WO2001060999A1 - Sequences d'acides nucleiques caracteristiques de cellules souches hematopoietiques - Google Patents
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- WO2001060999A1 WO2001060999A1 PCT/US2001/004700 US0104700W WO0160999A1 WO 2001060999 A1 WO2001060999 A1 WO 2001060999A1 US 0104700 W US0104700 W US 0104700W WO 0160999 A1 WO0160999 A1 WO 0160999A1
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Classifications
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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
Definitions
- hematopoiesis There are various types of blood cells, such as red blood cells (erythrocytes), white blood cells (e.g., neutrophils, basophils, and eosinophils and monocytes), lymphocytes, and platelets.
- Red blood cells are involved in respiratory gas (oxygen and carbon dioxide) transport.
- White blood cells function to protect the body from infectious diseases and to provide immunity to certain diseases.
- Monocytes are involved in phagocytosis of pathogens and can develop into macrophages in tissues. Lymphocytes can mount immune responses by direct cell attack or by producing antibodies. Platelets are instrumental in blood clotting and can seal small tears in blood vessels; (Bondurant, M.C.
- Early stage hematopoiesis involves the infrequent division of early stem cells to generate more stem cells, i.e. self-renewal.
- the stem cells differentiate into progenitor cells that are irreversibly committed to produce only one or a few lineages of blood cells.
- the progenitor cells progressively proliferate and develop into terminally-differentiated and mature blood cells of a single lineage.
- hematopoiesis appears to start in blood islands found in the fetal yolk sac during the first trimester of pregnancy. At about six weeks of gestation, hematopoiesis occurs predominantly in the fetal liver. In the beginning of the midtrimester, the bone marrow becomes the main site for hematopoiesis.
- Hematopoietic stem cells are operationally defined as cells capable of self-renewal, and of differentiating into all types of mature blood cells. As a result, hematopoietic stem cells are capable of providing long-term hematopoietic reconstitution of ablated animals, including the repopulation of all myeloid and lymphoid cell lines.
- Hematopoietic stem cells are morphologically small to medium mononuclear (lymphocyte-like) cells. They have a large nuclear/cytoplasmic ratio, with prominent nucleoli and non-basophilic and agranular cytoplasm.
- stem cells As stem cells reach the intermediate stage of hematopoiesis, they have less capacity for self-renewal and greater capacity for differentiation. Eventually, they become committed to develop into mature blood cells of a single lineage.
- the late stage of hematopoiesis involves the formation of mature blood cells.
- the mature blood cells are not mitotic, and do not self-renew or differentiate. Each particular type of mature blood cell is ultimately committed, terminally differentiated, and limited to a single lineage.
- hematopoietic stem cells In order to function properly, hematopoietic stem cells must be in intimate contact with stromal cells (Dexter, T.M., L.H., Spooncer, E., Heyworth, CM., Daniel, C.P., Schiro, R., Chang, J., and Allen, T.D. 1990. "Stromal Cells in Hematopoiesis” in: Bock, G., Marsh, J. (eds), Symposium on Molecular Control of Haemopoiesis held at Ciba Foundation, London, John Wiley & Sons Ltd., Chichester, p 76-95). Stromal cells are produced in various organs where stem cells exist.
- T-GPCRs seven-transmembrane G-protein coupled receptors. Over 1000 members of this family of receptors have been identified and some have served as targets for developing therapeutic agents that block or enhance their function. Members of this receptor family are characterized by an extracellular N- terminus, seven membrane-spanning domain and a cytoplasmic C-terminus.
- 7TM-GPCRs The primary function of 7TM-GPCRs is to identify a specific signaling molecule or ligand from a large array of chemically diverse extracellular substances. Once identified, these cell-surfaced receptors can activate an effector-signaling cascade that triggers an intracellular response and eventually a biological effect.
- 7TM-GPCRs undergo a conformational change upon binding with a ligand.
- the conformational change allows the receptors to associate with, and activate, heterotrimeric G- proteins.
- the G-proteins bind guanine and act to modulate intracellular signal pathways by interacting with a variety of effector molecules.
- the signals transduced lead to the regulation of important biological processes such as cell growth and differentiation. Disregulated cellular signaling through 7TM-GPCRs can contribute to human disease.
- 7TM-GCPRs are involved in cellular signaling. Increased knowledge of 7TM-GPCRs would lead to a better understanding of cellular signaling. Cellular signaling leads to the regulation of important biological processes such as growth and differentiation. Additional information on the regulation of biological processes such as growth and differentiation would be very beneficial in developing treatments for various human diseases.
- nucleic acid molecule comprising a nucleic acid sequence selected from the group consisting of SEQ ID NO: 1 to SEQ ID NO: 387.
- the invention further includes vectors and host cells comprising such nucleic acid molecules.
- hematopoietic stem cells are cells that are capable of differentiating at least into erythrocytes, neutrophils, basophils, eosinophils, monocytes, lymphocytes, and platelets.
- Mature hematopoietic cells are progenitor cells that are committed to a particular type of hematopoietic cell or to fully differentiated hematopoietic cells.
