Definitions

• the present invention relates to a novel CD44-like protein receptor.
• isolated nucleic acid molecules are provided encoding the CD44-like protein.
• CD44-like protein polypeptides are also provided, as are screening methods for identifying agonists and antagonists capable of enhancing or inhibiting CD44-like protein-mediated signaling.
• the invention further concerns therapeutic methods for treating diseases associated with processes mediated by CD44-like protein signaling.
• CD44 also known as Pgp-1, Hermes-3, HCAM, ECMR III
• Pgp-1 Hermes-3
• HCAM Hema-1
• ECMR III ECMR III
• CD44 is involved in a diverse range of biological functions such as lymphocyte binding to high endothelial venules (Jalkanen et al, J. Cell. Biol. 705:983-990 (1987)), lymphopoiesis (Miyake et al, J. Exp. Med. 777:477-488 (1990)) and activation of leukocytes (Webb et al, Science 249:1295-1291 (1990)).
• CD44 has also been shown to play a role in extracellular matrix binding, cell migration, lymphocyte activation, lymphocyte homing, and proliferation of bronchial smooth muscle cell (Herrlich et al., Immunology Today, 14 (8) : 395-3 9, (1993); Lesley etal., Immunology, 54:211-355, (1993); Lazzar et al, Journal of Experimental Medicine, 750:807-816 (1994)).
• a splice variant of CD44, CD44-V6 has been shown to play a role in tumor cell metastasis (Gunthert et al., Cell, 65: 13-24
• the CD44 cDNA sequence has revealed a domain of some 90 amino acids near the N-terminus bearing a significant similarity to the link and core proteins in proteoglycan (Goldstein et al.. Cell 56:1063-1072 ( 1989)), leading to the determination that hyaluronate, a major component of the extracellular matrix, is a ligand for CD44 (Miyake et al, J. Exp. Med 172:69-15 ( 1990); Aruffo et al, Cell 67:1303-1313 (1990)). Fibronectin and collagen type I and VI have also been shown to interact with CD44 (Carter et al, J. Biol. Chem. 265:4193-4201 (1988)).
• CD44 is a widely distributed heterogenous population of cell surface adhesion molecules.
• the CD44 gene has twenty exons, ten of which encode the sequence for standard CD44. An additional ten variable exons are inserted by alternative splicing.
• the standard 85 kDa CD44 is broadly expressed by many different types of cells, the expression of CD44 variants is rather limited. Arch et al. have demonstrated a transient expression of a CD44 variant, V6, in leukocytes from animals after allogeneic immunization (Arch et al., Science 257:682-685 (1992)), indicating a possible role for this variant in mediating leukocyte trafficking.
• CD44H the major CD44 isoform present on resting hemopoeitic cells
• CD44R1 a differentially expressed isoform containing a 132 amino acid insertion coded by the alternatively spliced exons v8-vl0, can bind both immobilized and soluble hyaluronan when transfected into the CD44-negative murine lymphoma cell line
• CD44R1 can induce these cells to homotypically aggregate. This homotypic aggregation may be mediated by the adhesive interaction between a determinant encoded by the insertion region present in CD44R1 and a common region present in both CD44R1 and CD44H.
• CD44 isoforms of CD44 are often preferentially expressed by certain cell types.
• a widely expressed CD44 isoform is the "standard” or "hematopoetic"
• CD44 CD44s molecule, which is encoded by exons 1-5, 15-17 and 19 of the
• CD44 gene This 85-95 kDa molecule is the principle CD44 isoform on hematopoetic cells and lymphocytes. Larger CD44 variants that contain different combinations of alternatively spliced exons are preferentially expressed on epithelial cells, but they can also be found on activated lymphocytes and high grade malignant lymphomas.
• the present invention provides isolated nucleic acid molecules comprising a nucleic acid sequence encoding a CD44-like protein whose amino acid sequence is shown in FIG. 1 [SEQ ID NO:2] or a fragment of the polypeptide.
• the CD44- like protein gene contains an open reading frame encoding a protein of about 322 amino acid residues whose initiation codon is at position 91-93 of the nucleotide sequence shown in FIG. 1 [SEQ ID NO. 1], with a leader sequence of about 21 amino acid residues, and a deduced molecular weight of about 35 kDa.
• the invention provides isolated nucleic acid molecules encoding the CD44-like polypeptide having an amino acid sequence encoded by the cDNA of the clone deposited as ATCC Deposit No. 97520 on April 25, 1996.
• the nucleic acid molecule will encode the mature polypeptide encoded by the above-described deposited cDNA.
• Preferred nucleic acid fragments of the present invention include nucleic acid molecules encoding: a polypeptide comprising the CD44-like protein extracellular domain (amino acid residues from about 22 to about 238 in FIG. 1 [SEQ ID NO 2]); a polypeptide comprising the CD44-like protein transmembrane domain (amino acid residues from about 239 to about 266 in FIG. 1 [SEQ ID NO 2]; a polypeptide comprising the CD44-like protein intracellular domain (amino acid residues from about 267 to about 322 in FIG. 1 [SEQ ID NO 2]; and a polypeptide comprising the extracellular and intracellular domain having all or part of the transmembrane region deleted.
• nucleic acid molecules having a nucleotide sequence that is least 90% identical, and more preferably at least 95%, 96%, 97%, 98% or 99% identical to the nucleotide sequence of any of the nucleic acid molecules described herein.
• the present invention also relates to vectors which include the isolated DNA molecules of the present invention, host cells which are genetically engineered with the recombinant vectors, and the production of CD44-like polypeptides or fragments thereof by recombinant techniques.
• polypeptides of the present invention include the polypeptide encoded by the deposited cDNA, the polypeptide of SEQ ID NO: 2 (in particular the mature polypeptide), as well as polypeptides having an amino acid sequence with at least 90% similarity, more preferably at least 95% similarity to the amino acid sequence of the polypeptide encoded by the deposited cDNA or the polypeptide of SEQ ID NO:2.
• polypeptides of the present invention include polypeptides having an amino acid sequence at least 80% identical, more preferably, at least 90% or 95% identical, to the amino acid sequence of the polypeptide encoded by the deposited cDNA or the polypeptide of SEQ ID NO:2.
• Preferred polypeptide fragments according to the present invention include a polypeptide comprising: the CD44-like protein extracellular domain, the CD44- like protein transmembrane domain; the CD44-like protein intracellular domain; or the CD44-like protein extracellular domain and intracellular domain having the transmembrane domain deleted.
• the invention also provides methods for administering an antagonist of the
• the method for inhibiting cell migration relates to treating cancer, metastasis, arteriosclerosis, or vascular restenosis and the method for inhibiting inflammation relates to treating graft versus host disease and rheumatoid arthritis.
• the invention further provides a method for increasing cell migration by administering a therapeutically effective amount of an agonist of the CD44-like protein mediated signaling pathway.
• the method of increasing cell migration is used to improve wound healing.
• the invention further provides a screening method for identifying compounds capable of enhancing or inhibiting a cellular response induced by the CD44-like protein receptor-mediated signaling pathway, which involves contacting cells which express the CD44-like protein receptor with the candidate compound, assaying a cellular response, and comparing the cellular response to a standard cellular response, the standard being assayed when contact is made in absence of the candidate compound; whereby, an increased cellular response over the standard indicates that the candidate compound is an agonist and a decreased cellular response over the standard indicates that the candidate compound is an antagonist.
• a screening assay for agonists and antagonists involves determining the effect a candidate compound has on ligand binding to the CD44-like protein receptor.
• the method involves contacting the CD44-like protein receptor with a ligand and a candidate compound and determining whether ligand binding to the CD44-like protein receptor is increased or decreased due to the presence of the candidate compound.
• FIG. 1 shows the nucleotide and deduced amino acid sequence of the CD44-Iike protein.
• Amino acids 1 to 21 represent the putative signal peptide (first underlined sequence), amino acids 22 to 238 the extracellular domain (sequence between the first and second underlined sequences), amino acids 239 to 266 the transmembrane domain (second underlined sequence), and amino acids 267 to 322 the intracellular domain (the remaining sequence).
• FIG. 2 shows the regions of similarity between the amino acid sequences of CD44-like protein and rat CD44 (deposited in Genbank with the accession number A38745).
• FIGs. 3 a and 3b are Northern blots showing the tissue distribution of human CD44-like mRNA expression.
• Fig 3a lane 1, spleen; lane 2, lymph node; lane 3, thymus; lane 4, appendix; lane 5, peripheral blood leukocyte; lane 6, bone marrow; lane 7, fetal liver.
• Figure 3b lane 1, heart; lane 2, brain; lane 3, placenta; lane 4, lung; lane 5, liver; lane 6, skeletal muscle; lane 7, kidney; lane 8, pancreas.
• the present invention provides isolated nucleic acid molecules comprising a polynucleotide encoding a CD44-like protein having an amino acid sequence shown in FIG. 1 [SEQ ID NO:2], which was determined by sequencing a cloned cDNA.
• the CD44-like protein of the present invention shares sequence homology with the rat cell adhesion molecule CD44 precursor (FIG. 2).
• the nucleotide sequence shown in FIG. 1 [SEQ ID NO: l] was obtained by sequencing the HUVDE75 clone, which was deposited on April 25, 1996, at the American Type Culture Collection, 12301 Park Lawn Drive, Rockville, Maryland 20852, and given accession number 97520.
• nucleotide sequences determined by sequencing a DNA molecule herein were determined using an automated DNA sequencer (such as the Model 373 from Applied Biosystems, Inc.), and all amino acid sequences of polypeptides encoded by DNA molecules determined herein were predicted by translation of a DNA sequence determined as above. Therefore, as is known in the art for any DNA sequence determined by this automated approach, any nucleotide sequence determined herein may contain a some errors. Nucleotide sequences determined by automation are typically at least about 90% identical, more typically at least about 95% to at least about 99.9% identical to the actual nucleotide sequence of the sequenced DNA molecule. The actual sequence can be more precisely determined by other approaches including manual DNA sequencing methods well known in the art.
• a single insertion or deletion in a determined nucleotide sequence compared to the actual sequence will cause a frame shift in translation of the nucleotide sequence such that the predicted amino acid sequence encoded by a determined nucleotide sequence will be completely different from the amino acid sequence actually encoded by the sequenced DNA molecule, beginning at the point of such an insertion or deletion.
• nucleotide sequence set forth herein is presented as a sequence of deoxyribonucleotides (abbreviated A, G , C and T).
• nucleotide sequence of a nucleic acid molecule or polynucleotide is intended, for a DNA molecule or polynucleotide, a sequence of deoxyribonucleotides, and for an RNA molecule or polynucleotide, the corresponding sequence of ribonucleotides (A, G, C and U) where each thymidine deoxynucleotide (T) in the specified deoxynucleotide sequence in is replaced by the ribonucleotide uridine (U).
• RNA molecule having the sequence of SEQ ID NO: l set forth using deoxyribonucleotide abbreviations is intended to indicate an RNA molecule having a sequence in which each deoxynucleotide A, G or C of SEQ ID NO: 1 has been replaced by the corresponding ribonucleotide A, G or C, and each deoxynucleotide T has been replaced by a ribonucleotide U.
• isolated nucleic acid molecules are provided which encode the CD44-like protein.
• isolated nucleic acid molecule(s) is intended a nucleic acid molecule, DNA or RNA, which has been removed from its native environment.
• isolated DNA molecules include recombinant DNA molecules maintained in heterologous host cells or purified (partially or substantially) DNA molecules in solution.
• isolated RNA molecules include in vitro RNA transcripts of the DNA molecules of the present invention. Isolate nucleic acid molecules according to the present invention further include such molecules produced synthetically.
• nucleic acid molecule of the present invention encoding a CD44- like protein may be obtained using standard cloning and screening procedures, such as those for cloning cDNAs using mRNA as starting material.
• standard cloning and screening procedures such as those for cloning cDNAs using mRNA as starting material.
• the nucleic acid molecule described in FIG. 1 was discovered in a cDNA library derived from human umbilical vein endothelial cells.
• the gene was also found in cDNA libraries derived from the following types of human cells: ovarian cancer, breast cancer, prostate, 12 week old early stage fetus, fetal heart, bone cancer, 8 week whole embryo, fetal liver, dura mater, umbilical vein endothelium, placenta, lung, and fetal liver-spleen.
• the CD44-like protein gene contains an open reading frame encoding a protein of about 322 amino acid residues whose initiation codon is at position 91- 93 of the nucleotide sequence shown in FIG. 1 [SEQ ID NO. 1], with a predicted leader sequence of about 21 amino acid residues, and a deduced molecular weight of about 35 kDa.
• the amino acid sequence of the predicted mature CD44-like protein is shown in FIG. 1 [SEQ ID NO: 1] from amino acid residue 22 to residue 322.
• the mature CD44-like protein has three main structural domains. These include the extracellular domain, which includes the ligand binding domain, and is predicted to correspond to amino acid residues from about 22 to about 238 in FIG. 1 [SEQ ID NO: 1].
• the mature extracellular domain is predicted to be about
• Another domain is the transmembrane domain, which has been predicted to correspond to about residues 239 to about 266 in FIG. 1 [SEQ ED NO: 1].
• Another domain is the intracellular domain, which has been predicted to correspond to amino acid residue 267 to about 322 in FIG. 1 [SEQ ID NO: 1].
• the CD44-like protein shown in FIG. 1 [SEQ ID NO 2] is about 24% identical and about 46% similar to the rat cell adhesion molecule CD44 precursor, which can be accessed on GenBank as Accession No. A38745.
• the actual full- length CD44-like protein (including the leader) encoded by the deposited cDNA comprises about 322 amino acids, but may be anywhere in the range of about 318- 328 amino acids; and the actual leader sequence of this protein is about 21 amino acids, but may be anywhere in the range of about 17 to about 25 amino acids.
