene targeting within the respective target genome. The present invention also extends to animal cells, non-human transgenic embryos, non-human transgenic animals (such as founder animals and transgenic littermates) which are heterozygous for a functional RAMP1 gene native to that animal. Namely, the heterozygosity referring the one functional, endogenous RAMP1 gene and one functional, humanized RAMP1 gene. Also, the present invention relates to animal cells, non-human transgenic embryos and non-human transgenic littermates having at least one and possibly multiple humanized RAMP1 genes being randomly inserted within the target genome, such that both functional endogenous and humanized RAMP1 proteins may be expressed. The transgenic animals of the present invention can be used in the study of the effect of modulators, especially antagonists, of the CGRP receptor. Such a transgenic non-human animal will be especially useful for in vivo efficacy and receptor occupancy studies for testing of CGRP receptor modulators, especially antagonists, for treatment of various disorders, including but not limited to migraine headaches, pain, menopausal hot flash, migraine prophylaxis, chronic tension type headache, cluster headache, neurogenic or chronic inflammation, gastrointestinal disorders, type 2 diabetes and cardiovascular disorders (via agonizing the CGRP receptor). Generation of a genetically engineered mouse expressing a human-like mutant RAMP1 will result in a species in which small molecule CGRP receptor antagonists display potency similar to that for the human CGRP receptor. The non-human transgenic animal of the present invention may also provide cells for culture, for in vitro studies. Therefore, in particular embodiments of the present invention, cell lines are produced and cells isolated from any of the animals produced in the steps described herein.

An aspect of this portion of the invention is a method to obtain an animal wherein the endogenous RAMP1 gene native to the animal has been replaced by "knock-in" technology such that a humanized form of RAMP1 has replaced the endogenous RAMP1 allele(s). A RAMP1 gene that naturally occurs in the animal is referred to as the native gene, endogenous gene and/or "wild-type" gene. It is preferred that expression of a non-native RAMP1 gene (e.g., a "humanized" RAMP1 gene) take place in a transgenic animal in the absence of a native RAMP1 gene. Such a transgenic "knock-in" non-human animal (such as a transgenic mouse) will be especially useful in animal studies to mimic pharmacology of the human CGRP receptor while utilizing an endogenous CRLR gene and a "humanized" RAMP1 gene. The method includes providing a gene for a humanized form of RAMP1 in the form of a transgene and targeting the transgene into a chromosome of the animal at the place of the native RAMP1 gene or at another chromosomal location. The transgene can be introduced into the embryonic stem cells by a variety of methods known in the art, including electroporation, microinjection, and lipofection. Cells carrying the transgene can then be injected into blastocysts which are then implanted into pseudopregnant animals. In alternate embodiments, the transgene-targeted embryonic stem cells can be co-incubated with fertilized eggs or morulae followed by implantation into females. After gestation, the animals obtained are chimeric founder transgenic animals. The founder animals can be used in further embodiments to cross with wild-type animals to produce F1 animals heterozygous for the altered RAMP1 gene. In further embodiments, these heterozygous animals can be interbred to obtain the viable transgenic embryos whose somatic and germ cells are homozygous for the altered, humanized RAMP1. In other embodiments, the heterozygous animals can be used to produce cells lines. In preferred embodiments, the animals are mice or rat. Therefore, a preferred aspect of this portion of the present invention is a transgenic non-human animal which expresses a non-native, humanized RAMP1 protein on a native RAMP1 null background. As noted above, the animal can be heterozygous (i.e., having a different allelic representation of a gene on each of a pair of chromosomes of a diploid genome, such as native RAMP1/humanized RAMP1), homozygous (i.e., having the same representation of a gene on each of a pair of chromosomes of a diploid genome, such as humanized RAMP1/humanized RAMP1) for the altered RAMP1 gene, hemizygous (i.e., having a gene represented on only one of a pair of chromosomes of a diploid genome, preferably a humanized version of RAMP1), or homozygous for the humanized RAMP1 gene. In preferred embodiments, the animal is a mouse or a rat, with mouse being especially preferred. In a further embodiment, the targeted or randomly inserted humanized RAMP1 gene may be operably linked to a promoter. As used herein, operably linked is used to denote a functional connection between two elements whose orientation relevant to one another can vary. In this particular case, it is understood in the art that a promoter can be operably linked to the coding sequence of a gene to direct the expression of the coding sequence while placed at various distances from the coding sequence in a genetic construct. Further embodiments are cell lines and cells derived from animals of this aspect of the invention.

The non-human transgenic animals of the present invention include non-human mammalian species which are candidates for humanization, including but not limited to transgenic mice, transgenic rats, as well as non-human primates which are candidates for RAMP1 humanization. Transgenic mice are preferred.