- Nucleic acid molecules include both DNA and RNA.
- a nucleic acid molecule is isolated if it is removed from a cell, such as, for example, the cell in which the nucleic acid molecule is naturally found, and at least some of the other nucleic acid molecules that are found in the cell.
- the nucleic acid molecule is purified.
- a nucleic acid molecule of the invention is considered purified if it is substantially free from other nucleic acid molecules that are found naturally in the same cell as the nucleic acid molecule of the invention.
- a nucleic acid molecule of the invention is considered substantially free from other nucleic acid molecules if the nucleic acid molecule of the invention constitutes at least about 50%, preferably at least about 60%, more preferably at least about 70%, most preferably at least about 80%, and optimally at least about 90%, or even higher, such as about 95%, about 98%, or even about 99% of the total weight of a mixture of nucleic acid molecules.
- the nucleic acid molecule may consist of the nucleotides shown in a particular sequence, i.e., not contain any nucleotides other than those shown in SEQ ID NO: 1-387.
- the nucleic acid molecule may comprise the nucleotides shown in a particular sequence, i.e., include the nucleotides shown in SEQ ID NO: 1-387 as well as nucleotides other than those shown in the SEQ ID NO: 1-387 at either, or both, the 5' or the 3' end of the nucleic acid molecule.
- Such other nucleotides may include, for example, regulatory elements, such as transcription factors, translation factors, polyadenylation signals, transcription termination sequences, translation termination sequences, transport signals, promoters, transcription enhancers, activation sequences, and the like.
- the nucleic acid molecule may be incorporated into a vector.
- a vector is a nucleic acid molecule that comprises an isolated nucleic acid molecule according to the invention, and that is useful for transfecting cells.
- the vector may be linear or circular, i.e. a plasmid.
- Some examples of vectors include cloning vectors and expression vectors.
- the invention also includes the nucleic acid molecule shown in SEQ ID NO: 386.
- This nucleic acid molecule is a full reading frame that encodes a 7TM-GPCR when expressed. (SEQ NO ID: 389)
- the invention includes the nucleic acid molecule shown in SEQ ID NO: 387.
- This nucleic acid molecule represents the full reading frame of SEQ ID NO: 173.
- the reading frame for SEQ ID NO: 387 encodes a 7TM-GPCR when expressed. (SEQ ID NO: 388)
- the invention also includes antibodies generated against the proteins represented by SEQ. ID NO: 388 or SEQ. ID NO: 389.
- the antibodies may be polyclonal or monoclonal.
- Northern analysis of the nucleic acid molecule of SEQ ID NO: 386 reveals wide expression in the tissue of the brain, heart, lung and kidneys, as well as murine thymus and whole bone marrow.
- Northern analysis reveals the nucleic acid molecule of SEQ ID NO: 387 to be expressed in murine bone marrow, thymus and a monoblastic leukemia cell line.
- the nucleic acid of SEQ ID NO. 387 appears to be restricted to hematopoietic tissues. Further studies reveal that the nucleic acid of SEQ ID NO. 387 is expressed in myeloblastic leukemia (Ml) cells.
- Ml myeloblastic leukemia
- the invention further includes host cells comprising a nucleic acid molecule according to the invention.
- the nucleic acid molecule may be in a vector that has been transfected into the host cell, such as a cloning vector or an expression vector.
- the nucleic acid molecule may be incorporated into the genome of the host cell.
- the host cell preferably replicates or expresses the nucleic acid molecule, and more preferably both replicates and expresses the nucleic acid molecule.
- the host cells may be used to express the polypeptides and proteins encoded by the nucleic acid molecules of the invention. Further, antibodies may be raised against epitopes of the expression products.
- the nucleic acid molecules of the invention include homologs of the nucleic acid sequences provided in this application.
- the sequence of a first nucleotide sequence is considered homologous to that of a second nucleotide sequence if the first sequence is at least about 60% identical, preferably at least about 70% identical, and more preferably at least about 75% identical to the second nucleotide sequence.
- the first sequence is at least about 80%, preferably at least about 85%, more preferably at least about 95%, and optimally at least about 98% or 99% identical to the second nucleotide sequence.
- sequences are aligned so as to maximize the number of identical nucleotides.
- the sequences of homologous nucleic acid molecules can usually be aligned by visual inspection. If visual inspection is insufficient, the nucleic acid molecules may be aligned in accordance with the methods described by George, D.G. et al., in Macromolecular Sequencing and Synthesis, Selected Methods and Applications, pages 127-149, Alan R. Liss, Inc. (1988), such as formula 4 at page 137 using a match score of 1, a mismatch score of 0, and a gap penalty of -1.
- An alternative test for homology of two nucleic acid sequences is whether they hybridize under normal hybridization conditions, preferably under stringent hybridization conditions.
- stringent conditions is equivalent to “high stringent conditions” and “high stringency.”
- High stringent conditions are defined in a number of ways. In one definition, stringent conditions are selected to be about 25°C lower than the thermal melting point (T m ) for DNA or RNA hybrids longer than 70 bases, and 5°C lower than the T m for shorter oligonucleotides (11-70 bases long).
