KR20000012151A - Ret ligand to stimulate cultivation of neuron and kidney cell - Google Patents

Abstract

PURPOSE: A nucleotide sequence and a stimulating method of cultivation of neuron and kidney cell by treating a mammal cell with RET ligand or protein. CONSTITUTION: The RET ligand has a nucleotide sequence and provides a purified and separated DNA molecular encoding RetL containing rat RetL1 cDNA(seq. no. 1), partial human retL cDNA(seq. no. 8), full length of human RetL cDNA(seq. no. 10), human RetL cDNA(seq. no. 12), rat RetL3 cDNA(seq. nl. 16), partial human RetL3 cDNA(seq. no. 18) and human RetL3 cDNA(seq. no. 20). The Ret ligand also has a RetL protein containing retL protein sequence of rat RetL1(seq. no. 1), partial human RetL1(seq. no. 9), full length of human RetL1(seq. no. 11), human RetL2(seq. no. 13), rat RetL3(seq. no. 17), partial human RetL3(seq. no. 19) or human Ret L3(seq. no. 21). The neuron and kidney cell is cultivated by introducing the encoding vector of the Ret L.

Description

RET ligands to stimulate nerve and kidney proliferation

One of the goals of existing research on cell signaling and receptor activation is to therapeutically control the processes involved in cell proliferation and survival. This process determines the outcome of various medical symptoms, including organ failure, fetal development and tumor proliferation. Each of these symptoms is of clinical significance worldwide, but there are limited effective treatment options. It is an object of the present invention to provide compositions and methods for promoting the regeneration or survival of damaged tissue and for treating diseases including abnormal proliferation and development of tissue.

Tissue loss or end stage organ dysfunction occurs in millions of populations each year worldwide, substantially increasing health care costs. In general, organ or tissue loss is treated by transplanting an organ from a donor, by reconstructive surgery, or by mechanical device. However, each of these therapies has a drawback. Transplantation is limited in the lack of donors, reconstructive surgery can lead to a number of long-term problems, and mechanical devices cannot perform all the functions of a single organ, preventing progressive degeneration. Therefore, in order to solve these problems newly, there is a demand in the real medical field.

Protein factors that affect the proliferation, differentiation and / or survival of cells can be useful for treating diseases of organs containing responsive cells. Of particular interest are factors or ligands that interact with receptors of the receptor protein tyrosine kinase (RPTK). These receptors are involved in several cellular programs, including cell proliferation and differentiation, and are also involved in the development of several tumor formations. Thus, factors or ligands that interact with these receptors may prove useful for treating diseases of certain organs in which tissue is damaged. Alternatively, it may be useful to block the interaction of receptors with factors to block tumor proliferation.

Ret proto-oncogenes encode receptor tyrosine kinases that are expressed in the development of various tissues, including the peripheral nervous system and the central nervous system and kidneys. Aberrations present in ret null mice suggest that Ret is important for small intestinal neuron migration and nerve domination into the posterior ileum and is important for proliferation and branching of ureteric epithelial cells during kidney development (Nature 367, 380-). 383, 1994). Investigation of important components of the Ret signaling pathway, namely Ret ligands, has been intensively studied.

The present invention relates to a nucleotide sequence encoding Ret Ligand (RetL) and a method for stimulating nerve and kidney proliferation by treating cell and mammalian specimens with RetL DNA or protein.

1 shows the cDNA sequence (SEQ ID NO: 1) and the deduced amino acid sequence (SEQ ID NO: 2) of rat RetL1. The nucleotide sequence extends from base pair 201 of SEQ ID NO: 1 to base pair 1700 and encompasses the entire open reading frame.

2A shows the partial cDNA sequence (SEQ ID NO: 8) and the deduced amino acid sequence (SEQ ID NO: 9) of human RetL1. The sequence is the sequence of the insert of clone HRL20 deposited with ATCC No. 97604.

2B shows the complex full length DNA sequence (SEQ ID NO: 10) and the deduced amino acid sequence (SEQ ID NO: 11) of human RetL1.

3A compares the nucleotide sequence of human RetL1 (top row of sequences) and the rat RetL1 sequence (bottom row of sequences). Vertical lines between nucleotides indicate identity at two positions and dots indicate difference at that position.

3B compares the nucleotide sequence of human RetL1 (top row of sequences) and the rat RetL1 sequence (bottom row of sequences). Vertical lines between the corresponding amino acids show the identity of the residues, with dots representing conservative substitutions at that residue.

4A is a schematic diagram showing the possible role of Ret and RetL in the interaction between ureterocytes and epinephal mesenchymal cells.

4B is a schematic diagram of a method for selecting a transfectant of a cDNA library of clones expressing RetL. The presence of RetL expressed in the transfectant is detected by assessing binding by the transfectant of the Ret / IgG fusion protein or the Ret / alkaline phosphatase fusion protein.

5 is a schematic showing the construction of plasmids used to express rat Ret / IgG fusion proteins.

6 is a schematic showing the construction of a plasmid used to express human Ret / IgG fusion protein.

FIG. 7 is the cDNA sequence of human retL2 (SEQ ID NO: 12) and the inferred amino acid sequence (SEQ ID NO: 13) found in clone DSW240. The protein translation frame is contained between nucleotides 25-1416.

8 compares the amino acid sequence of human RetL2 (top row of sequences) and the human RetL1 sequence (bottom row of sequences). The vertical line between the corresponding amino acids shows the identity at that position, and the dots indicate the difference at that position.

9 shows the cDNA sequence (SEQ ID NO: 16) and the deduced amino acid sequence (SEQ ID NO: 17) of murine RetL3.

10 shows the cDNA sequence (SEQ ID NO: 20) and the deduced amino acid sequence (SEQ ID NO: 21) of human RetL3.

Detailed description of the invention

Sequence number

Nucleotide and amino acid sequences herein are represented by the following sequence numbers.

SEQ ID NO: 1 rat retL1 cDNA

SEQ ID NO: 2 Rat RetL1 Amino Acid

SEQ ID NO: 3 oligomer kid-13

SEQ ID NO: 4 oligomer kid-14

SEQ ID NO: 5 oligomer kid-15

SEQ ID NO: 6 extracellular rat retL1 cDNA

SEQ ID NO: 7 Extracellular Rat RetL1 Amino Acid

SEQ ID NO: 8 partial human retL1 cDNA

SEQ ID NO: 9 partial human RetL1 amino acid

SEQ ID NO: 10 human retL1 cDNA

SEQ ID NO: 11 human RetL1 amino acid

SEQ ID NO: 12 human retL2 cDNA

SEQ ID NO: 13 Human RetL2 amino acid

SEQ ID NO: 14 partial rat retL3 cDNA (EST AA50083)

SEQ ID NO: 15 Partial Mouse RetL3 Amino Acid

SEQ ID NO: 16 murine retL3 cDNA

SEQ ID NO: 17 Rat RetL3 amino acid

SEQ ID NO: 18 partial human retL3 cDNA

SEQ ID NO: 19 partial human RetL3 amino acid

SEQ ID NO: 20 human retL3 cDNA

SEQ ID NO: 21 Human retL3 amino acid

Justice

By “RetL” herein is meant any protein that specifically interacts with the receptor protein Ret and, when interacting with Ret, triggers autophosphorylation of the tyrosine kinase domain of Ret and / or dimerization of Ret. DNA sequences encoding RetL and Ret are referred to as "retL" and "ret", respectively. The ligand may be soluble and may exist as a membrane binding molecule on the same or different cells as the Ret molecule that triggers autophosphorylation. In certain applications or interactions with Ret, ligands may require additional molecules that can trigger autophosphorylation. Ligands of the invention include co-receptor or auxiliary ligand cofactors. The ligands of the invention also include anti-Ret mAbs that act as Ret antagonists, which triggers Ret dimerization and autophosphorylation. Ligands can also be modified by inserting as part of a variety of methods, such as fusion proteins with toxins or radionuclides.

"Sequence alignment" means that one of the sequences of nucleotides or amino acids is aligned with the sequence of the other so that sequences of related parts can be compared. An example of one method of this process is described in Needleman et al., J. Mol. Biol. 48: 443-453, 1970. This method can usually be performed by a computer program, such as an alignment program (DNAstar Inc.). As will be appreciated by those skilled in the art, homologous or functionally equivalent sequences include functionally equivalent arrangements of cysteine residues within the conserved cysteine backbone, including amino acid insertions or deletions that alter the linear alignment of cysteine, but are not It doesn't really compromise the relevance. Thus, internal differences and amino acid sequence insertions in the sequence are ignored in calculating the degree of amino acid sequence homology or identity between the sequence and the reference sequence. A feature often used to establish protein identity is the similarity in the number and position of cysteine residues between one protein and another.

"Clonening" means inserting a particular gene or other DNA sequence into a vector molecule using in vitro recombinant techniques. In order to successfully clone the desired gene, the steps of generating a DNA fragment, linking the fragment into a vector, introducing a complex DNA molecule into a replicable host cell, and selecting a clone carrying the target gene from a recipient host cell There is a need to use a method that includes steps.

"cDNA" means complementary DNA or copy DNA produced from an RNA template by the action of an RNA dependent DNA polymerase (reverse transcriptase). Thus, "cDNA clone" refers to a double-stranded DNA sequence that is complementary to the RNA molecule of interest contained in the cloning vector.

A "cDNA library" is a collection of recombinant DNA molecules containing cDNA inserts that together include representative mRNA molecules present in whole organism or tissue, depending on the source of RNA template. This cDNA library can be prepared by methods known to those skilled in the art and as disclosed, for example, in Maniatis et al., Molecular Cloning: A Laboratory Manual, supra. In general, RNA is first isolated from cells of an organism if it is desired to clone a particular gene from the genome. For the purposes of the present invention, mammalian cell lines are preferred, and human cell lines are particularly preferred. Alternatively, RNA can be isolated from animal tumors, particularly tumor cells derived from human tumors. Thus, libraries can be prepared, for example, from human adrenal tumors, but any tumor can be used.

As used herein, “DNA polymorphism” refers to conditions under which two or more different nucleotide sequences may be present at a particular site of DNA.

"Expression vector" means a vector capable of expressing a DNA sequence contained in a vector, ie, a vector in which the coding sequence is operably bound to other sequences capable of expression. Although not always explicitly stated, these expression vectors must replicate in the host organism as endogenous portions of episomal or chromosomal DNA. A non-essential but useful factor of an effective expression vector is a marker coding sequence, which encodes a protein that results in the phenotypic characteristics of the cell (eg tetracycline resistance) containing the protein that allows for easy identification of the cell. In short, an "expression vector" is a functional definition, and when applied to a particular sequence, any DNA sequence capable of expressing a particular included DNA code is included in this term. This vector is often in the form of a plasmid, and "plasmid" and "expression vector" can often be used interchangeably. However, the present invention includes forms of other expression vectors, including phage, which have equivalent functions and may sometimes be known in the art.

Similarly, "functional derivatives" of genes of any protein of the invention include "fragments", "variants" and "analogs" of the genes, which may be "substantially similar" in nucleotide sequence and exhibit similar activity. Encrypt the molecule you hold.

"GDNF related molecule" means any molecule that is at least 40% homologous to GDNF or neutrin and can specifically bind to RetL.

"Gene" means a polynucleotide encoding a peptide.

By “homology” is meant that when referring to a peptide or DNA sequence, the primary molecular structure (ie, amino acid or nucleotide sequence) of almost all molecules present in the composition is identical under various considerations.

As used herein, "oligonucleotide" as used to refer to a probe, an oligomer fragment to be detected, an oligomer control, an unlabeled blocking oligomer and a primer for amplification of a sequence means a molecule consisting of three or more deoxyribonucleotides or ribonucleotides. . The exact size depends on several factors and depends on the ultimate function or use of the oligonucleotide.

"Probe" means a known amount of ligand that can selectively bind to a target antiligand. When used in nucleic acids, "probe" refers to a strand of nucleic acid having a nucleotide sequence complementary to the target strand.

"Recombinant host cell" means a cell transformed with a vector constructed using recombinant DNA technology. As defined herein, an antibody or variant thereof formed by a recombinant host cell, in small amounts, more generally less than detectable, may be produced by a transformant rather than produced by an untransformed host. It was created.

As used herein, "limiting endonuclease" and "limiting enzyme" refer to bacterial enzymes, each of which cleaves double stranded DNA at or near a particular nucleotide sequence.

"Restriction fragment length polymorphism" ("RFLP") means that the particular restriction fragments have different lengths.

A molecule is said to be "substantially similar" to another molecule when the sequences of amino acids in both molecules are nearly similar and when the two molecules possess similar biological activity. Thus, given two molecules with similar activity, one molecule may further comprise amino acid residues that are not found in another molecule, or variants even when the amino acid residue sequences are not identical. As used herein, when it includes additional chemical moieties that are not usually part of a molecule, it is referred to as a "chemical derivative" of another molecule. This part can improve the solubility of molecules, adsorption, biological half-life, and so on. In addition, this portion may reduce the toxicity of the molecule and eliminate or attenuate any undesirable side effects of the molecule and the like. Portions that can mediate this effect are described, for example, in Remington's Pharmaceutical Sciences, 16th Edition, Mack Publishing Co., Easton, Penn. (1980).

"Vector" refers to a DNA molecule derived from a plasmid or bacteriophage capable of inserting or cloning a DNA fragment. The vector contains one or more unique restriction sites and can autonomously replicate in a defined host or mediated organism to propagate cloned sequences.