The present invention especially relates to analysis of the complex function(s) of the CGRP receptor. The native wild type gene is selectively replaced via targeted gene delivery or it resides within the same genome by random integration of a humanized RAMP1 gene in totipotent ES cells and used to generate the transgenic mice of the present invention. Techniques are available to replace an endogenous homologue or to randomly insert such a homologue into the endogenous genomic background by using known targeted homologous recombination or random integration, respectively, to generated genotypic changes into chromosomal alleles. Therefore, as noted above, the present invention relates to diploid animal cells, non-human transgenic embryos, non-human transgenic animals and non-human transgenic founders and/or transgenic littermates which are heterozygous or homozygous for a disrupted RAMP1 gene and/or insertion of a humanized RAMP1 gene. The cells, embryos and non-human transgenic animals contain two chromosome alleles for humanized RAMP1 wherein at least one of the wild type RAMP1 alleles is mutated such that less than wild-type levels of RAMP1 activity is produced. The diploid cell, embryo or non-human transgenic animal homozygous for a humanized RAMP1 gene, wherein a humanized RAMP1 gene has been targeted to replace the wild type allele, may show at least from about 50%, and preferably about 100% reduction in wild type RAMP1 activity (measured by the loss of "wild type" pharmacological characteristics of the endogenous CGRP receptor) and a concomitant CGRP receptor activity which mimics human CGRP receptor pharmacology, as compared to a wild type diploid cell. A diploid mouse cell, embryo or non-human transgenic mouse generated herein which is heterozygous for a disrupted RAMP1 gene (i.e., wtRAMP1/humanized RAMP1) gene may show at least from about 10% to about 100% reduction in endogenous RAMP1 activity compared to a wild type diploid cell. It is within the purview of the artisan of ordinary skill to use known molecular biology techniques to measure the level of transcription, expression and/or functional CRLR/RAMP1 activity in mouse cell homozygous, heterozygous or hemizygous for a humanized RAMP1 gene. Therefore, the present invention especially relates to analysis of the complex function(s) of the CGRP receptor by generating homozygous, heterozygous or hemizygous transgenic mice and studying how various potential modulators interact within these manipulated animals. In a preferred embodiment, the assay is performed by providing an animal of the present invention (especially a transgenic animal wherein a humanized RAMP1 gene has replaced the endogenous RAMP1 gene at both alleles), exposing the animal to a compound (preferably a potential antagonist of CGRP receptor activity), and measuring the effect of said compound on biochemical and physiological responses related to CGRP activity, or lack thereof. The measurement can be compared to these measurements in a genetically similar or identical animal that is not exposed to the compound.

As introduced above, a type of target cell for transgene introduction is preferably the embryonic stem cell (ES), especially when generating a transgenic mouse, where culturing of ES cells has been particularly successful. ES cells can be obtained from pre-implantation embryos cultured in vitro and fused with embryos (Evans et al., 1981, Nature 292: 154 156; Bradley et al., 1984, Nature 309: 255 258; Gossler et al., 1986, Proc. Natl. Acad. Sci. USA 83: 9065 9069; and Robertson et al., 1986, Nature 322: 445 448). Transgenes can be efficiently introduced into the ES cells by a variety of standard techniques such as DNA transfection, microinjection, or by retrovirus-mediated transduction. The resultant transformed ES cells can thereafter be combined with blastocysts from a non-human animal. The introduced ES cells thereafter colonize the embryo and contribute to the germ line of the resulting chimeric animal (Jaenisch, 1988, Science 240: 1468 1474). The use of gene-targeted ES cells in the generation of gene-targeted transgenic mice was described in 1987 (Thomas et al., Cell 51:503 512, (1987)) and is reviewed elsewhere (Frohman et al., Cell 56:145 147 (1989); Capecchi, Trends in Genet. 5:70 76 (1989); Baribault et al., Mol. Biol. Med. 6:481 492, (1989); Wagner, EMBO J. 9:3025 3032 (1990); Bradley et al., Bio/Technology 10:534 539 (1992)). See also, U.S. Pat. No. 5,464,764, issued to Cappecchi and Thomas on Nov. 7, 1995; U.S. Pat. No. 5,789,215, issued to Berns et al on Aug. 4, 1998, both of which are hereby incorporated by reference). Therefore, techniques are available in the art to generate the transgenic animal cells, non-human transgenic embryos, non-human transgenic animals and non-human transgenic littermates of the present invention. The methods for evaluating the targeted recombination events as well as the resulting knockout mice are also readily available and known in the art. Such methods include, but are not limited to DNA (Southern) hybridization to detect the targeted allele, polymerase chain reaction (PCR), polyacrylamide gel electrophoresis (PAGE), in situ hybridization, RNA/Northern hybridization and Western blots to detect DNA, RNA and protein.

It is now well known in the art that various strategies are readily available to the artisan to generated transgenic animals, such as transgenic "knock-in" animals. For example, BAC recombination technologies, the following of which are expressly incorporated by reference in their entirety, include but are not limited to the teachings of Shizuya, et al., 1992, Proc. Natl. Acad. Sci. USA 89: 8794 8797 (introduction of BAC vectors); Zhang et al., 1998, Nature Genetics 20: 123 128 and Muyrers, et al., 2001, Nucleic Acids Research 27(6): 1555 1557 (modification of BAC clones via plasmid based expression of recA/recT proteins from the Rac phage or rad.alpha. or rad.beta. from .lamda. phage, respectively, for a review see also Muyrers et al. 2001, Trends in Biochemical Sciences 26(5): 325 331); Yu et al. 2000, Proc. Natl. Acad. Sci. USA 97(11): 5978 5983 and Lee et al., 2001, Genomics 73: 56 65 (use of a defective .lamda. prophage to provide for rad.alpha. or rad.beta. proteins to promote BAC-based recombination). These technologies allow for the efficient engineering and manipulation of BAC clones to generate an appropriate targeting vector delivery to and recombination within ES cells or harvested pronuclie. It is also known that techniques are readily available that promote site specific recombination, allowing for precise chromosome and transgene engineering. For a review of two well known systems, the FLP recombinase from yeast and Cre recombinase system from bacteriophage P1, see Kilby et al., 1993, Trends Genetics 9: 413 421, as well as U.S. Pat. Nos. 5,654,182; 5,677,177; and 5,885,836 (FLP/frt) and U.S. Pat. No. 4,959317 (Cre/loxP), each U.S. patent which is hereby incorporated by reference in their entirety. Therefore, this technology may be utilized to identify a RAMP1 genomic clone (such as a mouse genomic clone), modifying such a genomic clone so as to humanize the coding sequence (i.e., Lys to Trp at amino acid residue 74; where, for example, in generating a transgenic mouse, the only modification required will be the mutagenesis of 2 nucleotides to change the Lysine (AAG) to a Tryptophan (TGG), which results in introduction of a BstNI restriction site [from CCAAG to CCTGG], which is helpful for screening purposes) and to then deliver and stably incorporate, either by homologous or non-homologous recombination, to an ES cell or pronucleus. To provide guidance in developing a humanized mouse "knock in" strategy, the mouse sequence consortium (MSC) database is queried with RAMP1 nucleotide sequence. An initial search resulted in 2 mouse genomic sequence "hits" which were identified as mouse RAMP1. These 2 hits encoded putative exons 2 and 3 of mouse RAMP1. Putative exons 2 and 3 were found on a 712 bp fragment and a 1339 bp contig of 2 fragments, respectively. Putative exon 3 contains amino acid residue 74. This information can be utilized to design a probe for mouse BAC library screening to obtain putative exon 3 and the surrounding intronic sequence for targeting vector construction. The genomic organization appears to be conserved between human and mouse with intron/exon borders located at similar residues (Derst et al., 2000, Cytogenet Cell Genet 90: 115 118).

