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Patent US5843423 - Methods of stimulating hematopoietic cells with flt3-ligand - Google PatentsSearch Images Maps Play YouTube News Gmail Drive More »Sign inPatentsLigands for flt3 receptors capable of transducing self-renewal signals to regulate the growth, proliferation or differentiation of progenitor cells and stem cells are disclosed. The invention is directed to flt3-L as an isolated protein, the DNA encoding the flt3-L, host cells transfected with cDNAs...http://www.google.com/patents/US5843423?utm_source=gb-gplus-sharePatent US5843423 - Methods of stimulating hematopoietic cells with flt3-ligandAdvanced Patent SearchTry the new Google Patents, with machine-classified Google Scholar results, and Japanese and South Korean patents.Publication numberUS5843423 APublication typeGrantApplication numberUS 08/993,962Publication dateDec 1, 1998Filing dateDec 18, 1997Priority dateMay 24, 1993Fee statusPaidAlso published asCA2162397A1, CA2162397C, CN1123574C, CN1125479A, US5554512, US6919206, US20030148516, WO1994028391A1Publication number08993962, 993962, US 5843423 A, US 5843423A, US-A-5843423, US5843423 A, US5843423AInventorsStewart D. Lyman, M. Patricia BeckmannOriginal AssigneeImmunex CorporationExport CitationBiBTeX, EndNote, RefManPatent Citations (15), Non-Patent Citations (15), Referenced by (85), Classifications (57), Legal Events (5) External Links: USPTO, USPTO Assignment, EspacenetMethods of stimulating hematopoietic cells with flt3-ligand
US 5843423 AAbstract
1. A method for stimulating the proliferation of hemapoeitic stem or progenitor cells in a patient in need thereof, comprising administering a hemapoeitic stem or progenitor cell-stimulating amount of a human flt3-L polypeptide, and optionally, a hemapoeitic stem or progenitor cell-stimulating amount of at least one growth factor selected from the group consisting of CSF-1, GM-CSF, SF, G-CSF, EPO, IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, IL-14, IL-15, GM-CSF/IL-3 fusion proteins, LIF and FGF.
This application is a continuation of U.S. application No. 08/444,625 filed May 19, 1995, abandoned, which is a divisional of U.S. application No. 08/243,545, filed May 11, 1994, now U.S. Pat. No. 5,554,512 issued Sep. 6, 1996, which is a continuation-in-part of U.S. application No. 08/209,502, filed Mar. 7, 1994, abandoned, which is a continuation-in-part of U.S. application No. 08/162,407, filed Dec. 3, 1993, abandoned, which is a continuation-in-part of U.S. application No. 08/111,758 filed Aug. 25, 1993, abandoned, which is a continuation-in-part of U.S. application No. 08/106,463, filed Aug. 12, 1993, abandoned, which is a continuation-in-part of U.S. application No. 08/068,394, filed May 24, 1993.
Blood cells originate from hematopoietic stem cells that become committed to differentiate along certain lineages, i.e., erythroid, megakaryocytic, granulocytic, monocytic, and lymphocytic. Cytokines that stimulate the proliferation and maturation of cell precursors are called colony stimulating factors ("CSFs"). Several CSFs are produced by T-lymphocytes, including interleukin-3 ("IL-3"), granulocyte-monocyte CSF (GM-CSF), granulocyte CSF (G-CSF), and monocyte CSF (M-CSF). These CSFs affect both mature cells and stem cells. Heretofore no factors have been discovered that are able to predominantly affect stem cells.
Nucleic acid sequences within the scope of the invention include isolated DNA and RNA sequences that hybridize to the native flt3-L nucleotide sequences disclosed herein under conditions of moderate or severe stringency, and which encode biologically active flt3-L. Conditions of moderate stringency, as defined by Sambrook et al. Molecular Cloning: A Laboratory Manual, 2 ed. Vol. 1, pp. 1.101-104, Cold Spring Harbor Laboratory Press, (1989), include use of a prewashing solution of 5� SSC, 0.5% SDS, 1.0 mM EDTA (pH 8.0) and hybridization conditions of about 55-- C, 5� SSC, overnight. Conditions of severe stringency include higher temperatures of hybridization and washing. The skilled artisan will recognize that the temperature and wash solution salt concentration may be adjusted as necessary according to factors such as the length of the probe.
Alternatively, mixtures of cells suspected of containing flt3+ cells first can be incubated with a biotinylated flt3-binding protein. Incubation periods are typically at least one hour in duration to ensure sufficient binding to flt3. The resulting mixture then is passed through a column packed with avidin-coated beads, whereby the high affinity of biotin for avidin provides the binding of the cell to the beads. Use of avidin-coated beads is known in the art. See Berenson, et al. J. Cell. Biochem., 10D: 239 (1986). Wash of unbound material and the release of the bound cells is performed using conventional methods.
As described above, flt3-L of the invention can be used to separate cells expressing flt3 receptors. In an alternative method, flt3-L or an extracellular domain or a fragment thereof can be conjugated to a detectable moiety such as 125 I to detect flt3 expressing cells. Radiolabeling with 125 I can be performed by any of several standard methodologies that yield a functional 125 I-flt3-L molecule labeled to high specific activity. Or an iodinated or biotinylated antibody against the flt3 region or the Fc region of the molecule could be used. Another detectable moiety such as an enzyme that can catalyze a colorimetric or fluorometric reaction, biotin or avidin may be used. Cells to be tested for flt3 receptor expression can be contacted with labeled flt3-L. After incubation, unbound labeled flt3-L is removed and binding is measured using the detectable moiety.
The binding characteristics of flt3-L (including variants) may also be determined using the conjugated, soluble flt3 receptors (for example, 125 I-flt3:Fc) in competition assays similar to those described above. In this case, however, intact cells expressing flt3 receptors, or soluble flt3 receptors bound to a solid substrate, are used to measure the extent to which a sample containing a putative flt3-L variant competes for binding with a conjugated a soluble flt3 to flt3-L.