"Substantially pure" means a gene or any protein of the invention, each of which encodes any protein with little or no other protein or gene, or little other contaminant commonly found in nature, and in such a form Not found

Compound of the Invention

The present invention includes cDNAs encoding RetL, such as nucleotide sequences of rat retL1 cDNA, partial human retL1 cDNA, full length human retL1 cDNA, human retL2 cDNA, murine retL3 cDNA or human retL3 cDNA. In addition, the compounds of the present invention include sequences comprising derivatives of the above sequences or one of these sequences. The present invention also encompasses vectors, liposomes and other carrier media comprising derivatives of one of these sequences or of one of these sequences. The present invention includes, but is not limited to, rat RetL1, partial human RetL1, full length human RetL1, human RetL2, rat RetL3 or human RetL3, rat retL1 cDNA, partial human retL1 cDNA, full human retL2 cDNA, partial human retL2 cDNA, rat retL3 Also included are proteins transcribed and translated from cDNA or human retL3 cDNA, as well as derivatives and variants thereof.

One aspect of the invention includes soluble variants of RetL. Soluble variants lack at least some of the dural portion of native RetL. In some instances, the soluble variant lacks the phosphatidylinositol glycan bond of the native RetL. Soluble variants include fusion proteins comprising derivatives of RetL lacking a phosphatidylinositol motif.

Variants may differ from naturally occurring RetL in an amino acid sequence or in a manner that does not include sequences, or both. Substitution of one or more amino acids of naturally occurring RetL with another natural amino acid, amino acid derivative or non-natural amino acid forms a variant of the amino acid sequence. Particularly preferred variants include naturally occurring RetL, or biologically active fragments of naturally occurring RetL, wherein the sequence of RetL differs from the wild-type sequence by one or more conservative amino acid substitutions, and depends on the hydrophobicity and secondary structure of the protein or peptide. Minimal impact is common. In addition, variants may include sequences that are varied by one or more non-conservative amino acid substitutions, deletions, or insertions that do not disrupt RetL biological activity. Conservative substitutions usually include, for example, replacing one amino acid with another amino acid having similar properties in the following group; Valine, glycine; Glycine, alanine; Valine, isoleucine; Aspartamic acid, glutamic acid; Asparagine, glutamine; Serine, threonine; Lysine, arginine; And phenylalanine, tyrosine. Nonpolar (hydrophobic) amino acids include alanine, leucine, isoleucine, valine, proline, phenylalanine, tryptophan and methionine. Polar neutral amino acids include glycine, serine, threonine, cysteine, tyrosine, asparagine and glutamine. Positively charged (basic) amino acids include arginine, lysine and histidine. Negatively charged (acidic) amino acids include aspartamic acid and glutamic acid.

Other conservative substitutions are taken from the table below and others are disclosed in Dayhoff's Atlas of Protein Sequence and Structure (1988).

Conservative Amino Acid Substitutions amino acid code Replace with any of the following selected Alanine A D-Ala, Gly, Beta-Ala, L-Cys, D-Cys Arginine R D-Arg, Lys, Homo-Arg, D-Homo-Arg, Met, D-Met, Ile, D-Ile, Orn, D-Orn Asparagine N D-Asn, Asp, D-Asp, Glu, D-Glu, Gln, D-Gln Aspartamic acid D D-Asp, D-Asn, Asn, Glu, D-Glu, Gln, D-Gln Cysteine C D-Cys, S-Me-Cys, Met, D-Met, Thr, D-Thr Glutamine Q D-Gln, Asn, D-Asn, Glu, D-Glu, Asp, D-Asp Glutamic acid E D-Glu, D-Asp, Asp, Asn, D-Asn, Gln, D-Gln Glycine G Ala, D-Ala, Pro, D-Pro, Beta-Ala, Acp Isoleucine I D-Ile, Val, D-Val, Leu, D-Leu, Met, D-Met Leucine L D-Leu, Val, D-Val, Met, D-Met Lee Sin K D-Lys, Arg, D-Arg, Homo-Arg, D-Homo-Arg, Met, D-Met, Ile, Orn, D-Orn Methionine M D-Met, S-Me-Cys, Ile, D-Ile, Leu, D-Leu, Val, D-Val, Norleu Phenylalanine F D-Phe, Tyr, D-Thr, L-Dopa, His, D-His, Trp, D-Trp trans 3,4 or 5-phenylproline, cis 3,4, or 5-phenylproline Proline P D-Pro, L-I-thioazolidine-4-carboxylic acid, D-1-oxazolidine-4-carboxylic acid or L-1-oxazolidine-4-carboxylic acid Serine S D-Ser, Thr, D-Thr, Allo-Thr, Met, D-Met, Met (O), D-Met (O), Val, D-Val Threonine T D-Thr, Ser, D-Ser, Allo-Thr, Met, D-Met, Met (O), D-Met (O), Val, D-Val Tyrosine Y D-Tyr, Phe, D-Phe, L-Dopa, His, D-His Valine V D-Val, Leu, D-Leu, Ile, D-Ile, Met, D-Met

Other variants of the invention have been modified to increase peptide stability. This variant may, for example, comprise one or more non-peptide bonds (replacement of peptide bonds) in the peptide sequence. Also included are variants and cyclic variants, including residues other than naturally occurring L-amino acids, such as D-amino acids or non-naturally occurring amino acids or synthetic amino acids such as beta amino acids or gamma amino acids. Incorporation of D-amino acids into polypeptides instead of L-amino acids can increase resistance to proteases. See, eg, US Pat. No. 5,219,990.

The peptides of the present invention may be modified by various changes, such as conservative or non-conservative insertions, deletions and substitutions, provided that changes in insertions, deletions and substitutions provide particular advantages for their use. In particular, splice variants are included in the present invention.

In addition to substantial full-length polypeptides, the present invention provides biologically active fragments of the polypeptide. If the RetL polypeptide or fragment exhibits the biological activity of naturally occurring RetL, it is biologically active. This biological activity includes the ability to specifically bind to the extracellular portion of Ret and exhibits at least 50%, preferably approximately the same affinity, of naturally occurring RetL to the extracellular portion of Ret. . Another biological activity is the ability to bind to antibodies derived from epitopes present in naturally occurring RetL.

In another embodiment, variants with less conservative amino acid substituents can, for example, change the charge, structure and other biological properties to form the desired derivatives. These substitutions include, for example, substitution of hydrophobic residues with hydrophilic residues, substitution of other residues with cysteine or proline, substitution of residues with large side chains with residues with small side chains, or residues with positive charges with residues with positive charges. Substitutions may be included. If the outcome of a given substitution cannot be reliably predicted, the derivative can be readily analyzed according to the methods disclosed herein to determine the presence or absence of the desired property.

In general, substitutions that can be expected to cause changes in the functional properties of Ret polypeptides include (i) substitution of hydrophilic residues such as serine or threonine with hydrophobic residues such as leucine, isoleucine, phenylalanine or alanine; (ii) replacing the cysteine residue in place of (by residue) any other residue; (iii) a residue having a positive side chain, such as lysine, arginine or histidine, is substituted for (by) a residue having a negative charge, such as glutamic acid or aspartamic acid; Or (iv) a residue having a large side chain, such as phenylalanine, in place of (by) a residue having no such side chain, such as glycine.

Variants of the invention include rat RetL1 (SEQ ID NO: 2), partial human RetL1 (SEQ ID NO: 9), full length human RetL1 (SEQ ID NO: 11), human RetL2 (SEQ ID NO: 13), rat RetL3 (SEQ ID NO: 17), partial human RetL3 (SEQ ID NO: 19) or an amino acid sequence, protein and peptide having at least 60% homology with human RetL3 (SEQ ID NO: 21). More preferably, sequence homology is at least 80%, at least 90%, at least 95%. The length of the comparative sequence for comparing homology is generally at least 8 amino acid residues, usually 20 amino acid residues. Variants of the compounds of the invention 1) have an amino acid sequence that is at least 40% homologous to the RetL protein of the invention, and 2) are placed in optimal alignment with the RetL sequence (shown in RetL1 and RetL2 in FIG. 8), The RetL protein of the invention can include any protein having at least 80% cysteine residues aligned with cysteine.

As can substitute for substituents on the backbone, groups characterized by similar features can be used to substitute functional groups bound to the backbone. This modification does not alter the primary sequence. Initially they are conservative. That is, the substituent has the same size, shape, hydrophobicity and charge as the original group. Nonsequence modifications include, for example, chemical derivatization of naturally occurring RetL moieties in vivo or in vitro, as well as changes in acetylation, methylation, phosphorylation, carboxylation or glycosylation.

The present invention includes a protein of the present invention, an agent capable of specifically binding to a fragment of the protein. These formulations include Ig fusion proteins and antibodies (including single-stranded, double-stranded, Fab fragments or the like, whether natural, humanized, sensitized or chimeric). These categories of formulations are described in detail in PCT Application 95/16709, which is incorporated herein by reference.

Summary of the Invention

The present invention has the nucleotide sequence of any RetL, but in particular rat retL1 cDNA (SEQ ID NO: 1), partial human retL1 cDNA (SEQ ID NO: 8), full length human retL1 cDNA (SEQ ID NO: 10), human retL2 cDNA (SEQ ID NO: 12) Provided are purified and isolated DNA molecules encoding RetL, including murine retL3 cDNA (SEQ ID NO: 16), partial human retL3 cDNA (SEQ ID NO: 18), and human retL3 cDNA (SEQ ID NO: 20). In addition, the present invention provides the amino acid sequence of rat RetL1 (SEQ ID NO: 1), partial human RetL1 (SEQ ID NO: 9), full length human RetL1 (SEQ ID NO: 11), human RetL2 (SEQ ID NO: 13), rat RetL3 (SEQ ID NO: 17), Provided is a RetL protein comprising the amino acid sequence of partially human RetL3 (SEQ ID NO: 19) or human RetL3 (SEQ ID NO: 21).

In one aspect of the invention, the invention provides a sequence of insertion DNA of clone HRL20 (part human retL1 cDNA (SEQ ID NO: 8)), or ATCC number 97604, or insert DNA of clone # 230-5A-86-17 with ATCC number 98047. DNA sequence comprising the sequence (rat retL1 cDNA (SEQ ID NO: 1)).

In another aspect of the invention, a purified and isolated DNA molecule for use in maintaining expression in a prokaryotic or eukaryotic host cell of a polypeptide product retains at least some of the primary structural forms of RetL and biological activity; DNA is a) rat retL1 cDNA, partial human retL1 cDNA, full length human retL1 cDNA, human retL2 cDNA, mouse retL3 cDNA or human retL3 cDNA, or rat retL1 cDNA, partial human retL1 cDNA, full length human retL1 cDNA, human DNA molecules comprising the complementary strand of murine retL3 cDNA or human retL3 cDNA; b) a DNA molecule hybridized to a DNA molecule or fragment thereof as defined in a) under stringent conditions; Or c) a DNA molecule that hybridizes to the DNA molecule as defined in a) and b) if there is no denaturation of the genetic code. Purified and isolated DNA molecules encoding polypeptide fragments or variants of human RetL that retain the biological activity of RetL also belong to the invention.

Any recombinant DNA molecule of the invention can be operably linked to expression control sequences.

Also included in the present invention are vectors and delivery systems comprising a DNA molecule or construct as defined herein. The vector may comprise a DNA molecule encoding RetL or a variant of RetL.

The present invention includes eukaryotic host cells or prokaryotic host cells stably transformed or transfected by a vector comprising a DNA molecule encoding a native RetL or modified RetL.

Like the processes for producing polypeptide products that retain some or all of the primary structural form and biological activity of RetL, purified and isolated human RetL, which is almost free of other human proteins, is particularly included in the present invention. Such processes may include propagating prokaryotic or eukaryotic host cells transformed or transfected with any of the DNA molecules of the invention under appropriate culture conditions, expressing some degree of polypeptide product, and recovering RetL. Can be. Polypeptide products expressed in prokaryotic or eukaryotic host cells of DNA also belong to the invention.

The present invention also encompasses proteins and fragments, variants and derivatives of proteins, whether soluble or membrane bound. In certain embodiments, the protein has an amino acid sequence comprising rat RetL1, partial human RetL1, full length human RetL1, human RetL2, murine RetL3, or human RetL3, or is a variant of one of these sequences. In another embodiment, the protein is a fusion protein comprising RetL or Ret fused to other molecules or molecular fragments, such as immunoglobulins, toxins, imaging compounds or radionuclides. Chimeric molecules of RetL also belong to the present invention.

Another aspect of the invention includes specific monoclonal antibodies against RetL of the invention. This antibody can bind to toxins, imaging compounds or radionuclides. The invention also relates to AA.FF9, AA.HE3, AF.E9, BA.B1, BB.B6, AA.GE7, CD.F11, AH.E3, CD.G4, AG.E7, BD.G6 and BH. Hybridoma cell lines that produce specific antibodies to Ret, including G8, and subclones of these hybridomas, and antibodies produced by these hybridomas or subclones of hybridomas.

In addition, the present invention comprises administering to the sample a therapeutically effective amount of a compound that induces autophosphorylation of Ret by interacting with the cell Ret to promote proliferation of new tissue or to promote survival of the damaged tissue of the sample. It includes a method. The compound may be RetL1, RetL2 or RetL3, a fragment of full length RetL or an antibody that binds to Ret. This compound may be administered simultaneously with a therapeutically effective amount of a second compound, such as GDNF, neurturin or a GDNF related molecule. The tissue of interest for these methods may include any tissue, but renal tissue, neural tissue, heart, stomach, small intestine, spinal cord or lung are preferred. In one embodiment, RetL is soluble RetL. In these methods the sample is a human.