It will be within the scope of the invention to submit screened compounds which show an in vitro modulation effect on humanized CGRP receptor to in vivo analysis, preferably by administering the compound of interest to either a transgenic or wild-type animal as described herein to measure in vivo effects of the compound on this humanized CGRP receptor and to further measure biological and physiological effects of compound administration on the non-human animal. These in vivo studies may be done either alone or in combination with a known RAMP1.

The transgenic non-human animal models as described herein will be useful to screen any potential modulator of CGRP receptor activity (e.g., antagonists or agonists), including but not necessarily limited to peptides, proteins, or non-proteinaceous organic or inorganic molecules. To this end, the present invention relates to processes for the production of the transgenic animals of the present invention and their offspring and their use for pharmacological testing. The invention further relates to methods of determining the selectivity and activity of potential modulators (especially antagonists) of humanized CGRP receptors expressed within transgenic animals of the present invention by administering a test compound or compounds to the transgenic animal and measuring the effect of the compound on the activity of the humanized CGRP receptor. To this end, the present invention relates to various occupancy assays which may be run in conjunction with the transgenic non-human animals of the present invention.

As used and exemplified herein, a transgene is a genetic construct including a gene. The transgene of interest is incorporated into the target genome of the target cell, thus being introduced into their germ cells and/or somatic cells such that it is stably incorporated and is capable of carrying out a desired function. While a chromosome is the preferred target for stable incorporation of a transgene into the target animal, the term "genome" refers to the entire DNA complement of an organism, including nuclear DNA (chromosomal or extrachromosomal DNA) as well as mitochondrial DNA, which is localized within the cytoplasm of the cell. Thus, the transgenic non-human animal of the present invention will stably incorporate one or more transgenes in either/or of the mouse germ cells or somatic cells (preferably both), such that the expression of the transgene (e.g., a humanized form of mammalian RAMP1) achieves the desired effect of presenting a specific receptor occupancy model for modulators of human RAMP1 as well as providing for an pharmacodynamic animal model system to study the selectivity of test compounds to modulate the human RAMP1 receptor. It is preferable to introduce the transgene into a germ line cell, thereby conferring the ability to transfer the information to offspring. If offspring in fact possess some or all of the genetic information, then they, too, are transgenic animals.

As used herein, the term "animal" may include all mammals, except that when referring to transgenic animals, the use of this term excludes humans. It also includes an individual animal in all stages of development, including embryonic and fetal stages. A "transgenic animal" is an animal containing one or more cells bearing genetic information received, directly or indirectly, by deliberate genetic manipulation at a subcellular level, such as by microinjection, targeted gene delivery such as by homologous recombination, or infection with recombinant virus. As noted above, this introduced DNA molecule (i.e., transgene) can be integrated within a chromosome, or it can be extra-chromosomally replicating DNA.

As used herein in reference to transgenic animals of this invention, we refer to "transgenes" and "genes". A gene is a nucleotide sequence that encodes a protein, or structural RNA. The gene and/or transgene may also include genetic regulatory elements and/or structural elements known in the art. As used and exemplified herein, a transgene is a genetic construct including a gene. The transgene is integrated into one or more chromosomes in the cells in an animal by methods known in the art. Once integrated, the transgene is carried in at least one place in the genome, preferably a chromosome, of a transgenic animal.

As used herein, "founder" refers to a transgenic animal which develops from the microinjected egg. The founders are tested for expression of a functional gene by any suitable assay of the gene product.

As used herein, the term "line" refers to animals that are direct descendants of one founder and bearing one transgene locus stably integrated into their germline.

As used herein, the term "inbred line" refers to animals which are genetically identical at all endogenous loci. As used in the art, inbred lines may be used for including reproducibility from one animal to the next, ability to transfer cells or tissue among animals, and the ability to carry out defined genetic studies to identify the role of endogenous genes. Such inbred lines may be developed from such lines wherein the mice that are used for microinjection are members of established inbred strains.

As used herein, the term "genotype" is the genetic constitution of an organism.

As used herein, the term "phenotype" is a collection of morphological, physiological and/or biochemical traits possessed by a cell or organism that results from the interaction of the genotype and the environment. Included in this definition of phenotype is a biochemical trait wherein a non-native transgene has been introduced into the animal, thus altering its the genotypic profile, and whereby expression of this transgene(s) within the animal results in a new pharmacological selectivity to one or more chemical compounds, such a selectivity based on functional expression of the transgene(s) of interest. To this end, the term "phenotypic expression" relates to the expression of a transgene or transgenes which results in the production of a product, e.g., a polypeptide or protein, or alters the expression of the zygote's or the organism's natural phenotype.