Promoter sequences commonly used for recombinant prokaryotic host cell expression vectors include β-lactamase (penicillinase), lactose promoter system (Chang et al., Nature 275: 615, 1978; and Goeddel et al., Nature 281: 544, 1979), tryptophan (trp) promoter system (Goeddel et al., Nucl. Acids Res. 8: 4057, 1980; and EP-A-36776) and tac promoter (Maniatis, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, p. 412, 1982). A particularly useful prokaryotic host cell expression system employs a phage λ PL promoter and a cI857ts thermolabile repressor sequence. Plasmid vectors available from the American Type Culture Collection which incorporate derivatives of the λ PL promoter include plasmid pHUB2 (resident in E. coli strain JMB9 (ATCC 37092)) and pPLc28 (resident in E. coli RR1 (ATCC 53082)).
Flt3-L polypeptides alternatively may be expressed in yeast host cells, preferably from the Saccharomyces genus (e.g., S. cerevisiae). Other genera of yeast, such as Pichia, K. lactis or Kluyveromyces, may also be employed. Yeast vectors will often contain an origin of replication sequence from a 2μ yeast plasmid, an autonomously replicating sequence (ARS), a promoter region, sequences for polyadenylation, sequences for transcription termination, and a selectable marker gene. Suitable promoter sequences for yeast vectors include, among others, promoters for metallothionein, 3-phosphoglycerate kinase (Hitzeman et al., J. Biol. Chem. 255: 2073, 1980) or other glycolytic enzymes (Hess et al., J. Adv. Enzyme Reg. 7: 149, 1968; and Holland et al., Biochem. 17: 4900, 1978), such as enolase, glyceraldehyde-3-phosphate dehydrogenase, hexokinase, pyruvate decarboxylase, phosphofructokinase, glucose-6-phosphate isomerase, 3-phosphoglycerate mutase, pyruvate kinase, triosephosphate isomerase, phosphoglucose isomerase, and glucokinase. Other suitable vectors and promoters for use in yeast expression are further described in Hitzeman, EPA-73,657 or in Fleer et. al., Gene, 107: 285-195 (1991); and van den Berg et. al., Bio/Technology, 8: 135-139 (1990). Another alternative is the glucose-repressible ADH2 promoter described by Russell et al. (J. Biol. Chem. 258: 2674, 1982) and Beier et al. (Nature 300: 724, 1982). Shuttle vectors replicable in both yeast and E. coli may be constructed by inserting DNA sequences from pBR322 for selection and replication in E. coli (Ampr gene and origin of replication) into the above-described yeast vectors.
Yeast transformation protocols are known to those of skill in the art. One such protocol is described by Hinnen et al., Proc. Natl. Acad. Sci. USA 75: 1929, 1978. The Hinnen et al. protocol selects for Trp+ transformants in a selective medium, wherein the selective medium consists of 0.67% yeast nitrogen base, 0.5% casamino acids, 2% glucose, 10 μg/ml adenine and 20 μg/ml uracil.
Antisense or sense oligonucleotides may be introduced into a cell containing the target nucleic acid sequence by any gene transfer method, including, for example, CaPO4 -mediated DNA transfection, electroporation, or by using gene transfer vectors such as Epstein-Barr virus. Antisense or sense oligonucleotides are preferably introduced into a cell containing the target nucleic acid sequence by insertion of the antisense or sense oligonucleotide into a suitable retroviral vector, then contacting the cell with the retrovirus vector containing the inserted sequence, either in vivo or ex vivo. Suitable retroviral vectors include, but are not limited to, the murine retrovirus M-MuLV, N2 (a retrovirus derived from M-MuLV), or or the double copy vectors designated DCT5A, DCT5B and DCT5C (see PCT U.S. application Ser. No. 90/02,656).
This example describes the cloning of murine flt3 cDNA, and the construction of an expression vector encoding a soluble murine flt3-receptor:Fc fusion protein for use in detecting cDNA clones encoding flt3-L. Polymerase chain reaction (PCR) cloning of the flt3 cDNA from a murine T-cell was accomplished using the oligonucleotide primers and the methods as described by Lyman et al., Oncogene, 8: 815-822, (1993), incorporated herein by reference. The cDNA sequence and encoded amino acid sequence for mouse flt3 receptor is presented by Rosnet et el., Oncogene, 6: 1641-1650, (1991), hereby incorporated by reference. The mouse flt3 protein has a 542 amino acid extracellular domain, a 21 amino acid transmembrane domain, and a 437 amino acid cytoplasmic domain.
Prior to fusing the murine flt3 cDNA to the N-terminus of cDNA encoding the Fc portion of a human IgG1 molecule, the amplified mouse flt3 cDNA fragment was inserted into Asp718-NotI site of pCAV/NOT, described in PCT Application WO 90/05183. DNA encoding a single chain polypeptide comprising the Fc region of a human IgG1 antibody was cloned into the SpeI site of the pBLUESCRIPT SK� vector, which is commercially available from Stratagene Cloning Systems, La Jolla, Calif. This plasmid vector is replicable in E. coli and contains a polylinker segment that includes 21 unique restriction sites. A unique BglII site was introduced near the 5' end of the inserted Fc encoding sequence, such that the BglII site encompasses the codons for amino acids three and four of the Fc polypeptide.