• reasonable persons of skill in the art may disagree, depending on the criteria used, concerning the exact 'address' of the above described CD44-like protein domains.
• the exact location of the CD33-like protein extracellular, intracellular and transmembrane domains in FIG. 1 [SEQ ID NO. 2] may vary slightly (e.g., the exact 'address' may differ by about 1 to about 5 residues compared to that shown in FIG. 1) depending on the criteria used to define the domain.
• nucleic acid molecules of the present invention may be in the form of RNA, such as mRNA, or in the form of DNA, including, for instance, cDNA and genomic DNA obtained by cloning or produced synthetically.
• the DNA may be double-stranded or single-stranded.
• Single-stranded DNA may be the coding strand, also known as the sense strand, or it may be the non-coding strand, also referred to as the anti-sense strand.
• Isolated nucleic acid molecules of the present invention include DNA molecules comprising an open reading frame (ORF) whose initiation codon is at position 91-93 of the nucleotide sequence shown in FIG. 1 [SEQ ID NO 1] and further include DNA molecules which comprise a sequence substantially different than all or part of the ORF whose initiation codon is at position 91-93 of the nucleotide sequence shown in FIG. 1 [SEQ ID NO 1] but which, due to the degeneracy of the genetic code, still encode the CD44-like protein or a fragment thereof.
• the genetic code is well known in the art. Thus, it would be routine for one skilled in the art to generate the degenerate variants described above.
• the invention provides isolated nucleic acid molecules encoding the CD44-like polypeptide having an amino acid sequence encoded by the cDNA of the clone deposited as ATCC Deposit No. 97520 on April 25, 1996.
• the nucleic acid molecule will encode the mature polypeptide encoded by the above-described deposited cDNA.
• the invention further provides an isolated nucleic acid molecule having the nucleotide sequence shown in FIG. 1 [SEQ ID NO 1 ] or the nucleotide sequence of the CD44-like protein gene contained in the above-described deposited cDNA, or a nucleic acid having a sequence complementary to one of the above sequences.
• isolated nucleic acid molecules particularly DNA molecules, are useful as probes for gene mapping by in situ hybridization with chromosomes and for detecting expression of the CD44-like protein gene in human tissue by Northern blot analysis. As described in detail below, detecting enhanced CD44-like protein gene expression in certain tissues is indicative of certain disorders.
• the present invention is further directed to fragments of the isolated nucleic acid molecules described herein.
• DNA fragments at least 15 nt, and more preferably at least 20 nt, still more preferably at least 30 nt in length which are useful as diagnostic probes and primers as discussed above and in more detail below.
• larger DNA fragments 50-1500 nt in length are also useful as probes according to the present invention as are DNA fragments corresponding to most, if not all, of the nucleotide sequence of the deposited cDNA or the nucleotide sequence as shown in FIG. 1 [SEQ ID NO 1]
• fragments at least 20 nt in length for example, is intended fragments which include
• nucleic acid fragments of the present invention include nucleic acid molecules encoding: a polypeptide comprising the CD44-like protein extracellular domain (amino acid residues from about 22 to about 238 in FIG. 1
• the invention provides an isolated nucleic acid molecule comprising a polynucleotide which hybridizes under stringent hybridization conditions to a portion of the polynucleotide in a nucleic acid molecule of the invention described above, for instance, the cDNA clone contained in ATCC
• stringent hybridization conditions is intended overnight incubation at 42°C in a solution comprising: 50% formamide, 5x SSC (150 mM NaCl, 15mM trisodium citrate), 50 mM sodium phosphate (pH7.6), 5x Denhardt's solution, 10% dextran sulfate, and 20 ⁇ g/ml denatured, sheared salmon sperm DNA, followed by washing the filters in O. lx SSC at about 65°C.
• a polynucleotide which hybridizes to a "portion" of a polynucleotide is intended a polynucleotide (either DNA or RNA) hybridizing to at least about 15 nucleotides (nt), and more preferably at least about 20 nt, still more preferably at least about 30 nt, and even more preferably about 30-70 nt of the reference polynucleotide. These are useful as diagnostic probes and primers as discussed above and in more detail below.
• polynucleotides hybridizing to a larger portion of the reference polynucleotide e.g., the deposited cDNA clone
• a portion 50-750 nt in length, or even to the entire length of the reference polynucleotide also are useful as probes according to the present invention, as are polynucleotides corresponding to most, if not all, of the nucleotide sequence of the deposited cDNA or the nucleotide sequence as shown in FIG. 1 [SEQ ID NO:l].
• such portions are useful diagnostically either as a probe according to conventional DNA hybridization techniques or as primers for amplification of a target sequence by the polymerase chain reaction (PCR), as described, for instance, in Molecular Cloning, A Laboratory Manual, 2nd. edition, edited by Sambrook, J., Fritsch, E. F. and Maniatis, T., (1989), Cold Spring Harbor Laboratory Press, the entire disclosure of which is hereby incorporated herein by reference.
• a polynucleotide which hybridizes only to a poly A sequence such as the 3' terminal poly(A) tract of the CD44-like cDNA shown in FIG. 1 [SEQ ID NO:l]), or to a complementary stretch of T (or U) resides, would not be included in a polynucleotide of the invention used to hybridize to a portion of a nucleic acid of the invention, since such a polynucleotide would hybridize to any nucleic acid molecule contain a poly (A) stretch or the complement thereof (e.g., practically any double-stranded cDNA clone).
• nucleic acid molecules of the present invention which encode the CD44-like protein may include, but are not limited to the coding sequence for the mature polypeptide, by itself; the coding sequence for the mature polypeptide and additional sequences, such as those encoding the about 21 amino acid leader or secretory sequence, such as a pre-, or pro- or prepro- protein sequence; the coding sequence of the mature polypeptide, with or without the aforementioned additional coding sequences, together with additional, non-coding sequences, including for example, but not limited to introns and non-coding 5' and 3' sequences, such as the transcribed, non-translated sequences that play a role in transcription, mRNA processing - including splicing and polyadenylation signals, for example - ribosome binding and stability of mRNA; additional coding sequence which codes for additional amino acids, such as those which provide additional functionalities.
• the polypeptide may be fused to a marker sequence, such as a peptide, which facilitates purification of the fused polypeptide.
• the marker sequence is a hexa-histidine peptide, such as the tag provided in a pQE vector (Qiagen, Inc.), among others, many of which are commercially available.
• hexa-histidine provides for convenient purification of the fusion protein.
• the HA tag corresponds to an epitope derived of influenza hemagglutinin protein, which has been described by Wilson et al, Cell 37:161 (1984), for instance.
• Other such fusion proteins include the CD44-like protein fused to Fc at the N- or C-terminus.
• the extracellular CD44-like protein domain described above has been fused, together with the leader sequence, to Fc at its C-terminus. This construct was successfully expressed recombinantly in CHO cells.
• the polypeptide may be expressed in a modified form, such as a fusion protein, and may include not only secretion signals but also additional heterologous functional regions.
• a region of additional amino acids, particularly charged amino acids may be added to the N-terminus of the polypeptide to improve stability and persistence in the host cell, during purification or during subsequent handling and storage.
• a region(s) also may be added to the polypeptide to facilitate purification. Such regions may be removed prior to final preparation of the polypeptide.
• the addition of peptide moieties to polypeptides to engender secretion or excretion, to improve stability and to facilitate purification, among others, are familiar and routine techniques in the art.
• a preferred fusion protein comprises a heterologous region from immunoglobulin that is useful to solubilize receptors.
• EP-A-O 464 533 (Canadian counterpart 2045869) discloses fusion proteins comprising various portions of constant region of immunoglobin molecules together with another human protein or part thereof.
• the Fc part in the fusion protein is thoroughly advantageous for use in therapy and diagnosis and thus results, for example, in improved pharmacokinetic properties (EP-A 0232 262).
• the present invention further relates to variants of the nucleic acid molecules of the present invention, which encode for fragments, analogs or derivatives of the CD44-like protein Variants may occur naturally, such as an allelic variant.
• allelic variant is intended one of several alternate forms of a gene occupying a given locus on a chromosome of an organism
• Non-naturally occurring variants may be produced using art-known mutagenesis techniques
• variants include those produced by nucleotide substitutions, deletions or additions.
• the substitutions, deletions or additions may involve one or more nucleotides.
• the variants may be altered in coding or non-coding regions or both Alterations in the coding regions may produce conservative or non-conservative amino acid substitutions, deletions or additions
• nucleic acid molecules that are at least 90% identical, and more preferably at least 95%, 96%, 97%, 98% or 99% identical to- (a) a nucleotide sequence encoding the full-length
• CD44-like polypeptide having the complete amino acid sequence (including the predicted leader sequence) shown in FIG. 1 [SEQ ID NO 2] or as encoded by the cDNA clone contained in ATCC Deposit No 97520, (b) a nucleotide sequence encoding the mature CD44-like polypeptide (full-length polypeptide with the leader removed) having an amino acid sequence shown in FIG 1 [SEQ ID NO 2] or as encoded by the cDNA clone contained in ATCC Deposit No 97520; (c) a nucleotide sequence encoding the CD44-like protein extracellular domain having an amino acid sequence shown in FIG. 1 [SEQ ID NO 2] or as encoded by the cDNA clone contained in ATCC Deposit No.
• nucleotide sequence of the polynucleotide is identical to the reference sequence except that the polynucleotide sequence may include up to five point mutations per each 100 nucleotides of the reference nucleotide sequence encoding the CD44-like polypeptide.
• a polynucleotide having a nucleotide sequence at least 95% identical to a reference nucleotide sequence up to 5% of the nucleotides in the reference sequence may be deleted or substituted with another nucleotide, or a number of nucleotides up to 5% of the total nucleotides in the reference sequence may be inserted into the reference sequence.
• These mutations of the reference sequence may occur at the 5' or 3' terminal positions of the reference nucleotide sequence or anywhere between those terminal positions, interspersed either individually among nucleotides in the reference sequence or in one or more contiguous groups within the reference sequence.
• nucleotide sequence at least 90%, 95%, 96%, 97%, 98% or 99% identical to, for instance, the nucleotide sequence shown in FIG. 1 or to the nucleotide sequence of the deposited cDNA clone can be determined conventionally using known computer programs such as the Bestfit program (Wisconsin Sequence Analysis Package, Version 8 for Unix, Genetics Computer Group, University Research Park, 575 Science Drive, Madison, WI 5371 1). Bestfit uses the local homology algorithm of Smith and Waterman (Advances in Applied Mathematics 2: 482-489, 1981) to find the best segment of homology between two sequences.
• the parameters are set, of course, such that the percentage of identity is calculated over the full length of the reference nucleotide sequence and that gaps in homology of up to 5% of the total number of nucleotides in the reference sequence are allowed.
• nucleic acid molecules which are at least 90%, 95%, 96%, 97%, 98% or 99% identical to a nucleic acid sequence described above irrespective of whether they encode a polypeptide having CD44-like protein activity. This is because, even where a particular nucleic acid molecule does not encode a polypeptide having CD44-like protein activity, one of skill would still know how to use the nucleic acid molecule, for instance, as a hybridization probe or a polymerase chain reaction (PCR) primer.
• PCR polymerase chain reaction
• nucleic acid molecules of the present invention that do not encode a polypeptide having CD44-like protein activity include, inter alia, (1) isolating the gene encoding the CD44-like protein, or allelic variants thereof from a cDNA library; (2) in situ hybridization (FISH) to metaphase chromosomal spreads to provide precise chromosomal location of the CD44-like gene as described in Ver a et al. ,
• nucleic acid molecules which are at least 90%, 95%, 96%, 97%, 98% or 99% identical to the nucleic acid sequence shown in
• FIG. 1 or to the nucleic acid sequence of the deposited cDNA which do, in fact, encode a polypeptide having CD44-like protein activity.
• a polypeptide having CD44-like protein activity is intended polypeptides exhibiting similar, but not necessarily identical, activity as compared to the CD44-like protein as measured in a particular biological assay.
• CD44 and fragments thereof are capable of inhibiting T cell receptor-mediated T cell activation (Denning et al., Immunol 798- 801 (1989); Shimazu et al. J. Immunol. 43:2451-2463 (1989); WO 94/09811).
• the assay involves assessing the effect of soluble CD44-like protein on T cell activation by incorporating affinity purified CD44-like protein into liposomes, incubating the liposomes with peripheral blood mononuclear cells prior to stimulation with CD2 antibodies (which are potent stimulators of T cell activation).
• CD2 antibodies which are potent stimulators of T cell activation.
• the CD44-like protein of the present invention will significantly decrease T-cell proliferation when added to T cells immediately before the addition of CD2 antibodies, it will thus be possible to determine whether a candidate polypeptide has CD44-like protein activity.
• CD44 is known to act as a receptor for hyaluronan, and is thus proposed to be important for cell-extracelluar matrix interaction.
• a hyaluronan binding assay is described in Telen et al., J. Bio Chem. 271 :7147- 7153 (1996) and in WO 94/09811. Briefly the assay involves contacting either ( 1 ) cells which express CD44 on their surface or (2) cells which do not express CD44 with hyaluronan that has been labeled with FITC, washing the cells, resuspending the cells in PBS containing 0.3% paraformaldehyde, and analyzing the cells for FITC labeling by flow cytometry.
• CD44-like protein of the present invention will bind hyaluronan.
• a "polypeptide having CD44-like protein activity" includes polypeptides that are capable of specific binding of hyaluronan.
