WO2001051518A2 - Molecules d'acide nucleique isolees codant pour une molecule de semaphorine humaine et utilisations correspondantes - Google Patents

Molecules d'acide nucleique isolees codant pour une molecule de semaphorine humaine et utilisations correspondantes Download PDF

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WO2001051518A2
WO2001051518A2 PCT/US2001/001275 US0101275W WO0151518A2 WO 2001051518 A2 WO2001051518 A2 WO 2001051518A2 US 0101275 W US0101275 W US 0101275W WO 0151518 A2 WO0151518 A2 WO 0151518A2
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seq
nucleic acid
acid molecule
isolated nucleic
nucleotides
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PCT/US2001/001275
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WO2001051518A3 (fr
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Andrew John George Simpson
Ricardo Correa
Sandro De Souzo
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Ludwig Institute For Cancer Research
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Publication of WO2001051518A3 publication Critical patent/WO2001051518A3/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
    • C07K14/4702—Regulators; Modulating activity
    • C07K14/4703—Inhibitors; Suppressors
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • the mvention relates to isolated nucleic acid molecules which encode human analogs of semaphorin, the proteins encoded thereby, as well as their use.
  • the molecules described herein were isolated and identified using the ORESTES method, which is described herein.
  • a second approach which has found more widespread acceptance, is to cleave the genome into relatively large fragments, and then to "map" the larger, non-sequenced fragments to show overlap prior to sequencing the material. After this overlapping, which results in a physical map of the genome, the segments are fragmented, and sequenced. While this approach should, in theory, eliminate the gaps in the sequence, it is time consuming and costly. Further, both of these approaches suffer from a fundamental drawback, as will all approaches which begin with eukaryotic genomic DNA, as will now be explained.
  • Eukaryotic DNA consists of both "coding” and “non-coding” DNA.
  • coding DNA is under consideration, as it is this material which is transcribed and then translated into proteins.
  • This coding DNA is sometimes referred to as "open reading frames” or “ORFs”, and this terminology will be used hereafter.
  • eukaryotic DNA has a much more complex structure.
  • Genes generally consist of a non-coding, regulatory portion of hundreds of nucleotides followed by coding regions ("exons"), separated by non-coding regions ("introns").
  • exons coding regions
  • introns non-coding regions
  • U.S. Patent No. 5,487,985 to McClelland, et al. teaches a method referred to as "AP-PCR", or arbitrarily primed polymerase chain reaction.
  • the method employs a single primer designed so that there is a degree of internal mismatch between the primer and the template.
  • a second PCR is carried out.
  • the amplification products are separated on a gel to yield a so-called "fingerprint" of the organism or individual under study.
  • the '985 patent does not discuss the identification of internal portions of open reading frames, nor does it discuss the analysis of sequences to develop contigs.
  • the semaphorins are one of the most prominent of the conserved families of axon guidance molecules. As described by, e.g., Van Vactor et al., Curr Biol 25(9): R201- 204(1999) they are expressed in many different regions of the developing nervous system, and are known to play important roles in establishing accurate axonal projections.
  • the semaphorins have been divided into 7 different classes, depending upon whether they are secreted molecules or transmembrane molecules.
  • the membranes of the Class III semaphorin family are secreted molecules, known to act as repulsive factors for specific axonal populations.
  • "Semaphorin III” which has also been referred to as “Collapsin- I” or “Sema D,” causes growth cone collapse, as well as axonal retraction and repulsion, in sensory and sympathetic axons in culture. See, e.g., Yu et al., Neuron22(l):l 1-14(1999).
  • SemaVIb murine semaphorin cDNA
  • the molecule comprises a characteristic, extracellular semaphorin domain, but lacks both the immunoglobin domain and thrombospondin repeats that have been observed in other vertebrate, transmembrane semaphorins.
  • Sema Nib is expressed in subregions of the nervous system during development, and is especially prominent in muscle tissue. Sema Nib mR A is expressed ubiquitously in adulthood. Studies carried out in vitro have shown that this molecule binds the SH3 domain of c-src. This may indicate a role in intracellular signaling via an src-related cascade.