An Asp718 restriction endonuclease cleavage site was introduced upstream of the flt3 coding region. An Asp 718-NotI fragment of mouse flt3 cDNA (comprising the entire extracellular domain, the transmembrane region, and a small portion of the cytoplasmic domain) was isolated. The above-described Asp718-NotI flt3 partial cDNA was cloned into the pBLUESCRIPT SK� vector containing the Fc cDNA, such that the flt3 cDNA is positioned upstream of the Fc cDNA. Single stranded DNA derived from the resulting gene fusion was mutagenized by the method described in Kunkel (Proc. Natl. Acad. Sci. USA 82: 488, 1985) and Kunkel et al. (Methods in Enzymol. 154: 367, 1987) in order to perfectly fuse the entire extracellular domain of flt3 to the Fc sequence. The mutagenized DNA was sequenced to confirm that the proper nucleotides had been removed (i.e., transmembrane region and partial cytoplasmic domain DNA was deleted) and that the flt3 and Fc sequences were in the same reading frame. The fusion cDNA was then excised and inserted into a mammalian expression vector designated sfHAV-EO 409 which was cut with SalI-NotI, and the SalI and Asp718 ends blunted. The sfHAV-EO vector (also known as pDC406) is described by McMahan et al. (EMBO J., 10; No. 10: 2821-2832 (1991)).
A murine T-cell cDNA library of cell line P7B-0.3A4 was chosen as a possible source of flt3-L cDNA. P7B-0.3A4 is a murine T cell clone that is Thy1.2+, CD4-, CD8-, TCRab.sup.�, CD44+. It was originally cloned at a cell density of 0.33 cells/well in the presence of rHuIL-7 and immobilized anti-CD3 MAb, and was grown in continuous culture for more than 1 year by passage once a week in medium containing 15 ng/ml rHuIL-7. The parent cell line was derived from lymph node cells of SJL/J mice immunized with 50 nmoles PLP139-151 peptide and 100 μg Mycobacterium tuberculosis H37Ra in Incomplete Freund's Adjuvant. PLP is the proteolipid protein component of the myelin sheath of the central nervous system. The peptide composed of amino acids 139-151 has previously been shown to be the encephalogenic peptide in experimental autoimmune encephalomyelitis (EAE), a murine model for multiple sclerosis in SJL/J mice. (Touhy, V. K., Z. Lu, R. A. Sobel, R. A. Laursen and M. B. Lees; 1989. Identification of an encephalitogenic determinant of myelin proteolipid protein for SJL mice. J. Immunol. 142: 1523.) After the initial culture in the presence of antigen, the parent cell line, designated PLP7, had been in continuous culture with rHuIL-7 (and without antigen) for more than 6 months prior to cloning.
Double-stranded, blunt-ended, random-primed cDNA was prepared from 0.3A4 poly (A)+ RNA essentially as described by Gubler and Hoffman, Gene, 25: 263-269 (1983), using a Pharmacia DNA kit. The above adapters were added to the cDNA as described by Haymerle et al.. Low molecular weight material was removed by passage over Sephacryl S-1000 at 65-- C, and the cDNA was ligated into sfHAV-EO410, which had previously been cut with SalI and ligated to the same oligonucleotide pair. This vector is designated as sfHAV-EO410. DNA was electroporated (Dower et al., Nucleic Acids Res., 16: 6127-6145, (1988) into E. coli DH10B, and after one hour growth at 37-- C, the transformed cells were frozen in one milliliter aliquots in SOC medium (Hanahan et al., J. Mol. Biol., 166: 557-580, (1983) containing 20% glycerol. One aliquot was titered to determine the number of ampcillin-resistant colonies. The resulting 0.3A4 library had 1.84 million clones.
Transfected monolayers of CV-1/EBNA-1 cells were assayed for expression of flt3-L by slide autoradiography essentially as described by Gearing et al. (EMBO J. 8: 3667, 1989). Transfected CV-1/EBNA-1 cells (adhered to chambered slides) were washed once with binding medium with nonfat dry milk (BM-NFDM) (RPMI medium 1640 containing 25 mg/ml bovine serum albumin (BSA), 2 mg/ml sodium azide, 20 mM HEPES, pH 7.2, and 50 mg/ml nonfat dry milk). Cells were then incubated with flt3:Fc in BM-NFDM (1 μg/ml) for 1 hour at room temperature. After incubation, the cell monolayers in the chambered slides were washed three times with BM-NFDM to remove unbound flt3:Fc fusion protein and then incubated with 40 ng/ml 125 I-mouse anti-human Fc antibody (described below) (a 1:50 dilution) for 1 hour at room temperature. The cells were washed three times with BM-NFDM, followed by 2 washes with phosphate-buffered saline (PBS) to remove unbound 125 I-mouse anti-human Fc antibody. The cells were fixed by incubating for 30 minutes at room temperature in 2.5% glutaraldehyde in PBS, pH 7.3, washed twice in PBS and air dried. The chamber slides containing the cells were exposed on a Phophorimager (Molecular Dynamics) overnight, then dipped in Kodak GTNB-2 photographic emulsion (6� dilution in water) and exposed in the dark for 3-5 days at 4-- C in a light proof box. The slides were then developed for approximately 4 minutes in Kodak D19 developer (40 g/500 ml water), rinsed in water and fixed in Agfa G433C fixer. The slides were individually examined with a microscope at 25-40� magnification and positive cells expressing flt3-L were identified by the presence of autoradiographic silver grains against a light background.
The mouse anti-human Fc antibody was obtained from Jackson Laboratories. This antibody showed minimal binding to Fc proteins bound to the Fcγ receptor. The antibody was labeled using the Chloramine T method. Briefly, a Sephadex G-25 column was prepared according to the manufacturer's instructions. The column was pretreated with 10 column volumes of PBS containing 1% bovine serum albumin to reduce nonspecific adsorption of antibody to the column and resin. Nonbound bovine serum albumin was then washed from the column with 5 volumes of PBS lacking bovine serum albumin. In a microfuge tube 10 μg of antibody (dissolved in 10 μl of PBS) was added to 50 μl of 50 mM sodium phosphate buffer (pH 7.2) 2.0 mCi of carrier-free Na125 I was added and the solution was mixed well. 15 μl of a freshly prepared solution of chloramine-T (2 mg/ml in 0.1M sodium phosphate buffer (pH 7.2)) was then added and the mixture was incubated for 30 minutes at room temperature, and the mixture then was immediately applied to the column of Sephadex G-25. The radiolabelled antibody was then eluted from the column by collecting 100-150 μl fractions of eluate. Bovine serum albumin was added to the eluted fractions containing the radiolabeled antibody to a final concentration of 1%. Radioiodination yielded specific activities in the range of 5-10�1015 cpm/nmol protein.