• nucleic acid molecules at least 90%, 95%, 96%, 97%, 98% or 99% identical to the nucleic acid sequence of the deposited cDNA or the nucleic acid sequence shown in FIG. 1
• SEQ ID NO: 1 will encode a polypeptide "having CD44-like protein activity.” In fact, since degenerate variants all encode the same polypeptide, this will be clear to the skilled artisan even without performing the above described comparison assay. It will be further recognized in the art that, for such nucleic acid molecules that are not degenerate variants, a reasonable number will also encode a polypeptide having CD44-like protein activity. This is because the skilled artisan is fully aware of amino acid substitutions that are either less likely or not likely to significantly effect protein function (e.g., replacing one aliphatic amino acid with a second aliphatic amino acid).
• the present invention also relates to vectors which include the isolated
• DNA molecules of the present invention host cells which are genetically engineered with the recombinant vectors, and the production of CD44-like polypeptides or fragments thereof by recombinant techniques.
• Recombinant constructs may be introduced into host cells using well known techniques such as infection, transduction, transfection, transvection and transformation.
• the vector may be, for example, a plasmid, plasmid, viral or retroviral vector.
• Retroviral vectors may be replication competent or replication defective. In the latter case, retroviral propagation generally will occur only in complementing host cells.
• the polynucleotides may be joined to a vector containing a selectable marker for propagation in a host.
• a plasmid vector is introduced in a precipitate, such as a calcium phosphate precipitate, or in a complex with a charged lipid. If the vector is a virus, it may be packaged in vitro using an appropriate packaging cell line and then transfected into host cells.
• vectors comprising cis-acting control regions to the polynucleotide of interest.
• Appropriate trans-acting factors either are supplied by the host, supplied by a complementing vector or supplied by the vector itself upon introduction into the host.
• the vectors provide for specific expression, which may be inducible and or cell type-specific. Particularly preferred among inducible vectors are vectors that can be induced for expression by environmental factors that are easy to manipulate, such as temperature and nutrient additives.
• Expression vectors useful in the present invention include chromosomal-, episomal- and virus-derived vectors e.g., vectors derived from bacterial plasmids, bacteriophage, yeast episomes, yeast chromosomal elements, viruses such as baculoviruses, papova viruses, vaccinia viruses, adenoviruses, fowl pox viruses, pseudorabies viruses and retroviruses, and vectors derived from combinations thereof, such as those derived from plasmid and bacteriophage genetic elements, such as cosmids and phagemids.
• vectors derived from bacterial plasmids, bacteriophage, yeast episomes, yeast chromosomal elements, viruses such as baculoviruses, papova viruses, vaccinia viruses, adenoviruses, fowl pox viruses, pseudorabies viruses and retroviruses and vectors derived from combinations thereof, such as those derived from plasmid and bacter
• the DNA insert should be operatively linked to an appropriate promoter, such as the phage lambda PL promoter, the E. coli lac, trp and tac promoters, the SV40 early and late promoters and promoters of retroviral LTRs, to name a few.
• an appropriate promoter such as the phage lambda PL promoter, the E. coli lac, trp and tac promoters, the SV40 early and late promoters and promoters of retroviral LTRs, to name a few.
• the expression constructs will contain sites for transcription initiation, termination, and, in the transcribed region, a ribosome binding site for translation.
• the coding portion of the mature transcripts expressed by the constructs will include a translation initiating AUG at the beginning and a termination codon appropriately positioned at the end of the polypeptide to be translated.
• the expression vectors will preferably contain selectable markers.
• markers include dihydrofolate reductase or neomycin resistance for eukaryotic cell culture and tetracycline or ampicillin resistance for culturing in E. coli and other bacteria.
• Representative examples of appropriate hosts include bacterial cells, such as E. coli, Streptomyces and Salmonella typhimurium; fungal cells, such as yeast; insect cells such as Drosophila S2 and Spodoptera Sf9; animal cells such as CHO, COS and Bowes melanoma; and plant cells. Appropriate culture mediums and conditions for the above-described host cells are known in the art.
• vectors preferred for use in bacteria are pQE70, pQE60 and pQE-9, available from Qiagen; pBS vectors, Phagescript vectors, Bluescript vectors, pNH8A, pNHl ⁇ a, pNH18A, pNH46A, available from Stratagene; and ptrc99a, pKK223-3, pKK233-3, pDR540, pRIT5 available from Pharmacia.
• preferred eukaryotic vectors are pWLNEO, pSV2CAT, pOG44, pXTl and pSG available from Stratagene; and ⁇ SVK3, pBPV, pMSG and pSVL available from Pharmacia.
• Other suitable vectors will be readily apparent to the skilled artisan.
• bacterial promoters for use in the present invention include E coli lacl and lacZ promoters, the T3 and T7 promoters, the gpt promoter, the lambda PR, PL promoters and the trp promoter.
• Suitable eukaryotic promoters include the CMV immediate early promoter, the HSV thymidine kinase promoter, the early and late SV40 promoters, the promoters of retroviral LTRs, such as those of the Rous sarcoma virus ("RSV”), and metallothionein promoters, such as the mouse metallothionein-I promoter.
• Introduction of the construct into the host cell can be effected by calcium phosphate transfection, DEAE-dextran mediated transfection, cationic lipid- mediated transfection, electroporation, transduction, infection or other methods. Such methods are described in many standard laboratory manuals, such as Davis et al. , Basic Methods in Molecular Biology ( 1986).
• Enhancers are cis-acting elements of DNA, usually about from 10 to 300 bp that act to increase transcriptional activity of a promoter in a given host cell-type.
• enhancers include the SV40 enhancer, which is located on the late side of the replication origin at bp 100 to 270, the cytomegalovirus early promoter enhancer, the polyoma enhancer on the late side of the replication origin, and adenovirus enhancers.
• secretion signals may be incorporated into the expressed polypeptide.
• the signals may be endogenous to the polypeptide or they may be heterologous signals.
• the polypeptide may be expressed in a modified form, such as a fusion protein, and may include not only secretion signals but also additional heterologous functional regions.
• a region of additional amino acids, particularly charged amino acids may be added to the N-terminus of the polypeptide to improve stability and persistence in the host cell, during purification or during subsequent handling and storage.
• peptide moieties may be added to the polypeptide to facilitate purification.
• the CD44-like protein can be recovered and purified from recombinant cell cultures by well-known methods including ammonium sulfate or ethanol precipitation, acid extraction, anion or cation exchange chromatography, phosphocellulose chromatography, hydrophobic interaction chromatography, affinity chromatography, hydroxylapatite chromatography and lectin chromatography. Most preferably, high performance liquid chromatography ("HPLC") is employed for purification.
• Polypeptides of the present invention include naturally purified products, products of chemical synthetic procedures, and products produced by recombinant techniques from a prokaryotic or eukaryotic host, including, for example, bacterial, yeast, higher plant, insect and mammalian cells.
• polypeptides of the present invention may be glycosylated or may be non-glycosylated.
• polypeptides of the invention may also include an initial modified methionine residue, in some cases as a result of host-mediated processes.
• the invention further provides an isolated CD44-like polypeptide having the amino acid sequence encoded by the deposited cDNA, or the amino acid sequence as shown in FIG. 1 [SEQ ID NO 2], or a peptide or polypeptide comprising a portion of the above polypeptides.
• the terms "peptide” and “oligopeptide” are considered synonymous (as is commonly recognized) and each term can be used interchangeably as the context requires to indicate a chain of at least two amino acids coupled by peptidyl linkages.
• polypeptide is used herein for chains containing more than ten amino acid residues. All oligopeptide and polypeptide formulas or sequences herein are written from left to right and in the direction from amino terminus to carboxy terminus.
• isolated polypeptide or protein is intended a polypeptide or protein removed from its native environment.
• recombinantly produced polypeptides and proteins expressed in host cells are considered isolated for purposed of the invention as are native or recombinant polypeptides which have been substantially purified by any suitable technique such as, for example, the single-step purification method disclosed in Smith and Johnson, Gene 67.31-40
• the invention further includes variations of the CD44-like polypeptide which show substantial CD44-like polypeptide activity or which include regions of CD44-like protein such as the protein fragments discussed below.
• Such mutants include deletions, insertions, inversions, repeats, and type substitutions (for example, substituting one hydrophilic residue for another, but not strongly hydrophilic for strongly hydrophobic as a rule). Small changes or such "neutral" amino acid substitutions will generally have little effect on activity.
• conservative substitutions are the replacements, one for another, among the aliphatic amino acids Ala, Val, Leu and He; interchange of the hydroxyl residues Ser and Thr, exchange of the acidic residues Asp and Glu, substitution between the amide residues Asn and Gin, exchange of the basic residues Lys and Arg and replacements among the aromatic residues Phe, Tyr.
• polypeptides of the present invention include the polypeptide encoded by the deposited cDNA including the leader, the mature polypeptide encoded by the deposited cDNA minus the leader (i.e., the mature protein), the polypeptide of FIG. 1 [SEQ ID NO:2] including the leader, the polypeptide of FIG. 1 [SEQ ID NO. 2] minus the leader, as well as polypeptides which have at least 90% similarity, more preferably at least 95% similarity, and still more preferably at least 96%, 97%, 98% or 99% similarity to those described herein.
• polypeptides of the present invention include polypeptides at least 80% identical, more preferably at least 90% or 95% identical, still more preferably at least 96%, 97%, 98% or 98% identical to a polypeptide described herein and also include portions of such polypeptides with at least 30 amino acids and more preferably at least 50 amino acids.
• % similarity for two polypeptides is intended a similarity score produced by comparing the amino acid sequences of the two polypeptides using the Bestfit program (Wisconsin Sequence Analysis Package, Version 8 for Unix, Genetics Computer Group, University Research Park, 575 Science Drive, Madison, WI 53711) and the default settings for determining similarity. Bestfit uses the local homology algorithm of Smith and Waterman, Advances in Applied
• polypeptide having an amino acid sequence at least, for example, 95% "identical" to a reference amino acid sequence of a CD44-like polypeptide is intended that the amino acid sequence of the polypeptide is identical to the reference sequence except that the polypeptide sequence may include up to five amino acid alterations per each 100 amino acids of the reference amino acid of the CD44s-like polypeptide.
• up to 5% of the amino acid residues in the reference sequence may be deleted or substituted with another amino acid, or a number of amino acids up to 5% of the total amino acid residues in the reference sequence may be inserted into the reference sequence.
• These alterations of the reference sequence may occur at the amino or carboxy terminal positions of the reference amino acid sequence or anywhere between those terminal positions, interspersed either individually among residues in the reference sequence or in one or more contiguous groups within the reference sequence.
• any particular polypeptide is at least 90%, 95%, 96%, 97%, 98% or 99% identical to, for instance, the amino acid sequence shown in FIG. 1 [SEQ ID NO:2] or to the amino acid sequence encoded by deposited cDNA clone or a portion thereof can be determined conventionally using known computer programs such the Bestfit program (Wisconsin Sequence Analysis Package, Version 8 for Unix, Genetics Computer Group, University Research Park, 575 Science Drive, Madison, WI 53711.
• the parameters are set, of course, such that the percentage of identity is calculated over the full length of the reference amino acid sequence and that gaps in homology of up to 5% of the total number of amino acid residues in the reference sequence are allowed.
• the invention provides a peptide or polypeptide comprising an epitope-bearing portion of a polypeptide of the invention.
• the epitope of this polypeptide portion is an immunogenic or antigenic epitope of a polypeptide of the invention.
• immunogenic epitope is defined as a part of a protein that elicits an antibody response when the whole protein is the immunogen. These immunogenic epitopes are believed to be confined to a few loci on the molecule. On the other hand, a region of a protein molecule to which an antibody can bind is defined as an "antigenic epitope.” The number of immunogenic epitopes of a protein generally is less than the number of antigenic epitopes. See, for instance, Geysen, H. M. et al, Proc. Natl. Acad Sci. USA 57:3998-4002 (1984).
• peptides or polypeptides bearing an antigenic epitope i.e., that contain a region of a protein molecule to which an antibody can bind
• relatively short synthetic peptides that mimic part of a protein sequence are routinely capable of eliciting an antiserum that reacts with the partially mimicked protein. See, for instance, Sutcliffe, J. G. et al,
• Peptides capable of eliciting protein-reactive sera are frequently represented in the primary sequence of a protein, can be characterized by a set of simple chemical rules, and are confined neither to immunodominant regions of intact proteins (i.e., immunogenic epitopes) nor to the amino or carboxyl terminals. Peptides that are extremely hydrophobic and those of six or fewer residues generally are ineffective at inducing antibodies that bind to the mimicked protein; longer, soluble peptides, especially those containing proline residues, usually are effective. Sutcliffe etal, supra, at 661.
• 18 of 20 peptides designed according to these guidelines containing 8-39 residues covering 75% of the sequence of the influenza virus hemagglutinin HA1 polypeptide chain, induced antibodies that reacted with the HA1 protein or intact virus; and 12/12 peptides from the MuLV polymerase and 18/18 from the rabies glycoprotein induced antibodies that precipitated the respective proteins.
• Antigenic epitope-bearing peptides and polypeptides of the invention are therefore useful to raise antibodies, including monoclonal antibodies, that bind specifically to a polypeptide of the invention.
• a high proportion of hybridomas obtained by fusion of spleen cells from donors immunized with an antigen epitope-bearing peptide generally secrete antibody reactive with the native protein.
• the antibodies raised by antigenic epitope- bearing peptides or polypeptides are useful to detect the mimicked protein, and antibodies to different peptides may be used for tracking the fate of various regions of a protein precursor which undergoes posttranslation processing.
• the peptides and anti-peptide antibodies may be used in a variety of qualitative or quantitative assays for the mimicked protein, for instance in competition assays since it has been shown that even short peptides (e.g., about 9 amino acids) can bind and displace the larger peptides in immunoprecipitation assays. See, for instance, Wilson, I.A., et al., Cell 37161-11% at 777 (1984).