- T m is the temperature (under defined ionic strength and pH) at which 50% of the target sequence hybridizes to a perfectly matched sequence.
- Typical stringent conditions are those in which the salt concentration is about 0.02 M at pH 7.0 and the temperature is calculated as described below.
- T m 81.5°C + 16.6 log [M + ] + 41 (%G + C) - 0.63(% formamide) - (600/Z).
- T m 79.8°C + 18.5 log[M + ] + 58.4(%G + C) + 11.8(%G + C) 2 - 0.5(% formamide) - 820/X.
- T m 81.5°C + 16.6 log [M + ] + 41 (%G + C) - 600/1.
- T m 4(%G + C) + 2(%A + TAJ).
- T m thermal melting temperature
- %G+C percentage of total guanine and cytosine bases in the DNA, usually
- [M + ] monovalent cation concentration, usually sodium, expressed in molarity in the range of 0.01 M to 0.4 M;
- L length of the hybrid in base pairs
- %A+T mole fraction of total adenine and thymine bases in the DNA.
- %A+T/U mole fraction of total adenine and thymine or uracil bases in the DNA or
- stringent conditions useful in the present invention include overnight incubation at a hybrid temperature determined as described above in a solution comprising: 20% formamide, 5 x SSC (750 mM NaCl, 75 mM trisodium citrate), 50 mM sodium phosphate (pH 7.6), 5 x Denhardt's solution, 10% dextran sulfate, and 20 ⁇ g/ml denatured, sheared salmon sperm DNA.
- the stringent conditions are characterized by a hybridization buffer comprising 30% formamide in 5 x SSPE (0.75 M NaCl, 0.05 M NaPO 4 , pH 7.7, 5 mM EDTA) buffer at a temperature determined as described above and subsequent washing at the same temperature with 0.2 x SSPE.
- stringent conditions involve the use of a hybridization buffer comprising 50% formamide in 5 x SSPE at a temperature determined as described above and washing at the same temperature with 0.2 x SSPE (0.03 M NaCl, 2 mM NaPO 4 , pH 7.7, 0.2 mM EDTA).
- SEQ ID NOS: 1-387 were derived from murine hematopoietic stem cells (SEQ ID NO: 1- 248), or stromal cells (SEQ ID NO: 249-387).
- the nucleic acid molecules of the invention further include homologous sequences found in humans in accordance with the definitions of homology described above.
- the human sequences are derived from the same types of cells as the corresponding murine sequences, share homology, generally high homology, with the corresponding murine sequences, and are useful in the same ways as the corresponding murine sequences.
- Positive human cDNA clones may be isolated and sequence analysis of the clones may be performed by methods known in the art. Sequence comparisons may be carried out by screening several databases, e.g., dbEST, GenBank, Swiss-Prot, and EMBL.
- the human nucleic acid molecules screened by the methods described above may be from human cDNA or genomic libraries, preferably derived from hematopoietic cells or cell lineages.
- Particularly useful cells for this purpose include hematopoietic cells that are CD34+, CD38-, lin-, or any combination thereof.
- Such cells may be enriched for hematopoietic stem cells from bone marrow cells by standard methods well known in the art by which nucleated non-adherent cells are prepared to provide a rich source of hematopoietic stem cells.
- Stromal cells may also be prepared from bone marrow cells by a similar process, this time selecting adherent cells. Larger numbers of stromal cells may also be prepared from umbilical cord blood. Another commonly used and readily available source of human cDNAs is the commercially available human liver cDNA libraries.
- nucleic acid molecules of the invention are useful in numerous ways. For example, since hematopoietic stem cells selectively express the nucleic acid molecules of the invention, assays that are capable of determining the presence of the nucleic acid molecules in a sample are capable of distinguishing such cells from most, if not all, other types of cells, such as mature hematopoietic cells and non-hematopoietic cells.
- a nucleic acid molecule of the invention, or its complement is expressed in a cell, the cell is considered to have a high likelihood of being a hematopoietic stem cell.
- Methods for determining whether a nucleic acid molecule is expressed in a cell are known in the art. For example, high molecular weight DNA from a cell can be restricted with restriction enzymes and fractionated by agarose gel electrophoresis. The restricted fragments are denatured, transferred to a nitrocellulose filter, and immobilized (Southern transfer). A labeled nucleic acid molecule of the invention or its complement is prepared and used as a probe. The presence of the labeled probe hybridized to an immobilized nucleic acid molecule indicates the presence of the nucleic acid molecule in the sample.
- the labeled probe is applied directly to fixed, denatured, and dehydrated hematopoietic cells to localize the cellular transcripts in the cells and to identity the cell types that transcribe the mRNA of interest (in situ hybridization). Examples of these methods are described by Ausubel, F.M. et al. (eds), Current Protocols in Molecular Biology, John Wiley & Sons, Inc., New York (1999).
- Nucleic acid molecules of the invention can be labeled by methods known in the art.
- the label may be a radioactive atom, an enzyme, or a chromophoric moiety.
- the label may be radioactive.