A cDNA encoding human flt3-L was cloned from a human clone 22 T cell λgt10 random primed cDNA library as described by Sims et al., PNAS, 86: 8946-8950 (1989). The library was screened with a 413 bp Ple I fragment corresponding to the extracellular domain of the murine flt3-L (nucleotides 103-516 of SEQ ID NO:1). The fragment was random primed, hybridized overnight to the library filters at 55-- C in oligo prehybridization buffer. The fragment was then washed at 55-- C at 2� SSC/0.1% SDS for one hour, followed by 1� SSC/0.1% SDS for one hour and then by 0.5� SSC/0,1% SDS for one hour. The DNA from the positive phage plaques was extracted, and the inserts were amplified by PCR using oligonucleotides specific for the phage arms. The DNA then was sequenced, and the sequence for clone #9 is shown in SEQ ID NO:5. Additional human flt3-L cDNA was isolated from the same λgt10 random primed cDNA library as described above by screening the library with a fragment of the extracellular domain of the murine clone #5H cDNA comprising a cDNA sequence essentially corresponding to nucleotides 128-541 of SEQ ID NO:1.
For expression of soluble flt3-L in yeast, synthetic oligonucleotide primers were used to amplify via PCR (Mullis and Faloona, Meth. Enzymol. 155: 335-350, 1987) the entire extracellular coding domain of flt3-L between the end of the signal peptide and the start of the transmembrane segment. The 5' primer (5'-AATTGGTACCTTTGGATAAAAGAGACTACAAGGACGACGATGACAAGACACCTGACTGTTACTTCAGCCAC-3') SEQ ID NO:7 encoded a portion of of the alpha factor leader and an antigenic octapeptide, the FLAG sequence fused in-frame with the predicted mature N-terminus of flt3-L. The 3' oligonucleotide (5'-ATATGGATCCCTACTGCCTGGGCCGAGGCTCTGGGAG-3') SEQ ID NO:8 created a termination codon following Gln-189, just at the putative transmembrane region. The PCR-generated DNA fragment was ligated into a yeast expression vector (for expression in K. lactis) that directs secretion of the recombinant product into the yeast medium (Fleer et. al., Gene, 107: 285-195 (1991); and van den Berg et. al., Bio/Technology, 8: 135-139 (1990)). The FLAG:flt3-L fusion protein was purified from yeast broth by affinity chromatography as previously described (Hopp et. al., Biotechnology, 6: 1204-1210, 1988).
AA4.1-positive (AA4.1+) expressing cells were isolated from the livers of day 14 fetal C57BL/6 mice by cell panning in Optilux 100 mm plastic Petri dishes (Falcon No. 1001, Oxnard, Calif.). Plates were coated overnight at 4-- C in PBS plus 0.1% fetal bovine serum (FBS) containing 10 μg/ml AA4.1 antibody (McKearn et. al., J. Immunol., 132: 332-339, 1984) and then washed extensively with PBS plus 1% FBS prior to use. A single cell suspension of liver cells was added at 107 cells/dish in PBS plus 1% FBS and allowed to adhere to the plates for two hours at 4-- C. The plates were then extensively washed, and the adhering cells were harvested by scraping for analysis or further use in the hematopoiesis assays described below. FACS analysis using AA4.1 antibody demonstrated a >95% AA4.1+ cell population.
C-kit+ pluripotent stem cells were purified from adult mouse bone marrow (de Vries et. al., J. Exp. Med., 176: 1503-1509, 1992; and Visser and de Vries, Methods in Cell Biol., 1993, submitted). Low density cells (2 1.078 g/cm3) positive for the lectin wheat germ agglutinin and negative for the antigens recognized by the B220 and 15-1.4.1 (Visser et. al., Meth. in Cell Biol., 33: 451-468, 1990) monoclonal antibodies, could be divided into sub-populations of cells that do and do not express c-kit by using biotinylated Steel factor. The c-kit+ fraction has been shown to contain pluripotent hematopoietic stem cells (de Vries et. al., Science 255: 989-991, 1992; Visser and de Vries, Methods in Cell Biol., 1993, submitted; and Ware et. al., 1993, submitted).
The proliferation of c-kit+ stem cells, fetal liver AA4.1+ cells was assayed in 3H!-thymidine incorporation assays as essentially described by deVries et. al., J. Exp. Med., 173: 1205-1211, 1991. Purified c-kit+ stem cells were cultured at 37-- C in a fully humidified atmosphere of 6.5% CO2 and 7% O2 in air for 96 hours. Murine recombinant IL-3 was used at a final concentration of 100 ng/ml. Subsequently, the cells were pulsed with 2 μCi per well of 3 H!-thymidine (81 Ci/mmol; Amersham Corp., Arlington Heights, Ill.) and incubated for an additional 24 hours. AA4.1+ cells (approximately 20,000 cells/well) were incubated in IL-7, flt3-L and flt3-L+IL-7 for 48 hours, followed by 3 H!-thymidine pulse of six hours. The results of flt3-L and IL-7 are shown in Table I, and results of flt3-L and IL-3 are shown in Table II.