  • Semaphorin R a human molecule was identified and referred to as Semaphorin R. See, e.g., U.S. Patent application Serial No. 09/483,618, filed January 14, 2000, the disclosure of which is incorporated by reference. Recently, Bamberg et al., "Unified Nomenclature for the Semaphorins/Collapsins," Cell 97:551-552 (May 28, 1999), the disclosure of which is incorporated by reference, suggest renaming the members of the semaphorin family and, in accordance with this system, Semaphorin R would now be referred to as Semaphorin 6B.
  • FIGS 1A and IB both show, schematically, prior art genome sequencing approaches.
  • FIG. 1C shows the invention, schematically.
  • Figure 2 presents both a theoretical probability curve (dark ovals) and actual results (white ovals), obtained when practicing the invention.
  • the data points refer to the probability of securing the sequence of a particular portion of cDNA molecule when practicing the invention.
  • Figure 3 shows construction of a contig, using the invention.
  • mRNA messenger RNA
  • the extraction of mRNA is a standard technique, the details of which are well known by the artisan of ordinary skill.
  • eukaryotic mRNA as compared to other forms of RNA, is characterized by a "poly A" tail.
  • poly A poly A tail
  • oligo dT molecules hybridize to the poly A sequences on the mRNA molecules, and these then remain on the column.
  • Other approaches to separation of mRNA are known. All can be used. If prokaryotic mRNA is being considered, separation using poly A/poly T hybridization is not carried out. It is preferred to treat the resulting material to reduce or to eliminate contamination by DNA. Adding a DNA degrading enzyme, such as DNA ase is preferred. This is carried out prior to contact with the column. It is also preferred to pas the purified RNA over the column at least twice.
  • the separated mRNA is then used to prepare a cDNA.
  • the preparation of the cDNA represents the first inventive step in the method of the invention.
  • the mRNA is combined with a sample of a single, arbitrary primer.
  • arbitrary is meant that the primer used does not have to be designed to correspond to any particular mRNA molecule. Indeed, it should not be, because the primer is going to be used to make all of the cDNA. Details on the design of arbitrary primers can be found in Dias-Neto, et al., supra. McClelland, et al, supra, and Serial No. 08/907,129 filed August 6, 1997 and incorporated by reference.
  • the primer is preferably at least 15 nucleotides long. Theoretically, it should not exceed about 50 nucleotides, but it can. Most preferably, the primer is 15-30 nucleotides long. While the sequence of the primer can be totally arbitrary, it is preferred that the total content of nucleotides "G” and "C” in the primer be compatible with the "G” and "C” content of the open reading frames of the organism under consideration. It is found that this favors amplification of the desired sequences. General rules of primer construction favor a G and C content of at least 50%.
  • “Arbitrary primer” as used herein does not exclude specific design choices within the primers.
  • the four bases at the 3' end of a given primer are generally considered the most important portion for hybridization.
  • a "marker" sequence can be used, i.e., a stretch of predefined nucleotides.
  • the remainder of the primer should be selected to correspond to overall GC usage, as described supra. Hence, for a primer 25 nucleotides long, the first 17 should correspond to GC usage for the organism in question.
  • Nucleotides 18-21 would be a "tag", such as "GGCC.” Then, all possible combinations of four nucleotides would follow, to produce 256 primers, which contain a known marker. This procedure could be repeated with a second set of primers, where the marker at 18-21 is different.
  • each set of variants is used with mRNA from a single source, and would permit the artisan to mark all sequences from a source, and still permit pooling.
  • the primer is combined with the mRNA under low stringency conditions. What is meant by this is that the conditions are selected so that the primer will hybridize to partially, rather than to only completely complementary sequences. Again, this is necessary because the primer will amplify an arbitrary sample of the mRNA pool, not just one sequence.
  • the arbitrary primer and mRNA are mixed with appropriate reagents, such as reverse transcriptase, a buffer, and dNTPs, to yield a pool of single stranded, cDNA molecules.
  • appropriate reagents such as reverse transcriptase, a buffer, and dNTPs
  • the single stranded cDNA is prepared, it is used in an amplification reaction.
  • the single primer used is identical to the first primer, as described supra, and that low stringency conditions be employed. Using identical primers tends to produce longer products, but this is not required.
  • the result of this amplification is a mini library.
  • Four pools of single stranded cDNA are then produced, i.e, "A”, "B”, “C” and “D”.