In another embodiment of the present invention, Ret signal transduction between RetL expressing first and second cells is inhibited by contacting the first cell with an antibody to soluble Ret protein or RetL. This soluble Ret protein may be a fusion protein.

The invention also provides a method of targeting a toxin, imaging compound or radionuclide to Ret expressing cells, comprising contacting the cells with an anti-Ret antibody or RetL fusion protein conjugated to the toxin, imaging compound or radionuclide. Include. RetL may be RetL1, RetL2 or RetL3. In another method, contacting the cell with a toxin or radionuclide and a fusion protein of RetL, or an anti-Ret antibody conjugated to the toxin or radionuclide, inhibits proliferation of Ret-expressing tumor cells. The cells may be in the sample and the protein or conjugated antibody is administered to the sample.

Fusion protein comprising Ret and a toxin, an imaging compound or radionuclide, or toxins, imaging of a RetL-expressing cell, comprising contacting the cell with an anti-RetL antibody conjugated to the toxin, imaging compound or radionuclide Methods of targeting compounds or radionuclides also belong to the invention. In another embodiment, the present invention provides a method for inhibiting proliferation of RetL-expressing tumor cells, comprising contacting the cells with a fusion protein of Ret and a toxin or radionuclide, or an anti-RetL antibody conjugated to the toxin or radionuclide. A method; The cells may be present in the sample and the protein is administered to the sample.

RetL for use in any of the methods of the invention may be RetL1, RetL2 or RetL3, or may be a variant or fragment of RetL1, RetL2 or RetL3.

Gene therapy methods also belong to the present invention. In one aspect, the invention provides a method of treating a subject having a Ret metabolic disease, comprising administering to the subject a vector comprising a DNA molecule encoding RetL, and administering the vector to the subject. Provides a way to promote the proliferation of new tissue. In another embodiment, the present invention provides a method of promoting survival of a damaged tissue of a subject, comprising administering to the subject a therapeutically effective amount of a vector encoding RetL.

Outline method

The general method used to clone RetL1 is shown in FIGS. 4A and 4B. This method was based on the premise that at least RetL is expressed in the posterior mesenchymal of the developing kidney as a membrane protein (although it is possible to express the ligand in soluble form; FIG. 4A). RetL interacts with Ret receptors on ureter cells to activate the tyrosine kinase cytoplasmic domain and send signals to the nucleus, activating genes involved in the proliferation and branching of ureter cells. Thus, proteins containing extracellular domains of Ret fused to the Fc portion of human immunoglobulin G1 (IgG1) or alkaline phosphatase (AP) can be used as part of a method of cloning RetL as shown in FIG. 4B. . Fusion proteins, expression libraries, and other agents used for cloning RetL1 are described below.

We first isolated the cDNA for rat RetL1 and then use it as a probe to separate the cDNA for human RetL1. The cDNA is then separated for RetL2 and RetL3.

Preparation of Reagents for Direct Expression Cloning of Ret Ligands

1. Isolation of cDNA Encoding Rat Ret Extracellular Domain

To identify RetL1, a protein is formed, ie a fusion protein consisting of a human Fc portion of IgG1 in one example and an extracellular domain of rat Ret or human Ret fused to alkaline phosphatase in another example. Fusion pairs can be easily analyzed to detect cells expressing ligand as illustrated in FIG. 4B.

Since no cDNA encoding rat Ret is disclosed, the reverse transcriptase-polymerase chain reaction (RT-PCR) method is used to isolate the cDNA encoding the extracellular region of the rat Ret receptor. Two nucleotide sequences of human (Genbank Accession Nos. M57464 and X15262) and rat (Genbank Accession No. X67812) ret are compared and the oligonucleotide primers obtained from the high identity region between the two sequences are designed. A sense oligomer called kid-013 (SEQ ID NO: 3; comprising nucleotides 150-169 of Genbank sequence X15262) is selected from the 5 'end of the human ret cDNA sequence overlapping the ATG initiation codon. This includes nucleotides on the 5 'end that encode the Not1 restriction site for cloning. two antisense called kid-014 (SEQ ID NO: 4; including complement of nucleotides 1819-1839 of GenBank sequence M57464) and kid-015 (SEQ ID NO: 5; including complement of nucleotides 1894-1914 of GenBank sequence X67812) The oligomers are each selected from human and murine cDNA sequences just 5 'of the sequence encoding the transmembrane domain. The oligomers kid-014 and kid-015 contain additional nucleotides at the 5 'end encoding the Sal1 restriction site for cloning.

Total RNA is isolated from 14 day old embryonic rat kidneys and mRNA is purified by oligo-dT chromatography. mRNA is converted to cDNA using AMV reverse transcriptase, cDNA is converted to double-stranded cDNA and amplified with Taq polymerase in standard polymerase chain reaction with oligomers kid-013 and kid-105. Synthesis of a 1942 bp PCR fragment is confirmed by passing an aliquot of the PCR reaction on a 1% agarose gel. The remaining PCR fragment is digested with Notl and Sall and cloned into pSAB132 previously digested with Notl and Sall. The resulting plasmid is called pJC011. The entire insert of plasmid pJC011 contained between the Not1 and Sal1 positions is sequenced and shown as extracellular rat retL1 cDNA (SEQ ID NO: 6). Translation of this sequence shows the peptide sequence (SEQ ID NO: 7) for extracellular rat Ret1. Since the oligomers for RCR were selected from the human and mouse sequences of ret, the nucleotide sequence shown as that of the rat ret cDNA of the cell and the peptide sequence shown as that of the extracellular rat Ret are in the region of the kid-013 and kid-015 sequences. It may differ from native rat ret nucleotide and Ret peptide sequences. Thus, ret cDNA clones are isolated from 18 day embryonic embryonic cDNA libraries and some nucleotide changes are observed in the primer region with two amino acid changes. One is the change in signal sequence (change of arginine to threonine at position 5) and the other near the end of the extracellular domain (change of glutamic acid to alanine at position 633). All of these changes do not affect ligand binding.

2. Ret / IgG Fusion Proteins

A fusion protein consists of a rat (amino acid residues # 1-637) fused to the Fc portion of human IgG1 and an extracellular domain of the human (amino acid residues # 1-636) Ret receptor.

The construction of the plasmids used to express the rat Ret / IgG fusion protein is shown schematically in FIG. 5. To construct a gene encoding the rat Ret / IgG fusion protein, pJC011 (tactic) containing the rat Ret extracellular domain was digested with Sal1 and ligated to a 700 bp Sal1 fragment obtained from plasmid 2-4 to plasmid pJC012. Make. This Sal1 fragment comprises part of the Fc domain of human IgG1 originally derived from plasmid pSAB144. Plasmid 2-4 was prepared in advance by ligation of three methods: Not1-Sal1 fragments generated by PCR comprising an extracellular domain of rabbit TGF-beta type II receptor; 693 bp Sal1-Not1 fragment from pSAB144 comprising the Fc domain portion of human IgG1; PSAB132 digested with Not1. As shown in FIG. 5, fragments comprising an Fc domain, such as a 700 bp Sal fragment, can be released from 2-4 plasmids. pJC012 is transfected into COS cells and rat Ret / IgG fusion proteins are purified from the medium after 48 hours using Protein-A Sepharose chromatography. To create a stable cell line producing Ret / IgG protein, 2612 bp Not1 fragments obtained from pJC012 containing the whole rat Ret / IgG fusion protein were isolated and cloned to the Not1 position of the expression vector pMDR901. The resulting plasmid is called pJC022. Plasmid pJC022 is transfected into CHO cells to form a stable cell line. The best producing cell line is applied to the suspension. Typical yield of rat Ret / IgG CHO strain is 75 mg / l.

The construction of plasmids used to express human Ret / IgG fusion proteins is schematically illustrated in FIG. 6. To construct a gene encoding a human Ret / IgG fusion protein, a plasmid containing a cDNA encoding a human receptor was obtained from Dr. M. Takahashi (Department of Pathology, College of Pharmacy, Nagoya University, Nagoya, Japan). PCR fragments are prepared from this plasmid using oligomers kid-013 and kid-014. The PCR fragment was treated with a Cleno fragment and then digested with Not1 to form an RCR fragment with a sticky Not1 end and one smooth end. This fragment was cloned into the vector pGEM11zf (+) previously decomposed with EcoR1, treated with the Klenow fragment, and digested with Not1 to form one smooth end with the sticky Not1 end. The resulting plasmid is called pJC013. The 1916 bp Not1-Sal fragment obtained from pJC013 was completely digested with Not1, partially digested with Sal1 and then isolated, and the pSAB132 expression vector digested with 693 bp Sal1-Not1 fragment and Not1 from pSAB144 containing part of the Fc domain of human IgG1. Ligation on. The resulting plasmid is called pJC015. By sequencing the insert in plasmid pJC013, it was found that this insert contained a single nucleotide difference that changed one amino acid in the extracellular domain of human Ret (GenBank sequence M57464 had a C at position 812, pJC013 has a T at that position; this results in a change of amino acid from alanine to valine at position 294 of the human Ret protein sequence). This nucleotide is corrected back to the C residue specified by GenBank sequence M57464 by site specific mutagenesis of plasmid pJC013 to generate plasmid pJC023. The 585 bp BstE2 fragment derived from pJC023 containing the restored nucleotide sequence is isolated and cloned into plasmid pJC015 from which the 585 bp BstE2 fragment comprising the modified nucleotide is removed. The new plasmid is called pJC024. The 2609 pb Notl fragment obtained from pJC024 containing the whole human Ret / IgG fusion protein is isolated and cloned into the Notl position of the expression vector pMDR 901. The resulting plasmid is called pJCO25. Plasmid pJC025 is transfected into CHO cells to form a stable cell line. The highest producing cell line is applied to the suspension. Typical yield of human Ret / IgG CHO cells is 6 mg / l.

Details of the preparation of the vectors used in the methods of the present invention are disclosed in PCT applications 94/01456 and 92/02050, incorporated herein by reference.

3. Bioactivity of Ret / IgG Fusion Proteins

In order to determine if the Ret / IgG fusion proteins produced by the inventors are bioactive and are therefore good screening reagents for the cloning of RetL, several organ culture assays for bioactivity are performed. Organ culture analytes consist of embryonic rat kidneys grown 13-14 days in organ culture in the presence of Ret / Ig fusion protein at a concentration of 50 μg / ml for 3-5 days. Kidneys are also cultured in the presence of LFA-3TIP / IgG or mediator buffer. After the incubation period, some kidneys are stained with fluorescent lectin Dolichos Biflorus Agglutinin (DB lectin), which stains the collecting duct tissue, epithelial cells derived from ureteral cells. Since Ret is expressed in ureterocytes and epithelial cell derivatives thereof, these "DB" positive cells represent Ret positive cells. This allows an overall assessment of the effect of Ret / IgG fusion proteins on the growth and development of embryonic kidneys. There is a clear difference in proliferation and collection morphology between kidneys cultured with LFA-3TIP and kidneys cultured with rat Ret / IgG fusion proteins. Ret / IgG treated kidneys are significantly less branched and generally have smaller collecting canals.

Paraffin moieties are prepared from other kidneys for histological examination. Embryonic kidneys are treated with control buffer or Ret / IgG and then stained with hematoxylin and eosin. Embryo kidneys treated with Ret / Ig had less branching than embryonic kidneys treated with control buffer. In addition, kidneys treated with Ret / IgG have almost no tubules. We also observed using human Ret / IgG fusion proteins. These observations are consistent with the fusion protein blocking inducible signals between mesenchymal and ureter cells. Thus, we concluded that the fusion protein is an excellent reagent for cloning RetL.

4. Ret / alkaline phosphatase fusion protein

Successfully identified ligands for c-kit (Cell 63: 185, 1990) and ligands for eph family of orphan receptors (Cell 79: 157, 1994) using receptor / alkaline phosphatase (AP) fusion proteins And recently cloned the receptor of leptin, the product of the ob gene (Cell 83: 1263, 1995). Plasmids encoding the rat Ret / AP fusion protein are constructed and rat Ret / AP protein is prepared in COS7 cells of the cell plant. Subsequently, a stable NIH3T3 cell line expressing an average of 10 mg / l fusion protein is prepared. SDS-PAGE analysis of the rat Ret / AP protein showed that the size of this protein was consistent with the estimated molecular weight, and gel filtration analysis showed that the protein was produced as a dimer. Affinity chromatography is performed on the anti-AP column to effect partial purification.

5. Anti-Ret Antibodies

Rabbit polyclonal antibodies are prepared against rat Ret / IgG fusion proteins. Antibodies are subjected to Western blot, FACS analysis of Ret positive cell lines and immunohistochemistry of embryonic kidneys.

Form a panel of hamster antirat Ret monoclonal antibodies. Rat Ret / IgG fusion proteins coupled to Protein A Sepharose are used to immunize Armenian hamsters. 316 clones are obtained after fusion and the ELISA assay detects the ability to bind rat Ret fusion proteins and / or human IgG. Eleven clones produce antibodies that bind only to Rat Ret / IgG (and Rat Ret / AP) but not to human IgG. Cross reactivity for human Ret is analyzed by FACS; Four clones produce antibodies capable of binding to the Ret positive human cell line THP-1. The following table summarizes the Ret binding properties of 12 monoclonal antibodies.

