EP1278773A2 - Genes marqueurs de surface selectionnables - Google Patents

Genes marqueurs de surface selectionnables

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Publication number
EP1278773A2
EP1278773A2 EP00988723A EP00988723A EP1278773A2 EP 1278773 A2 EP1278773 A2 EP 1278773A2 EP 00988723 A EP00988723 A EP 00988723A EP 00988723 A EP00988723 A EP 00988723A EP 1278773 A2 EP1278773 A2 EP 1278773A2
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European Patent Office
Prior art keywords
cells
mutated
ptkr
musk
cell
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EP00988723A
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German (de)
English (en)
Inventor
Susanne Dagmar Pippig
Gabor Veres
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Novartis Pharma GmbH
Novartis AG
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Novartis Erfindungen Verwaltungs GmbH
Novartis AG
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Publication of EP1278773A2 publication Critical patent/EP1278773A2/fr
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/10—Transferases (2.)
    • C12N9/12—Transferases (2.) transferring phosphorus containing groups, e.g. kinases (2.7)
    • C12N9/1205—Phosphotransferases with an alcohol group as acceptor (2.7.1), e.g. protein kinases
    • 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
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/10—Transferases (2.)
    • C12N9/12—Transferases (2.) transferring phosphorus containing groups, e.g. kinases (2.7)
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
    • G01N33/56966—Animal cells

Definitions

  • This invention relates to a method of identifying genetically modified cells using a mutated protein-tyrosine kinase receptor (PTKR), particularly a mutated epidermal growth factor receptor (EGFR) family member or a mutated muscle specific kinase (MuSK) family member as a selectable cell marker.
  • PTKR protein-tyrosine kinase receptor
  • EGFR epidermal growth factor receptor
  • MoSK muscle specific kinase
  • selectable markers are well known for the identification of prokaryotic and eukaryotic cells, and the use is essential because frequently when a DNA sequence of interest is introduced into a cell it will not necessarily lead to a phenotype that is readily determined.
  • the number of selectable markers used in identifying eukaryotic cells and especially mammalian cells has been limited. In the past, selectable markers that conferred drug resistance were employed (i.e. G-418 and hygromycin). More recently, selectable markers that are combined with fluorescence activated cell sorting (FACS) have been used, for example, green fluorescent protein (GFP).
  • FACS fluorescence activated cell sorting
  • FACS fluorescence activated cell sorting
  • antibodies that recognize a cell surface molecule may be coupled to a fluorophore to help identify the cells of interest.
  • NGFR Low-Affinity Nerve Growth Factor Receptor
  • Some of these cell surface molecules have been mutated in their intracellular domain to avoid signaling of the molecule when binding to their ligand. However upon ligand binding some of the intracellularly mutated molecules may homo-, heterodimerize or trimerize. If a newly introduced molecule in the cell should heterodimerize with endogenous receptors a dominant negative effect may result.
  • the present invention provides cell surface markers that do not heterodimerize with endogenous receptors.
  • the present invention provides a method of identifying genetically modified mammalian cells comprising introducing a nucleic acid sequence encoding a mutated protein-tyrosine kinase receptor (PTKR) operatively linked to an expression control sequence into a cell to form a genetically modified cell; allowing expression of the mutated PTKR in the genetically modified cell; and identifying said genetically modified cell expressing the mutated PTKR.
  • the mutated PTKR is a mutated epidermal growth factor receptor (EGFR) family member (or MuSK family member), particularly preferred is a mutated EGFR1 and more specifically the sequences given the designation EGFRl-I and EGFRl-II.
  • the introducing step is accomplished by incorporating the nucleic acid sequence encoding the mutated PTKR, and particularly the mutated EGFR into a vector and introducing the vector into a cell.
  • the preferred vectors are retroviral vectors.
  • the mammalian cells are human cells, particularly hematopoietic cells, liver cells, endothelial vascular cells, and smooth muscle cells, more particularly hematopoietic cells.
  • a heterologous gene is incorporated into the construct or vector comprising the mutated PTKR marker sequence.
  • the identifying step is accomplished by contacting the genetically modified cells with an antibody that recognizes and binds to the mutated PTKR.
  • the identifying step separates the genetically modified cells from the non-genetically modified cells.
  • the invention provides a method of identifying genetically modified mammalian cells comprising the steps of incorporating into a vector a nucleic acid sequence encoding a mutated epidermal growth factor receptor (EGFR) family member operatively linked to an expression control sequence; introducing the vector into a mammalian cell to form a genetically modified cell; allowing expression of the mutated EGFR in the genetically modified cell; and identifying said genetically modified cell expressing the mutated EGFR.
  • the mutated EGFR is EGFRl-I or EGFRl-II and the vector is a retroviral vector.
  • the invention is directed to a method for the immunoselection of transduced mammalian cells comprising, retrovirally transducing mammalian cells with a nucleic acid sequence encoding a mutated epidermal growth factor receptor (EGFR) family member operatively linked to an expression control sequence; incubating the transduced cells with a marked antibody that recognizes and binds specifically to the mutated EGFR; and identifying the marked transduced cells.
  • the cells are human cells, particularly hematopoietic cells.