  • Each pool is then amplified using each of the four primers, to generate mini-libraries AA, AB, AC, AD, BA, BB, BC, BD, CA, CB, CC, CD, DA, DB, DC, and DD.
  • mini-libraries are used in the sequencing reaction which follows.
  • the resulting products are isolated, such as by size fractionation on a gel.
  • the resulting bands can be removed from the gel, such as by elution, and then subjected to standard methodologies for cloning and sequencing.
  • the highest probability for inclusion within amplified cDNA is the exact middle of the molecule. Lowest priority, in contrast, is at the extreme 5' and 3' ends.
  • Lowest priority in contrast, is at the extreme 5' and 3' ends.
  • a point directly in the middle of a cDNA molecule i.e., if the molecule is "x + 1" nucleotides long, .5x nucleotides precede the midpoint, and .5x nucleotides follow it.
  • the likelihood of a primer hybridizing to a point on the molecule, preceding the middle is .5x, and following it is also .5x. If "x" is 1, then the probability of hybridization surrounding the midpoint is .5(1 -.5), or .25, i.e., 25%).
  • a further aspect of the invention is the construction of contigs, once the sequence information has been determined.
  • the last 300 nucleotides of a sequence may be identical to the first 300 nucleotides of a second sequence.
  • the artisan can essentially splice the first and second sequences together, to produce a longer one.
  • the splicing can be done with two or more sequences found in the particular experiment that is carried out, or by comparing deduced sequences to sequences which are available in a public database, a private database, a journal, or any other source of sequence information.
  • a further aspect of the invention is the ability to compare information obtained using the inventive method to pre-existing information, in order to determine if a known nucleotide sequence is an internal sequence of a particular gene. This can be done because, as explained supra, the method described herein generates an extremely high percentage of internal sequences, with a very low percentage of sequences at the ends of a given molecule.
  • the prior art methods either generate predominantly terminal sequences, or internal sequences on a completely random basis. Hence, it is probable that nucleotide sequences of unknown origin are contained within various sources of sequence information. Data generated using the methods of this invention can be compared to this pre-existing information very easily, and can result in a determination that a particular nucleotide sequence is, in fact, an internal sequence.
  • the practice of the invention and how it is achieved will be seen in the examples which follow.
  • This example describes the generation of a cDNA library in accordance with the invention. While colon cancer cells from a human were used, any cell could also be treated in the manner described herein.
  • the mRNA was extracted from a sample of colon cancer cells, in accordance with standard methods well known to the artisan, and not repeated here. It was then divided into approximately 5 ⁇ H aliquots, which contained anywhere from 1 to lOng of mRNA. The samples were then stored at -70 °C until used.
  • a sample of lul of single stranded cDNA was combined, together with the same primer that had been used to generate the cDNA.
  • Amplification was carried out, using 12uM of primer, 200 uM of each dNTP, 1.5mM MgCl 2 , 1 unit of DNA polymerase, and buffer (50mM KC1, lOmM Tris-HCl, ⁇ H9.0, and 0.1 % Triton X-100), to reach a final volume of 15ul.
  • the cDNAs generated in the preceding examples were mixed, by pooling 10-20ul of each set of products into a final volume of 60ul, followed by electrophoresis through a 1 % low melting point agarose gel containing ethidium bromide to stain the cDNA fragments.
  • Known DNA size standards were also provided.
  • the gel portions containing fragments between 0.25 and 1.5 kilobases were excised, using a sterile razor blade.
  • Excised agarose was then heated to 65 °C for 10 minutes, in 1/10 volume of NaOAc (3mM, pH 7.0), and cDNA was recovered via standard phenol/chloroform extraction and ethanol precipitation, followed by resuspension in 40ul of water. The thus recovered cDNA was used in the following experiments.
  • the cDNA extracted supra was treated with 10 units of Klenow fragment cDNA polymerase, and 10 units of T4 polynucleotide kinase, for 45 minutes at 37 °C.
  • the reaction mixture was then extracted, once, with phenol, and the DNA was then recovered by passage through a standard Sephacryl S-200 column. Recovered cDNA was then ligated into the commercially available plasmid pUC18, and the plasmids were used to transform receptive E. coli, using standard methodologies. This resulted in sufficient amounts of individual cDNA molecules for the experiments which follow.
  • This example shows the use of the invention as applied to breast cancer cells.