Clone ELISA Rat Ret / Ig FACS Human THP-1 AA.FF9.5 + - AA.HE3.7 + + AF.E9.5 + - BA.B1.16 + - BB, B6 + - AA.GE7.3 + - CD.F11.2 + - AH, E3.11 + + CD.G4.2 + + AG.E7.9 + - BD.G6 + + BH.G8 - -

6. cDNA Expression Library

We prepare a cDNA library from rat embryonic extension in a CDM8 vector that can use the SV40 origin for amplification and pCEP4, a modified invitrogen vector that uses the EBV origin for amplification. The modified vector, CH269, has had its EBNA-1 gene sequence removed. EBNA-1 protein interacts with the EBV origin, but if cells are used that stably express the EBNA protein, this gene is not needed on the vector. The library of CDM8 vectors contains 1.5 × 10 ⁶ clones with an average insert size of 1.18 kb, while the library in the CH269 vector contains about 1 × 10 ⁶ clones with an average insert size of 1.5 kb.

Expression Cloning of Ret Ligand RetL1

A. Cloning of Rat Ret Ligand RetL1

1. Initial Attempt at Cloning Expression of Ret Ligand RetL1

Various direct expression methods were attempted to clone RetL1. All these methods are based on the concept illustrated in Fig. 4B. cDNA from the cDNA library is introduced into the mammal; Cells that receive RetL1 can be identified using Ret fusion proteins. Although the three approaches described below have not been successful, they have gained significant knowledge and experience and have evolved into a successful approach.

a. Panning method using Ret / IgG

Rat Ret / IgG fusion proteins are used in an attempt to isolate RetL1 by direct expression cloning using the panning method (Aruffo and Seed, Proc. Natl. Acad. Sci. 84: 8753-8757 (1987)). An 18-day old rat kidney cDNA library in CDM8 is used for panning. CDNA pools (5,000-10,000 cDNAs per pool) from this library are introduced into COS cells using the DEAE-dextrin method. After 48 hours, cells are removed from the plate using EDTA, incubated with fusion proteins, and then selected on plates coated with anti-human IgG antibody. DNA is recovered from adherent cells, and After transformation into E. Coli , it is isolated for second panning. No binding of any cells was observed after the third panning, and very few clones were obtained after transforming Hirt DNA back into E. coli. Dilutions of the VCAM cDNA used for conjugation with anti-VCAM monoclonal antibodies as a positive control in a ratio of 1: 100 were still detected, indicating that the pool size was too large. Analysis of some clones obtained after the second panning indicates that the clones undergo rearrangement and deletion.

b. Separation FACS Method Using Ret / IgG

80,000 cDNA clones obtained from an 18-day embryonic rat kidney library (CDM8 vector) are introduced into COS7 and isolated FACS is performed using rat Ret / IgG proteins followed by secondary antibodies tagged with phosphors. Top 0.5% and 0.9% of fluorescent cells are recovered and plasmid DNA is recovered by Hirt cell lysis. Electroporation of DNA back into E. coli: Get 228 clones for 0.5% pool and 752 clones for 0.9% pool. DNA is recovered from bacterial clones and secondary isolation FACS is performed. Plasmids recovered from bacterial clones after the second FACS run were analyzed to include large deletions and rearrangements.

c. Colorimetric Detection Using Ret / AP

COS cells are transfected with 400 pools (1000 clones per pool) of cDNA clones obtained from an 18-day old rat embryonic kidney cDNA library (CDM8 vector) and stained using colorimetric substrates for Ret / AP protein and alkaline phosphatase. Transfected cells are examined under a microscope for positive signals. In one experiment, five possible positives were reanalyzed, but all were negative.

As a control of the Ret / AP protein, the VCAM / AP protein is prepared by fusing the first two domains of human VCAM to the N terminus of placental AP. (VCAM binds to integrin VLA4 consisting of two chains, α-4 and β-1). Transient transfection of COS cells produces sufficient VCAM / AP protein for control experiments. COS cells transfected with α-4 chain cDNA compared to VCAM / IgG directly coupled to AP and VCAM / IgG coupled to AP + VCAM / IgG (COS cells already have β-1 chain Express VLA4). These results show that VLA4 can be detected on cells transfected with the VCAM / AP protein, but the best detection results can be obtained using the VCAM / IgG protein with the AP coupled secondary antibody.

d. Methodological conclusion

Three major conclusions were drawn from these initial cloning studies.

1) This method, which requires the recovery of plasmid DNA in successive rounds (ie panning and isolation FACS), is not appropriate when the target cDNA is not abundant because of rearrangements and deletions that occur in successive rounds. . Based on the low expression level of Ret, it was assumed that the expression level of RetL1 is also low. The preferred approach is to use a detection method capable of transfecting in pools and identifying positive pools. Existing pools can then be sorted and there is no need to recover transiently expressed DNA from the transfected cells.

2) Ret / IgG protein shows better detection than Ret / AP protein when coupled to a secondary reagent.

3) Control experiments with VCAM / IgG control protein (and AP coupled secondary antibody) and α-4 integrin cDNA (diluted into CDM8 vector and transfected into COS cells) resulted in a detection capability of about 1 / 1000 (ie, pool size cannot exceed 1000 clones). To obtain an improved degree of sensitivity, change is made from SV40 origin vector (expressed in COS cells) to EBV origin vector (EBNA positive cell line). The EBV origin vector was maintained as an episome and did not show toxicity to cells like the SV40 origin vector after amplification. There is an obvious increase that genes can be expressed in large quantities in these vectors and can be detected even by further dilution of cDNA (ie up to 1-80,000).

2. Retrieve the pool from the EBV origin cDNA library

Rat Ret / IgG fusion proteins are used to retrieve pools of clones obtained from an 18-day old rat embryonic cDNA library (CH269 vector with EVB origin). In the experiment, each pool produces 256 pools containing 5000 clones. In brief, aliquots of the cDNA library are titrated, 5000 cells are plated (256 times) and grown overnight. Collect colonies into medium: Some cultures are used to form glycerol stores for pools (stored at −70 ° C.) and some are used for plasmid preparation. DNA from 256 pools is transfected into 293 / EBNA cells (8 × 10 ⁵ ) on a 60 mm plate, respectively, using the lipofection method. After 48 hours, cells were washed twice with HBHA buffer (0.5 mg / ml BSA, 0.1% NaN ₃ 20 mM HEPES, pH 7.0), Tris buffered saline and 1 mM MgCl ₂ and CaCl for 60-90 minutes at room temperature. of ₂ 20 ㎍ / ㎖ rats incubated with the Ret / IgG. After incubation, cells are washed four times with HBHA buffer and fixed in 60% acetone / 3% formaldehyde / 20 mM HEPES (pH 7.0) for 30 seconds. After washing twice with HBS buffer (150 mM NaCl, 20 mM HEPES, pH 7.0), the AP-coupled secondary antibody (goat anti-human IgG Fc-gamma-specific F (ab ') ₂ ) ( Jackson Immuno Research Laboratories; Catalog # 109-056-098; 1: 5000 dilution in Tris buffered saline with 1 mM MgCl ₂ and CaCl ₂ ) incubated at room temperature for 60 minutes. The cells were then washed twice with HBS buffer, followed by AP substrate buffer (100 mM Tris-HCl, pH 9.5), 100 mM NaCl, 5 mM MgCl ₂ containing ₂ × Pierce Immuno Pure ^R phosphatase inhibitor (Catalog # 35002). Wash twice with). The final wash is maintained for 15 minutes. AP substrate NBT (0.33 mg / ml) and BCIP (0.17 mg / ml) are then added to AP substrate buffer containing Pierce AP inhibitor and incubated with cells for 5-20 minutes. The plate is then washed twice with water. The plate is then examined under a dissecting microscope to confirm the presence of purple stained cells.

From the analysis of 256 pools, 17 positive pools are identified in the first search. DNA from each positive pool is retransfected into 293 / EBNA cells and the procedure is repeated with some additional control experiments to confirm that the observed dye is Ret / IgG specific. Only 10 of the 17 positive pools showed staining with the Ret / IgG fusion protein, but not with other IgG fusion proteins.

3. Classification of Pool # 230

As an example, one of the positive pools described above is named # 230 and the cDNA is identified within the pool that confers binding to the Ret / IgG fusion protein by classifying it into smaller sub pools. 600 cells are plated (10 times) from the glycerol stock for pool # 230 and grown overnight. Collect colonies of these plates into the medium: 1/10 of the culture is used to make a glycerol stock and the remainder is used for DNA preparation. Ten pools of 600 clones are named 230-1A to 230-5A and 230-1B to 230-5B. The above procedure is repeated to transfect the DNA of these subpools into 293 / EBNA cells and stain with Ret / IgG fusion proteins. One subpool # 230-5A is positive for staining with Ret / IgG protein.

Further classify pool # 230-5A to identify cDNA with subpools that confer binding to the Ret / IgG fusion protein. Cells from glycerol stock of pool 230-5A are plated and allowed to grow overnight. Colonies are taken and transferred into wells of seven 96 well Bioblocks (registered trademark) and grown overnight. Four pools of 20 clones and one pool of 16 clones are made from each 96 well bioblock. Thus, 35 pools were formed from the seven bioblocks, designated 230-5A-71 to 230-5A-105. DNA is prepared from each of these pools and transfected into 293 / EBNA cells and reanalyzed using the Ret / IgG fusion protein as described above. Pool # 230-5A-86 is positive.

Pool # 230-5A-86 again identifies and classifies 20 clones that run on the bioblock and are mixed together to produce this pool. DNA is made from all 20 clones and each transfected into 293 / EBNA cells and re-analyzed for Ret / IgG as described above. Pool # 230-5A-86-17 was found to be positive.

4. Characterization of Clone # 230-5A-86-17

Clone # 230-5A-86-17 (referred to as retL-17 or clone 17, deposited with ATCC 98047) is further analyzed by DNA sequencing. The full nucleotide sequence of the insert of this clone is SEQ ID NO: 1 (rat retL1 cDNA), with a portion of the nucleotide sequence shown in FIG. 1. Within this nucleotide sequence, a translation frame encoding a 468 amino acid protein (rat RetL1) was found. The predicted protein has a signal sequence that is assumed to be cleaved behind amino acid 24 (Von Heijne et al., Nucl. Acid Res. 14: 14683 (1986)). The hydrophobic C-terminus shows that the protein can bind to cells via phosphatidylinositol glycan binding. There are three expected N-linked glycosylation sites. These properties are in line with what we expect for Ret's ligand.

We can express the soluble form of the rat RetL1 protein by truncating the gene before the hydrophobic C terminus. For example, this can be done truncated behind lysine 435 (rat RetL1). The truncation upstream of the amino acid can express the soluble form of the rat RetL1 protein. Soluble rat RetL1 protein can be expressed by itself or as part of a fusion with human immunoglobulins, as a small epitope recognized by histidine tags or antibodies.

B. Cloning of Human Ret Ligand RetL1

Human embryonic kidney cDNA libraries in vector lambda gt10 are purchased from Clontech (catalog # HL5004A). Units forming one million plaques from phage strains are plated onto 10 Nunc ^™ plates. Replica plaque lifts are made on Schreicher and Schiel Optitran ^™ filters.

After digesting the plasmid rat RetL1 with restriction enzyme PvuII, a probe is formed by agarose gel separation of 1.34 kb fragment corresponding to nucleotides 242-1582 of the rat RetL nucleotide sequence (rat retL1 cDNA). This coding region probe is randomly primed to stimulate P ³² labeling (Feinberg and Vogelstein, Anal. Biochem. 137: 266-267, 1984). The filtrate was washed with 300 ml plaque search PBS buffer (50 mM Tris pH 7.5, 1M NaCl, 0.1% sodium pyrophosphate) at 55 ° C. containing 10% dextran sulfate, 100 μg / ml tRNA and 6.7 × 10 ⁷ CPM of rat probe. , 0.2% PVP and 0.2% picol) overnight. It is washed twice with plaque search buffer at 55 ° C. and twice with 2 × SSC / 1% SDS and then exposed to the film at −70 ° C. using a sensitivity enhancing screen.

Replication positives are subtracted into SM (100 mM NaCl, 10 mM SO ₄ , 50 mM Tris pH 7.5) and gelatin in a master plate. These 24 positives are plaque purified. Lambda miniprep DNA obtained from purified subject plaques is digested with Not1, electrophoresed on 1% agarose gel and Southern blot. Southern blot hybridizes with the region probe encoding rat RetL1. Clone HRL20 has the longest insert (4.4 kb) that hybridizes strongly to rat probes. DNA sequences (partial human retL1 cDNA; SEQ ID NO: 8; FIG. 2A) and inferred peptide sequences (partial human RetL1; SEQ ID NO: 9; FIG. 2A) were obtained from this clone and identified as human homologues. This clone encodes most cryptographic domains, including the 3 'end of the cryptographic domain.