- Some examples of useful radioactive labels include P, 125 1, 131 1, 35 S, 14 C, and 3 H. Uses of radioactive labels have been described in U.K. 2,034,323, U.S. 4,358,535, and U.S. 4,302,204.
- the label may be non-radioactive.
- non-radioactive labels include enzymes, chromophores, atoms and molecules detectable by electron microscopy, and metals detectable by their magnetic properties.
- Some useful enzymatic labels include enzymes that cause a detectable change in a substrate. These useful enzymes and their substrates include, for example, horseradish peroxidase (pyrogallol and o-phenylenediamine), beta-galactosidase (fluorescein beta-D- galactopyranoside), and alkaline phosphatase (5-bromo-4-chloro-3-indolyl phosphate/nitro blue tetrazohum).
- horseradish peroxidase pyrogallol and o-phenylenediamine
- beta-galactosidase fluorescein beta-D- galactopyranoside
- alkaline phosphatase 5-bromo-4-chloro-3-indolyl phosphate/nitro blue tetrazohum.
- the use of enzymatic labels have been described in U.K. 2,019,404, EP 63,879, in Ausubel
- Useful chromophores include, for example, fluorescent, chemiluminescent, and bioluminescent molecules, as well as dyes.
- Some specific chromophores useful in the present invention include, for example, fluorescein, rhodamine, Texas red, phycoerythrin, umbelliferone, luminol.
- the labels may be conjugated to the nucleotide probe by methods that are well known in the art.
- the labels may be directly attached through a functional group on the probe.
- the probe either contains or can be caused to contain such a functional group.
- suitable functional groups include, for example, amino, carboxyl, sulfhydryl, maleimide, isocyanate, isothiocyanate.
- nucleic acid molecules of the invention are also useful in ways other than those described above.
- the nucleic acid molecules are useful in identifying nucleic acid molecules that comprise one or more of the sequences in SEQ. ID NO 1 to SEQ. ID NO 387 and additional nucleotides at the 5' end, the 3' end, or both the 5' and a 3' ends.
- Such longer nucleic acid molecules express proteins that are involved in hematopoiesis. Such proteins are important to improve the understanding of hematopoiesis, and may be important in treating conditions characterized by abnormal hematopoiesis.
- PCR polymerase chain reaction
- the RACE (rapid amplification of cDNA ends) method for 5'-end extension is particularly useful.
- RACE rapid amplification of cDNA ends
- PCR Protocols A Guide to methods and Applications (Innes, M.A. ed.) Academic Press, San Diego, pp28-38 (1990).
- primers oriented in the 5' and 3' directions are chosen to produce overlapping cDNAs when fully extended.
- the overlapping 5' and 3' end RACE products are ligated to produce longer nucleic acid molecules, such as full length cDNAs and genes.
- Frohman, M.A. Rapid Amplification of cDNA Ends
- nucleic acid molecules of the invention are used as primers for PCR amplification.
- the oligonucleotide primers may be synthesized by methods known in the art. Suitable methods include those described by Caruthers in Science 230:281-285 (1985) and DNA Structure, Part A: Synthesis and Physical Analysis of DNA, Lilley, D.M.J. and Dahlberg, J.E. (eds), Methods Enzymol., 211, Academic Press, Inc., New York (1992).
- the amplified fragment may be cloned, sequenced and further amplified to obtain longer nucleic acid molecules that comprise one or more of the sequences in SEQ. ID NO: 1 to SEQ. ID NO: 385.
- the labeled murine probes can be used by various methods known in the art to screen human hematopoietic tissues or cells. Such screening can, for example, show where and how the human gene is being expressed and transcribed (northern blot analysis). See Sambrook, J. et al. (eds), Molecular Cloning, Second Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York (1989) and Ausubel, F.M. et al. (eds), Current Protocols in Molecular Biology, John Wiley & Sons, New York (1999).
- the murine nucleic acid molecules of the invention may also be used as probes and used to isolate the corresponding human homolog by methods known in the art.
- the murine nucleic acid molecules may be used as PCR primers to screen a human DNA (cDNA or genomic) library.
- the PCR methods described above, such as RACE, are suitable for this purpose. See, for example, Zeng et al., Biochem. Biophys. Res. Comm., 236, 389-395 (1997), and Morita et al., Biochem. Biophys. Res. Comm., 248, 307-314 (1998). See also Buanne et al., Genomics 51, 233-242 (1998).
- the nucleic acid molecules may be immobilized and used to capture human nucleic acid molecules having homology, preferably high homology, as described above from an appropriate human nucleic acid library.
- Methods for using nucleic acid molecules to immobilize and capture homologous nucleic acid molecules are known in the art. The conditions used in the capture procedure are highly stringent, as described above.
- nucleic acid molecules of the invention Another utility of the nucleic acid molecules of the invention is the mapping of DNA sequences specific to hematopoietic stem cells to chromosomes.
- the sequences may be mapped to a particular chromosome, or to a specific region of the chromosome by techniques known in the art.
- An example of a useful technique is fluorescent in situ hybridization (FISH) analysis.