  • a sample of an infiltrative breast carcinoma with attached portions of normal tissues was operatively resected from a subject.
  • the material was kept at -70 °C until used.
  • the sample was characterized, inter alia, by a large tumor mass and a very small amount of normal tissue.
  • Reverse transcription was carried out as with the colon cancer sample, as described supra. Then, PCR amplification was carried out by combining 12.8uM of the same primer used in the reverse transcription 125uM of each dNTP, 1.5 mM MgCl 2 , 1 unit of thermostable DNA polymerase, and buffer (5 OmMKCl, 10mMTris-HCl,pH9.0, and 0.1% Triton X- 100), to a final volume of 20ul.
  • Amplification was carried out by executing 1 cycle (denaturation at94°C for 1 minute, annealing at 37°C for 2 minutes, and extension at 72 °C, for 2 minutes), followed by 34 cycles at 94 ° C for 45 seconds, annealing at 55 ° C for 1 minute and extension at 72 ° C for 5 minutes.
  • 1 cycle denaturation at94°C for 1 minute, annealing at 37°C for 2 minutes, and extension at 72 °C, for 2 minutes
  • 34 cycles at 94 ° C for 45 seconds
  • annealing at 55 ° C for 1 minute and extension at 72 ° C for 5 minutes.
  • the products were eluted from their gels, cloned into pUC-18, and the plasmids were transformed into E. coli strain DH5 ⁇ , all as described supra. Plasmids were subjected to minipreparation, using the known alkaline lysis method, and then about 150 of the molecules were sequenced. Of these, 69% were not found in any databank consulted, and appear to represent new sequences. A total of 22% was characterized by large quantities of repetitive elements and retroviral sequences. A total of 4% corresponded to known human sequences, another 4% to ribosomal RNA and mitochondrial sequences, and 8% were redundant sequences.
  • the darker portion is a sequence obtained in accordance with the invention.
  • the first sequence is a tentative human consensus sequence, as taught by Adams, et al., Nature 377: 3-17 (1995), while the third sequence is an EST obtained from human gall bladder cells, identified as human gall bladder EST 51121.
  • RACE-PCR in accordance with Frohman, et al., Meth. Enzymol 218 : 340- 356 (1993), incorporated by reference, was carried out on a pool of human mammary gland cDNA.
  • RT-PCR was carried out, and this resulted in an 1801 nucleotide product.
  • RT-PCR was carried out, as described supra, using an aliquot of 5ng of cDNA from a pool of mRNA from three isolated tumor breast tissues.
  • SEQ ID NO: 4 or 5 was used with SEQ ID NO: 2 or 3, and these experiments generated sequence products approximately 0.8 and 1.0 Kb long.
  • the fragment products were separated by agarose gel electrophoresis, purified, cloned into pUC 18 , and sequenced using standard methods .
  • the resulting 1.8kb sequence is set forth as SEQ ID NO: 6 and represents the complete cDNA sequence .
  • This sequence contains the entire 568 nucleotide fragment referred to supra (i.e., that obtained via RACE-PCR).
  • the sequence exhibited 84% homology to murine semaphorin Z and semaphorin Nib which, together with presence of a sema domain, led to the conclusion that the molecule encodes a human semaphorin molecule.
  • the putative amino acid sequence of the protein encoded thereby is presented as SEQ ID NO: 7.
  • a start codon is presumed to be at nucleotides 4-7 of SEQ ID NO: 6, and nucleotides 1555 represents the termination of the open reading frame.
  • the open reading frame is 1551 base pairs long, spans 13 exons (exons 1-12 and exon 17), and ends with a 250 base pair 3' untranslated region.
  • sequence flanked by nucleotides 9 and 1394 of SEQ ID NO: 6 is 84% homologous to a sequence flanked by nucleotides 218 and 1606 of semaphorin Z, while the sequence flanked by nucleotides 1704 and 1801 of SEQ ID NO: 6 are 85% identical to nucleotides 3682 and 3779 of semaphorin Z.
  • amino acid 1-169 of SEQ ID NO: 7 is 87% homologous to amino acids 1-166 of semaphorin Z.
  • the region defined by nucleotides 321-566 of SEQ ID NO:6 showed no homology to any other molecule in publicly accessible data bases. Further, sequences defined by nucleotide fragments 72-566, 685-835, and 902-1410 were non- homologous with any ESTs associated with cancer.