  • the cells are transduced by a retroviral vector derived from the group consisting of moloney murine leukemia virus (MoMLV), myeloproliferative sarcoma virus (MPSN), murine embryonic stem cell virus (MESN), murine stem cell virus (MSCN) and spleen focus forming virus (SFFN).
  • MoMLV moloney murine leukemia virus
  • MPSN myeloproliferative sarcoma virus
  • MSN murine embryonic stem cell virus
  • MSCN murine stem cell virus
  • SFFN spleen focus forming virus
  • the cells are transduced with a vector derived from a lentivirus.
  • the method includes the step of separating the identified marked transduced cells from non-marked cells.
  • the method includes the step of expanding the marked transduced cells.
  • the invention is directed to a method of identifying mammalian cells expressing a protein of interest, comprising the steps of, introducing into a mammalian cell a nucleic acid sequence encoding a mutated PTKR operatively linked to an expression control sequence and a nucleic acid sequence encoding a protein of interest; culturing the resulting mammalian cells; and identifying cells which express the mutated PTKR thereby obtaining cells which express the protein of interest.
  • Figure 1 A is a schematic representation of a wild-type (WT) EGFR molecule and two mutated EGFRs: EGFRl-I with amino acid residues deleted from the cytoplasmic domain and EGFRl-II with amino acid residues deleted from both the cytoplasmic and extracellular domains.
  • Fig. IB illustrates the wild-type and the mutated MuSK.
  • Figure 2 illustrates the amino acid sequence of a wild -type (WT) EGFR1 and corresponds to SEQ ID NO: 2.
  • the signal sequence is represented as amino acid residue 1 to 24.
  • the extracellular domain includes amino acid residues 25 through 645, the transmembrane domain includes amino acid residues 646 through 668, and the cytoplasmic domain includes amino acid residues 669 through 1210.
  • the tyrosine kinase domain is located at amino acid residues 718 though 964, and the threonine phosphorylation site is located at amino acid residue 678.
  • Figure 3 illustrates the nucleotide sequence of the wild-type (WT) EGFR1 encoding the amino acid sequence of Figure 2 and corresponds to SEQ ID NO: 1.
  • Figure 4 illustrates the general scheme used to generate a deletion in the extracellular domain and intracellular domain of the EGFR sequence depicted in Figure 2 and 3.
  • Figure 5 illustrates expression of EGFRl-II on primary T cells and CD34 + cells after transduction with supernatants that were generated from PPA-6 cells.
  • Panel A corresponds to untransduced cells
  • panel B illustrates expression after transduction
  • panel C illustrates expression after immuno-magnetic beadselection for mutated EGFR expression on T-cells.
  • Panel D corresponds to untransduced cells and panel E illustrates expression of CD34 + cells.
  • Figure 6 illustrates a MuSK-R designated hMuSK-R and corresponds to the nucleic acid sequence as set forth in SEQ ID NO:7 and the amino acid sequence as set forth in SEQ ID NO: 8.
  • a stem cell includes a plurality of stem cells.
  • the selectable marker of the instant invention is a mutated protein-tyrosine kinase receptor (PTKR) molecule.
  • PTKR molecules are activated by polypeptide ligands, and are closely related in their catalytic domains. They are Type I transmembrane proteins, with their N- termini outside the cell and single membrane-spanning regions.
  • PTKRs include but are not limited to the following subfamilies, MuSK-R which is believed to initiate the formation of neuromuscular junctions in response to agrin (Glass, et al. Cell 85:513 (1996), epidermal growth factor receptor (EGFR), platelet-derived growth factor receptor (PDGFR), insulin receptor (INSR), nerve growth factor receptor (NGFR), fibroblast growth factor receptor (FGFR), and EPH.
  • MuSK-R which is believed to initiate the formation of neuromuscular junctions in response to agrin
  • EGFR epidermal growth factor receptor
  • PDGFR platelet-derived growth factor receptor
  • INSR insulin receptor
  • NGFR nerve growth factor receptor
  • FGFR fibroblast growth factor receptor
  • EPH EPH
  • the PDGFR family includes PDGF receptor , PDGF receptor ⁇ , SCF receptor (c-Kit), CSF-1 receptor (c-Fms), Flk2 (Flt3), FLT1 (Quekl), Flkl (KDR), and FLT4 (Quk2).
  • the INSF subfamily includes insulin receptor, IGF-1 receptor, IRR, ROS and Ltk.
  • the NGFR subfamily includes NGFR receptor (TrkA), TrkB, and TrkC.
  • the EGFR family includes EGFR1, EGFR2, EGFR3, and EGFR4.
  • EGFR1 has been referred to as EGFR, HER, c-ErbB, and ErbB-1 ;
  • EGFR2 has been referred to as HER2, Neu and ErbB-2;
  • EGFR3 has been referred to as HER3 and ErbB-3; and
  • EGFR4 has been referred to as HER4 and ErbB-4. (Ullrich and Schlessinger, Cell, 61 : 203-212 (1990)).
  • the mutated PTKRs of the present invention include a modification in a PTKR molecule so that the receptor no longer possesses the signaling activity of the corresponding unmodified (wild type) receptor.