• the anti-peptide antibodies of the invention also are useful for purification of the mimicked protein, for instance, by adsorption chromatography using methods well known in the art.
• Antigenic epitope-bearing peptides and polypeptides of the invention designed according to the above guidelines preferably contain a sequence of at least seven, more preferably at least nine and most preferably between about 15 to about 30 amino acids contained within the amino acid sequence of a polypeptide of the invention.
• peptides or polypeptides comprising a larger portion of an amino acid sequence of a polypeptide of the invention, containing about 30 to about 50 amino acids, or any length up to and including the entire amino acid sequence of a polypeptide of the invention also are considered epitope-bearing peptides or polypeptides of the invention and also are useful for inducing antibodies that react with the mimicked protein.
• the amino acid sequence of the epitope-bearing peptide is selected to provide substantial solubility in aqueous solvents (i.e., the sequence includes relatively hydrophilic residues and highly hydrophobic sequences are preferably avoided); and sequences containing proline residues are particularly preferred.
• the epitope-bearing peptides and polypeptides of the invention may be produced by any conventional means for making peptides or polypeptides including recombinant means using nucleic acid molecules of the invention. For instance, a short epitope-bearing amino acid sequence may be fused to a larger polypeptide which acts as a carrier during recombinant production and purification, as well as during immunization to produce anti-peptide antibodies. Epitope-bearing peptides also may be synthesized using known methods of chemical synthesis.
• Houghten has described a simple method for synthesis of large numbers of peptides, such as 10-20 mg of 248 different 13 residue peptides representing single amino acid variants of a segment of the HA1 polypeptide which were prepared and characterized (by ELISA-type binding studies) in less than four weeks.
• Houghten R. A., Proc. Natl. Acad. Sci. USA 52:5131-5135 (1985).
• This "Simultaneous Multiple Peptide Synthesis (SMPS)" process is further described in U.S. Patent No 4,631,211 to Houghten et al.
• Epitope-bearing peptides and polypeptides of the invention are used to induce antibodies according to methods well known in the art. See, for instance, Sutcliffe etal, supra; Wilson et al, supra, Chow, M. etal, Proc. Natl. Acad Sci. USA 52:910-914; and Bittle, F.J. et al, J. Gen. Virol. 66:2347-2354 (1985).
• animals may be immunized with free peptide; however, anti-peptide antibody titer may be boosted by coupling of the peptide to a macromolecular carrier, such as keyhole limpet hemacyanin (KLH) or tetanus toxoid.
• KLH keyhole limpet hemacyanin
• peptides containing cysteine may be coupled to carrier using a linker such as m- maleimidobenzoyl-N-hydroxysuccinimide ester (MBS), while other peptides may be coupled to carrier using a more general linking agent such as glutaraldehyde.
• Animals such as rabbits, rats and mice are immunized with either free or carrier- coupled peptides, for instance, by intraperitoneal and/or intradermal injection of emulsions containing about 100 ⁇ g peptide or carrier protein and Freund's adjuvant. Several booster injections may be needed, for instance, at intervals of about two weeks, to provide a useful titer of anti-peptide antibody which can be detected, for example, by ELISA assay using free peptide adsorbed to a solid surface.
• the titer of anti-peptide antibodies in serum from an immunized animal may be increased by selection of anti-peptide antibodies, for instance, by adsorption to the peptide on a solid support and elution of the selected antibodies according to methods well known in the art.
• Immunogenic epitope-bearing peptides of the invention i.e., those parts of a protein that elicit an antibody response when the whole protein is the immunogen, are identified according to methods known in the art. For instance, Geysen et al, 1984, supra, discloses a procedure for rapid concurrent synthesis on solid supports of hundreds of peptides of sufficient purity to react in an enzyme-linked immunosorbent assay.
• a peptide bearing an immunogenic epitope of a desired protein may be identified routinely by one of ordinary skill in the art.
• the immunologically important epitope in the coat protein of foot-and-mouth disease virus was located by Geysen et al with a resolution of seven amino acids by synthesis of an overlapping set of all 208 possible hexapeptides covering the entire 213 amino acid sequence of the protein.
• a complete replacement set of peptides in which all 20 amino acids were substituted in turn at every position within the epitope were synthesized, and the particular amino acids conferring specificity for the reaction with antibody were determined.
• peptide analogs of the epitope-bearing peptides of the invention can be made routinely by this method.
• U.S. Patent No. 4,708,781 to Geysen (1987) further describes this method of identifying a peptide bearing an immunogenic epitope of a desired protein.
• U.S. Patent No. 5,194,392 to Geysen (1990) describes a general method of detecting or determining the sequence of monomers (amino acids or other compounds) which is a topological equivalent of the epitope (i.e., a "mimotope") which is complementary to a particular paratope (antigen binding site) of an antibody of interest. More generally, U.S. Patent No. 4,433,092 to Geysen (1989) describes a method of detecting or determining a sequence of monomers which is a topographical equivalent of a ligand which is complementary to the ligand binding site of a particular receptor of interest. Similarly, U.S. Patent No. 5,480,971 to Houghten, R. A.
• the CD44-like polypeptide is a 322 residue protein exhibiting three main structural domains.
• the extracellular domain (which inludes the ligand binding domain) was identified within residues from about 22 to about 238 in FIG. 1 [SEQ ID NO 2].
• the mature extracellular domain has been predicted by the inventors as being about 216 amino acids in length with a molecular weight of about 23.5 kDa.
• the transmembrane domain was identified within residues from about 239 to about
• the invention further provides preferred CD44-like protein fragments comprising a polypeptide selected from: the mature CD44-like protein; the CD44- like polypeptide extracellular domain; the CD44-like polypeptide transmembrane domain; the CD44-like polypeptide intracellular domain; or the CD44-like protein extracellular domain and intracellular domain with part or all of the transmembrane domain deleted.
• the extracellular domain of the CD44-like receptor of the present invention can be combined with parts of the constant domain of immunoglobulins (IgG), resulting in chimeric polypeptides.
• IgG immunoglobulins
• These fusion proteins show an increased half-life in vivo. This has been shown, e.g., for chimeric proteins consisting of the first two domains of the human CD4-polypeptide and various domains of the constant regions of the heavy or light chains of mammalian immunoglobulins (EPA 394,827; Traunecker et al, Nature 557:84-86 (1988)).
• Fusion proteins that have a disulfide-linked dimeric structure due to the IgG part can also be more efficient in binding and neutralizing the ligands that the monomeric extracellular domains alone (Fountoulakis et al, J Biochem 270:3958- 3964 (1995)).
• the polypeptides of the present invention and fragments thereof can be used to raise polyclonal and monoclonal antibodies, which are useful in diagnostic assays for detecting CD44-like protein expression as described below or as agonists and antagonists capable of enhancing or inhibiting the CD44-like protein receptor signaling pathway.
• Such fragments can be used in the yeast two-hybrid system to "capture" CD44-like protein binding proteins which are also candidate agonist and antagonist according to the present invention.
• the yeast two hybrid system which is discussed in more detail below, is described in Fields and Song, Nature 340:245-246 (1989). Further, the extracellular domain in soluble form is itself useful as an antagonist capable of inhibiting CD44-like protein signaling.
• tissues in mammals with cancer contain significantly greater CD44-like protein gene copy number and express significantly enhanced levels of the CD44-like protein and mRNA encoding the CD44-like protein when compared to a corresponding "standard" mammal, i.e., a mammal of the same species not having the cancer.
• enhanced levels of the CD44- like protein can be detected in certain body fluids (e.g., sera, plasma, urine, and spinal fluid) from mammals with cancer when compared to sera from mammals of the same species not having the cancer.
• the invention provides a diagnostic method useful during tumor diagnosis, which involves assaying the expression level of the gene encoding the CD44-like protein or the gene copy number in mammalian cells or body fluid and comparing the gene expression level or gene copy number with a standard CD44-like protein gene expression level or gene copy number, whereby an increase in the gene expression level or gene copy number over the standard is indicative of certain tumors.
• the present invention is useful as a prognostic indicator, whereby patients exhibiting enhanced CD44-like protein gene expression will experience a worse clinical outcome relative to patients expressing the gene at a lower level.
• assaying the expression level of the gene encoding the CD44-like protein is intended qualitatively or quantitatively measuring or estimating the level of the CD44-like protein or the level of the mRNA encoding the CD44-like protein in a first biological sample either directly (e.g., by determining or estimating absolute protein level or mRNA level) or relatively (e.g., by comparing to the CD44-like protein level or mRNA level in a second biological sample).
• “assaying the copy number of the gene encoding the CD44-like protein” is intended qualitatively or quantitatively measuring or estimating the gene copy number in a first biological sample either directly (e.g., by determining or estimating absolute gene copy number) or relatively (e.g., by comparing to the CD44-like protein gene copy number in a second biological sample).
• the CD44-like protein level, mRNA level, or gene copy number in the first biological sample is measured or estimated and compared to a standard CD44-like protein level, mRNA level, or gene copy number, the standard being taken from a second biological sample obtained from an individual not having the cancer.
• a standard CD44- like protein level, mRNA level, or gene copy number is known, it can be used repeatedly as a standard for comparison.
• biological sample any biological sample obtained from an individual, cell line, tissue culture, or other source which contains CD44-like protein or mRNA.
• Biological samples include mammalian body fluids (such as sera, plasma, urine, and spinal fluid) which contain secreted mature CD44-like protein or its soluble extracellular domain, and ovarian, prostate, heart, placenta, pancreas liver, spleen, lung, breast and umbilical tissue. Other biological samples include bone marrow. Methods for obtaining tissue biopsies and body fluids from mammals are well known in the art. Where the biological sample is to include mRNA, a tissue biopsy is the preferred source.
• the present invention is useful for detecting cancer in mammals.
• the invention is useful during diagnosis of the of following types of cancers in mammals: breast, ovarian, prostate, bone, liver, lung, pancreatic, and spleenic.
• Preferred mammals include monkeys, apes, cats, dogs, cows, pigs, horses, rabbits and humans. Particularly preferred are humans.
• Total cellular RNA can be isolated from a biological sample using any suitable technique such as the single-step guanidinium-thiocyanate-phenol- chloroform method described in Chomczynski and Sacchi, Anal Biochem. 762:156-159 (1987). Levels of mRNA encoding the CD44-like protein are then assayed using any appropriate method. These include Northern blot analysis, S 1 nuclease mapping, the polymerase chain reaction (PCR), reverse transcription in combination with the polymerase chain reaction (RT-PCR), and reverse transcription in combination with the ligase chain reaction (RT-LCR). Northern blot analysis can be performed as described in Harada et al, Cell
• RNA is prepared from a biological sample as described above.
• the RNA is denatured in an appropriate buffer (such as glyoxal/dimethyl sulfoxide/sodium phosphate buffer), subjected to agarose gel electrophoresis, and transferred onto a nitrocellulose filter.
• an appropriate buffer such as glyoxal/dimethyl sulfoxide/sodium phosphate buffer
• the filter is prehybridized in a solution containing formamide, SSC, Denhardt's solution, denatured salmon sperm, SDS, and sodium phosphate buffer.
• CD44-like protein cDNA labeled according to any appropriate method such as the 32 P-multiprimed DNA labeling system (Amersham) is used as probe. After hybridization overnight, the filter is washed and exposed to x-ray film.
• cDNA for use as probe according to the present invention is described in the sections above and will preferably at least 15 bp in length.
• SI mapping can be performed as described in Fujita et al, Cell 49:351- 367 (1987).
• probe DNA for use in SI mapping, the sense strand of above-described cDNA is used as a template to synthesize labeled antisense DNA.
• the antisense DNA can then be digested using an appropriate restriction endonuclease to generate further DNA probes of a desired length.
• Such antisense probes are useful for visualizing protected bands corresponding to the target mRNA (i.e., mRNA encoding the CD44-like protein).
• Northern blot analysis can be performed as described above.
• levels of mRNA encoding the CD44-like protein are assayed using the RT-PCR method described in Makino et al, Technique 2:295-301 (1990).
• the radioactivities of the "amplicons" in the polyacrylamide gel bands are linearly related to the initial concentration of the target mRNA.
• this method involves adding total RNA isolated from a biological sample in a reaction mixture containing a RT primer and appropriate buffer. After incubating for primer annealing, the mixture can be supplemented with a RT buffer, dNTPs, DTT, RNase inhibitor and reverse transcriptase. After incubation to achieve reverse transcription of the RNA, the RT products are then subject to PCR using labeled primers.
• a labeled dNTP can be included in the PCR reaction mixture.
• PCR amplification can be performed in a DNA thermal cycler according to conventional techniques. After a suitable number of rounds to achieve amplification, the PCR reaction mixture is electrophoresed on a polyacrylamide gel. After drying the gel, the radioactivity of the appropriate bands (corresponding to the mRNA encoding the CD44-like protein)) is quantified using an imaging analyzer.
• RT and PCR reaction ingredients and conditions, reagent and gel concentrations, and labeling methods are well known in the art. Variations on the RT-PCR method will be apparent to the skilled artisan.
• oligonucleotide primers which will amplify reverse transcribed target mRNA can be used and can be designed as described in the sections above.
• Assaying CD44-like protein gene copy number can occur according to any known technique, such as for example, in situ hybridization of tissue samples with a cDNA probe described above.
• Assaying CD44-like protein levels in a biological sample can occur using any art-known method.
• Preferred for assaying CD44-like protein levels in a biological sample are antibody-based techniques.
• CD44-like protein expression in tissues can be studied with classical immunohistological methods.