- FISH fluorescent in situ hybridization
- the DNA of SEQ. ID NOS: 386 and 387 and the proteins corresponding to them in SEQ. ID NOS: 388 and 389 may be used to identify the types of cells in which they are expressed. These sequences can also be used to investigate cellular signaling between 7TM-GPCRs. Furthermore, the sequences may be helpful in the identification of new 7TM-GPCRs. Additionally, the DNA of SEQ ID NOS. 386 and 387 encode for 7TM-GCPRs. 7TM- GPCRs are expressed on stem cells. Therefore, the DNA of SEQ ID NOS. 386 and 387 could serve as markers that can be used to sort stem cells. Sorted stem cells may be therapeutically useful, for example, in transplantations.
- the 7TM-GCPRs corresponding to the DNA of SEQ ID NOS. 386 and 387 are expressed in myeloblastic leukemia (Ml) cells, and play a role in leukemia.
- Ml myeloblastic leukemia
- the development of inhibitors to the 7TM-GPCRs that correspond to the DNA of SEQ ID NOS. 386 and 387 provide a promising treatment to inhibit leukemia progression. This inhibition can occur via growth or differentiation inhibition.
- the DNA of SEQ ID NOS. 386 and 387 and the proteins corresponding to them in SEQ. ID. NOS 388 and 389 may be used in the development of inhibitors of leukemic progression.
- the 7TM-GPCR corresponding to SEQ ID NOS. 386-389 is expressed in leukemia cells, particularly, myeloblastic leukemia (Ml) cells. Therefore, development of inhibitors to the 7TM-GPCR will also inhibit leukemic progression.
- the inhibition of leukemic cells can occur via agonists or antagonists of the 7TM-GPCR that may induce differentiation as an anti-leukemia therapy.
- the present invention provides antisense or sense oligonucleotides capable of binding to a target mRNA or to the sequence in the double stranded DNA helix of SEQ ID NOS. 386 and 387.
- Antisense or sense oligomers according to the present invention comprise a fragment of the coding region of the cDNA of SEQ ID NOS. 386 and 387. Such a fragment generally comprises at least 14 nucleotides, preferably about 14 to 30 nucleotides.
- Stein and Cohen Cancer Res. 48:2659, 1988 and van der Krol,et al., BioTechniques 6:958, 1988.
- binding of antisense or sense oligonucleotides to target nucleic acid sequences results in the formation of complexes that block translation (RNA) or transcription(DNA) by one of several means, including enhanced degradation of the duplexes, premature termination of transcription or translation, or by other means.
- the antisense oligonucleotides thus may be used to block expression of the proteins of SEQ ID NOS. 388 and 389 which correspond to the nucleic acids of SEQ ID NOS. 386 and 387.
- the antisense or sense oligonucleotides of the present invention further comprise oligonucleotides having modified sugar-phosphodiester backbones or other sugar linkages, which resist enzymatic degradation but retain sequence specificity to be able to target nucleotide sequences.
- Antisense or sense oligonucleotides of the present invention may be introduced into a cell containing the target nucleic acid sequence by any gene transfer method, including for example, CaPO 4 -medited DNA transfection, electroporation, or by using gene transfer vectors.
- Antisense or sense oligonucleotides of the present invention also may be introduced into a cell containing the target nucleotide sequence by formation of a conjugate with a ligand binding molecule, for example, cell surface receptors, growth factors, other cytokines or ligands that bind cell surface receptors.
- Isolated and purified DNA of SEQ ID NOS. 386 and 387, or fragments thereof, may also be useful themselves as therapeutic agents in regulating the corresponding proteins of SEQ ID NOS. 388 and 389, which belong to the family of 7TM-GCPRs.
- the present invention provides antibodies and/or functional equivalents of antibodies raised against the proteins represented by the amino aid sequences of SEQ. ID NO: 388 and SEQ. ID NO: 389.
- An antibody is defined as a protein that binds specifically to an epitope.
- the antibody may be polyclonal or monoclonal.
- the invention further includes isolating neutralizing antibodies that specifically recognize and bind to the proteins and functional analogs of the invention.
- an antibody is preferably a chimerized or humanized antibody.
- a chimerized antibody comprises the variable region of a non-human antibody and the constant region of a human antibody.
- a humanized antibody comprises the hypervariable region (CDRs) of a non-human antibody.
- the variable region other than the hypervariable region, e.g. the framework variable region, and the constant region of a humanized antibody are those of a human antibody.
- Suitable variable and hypervariable regions of non-human antibodies may be derived from antibodies produced by any non-human mammal in which monoclonal antibodies are made. Suitable examples of mammals other than humans include, for example, rabbits, rats, mice, horses, goats, or primates. Preferably, the antibodies are human antibodies. The antibodies may be produced in a transgenic mouse. An example of such a mouse is the so-called XenoMouseTM (Abgenix, Freemont, CA) described by Green, LL., "Antibody Engineering Via Genetic Engineering of the Mouse: XenoMouse Stains are a Vehicle for the Facile Generation of Therapeutic Human Monoclonal Antibodies," J. Immunol. Methods," 10;231(1-2):11-23(1999).