  • "Wild-Type PTKRs” include not only naturally occurring PTKRs but also may include genetically engineered PTKRs molecules wherein the PTKR molecule has undergone changes in the DNA sequence that do not significantly effect the properties of the protein tyrosine kinase receptor molecule. These changes include ones that do not change the encoded amino acid sequence, ones that result in conservative substitutions of amino acid sequences, or ones that result in one or a few amino acid deletions or additions. Suitable substitutions are known by those skilled in the art.
  • Amino acid residues which can be conservatively substituted for one another include, but are not limited to, glycine/alanine; valine/isoleucine/leucine, asparagine/glutamine; asparatic acid/glutamic acid; serine/threonine; lysine/arginine; and phenylalanine/tyrosine.
  • a modification of the wild-type PTKR molecule may include deletions, truncations and the like. More specifically, mutated PTKRs of the invention include a modification, however, obtained, in the cytoplasmic domain causing the molecule to be devoid of signaling activity. Signaling activity may be generally defined as activity that triggers a response pathway in the cytosol to the nucleus that ultimately leads to activation of transcription.
  • the mutated PTKRs according to the invention may include a modification in the extracellular domain. However, preferably the extracellular domain should still be capable of binding an antibody. In general, the smallest peptide fragment of the extracellular domain capable of binding an antibody would be approximately 15 amino acid residues, more preferably at least 50 amino acid residues.
  • the selective marker PTKR is a mutated member of the EGFR family.
  • the members of the EGFR family have similar overall topology and significant relatedness in amino acid sequence. Members of this family are not limited to the above- enumerated members, but include new members sharing common features, such as a single transmembrane domain; two cysteine rich regions of approximately 100 to 200 amino acid residues, and a single tyrosine kinase domain. Further, members of the EGFR family can heterodimerize with other members of the EGFR family.
  • a preferred mutated EGFR family member is EGFR1.
  • EGFR1 is a high affinity receptor for a number of ligands including but not limited to epidermal growth factor (EGF), transforming growth factor ⁇ (TGF ⁇ ) and amphiregulin.
  • EGF epidermal growth factor
  • TGF ⁇ transforming growth factor ⁇
  • amphiregulin Various EGFR sequences have been identified and reference is made to EMBL/GenBank Accession Numbers: X00588/X06370 (Ullrich, et al., Nature 309:418 (1984)); K01885/K02047, (Lin et al., Science 224:843 (1984)); K03193 (Merlino, et al., Mol. Cell.
  • EGFR is comprised of a signal peptide that targets the protein to the secretory pathway.
  • the extracellular domain follows the signal sequence. This domain is made up of several hundred amino acids.
  • the extracellular domain means the part of the receptor that normally projects from the cell into the extracellular environment and in the EGFR it contains distinctive Cys residues.
  • the transmembrane domain is generally localized in the cell membrane and consists of a stretch of hydrophobic residues followed by several basic residues.
  • the cytoplasmic domain (also referred to as the intracellular domain) includes the catalytic part of the molecule and is positioned within the cell. This domain contains the substrate binding site and regulatory tyrosine and threonine phosphorylation sites.
  • HER721 A is a point mutation in the cytoplasmic domain of EGFR 1 at amino acid position 721. The lysine is changed to alanine.
  • a second mutation (referred to as HERCD-533) involves the C-terminal end of the molecule wherein the intracellular domain was deleted but the transmembrane domain was not.
  • a third mutation (referred to as HERCD-566) includes the C- terminal deletion of 566 amino acids, both the intracellular and transmembrane domains were deleted.
  • Type I (EGFRvIII) has a large deletion of the extracellular domain (amino acids 6 - 273) and does not bind EGF (Moscatello, D.K. et al., Oncogene 13:85 (1996)).
  • Type II contains an in-frame deletion of 83 amino acids (520-603) in extracellular domain IV that does not prevent EGF and TGF ⁇ binding (Humphrey, P.A. et al., Biophys. Res. Commun. 178:1413 (1991)).
  • Type IJJ has an in-frame deletion of 267 amino acids (29-296) in extracellular domains II and III. These two mutations prevent ligand binding (Humphrey, P.A. et al., Proc. Natl. Acad. Sci. USA 87:4207 (1990)).
  • modifications to the PTKR molecules and particularly to EGFR molecules include modifications to the cytoplasmic domain, such as deletions of at least 150, preferably at least 250, more preferably at least 400, and still more preferably at least 500 amino acids of the cytoplasmic domain.
  • the deletions will consist of a truncation of the molecule. Truncations may include deletion of tyrosine phosphorylation sites in the range of 1 to 15 sites and deletion of the kinase catalytic site.
  • a mutated EGFR useful as selectable marker in the invention may include more than 1 and fewer than 15 deletions of tyrosine phosphorylation sites. Additionally, the above modifications may include in-frame deletions, such as deletions of at least 50 amino acids of the cytoplasmic domain. These deletions may include deletion of the protein tyrosine-kinase activity. Particularly preferred selectable marker sequences are mutated EGFRl, EGFR2 and EGFR3 molecules wherein at least 400 and preferably 500 amino acids of the cytoplasmic domain are deleted from the corresponding wild-type molecule.
  • a preferred mutated EGFR molecule is a mutated EGFRl .
  • Particularly preferred is a modified sequence derived from EGFRl as illustrated in Figures 2 and 3 (SEQ ID Nos. 1 and 2).