• the specific recognition is provided by the primary antibody (polyclonal or monoclonal) but the secondary detection system can utilize fluorescent, enzyme, or other conjugated secondary antibodies.
• an immunohistological staining of tissue section for pathological examination is obtained.
• Tissues can also be extracted, e.g., with urea and neutral detergent, for the liberation of CD44-like protein for Western-blot or dot/slot assay (Jalkanen, M., et al, J. Cell Biol 707:976-985 (1985); Jalkanen, M., et al, J. Cell . Biol 705:3087-3096 (1987)).
• urea and neutral detergent for the liberation of CD44-like protein for Western-blot or dot/slot assay.
• CD44-like protein as a standard. This technique can also be applied to body fluids. With these samples, a molar concentration of CD44-like protein will aid to set standard values of CD44-like protein content for different body fluids, like serum, plasma, urine, spinal fluid, etc. The normal appearance of CD44-like protein amounts can then be set using values from healthy individuals, which can be compared to those obtained from a test subject.
• Other antibody-based methods useful for detecting CD44-like protein gene expression include immunoassays, such as the enzyme linked immunosorbent assay (ELISA) and the radioimmunoassay (RIA).
• a CD44-like protein-specific monoclonal antibodies can be used both as an immunoabsorbent and as an enzyme-labeled probe to detect and quantify the CD44-like protein.
• the amount of CD44-like protein present in the sample can be calculated by reference to the amount present in a standard preparation using a linear regression computer algorithm.
• Such an ELISA for detecting a tumor antigen is described in Iacobelli et al, Breast Cancer Research and Treatment 77:1 -30 (1988).
• two distinct specific monoclonal antibodies can be used to detect
• CD44-like protein in a body fluid in a body fluid.
• one of the antibodies is used as the immunoabsorbent and the other as the enzyme-labeled probe.
• the above techniques may be conducted essentially as a "one-step” or “two-step” assay.
• the "one-step” assay involves contacting CD44-like protein with immobilized antibody and, without washing, contacting the mixture with the labeled antibody.
• the "two-step” assay involves washing before contacting the mixture with the labeled antibody.
• Other conventional methods may also be employed as suitable. It is usually desirable to immobilize one component of the assay system on a support, thereby allowing other components of the system to be brought into contact with the component and readily removed from the sample.
• Suitable enzyme labels include, for example, those from the oxidase group, which catalyze the production of hydrogen peroxide by reacting with substrate.
• Glucose oxidase is particularly preferred as it has good stability and its substrate
• an oxidase label may be assayed by measuring the concentration of hydrogen peroxide formed by the enzyme-labelled antibody/substrate reaction.
• suitable labels include radioisotopes, such as iodine ( I25 I, 121 I), carbon ( 14 C), salphee ( 3S S), tritium ( 3 H), indium ( ll2 In), and technetium (“Tc), and fluorescent labels, such as fluorescein and rhodamine, and biotin.
• CD44-like protein can also be detected in vivo by imaging.
• Antibody labels or markers for in vivo imaging of CD44-like protein include those detectable by X-radiography, NMR or ESR.
• suitable labels include radioisotopes such as barium or caesium, which emit detectable radiation but are not overtly harmful to the subject.
• Suitable markers for NMR and ESR include those with a detectable characteristic spin, such as deuterium, which may be incorporated into the antibody by labeling of nutrients for the relevant hybridoma.
• a CD44-like protein-specific antibody or antibody fragment which has been labeled with an appropriate detectable imaging moiety such as a radioisotope (for example, 131 1, 112 In, "Tc), a radio-opaque substance, or a material detectable by nuclear magnetic resonance, is introduced (for example, parenterally, subcutaneously or intraperitoneally) into the mammal to be examined for cancer.
• an appropriate detectable imaging moiety such as a radioisotope (for example, 131 1, 112 In, "Tc), a radio-opaque substance, or a material detectable by nuclear magnetic resonance
• the size of the subject and the imaging system used will determine the quantity of imaging moiety needed to produce diagnostic images.
• the quantity of radioactivity injected will normally range from about 5 to 20 millicuries of ""Tc.
• the labeled antibody or antibody fragment will then preferentially accumulate at the location of cells which contain CD44-like protein.
• In vivo tumor imaging is described in S.W. Burchiel et al, "Immunopharmacokinetics of Radiolabelled Antibodies and Their Fragments" (Chapter 13 in Tumor Imaging: The
• CD44-like-protein specific antibodies for use in the present invention can be raised against the intact CD44-Iike protein or an antigenic polypeptide fragment thereof, which may presented together with a carrier protein, such as an albumin, to an animal system (such as rabbit or mouse) or, if it is long enough (at least about 25 amino acids), without a carrier.
• a carrier protein such as an albumin
• antibody As used herein, the term “antibody” (Ab) or “monoclonal antibody” (Mab) is meant to include intact molecules as well as antibody fragments (such as, for example, Fab and F(ab') 2 fragments) which are capable of specifically binding to
• Fab and F(ab') 2 fragments lack the Fc fragment of intact antibody, clear more rapidly from the circulation, and may have less non-specific tissue binding of an intact antibody (Wahl et al, J. Nucl. Med 24:316-325 (1983)). Thus, these fragments are preferred.
• the antibodies of the present invention may be prepared by any of a variety of methods.
• cells expressing the CD44-like protein or an antigenic fragment thereof can be administered to an animal in order to induce the production of sera containing polyclonal antibodies.
• a preparation of CD44-like protein is prepared and purified to render it substantially free of natural contaminants. Such a preparation is then introduced into an animal in order to produce polyclonal antisera of greater specific activity.
• the antibodies of the present invention are monoclonal antibodies (or CD44-like protein binding fragments thereof)
• monoclonal antibodies can be prepared using hybridoma technology (Kohler et al, Nature 256:495 (1975); Kohler et al, Eur. J. Immunol 6:511 (1976); Kohler et al, Eur. J. Immunol. 6:292 (1976); Hammerling et al, In: Monoclonal
• Such procedures involve immunizing an animal (preferably a mouse) with a CD44-like protein antigen or, more preferably, with a CD44-like protein- expressing cell. Suitable cells can be recognized by their capacity to bind anti- CD44-like protein antibody.
• Such cells may be cultured in any suitable tissue culture medium; however, it is preferable to culture cells in Earle's modified Eagle's medium supplemented with 10% fetal bovine serum (inactivated at about 56°C), and supplemented with about 10 ⁇ g/1 of nonessential amino acids, about 1,000 U/ml of penicillin, and about 100 ⁇ g/ml of streptomycin.
• the splenocytes of such mice are extracted and fused with a suitable myeloma cell line.
• Any suitable myeloma cell line may be employed in accordance with the present invention; however, it is preferable to employ the parent myeloma cell line (SP 2 O), available from the American Type Culture Collection, Rockville, Maryland.
• the resulting hybridoma cells are selectively maintained in HAT medium, and then cloned by limiting dilution as described by Wands et ⁇ l. (G ⁇ stro- enterology 50:225-232 (1981)). The hybridoma cells obtained through such a selection are then assayed to identify clones which secrete antibodies capable of binding the CD44-like protein antigen.
• additional antibodies capable of binding to the CD44-like protein antigen may be produced in a two-step procedure through the use of anti- idiotypic antibodies.
• Such a method makes use of the fact that antibodies are themselves antigens, and that, therefore, it is possible to obtain an antibody which binds to a second antibody.
• CD44-like-protein specific antibodies are used to immunize an animal, preferably a mouse.
• the splenocytes of such an animal are then used to produce hybridoma cells, and the hybridoma cells are screened to identify clones which produce an antibody whose ability to bind to the CD44-like protein-specific antibody can be blocked by the CD44-like protein antigen.
• Such antibodies comprise anti-idiotypic antibodies to the CD44-like protein-specific antibody and can be used to immunize an animal to induce formation of further CD44-like protein-specific antibodies.
• Fab and F(ab') 2 and other fragments of the antibodies of the present invention may be used according to the methods disclosed herein. Such fragments are typically produced by proteolytic cleavage, using enzymes such as papain (to produce Fab fragments) or pepsin (to produce F(ab') 2 fragments).
• CD44-like protein-binding fragments can be produced through the application of recombinant DNA technology or through synthetic chemistry.
• chimeric monoclonal antibodies can be produced using genetic constructs derived from hybridoma cells producing the monoclonal antibodies described above. Methods for producing chimeric antibodies are known in the art. See, for review, Morrison, Science 229:1202 (1985); Oi et al, BioTechniques 4:2X4 (1986); Cabilly et al, U.S. Patent No.
• suitable labels for the CD44-like protein-specific antibodies of the present invention are provided below.
• suitable enzyme labels include malate dehydrogenase, staphylococcal nuclease, delta-5-steroid isomerase, yeast- alcohol dehydrogenase, alpha-glycerol phosphate dehydrogenase, triose phosphate isomerase, peroxidase, alkaline phosphatase, asparaginase, glucose oxidase, beta- galactosidase, ribonuclease, urease, catalase, glucose-6-phosphate dehydrogenase, glucoamylase, and acetylcholine est erase.
• radioisotopic labels examples include 3 H, ⁇ l In, 12J I, ,31 1, 32 P, 35 S, ,4 C, 51 Cr, 57 To, 58 Co, 5 Fe, 75 Se, ,52 Eu, 90 Y, 67 Cu, 17 Ci, 211 At, 2i2 Pb, 47 Sc, 109 Pd, etc.
• lll In is a preferred isotope where in vivo imaging is used since its avoids the problem of dehalogenation of the I25 I or 131 I-labeled monoclonal antibody by the liver.
• this radionucleotide has a more favorable gamma emission energy for imaging (Perkins et al, Eur. J. Nucl.
• non-radioactive isotopic labels examples include 157 Gd, 55 Mn, 162 Dy, 52 Tr, and 56 Fe.
• fluorescent labels examples include an 15 Eu label, a fluorescein label, an isothiocyanate label, a rhodamine label, a phycoerythrin label, a phycocyanin label, an allophycocyanin label, an o-phthaldehyde label, and a fluorescamine label.
• suitable toxin labels include diphtheria toxin, ricin, and cholera toxin.
• chemiluminescent labels include a luminal label, an isoluminal label, an aromatic acridinium ester label, an imidazole label, an acridinium salt label, an oxalate ester label, a luciferin label, a luciferase label, and an aequorin label.
• nuclear magnetic resonance contrasting agents include heavy metal nuclei such as Gd, Mn, and iron.
• Typical techniques for binding the above-described labels to antibodies are provided by Kennedy et al, Clin. Chim. Acta 70: 1- 1 (1976), and Schurs et al, Clin. Chim. Acta 57: 1-40 (1977). Coupling techniques mentioned in the latter are the glutaraldehyde method, the periodate method, the dimaleimide method, the m- maleimidobenzyl-N-hydroxy-succinimide ester method, all of which methods are incorporated by reference herein.
• CD44 is a broadly distributed cell suface glycoprotein that has been shown to play an important role in many adhesion-dependent cellular processes including lymphocyte recirculation, hemopoiesis, NK-cell mediated killing, macrophage and lymphocyte activation, and tumor metastasis (Telen et al, J. Biol. Chem. 277(12): 7147-7153 (1996)). CD44 is known to function as a receptor for the glycosaminoglycan hyaluronan, which has been associated with wound repair, tumor invasion and immune recognition (Yang et al, J. Biol. Chem. 265(12):8617-8623 (1993)).
• CD44 has been shown to be capable of binding a number of extracellular matrix components besides hyaluronan, such as fibronectin, heparin sulfate, collagen types I and VI, and chondroitin 4- and 6-sulfates (Aruffo et al, Cell 67: 1303- 1313 (1990)).
• Another ligand of CD44 is the cytokine osteopontin, which interacts with CD44 surface receptors to induce cell migration (Biocentury, Jan 29, 1996 referencing Science).
• CD44 is a very polymorphic molecule; species ranging in size from 80 to
• CD44-like receptor protein of the present invention The various cellular responses in which CD44 is involved are induced by the binding of a CD44 ligand to a CD44 receptor, including the CD44-like receptor protein of the present invention.
• a cellular response to ligands of the CD44-like receptor is intended any genotypic, phenotypic and/or morphologic change to a cell, cell line, tissue, tissue culture or patient that is induced by such a ligand.
• one aspect of the present invention is directed to the identification of compounds capable of binding to the CD44-like protein of the invention.
• the CD44-like protein of the present invention is capable of binding to hyaluronan
• new ligands of the CD44-like protein can be identified by their ability to interfere with the binding of labeled hyaluronan to the CD44-like protein of the invention.
• An assay which may be utilized to determine hyaluronan binding is disclosed in Telen, et al, J. Biol. Chem. 277:7147-7153 (1996).
• this method involves comparing the degree to which FITC-hyaluronan binds to cells expressing the CD44-like protein of the invention, as measured by flow cytometry, to the degree to which the same cells bind FITC-hyaluronan when the cells are first incubated with a solution containing a proposed ligand of the CD44-like molecule.
• the present invention is directed to a method for enhancing a cellular response induced by the CD44-like protein-mediated signaling pathway, which involves administering to a cell which expresses the CD44-like protein an effective amount of an agonist to enhance CD44-like protein-mediated signaling.
• the present invention is directed to a method for inhibiting a cellular response induced by the CD44-like protein-mediated signaling pathway, which involves administering to a cell which expresses the CD44-like protein an effective amount of an antagonist capable of inhibiting CD44-like protein-mediated signaling.
• CD44-like protein-mediated signaling is inhibited to treat metastasis.
• An example of an antagonist capable of diminishing CD44-mediated cell migration is hyaluronate (Biocentury, Jan 29, 1996).
• CD44-like protein mediated signaling is decreased to inhibit graft versus host disease.