The transgene of interest is incorporated into the target genome of the target cell, thus being introduced into their germ cells and/or somatic cells such that it is stably incorporated and is capable of carrying out a desired function. While a chromosome is the preferred target for stable incorporation of a transgene into the target animal, the term "genome" refers to the entire DNA complement of an organism, including nuclear DNA (chromosomal or extrachromosomal DNA) as well as mitochondrial DNA, which is localized within the cytoplasm of the cell. Thus, as noted previously, the transgenic non-human animals of the present invention will stably incorporate one or more transgenes in either/or of the animal's germ cells or somatic cells (preferably both), such that the expression of the transgene (e.g., a functional, humanized version of RAMP1) achieves the desired effect of presenting a specific receptor occupancy model for modulators of a humanized CGRP receptor as well as providing for an pharmacodynamic animal model system to study the selectivity of test compounds to modulate a humanized CGRP receptor which comprises an endogenous CRLR protein and a humanized RAMP1 protein. It is preferable to introduce the transgene into a germ line cell, thereby conferring the ability to transfer the information to offspring. If offspring in fact possess some or all of the genetic information, then they, too, are transgenic animals.

As used herein, the term "animal" may include all mammals, except that when referring to transgenic animals, the use of this term excludes humans. It also includes an individual animal in all stages of development, including embryonic and fetal stages. A "transgenic animal" is an animal containing one or more cells bearing genetic information received, directly or indirectly, by deliberate genetic manipulation at a subcellular level, such as by microinjection, targeted gene delivery such as by homologous recombination, or infection with recombinant virus. As noted above, this introduced DNA molecule (i.e., transgene) can be integrated within a chromosome, or it can be extra-chromosomally replicating DNA. In a preferred aspect of the present invention a targeted "knock-in" is performed whereby a humanized version of RAMP1 is inserted and replaces the endogenous RAMP1 coding region.

The degeneracy of the genetic code is such that, for all but two amino acids, more than a single codon encodes a particular amino acid. This allows for the construction of synthetic DNA that encodes a humanized RAMP1 protein where the nucleotide sequence of the synthetic DNA differs significantly from the nucleotide sequences disclosed herein, as exemplification but not limitations, but still encodes a humanized RAMP1 protein. Such synthetic DNAs are intended to be within the scope of the present invention. If it is desired to express such synthetic DNAs in a particular host cell or organism, the codon usage of such synthetic DNAs can be adjusted to reflect the codon usage of that particular host, thus leading to higher levels of expression of the humanized RAMP1 protein in the host. In other words, this redundancy in the various codons which code for specific amino acids is within the scope of the present invention. Therefore, this invention is also directed to those DNA sequences which encode RNA comprising alternative codons which code for the eventual translation of the identical amino acid, as shown below: A=Ala=Alanine: codons GCA, GCC, GCG, GCU C=Cys=Cysteine: codons UGC, UGU D=Asp=Aspartic acid: codons GAC, GAU E=Glu=Glutamic acid: codons GAA, GAG F=Phe=Phenylalanine: codons UUC, UUU G=Gly=Glycine: codons GGA, GGC, GGG, GGU H=His=Histidine: codons CAC, CAU I=Ile=Isoleucine: codons AUA, AUC, AUU K=Lys=Lysine: codons AAA, AAG L=Leu=Leucine: codons UUA, UUG, CUA, CUC, CUG, CUU M=Met=Methionine: codon AUG N=Asp=Asparagine: codons AAC, AAU P=Pro=Proline: codons CCA, CCC, CCG, CCU Q=Gln=Glutamine: codons CAA, CAG R=Arg=Arginine: codons AGA, AGG, CGA, CGC, CGG, CGU S=Ser=Serine: codons AGC, AGU, UCA, UCC, UCG, UCU T=Thr=Threonine: codons ACA, ACC, ACG, ACU V=Val=Valine: codons GUA, GUC, GUG, GUU W=Trp=Tryptophan: codon UGG Y=Tyr=Tyrosine: codons UAC, UAU Therefore, the present invention discloses codon redundancy which may result in differing DNA molecules expressing an identical protein. For purposes of this specification, a sequence bearing one or more replaced codons will be defined as a degenerate variation. Another source of sequence variation may occur through RNA editing, as discussed infra. Such RNA editing may result in another form of codon redundancy, wherein a change in the open reading frame does not result in an altered amino acid residue in the expressed protein. Also included within the scope of this invention are mutations either in the DNA sequence or the translated protein which do not substantially alter the ultimate physical properties of the expressed protein. For example, substitution of valine for leucine, arginine for lysine, or asparagine for glutamine may not cause a change in functionality of the polypeptide.

It is known that DNA sequences coding for a peptide may be altered so as to code for a peptide having properties that are different than those of the naturally occurring peptide. Methods of altering the DNA sequences include but are not limited to site directed mutagenesis. Examples of altered properties include but are not limited to changes in the affinity of an enzyme for a substrate or a receptor for a ligand.