  • a BLAST search was then carried out, using the human sequence described herein, and the rat semaphorin Z sequence, against the HTGS database. This search indicated that there was a possible, alternatively spliced variant, with additional exons at the 3' end.
  • RT-PCR was carried out, using primers designed to amplify the potential, 3'- coding region that is absent from the initial sequence.
  • SEQ ID NO: 3 and gaggagtttg agacctaccg gc were used.
  • the results of the RT-PCR confirmed that there was, in fact, an alternate, longer human semaphorin sequence, the nucleotide sequence of which is set forth at SEQ ID NO : 16.
  • the sequence contains an open reading frame 2061 base pairs long, which contains 4 additional exons, plus a cryptic acceptor site in the middle of the last exon, i.e., exon 17, in the 3' region of the mRNA sequence. This permits the RNA processing and posterior translation of the middle portion of the final exon.
  • the isoform encoded by this nucleotide sequence may contain a transmembrane domain, and a short cytoplasmic domain.
  • the amino acid sequence is provided as SEQ ID NO: 17.
  • Glucocorticoid hormones or all-trans-retinoic acid (ATRA) were used to treat these cell lines for long periods of time, i.e., 24, 48 and 72 hours.
  • Glucocorticoid hormones are widely used as anti-inflammatory agents, and anti-tumoral agents, and are the only chemotherapeutic agents available for gliomas and glioblastomas).
  • Retinoids are known to inhibit proliferation and migration of primary cultures of human multiform glioblastoma strongly, supporting clinical trials for their use. See Bouterfa, et al, Neurosurgery 46(2):419- 430/2000)).
  • RNA (10 ug) samples were isolated from cells that were untreated received glucocorticord treatement, or that received 10 "5 M ATRA for the listed periods of time. Acid ribosomal phosphoprotein PO was used as an internal control for RNA loading. The results indicated that glucocorticoid hormones did not regulate the expression of the sequence; however, the all-trans-retinoic acid did so, in a time dependent manner. Specifically, the expression was inhibited by 2.5 fold after 24 hours, and 7 fold after 72 hours inT98G, and in A172, expression was inhibited by 2.5 and 10 fold, respectively, after 48 and 72 hours.
  • Human semaphorin R6B refers to a protein encoded by, if nucleotides 4-1555 of SEQ ID NO. 6, where nucleotides 4-7 constitute a start codon, and nucleotides 1553-1555 a termination signal, as well as any protein which is encoded by the nucleic acid molecules of the invention, or is equivalent thereto such as proteins encoded by SEQ ID NO: 16.
  • nucleic acid molecules which encode this protein such as the nucleotides which make up the 2061 base pair ORF of SEQ. ID NO:6, as well as nucleic acid molecules which comprise the nucleotide sequence set forth in SEQ ID NO: 1 or 6.
  • Expression vectors and recombinant cells which comprise these nucleic acid molecules are also a part of the invention.
  • "Expression vector” as used herein, refers to any vector wherein the nucleic acid molecule is operably linked to a promoter.
  • Recombinant cells in accordance with the invention are preferably eukaryotic cells, and may comprise the expression vector.
  • cDNA is preferred, but genomic DNA is also a part of the invention.
  • the proteins which are a part of this invention may be admixed with, e.g. , pharmaceutically acceptable adjuvants, such as those which are well known to the skilled artisan.
  • Such compositions can be used, e.g., but not exclusively, to produce antibodies, such as monoclonal antibodies.
  • These, as well as hybridomas producing them, are also a part of the invention.
  • These proteins include e.g., those having amino acid sequences as set forth at SEQ ID NO: 7 or SEQ ID NO: 17, as well as those proteins homologous thereto.
  • yet another aspect of the invention is a diagnostic method for determining the possible presence of cancer, breast cancer or glioblastoma in particular, by determining expression or presence of one or both of the nucleic acid molecules and the proteins of the invention.

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Abstract

L'invention concerne des molécules d'acide nucléique isolées codant pour la sémaphorine humaine R/6B. Ces molécules sont associées au cancer, tel que le cancer du sein. L'invention concerne également les protéines codées par ces molécules, ainsi que les oligonucléotides les hybridant. L'invention porte enfin sur différentes utilisations de ces molécules.