• agonist is intended naturally occurring and synthetic compounds capable of enhancing or potentiating a cellular response to a ligand of the CD44- like protein.
• antagonist is intended naturally occurring and synthetic compounds capable of inhibiting a cellular response to a CD44-like ligand.
• any candidate "agonist” or “antagonist” of the present invention can enhance or inhibit such a cellular response can be determined using art-known CD44-like protein ligand/receptor cellular response or binding assays, including those described in more detail below.
• One method involves screening for compounds which inhibit activation of the receptor polypeptide of the present invention by determining inhibition of binding of labeled ligand to cells which have the receptor on the surface thereof.
• Such a method involves transfecting a eukaryotic cell with DNA encoding the CD44-like protein receptor such that the cell expresses the receptor on its surface and contacting the cell with a candidate compound in the presence of a labeled form of a known ligand.
• the ligand can be labeled, e.g., by radioactivity.
• the amount of labeled ligand bound to the receptors is measured, e.g., by measuring radioactivity of the receptors. If the compound binds to the receptor as determined by a reduction of labeled ligand which binds to the receptors, the binding of labeled ligand to the receptor is inhibited.
• a screening method is provided for determining whether a candidate agonist or antagonist is capable of enhancing or inhibiting a cellular response induced by the CD44-like receptor pathway.
• the method involves contacting cells which express the CD44-like protein polypeptide with a candidate compound, assaying a cellular response, and comparing the cellular response to a standard cellular response, the standard being assayed in the absence of the candidate compound, whereby an increased cellular response over the standard indicates that the candidate compound is an agonist of the CD44-like protein receptor signaling pathway and a decreased cellular response compared to the standard indicates that the candidate compound is an antagonist of the
• CD44-like protein receptor signaling pathway By “assaying a cellular response” is intended qualitatively or quantitatively measuring a cellular response to a candidate compound and/or a CD44-like protein ligand.
• a cell expressing the CD44-like protein polypeptide can be contacted with either an endogenous or exogenously administered CD44 ligand.
• the assay includes the steps of providing a smooth muscle cell bearing the CD44-like molecule on its surface, contacting the smooth muscle cell with a candidate compound, and determining the amount of smooth muscle migration that occurs. A decrease in migration of the smooth muscle cell in the presence of a candidate compound compared to the amount in the absence of the candidate compound indicates that the candidate compound inhibits smooth muscle migration that is mediated by the CD44-like molecule.
• This assay is described in more detail in WO 95/19183 and Jain et al, J. Clin.
• Agonists according to the present invention include naturally occurring and synthetic compounds such as, for example, CD44 ligands and fragments thereof.
• Other preferred agonist includes polyclonal and monoclonal antibodies raised against the CD44-like receptor polypeptide, or a fragment thereof. Such agonist antibodies can be raised against the CD44-like protein as described in WO 95 2224913, WO 93/17047, and Moll et al, Journal of Immunology 2085-2094.
• Antagonists according to the present invention include naturally occurring and synthetic compounds such as, for example, the cytokine osteopontin (Weber et al. Science 277:509-512 (1996)).
• Other antagonists include polyclonal and monoclonal antagonist antibodies raised against the CD44-like protein or a fragment thereof.
• Antisense molecules can be used to control gene expression through antisense DNA or RNA or through triple-helix formation. Antisense techniques are discussed, for example, in Okano, J. Neurochem. 56:560 (1991); OLIGODEOXY- NUCLEOTIDES AS ANTISENSE INHIBITORS OF GENE EXPRESSION, CRC Press, Boca Raton, FL (1988). Triple helix formation is discussed in, for instance Lee et al, Nucleic Acids Research 10-1573 ( 1979); Cooney et al, Science 247:456 (1988); and Dervan et al. Science 257: 1360 (1991).
• the methods are based on binding of a polynucleotide to a complementary DNA or RNA.
• the 5' coding portion of a polynucleotide that encodes the mature polypeptide of the present invention may be used to design an antisense RNA oligonucleotide of from about 10 to 40 base pairs in length.
• a DNA oligonucleotide is designed to be complementary to a region of the gene involved in transcription thereby preventing transcription and the production of the receptor.
• the antisense RNA oligonucleotide hybridizes to the mRNA in vivo and blocks translation of the mRNA molecule into receptor polypeptide.
• the oligonucleotides described above can also be delivered to cells such that the antisense RNA or DNA may be expressed in vivo to inhibit production of the receptor.
• Further antagonists according to the present invention include soluble forms of CD44-like protein, e.g., CD44-like protein fragments that include the extracellular domain, which contains the ligand binding region.
• soluble forms of the receptor which may be naturally occurring or synthetic, antagonize CD44-like protein- mediated signaling by competing with the cell surface CD44- like protein receptor for binding to CD44 receptor ligands.
• antibody or “monoclonal antibody” (mAb) as used herein is meant to include intact molecules as well as fragments thereof (such as, for example, Fab and F(ab fragments) which are capable of binding an antigen.
• Fab and F(ab') 2 fragments lack the Fc fragment of intact antibody, clear more rapidly from the circulation, and may have less non-specific tissue binding of an intact antibody (Wahl et al, J. Nucl. Med 24:316-325 (1983)).
• Antibodies according to the present invention may be prepared by any of a variety of methods using CD44-like protein immunogens of the present invention.
• CD44-like protein immunogens include the full length CD44-like protein polypeptide (which may or may not include the leader sequence) and CD44-like protein polypeptide fragments such as the extracellular domain, the transmembrane domain, and the intracellular domain.
• antibodies according to the present invention are mAbs.
• Such mAbs can be prepared using hybridoma technology as described above and in the following references: Kohler and Millstein, Nature 256:495-497 (1975) and U.S. Patent No. 4,376,110; Harlow et al, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1988; Monoclonal Antibodies and Hybridomas: A New Dimension in Biological
• chimeric antibodies produced using genetic constructs derived from hybridoma cells producing the mAbs described above.
• Methods for production of chimeric antibodies are known in the art. See, for review: Morrison, Science, 229: 1202- 1207 (1985); Oi et al, BioTechniques 4:214 (1986); see, also: Cabilly et al, U.S. Patent 4,816,567 (3/28/89); Taniguchi et al, EPO Patent Public. EP171496 (2/19/86); Morrison et al, EPO Patent Pub. EP173494 (3/5/86); Neuberger et al,
• PCT Pub. WO8601533 (3/13/86); Robinson et al, PCT Pub. WO 8702671 (5/7/87); Boulianne et al, Nature 312:643-646 (1984); Neuberger et al, Nature 314:268-270 (1985).
• Proteins and other compounds which bind the CD44-like protein domains are also candidate agonist and antagonist according to the present invention.
• binding compounds can be "captured” using the yeast two-hybrid system (Fields and Song, Nature 340:245-246 (1989)).
• yeast two-hybrid system Fields and Song, Nature 340:245-246 (1989)
• a modified version of the yeast two- hybrid system has been described by Roger Brent and his colleagues (Gyuris, J. et al. Cell 75:791-803 (1993); Zervos, A S. el al. Cell 72:223-232 (1993)). Briefly, a domain of the CD44-like protein polypeptide is used as bait for binding compounds.
• yeast two-hybrid system is used according to the present invention to capture compounds which bind to either the CD44-like protein ligand binding domain or to the CD44-like protein intracellular domain.
• Such compounds are good candidate agonist and antagonist of the present invention.
• This system has been used previously to isolate proteins which bind to the intracellular domain of the p55 and p75 TNF receptors (WO 95/31544)
• CD44-like protein receptor ligand By a "CD44-like protein receptor ligand" is intended naturally occurring, recombinant, and synthetic ligands that are capable of binding to the CD44-like protein receptor and inducing the receptor signaling pathway.
• ligands of the CD44-like protein receptor include, but are not limited to, hyaluronate and osteopontin.
• CD44-like protein receptor of the present invention uses include treatment or prevention of tissue necrosis, thermal injury, scarring, artereosclerosis, vascular restenosis, inflammation, graft versus host disease, cancer, and metastasis. More detail related to these representative therapeutic applications of the present invention is given below.
• One therapeutic use involving the CD44-like protein of the invention is a method for treating tissue necrosis, or loss, in an animal.
• Tissue necrosis is treated by providing the subject with a therapeutically effective amount of an antagonist of the CD44-like molecule of the invention, where the CD44-like molecule is expressed on the surface of inflammatory cells or endothelial cells.
• the antagonist will contain the progression of tissue injury by preventing the influx of neutrophils, especially polymorphonuclear neutrophils (which are inflammatory cells) to a burn site.
• a preferred application of this method will be in the treatment and containment of tissue necrosis surrounding a burn injury, such as a thermal injury resulting from skin contact with elevated temperatures, such as a heated stove, etc.
• the treatment with the agent capable of binding the CD44-like molecule of the invention should begin with measuring the tissue necrosis within one hour of a burn to provide a reference point of the burn size, treating the animal with a therapeutic amount of the agent, continuing to treat the animal with the agent for not more than 24 hours after the burn contact, and measuring the tissue necrosis after the treatment, wherein a treated size equal to the reference size indicates an inhibition of the progression of tissue necrosis (WO 93/17047). Modes of administration and dosages are discussed in detail below.
• the above method for the treatment of thermal injury may also be effective for decreasing scarring or the formation of scar tissue attendant the healing process at a burn site.
• scarring is defined as the formation of fibrous tissue at sites where normal tissue has been destroyed.
• the present invention thus also includes a method for decreasing scarring specifically at skin tissue areas of second or third degree burn.
• This method comprises treating an animal with a second or third degree burn with a therapeutically effective amount of a pharmaceutically acceptable preparation comprising an antagonist of the CD44-like molecule of the invention to decrease tissue scarring in an animal.
• a pharmaceutically acceptable preparation comprising an antagonist of the CD44-like molecule of the invention to decrease tissue scarring in an animal.
• Such an agent can of course include an antibody capable of binding the CD44-like molecule of the invention.
• Another therapeutic use involving the CD44-like molecule of the invention is a method for treating arteriosclerosis (the abnormal hardening or thickening of arterial walls) or vascular restenosis (the narrowing or constriction of blood vessel walls) in an animal by inhibiting smooth muscle migration or proliferation that is mediated by the CD44-like protein receptor of the invention.
• the method comprises inhibiting expression of the CD44-like molecule of the invention by introducing into the target cells of the patient by standard vectors and/or gene delivery systems, a therapeutically effective amount of an isolated antisense DNA of the CD44-like molecule.
• Suitable gene delivery systems may include liposomes, receptor-mediated delivery systems, naked DNA, and viral vectors such as herpes viruses, retroviruses and adenoviruses, among others.
• Yet another method of treatment included in the present invention comprises a method for treating graft-versus-host disease (GvHD).
• GvHD graft-versus-host disease
• One of the complications that may arise in tissue transplantation involves an immune response mounted by the graft tissue against the host. This is the principle complication involved in the use of ly phoid tissue in transplantation therapies, particularly bone marrow transplants (WO 95/24913).
• one aspect of the present invention is directed to administering to the recipient of a tissue transplant, preferably during a time period concurrent with the presence of a transplant, a therapeutic amount of a preparation comprising an antagonist of the CD44-like protein receptor.
• the donor tissue is exposed to the above-described pharmaceutical preparation prior to transplant for a period of time sufficient to inhibit or prevent a graft-versus-host response upon implantation (WO 95/24913).
• Another therapeutic use included in the invention is a method of treating inflammation or inhibiting cancer cell metastasis.
• CD44 is known to be a proinflammatory molecule involved in immune cell activation (reviewed in Haynes etal, Immunol. Today 70:423-428 (1989)) as well as metastasis of certain tumor cell types (Id, WO 94/09811).
• the CD44 molecule has been shown to be upregulated in rheumatoid arthritis on many synovial cell types; the level of CD44 present in synovial tissue is directly proportional to the degree of synovial inflammation.
• the present invention includes a method of treating inflammation in an animal comprising administering to the animal a therapeutically effective amount of an antagonist of the CD44-like protein of the invention, or a fragment thereof, in an amount sufficient to reduce the inflammation
• the invention relates to a method of transporting a drug or cytotoxic agent to a site of inflammation in an animal, comprising administering to the animal an anti-CD44-like protein monoclonal antibody linked to a drug or cytotoxic agent.
• the monoclonal antibody and the drug or cytotoxic agent (which, together, are referred to as an immunotoxin) are incorporated into a liposome for delivery to the site of inflammation.
• a similar principle can be utilized in order to prevent metastasis.
• the process of metastatic spread of tumor cells requires interactions between the tumor cells and the stromal cells, as well as recognition by cell surface receptors of the tumor cells of ligands comprising the extracellular matrix.
• CD44 is one of the receptors found to be expressed at high levels in tumor tissue, and as the CD44 ligand hyaluronate, which induces homotypic aggregation, is present in the extracellular matrix, it follows that the administration of an antagonist of the CD44-like protein would inhibit the interaction between CD44 and hyaluronate that occurs in metastasis (Weber et al, Science 277:509-512 (1996)).
• the present invention relates to a method of inhibiting binding of hyaluronan to the CD44-like molecule of the invention, comprising contacting the CD44-like molecule with a therapeutically effective amount of an antagonist under conditions such that binding of hyaluronan to the CD44-like molecule is inhibited.
• Yet another therapeutic method included in the invention is a method of treating cancer by administering to an animal suffering from cancer a pharmaceutically effective amount of an antagonist compound capable of binding to cancer cells, wherein the compound is associated with a substance capable of damaging cancer cells.
• T e agonist or antagonist described herein can be administered in vitro, ex vivo, or in vivo to cells which express the receptor of the present invention.