"Identity" is a measure of the identity of nucleotide sequences or amino acid sequences. In general, the sequences are aligned so that the highest order match is obtained. "Identity" per se has an art-recognized meaning and can be calculated using published techniques. See, e.g.,: (Computational Molecular Biology, Lesk, A. M., ed. Oxford University Press, New York, 1988; Biocomputing: Informatics and Genome Projects, Smith, D. W., ed., Academic Press, New York, 1993; Computer Analysis of Sequence Data, Part I, Griffin, A. M., and Griffin, H. G., eds. Humana Press, New Jersey, 1994; Sequence Analysis in Molecular Biology, von Heinje, G., Academic Press, 1987; and Sequence Analysis Primer, Gribskov, M. and Devereux, J., eds., M Stockton Press, New York, 1991). While there exists a number of methods to measure identity between two polynucleotide or polypeptide sequences, the term "identity" is well known to skilled artisans (Carillo and Lipton, 1988, SIAM J Applied Math 48:1073). Methods commonly employed to determine identity or similarity between two sequences include, but are not limited to, those disclosed in Guide to Huge Computers, Martin J. Bishop, ed., Academic Press, San Diego, 1994, and Carillo and Lipton, 1988, SIAM J Applied Math 48:1073. Methods to determine identity and similarity are codified in computer programs. Preferred computer program methods to determine identity and similarity between two sequences include, but are not limited to, GCG program package (Devereux, et al, 1984, Nucleic Acids Research 12(1):387), BLASTN, and FASTA (Altschul, et al., 1990, J Mol. Biol. 215:403).

As an illustration, by a polynucleotide having a nucleotide sequence having at least, for example, 95% "identity" to a reference nucleotide sequence of SEQ ID NO:1 is intended that the nucleotide sequence of the polynucleotide is identical to the reference sequence except that the polynucleotide sequence may include up to five point mutations or alternative nucleotides per each 100 nucleotides of the reference nucleotide sequence of SEQ ID NO:1. In other words, to obtain a polynucleotide having a nucleotide sequence at least 95% identical to a reference nucleotide sequence, up to 5% of the nucleotides in the reference sequence may be deleted or substituted with another nucleotide, or a number of nucleotides up to 5% of the total nucleotides in the reference sequence may be inserted into the reference sequence. These mutations or alternative nucleotide substitutions of the reference sequence may occur at the 5' or 3' terminal positions of the reference nucleotide sequence or anywhere between those terminal positions, interspersed either individually among nucleotides in the reference sequence or in one or more contiguous groups within the reference sequence. One source of such a "mutation" or change which results in a less than 100% identity may occur through RNA editing. The process of RNA editing results in modification of an mRNA molecule such that use of that modified mRNA as a template to generate a cloned cDNA may result in one or more nucleotide changes, which may or may not result in a codon change. This RNA editing is known to be catalyzed by an RNA editase. Such an RNA editase is RNA adenosine deaminase, which converts an adenosine residue to an inosine residue, which tends to mimic a cytosine residue. To this end, conversion of an mRNA residue from A to I will result in A to G transitions in the coding and noncoding regions of a cloned cDNA (e.g., see Hanrahan et al, 1999, Annals New York Acad. Sci. 868:51 66; for a review see Bass, 1997, TIBS 22: 157 162). Similarly, by a polypeptide having an amino acid sequence having at least, for example, 95% identity to a reference amino acid sequence of SEQ ID NO:2 is intended that the amino acid sequence of the polypeptide is identical to the reference sequence except that the polypeptide sequence may include up to five amino acid alterations per each 100 amino acids of the reference amino acid of SEQ ID NO:2. In other words, to obtain a polypeptide having an amino acid sequence at least 95% identical to a reference amino acid sequence, up to 5% of the amino acid residues in the reference sequence may be deleted or substituted with another amino acid, or a number of amino acids up to 5% of the total amino acid residues in the reference sequence may be inserted into the reference sequence. These alterations of the reference sequence may occur at the amino or carboxy terminal positions of the reference amino acid sequence of anywhere between those terminal positions, interspersed either individually among residues in the reference sequence or in one or more contiguous groups within the reference sequence. Again, as noted above, RNA editing may result in a codon change which will result in an expressed protein which differs in "identity" from other proteins expressed from "non-RNA edited" transcripts, which correspond directly to the open reading frame of the genomic sequence. Therefore, the concept of nucleic acid sequence identity is applicable to the present invention in the context that variations, other than "humanization" of amino acid residue 74, are within the scope of the present invention so long as those variations do not significantly effect the ability of the respective expressed RAMP1 protein to mimic human RAMP1 when associated with a mammalian CRLR protein.