- XenoMouseTM Abgenix, Freemont, CA
- Functional equivalents of antibodies further include fragments that have binding characteristics that are the same as, or are comparable to, those of the whole antibody.
- Suitable fragments of the antibody include any fragment that comprises a sufficient portion of the hypervariable (i.e. complementary determining) region to bind specifically, and with sufficient affinity, to 7TM-GPCR.
- the preferred fragments are single chain antibodies.
- Single chain antibodies are polypeptides that comprise at least the variable region of the heavy chain of the antibody and the variable region of the light chain, with or without an interconnecting linker.
- the antibodies and functional equivalents may be members of any class of immunoglobins, such as: IgG, IgM, IgA, IgD or IgE, and the subclass thereof.
- the functional equivalents may also be equivalents of combinations of any of the above classes and subclasses.
- Methods for making monoclonal antibodies include, for example, the immunological method described by Kohler and Milstein in Nature 256:495-497 (1975) and by Camplbell in "Monoclonal Antibody Technology, The Production and Characterization of Rodent and Human Hybridomas" in Burdon, et al., Eds, Laboratory Techniques in Biochemistry and Molecular Biology, Volume 13, Elsevier Science Publishers, Amsterdam (1985).
- the recombinant DNA method described by Huse, et al. in Science 246:1275-1281 (1989) is also suitable.
- a host mammal inoculated with a receptor or a fragment of a receptor, as described above, and t hen, optionally, boosted.
- the receptor fragment must contain sufficient amino acid residues to define the epitope of the molecule being detected. If the fragment is too short to be immunogenic, it may be conjugated to a carrier molecule.
- suitable carrier molecules include keyhold limpet hemocyanin and bovine serum albumin. Conjugation may be carried out by methods known in the art. One such method is to combine a cysteine residue of the fragment with a cysteine residue on the carrier molecule.
- Spleens are collected from the inoculated mammals a few days after the final boost. Cell suspensions from the spleen are fused with a tumor cell. The resulting hybridoma cells that express the antibodies are isolated, grown and maintained in culture.
- Suitable monoclonal antibodies as well as growth factor receptor tyrosine kinases for making them are also available from commercial sources, for example, from Upstate Biotechnology, Santa Cruz Biotechnology of Santa Cruz, California, Transduction Laboratories of Lexington, Kentucky, R&D Systems Inc of Minneapolis, Minnesota, and Dako Corporation of Carpinteria, California.
- Methods for making chimeric and humanized antibodies are also known in the art.
- methods for making chimeric antibodies include those described in U.S. patents by Boss (Celltech) and by Cabilly (Genentech). See U.S. Patent Nos. 4,816,397 and 4,816,567, respectively.
- Methods for making humanized antibodies are described, for example, in Winter, U.S. Patent No. 5,225,539.
- Antibodies or antibody fragments can also be isolated from antibody phage libraries generated using techniques, for example, described in McCafferty et al., Nature, 348: 552-554 (1990), using the antigen of interest to select for a suitable antibody or antibody fragment.
- Clackson et al., Nature, 352: 624-628 (1991) and Marks et al., J. Mol. Biol., 222: 581-597 (1991) describe the isolation of murine and human antibodies, respectively, using phage libraries.
- Subsequent publications describe the production of high affinity (nM range) human antibodies by chain shuffling (Mark et al., Bio/Technol.
- CDR-grafting The preferred method for the humanization of antibodies is called CDR-grafting.
- CDR-grafting the regions of the mouse antibody that are directly involved in binding to antigen, the complementarity determining region or CDRs, are grafted into human variable regions to create "reshaped human” variable regions. These fully humanized variable regions are then joined to human constant regions to create complete "fully humanized” antibodies.
- the human variable regions into which the CDRs will be grafted should be carefully selected, and it is usually necessary to make a few amino acid changes at critical positions within the framework regions (FRs) of the human variable regions.
- the reshaped human variable regions may include up to ten amino acid changes in the FRs of the selected human light chain variable region, and as many as twelve amino acid changes in the FRs of the selected human heavy chain variable region.
- the DNA sequences coding for these reshaped human heavy and light chain variable region genes are joined to DNA sequences coding for the human heavy and light chain constant region genes, preferably ⁇ l and K, respectively.
- the reshaped humanized antibody is then expressed in mammalian cells and its affinity for its target compared with that of the corresponding murine antibody and chimeric antibody.
- Methods for making single chain antibodies are also known in the art. Such methods include screening phage libraries transfected with immunoglobulin genes described in U.S. Patent 5,565,332; U.S. Patent 5,5837,242; U.S. Patent 5,855,885; U.S. Patent 5,885,793; and U.S. Patent 5,969,108. Another method includes the use of a computer-based system for designing linker peptides for converting two separate polypeptide chains into a single chain antibody described in U.S. Patent 4,946,778; U.S. Patent 5,260,203; U.S. Patent 5,455,030; and U.S. Patent 5,518,889.
- the antibodies that bind specifically to the proteins comprising the amino acid sequences of SEQ ID NOS: 388 and 389 can be used to detect the presence of said proteins.