• an "effective amount" of an agonist or antagonist is intended an amount of the compound that is sufficient to enhance or inhibit a cellular response to a CD44-like protein receptor ligand.
• effective amounts of an agonist or antagonist can be determined empirically and may be employed in pure form or, where such forms exist, in pharmaceutically acceptable salt, ester or prodrug form.
• the agonist or antagonist my be administered in compositions in combination with one or more pharmaceutically acceptable excipients.
• the total daily usage of the compounds and compositions of the present invention will be decided by the attending physician within the scope of sound medical judgement.
• the specific therapeutically effective dose level for any particular patient will depend upon a variety of factors including the type and degree of the cellular response to be achieved; activity of the specific agonist or antagonist employed; the specific composition employed; the age, body weight, general health, sex and diet of the patient; the time of administration, route of administration, and rate of excretion of the agonist or antagonist; the duration of the treatment; drugs used in combination or coincidental with the specific agonist or antagonist; and like factors well known in the medical arts.
• a antagonist or agonist for example, satisfactory results are obtained by oral administration of a antagonist or agonist at dosages on the order of from 0.05 to 10 mg/kg/day, preferably 0.1 to 7.5 mg kg/day, more preferably 0.1 to 2 mg/kg/day, administered once or, in divided doses, 2 to 4 times per day
• dosages on the order of from 0.01 to 5 mg/kg/day, preferably 0.05 to 1.0 mg/kg/day and more preferably 0.1 to 1.0 mg/kg/day can be used.
• Suitable daily dosages for patients are thus on the order of from 2.5 to 500 mg p.o., preferably 5 to 250 mg p.o., more preferably 5 to 100 mg p.o., or on the order of from 0.5 to 250 mg i.v., preferably 2.5 to 125 mg i.v. and more preferably 2.5 to 50 mg i.v.
• Dosaging may also be arranged in a patient specific manner to provide a predetermined concentration of an agonist or antagonist in the blood, as determined by the RIA technique.
• patient dosaging may be adjusted to achieve regular on-going trough blood levels, as measured by RIA, on the order of from 50 to 1000 ng/ml, preferably 150 to 500 ng/ml.
• pharmaceutical compositions are provided comprising an agonist or antagonist and a pharmaceutically acceptable carrier or excipient, which may be administered orally, rectally, parenterally, intracisternally, intravaginally, intraperitoneally, topically (as by powders, ointments, drops or transdermal patch), bucally, or as an oral or nasal spray.
• agonist can be "co-administered" either before, after, or simultaneously with the CD44-like receptor ligand, depending on the exigencies of a particular therapeutic application.
• pharmaceutically acceptable carrier is meant a non-toxic solid, semisolid or liquid filler, diluent, encapsulating material or formulation auxiliary of any type.
• parenteral refers to modes of administration which include intravenous, intramuscular, intraperitoneal, intrasternal, subcutaneous and intraarticular injection and infusion.
• compositions of the present invention for parenteral injection can comprise pharmaceutically acceptable sterile aqueous or nonaqueous solutions, dispersions, suspensions or emulsions as well as sterile powders for reconstitution into sterile injectable solutions or dispersions just prior to use.
• suitable aqueous and nonaqueous carriers, diluents, solvents or vehicles include water, ethanol, polyols (such as glycerol, propylene glycol, polyethylene glycol, and the like), carboxymethylceuulose and suitable mixtures thereof, vegetable oils (such as olive oil), and injectable organic esters such as ethyl oleate.
• Proper fluidity can be maintained, for example, by the use of coating materials such as lecithin, by the maintenance of the required particle size in the case of dispersions, and by the use of surfactants.
• compositions of the present invention may also contain adjuvants such as preservatives, wetting agents, emulsifying agents, and dispersing agents. Prevention of the action of microorganisms may be ensured by the inclusion of various antibacterial and antifimgal agents, for example, paraben, chlorobutanol, phenol sorbic acid, and the like. It may also be desirable to include isotonic agents such as sugars, sodium chloride, and the like. Prolonged absorption of the injectable pharmaceutical form may be brought about by the inclusion of agents which delay absorption such as aluminum monostearate and gelatin.
• the absorption from subcutaneous or intramuscular injection in order to prolong the effect of the agonist or antagonist, it is desirable to slow the absorption from subcutaneous or intramuscular injection. This may be accomplished by the use of a liquid suspension of crystalline or amorphous material with poor water solubility. The rate of absorption of the drug then depends upon its rate of dissolution which, in turn, may depend upon crystal size and crystalline form. Alternatively, delayed absorption of a parenterally administered drug form is accomplished by dissolving or suspending the drug in an oil vehicle.
• Injectable depot forms are made by forming microencapsule matrices of the drug in biodegradable polymers such as polylactide-polyglycolide. Depending upon the ratio of drug to polymer and the nature of the particular polymer employed, the rate of drug release can be controlled. Examples of other biodegradable polymers include poly(orthoesters) and poly(anhydrides). Depot injectable formulations are also prepared by entrapping the drug in liposomes or rnicroemulsions which are compatible with body tissues.
• the injectable formulations can be sterilized, for example, by filtration through a bacterial-retaining filter, or by incorporating sterilizing agents in the form of sterile solid compositions which can be dissolved or dispersed in sterile water or other sterile injectable medium just prior to use.
• Solid dosage forms for oral administration include capsules, tablets, pills, powders, and granules.
• the active compounds are mixed with at least one item pharmaceutically acceptable excipient or carrier such as sodium citrate or dicalcium phosphate and/or a) fillers or extenders such as starches, lactose, sucrose, glucose, mannitol, and silicic acid, b) binders such as, for example, carboxymethylcerulose, alginates, gelatin, polyvinylpyrrolidone, sucrose, and acacia, c) humectants such as glycerol, d) disintegrating agents such as agar, calcium carbonate, potato or tapioca starch, alginic acid, certain silicates, and sodium carbonate, e) solution retarding agents such as paraffin, f) absorption accelerators such as quaternary ammonium compounds, g) wetting agents such as, for example, cetyl alcohol and glycerol monoste
• compositions of a similar type may also be employed as fillers in soft and hard filled gelatin capsules using such excipients as lactose or milk sugar as well as high molecular weight polyethylene glycols and the like.
• the solid dosage forms of tablets, dragees, capsules, pills, and granules can be prepared with coatings and shells such as enteric coatings and other coatings well known in the pharmaceutical formulating art. They may optionally contain opacifying agents and can also be of a composition that they release the active ingredient(s) only, or preferentially, in a certain part of the intestinal tract, optionally, in a delayed manner. Examples of embedding compositions which can be used include polymeric substances and waxes.
• the active compounds can also be in micro-encapsulated form, if appropriate, with one or more of the above-mentioned excipients.
• Liquid dosage forms for oral administration include pharmaceutically acceptable emulsions, solutions, suspensions, syrups and elixirs.
• the liquid dosage forms may contain inert diluents commonly used in the art such as, for example, water or other solvents, solubilizing agents and emulsifiers such as ethyl alcohol, isoptopyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butylene glycol, dimethyl formamide, oils (in particular, cottonseed, groundnut, corn, germ, olive, castor, and sesame oils), glycerol, tetrahydrofurfuryl alcohol, polyethylene glycols and fatty acid esters of sorbitan, and mixtures thereof.
• inert diluents commonly used in the art such as, for example, water or other solvents, solubilizing agents and emulsifiers
• the oral compositions can also include adjuvants such as wetting agents, emulsifying and suspending agents, sweetening, flavoring, and perfuming agents.
• Suspensions in addition to the active compounds, may contain suspending agents as, for example, ethoxylated isostearyl alcohols, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum metahydroxide, bentonite, agar-agar, and tragacanth, and mixtures thereof.
• Topical administration includes administration to the skin or mucosa, including surfaces of the lung and eye.
• Compositions for topical administration may be prepared as a dry powder which may be pressurized or non-pressurized.
• the active ingredient in finely divided form may be used in admixture with a larger- sized pharmaceutically acceptable inert carrier comprising particles having a size, for example, of up to 100 ⁇ m in diameter.
• suitable inert carriers include sugars such as lactose. Desirably, at least 95% by weight of the particles of the active ingredient have an effective particle size in the range of 0.01 to 10 ⁇ m.
• the composition may be pressurized and contain a compressed gas, such as nitrogen or a liquefied gas propellant.
• a compressed gas such as nitrogen or a liquefied gas propellant.
• the liquefied propellant medium and indeed the total composition is preferably such that the active ingredient does not dissolve therein to any substantial extent.
• the pressurized composition may also contain a surface active agent.
• the surface active agent may be a liquid or solid non-ionic surface active agent or may be a solid anionic surface active agent. It is preferred to use the solid anionic surface active agent in the form of a sodium salt.
• compositions for rectal or vaginal administration are preferably suppositories which can be prepared by mixing the agonist or antagonist with suitable non-irritating excipients or carriers such as cocoa butter, polyethylene glycol or a suppository wax which are solid at room temperature but liquid at body temperature and therefore melt in the rectum or vaginal cavity and release the drugs.
• suitable non-irritating excipients or carriers such as cocoa butter, polyethylene glycol or a suppository wax which are solid at room temperature but liquid at body temperature and therefore melt in the rectum or vaginal cavity and release the drugs.
• the agonist or antagonist can also be administered in the form of liposomes.
• liposomes are generally derived from phospholipids or other lipid substances. Liposomes are formed by mono- or multi-lameflar hydrated liquid crystals that are dispersed in an aqueous medium. Any non-toxic, physiologically acceptable and metabolizable lipid capable of forming liposomes can be used.
• the present compositions in liposome form can contain, in addition to the agonist or antagonist, stabilizers, preservatives, excipients, and the like.
• the preferred lipids are the phospholipids and the phosphatidyl choaes (lecithins), both natural and synthetic.
• the DNA sequence encoding the CD44-like protein in the deposited polynucleotide is amplified using PCR oligonucleotide primers specific to the amino acid carboxyl terminal sequence of the CD44-like protein protein and to vector sequences 3' to the gene. Additional nucleotides containing restriction sites to facilitate cloning are added to the 5' and 3' sequences respectively.
• the 5' oligonucleotide primer has the sequence 5'
• the restrictions sites are convenient to restriction enzyme sites in the bacterial expression vector pQE-60, which is used for bacterial expression in these examples. (Qiagen, Inc. 9259 Eton Avenue, Chatsworth, CA, 91311). pQE60 encodes ampicillin antibiotic resistance ("Ampr”) and contains a bacterial origin of replication ("ori"), an EPTG inducible promoter, a ribosome binding site ("RBS”), a 6-His tag and restriction enzyme sites.
• Ampr ampicillin antibiotic resistance
• ori bacterial origin of replication
• RBS ribosome binding site
• 6-His tag a 6-His tag and restriction enzyme sites.
• Insertion of the CD44-Iike protein DNA into pQE60 places the CD44-like protein coding region downstream of and operably linked to the vector's IPTG-inducible promoter, and in-frame with an initiating AUG appropriately positioned for translation of the CD44-like protein.
• the ligation mixture is transformed into competent E. coli cells using standard procedures. Such procedures are described in Sambrook et al, MOLECULAR CLONING: A LABORATORY MANUAL, 2nd Ed.; Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N Y. (1989). E. coli strain
• M15/rep4 containing multiple copies of the plasmid pREP4, which expresses lac repressor and confers kanamycin resistance (“Kanr"), is used in carrying out the illustrative example described here.
• This strain which is only one of many that are suitable for expressing the CD44-like protein, is available commercially from Qiagen.
• Transformants are identified by their ability to grow on LB plates in the presence of ampicillin. Plasmid DNA is isolated from resistant colonies and the identity of the cloned DNA is confirmed by restriction analysis.
• Clones containing the desired constructs are grown overnight ("O/N") in liquid culture in LB media supplemented with both ampicillin (100 ug/ml) and kanamycin (25 ug/ml).
• the O/N culture is used to inoculate a large culture, at a dilution of approximately 1:100 to 1:250.
• the cells are grown to an optical density at 600nm ("OD600") of between 0.4 and 0.6.
• Isopropyl-B-D-thiogalactopyranoside (“IPTG”) is then added to a final concentration of 1 mM to induce transcription from lac repressor sensitive promoters, by inactivating the lad repressor.
• Cells subsequently are incubated further for 3 to 4 hours.
• Cells are then harvested by centrifugation and disrupted, by standard methods.
• Inclusion bodies are purified from the disrupted cells using routine collection techniques, and protein is solubilized from the inclusion bodies into 8M urea.
• the 8M urea solution containing the solubilized protein is passed over a PD-10 column in 2X phosphate buffered saline ("PBS"), thereby removing the urea, exchanging the buffer and refolding the protein.
• PBS 2X phosphate buffered saline
• the protein is purified by a further step of chromatography to remove endotoxin. Then, it is sterile filtered.
• the sterile filtered protein preparation is stored in 2X PBS at a concentration of 95 micrograms per mL.
• Example 2 Expression in Mammalian Cells (CHO, COS and others).
• vectors used for the transient expression of the CD44-like protein gene sequence in mammalian cells should carry the SV40 origin of replication. This allows the replication of the vector to high copy numbers in cells (e.g. COS cells) which express the T antigen required for the initiation of viral
• DNA synthesis Any other mammalian cell line can also be utilized for this purpose.
• a typical mammalian expression vector contains the promoter element, which mediates the initiation of transcription of mRNA, the protein coding sequence, and signals required for the termination of trancription and polyadenylation of the transcript. Additional elements include enhancers, Kozak sequences and intervening sequences flanked by donor and acceptor sites for RNA splicing. Highly efficient transcription can be achieved with the early and late promoters from SV40, the long terminal repeats (LTRs) from Retroviruses, e.g. RSV, HTLVI, HI VI and the early promoter of the cytomegalovirus (CMV).