As stated earlier in this section, the present invention also relates to recombinant vectors and recombinant hosts, both prokaryotic and eukaryotic, which contain the substantially purified nucleic acid molecules disclosed throughout this specification. The nucleic acid molecules of the present invention encoding a RAMP1 protein, in whole or in part, can be linked with other DNA molecules, i.e, DNA molecules to which the RAMP1 coding sequence are not naturally linked, to form "recombinant DNA molecules" which encode a respective RAMP1 protein. The DNA molecules of the present invention can be inserted into vectors which comprise nucleic acids encoding RAMP1 or a functional equivalent. These vectors may be comprised of DNA or RNA; for most cloning purposes DNA vectors are preferred. Typical vectors include plasmids, modified viruses, bacteriophage, cosmids, yeast artificial chromosomes, and other forms of episomal or integrated DNA that can encode a RAMP1 protein. It is well within the purview of the skilled artisan to determine an appropriate vector for a particular gene transfer or other use. Therefore, as with many proteins, it is possible to modify many of the amino acids of RAMP1 protein and still retain substantially the same biological activity as the wild type protein. Thus this invention includes modified RAMP1 polypeptides which have amino acid deletions, additions, or substitutions but that still retain substantially the same biological activity as a respective, corresponding humanized RAMP1 (i.e, wherein amino acid 74 is a tryptophan residue and any other changes do not significantly effect the ability of the altered RAMP1 to mimic human pharmacological characteristics as the human CGRP receptor). It is disclosed herein that the essence of the present invention is the ability to humanize a vertebrate RAMP1 protein by altering the vertebrate RAMP1 amino acid sequence at residue 74 to a tryptophan residue. Therefore, alteration of just a single amino acid resulted in a completely different, and now predictable, pharmacological profile for such a mutated protein. This is a surprising result given that historically it was generally accepted that single amino acid substitutions do not usually alter the biological activity of a protein (see, e.g., Molecular Biology of the Gene, Watson et al., 1987, Fourth Ed., The Benjamin/Cummings Publishing Co., Inc., page 226; and Cunningham & Wells, 1989, Science 244:1081 1085). To this end, the present invention also discloses that minor additional alterations (such as one, two or several non-silent codon changes) will not effect the humanizing characteristic of a RAMP1 protein with the Trp74 modification, and accordingly, such mutant protein are within the scope of the present invention. Therefore, the present invention includes polypeptides where one or more additional amino acid substitutions has been made in SEQ ID NOs:2, 4, 6, and/or 8, wherein the polypeptides still retain substantially the same biological activity as a corresponding RAMP1 protein. For example, mutation of rat K74W to include a mutation at Lys 103 to a Ser residue. This humanized double mutant shows the same "humanized" pharmacological profile as rat K74W, showing that an additional amino acid substitution does not deleteriously effect the ability of the K74W to "humanize" the RAMP1 protein. The present invention also includes polypeptides where two or more amino acid substitutions have been made in SEQ ID NOs:2, 4, 6, or 8, wherein the polypeptides still retain substantially the same biological activity as a corresponding RAMP1 protein. In particular, the present invention includes embodiments where the above-described substitutions are conservative substitutions. To this end, one of ordinary skill in the art would also recognize that polypeptides that are functional equivalents of RAMP1 and have changes from the RAMP1 amino acid sequence that are small deletions or insertions of amino acids could also be produced by following the same guidelines, (i.e, minimizing the differences in amino acid sequence between RAMP1 and related proteins. Small deletions or insertions are generally in the range of about 1 to 5 amino acids. The effect of such small deletions or insertions on the biological activity of the modified RAMP1 polypeptide can easily be assayed by producing the polypeptide synthetically or by making the required changes in DNA encoding RAMP1 and then expressing the DNA recombinantly and assaying the protein produced by such recombinant expression. For instance, as long as amino acid residue 74 remains in a "humanized" form (i.e., Trp), then minor modifications to the remainder of the RAMP1 sequence may be generated and are in turn easily tested alongside an expressed CRLR receptor to determine if the expected human pharmacological profile remains. Furthermore, the present invention also includes truncated forms of RAMP1. Such truncated proteins are useful in various assays described herein, for crystallization studies, and for structure-activity-relationship studies.

The present invention also relates to isolated nucleic acid molecules which are fusion constructions expressing fusion proteins useful in assays to identify compounds which modulate wild-type RAMP1 activity, as well as generating antibodies against RAMP1. One aspect of this portion of the invention includes, but is not limited to, glutathione S-transferase (GST)-RAMP1 fusion constructs. Recombinant GST-RAMP1 fusion proteins may be expressed in various expression systems, including Spodoptera frugiperda (Sf21) insect cells (Invitrogen) using a baculovirus expression vector (pAcG2T, Pharmingen). Another aspect involves RAMP1 fusion constructs linked to various markers, including but not limited to GFP (Green fluorescent protein), the MYC epitope, His-tag, and GST. Again, any such fusion constructs may be expressed in the cell line of interest and used to screen for modulators of one or more of the RAMP1 proteins disclosed herein, as well as being expressed and purified.

Any of a variety of procedures may be used to clone and generate a vertebrate or mammalian RAMP1, such as rat, mouse, human, etc., RAMP1. These methods include, but are not limited to, (1) a RACE PCR cloning technique (Frohman, et al., 1988, Proc. Natl. Acad. Sci. USA 85: 8998 9002). 5' and/or 3' RACE may be performed to generate a full-length cDNA sequence. This strategy involves using gene-specific oligonucleotide primers for PCR amplification of RAMP1 cDNA. These gene-specific primers are designed through identification of an expressed sequence tag (EST) nucleotide sequence which has been identified by searching any number of publicly available nucleic acid and protein databases; (2) direct functional expression of the RAMP1 cDNA following the construction of a RAMP1-containing cDNA library in an appropriate expression vector system; (3) screening a RAMP1-containing cDNA library constructed in a bacteriophage or plasmid shuttle vector with a labeled degenerate oligonucleotide probe designed from the amino acid sequence of the RAMP1 protein; (4) screening a RAMP1-containing cDNA library constructed in a bacteriophage or plasmid shuttle vector with a partial cDNA encoding the RAMP1 protein. This partial cDNA is obtained by the specific PCR amplification of RAMP1 DNA fragments through the design of degenerate oligonucleotide primers from the amino acid sequence known for other proteins which are related to the RAMP1 protein; (5) screening a RAMP1-containing cDNA library constructed in a bacteriophage or plasmid shuttle vector with a partial cDNA or oligonucleotide with homology to a RAMP1 protein. This strategy may also involve using gene-specific oligonucleotide primers for PCR amplification of RAMP1 cDNA identified as an EST as described above; or (6) designing 5' and 3' gene specific oligonucleotides using any of the disclosed mammalian RAMP1 sequences as a template so that either the full-length cDNA may be generated by known RACE techniques, or a portion of the coding region may be generated by these same known RACE techniques to generate and isolate a portion of the coding region to use as a probe to screen one of numerous types of cDNA and/or genomic libraries in order to isolate a full-length version of the nucleotide sequence encoding RAMP1. It is readily apparent to those skilled in the art that other types of libraries, as well as libraries constructed from other cell types-or species types, may be useful for isolating a RAMP1-encoding DNA or a RAMP1 homologue. Other types of libraries include, but are not limited to, cDNA libraries derived from other brown dog tick cell types.