TABLE I______________________________________Effect of Flt3-L and IL-7 on Proliferation of AA4.1 + Fetal LiverCells.     Factor     Control           flt3-L   IL-7   flt3-L + IL-7______________________________________ 3 H!-thymidine       100     1000     100  4200incorporation(CPM)______________________________________
TABLE II______________________________________Effect of Flt3-L and IL-3 on Proliferation of C-kit + Cells.  Factor  Control (vector alone)              flt3-L  IL-3   flt3-L + IL-3______________________________________ 3 H!-thymidine    100           1800    3000 9100incorporation(CPM)______________________________________
This example describes a method for constructing a fusion protein comprising an extracellular region of the flt3-L and the Fc domain of a human immunoglobulin. The methods are essentially the same as those described in Example 1 for construction of a flt3:Fc fusion protein.
Prior to fusing a flt3-L cDNA to the N-terminus of cDNA encoding the Fc portion of a human IgG1 molecule, the flt3-L cDNA fragment is inserted into Asp718-NotI site of pCAV/NOT, described in PCT Application WO 90/05183. DNA encoding a single chain polypeptide comprising the Fc region of a human IgG1 antibody is cloned into the SpeI site of the pBLUESCRIPT SK� vector, which is commercially available from Stratagene Cloning Systems, La Jolla, Calif. This plasmid vector is replicable in E. coli and contains a polylinker segment that includes 21 unique restriction sites. A unique BglII site is then introduced near the 5' end of the inserted Fc encoding sequence, such that the BglII site encompasses the codons for amino acids three and four of the Fc polypeptide.
An Asp718-StuI partial cDNA of flt3-L in pCAV/NOT can be cloned into a Asp718-SpeI site of pBLUESCRIPT SK� vector containing the Fc cDNA, such that the flt3-L cDNA is positioned upstream of the Fc cDNA. The sequence of single stranded DNA derived from the resulting gene fusion can be affected by template-directed mutagensis described by Kunkel (Proc. Natl. Acad. Sci. USA 82: 488, 1985) and Kunkel et al. (Methods in Enzymol. 154: 367, 1987) in order to perfectly fuse the entire extracellular domain of flt3-L to the Fc sequence. The resulting DNA can then be sequenced to confirm that the proper nucleotides are removed (i.e., transmembrane region and partial cytoplasmic domain DNA are deleted) and that flt3-L and Fc sequences are in the same reading frame. The fusion cDNA is then excised and inserted using conventional methods into the mammalian expression vector pCAV/NOT which is cut with Asp 718-NotI.
This example describes a procedure used to generate transgenic mice that overexpress flt3-L. Flt3-L-overexpressing transgenic mice were studied to determine the biological effects of overexpression. Mouse (B 16/J) pronuclei were microinjected with flt3-L DNA according to the method described by Gordon et al., Science 214: 1244-1246, (1981). In general, fertilized mouse eggs having visible pronuclei were first placed on an injection chamber and held in place with a small pipet. An injection pipet was then used to inject the gene encoding the flt3-L (clone #6C) into the pronuclei of the egg. Injected eggs were then either (i) transferred into the oviduct of a 0.5 day p.c. pseudopregnant female; (ii) cultured in vitro to the two-cell stage (overnight) and transferred into the oviduct of a 0.5 day p.c. pseudopregnant female; or (iii) cultured in vitro to the blastocyst stage and transferred into the uterus of a 2.5 day p.c. pseudopregnant female. Preferably, either of the first two options can be used since they avoid extended in vitro culture, and preferably, approximately 20-30 microinjected eggs should be transferred to avoid small litters.
The above data indicate that flt3-L overexpression in mice leads to an increase in the number of B cells, as indicated by the increase B220+ cells and SCA-1+ cells. Analysis of B220+ cells by FACS indicated an increase in proB cells (HSA-, S7+). The increase in CD4+ cells indicated an approximate two-fold increase in T cells and stem cells. The decrease in cells having the sIgM marker indicated that flt3-L does not stimulate proliferation of mature B cells. These data indicate that flt3-L increases cells with a stem cell, T cell or an early B cell phenotype, and does not stimulate proliferation of mature B cells or macrophages.
FACS analysis demonstrated that no total change in cell number occurred and that the mice showed no change in the ratios of maturing thymocytes using the markers: CD4 vs. CD8; CD3 vs. αβTCR (T cell receptor); and CD3 vs. γδTCR (T cell receptor). However, a change in the ratios of certain cell types within the CD4- and CD8- compartment (i.e., the earliest cells with respect to development; which represent approximately 2% to 3% of total thymus cells) occurred. Specifically, CD4- and CD8- cells in the thymus develop in three stages. Stage 1 represents cells having the Pgp-1++, HSA+ and IL-2 receptor-negative ("IL-2R-") markers. After stage 1, thymic cells develop to stage 2 consisting of cells having Pgp-1+, HSA++, and IL-2R++ markers, and then to stage 3, characterized by cells having Pgp-1.sup.�, HSA++, and IL-2R- markers. Thymic cells in stage 2 of the transgenic mice were reduced by about 50%, while the population of cells in stage 3 was proportionately increased. These data suggest that flt3-L drives the thymic cells from stage 2 to stage 3 of development, indicating that flt3-L is active on early T cells.