• LTRs long terminal repeats
• cellular signals can be used (e.g. human actin, promoter).
• Suitable expression vectors for use in practicing the present invention include , for example, vectors such as pSVL and pMSG (Pharmacia, Uppsala, Sweden), pRSVcat (ATCC 37152), pSV2dhfr (ATCC 37146) and pBC12MI (ATCC67109).
• Mammalian host cells that could be used include, human Hela,
• the gene can be expressed in stable cell lines that contain the gene integrated into a chromosome.
• the co-transfection with a selectable marker such as dhfr, gpt, neomycin, hygromycin allows the identification and isolation of the transfected cells.
• the transfected gene can also be amplified to express large amounts of the encoded protein.
• the DHFR dihydrofolate reductase is a useful marker to develop cell lines that carry several hundred or even several thousand copies of the gene of interest.
• the mammalian cells are grown in increasing amounts of methotrexate for selection and the cells with the highest resistance are selected.
• These cell lines contain the amplified gene(s) integrated into a chromosome.
• Chinese hamster ovary (CHO) cells are often used for the production of proteins.
• the expression vectors pC 1 and pC4 contain the strong promoter (LTR) of the Rous Sarcoma Virus (Cullen et al. , Molecular and Cellular Biology, 438-4470 (March, 1985)) plus a fragment of the CMV-enhancer (Boshart et al,
• the vectors contain in addition the 3' intron, the polyadenylation and termination signal of the rat preproinsulin gene.
• Example 2A Expression of extracellular soluble domain of CD44-like protein in COS cells
• the expression plasmid, CD44-like protein HA is made by cloning a cDNA encoding CD44-like protein into the expression vector pcDNAI Amp (which can be obtained from Invitrogen, Inc.).
• the expression vector pcDNAI amp contains: (1) an E.coli origin of replication effective for propagation in E.
• coli and other prokaryotic cell (2) an ampicillin resistance gene for selection of plasmid-containing prokaryotic cells; (3) an SV40 origin of replication for propagation in eukaryotic cells; (4) a CMV promoter, a polylinker, an SV40 intron, and a polyadenylation signal arranged so that a cDNA conveniently can be placed under expression control of the CMV promoter and operably linked to the SV40 intron and the polyadenylation signal by means of restriction sites in the polylinker.
• a DNA fragment encoding the entire CD44-like protein precursor and a HA tag fused in frame to its 3' end is cloned into the polylinker region of the vector so that recombinant protein expression is directed by the CMV promoter.
• the HA tag corresponds to an epitope derived from the influenza hemagglutinin protein described by Wilson et al, Cell 37:161 (1984).
• the fusion of the HA tag to the target protein allows easy detection of the recombinant protein with an antibody that recognizes the HA epitope.
• the plasmid construction strategy is as follows.
• the CD44-like protein cDNA of the deposit clone is amplified using primers that contained convenient restriction sites, much as described above regarding the construction of expression vectors for expression of CD44-like protein in E. coli and S. fugiperda.
• primers that contained convenient restriction sites, much as described above regarding the construction of expression vectors for expression of CD44-like protein in E. coli and S. fugiperda.
• one of the primers contains a hemagglutinin tag ("HA tag”) as described above.
• Suitable primers include that following, which are used in this example; the 5' primer CGC GGA TCC GCC ATC ATG GCC AGG TGC TTC AGC 3 '[SEQ ID NO.:6] contains the underlined Bam HI site, an ATG start codon and 15 codons thereafter.
• I site, stop codon, hemagglutinin tag and last 15 bp preceding transmembrane domain (at the 3' end) has the following sequence. 5' CGC TCT AGA TCA AGC GTAGTC TGGGAC GTC GTATGGGTA AGC CTG GGG GAC ACC TC 3'
• the PCR amplified DNA fragment and the vector, pcDNAI/Amp, are digested with Bam HI and Xba I and then ligated.
• the ligation mixture is transformed into E. coli strain SURE (available from Stratagene Cloning Systems, 1 1099 North Torrey Pines Road, La Jolla, CA 92037), and the transformed culture is plated on ampicillin media plates which then are incubated to allow growth of ampicillin resistant colonies. Plasmid DNA is isolated from resistant colonies and examined by restriction analysis and gel sizing for the presence of the CD44-Iike protein-encoding fragment.
• COS cells are transfected with an expression vector, as described above, using DEAE-
• the cells and the media are collected, and the cells are washed and the lysed with detergent-containing RIPA buffer: 150 mM NaCl, 1% NP-40, 0.1% SDS, 1% NP-40, 0.5% DOC, 50 mM TRIS, pH 7.5, as described by Wilson et al. cited above.
• Proteins are precipitated from the cell lysate and from the culture media using an HA-specific monoclonal antibody. The precipitated proteins then are analyzed by SDS-PAGE gels and autoradiography.
• Example 2B Expression and purification of human CD44-like protein using the CHO Expression System
• the DNA sequence encoding CD44-like protein in the deposited polynucleotide is amplified using PCR oligonucleotide primers specific to the carboxyl terminal sequence of the CD44-like protein protein and to vector sequences 3' to the gene. Additional nucleotides containing restriction sites to facilitate cloning are added to the 5' and 3' sequences respectively.
• the 5' primer has the sequence 5' CGC GGA TCC GCC ATC ATG GCC AGG TGC TTC AGC 3' [SEQ ID NO.: 09] containing the underlined Bam HI restriction enzyme site followed by Kozak sequence and 18 bases of the sequence of CD44-like protein receptor of FIG. 1.
• the 3' primer has the full length sequence CGC GGT ACC TCA ACC AGC CTC AGG TGT [SEQ ID NO.: 8, containing the underlined Asp718 restriction followed by nucleotides complementary to 18 bp preceding the transmembrane domain of the CD44-like protein receptor set out in FIG. 1, including the stop codon.
• the 3' primer has the sequence of 5' CGC
• the restrictions sites are convenient to restriction enzyme sites in the CHO expression vectors PC4.
• the amplified CD44-like protein DNA and the vector PC4 both are digested with BamHI and the digested DNAs then ligated together. Insertion of the CD44-like protein DNA into the BamHJ restricted vector placed the CD44- like protein coding region downstream of and operably linked to the vector's promoter.
• the ligation mixture is transformed into competent E. coli cells using standard procedures. Such procedures are described in Sambrook et al, MOLECULAR CLONING: A LABORATORY MANUAL, 2nd Ed.; Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1989).
• Example 3 Cloning and expression of the soluble extracellular domain of CD44-like protein in a baculovirus expression system
• the cDNA sequences encoding either the soluble extracellular domain or the full length protein of the CD44-like protein receptor protein in the deposited clone is amplified using PCR oligonucleotide primers corresponding to the 5' and
• the 5' primer for expression of either the extracellular domain or the full length protein has the sequence 5' CGC GGA TCC GCC ATC ATG GCC AGG TGC TTC AGC 3' [SEQ ID NO.: 09] containing the underlined Bam HI restriction enzyme site followed by Kozak sequence and 18 bases of the sequence of CD44-like protein receptor of FIG. 1. Inserted into an expression vector, as described below, the 5' end of the amplified fragment encoding CD44-like protein receptor provides an efficient signal peptide. An efficient signal for initiation of translation in eukaryotic cells, as described by Kozak, M., J. Mol. Biol 196: 947- 950 (1987) is appropriately located in the vector portion of the construct.
• the 3' primer has the full length sequence CGC
• the 3' primer has the sequence of 5' CGC GGT ACC TCA AGC CGT GGG GAC ACC TC 3'[SEQ ID NO. 8
• the amplified fragment is isolated from a 1% agarose gel using a commercially available kit ("Geneclean,” BIO 101 Inc., La Jolla, Ca ). The fragment then is digested with BamHI and Asp718 and again is purified on a 1% agarose gel. This fragment is designated herein F2
• the vector pA2 is used to express the CD44-like protein protein in the baculovirus expression system, using standard methods, such as those described in Summers et al, A MANUAL OF METHODS FOR BACULOVIRUS
• This expression vector contains the strong polyhedrin promoter of the Autographa californica nuclear polyhedrosis virus (AcMNPV) followed by convenient restriction sites.
• AcMNPV Autographa californica nuclear polyhedrosis virus
• the beta-galactosidase gene from E.coli is inserted in the same orientation as the polyhedrin promoter and is followed by the polyadenylation signal of the polyhedrin gene.
• the polyhedrin sequences are flanked at both sides by viral sequences for cell-mediated homologous recombination with wild-type viral DNA to generate viable virus that express the cloned polynucleotide.
• baculovirus vectors could be used in place of pA2, such as pAc373, pVL941 and pAcIMl provided, as those of skill readily will appreciate, that construction provides appropriately located signals for transcription, translation, trafficking and the like, such as an in-frame AUG and a signal peptide, as required.
• pA2 pAc373, pVL941 and pAcIMl provided, as those of skill readily will appreciate, that construction provides appropriately located signals for transcription, translation, trafficking and the like, such as an in-frame AUG and a signal peptide, as required.
• Such vectors are described in Luckow et al, Virology 170: 31-39, among others.
• the plasmid is digested with the restriction enzymes Sma I and Asp718 and then is dephosphorylated using calf intestinal phosphatase, using routine procedures known in the art.
• the DNA is then isolated from a 1% agarose gel using a commercially available kit ("Geneclean" BIO 101 Inc., La Jolla, Ca ). This vector DNA is designated herein " V2" .
• Fragment F2 and the dephosphorylated plasmid V2 are ligated together with T4 DNA ligase.
• E.coli HB101 cells are transformed with ligation mix and spread on culture plates.
• Bacteria are identified that contain the plasmid with the human CD44-like protein gene by digesting DNA from individual colonies using Bam HI and Asp718 and then analyzing the digestion product by gel electrophoresis. The sequence of the cloned fragment is confirmed by DNA sequencing. This plasmid is designated herein pBac/CD44-like protein.
• 5 ⁇ g of the plasmid pBac/CD44-like protein is co-transfected with 1.0 ⁇ g of a commercially available linearized baculovirus DNA ("BaculoGoldTM baculovirus DNA", Pharmingen, San Diego, CA.), using the lipofection method described by Feigner et al, Proc. Natl. Acad. Sci. USA 54:7413-7417 (1987) l ⁇ g of BaculoGoldTM virus DNA and 5 ⁇ g of the plasmid pBac/CD44-like protein are mixed in a sterile well of a microtiter plate containing 50 ⁇ l of serum free Grace's medium (Life Technologies Inc., Gaithersburg, MD).
• plaque assay After four days the supernatant is collected and a plaque assay is performed, as described by Summers and Smith, cited above. An agarose gel with "Blue Gal” (Life Technologies Inc., Gaithersburg) is used to allow easy identification and isolation of gal-expressing clones, which produce blue-stained plaques. (A detailed description of a "plaque assay” of this type can also be found in the user's guide for insect cell culture and baculovirology distributed by Life Technologies Inc., Gaithersburg, page 9-10).
• the virus is added to the cells. After appropriate incubation, blue stained plaques are picked with the tip of an
• V-CD44-like protein A clone containing properly inserted CD44-like protein receptor is identified by DNA analysis including restriction mapping and sequencing. This is designated herein as V-CD44-like protein.
• Sf9 cells are grown in Grace's medium supplemented with 10% heat- inactivated FBS.
• the cells are infected with the recombinant baculovirus V-
• CD44-like protein at a multiplicity of infection ("MOI") of about 2 (about 1 to about 3).
• MOI multiplicity of infection
• the medium is removed and is replaced with SF900 II medium minus methionine and cysteine (available from Life Technologies Inc., Gaithersburg). 42 hours later, 5 ⁇ Ci of 35S-methionine and 5 ⁇ Ci 35S cysteine (available from Amersham) are added.
• the cells are further incubated for 16 hours and then they are harvested by centrifugation, lysed and the labeled proteins are visualized by SDS-PAGE and autoradiography.
• Northern blot analysis was carried out to examine the levels of expression of the gene encoding the CD44-like protein in human tissues, using methods described by, among others, Sambrook et al, cited above.
• a cDNA probe containing the entire nucleotide sequence of the CD44-like protein of the present invention (SEQ ID NO: l) was labeled with "P using the ra rprimeTM DNA labelling system (Amersham Life Science), according to manufacturer's instructions. After labelling, the probe was purified using a CHROMA SPIN-
• MTN Multiple Tissue Northern
• H human tissues
• IM human immune system tissues
• CD44-like protein of the present invention was detected in most human tissues.
• the levels of CD44-like mRNA were substantial in spleen, lymph node, fetal liver and placenta ( Figure 3 a, lanes 1, 2 and 7, and Figure 3 b, lane 3, respectively), and lower levels of expression were also observed in appendix, bone marrow, lung, liver and skeletal muscle ( Figure 3 a, lanes 4 and 6, and Figure 3b, lanes 4, 5 and
• ADDRESSEE STERNE, KESSLER, GOLDSTEIN & FOX P.L.L.C.
• NAME GOLDSTEIN, JORGE A.
• MOLECULE TYPE DNA (genomic)
• FEATURE FEATURE
• AATAATATCC CACTGGGAGA AAGGAGTTTT GCAAAGTGCA AGGACCTAAA ACATCTCATC 1376
• MOLECULE TYPE protein
• MOLECULE TYPE DNA (genomic)
• MOLECULE TYPE DNA (genomic)
• MOLECULE TYPE DNA (genomic)
• MOLECULE TYPE DNA (genomic)
• MOLECULE TYPE DNA (genomic)
• MOLECULE TYPE DNA (genomic)
• MOLECULE TYPE DNA (genomic)

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