It is readily apparent to those skilled in the art that suitable cDNA libraries may be prepared from cells or cell lines which have RAMP1 activity. The selection of cells or cell lines for use in preparing a cDNA library to isolate a cDNA encoding RAMP1 may be done by first measuring cell-associated RAMP1 activity using any known assay available for such a purpose.

Preparation of cDNA libraries can be performed by standard techniques well known in the art. Well known cDNA library construction techniques can be found for example, in Sambrook et al., 1989, Molecular Cloning: A Laboratory Manual; Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. Complementary DNA libraries may also be obtained from numerous commercial sources, including but not limited to Clontech Laboratories, Inc. and Stratagene.

This invention also includes vectors containing a humanized RAMP1 gene, host cells containing the vectors, and methods of making substantially pure humanized RAMP1 protein comprising the steps of introducing the humanized RAMP1 gene into a host cell, and cultivating the host cell under appropriate conditions such that humanized RAMP1 is produced. The humanized RAMP1 so produced may be harvested from the host cells in conventional ways. Therefore, the present invention also relates to methods of expressing the humanized RAMP1 protein and biological equivalents disclosed herein, assays employing these gene products, recombinant host cells which comprise DNA constructs which express these proteins, and compounds identified through these assays which act as agonists or antagonists of humanized RAMP1 activity.

The cloned humanized RAMP1 cDNA obtained through the methods described above may be recombinantly expressed by molecular cloning into an expression vector (such as pcDNA3.neo, pcDNA3.1, pCR2.1, pBlueBacHis2, pLITMUS28, the pIRES series from Clontech, as well as other examples, listed infra) containing a suitable promoter and other appropriate transcription regulatory elements, and transferred into prokaryotic or eukaryotic host cells to produce recombinant humanized RAMP1. Expression vectors are defined herein as DNA sequences that are required for the transcription of cloned DNA and the translation of their mRNAs in an appropriate host. Such vectors can be used to express eukaryotic DNA in a variety of hosts such as bacteria, blue green algae, plant cells, insect cells and animal cells. Specifically designed vectors allow the shuttling of DNA between hosts such as bacteria-yeast or bacteria-animal cells. An appropriately constructed expression vector should contain: an origin of replication for autonomous replication in host cells, selectable markers, a limited number of useful restriction enzyme sites, a potential for high copy number, and active promoters. A promoter is defined as a DNA sequence that directs RNA polymerase to bind to DNA and initiate RNA synthesis. A strong promoter is one which causes mRNAs to be initiated at high frequency. To determine the humanized RAMP1 cDNA sequence(s) that yields optimal levels of humanized RAMP1, cDNA molecules including but not limited to the following can be constructed: a cDNA fragment containing the full-length open reading frame for humanized RAMP1 as well as various constructs containing portions of the cDNA encoding only specific domains of the protein or rearranged domains of the protein. All constructs can be designed to contain none, all or portions of the 5' and/or 3' untranslated region of a humanized RAMP1 cDNA. The expression levels and activity of RAMP1 can be determined following the introduction, both singly and in combination, of these constructs into appropriate host cells. Following determination of the humanized RAMP1 cDNA cassette yielding optimal expression in transient assays, this humanized RAMP1 cDNA construct is transferred to a variety of expression vectors (including recombinant viruses), including but not limited to those for mammalian cells, plant cells, insect cells, oocytes, bacteria, and yeast cells. Techniques for such manipulations can be found described in Sambrook, et al., supra, are well known and available to the artisan of ordinary skill in the art. Therefore, another aspect of the present invention includes host cells that have been engineered to contain and/or express DNA sequences encoding the humanized RAMP1. An expression vector containing DNA encoding a humanized RAMP1-like protein may be used for expression of humanized RAMP1 in a recombinant host cell. Such recombinant host cells can be cultured under suitable conditions to produce humanized RAMP1 or a biologically equivalent form. Expression vectors may include, but are not limited to, cloning vectors, modified cloning vectors, specifically designed plasmids or viruses. Commercially available mammalian expression vectors which may be suitable for recombinant humanized RAMP1 expression, include but are not limited to, pcDNA3.neo (Invitrogen), pcDNA3.1 (Invitrogen), pCI-neo (Promega), pLITMUS28, pLITMUS29, pLITMUS38 and pLITMUS39 (New England Bioloabs), pcDNAI, pcDNAIamp (Invitrogen), pcDNA3 (Invitrogen), pMClneo (Stratagene), pXT1 (Stratagene), pSG5 (Stratagene), EBO-pSV2-neo (ATCC 37593) pBPV-1(8-2) (ATCC 37110), pdBPV-MMTneo(342-12) (ATCC 37224), pRSVgpt (ATCC 37199), pRSVneo (ATCC 37198), pSV2-dhfr (ATCC 37146), pUCTag (ATCC 37460), 1ZD35 (ATCC 37565) and the pIRES series (Clontech). Also, a variety of bacterial expression vectors may be used to express recombinant humanized RAMP1 in bacterial cells. Commercially available bacterial expression vectors which may be suitable for recombinant humanized RAMP1 expression include, but are not limited to pCR2.1 (Invitrogen), pET11a (Novagen), lambda gt11 (Invitrogen), and pKK223-3 (Pharmacia). In addition, a variety of fungal cell expression vectors may be used to express recombinant RAMP1 in fungal cells. Commercially available fungal cell expression vectors which may be suitable for recombinant humanized RAMP1 expression include but are not limited to pYES2 (Invitrogen) and Pichia expression vector (Invitrogen). Also, a variety of insect cell expression vectors may be used to express recombinant protein in insect cells. Commercially available insect cell expression vectors which may be suitable for recombinant expression of humanized RAMP1 include but are not limited to pBlueBacIII and pBlueBacHis2 (Invitrogen), and pAcG2T (Pharmingen).