PCT/US2001/001275 2000-01-14 2001-01-12 Molecules d'acide nucleique isolees codant pour une molecule de semaphorine humaine et utilisations correspondantes WO2001051518A2 (fr)

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Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2002074994A2 (fr) * 2000-11-07 2002-09-26 Ludwig Institute For Cancer Research Methode de sequençage orestes amelioree
EP1248801A1 (fr) * 2000-01-18 2002-10-16 Human Genome Sciences, Inc. Polynucleotides, polypeptides et anticorps humains
WO2004006898A2 (fr) * 2002-07-11 2004-01-22 Sema Aps Utilisation de composes pouvant inhiber le traitement proteolytique de semaphorines pour la prevention, le traitement, le diagnostic et le pronostic d'une maladie invasive
US7781413B2 (en) 2001-10-31 2010-08-24 Board Of Regents, The University Of Texas System SEMA3B inhibits tumor growth and induces apoptosis in cancer cells

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EP0896058A1 (fr) * 1995-12-06 1999-02-10 Sumitomo Pharmaceuticals Company, Limited Nouvelle semaphorine z et gene la codant

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EP0896058A1 (fr) * 1995-12-06 1999-02-10 Sumitomo Pharmaceuticals Company, Limited Nouvelle semaphorine z et gene la codant

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Title
CORREA ET AL: "Human semaphorin 6B Ä(HSA)SEMA6BÜ, a novel human class 6 semaphorin gene: alternative splicing and all-trans-retinoic acid-dependent downregulation in glioblastoma cell lines" EMBL DATA BASE - EMBL:AF216389, 29 July 2000 (2000-07-29), XP002171337 *
CORREA ET AL: "Human Semaphorin 6B Ä(HSA)SEMA6BÜ, a novel human class 6 semaphorin gene: Alternative splicing and all-trans-retinoic acid-dependent downregulation in glioblastoma cell lines" GENOMICS, vol. 73, 12 April 2001 (2001-04-12), pages 343-348, XP002171339 *
ECKHARDT F ET AL: "A NOVEL TRANSMEMBRANE SEMAPHORIN CAN BIND C-SRC" MOLECULAR AND CELLULAR NEUROSCIENCES,SAN DIEGO,US, vol. 9, 1997, pages 409-419, XP000996620 ISSN: 1044-7431 *
KLOSTERMANN ET AL: "The orthologous human and murine semaphorin 6A-1 proteins (SEMA6A-1/Sema6A-1) bind to the enabled/vasodilator-stimulated phosphoprotein-like protein (EVL) via a novel carboxy-terminal zyxin-like domain " JOURNA LOF BIOLOGICAL CHEMISTRY, vol. 275, 15 December 2000 (2000-12-15), pages 39647-39653, XP002171338 *
SIMPSON: "The FAPESP/LICR human cancer genome project; http://www.ludwig.org.br/ORESTES" EMBL DATA BASE - EMBL:AW054434, 27 September 1999 (1999-09-27), XP002171336 *

Cited By (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP1248801A1 (fr) * 2000-01-18 2002-10-16 Human Genome Sciences, Inc. Polynucleotides, polypeptides et anticorps humains
EP1248801A4 (fr) * 2000-01-18 2003-08-27 Human Genome Sciences Inc Polynucleotides, polypeptides et anticorps humains
WO2002074994A2 (fr) * 2000-11-07 2002-09-26 Ludwig Institute For Cancer Research Methode de sequençage orestes amelioree
WO2002074994A3 (fr) * 2000-11-07 2003-08-14 Ludwig Inst Cancer Res Methode de sequençage orestes amelioree
US7781413B2 (en) 2001-10-31 2010-08-24 Board Of Regents, The University Of Texas System SEMA3B inhibits tumor growth and induces apoptosis in cancer cells
WO2004006898A2 (fr) * 2002-07-11 2004-01-22 Sema Aps Utilisation de composes pouvant inhiber le traitement proteolytique de semaphorines pour la prevention, le traitement, le diagnostic et le pronostic d'une maladie invasive
WO2004006898A3 (fr) * 2002-07-11 2004-02-17 Sema Aps Utilisation de composes pouvant inhiber le traitement proteolytique de semaphorines pour la prevention, le traitement, le diagnostic et le pronostic d'une maladie invasive

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