Prior to cell collection, it may be desirable to mobilize or increase the numbers of circulating PBPC and PSC. Mobilization can improve PBPC and PSC collection, and is achievable through the intravenous administration of flt3-L to the patients prior to collection of such cells. Other growth factors such as CSF-1, GM-CSF, SF, G-CSF, EPO, IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, IL-14, IL-15, GM-CSF/IL-3 fusion proteins, LIF, FGF and combinations thereof, can be likewise administered in sequence, or in concurrent combination with flt3-L. Mobilized or non-mobilized PBPC and PSC are collected using apheresis procedures known in the art. See, for example, Bishop et al., Blood, vol. 83, No. 2, pp. 610-616 (1994). Briefly, PBPC and PSC are collected using conventional devices, for example, a Haemonetics Model V50 apheresis device (Haemonetics, Braintree, Mass.). Four-hour collections are performed typically no more than five times weekly until approximately 6.5�108 mononuclear cells (MNC)/kg patient are collected. Aliquots of collected PBPC and PSC are assayed for granulocyte-macrophage colony-forming unit (CFU-GM) content by diluting approximately 1:6 with Hank's balanced salt solution without calcium or magnesium (HBSS) and layering over lymphocyte separation medium (Organon Teknika, Durham, N.C.). Following centrifugation, MNC at the interface are collected, washed and resuspended in HBSS. One milliliter aliquots containing approximately 300,000 MNC, modified McCoy's 5A medium, 0.3% agar, 200 U/mL recombinant human GM-CSF, 200 u/mL recombinant human IL-3, and 200 u/mL recombinant human G-CSF are cultured at 37-- C in 5% CO2 in fully humidified air for 14 days. Optionally, flt3-L or GM-CSF/IL-3 fusion molecules (PIXY 321) may be added to the cultures. These cultures are stained with Wright's stain, and CFU-GM colonies are scored using a dissecting microscope (Ward et al., Exp. Hematol., 16: 358 (1988). Alternatively, CFU-GM colonies can be assayed using the CD34/CD33 flow cytometry method of Siena et al., Blood, Vol. 77, No. 2, pp 400-409 (1991), or any other method known in the art.
This Example describes a method for purifying hematopoietic progenitor cells and stem cells from a suspension containing a mixture of cells. Cells from bone marrow and peripheral blood are collected using conventional procedures. The cells are suspended in standard media and then centrifuged to remove red blood cells and neutrophils. Cells located at the interface between the two phases (also known in the art as the buffy coat) are withdrawn and resuspended. These cells are predominantly mononuclear and represent a substantial portion of the early hematopoietic progenitor and stem cells. The resulting cell suspension then is incubated with biotinylated flt3-L for a sufficient time to allow substantial flt3:flt3-L interaction. Typically, incubation times of at least one hour are sufficient. After incubation, the cell suspension is passed, under the force of gravity, through a column packed with avidin-coated beads. Such columns are well known in the art, see Berenson, et al., J. Cell Biochem., 10D: 239 (1986). The column is washed with a PBS solution to remove unbound material. Target cells can be released from the beads and from flt3-L using conventional methods.
__________________________________________________________________________SEQUENCE LISTING(1) GENERAL INFORMATION:(iii) NUMBER OF SEQUENCES: 8(2) INFORMATION FOR SEQ ID NO:1:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 879 base pairs(B) TYPE: nucleic acid(C) STRANDEDNESS: single(D) TOPOLOGY: linear(ii) MOLECULE TYPE: cDNA to mRNA(iii) HYPOTHETICAL: NO(iv) ANTI-SENSE: NO(ix) FEATURE:(A) NAME/KEY: misc-- feature(B) LOCATION: 1..25(ix) FEATURE:(A) NAME/KEY: misc-- feature(B) LOCATION: 855..879(ix) FEATURE:(A) NAME/KEY: CDS(B) LOCATION: 