- Assays for detecting the presence of proteins with antibodies can be performed using known formats such as standard blot and ELISA formats.
- the antibodies that bind specifically to the protein comprising the amino acid sequence of SEQ ID NO. 388 can be used to identify murine bone marrow and thymus cells as well as cells of the monoblastic leukemia cell line in which it is expressed.
- the antibodies corresponding to the protein comprising the amino acid sequence of SEQ ID NO. 389 can be used to identify brain, heart, lung and kidney cells.
- the antibodies of the invention may be used therapeutically as cytotoxic agents against, for example, leukemia cells. Antibodies with proper biological properties are useful directly as therapeutic agents. See, for example, U.S. Patent No. 5,134,075. Alternatively, the antibodies can be bound to a toxin to form an immunotoxin or to a radioactive material or drug to form a radiopharmaceutical or pharmaceutical. Methods for producing immunotoxins and radiopharmaceuticals of antibodies are well-known. See, for example, Cancer Treatment Reports (9184) 68: 317-328.
- AA4+Scal+c+kit+Lin-/lo (hematopoietic stem cell-enriched) and AA4- (hematopoietic stem cell depleted) cells were isolated by fluorescence activated cell sorting from the fetal liver of C57BL/6j-Ly-5.2 mice on day 14 of gestation as follows: quantitative enrichment of stem cell fractions was performed according to described protocols. Fetal livers were dissected from day 14 embryos in a sterile environment. Single cell suspensions were subjected to immunopanning with AA4.1 antibody.
- the AA4.1 positive fraction was then stained with fluorescein isothiocyanate (FITC)-labeled rat anti-CD3, CD4, CD5, CD8, CD45R, Gr-1 and TER-119 antibodies (called the "lin” set of antibodies).
- FITC fluorescein isothiocyanate
- Cells were also stained with phycoerythrin (PE)- conjugated anti-Sca-1 (Ly-6A/E) and allophycocyanin (APCO-conjugated anti-c-kit. All antibodies were obtained from Pharmingen (San Deigo, CA). Stained cells were separated on a dual laser EPICS 753 cell sorter (Beckman Coulter, San Diego, CA) by selection of high PE and APC as well as low FITC fluorescence. Cells used for cDNA library production were tested for their stem cell potential in competitive repopulation assays that test the ability of stem cells to reconstitute the radioblated hematopoietic system of recipient mice.
- This transplantation assay was performed essentially by methods known in the art.
- specified numbers of cells 25, 50, 100 or 250
- C57Bl/6-Ly-5.1 whole bone marrow cells were mixed with 400,000 C57Bl/6-Ly-5.1 whole bone marrow cells and injected into the retro-orbital sinus of each irradiated (10 Gy) mouse.
- the level of engraftment was measured at different timepoints as the percent C57bl/6J-Ly-5.2 (donor) cells in the peripheral blood of transplanted Ly-5.1 animals. Animals demonstrating engraftment at least two standard deviations higher than the mean of negative controls were considered positive.
- Sorted cells were placed directly into Trizol (BRL-Gibco) reagent and treated with RNAse-free DNAse I according to the manufacturers protocols.
- the polyA-plus fraction was purified on oligo-dT cellulose (New England Biolabs).
- the mRNA was denatured using methyl-mercury hydroxide.
- the first and second strand cDNA was synthesized using the reverse transcriptase Superscript II, DNA polymerasel, RNAseH and E.coli ligase.
- the first-strand primer was oligo-dT with a 5' Notl site.
- Double-stranded cDNA was blunt-ended with T4-DNA polymerase and ligated to a Sail adapter with T4-DNA ligase.
- the ligated cDNA was digested with Notl and size fractionated on columns. Size fractions 1 kilobase pair (kbp.) and greater in length were pooled and ligated in to the Sall-Notl sites of either pSport- 1 (stem cell library) or pSport-2 (mature cell library).
- the ligations were electroporated into E.coli strain DH12S, titered and amplified on agar plates. Except where indicated, all of the reagents and protocols for the construction of the cDNA were from BRL-Gibco.
- the stem cell library was converted into single stranded molecules by in vivo infection with helper phage according to BRL-Gibco protocols.
- Cesium gradient banded DNA from the mature cell library was linearized with Sail and used as template for the synthesis of biotinylated RNA using T7-polymerase.
- the use of the two different cloning plasmids insures that the RNA is complementary to the single-stranded cDNA inserts in the stem cell library.
- Hybridization conditions and the post-hybridization processing of the reactions were essentially as described in the BRL-Gibco protocols.
- the resultant subtracted single-stranded DNA populations were repaired to a double-stranded from and amplified in E.coli DH12S.
- the DNA populations representing the subtracted libraries were purified on Cesium gradients.
- the library DNAs were linearized with Notl and subjected to four successive agarose gel electrophoresis fractionations. After each electrophoresis gel the DNA smear corresponding in size to vector plus 1 kbp and greater was excised and eluted using the sodium iodide/glass bead procedure. After four such fractionations essentially all empty plasmids were eliminated.