  • the extracellular domain is encoded by nucleotides 1 through 1935
  • the transmembrane domain is encoded by nucleotides 1938 through 2004,
  • the intracellular domain is coded by nucleotides 2007 to 3630.
  • One non-limiting example of a preferred mutated EGFRl marker is designated EGFRl-I wherein amino acid sequence 679 to 1210 of the cytoplasmic domain as illustrated in Figure 2 is deleted.
  • the selective marker PTKR is a mutated member of the MuSK-R family.
  • MuSK-R is comprised of a signal sequence or leader sequence that targets the protein to the secretory pathway.
  • the extracellular domain follows the signal sequence. This domain is made up of several hundred amino acids, and while the exact number of amino acid residues varies, typically the extracellular domain includes around 500 amino acids.
  • the extracellular domain is the part of the receptor that normally projects from the cell into the extracellular environment and includes a ligand binding region.
  • the extracellular domain is one of the most distinctive features of the kinase receptors.
  • the extracellular domain contains immunoglobulin-like (Ig-like) regions. Typically four Ig-like regions are found.
  • the extracellular domain may include 6 contiguous cysteine residues known as a C6-box. While the location of the C6-box may vary depending on the particular MuSK-R, in certain MuSK-Rs it is found approximately at amino acid residues 373 - 382.
  • the transmembrane domain is generally localized in the cell membrane and consists of a stretch of hydrophobic residues followed by several basic residues.
  • the intracellular domain (used interchangeably with the cytoplasmic domain) includes the catalytic part of the molecule and is positioned within the cell.
  • MuSK-Rs are also known in the art as denervated muscle kinase receptors and have been referred to as DmKs (see U.S. Pat. No. 5,656,473 and particularly SEQ ID NOS: 16 and 17 therein). MuSK-R sequences have been isolated and identified from humans, rats, mice, and xenopus. Closely related to human MuSK-R is a receptor isolated from the electric ray Torpedo californica designated Torpedo tyrosine kinase receptor, and ROR tyrosine kinase receptors (Jennings, et al. Proc. Natl. Acad. Sci. USA 90:2895 (1993); Masiakowski et al., J. Biol.
  • MuSK-Rs available from public depositories such as GeneBank and ATCC include accession numbers: ⁇ M005592; AF006464; A448972; AI800924; AI700028; AI341265; AI341122; AD02067; U34985; AA448972; and ATCC 75498. As mentioned above MuSK-R is specific to the skeletal muscle lineage.
  • MuSK-R as used in the present specification and claims is broadly defined to include the known MuSK-Rs (including DmK receptors), isoforms or variants of known MuSK- Rs having similar structure, tyrosine kinase receptors that are functionally similar to known MuSK-Rs and novel MuSK-Rs not previously described that are identified using screening techniques well known to those in the art. Such techniques may include the use of degenerate oligodeoxyribonucleotide primers.
  • the term MuSK-R when referring to a nucleic acid molecule includes (a) nucleic acid sequences comprising a coding region of a known mammaUan MuSK-R; (b) a nucleic acid sequence which hybridizes under stringent conditions to the nucleic acid of (a) and which encodes a mammalian MuSK-R; and (c) a degenerate MuSK-R wherein the MuSK-R has undergone changes in its nucleic acid sequence that does not significantly effect the properties of the MuSK-R protein encoded by the polynucleotide.
  • MuSK-R includes not only naturally occurring MuSK-Rs but also may include genetically engineered MuSK-Rs.
  • MuSK-R when referring to a polypeptide encompasses known MuSK receptors, isoforms or variants of MuSK-Rs, and functionally equivalent receptors.
  • a functionally equivalent receptor is a MuSK-R that can compete with a known MuSK-R for binding. More specifically, a functionally equivalent MuSK-R has at least 40%, preferably at least 60%, and more preferably at least 80% identical amino acids to the sequence set forth in SEQ ID NO: 8 and can compete with the MuSK-R illustrated in figure 6 for ligand or substrate binding.
  • mutated MuSK-R are used as selective markers to identify genetically modified cells. The marker is introduced on a nucleic acid construct into a target cell that normally does not express a MuSK-R.
  • the term "introduced” is broadly used herein to include inserted, incorporated and the like.
  • the molecule no longer possesses signaling activity.
  • Signaling activity has be generally defined as triggering a response pathway in the cytosol to the nucleus which ultimately leads to activation of transcription.
  • the lack of signaling activity may be due to a) use of a MuSK-R in tissue or cells other than muscle (Glass et al., Cell 85:513-523 (1996)) or b) use of a mutated MuSK-R.
  • MuSK-R While modifications of MuSK-R may be known, the method of identifying genetically modified cells comprising using a mutated MuSK-R as a selectable marker is not known.
  • MuSK-R may be used as a selective marker in tissue other than muscle.
  • the modifications to a MuSK-R include truncations and/or deletions of MuSK-Rs.
  • the mutation may occur in the extracellular domain and/or the intracellular domain by means well known in the art. The mutation causes the molecule to be devoid of signaling activity. Preferably the extracellular domain should still be capable of binding an antibody.