57..752(xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:GTCGACTGGAACGAGACGACCTGCTCTGTCACAGGCATGAGGGGTCCCCGGCAGAG56ATGACAGTGCTGGCGCCAGCCTGGAGCCCAAATTCCTCCCTGTTGCTG104MetThrValLeuAlaProAlaTrpSerProAsnSerSerLeuLeuLeu151015CTGTTGCTGCTGCTGAGTCCTTGCCTGCGGGGGACACCTGACTGTTAC152LeuLeuLeuLeuLeuSerProCysLeuArgGlyThrProAspCysTyr202530TTCAGCCACAGTCCCATCTCCTCCAACTTCAAAGTGAAGTTTAGAGAG200PheSerHisSerProIleSerSerAsnPheLysValLysPheArgGlu354045TTGACTGACCACCTGCTTAAAGATTACCCAGTCACTGTGGCCGTCAAT248LeuThrAspHisLeuLeuLysAspTyrProValThrValAlaValAsn505560CTTCAGGACGAGAAGCACTGCAAGGCCTTGTGGAGCCTCTTCCTAGCC296LeuGlnAspGluLysHisCysLysAlaLeuTrpSerLeuPheLeuAla65707580CAGCGCTGGATAGAGCAACTGAAGACTGTGGCAGGGTCTAAGATGCAA344GlnArgTrpIleGluGlnLeuLysThrValAlaGlySerLysMetGln859095ACGCTTCTGGAGGACGTCAACACCGAGATACATTTTGTCACCTCATGT392ThrLeuLeuGluAspValAsnThrGluIleHisPheValThrSerCys100105110ACCTTCCAGCCCCTACCAGAATGTCTGCGATTCGTCCAGACCAACATC440ThrPheGlnProLeuProGluCysLeuArgPheValGlnThrAsnIle115120125TCCCACCTCCTGAAGGACACCTGCACACAGCTGCTTGCTCTGAAGCCC488SerHisLeuLeuLysAspThrCysThrGlnLeuLeuAlaLeuLysPro130135140TGTATCGGGAAGGCCTGCCAGAATTTCTCTCGGTGCCTGGAGGTGCAG536CysIleGlyLysAlaCysGlnAsnPheSerArgCysLeuGluValGln145150155160TGCCAGCCGGACTCCTCCACCCTGCTGCCCCCAAGGAGTCCCATAGCC584CysGlnProAspSerSerThrLeuLeuProProArgSerProIleAla165170175CTAGAAGCCACGGAGCTCCCAGAGCCTCGGCCCAGGCAGCTGTTGCTC632LeuGluAlaThrGluLeuProGluProArgProArgGlnLeuLeuLeu180185190CTGCTGCTGCTGCTGCCTCTCACACTGGTGCTGCTGGCAGCCGCCTGG680LeuLeuLeuLeuLeuProLeuThrLeuValLeuLeuAlaAlaAlaTrp195200205GGCCTTCGCTGGCAAAGGGCAAGAAGGAGGGGGGAGCTCCACCCTGGG728GlyLeuArgTrpGlnArgAlaArgArgArgGlyGluLeuHisProGly210215220GTGCCCCTCCCCTCCCATCCCTAGGATTCGAGCCTTGTGCATCGTTGACTC779ValProLeuProSerHisPro225230AGCCAGGGTCTTATCTCGGTTACACCTGTAATCTCAGCCCTTGGGAGCCCAGAGCAGGAT839TGCTGAATGGTCTGGAGCAGGTCGTCTCGTTCCAGTCGAC879(2) INFORMATION FOR SEQ ID NO:2:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 231 amino acids(B) TYPE: amino acid(D) TOPOLOGY: linear(ii) MOLECULE TYPE: protein(xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:MetThrValLeuAlaProAlaTrpSerProAsnSerSerLeuLeuLeu151015LeuLeuLeuLeuLeuSerProCysLeuArgGlyThrProAspCysTyr202530PheSerHisSerProIleSerSerAsnPheLysValLysPheArgGlu354045LeuThrAspHisLeuLeuLysAspTyrProValThrValAlaValAsn505560LeuGlnAspGluLysHisCysLysAlaLeuTrpSerLeuPheLeuAla65707580GlnArgTrpIleGluGlnLeuLysThrValAlaGlySerLysMetGln859095ThrLeuLeuGluAspValAsnThrGluIleHisPheValThrSerCys100105110ThrPheGlnProLeuProGluCysLeuArgPheValGlnThrAsnIle115120125SerHisLeuLeuLysAspThrCysThrGlnLeuLeuAlaLeuLysPro130135140CysIleGlyLysAlaCysGlnAsnPheSerArgCysLeuGluValGln145150155160CysGlnProAspSerSerThrLeuLeuProProArgSerProIleAla165170175LeuGluAlaThrGluLeuProGluProArgProArgGlnLeuLeuLeu180185190LeuLeuLeuLeuLeuProLeuThrLeuValLeuLeuAlaAlaAlaTrp195200205GlyLeuArgTrpGlnArgAlaArgArgArgGlyGluLeuHisProGly210215220ValProLeuProSerHisPro225230(2) INFORMATION FOR SEQ ID NO:3:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 24 base pairs(B) TYPE: nucleic acid(C) STRANDEDNESS: single(D) TOPOLOGY: linear(iii) HYPOTHETICAL: NO(iv) ANTI-SENSE: NO(xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:TCGACTGGAACGAGACGACCTGCT24(2) INFORMATION FOR SEQ ID NO:4:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 20 base pairs(B) TYPE: nucleic acid(C) STRANDEDNESS: single(D) TOPOLOGY: linear(iii) HYPOTHETICAL: NO(iv) ANTI-SENSE: NO(xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:AGCAGGTCGTCTCGTTCCAG20(2) INFORMATION FOR SEQ ID NO:5:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 988 base pairs(B) TYPE: nucleic acid(C) STRANDEDNESS: single(D) TOPOLOGY: linear(ii) MOLECULE TYPE: cDNA to mRNA(iii) HYPOTHETICAL: NO(iv) ANTI-SENSE: NO(ix) FEATURE:(A) NAME/KEY: CDS(B) LOCATION: 30..734(xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:CGGCCGGAATTCCGGGGCCCCCGGCCGAAATGACAGTGCTGGCGCCAGCCTGG53MetThrValLeuAlaProAlaTrp15AGCCCAACAACCTATCTCCTCCTGCTGCTGCTGCTGAGCTCGGGACTC101SerProThrThrTyrLeuLeuLeuLeuLeuLeuLeuSerSerGlyLeu101520AGTGGGACCCAGGACTGCTCCTTCCAACACAGCCCCATCTCCTCCGAC149SerGlyThrGlnAspCysSerPheGlnHisSerProIleSerSerAsp25303540TTCGCTGTCAAAATCCGTGAGCTGTCTGACTACCTGCTTCAAGATTAC197PheAlaValLysIleArgGluLeuSerAspTyrLeuLeuGlnAspTyr455055CCAGTCACCGTGGCCTCCAACCTGCAGGACGAGGAGCTCTGCGGGGGC245ProValThrValAlaSerAsnLeuGlnAspGluGluLeuCysGlyGly606570CTCTGGCGGCTGGTCCTGGCACAGCGCTGGATGGAGCGGCTCAAGACT293LeuTrpArgLeuValLeuAlaGlnArgTrpMetGluArgLeuLysThr758085GTCGCTGGGTCCAAGATGCAAGGCTTGCTGGAGCGCGTGAACACGGAG341ValAlaGlySerLysMetGlnGlyLeuLeuGluArgValAsnThrGlu9095100ATACACTTTGTCACCAAATGTGCCTTTCAGCCCCCCCCCAGCTGTCTT389IleHisPheValThrLysCysAlaPheGlnProProProSerCysLeu105110115120CGCTTCGTCCAGACCAACATCTCCCGCCTCCTGCAGGAGACCTCCGAG437ArgPheValGlnThrAsnIleSerArgLeuLeuGlnGluThrSerGlu125130135CAGCTGGTGGCGCTGAAGCCCTGGATCACTCGCCAGAACTTCTCCCGG485GlnLeuValAlaLeuLysProTrpIleThrArgGlnAsnPheSerArg140145150TGCCTGGAGCTGCAGTGTCAGCCCGACTCCTCAACCCTGCCACCCCCA533CysLeuGluLeuGlnCysGlnProAspSerSerThrLeuProProPro155160165TGGAGTCCCCGGCCCCTGGAGGCCACAGCCCCGACAGCCCCGCAGCCC581TrpSerProArgProLeuGluAlaThrAlaProThrAlaProGlnPro170175180CCTCTGCTCCTCCTACTGCTGCTGCCCGTGGGCCTCCTGCTGCTGGCC629ProLeuLeuLeuLeuLeuLeuLeuProValGlyLeuLeuLeuLeuAla185190195200GCTGCCTGGTGCCTGCACTGGCAGAGGACGCGGCGGAGGACACCCCGC677AlaAlaTrpCysLeuHisTrpGlnArgThrArgArgArgThrProArg205210215CCTGGGGAGCAGGTGCCCCCCGTCCCCAGTCCCCAGGACCTGCTGCTT725ProGlyGluGlnValProProValProSerProGlnAspLeuLeuLeu220225230GTGGAGCACTGACCTGGCCAAGGCCTCATCCTGCGGAGCCTTAAACAAC774ValGluHis235GCAGTGAGACAGACATCTATCATCCCATTTTACAGGGGAGGATACTGAGGCACACAGAGG834GGAGTCACCAGCCAGAGGATGTATAGCCTGGACACAGAGGAAGTTGGCTAGAGGCCGGTC894CCTTCCTTGGGCCCCTCTCATTCCCTCCCCAGAATGGAGGCAACGCCAGAATCCAGCACC954GGCCCCATTTACCCAACTCTGAACAAAGCCCCCG988(2) INFORMATION FOR SEQ ID NO:6:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 235 amino acids(B) TYPE: amino acid(D) TOPOLOGY: linear(ii) MOLECULE TYPE: protein(xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:MetThrValLeuAlaProAlaTrpSerProThrThrTyrLeuLeuLeu151015LeuLeuLeuLeuSerSerGlyLeuSerGlyThrGlnAspCysSerPhe202530GlnHisSerProIleSerSerAspPheAlaValLysIleArgGluLeu354045SerAspTyrLeuLeuGlnAspTyrProValThrValAlaSerAsnLeu505560GlnAspGluGluLeuCysGlyGlyLeuTrpArgLeuValLeuAlaGln65707580ArgTrpMetGluArgLeuLysThrValAlaGlySerLysMetGlnGly859095LeuLeuGluArgValAsnThrGluIleHisPheValThrLysCysAla100105110PheGlnProProProSerCysLeuArgPheValGlnThrAsnIleSer115120125ArgLeuLeuGlnGluThrSerGluGlnLeuValAlaLeuLysProTrp130135140IleThrArgGlnAsnPheSerArgCysLeuGluLeuGlnCysGlnPro145150155160AspSerSerThrLeuProProProTrpSerProArgProLeuGluAla165170175ThrAlaProThrAlaProGlnProProLeuLeuLeuLeuLeuLeuLeu180185190ProValGlyLeuLeuLeuLeuAlaAlaAlaTrpCysLeuHisTrpGln195200205ArgThrArgArgArgThrProArgProGlyGluGlnValProProVal210215220ProSerProGlnAspLeuLeuLeuValGluHis225230235(2) INFORMATION FOR SEQ ID NO:7:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 71 base pairs(B) TYPE: nucleic acid(C) STRANDEDNESS: single(D) TOPOLOGY: linear(ii) MOLECULE TYPE: cDNA to mRNA(iii) HYPOTHETICAL: NO(iv) ANTI-SENSE: NO(xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:AATTGGTACCTTTGGATAAAAGAGACTACAAGGACGACGATGACAAGACACCTGACTGTT60ACTTCAGCCAC71(2) INFORMATION FOR SEQ ID NO:8:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 37 base pairs(B) TYPE: nucleic acid(C) STRANDEDNESS: single(D) TOPOLOGY: linear(ii) MOLECULE TYPE: cDNA to mRNA(iii) HYPOTHETICAL: NO(iv) ANTI-SENSE: NO(xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:ATATGGATCCCTACTGCCTGGGCCGAGGCTCTGGGAG37__________________________________________________________________________
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ClassificationA61K38/00, C07K2319/30, C07K2319/00, C12N2501/23, C12N2501/26, A61K48/00, C12N5/0647, C07K14/475, A01K2217/05, A01K2267/03, A01K2227/105, A01K2267/0381, A01K2217/30, C12N15/8509, A01K67/0271, C07K14/70596, A01K67/0275, C12N2501/22, C12N2510/00, A01K2267/0331European ClassificationA61K38/19, C12N5/06B11P, A01K67/027M, C12N15/85A, C07K14/475, A01K67/027B, C07K14/705Z, A61K38/18Legal EventsDateCodeEventDescriptionMay 9, 2002FPAYFee paymentYear of fee payment: 4May 5, 2006FPAYFee paymentYear of fee payment: 8Dec 16, 2009ASAssignmentOwner name: IMMUNEX CORPORATION, WASHINGTONFree format text: ASSIGNMENT OF ASSIGNORS INTEREST;ASSIGNORS:LYMAN, STEWART D.;BECKMANN, M. PATRICIA;REEL/FRAME:023660/0600;SIGNING DATES FROM 19940506 TO 19940511Dec 22, 2009ASAssignmentOwner name: CELLDEX THERAPEUTICS, INC., NEW JERSEYFree format text: ASSIGNMENT OF ASSIGNORS INTEREST;ASSIGNOR:IMMUNEX CORPORATION;REEL/FRAME:023679/0893Effective date: 20090317Jun 1, 2010FPAYFee paymentYear of fee payment: 12RotateOriginal ImageGoogle Home - Sitemap - USPTO Bulk Downloads - Privacy Policy - Terms of Service - About Google Patents - Send FeedbackData provided by IFI CLAIMS Patent Services