- the resultant DNA population was introduced into E.coli DH12S and individual clones were robotically picked into 384-well plates. A separate aliquot of the library was amplified as a population and used to prepare DNA. The subtraction efficiencies were verified by monitoring the clone number reduction as a function of hybridization with biotinylated RNA in comparison with mock hybridizations lacking RNA. Generally, this was on the order of 100-fold. A more direct measure of subtraction efficiency was obtained by hybridizing pre and post-subtraction cDNA Southern blots with probes such as beta-actin, CD34 and flk-2. The DNA populations on the blots represent approximately equal numbers of individual clones.
- the cDNAs corresponding to the CD34 and flk-2 molecules are enriched or at least retained in the subtracted libraries. Special care was taken to carefully monitor the minimal number of PCR cycles necessary to produce the required amount of amplified product. This was done by performing pilot reactions using 2 cycle increments.
- the amplified material representing different numbers of PCR cycles were analyzed in triplicate Southern blots that were hybridized with probes representative of different mRNA size classes. In various experiments these included GAPDH, beta-actin, flk-2, CD34 and ckit. Cycle numbers where discrete full-length cDNA products were observed with little or no detectable lower molecular weight material were determined and, in general, the preparative amplifications employed 1 fewer cycles.
- the amplified cDNA material was used to generate libraries as described above. Alternatively, the cDNA was used for PCR-Select subtractions. These methods were performed by methods well known in the art. DNA Sequencing
- DNA sequences and conceptual translations were compared with known nucleotide and protein sequences using the BLAST algorithm (blastn for nucleotide and blastx for protein databases).
- BLAST algorithm blastn for nucleotide and blastx for protein databases.
- Six publicly-accessible databases were searched: SwissProt, Genbank nr protein, Genbank nr nucleotide, dbEST expressed sequence tags, and the murine and human DOTS databases of EST contigs. Sequences were also compared with those in SCBD itself as a measure of internal redundancy.
- Potential open reading frames (ORFs) were located using ORF finder (NCBI, Bethesda, MD). Protein motif searches were performed using five different motif identification programs: Prosite (PBIL, France), Pfam (Washington University, St.
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Cited By (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2002002602A3 (fr) * | 2000-06-30 | 2002-08-22 | Millennium Pharm Inc | Nouvelle proteine slgp et nouvelles molecules d'acide nucleique et leurs utilisations |
US7510845B2 (en) | 2000-05-04 | 2009-03-31 | California Institute Of Technology | Assay employing G protein-coupled receptor expressed in dorsal root ganglia |
US7628989B2 (en) | 2001-04-10 | 2009-12-08 | Agensys, Inc. | Methods of inducing an immune response |
US8916355B2 (en) | 2000-05-04 | 2014-12-23 | California Institute Of Technology | Pain signaling molecules |
-
2001
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- 2001-02-14 WO PCT/US2001/004700 patent/WO2001060999A1/fr active Application Filing
Non-Patent Citations (4)
Title |
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LELIAS J. ET AL.: "cDNA cloning of a human mRNA preferentially expressed in hematopoietic cells and with homology to a GDP-dissociation inhibitor of the rho GTP-binding proteins", PROC. NATL. ACAD. USA, vol. 90, February 1993 (1993-02-01), pages 1479 - 1483, XP002942874 * |
MIRAGLIA S. ET AL.: "A Novel Five-Transmembrane Hematopoietic Stem Cell Antigen: Isolation, Characterization, and Molecular Cloning", BLOOD, vol. 90, no. 12, 15 December 1997 (1997-12-15), pages 5013 - 5021, XP002942875 * |
MOORE K.A. ET AL.: "Hematopoietic activity of a stromel cell transmembrane protein containing epidermal growth factor-like repeat motifs", PROC. NATL. ACAD. SCI. USA, vol. 94, April 1997 (1997-04-01), pages 4011 - 4016, XP002942876 * |
PHILLIPS R.L. ET AL.: "The Genetic Program of Hematopoietic Stem Cells", SCIENCE, vol. 288, 2 June 2000 (2000-06-02), pages 1635 - 1640, XP002942873 * |
Cited By (6)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US7510845B2 (en) | 2000-05-04 | 2009-03-31 | California Institute Of Technology | Assay employing G protein-coupled receptor expressed in dorsal root ganglia |
US8916355B2 (en) | 2000-05-04 | 2014-12-23 | California Institute Of Technology | Pain signaling molecules |
WO2002002602A3 (fr) * | 2000-06-30 | 2002-08-22 | Millennium Pharm Inc | Nouvelle proteine slgp et nouvelles molecules d'acide nucleique et leurs utilisations |
US7628989B2 (en) | 2001-04-10 | 2009-12-08 | Agensys, Inc. | Methods of inducing an immune response |
US7641905B2 (en) | 2001-04-10 | 2010-01-05 | Agensys, Inc. | Methods of inducing an immune response |
US7736654B2 (en) | 2001-04-10 | 2010-06-15 | Agensys, Inc. | Nucleic acids and corresponding proteins useful in the detection and treatment of various cancers |
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