  • a preferred MuSK-R according to the invention is the sequence set forth in SEQ ID NOs: 7 and 8, designated herein as hMuSK-R.
  • the extracellular domain is encoded by nucleotides 1 through 1479
  • the transmembrane domain is encoded by nucleotides 1480 through 1545
  • the intracellular domain is encoded by nucleotides 1546 through 2607.
  • Other preferred MuSK-Rs are molecules closely related to the sequences set forth in SEQ ID NO: 8 and 7. Examples of closely related sequences are the sequences set forth in U.S. Pat No. 5,656,473 particularly NO: 16 and 17 disclosed therein.
  • MuSK-R Mutants of MuSK-R are known and reference is made to Apel et al., Neuron 18:623 - 635 (1997).
  • preferred modifications to a MuSK-R include modifications to the cytoplasmic domain such as deletions of at least 150, preferably at least 200, more preferably at least 250, more preferably 300, and most preferably at least 350 amino acids of the cytoplasmic domain.
  • the deletions are preferably truncations. Deletions or truncations may include deletion of tyrosine phosphorylation sites in the range of 1 to 19, preferably 2 - 15, more preferably 2 - 10 sites. Additionally the kinase catalytic site may be deleted from a MuSK-R.
  • the kinase catalytic site is found at approximately amino acid residues 672 to 691 of SEQ ID NO: 8. As long as the protein is stably expressed, there is no limitation to the number of sequences deleted or truncated in the cytoplasmic domain.
  • MuSK-Rs useful as selectable markers according to the invention include modifications to the MuSK-R sequence set forth in Figure 6 (SEQ ID NO: 8).
  • the MuSK-R is truncated by least 300 amino acid residues in the cytoplasmic domain.
  • One preferred embodiment includes the deletion of amino acid sequence 538 - 869 and is designated mMuSK-RI.
  • Another preferred embodiment includes the deletion of amino acid sequence 577 - 869 and is designated mMuSK-RJJ.
  • cytoplasmic domain mutations may be made in the extracellular domain.
  • the extracellular domain modification may include deletion of at least about 100 amino acids, preferably at least about 150 amino acids, more preferably at least about 200 amino acids, and still more preferably at least about 250 amino acids.
  • a mutated MuSK-R used as a selectable marker according to the invention preferably should contain an antibody- binding site in the extracellular domain.
  • a mutated PTKR, particularly a mutated EGFR or mutated MusSK-R useful as a selectable marker includes both modification of the cytoplasmic 12
  • the extracellular domain modifications may include deletion of at least about 100 amino acids, preferably at least about 150 amino acids, more preferably at least about 200 amino acids, and still more preferably at least about 250 amino acids.
  • the modification will include removal of one cysteine-rich area generally characterized by an area of about 100 amino acids.
  • Particularly preferred marker sequences are mutated EGFRl, EGFR2 and EGFR3 molecules having not only a truncation in the cytoplasmic domain but also a deletion of at least 200 and preferably at least 250 amino acids of the extracellular domain.
  • a mutated PTKR as a selectable marker useful in the methods of the invention should preferably include an antibody-binding site in the extracellular domain.
  • a preferred mutated EGFR molecule wherein both the extracellular domain and the cytoplasmic domain are modified is a sequence derived from WT EGFRl illustrated in Figure 2 (SEQ ID Nos. 1 and 2).
  • Particularly preferred is a mutated EGFRl sequence designated EGFRl-II wherein amino acid sequence 25 through 313 of the extracellular domain and amino acid sequence 679 through 1210 of the cytoplasmic domain of Figure 2 (SEQ ID NO. 2) are deleted.
  • mutated PTKR or a “mutated EGFR” or a “mutated MuSK-R” selectable marker refers to nucleotides or protein as appropriate from context.
  • Polynucleotides of the invention may be in the form of RNA or in the form of DNA, which DNA includes cDNA, genomic DNA or synthetic DNA.
  • PTKR molecules including members of the EGFR family may be obtained from sources such as various sequence databases including GenBank. Both random and site-directed mutagenesis methods may be effective to create mutations in wild type PTKRs. Random methods encompass altering the sequences within restriction endonuclease sites, inserting an oligonucleotide linker into a plasmid, using chemicals to damage plasmid DNA, and incorporating incorrect nucleotides during in vitro DNA synthesis. However, site-directed mutagenesis may be a more beneficial tool. Particularly preferred site-directed methods include 13
  • oligonucleotide-directed mutagenesis and polymerase chain reaction (PCR) -amplified oligonucleotide -directed mutagenesis.
  • PCR polymerase chain reaction
  • a mutated PTKR as a selectable marker concerns the ability to identify genetically modified mammalian cells in vitro, ex vivo, and in vivo.
  • the mutated PTKR sequence may be introduced into a target cell as part of a nucleic acid construct operatively linked to an expression control sequence
  • the construct including the mutated PTKR sequence is placed in a vector and then introduced into a target cell.
  • operatively linked refers to an arrangement of elements wherein the components are configured so as to perform their usual function. The control elements need not be contiguous with the coding sequence.