To obtain the 5 'end of human cDNA, a human fetal kidney Marathon-Ready ^™ cDNA kit is purchased from Clontech (Catalog # 7423-1). The antisense oligonucleotide Kid-155 corresponding to the complement of nucleotides 62-81 of SEQ ID NO: 8 (partial human retL1 cDNA) and the nucleotide 17-43 of SEQ ID NO: 8 (complement of nucleotides 17-43 of the partial human retL1 cDNA) The antisense oligonucleotide Kid-154 corresponding thereto is synthesized. PCR is performed using the Advantage ^™ cDNA PCR Kit (Clontech Catalog # 8417-1) with Marathon ^™ cDNA reagent and oligonucleotide Kid-155 or Kid-154. The first PCR reaction was set up as follows: 35.5 μl H ₂ O; 5.0 μl 10 × Klen Taq buffer; 1.0 μl 10 mM dNTP mix; 1.0 μl 50X Advantage ^™ Klen Taq Polymerase Mix. These reagents are mixed together. 5.0 μl of Marathon-Ready ^™ fetal kidney cDNA; Add 1.0 μl 10 μM AP1 primer and 1.5 μl 6.4 μM Kid-155 (final volume = 50 μl). PCR is performed in Perkin-Elmer Setus DNA Summer Cycler 480 with the following cycle conditions: 1 cycle at 94 ° C. for 1 minute; 30 cycles at 94 ° C. for 30 total, 55 ° C. for 30 seconds, 68 ° C. for 4 minutes. Nested PCR is performed using the product of the first PCR reaction. First, 5 μl of PCR product # 1 is diluted 50-fold with TE (final volume 250 μl). Nest PCR reactions were 35.5 μl H ₂ O; 5.0 μl 10 × Klen Taq buffer; 1.0 μl 10 mM dNTP mix; 1.0 μl 50 × Advantage ^™ Klen Taq Polymerase Mix. These reagents are mixed as described above. Then 5.0 μl of diluted PCR product # 1, 1.0 μl 10 μM AP2 primer and 1.5 μl 6.9 μM kid-154 are added. Cycle conditions are the same as described above. About 700 bp of product is purified on 1% low melt agarose gel and phenol extracted. Purified DNA is cloned into the EcoR5 site of pZErO ^™ (Invitrogen Catalog # K2510-01). Sequence information is obtained from several isolates, including clones called HRL7G6 and HRL7G8.

The sequence obtained from clone HRL7G8 as shown in FIG. 2B was found to overlap with the sequence of clone HRL20 (partial human retL1 cDNA) and is used to form the full length sequence of human RetL1 (full length human retL1 cDNA). Nucleotide sequences obtained from clone HRL7G8 represent nucleotides 1-502 of the full length human retL1 cDNA; Nucleotides obtained from clone HRL20 represent nucleotides 460-1682 of full length human retL1 cDNA. Sequences obtained from clone HRL7G8 are identified by sequencing another cDNA clone (GJ102) isolated from the human embryonic kidney lambda gt10 cDNA library described above using a probe derived from clone HRL7G6. Nucleotides 118-1497 comprise the protein translation frame of full length human retL1 cDNA.

The complete amino acid sequence of human RetL1 is shown in FIG. 2B. As shown by the BESTFIT analysis shown in FIG. 3A, human retL1 cDNA is 88.2% identical to rat retL1 cDNA. Peptide comparison (FIG. 3B) shows that the human putative peptide sequence is 93.3% identical and 97.2% similar to that of rat.

Cloning of Ret Ligand RetL2

A. Cloning of Human RetL2

The peptide sequence of rat RetL1 (rat RetL1) is used for gene bank database investigation using the program BLAST to identify related proteins (ie isologue). BLAST (ie, Basic Local Alignment Search Tool) is described by Altschul et al. [J. Mol. Biol. 215: 403-410, 1990] to find the similarity of all sequences to unknown sequences in a sequence database. The unknown sequence and the database to be searched can be any combination of peptides or nucleotides. If the rat RetL1 peptide sequence is unknown to the expressed sequence tag (EST) nucleotide database, two important counterparts are obtained. One is a 229 bp EST obtained from the mixed human fetal liver and spleen cDNA library, Genbank Accession # R02249, and the other is a 521 bp EST obtained from the human infant brain cDNA library, Genbank Accession # H12981. The two ESTs have 99% identity in the overlapping region, meaning that they come from the same cDNA. Oligonucleotides include H12981 EST: KID-228 (GAA TGA CAA CTG CAA GAA GCT GCG CTC CTC; corresponds to nucleotides 38-67 and 534-563 of SEQ ID NO: 12) and antisense oligonucleotide KID-229 (GTG TAC TCG CTG GGC ACC CG; corresponds to the complement of nucleotides 156-175 of SEQ ID NO: 12 and the complement of nucleotides 652-771.

1 × 10 ⁶ plaque forming units obtained from Clontech human fetal liver 5′-stretch plus lambda GT10 cDNA library (catalog # HL5003a) were selected for overlap in OPTITRAN ^™ filter. P in 400 ml of plaque search buffer (50 mM Tris pH 7.5, 1M NaCl, 0.1% sodium pyrophosphate, 0.2% polyvinylpyrrolidine and 0.2% picol) containing 10% dextran sulfate and 100 μg / ml tRNA ^{The 32} -labeled oligonucleotides KID-228 and KID-229 (each P ³² -labeled oligonucleotide is 80 pmol) and the filtrate are hybridized overnight at 65 ° C. Wash twice with 2xSSC / 1% SDS and twice with 1xSSC / 1% SDS and expose to film. Eleven replication positives are purified. DNA from each clone is analyzed by agarose gel electrophoresis and Southern blot after restriction enzyme digestion. The filtrate is hybridized with KID-228 and KID-229 to confirm that the insert hybridized to the probe. The insert of clone DSW240 was fully sequenced (human retL2 cDNA, SEQ ID NO: 12), which is shown in FIG. 7.

Nucleotide 25-1416 comprises a protein translation frame of human retL2 cDNA encoding a protein of 464 amino acids (human RetL2; SEQ ID NO: 13), which is shown in FIG. 7. As can be seen by the BESTFIT assay shown in FIG. 8, the human RetL2 protein is 49.1% identical and 63.7% similar to the human RetL protein. Like human RetL1, it shares a hydrophobic N-terminus that represents a signal sequence with a hydrophobic C-terminus that represents a phosphatidylinositol glycan binding motif. In addition, 30 of 31 cysteines present in each protein are conserved.

B. Prove that RetL2 is a ligand for Ret

We demonstrated that this is a ligand for Ret by transfecting 293 / EBNA with an expression plasmid comprising the insert of clone DSW240 and showing that the cells can bind to soluble Ret / IgG fusion proteins.

Inserts of DSW240 were removed using Not1 and cloned into expression vector CH269 containing the EBV origin and exhibiting high expression in EBNA positive cell lines. Limited digestion is performed to identify clones with the correct orientation. Plasmid DNA is prepared from clones with the correct orientation.

Plasmid DNA (expression plasmid comprising retreL2 expressing plasmid for positive control, retL1 expressing plasmid for positive control and unrelated protein for negative control) was transfected into 293 / EBNA cells (8 × 10 ⁵ on 60 mm plates) using lipofection method. Infect. After 48 hours, cells were washed twice with HBHA buffer (0.5 mg / ml BSA, 0.1% NaN ₃ , 20 mM HEPES, pH 7.0) and Tris buffered saline + 1 mM MgCl ₂ and CaCl for 60-90 minutes at room temperature. Incubate with 20 μg / ml rat Ret / IgG in ₂ . After incubation, cells are washed four times with HBHA buffer and then fixed for 30 seconds with 60% acetone / 3% formaldehyde / 20 mM HEPES, pH 7.0. AP-coupled secondary antibody (goat anti-human IgG Fc-gamma-specific F (ab ') ₂ ) after washing twice with HBS buffer (150 mM NaCl, 20 mM HEPES, pH 7.0). (Jackson Immuno Research Laboratories; Catalog No. 109-056-098; 1: 5000 dilution in Tris buffered saline with 1 mM MgCl ₂ and CaCl ₂ ) incubated at room temperature for 60 minutes. The cells were then washed twice with HBS buffer, followed by AP substrate buffer (100 mM Tris-HCl, pH 9.5), 100 mM NaCl, 5 mM MgCl containing 2 × Pierce Immuno Pure ^R phosphatase inhibitor (Catalog # 35002). Wash twice with ₂ ). The final wash is maintained for 15 minutes. AP substrate NBT (0.33 mg / ml) and BCIP (0.17 mg / ml) are then added to AP substrate buffer containing Pierce AP inhibitor and incubated with cells for 5-20 minutes. The plate is then washed twice with water. The plate is then examined under a dissecting microscope to confirm the presence of purple stained cells. The presence of purple stained cells means that the Ret / fusion protein binds to the cell and the RetL2 protein is a ligand for Ret. In addition, purple stained cells were observed after transfection with the retL1 expression vector, but not after transfection with a negative control vector.

Cloning of Ret Ligand RetL3

A. Rat RetL 3

Rat RetL1 amino acid sequence and EST database investigation revealed two rat ESTs with identity to the Ret ligand. These ESTs are AA049894 and AA050083 (which are partial murine retL3 cDNA, SEQ ID NO: 14). Plasmids encoding these ESTs were obtained as bacterial puncture from Genome Systems Inc. (catalogs # 475791 and # 475497). Plasmid DNA is prepared from a single colony obtained by plating a puncture on an LB Amp plate. Comparing the two sequences demonstrated that AA049894 with a 1.4 kb insert was included in AA050083 with a 1.9 kb insert. Translation of the DNA sequence obtained from AA050083 indicates that there is a continuous open reading frame from NT205 to NT1242 (partial murine RetL3; SEQ ID NO: 15). This ORF shows 37.5% identity to that of rat retL1 and 40.2% to that of rat retL2. However, the open reading frame does not encode the Met or signal sequence at the 5 'end. The 5 ′ ORF upstream of this region was examined and Met was found under the Ka2ak consensus sequence for translation initiation and the effective signal sequence for surface expression / secretion. This ORF is a downstream OFR outside the framework, meaning that EST AA050083 contains an effective mutation such as an insertion, deletion, intron or cloning artifact at the 5 'end.

To get the correct 5 'end, use the marathon RACE. Mouse 11-day-old embryos Marathon-Ready ^™ cDNA (catalog # 7458-1) and Advantage ^™ kit (catalog 8417-1) are purchased from Clontech. An antisense oligonucleotide Kid-366 corresponding to the complement of nucleotides 847-866 of SEQ ID NO: 14 and an antisense oligonucleotide Kid-365 corresponding to the complement of nucleotides 589-615 of SEQ ID NO: 14 are synthesized. PCR is performed using the Advantage ^™ cDNA PCR Kit (Clontech Catalog # 8417-1) with Marathon ^™ cDNA reagent and oligonucleotide Kid-366. The first PCR reaction was set up as follows: 35.3 μl H ₂ O; 5.0 μl 10 × Klen Taq buffer; 1.0 μl 10 mM dNTP mix; 1.0 μl 50X Advantage ^™ Klen Taq Polymerase Mix. These reagents are mixed together. 5.0 μl of Marathon-Ready ^™ mouse 11-day embryonic cDNA; Add 1.0 μl 10 μM AP1 primer and 1.7 μl 5.88 μM Kid-366 (final volume = 50 μl). PCR is performed in Perkin-Elmer Setus DNA Summer Cycler 480 with the following cycle conditions: 1 cycle at 94 ° C. for 1 minute; 5 cycles at 94 ° C. for 30 seconds and 72 ° C. for 4 minutes; 5 cycles at 94 ° C. for 30 seconds and 70 ° C. for 4 minutes; 25 cycles at 94 ° C. for 30 seconds and 68 ° C. for 4 minutes. Nested PCR is performed using the product of the first PCR reaction. First, 5 μl of PCR product # 1 is diluted 50-fold with TE (final volume 250 μl). Nest PCR reactions were 35.5 μl H ₂ O; 5.0 μl 10 × Klen Taq buffer; 1.0 μl 10 mM dNTP mix; 1.0 μl 50 × Advantage ^™ Klen Taq Polymerase Mix. These reagents are mixed as described above. Then 5.0 μl diluted PCR product # 1; Add 1.0 μl 10 μM AP2 primer and 3.6 μl 2.8 μM Kid-365. Cycle conditions are the same as described above. About 665 bp of product is purified on a 1% low melt agarose gel and extracted with QiaexII (Qiaex Catalog # 20021). Purified DNA is cloned into pNoTA / T7 ^™ using the PRIME PCR CLONER ^™ cloning system (5 Prime> 3 Prime Catalog # 1-725029). Sequence information is obtained from several isolates, including clones called DSW252 and DSW253.

The sequence of DSW252 was found to overlap with SEQ ID NO: 14 except that an additional T is present between NT252 and NT253 of SEQ ID NO: 14 sequence. This T is also present in the other isolates DSW251 and DSW253. Additional bases are inserted to correct the ORF to obtain a single 1191 bp ORF (counting from the first Met) encoding 397 amino acids. This ORF encodes Met under the canonical translation initiation consensus consensus sequence (Cossack) and includes a signal sequence for surface expression / secretion.

To obtain full-length murine clones that can be expressed, 630 bp Not1-BamH1 fragment of DSW252 and 1308 bp BamH1-Not1 fragment of AA050083 are purified and ligated to Not1 degradation expression vector CH269. This ligation is transformed into E. coli XL1-Blue (Strazen Catalog # 200236). Qiawell Ultra Minipreps are performed on production transformants. They are analyzed for correct orientation and size by limiting digestion and gel electrophoresis. This construct is named DSW254. The insert of DSW254 was totally sequenced (rat retL3; SEQ ID NO: 16), and the ORF was confirmed to encode a protein of 397 amino acids (rat RetL3; SEQ ID NO: 17). The sequence is shown in FIG. 9. The C terminus of RetL3 is hydrophobic, indicating a phosphatidylinositol glycan binding motif.