Recombinant host cells may be prokaryotic or eukaryotic, including but not limited to, bacteria such as E. coli, fungal cells such as yeast, mammalian cells including, but not limited to, cell lines of bovine, porcine, monkey and rodent origin; and insect cells. For instance, one insect expression system utilizes Spodoptera frugiperda (Sf21) insect cells (Invitrogen) in tandem with a baculovirus expression vector (pAcG2T, Pharmingen). Also, mammalian cells which may be suitable and which are commercially available, include but are not limited to, L cells L-M(TK.sup.-) (ATCC CCL 1.3), L cells L-M (ATCC CCL 1.2), Saos-2 (ATCC HTB-85), HEK 293 (ATCC CRL 1573), Raji (ATCC CCL 86), CV-1 (ATCC CCL 70), COS-1 (ATCC CRL 1650), COS-7 (ATCC CRL 1651), CHO-K1 (ATCC CCL 61), 3T3 (ATCC CCL 92), NIH/3T3 (ATCC CRL 1658), HeLa (ATCC CCL 2), C127I (ATCC CRL 1616), BS-C-1 (ATCC CCL 26), MRC-5 (ATCC CCL 171) CPAE (ATCC CCL 209), and 293 EBNA cells (Invitrogen).

Expression of humanized RAMP1 DNA may also be performed using in vitro produced synthetic mRNA. Synthetic mRNA can be efficiently translated in various cell-free systems, including but not limited to wheat germ extracts and reticulocyte extracts, as well as efficiently translated in cell based systems, including but not limited to microinjection into frog oocytes, with microinjection into frog oocytes being preferred.

Following expression of humanized RAMP1 in a host cell, humanized RAMP1 protein may be recovered to provide humanized RAMP1 protein in active form. Several humanized RAMP1 protein purification procedures are available and suitable for use. Recombinant humanized RAMP1 protein may be purified from cell lysates and extracts by various combinations of, or individual application of salt fractionation, ion exchange chromatography, size exclusion chromatography, hydroxylapatite adsorption chromatography, hydrophobic interaction chromatography, as well as metal chelate chromotography (e.g., for His-tagged proteins). In addition, recombinant humanized RAMP1 protein can be separated from other cellular proteins by use of an immunoaffinity column made with monoclonal or polyclonal antibodies specific for full-length humanized RAMP1 protein, or polypeptide fragments of humanized RAMP1 protein.

Expression of humanized RAMP1 DNA may also be performed using in vitro produced synthetic mRNA. Synthetic mRNA can be efficiently translated in various cell-free systems, including but not limited to wheat germ extracts and reticulocyte extracts, as well as efficiently translated in cell based systems, including but not limited to microinjection into frog oocytes, with microinjection into frog oocytes being preferred.

Following expression of humanized RAMP1 in a host cell, humanized RAMP1 protein may be recovered to provide humanized RAMP1 protein in active form. Several humanized RAMP1 protein purification procedures are available and suitable for use. Recombinant humanized RAMP1 protein may be purified from cell lysates and extracts by various combinations of, or individual application of salt fractionation, ion exchange chromatography, size exclusion chromatography, hydroxylapatite adsorption chromatography and hydrophobic interaction chromatography, and metal chelate chromatography. In addition, recombinant humanized RAMP1 protein can be separated from other cellular proteins by use of an immunoaffinity column made with monoclonal or polyclonal antibodies specific for full-length humanized RAMP1 protein, or polypeptide fragments of humanized RAMP1 protein.

The humanized RAMP1 proteins of the present invention may be generated by techniques known in the art, as shown in Example Sections 1 and 2, for use in an assay procedure with the CRLR GPCR to identify CGRP receptor modulators (e.g., antagonists of CGRP receptor activity. In general, an assay procedure to identify such receptor modulators will contain a humanized CGRP receptor of the present invention, and a test compound or sample which contains a putative CGRP receptor modulator. The test compounds or samples may be tested directly on, for example, purified receptor protein whether native or recombinant, subcellular fractions of receptor-producing cells whether native or recombinant, and/or whole cells expressing the receptor whether native or recombinant. The test compound or sample may be added to the receptor in the presence or absence of a known labeled or unlabelled receptor ligand. For instance, recombinant membrane fractions containing a humanized CRGP receptor can be used to screen for compounds which inhibit binding of .sup.125I-CGRP to the receptor in a radioligand biding assay. The modulating activity of the test compound or sample may be determined by, for example, analyzing the ability of the test compound or sample to bind to the receptor, activate the receptor, inhibit receptor activity, inhibit or enhance the binding of other compounds to the receptor, modify receptor regulation, or modify an intracellular activity.

The present invention is also directed to methods for screening for compounds which modulate the expression of DNA or RNA encoding a humanized CGRP receptor as well as the function of a humanized CGRP receptor in vivo. Compounds which modulate these activities may be DNA, RNA, peptides, proteins, or non-proteinaceous organic molecules. Compounds may modulate by increasing or attenuating the expression of DNA or RNA encoding CRLR and/or humanized RAMP1 receptor respectively, or the function either protein. Compounds that modulate the expression of DNA or RNA encoding the CGRP receptor or the function of this receptor may be detected by a variety of assays. The assay may be a simple "yes/no" assay to determine whether there is a change in expression or function. The assay may be made quantitative by comparing the expression or function of a test sample with the levels of expression or function in a standard sample.


Claim 1 of 8 Claims

1. A purified nucleic acid molecule encoding a mammalian receptor-activity modifying protein 1 which comprises a nucleotide sequence selected from the group consisting of SEQ ID NO: 1 and 3.
 

 

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