  • Vectors containing both a promoter and a cloning site into which a polynucleotide can be operatively linked are well known in the art. Such vectors are capable of transcribing RNA in vitro or in vivo, and are commercially available from sources such as Stratagene (La Jolla, CA) and Promega Biotech (Madison, WI). Examples of vectors include vectors derived from viruses, such as baculovirus, retrovimses, adenoviruses, adeno-associated viruses, and herpes simplex viruses; bacteriophages; cosmids; plasmid vectors; fungal vectors; synthetic vectors; and other recombination vehicles typically used in the art.
  • viruses such as baculovirus, retrovimses, adenoviruses, adeno-associated viruses, and herpes simplex viruses; bacteriophages; cosmids; plasmid vectors; fungal vectors; synthetic vectors;
  • the vector comprises a nucleic acid sequence coding for a selective marker according to the invention, operatively linked to an expression control sequence. Selection of appropriate control sequences is dependent on the target cell used and the choice is within the skill of one in the art.
  • expression control sequences also referred to as regulatory sequences, include promoters, enhancers, polyadenylation signals, RNA polymerase binding sequences, sequences conferring inducibility of transcription and other expression control elements, such as scaffold attachment regions (SARs).
  • the promoter may be either a prokaryotic or eukaryotic promoter.
  • the promoter may be a tissue specific promoter, inducible promoter, synthetic promoter, or hybrid promoter. More than one promoter may be placed in the construct.
  • promoters include but are not limited to the phage lamda (PL) promoter; SV40 early promoter; adenovirus promoters, such as adenovirus major late promoter (Ad MLP); herpes simplex virus (HSV) promoter; a cytomegalovirus (CMV) promoter; such as the human CMV immediate early promoter; a long terminal repeat (LTR) promoter, such as a MoMLV LTR; the U3 region promoter of the Moloney murine sarcoma virus; Granzyme A promoter; regulatory sequences of the metallothioein gene; CD34 promoter; CD8 promoter; thymidine kinase (TK) promoters; B19 parvovirus promoters; and rous sarcoma virus (RSV) promoter.
  • PL
  • promoter elements from yeast and other fungi may be used, such as Gal 4 promoter and the alcohol dehydrogenase (ADH) promoter. These promoters are available commercially from various sources such as Stratagene (La Jolla, CA). It is to be understood that the scope of the present invention is not to be limited to a specific promoter.
  • the vector may further comprise a polyadenylation signal that is positioned 3' of the carboxy-terminal amino acid. Vectors containing both a promoter and a cloning site into which a polynucleotide can be operably linked are well known in the art. Such vectors are capable of transcribing RNA in vitro or in vivo, and are commercially available.
  • exemplary vectors include the pCMV mammalian expression vectors, such as pCMV6b and pCMV6c (Chiron Corporation, CA), pSFFV-Neo, and pBluescript-SK+.
  • Retroviral vectors are retroviral vectors and reference is made to Coffin et al., "Retroviruses", (1997) Chapter 9 pp; 437-473 Cold Springs Harbor Laboratory Press. Retroviral vectors useful in the invention are produced recombinantly by procedures already taught in the art. WO94/29438, WO97 21824 and WO97 21825 describe the construction of retroviral packaging plasmids and packing cell lines. Common retroviral vectors are those derived from murine, avian or primate retroviruses. The most common retroviral vectors are those based on the Moloney murine leukemia virus (MoMLN) and mouse stem cell virus (MSCN).
  • MoMLN Moloney murine leukemia virus
  • MSCN mouse stem cell virus
  • Vectors derived from MoMLV include, LMily, LI ⁇ GFER, MI ⁇ GFR, M ⁇ D and MINT (Bender, et al., J. Virol. 61 :1639 -1649 (1987); Miller, et al., Biotechniques 7: 98-990 (1989); Robbins, et al., J. Virol. 71 :9466-9474 (1997) and U. S. Pat. No. 5,707,865).
  • Vectors derived from MSCV include MSCV-MiLy (Agarwal, et al., J. of Virology 72:3720).
  • vectors include those based on Gibbon ape leukemia virus (GALV), Moloney murine sacroma virus (MoMSV), myeloproliferative sarcoma virus (MPSV), murine embryonic stem cell virus (MESV), spleen focus forming virus (SFFV) and the lentiviruses, such as Human immunodeficiency virus (HJN-1 and HTV-2).
  • GALV Gibbon ape leukemia virus
  • MoMSV Moloney murine sacroma virus
  • MPSV myeloproliferative sarcoma virus
  • MMV murine embryonic stem cell virus
  • SFFV spleen focus forming virus
  • New vector systems are continually being developed to take advantage of particular properties of parent retroviruses such as host range, usage of alternative cell surface receptors and the like (See C. Baum et al., Chapter 4 in Gene Therapy of Cancer Cells eds., Lattime and Gerson (1998)).
  • the present invention is not limited to particular retroviral vectors, but may include any retroviral vector.
  • Particularly preferred vectors include DNA from a murine virus corresponding to two long terminal repeats, and a packaging signal.
  • the vector is a MoMLV or MSCV derived vector.
  • the vector is MND.
  • the viral gag, pol and env sequence will generally be removed from the virus, creating room for insertion of foreign DNA sequences.
  • Genes encoded by foreign DNA are usually expressed under the control a strong viral promoter in the long terminal repeat (LTR). While a LTR promoter is preferred, as mentioned above, numerous promoters are known.