B. Human RetL3

To find a target tissue for cloning human RetL3, Northern blots of mouse tissue are used to determine the expression pattern of murine RetL3. Among the tissues examined, RetL3 expression was highest in cardiac tissue. Human adult cardiac cDNA library in vector lambda gt10 is purchased from Clontech (catalog # HL3026a). One million plaque forming units obtained from phage stock are plated on 10 Nunc plates. Replica plaque lifts are made on Schreicher and Schiel Optitran ^™ filters.

Probes are formed by PCR with primers Kid-366 and Kid-367 corresponding to nucleotides 397-420 of the AA050083 sequence. PCR reactions were set up as follows; 10 μl 10X PFU buffer, 2.0 μl 10 mM dNTP mix, 72.1 μl H ₂ O, 3.1 μl 13.2 μM Kid-367, 6.8 μl 5.88 μM Kid-366, 5.0 μl 0.1 μg / μL AA050083 DNA and 2.0 μl 2.5 units / μL PFU Mix (Strazen Catalog # 600154). PCR is performed in Perkin-Elmer Setus DNA Summer Cycler 480 with the following cycle conditions: 25 cycles at 94 ° C. for 1 minute, 53 ° C. for 1 minute, 72 ° C. for 4 minutes. The product is extracted with 50: 49: 1 phenol, chloroform, isoamyl alcohol and purified by performing low melt agarose gel electrophoresis and QiaexII purification of the excised fragments. This coding region probe was randomly challenged to P ³² label (Feinberg and Vogelstein). The filtrate is hybridized overnight at 200 ° C. in 200 ml plaque search PBS buffer containing 10% dextran sulfate, 100 μg / ml tRNA and 6.7 × 10 ⁸ CPM of mouse probe. It is washed twice with plaque search buffer at 65 ° C., twice with 2 × SSC / 1% SDS, twice with 1 × SSC / 1% SDS and then exposed to the film at −70 ° C. using a sensitivity enhanced screen. Replication positives are plaque purified. Lambda miniprep DNA obtained from purified subject plaques is digested with EcoR1, electrophoresed on a 1% agarose gel and Southern blot. Southern blots hybridize with mouse probes. Clone GJ128 with an insert of 1.3 kb strongly hybridizes to the mouse cryptographic region probe. DNA sequences (partially human retL3 cDNA; SEQ ID NO: 18) and inferred peptide sequences (partially human RetL3; SEQ ID NO: 19) were obtained from this clone and identified as human homologues. This clone encodes most cryptographic domains, including the 3 'end of the cryptographic domain.

To obtain a clone with the 5 'end, the 1.3 kb insert from GJ128 was purified and labeled with P ³² and used to search the clonetech human adult heart library. Clones containing the 5 'end were not obtained in the search of 2x10 ⁶ plaques obtained from this library. Northern analysis of human adult tissue mRNA blots (clonetechnical catalogs # 7760-1, 7759-1 and 7767-1) hybridized with the same probe using a manufacturer-supplied protocol showed that human RetL3 was isolated from human adult spinal cord, stomach and heart. , Pancreas, small intestine, colon, prostate and testes. Clontech human adult spinal cord cDNA library (catalog # 5001a) is searched using a GJ128 insert. Three independent clones are purified and the longest GJ135 is sequenced. The insert sequence of GJ135 overlaps with the insert of GJ128, which forms a complex sequence of full length human retL3 cDNA (SEQ ID NO: 20) and determines full length human RetL3 (SEQ ID NO: 21). These sequences are shown in FIG. Human RetL3 is 34.3% and 34.9% identical for human RetL1 and human RetL2, respectively. Human RetL3 is 76.8% identical to rat RetL3.

Therapeutic Uses of Compounds of the Invention

Activation or blocking of Ret signaling pathways, stimulation of renal and / or neuronal cell proliferation or survival in the state of lost or impaired disease, or inhibition or proliferation of undesirable cells such as tumor cells expressing Ret or RetL If desired, anti-RetL antibodies of natural and modified RetL, anti-Ret antibodies, and fusion proteins of Ret and RetL may have therapeutic utility.

In general, compounds of the invention that bind to Ret induce dimerization and / or autophosphorylation of Ret, and are useful for promoting proliferation or limiting damage to Ret expressing tissue. The compounds of the present invention are useful for minimizing damage to kidney tissue after stimulating proliferation and / or survival of kidney tissue, supporting kidney function and various injuries. Specific symptoms that may be useful with treatment with the compounds of the present invention include acute renal failure, acute nephritis, chronic renal failure, nephrotic syndrome, tubular defects, kidney transplantation, toxic damage, hypoxic damage and trauma.

Tubular defects include hereditary or acquired diseases such as polycystic kidney disease, medulla cystic disease, and medulla spongy kidney. This example is not limited to, and may include many other renal diseases (eg, Harrison's Principles of Internal Medicine, 13th ed., 1994, incorporated herein by reference).

As another use, the genes and proteins of the present invention can be used to treat diseases that require neural proliferation and regeneration. Examples include Alzheimer's disease, Parkinson's disease, Huntington's disease, Tourette's disease, amyotrophic lateral sclerosis, as well as neurodegenerative diseases such as motor neuron disease, dehydration diseases such as multiple sclerosis, bacterial diseases such as meningitis, abscesses or sinusitis, Viral diseases such as HIV-associated myelopathy, and prion diseases such as Creutzfeldt-Jakob disease. It also includes damage disorders to nerve tissue, whether or not the causative factor is due to tumor invasion, trauma, or cerebrovascular cases such as bleeding or embolism. Specifically, it includes cranial nerve diseases or spinal cord diseases, including diseases associated with traumatic, inflammatory, congenital or vascular pathogenesis, diseases affecting the autonomic nervous system. It also includes neurological disorders such as mental retardation, autism, fetal alcohol syndrome, Down syndrome and cerebral palsy. The compounds of the present invention can be used to treat syndromes associated with the peripheral nervous system. Such diseases include diseases caused by the aforementioned etiologies, and specifically include Lyme disease, HIV-related neuropathy, multiple myositis, muscular dystrophy and myasthenia gravis.

The anti-RetL antibodies and Ret fusion proteins described herein that specifically bind to a protein or fragment of rat RetL, partial human RetL1, full length human RetL1, human RetL2, rat RetL3, or human RetL3 can be used in various methods. Can be. The compounds of the present invention can be used therapeutically to inhibit or block Ret receptor signaling, such as to block tumor growth dependent on the activation of Ret signaling upon growth. Such preparations can be fused to detection markers, such as X-ray fluoroscopy or opaque material for radiographic imaging, to image tissue expressing RetL by administration to the specimen. The agent can also be used in combination with a substance such as horseradish peroxidase, which can be used as an immunohistochemical stain to visualize RetL positive cell regions on tissue fragments. In this method, a specific antibody can be used alone, and the site to which the antibody binds can be visualized by a sandwich assay using an anti-immunoglobulin antibody bound to a detection marker. Specific antibodies against all RetLs are also useful for immunoassays to quantify substances that are specific for these antibodies. Antibodies specific for RetL can be used in conjunction with solid supports such as beads or dishes and can be used to remove ligands from solution or to remove proteins from solution. All of these techniques are common to those of ordinary skill in the art of immunology.

Other methods of the present invention include controlling Ret-RetL signaling by contacting Ret with an anti-Ret monoclonal antibody. This mAb-Ret contact has the effect of blocking or stimulating activation of the Ret signaling pathway depending on the interaction characteristics of Ret with each particular mAb. Certain mAbs interact with Ret as an agonist, and the binding of agonist mAb-Ret triggers dimerization and autophosphorylation of Ret. Other mAbs act as Ret antagonists. When Ret interacts with the antagonist mAb, Ret signaling activation is blocked by other RetL or complexes containing RetL that normally activate the Ret signaling pathway.

Antibodies to RetL and / or Ret or antibodies to Ret fusion proteins can be used to image tissues expressing Ret or in the immunohistologic or purification methods described above for antibodies to RetL.

Fusion proteins containing RetL and / or anti-Ret antibodies can be used to specifically target medical therapeutics for cancers and tumors expressing Ret. Such tumors include several other tumor phenotypes associated with mutations in Ret (N. Engl. J. Med. 335: 943-951, 1996; Nature 367: 319-320, 1996; Trends Gen. 12: 138-144 , 1996). Therapeutic involvement in tumors expressing RetL utilizes fusion proteins containing Ret and / or antiRetL antibodies. Anti-Ret antibodies or anti-RetL antibodies may exert their effects through antibody dependent cytolysis and complement dependent lysis that are themselves mediated by the Fc domain. Such hybrid ligands and antibodies can be used as delivery excipients for cytotoxic radionuclides such as tumor suppressor drugs, toxins and yttrium 90 to obtain more effective cancer therapeutics. Cytotoxic effector cells are tumors using heterozygous antibodies in which antibodies specific for Ret or RetL expressed by the tumor are covalently bound to antibodies directed against surface proteins on cytotoxic effector cells such as NK cells or CTLs. Can target cells.

An example of an anti-Ret antibody or RetL therapy is the conjugation of a toxic A chain of lysine or a modified full-length form of lysine (which can no longer bind to cells) to an antibody directed against RetL or a Ret polypeptide expressed on the surface of malignant cells. It is to let. In another embodiment, toxins can be bound to Ret or anti-RetL antibodies to selectively target and kill RetL positive cells, such as tumors expressing RetL. This method has proven successful with blocked lysine conjugated to monoclonal antibodies against the CD19 antigen expressed on most tumor cells (Grossbard et al., Blood 79: 576, 1992). All other toxins known in the art are equally useful. Examples include, but are not limited to, Pseudomonas exotoxin, diphtheria toxin, and saporin. This method is observed to be more successful when using RetL or anti-Ret antibodies as opposed to the known anti-CD19 antigens because Ret is expressed in a very limited number of tissues.

The methods described above can be used equally for toxin conjugates of RetL or anti-Ret antibodies, using fusions of lysine or other toxins, ie, useful for selectively targeting and killing Ret positive cells, such as tumor cells expressing Ret. .

Another method of using such medical therapeutics is to use radioisotope labeled RetL or anti-Ret antibodies. Such radiolabelled compounds are preferred because they target radioactivity only to tumor sites present in cells expressing Ret without affecting normal tissues. Depending on the radioactive isotope employed, radioactivity released from radiolabeled antibodies bound to tumor cells may kill cells in the vicinity of malignant tumor cells that do not express Ret. Various radionuclides can be used. Isotopes that release β particles (eg, ¹³¹ I) are useful when using monoclonal antibodies against CD20 present on B cell lymphoma [Kaminski et al., N. Engl. J. Med. 329: 459 (1993); Press et al., N. Engl. J. Med. 329: 1219 (1993). Radionuclides releasing β particles produce tumor-degradable radioactive releases over distances corresponding to different cell diameters to eradicate antigen-negative cells and reduce the consequences of heterogeneous deposition of antibodies or ligands in tumors.

Radionuclides emitting alpha particles can also be used. Low dose radiation generated by radionuclide labeled RetL or anti-Ret antibodies is therapeutically more effective than immediate externally delivered radiation in conventional radiation therapy. Low doses of radiation can induce apoptosis (planned cell death) in certain cell lines [Macklis et al., Radiat. Res. 130: 220 (1992); Maklis et al., Radiopharm. 5: 339 (1992).

The compounds of the present invention are administered in a therapeutically effective amount, ie, in an amount which produces a medically desired result or affects the particular disease to be treated.

As used herein, the term "sample" refers to any mammal to which a Ret ligand or gene is administered. Certain specimens to be treated in accordance with the methods of the invention include humans, non-human primates, sheep, horses, cattle, goats, pigs, dogs, cats, rabbits, guinea pigs, hamsters, gerbils, rats and mice, as well as these hosts. Organs, tumors, and cells derived from or arising from them.

Use of Compounds of the Invention in Gene Therapy

RetL genes of the present invention, when introduced into damaged tissues, can stimulate RetL production by transfected cells and promote the proliferation and / or survival of cells expressing Ret.

As a specific aspect of gene therapy, the RetL gene can be introduced into selected kidney or nerve target tissues. RetL can then be stably expressed to stimulate growth, division, differentiation and / or enhanced cell survival of Ret receptor positive cells. In addition, RetL genes are introduced into target cells using a variety of known methods using procedures based on viruses or non-viruses.

Non-viral methods include electropenetration, membrane fusion with liposomes, rapid bombardment with DNA-coated microinjections, incubation with calcium phosphate DNA precipitates, DEAE dextran-mediated transfection and direct microinjection into single cells. It includes. For example, the RetL gene can be introduced into cells via calcium phosphate co-precipitation (Pillicer et al., Science, 209: 1414-1422 (1980); mechanical microinjection and / or particle acceleration (Anderson et al., Proc. Natl). Acad. Sci. USA, 77: 5399-5403 (1980); liposome-based DNA transfer methods (e.g., LIPOFECTIN mediated transfection-Fefgner et al., Proc. Nat. Acad. Sci., USA, 84: 471-477. , 1987; Gao and Huang, Biochim. Biophys. Res. Comm., 179: 280-285, 1991); DEAE dextran-mediated transfection; electropermeation (US Pat. No. 4,956,288); or DNA conjugated to hepatocytes Polylysine-based methods for preferential delivery of DNA (Wolff et al., Science, 247: 465-468, 1990; Curiel et al., Human Gene Therapy 3: 147-154, 1992).