  • LTR long terminal repeat
  • Such a construct can be packaged into viral particles efficiently if the gag, pol and env functions are provided in trans by a packaging cell line. Therefore when the vector construct is introduced into the packaging cell, the gag-pol and env proteins produced by the cell, assemble with the vector RNA to produce infectious virions that are secreted into the culture medium. The virus thus produced can infect and integrate into the DNA of the target cell, but does not produce infectious viral particles since it is lacking essential packaging sequences. Most of the packaging cell lines currently in use have been transfected with separate plasmids, each containing one of the necessary coding sequences, so that multiple recombination events are necessary before a replication competent virus can be produced. Alternatively the packaging cell line harbors a provirus.
  • RNA produced from the recombinant virus is packaged instead. Therefore, the virus stock released from the packaging cells contains only recombinant virus.
  • retroviral packaging lines include PA12, PA317, PE501, PG13, ⁇ CRIP, RD114, GP7C-tTA-G10, ProPak-A (PPA-6), and PT67. Reference is made to Miller et al., Mol. Cell Biol.
  • Retroviral vector DNA can be introduced into packaging cells either by stable or transient transfection to produce vector particles.
  • vectors include adenoviral vectors (See Frey et al., Blood 91 :2781 (1998) and WO95/27071) and adeno-associated viral vectors (AAV) (See Chatteijee et al., Current Topics in Microbiol. and Immunol. 218:61 (1996). Reference is also made to Shenk, Chapter 6, 161- 78, Breakefield et al., Chapter 8 201-235; Kroner-Lux et al., Chapter 9, 235-256 in Stem Cell Biology and Gene Therapy, eds. Quesenberry et al., John Wiley & Sons, 1998 and U.S. Pat. Nos. 5,693,531 and 5,691,176.
  • adenovims derived vectors may be advantageous under certain situations because they are capable of infecting non-dividing cells, and unlike retroviral DNA, the adenoviral DNA is not integrated into the genome of the target cell. Further the capacity to carry foreign DNA is much larger in adenoviral vectors than retroviral vectors.
  • the adeno-associated viral vectors are another useful delivery system.
  • the DNA of these viruses may be integrated into non-dividing cells, and a number of polynucleotides have been successfully introduced into different cell types using adeno- associated viral vectors.
  • the vectors are capable of transducing several cell types including hematopoietic cells and epithelial cells.
  • Vectors may also include hybrid vectors of AAV and adenoviruses as described in WO96/13598 and WO99/47691 (The Trustees of the University of Pennsylvania), WO98/21345 (General Hospital), US5965441 (General Hospital), or WO99/58700 (Ariad Gne Therap.), the teaching of which being incorporated into the present invention in their entirety.
  • the construct or vector will include not only a nucleic acid sequence encoding a mutated PTKR or a mutated EGFR as a selectable marker but also a second nucleic acid sequence encoding a heterologous gene to be transferred into a target cell.
  • the nucleic acid molecules are DNA.
  • heterologous as it relates to nucleic acid sequences such as gene sequences denotes sequences that are not normally associated with a particular cell or vector and which are suitably inserted into a construct or vector under control of a promoter to permit expression in the target cell to be genetically modified.
  • the heterologous gene may be located 5' or 3' to the selectable marker of the invention.
  • Non-limiting preferred vector constructs comprise the general structure as outlined below 5' to 3':
  • LTR-X-I-M-SAR-LTR wherein LTR is a long terminal repeat, X is a heterologous gene for a desired protein, M is a selectable marker of the invention, I is an internal ribosomal entry site, SAR is a scaffold attachment region and p is a second promoter, preferably a CMV or PGK promoter.
  • a gene or coding sequence or a sequence that encodes a particular protein is a nucleic acid molecule that is transcribed and translated into a polypeptide in vitro or in vivo when placed under the control of appropriate regulatory sequences.
  • the heterologous gene may be any gene for which expression is desired.
  • heterologous genes include but are not limited to a therapeutic protein, a structural gene, a ribozyme, or an antisense sequence.
  • the heterologous gene may be the entire protein or only the functionally active fragment thereof.
  • the protein may include for example one that regulates cell differentiation or a therapeutic gene capable of compensating for a deficiency in a patient that arises from a defective endogenous gene.
  • a therapeutic protein or gene may be one that antagonizes production or function of an infectious agent, antagonizes pathological processes, improves a host's genetic makeup, or facilitates engraftment.
  • a therapeutic gene or gene sequences are ones effective in the treatment of adenosine deaminase deficiency (ADA); sickle cell anemia; recombinase deficiency; recombinase regulatory gene deficiency; HIV, such as an antisense or trans- dominant REV gene; or a gene carrying a herpes simplex virus thymidine kinase (HSV-tk)).
  • the heterologous gene may encode new antigens or drug resistant genes or may encode a toxin or an apoptosis inducer effective to specifically kill cancerous cells, or a specific suicide gene to hematopoietic cells may be included.
  • heterologous gene may be a therapeutic gene that is non-human such as a yeast gene (Seo et al., Proc. Natl. Acad. Sci. 95:9167 (1998)).
  • the vector or construct may also comprise a second heterologous gene in addition to the first heterologous gene encoding a protein of interest. More than one gene may be necessary for the treatment of a particular disease. Alternatively more than one gene can be delivered using several compatible vectors.