Target cells can be transfected by direct gene transfer using the genes of the invention (eg, Wolff et al., "Direct Gene Transfer Into Moose Muscle In Vivo", Science 247: 1465-1468, 1990). In most cases vector-mediated transfection is preferred. Any method known in the art for inserting polynucleotide sequences into vectors can be used (eg, Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY (1989) and Ausubel et al., Current Protocols in Molecualr Biology, J. Wiley & Sons, NY (1992), incorporated herein by reference. Promoter activation is induced or metabolized by metabolites or administered substances. Examples of such promoters / enhancers include native RetL promoters, cytomegalovirus contiguous early promoters / enhancers (Karasuyama et al., J. Exp. Med., 169: 13 (1989)); Human beta actin promoter [Gunning et al., Proc. Nat. Acad. Sci. USA, 84: 4831 (1987); Glucocorticoid inducible promoters present in mouse breast cancer virus long terminal repeats (MMTV LRT) (Klessig et al., Mol. Cell. Biol., 4: 1354 (1984)); Intestinal terminal repeat sequences of Molluni rat leukemia virus (MuLV LTR) (Weiss et al., RNA Tumor Viruses, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY (1985)); SV40 early region promoter (Bernoist and Chambon, Nature, 290: 304 (1981)); Lewis sarcoma virus (RSV) (Yamamoto et al., Cell, 22: 787 (1980)); Herpes simple virus (HSV) thymidine kinase promoter [Wagner et al., Proc. Nat. Acad. Sci. USA, 78: 1441 (1981); Adenovirus promoters [Yamada et al., Proc. Nat. Acad. Sci. USA, 82: 3567 (1985), but is not limited to such.

In addition, RetL genes can be introduced via certain viral vectors used in currently known gene transfer systems. Examples are disclosed in the following documents. Madzak et al., J. Gen. Virol., 73: 1533-1536, 1992 (Papovavirus SV40); Berkner et al., Curr. Top. Microbiol. Immunol., 158: 39-61, 1992 (adenovirus); Hofmann et al., Proc. Natl. Acad. Sci. 92: 10099-10103, 1995 (baculovirus); Moss et al., Curr. Top. Microbiol. Immunol., 158: 25-38, 1992 (headache virus); Muzyczka, Curr. Top. Microbiol. Immunol., 158: 97-123, 1992 (Adeno-associated virus); Margulskee, Curr. Top. Microbiol. Immunol., 158: 67-93, 1992 [Herpes Simplex Virus (HSV) and Epstein Barr Virus (HBV)]; Miller, Curr. Top. Microbiol. Immunol. 158: 1-24, 1992 (retrovirus); Brandyopadhyay et al., Mol. Cell. Biol., 4: 749-754, 1984 (retrovirus); Miller et al., Nature, 357: 455-450, 1992 (retrovirus); Anderson, Science, 256: 808-813, 1992 (Retrovirus), Current Protocols in Molecular Biology: Sections 9.10-9.14 (Ausubel et al., Eds.), Greene Publishing Associates, 1989 (all incorporated herein by reference).

Preferred vectors are adenoviruses (preferably Ad-2 or Ad-5 based vectors), baculoviruses, herpes viruses (preferably herpes simple virus based vectors) and parvoviruses (preferably "deleted" or non-autonomous) DNA virus comprising a parvovirus based vector, more preferably an adeno-associated viral vector, most preferably an AAV-2 based vector). Examples include those described in Ali et al., Gene Therapy 1: 367-384, 1994; US Pat. Nos. 4,797,368 and 5,399,346, as described below.

For example, a particular vector system for transferring RetL sequences is selected according to several factors. One important factor among them is the characteristics of the target cell population. Although retroviral vectors have been extensively studied and used in many gene therapy applications, they are generally not suitable for infecting non-dividing cells, but are useful for cancer therapy because they express only their own genes in cloned cells. Do. This vector is also useful in ex vivo methods because it integrates stably into the target cell genome.

Adenoviruses are eukaryotic DNA viruses that can be modified to effectively deliver therapeutic or reporter mutant genes to a variety of cell types. Common adenovirus types 2 and 5 (Ad2 and Ad5, respectively) that cause respiratory diseases in the human body are currently being developed for gene therapy of Duchenne muscular dystrophy (DMD) and cystic fibrosis (CF). Ad2 and Ad5 belong to the subfamily of adenoviruses not associated with human malignancies. Adenovirus vectors can deliver mutant genes with extremely high efficiency to virtually any cell type, regardless of mitosis stage. High titers of recombinant virus (10 ¹³ plaque forming units / ml) can be readily obtained in 293 cells (adenovirus transformed complementary human fetal kidney cell line: ATCC CRL 1573) and can be stored cold for prolonged periods without appreciable loss. Can be. Efficacy in in vivo delivery of therapeutic mutant genes to compensate for genetic imbalance in this system has been demonstrated in animal models of various diseases [Y. Watanabe, Atherosclerosis, 36: 261-268 (1986); K. Tanzawa et al., FEBS Letters, 118 (1): 81-84 (1980); JL Golasten et al., New Engl. J. Med., 309 (11983): 288-296 (1983); S. Ishibashi et al. J. Clin. Invest., 92: 883-893 (1993); And S. Ishibashi et al., J. Clin. Invest., 93: 1889-1893 (1994), all of which are incorporated herein by reference]. Indeed, cDNA encoding a recombinant replication-deficient adenovirus against cystic fibroblastic dura modulator (CFTR) has been used in two or more human CF clinical trials [J. Wilson, Nature, 369: 691-692 (Oct., 21, 1993). In addition, evidence that recombinant adenoviruses are safe for gene therapy is supported by the experience of using live adenovirus vaccines in humans.

Human adenovirus consists of a linear two-stranded DNA genome of about 36 kb divided into 100 map units (m.u.) each 360 bp in length. This DNA contains at each end of the genome a short reverse terminal repeat (ITR) required for viral DNA replication. This gene product consists of early (E1 to E4) and late (L1 to L5) regions based on whether they are expressed before or after the initiation of viral DNA synthesis [Horwitz, Virology, 2d edit., Ed. B. N. Fields, Raven Press Ltd., New York (1990)].

First generation recombinant replication deficiency adenoviruses developed as gene therapy for DMD and other genetic diseases have defects in the entire region of E1a and in some regions of E1b. This replication defective virus propagates in 293 cells that produce a functional adenovirus E1a gene that provides a transfunctional E1a protein. E1 deficient viruses are capable of replicating and producing infectious viruses among 293 cells of the gene product of the El and El regions in a trans manner. The resulting virus can infect many cell types and can express the transgene (if carrying its own promoter), but among the cells that do not carry the E1 region DNA unless the cells are infected with a very high multiplicity of infection. You cannot duplicate it. Adenoviruses have a broad host range and can infect resting cells, such as neurons, or terminally differentiated cells, and have the advantages of being observed substantially non-tumorigenic. Adenoviruses are not observed to integrate into the host genome. Thus, since the virus is extrachromosomal, the risk of insertional mutagenesis is greatly reduced (Ali et al., Supra, p. 373). Recombinant adenovirus (rAdV) has very high titers, moderate stability of viral particles, high expression rates and can infect a variety of cells. These natural host cells are airway epithelial cells and are therefore useful for treating lung cancer.

Baculovirus mediated transmission has several advantages. Baculovirus gene transfer can occur in cloned and nonreplicated cells, and can also occur in kidney cells, hepatocytes, nerve cells, spleen, skin and muscle. Baculoviruses are non-replicating and therefore non-pathogenic to mammalian cells. Humans do not have a prototype antibody against recombinant baculovirus that can block baculovirus infection. Baculoviruses can also be transduced by bottling very large DNA inserts.

In addition, adeno-associated virus (AAV) has been used as a vector for somatic gene therapy. AAV is a small single-stranded (ss) DNA virus with a simple genome construct (4.7 kb) that is an ideal substrate for genetic engineering. Two open reading frames encode a series of rep and cap polypeptides. rep polypeptides ( rep78 , rep68 , rep62 and rep40 ) are involved in the replication, rescue and integration of the AAV genome. cap proteins (VP1, VP2 and VP3) form virion capsids. The rep and cap open reading frames are adjacent to the 5 'and 3' ends of an inverted terminal repeat (ITR) of 145 bp, of which the first 125 bp will form a double strand structure of type Y and T. Can be. What is important in the development of AAV vectors is that the entire rep and cap domains can be cut out and replaced with therapeutic or reporter mutant genes [BJ Carter, in Handbook of Parvoviruses, ed., P. Tijsser, CRC Press, pp. 155-168 (1990). ITRs have been found to be the minimum sequences required for replication, rescue, packaging and integration of the AAV genome.

AAV living ring is a two-stage type consisting of incubation and lysis. During latent infection, AAV virions enter the cell as encapsulated ssDNA, which are then immediately delivered to the nucleus, stably integrating AAV DNA into the host chromosome without apparent host cell division. This integrated AAV genome remains dormant in the absence of a helper virus but can be activated and rescued. The lytic phase of the life cycle is initiated when cells containing AAV proviruses are secondary infected by herpesviruses or adenoviruses (encoding a helper function that helps to be recovered by AAV and cleaved from host chromatin) (BJCarter, supra). See literature). Infectious parent ssDNA becomes a double stranded clone (RF) DNA in a rep- dependent manner. The rescued AAV genome is packaged with a preformed protein capsid (symmetric icosahedron with a diameter of about 20 nm) and released into infectious virions that packaged the + or − ssDNA genome after cell lysis.

Adeno-associated virus (AAV) is very important for gene therapy. This virus particle is very stable and recombinant AAV (rAAV) has "drug-like" properties because it can be purified by CsCl gradient banding or pelletization. These particles are heat stable and can be lyophilized to powder and then rehydrated to show full activity. The DNA of these particles is stably integrated into the host chromosome and its expression is long term. The host range of these particles is wide and their recombinant vectors are nontoxic because AAV is not known to cause any disease.

After introduction into the target cell, the target sequence can be identified by conventional techniques such as nucleic acid hybridization using probes containing sequences homologous / complementary to the gene sequence inserted into the vector. Alternatively, the sequence may be identified by the presence or absence of a "marker" gene function (eg, thymidine kinase activity, antibiotic resistance, etc.) that appears upon introduction of the expression vector into the target cell.

Formulation and Administration Methods

The compounds of the present invention can be administered by any medically acceptable method. Examples include oral, nasal, ocular, rectal or topical administration, as well as injections via parenteral routes such as intravenous, intravascular, intraarterial, subcutaneous, intramuscular, intratumoral, intraperitoneal, intraventricular, intradural, etc. Include. Also included in the present invention are sustained release dosage forms, such as depot injections or erosive implants. Local delivery forms are particularly preferred, examples of which include delivery means such as catheter to one or more arteries, such as renal arteries or one or more arteries such as blood vessels located in local tumors.

The term "pharmaceutically acceptable carrier" means one or more organic or inorganic components, natural or synthesized, that are mixed with a mutant proontogene or mutant oncoprotein to facilitate administration. Suitable carriers include aqueous or non-aqueous isotonic sterile solutions and sterile suspensions known to those skilled in the art, although sterile saline is preferred. The term "carrier" also includes all plasmids and viral expression vectors as well as liposomes and HIV-1 tat proteins (Chen et al., Anal. Biochem. 227: 168-175, 1995). By "effective amount" is meant an amount that can mitigate or delay the progression of a disease, degeneration or impairment. The effective amount will vary from person to person, but in part based on the symptoms being treated and the desired outcome. In view of these factors one skilled in the art will be able to determine the effective amount without further general experimentation.

Liposome systems may use various monolayer lamellae vesicles, multilayer lamellae vesicles or stable multilayer lamellae vesicles, and are well known in the art, such as in US Pat. Nos. 5,169,637, 4,762,915, 5,000,958 or 5,185,154. It may be prepared and administered according to the teachings described. In addition, it may be desirable to express the novel polypeptides of the invention as well as other selected polypeptides as lipoproteins in order to enhance binding to liposomes. For example, treatment of human acute renal failure with liposome encapsulated RetL can be performed in vivo by introducing RetL with liposomes into cells in need of such treatment. This liposome can be delivered to the renal artery via a catheter. Recombinant RetL protein can be purified into, for example, CHO cells by immunoaffinity chromatography or other conventional method and then mixed into liposomes into liposomes with high efficiency. Encapsulated proteins are tested for their effect on stimulation of cell growth in vitro.

The novel polypeptides described in the present invention are preferably administered to an animal via a liposome delivery system to enhance its stability and / or immunogenicity. Delivery of new polypeptides via liposomes is particularly preferred because the liposomes are internalized by phagocytes in the treatment animal. These cells, upon digesting the liposome membrane, subsequently provide the polypeptide of the invention to the immune system along with other molecules necessary to elicit a strong immune response.

The novel RetL polypeptides of the present invention are available in pharmaceutically acceptable salt form. Suitable acids and bases capable of forming salts with the polypeptides of the invention are well known in the art, and examples include inorganic and organic acids and bases.

The present invention has been described above for the purpose of clearer understanding through some detailed description, but it will be apparent to those skilled in the art that any change and modification can be made within the scope of the present invention as defined by the appended claims. .

Sequence list

(1) General Information:

(i) Applicant: Doherty Jody

(ii) Name of the invention: Biogen patent application title

(iii) Number of sequences: 21

(iv) Letter Address:

(A) Name: Biogen Incorporated

(B) Street: Cambridge Center 14

(C) City: Cambridge

(D) Note: Massachusetts

(E) Country: United States

(F) ZIP code: 02142

(v) computer readable form:

(A) Medium type: floppy disk

(B) Computer: IBM PC compatible model

(C) operating system: PC-DOS / MS-DOS

(D) Software: PatentIn Release # 1.0, Version # 1.30

(vi) Prior application data:

(A) Application number:

(B) Application date: May 7, 1997

(C) Classification code:

(vii) prior application data:

(A) Application number: US 08 / 999,999

(B) Application date: January 1, 1996

(viii) Agent Information:

(A) Name: Levin Leslie M.

(B) Registration Number: 35,245

(C) Ref: A008 PCT CIP

(ix) Communication Information:

(A) Telephone: 617-679-2400

(B) Fax: 617-679-2838

(2) Information about SEQ ID NO: 1

(i) Sequence Properties:

(A) Length: 3616 base pairs

(B) Sequence type: nucleic acid

(C) Number of chains: 2 strands

(D) Form: straight chain

(ii) Type of sequence: cDNA

(iii) presumed sequence: no

(iv) Antisense: No

(ix) sequence characteristics

(A) Name / Key: CDS

(B) Location: 257..1660

(xi) SEQ ID NO 1:

(2) Information about SEQ ID NO:

(i) Sequence Properties:

(A) Length: 468 amino acids

(B) Sequence type: amino acid

(D) Form: straight chain

(ii) Type of sequence: protein

(xi) SEQ ID NO 2:

(2) Information about SEQ ID NO:

(i) Sequence Properties:

(A) Length: 39 base pairs

(B) Sequence type: nucleic acid

(C) Number of chains: 1 chain

(D) Form: straight chain

(ii) Type of sequence: cDNA

(xi) SEQ ID NO: 3:

(2) Information about SEQ ID NO:

(i) Sequence Properties:

(A) Length: 33 base pairs

(B) Sequence type: nucleic acid

(C) Number of chains: 1 chain

(D) Form: straight chain

(ii) Type of sequence: cDNA

(xi) SEQ ID NO 4:

(2) Information about SEQ ID NO:

(i) Sequence Properties:

(A) Length: 33 base pairs

(B) Sequence type: nucleic acid

(C) Number of chains: 1 chain

(D) Form: straight chain

(ii) Type of sequence: cDNA

(xi) SEQ ID NO: 5:

(2) Information about SEQ ID NO:

(i) Sequence Properties:

(A) Length: 1926 base pairs

(B) Sequence type: nucleic acid

(C) Number of chains: 1 chain

(D) Form: straight chain

(ii) Type of sequence: cDNA

(ix) sequence characteristics

(A) Name / Key: CDS

(B) Location: 10..1920

(xi) SEQ ID NO 6:

(2) Information about SEQ ID NO:

(i) Sequence Properties:

(A) Length: 637 amino acids

(B) Sequence type: amino acid

(D) Form: straight chain

(ii) Type of sequence: protein

(xi) SEQ ID NO 7:

(2) Information about SEQ ID NO:

(i) Sequence Properties:

(A) Length: 1223 base pairs

(B) Sequence type: nucleic acid

(C) Number of chains: 1 chain

(D) Form: straight chain

(ii) Type of sequence: cDNA

(ix) sequence characteristics

(A) Name / Key: CDS

(B) Location: 1..1038

(xi) SEQ ID NO: 8:

(2) Information about SEQ ID NO:

(i) Sequence Properties:

(A) Length: 346 amino acids

(B) Sequence type: amino acid

(D) Form: straight chain

(ii) Type of sequence: protein

(xi) SEQ ID NO: 9:

(2) Information about SEQ ID NO: 10:

(i) Sequence Properties:

(A) Length: 1682 base pairs

(B) Sequence type: nucleic acid

(C) Number of chains: 1 chain

(D) Form: straight chain

(ii) Type of sequence: cDNA

(ix) sequence characteristics

(A) Name / Key: CDS

(B) Location: 118..1497

(xi) SEQ ID NO: 10:

(2) Information about SEQ ID NO:

(i) Sequence Properties:

(A) Length: 460 amino acids

(B) Sequence type: amino acid

(D) Form: straight chain

(ii) Type of sequence: protein

(xi) SEQ ID NO: 11:

(2) Information about SEQ ID NO: 12:

(i) Sequence Properties:

(A) Length: 1888 base pairs

(B) Sequence type: nucleic acid

(C) Number of chains: 1 chain

(D) Form: straight chain

(ii) Type of sequence: cDNA

(ix) sequence characteristics

(A) Name / Key: CDS

(B) Location: 25..1416

(xi) SEQ ID NO: 12:

(2) Information about SEQ ID NO: 13

(i) Sequence Properties:

(A) Length: 464 amino acids

(B) Sequence type: amino acid

(D) Form: straight chain

(ii) Type of sequence: protein

(xi) SEQ ID NO: 13:

(2) Information about SEQ ID NO: 14:

(i) Sequence Properties:

(A) Length: 1878 base pairs

(B) Sequence type: nucleic acid

(C) Number of chains: 1 chain

(D) Form: straight chain

(ii) Type of sequence: cDNA

(ix) sequence characteristics

(A) Name / Key: CDS

(B) Location: 205..1242

(xi) SEQ ID NO: 14:

(2) Information about SEQ ID NO: 15:

(i) Sequence Properties:

(A) Length: 346 amino acids

(B) Sequence type: amino acid

(D) Form: straight chain

(ii) Type of sequence: protein

(xi) SEQ ID NO: 15:

(2) Information about SEQ ID NO: 16:

(i) Sequence Properties:

(A) Length: 1889 base pairs

(B) Sequence type: nucleic acid

(C) Number of chains: 1 chain

(D) Form: straight chain

(ii) Type of sequence: cDNA

(ix) sequence characteristics

(A) Name / Key: CDS

(B) Location: 41..1231

(xi) SEQ ID NO 16:

(2) Information about SEQ ID NO: 17:

(i) Sequence Properties:

(A) Length: 397 amino acids

(B) Sequence type: amino acid

(D) Form: straight chain

(ii) Type of sequence: protein

(xi) SEQ ID NO: 17:

(2) Information about SEQ ID NO: 18:

(i) Sequence Properties:

(A) Length: 1271 base pairs

(B) Sequence type: nucleic acid

(C) Number of chains: 1 chain

(D) Form: straight chain

(ii) Type of sequence: cDNA

(ix) sequence characteristics

(A) Name / Key: CDS

(B) Location: 2..946

(xi) SEQ ID NO: 18:

(2) Information about SEQ ID NO: 19:

(i) Sequence Properties:

(A) Length: 315 amino acids

(B) Sequence type: amino acid

(D) Form: straight chain

(ii) Type of sequence: protein

(xi) SEQ ID NO: 19:

(2) Information about SEQ ID NO: 20:

(i) Sequence Properties:

(A) Length: 1699 base pairs

(B) Sequence type: nucleic acid

(C) Number of chains: 1 chain

(D) Form: straight chain

(ii) Type of sequence: cDNA

(ix) sequence characteristics

(A) Name / Key: CDS

(B) Location: 175..1374

(xi) SEQ ID NO: 20:

(2) Information about SEQ ID NO: 21:

(i) Sequence Properties:

(A) Length: 400 amino acids

(B) Sequence type: amino acid

(D) Form: straight chain

(ii) Type of sequence: protein

(xi) SEQ ID NO: 21:

Claims (47)

Purified and isolated DNA molecule having a nucleotide sequence designated as rat retL1 cDNA, partial human retL1 cDNA, human retL1 cDNA, human retL2 cDNA, rat retL3 cDNA or human retL3 cDNA. A DNA molecule encoding the amino acid sequence of rat RetL1, partial human RetL1, full length human RetL1, human RetL2, murine RetL3 or human RetL3. a) DNA molecules of rat retL1 cDNA, partial human retL1 cDNA, human retL1 cDNA, human retL2 cDNA, mouse retL3 cDNA or human retL3 cDNA, or complementary strands thereof; b) a DNA molecule hybridized to a DNA molecule or fragment thereof as defined in a) under stringent conditions; c) if there is no denaturation of the genetic code, it is selected from DNA molecules that hybridize to the DNA molecules defined in a) and b), Purification and e. Isolated DNA Molecules. A purified and isolated DNA molecule having at least 80% homology to rat retL1 cDNA, partial human retL1 cDNA, human retL1 cDNA, human retL2 cDNA, rat retL3 cDNA or human retL3 cDNA. The DNA molecule of claim 4 further characterized by encoding a protein having a biological activity of rat RetL1, partial human RetL1, full length human RetL1, human RetL2, murine RetL3 or human RetL3. The recombinant DNA molecule of any one of claims 1-5, wherein said recombinant DNA molecule is operably linked to an expression control sequence. A vector comprising a DNA molecule encoding rat RetL1, partial human RetL1, full length human RetL1, human RetL2, rat RetL3, partial human RetL3 or full length human RetL3. A plasmid or viral DNA vector having a biological function, comprising the DNA molecule according to any one of claims 1 to 5. Eukaryotic host cell or prokaryotic host cell stably transformed or transfected with a vector comprising a DNA molecule encoding rat RetL1, partial human RetL1, full length human RetL1, human RetL2, rat RetL3 or human RetL3. Propagating prokaryotic or eukaryotic host cells transformed or transfected with the DNA molecule of any one of claims 1 to 5 under appropriate culture conditions, and expressing this polypeptide product to some extent And recovering rat RetL1, partial human RetL1, full length human RetL1, human RetL2, rat RetL3, or human RetL3, including rat RetL1, partial human RetL1, full length human RetL1, human RetL2, rat RetL3, partial human RetL3 or full length A method of producing a polypeptide product having the biological activity of human RetL3 and some or all of its primary structural forms. The polypeptide product expressed in the eukaryotic host cell or prokaryotic host cell of Claim 10. Purified and isolated rat RetL1, partial human RetL1, full length human RetL1, human RetL2, rat RetL3, or human RetL3, with little other human protein. A protein having an amino acid sequence comprising rat RetL1, partial human RetL1, full length human RetL1, human RetL2, rat RetL3, partial human RetL3 or full length human RetL3. A protein that is a variant of rat RetL1, partial human RetL1, full length human RetL1, human RetL2, rat RetL3, partial human RetL3 or full length human RetL3. Soluble variant of a protein according to any one of claims 11 to 14. IgG fusion protein comprising rat RetL1, partial human RetL1, full length human RetL1, human RetL2, rat RetL3, partial human RetL3 or full length human RetL3. The protein of claim 15 fused to a toxin, an imaging compound, or a radionuclide. The monoclonal antibody specific for the protein of any one of Claims 11-14. The antibody of claim 18, wherein the antibody is bound to toxins, imaging compounds, or radionuclides. A hybridoma cell line producing antibodies specific for rat RetL1, partial human RetL1, full length human RetL1, human RetL2, rat RetL3, partial human RetL3 or full length human RetL3. The antibody produced by the hybridoma of Claim 20. Promoting the survival of a damaged tissue of a sample or administering an effective amount of a polypeptide of rat RetL1, partial human RetL1, full-length human RetL1, human RetL2, rat RetL3, partial human RetL3, or full-length human RetL3 to a sample, How to promote proliferation. The method of claim 22, further comprising administering to the subject a therapeutically effective amount of a second polypeptide. The method of claim 23, wherein the second polypeptide is a GDNF, neurturin, or GDNF related polypeptide. The method of claim 22, wherein the tissue is renal tissue or neural tissue. The method of claim 22, wherein the polypeptide is membrane bound. The method of claim 22, wherein the polypeptide is soluble. The method of claim 27, wherein the polypeptide is a fusion protein. The method of any one of claims 22-27, wherein the sample is human. A method of inhibiting signal transduction associated with a RetL-expressing cell, comprising contacting the cell with an antibody against RetL. A method of inhibiting signal transduction associated with a cell expressing RetL, comprising contacting the soluble Ret protein with the cell. The method of claim 31, wherein the soluble Ret protein is a fusion protein. A method of targeting a toxin, imaging compound, or radionuclide to a Ret-expressing cell, comprising contacting the RetL fusion protein of claim 17 with the cell. A method of inhibiting proliferation of tumor cells expressing Ret comprising contacting a cell with a fusion protein of RetL and a toxin or radionuclide. 35. The method of any one of claims 30-34, wherein the cells are in the sample and the protein is administered to the sample. A method of targeting a toxin, imaging compound or radionuclide to a cell expressing RetL, comprising contacting the cell with an anti-RetL antibody conjugated with a toxin, imaging compound or radionuclide. A method of inhibiting proliferation of tumor cells expressing RetL, comprising contacting a cell with an anti-RetL antibody conjugated to a toxin or radionuclide. 38. The method of claim 36 or 37, wherein the cells are in the sample and the conjugated antibody is administered to the sample. A method of targeting a toxin, imaging compound or radionuclide to a cell expressing RetL, comprising contacting the cell with a Ret fusion protein comprising Ret and a toxin, imaging compound or radionuclide. The method of claim 39, further comprising contacting the GDNF, neutrin, or GDNF related polypeptide with the cell. A method of inhibiting proliferation of tumor cells expressing RetL, comprising contacting the cells with a fusion protein of RetL and a toxin or radionuclide. 42. The method of any one of claims 39-41, wherein the cells are in the sample and the protein is administered to the sample. A method of treating a subject with RetL metabolic disorders comprising administering to the subject a vector according to claim 7. A method for promoting the proliferation of new tissue in a sample, comprising administering to the sample a vector according to claim 7. 45. The method of claim 44, wherein the tissue is kidney tissue or neural tissue. A method of promoting survival of a damaged tissue of a sample, comprising administering to the sample a therapeutically effective amount of the vector according to claim 7. 47. The method of claim 46, wherein the tissue is kidney tissue or neural tissue.