  • the therapeutic gene can include regulatory and untranslated sequences. For human patients the therapeutic gene will generally be of human origin although genes of closely related species that exhibit high homology and biologically identical or equivalent function in humans may be used if the gene does not produce an adverse immune reaction in the recipient.
  • Nucleotide sequences of the heterologous gene encoding a protein of interest will generally be known in the art or can be obtained from various sequence databases such as GenBank.
  • GenBank One skilled in the art will readily recognize that any structural gene can be excised as a compatible restriction fragment and placed in a vector in such a manner as to allow proper expression of the structural gene in target cells.
  • the target cells of the invention are mammalian ceDs and these include but are not limited to humans, mice, monkeys, chimpanzees, farm animals; such as cattle, sheep, pigs, goats, and horses, sport animals, pets; such as dogs and cats, and other laboratory rodents and animals; such as mice, rats, guinea pigs and the like.
  • the target cells are human cells.
  • Preferred human cells include liver, hematopoietic, smooth muscle, neural, endothelial vascular cells, tumor cells and epithelial cells.
  • Hematopoietic cells are particularly preferred, and these cells encompass hematopoietic stem cells, erythrocytes, neutrophils, monocytes, platelets, mast cells, eosinophils and basophils, B and T lymphocytes, and NK cells as well as the respective lineage progenitor cells. Hematopoietic stem cells and T-cells are especially preferred. Hematopoietic stem cells (HSC) are defined as a population of hematopoietic cells containing long term multilineage repopulating potential. T-cells are defined as a type of lymphocyte and are thought to develop from hematopoietic stem cells. There are many types of T-cells including cytotoxic T-cells, helper T-cells, inducer T-cells and supressor T cells.
  • HSC Hematopoietic stem cells
  • T-cells are defined as a type of lymphocyte and are thought to develop from hematopoietic stem cells.
  • Non-hmiting sources of hematopoietic cells are bone marrow, embryonic yolk sac, fetal liver tissue, adult spleen, and blood such as adult peripheral blood and umbilical cord blood.
  • Bone marrow cells may be obtained from ilium, sternum, tibiae, femora, spine and other bone cavities. The manner in which target cells may be separated from other cells is not critical to this invention.
  • FACS fluorescence activated cell sorters
  • cell separation is not critical to the invention, and specific cell types may be separated either prior to genetic modification with the mutated PTKR or after genetic modification. Preferably cells are initially separated by a coarse separation followed by using positive and/or negative selection.
  • the surface antigen expression profile of an enriched hematopoietic stem cell population may be identified by CD34 + Thy-1 + Lin " .
  • Other nonlimiting enriched phenotypes may include: CD2 ' , CD3 " , CD4 " , CD8 “ , CD10 “ , CD14 “ , CD15 ⁇ CD19 “ , CDW109 + , glycophorin- , AC133 + , HLA-DR*' " , and EM + .
  • Lin " refers to a cell population selected on the basis of lack of expression of at least one lineage specific marker, such as, CD2, CD3, CD14, CD15 and CD56.
  • the combination of expression markers used to isolate and define an enriched HSC population may vary depending on various factors and may vary as other express markers become available.
  • Murine HSCs with similar properties to the human CD34 + Thy-1 + Lin " may be identified by kit + Thy-l .l to Lin ⁇ o Sca- (KTLS). Other phenotypes are well known.
  • KTLS Kit + Thy-l .l to Lin ⁇ o Sca-
  • Other phenotypes are well known.
  • TCR T cell antigen receptor
  • B cells may be selected, for example, by expression of CD 19 and CD20.
  • Myeloid cells may be selected for example, by expression of CD14, CD15 and CD16.
  • NK cells may be selected based on expression of CD56 and CD16.
  • Erythrocytes may be identified by expression of glycophorin A.
  • Neuronal cells may be identified by NCAM and LNGFR (Baldwin et al., J. Cell Biochem. 15:502 (1996)).
  • Vascular endothelial cells may be identified by VEGFR2, CD34, P-Selectin, VCAM-1, ELAM-1, and ICAM-l.(Horvathova et al., Biol. Trace Elem. Res., 69: 15-26 (1999).
  • the cells are cultured in a suitable medium comprising a combination of growth factors that are sufficient to maintain growth.
  • a suitable medium comprising a combination of growth factors that are sufficient to maintain growth.
  • Methods for culturing target cells are well known to those skilled in the art, and these methods are only briefly mentioned herein. Any suitable culture container may be used, and these are readily available from commercial vendors.
  • the seeding level is not critical and will depend on the type of cells used, but in general the seeding level for hematopoietic cells will be at least 10 cells per ml, more usually at least about 100 cells per ml and generally not more than 10 6 cells per ml when the cells express CD34.
  • Various culture media, solid or liquid can be used and non-limiting examples include
  • DMEM fetal calf serum
  • IMDM X-vivo 15
  • RPMI-1640 fetal calf serum
  • the medium can be serum free or supplemented with suitable amounts of serum such as fetal calf serum, autologous serum or plasma. If cells or cellular products are to be used in humans, the medium will preferably be serum free or supplemented with autologous serum or plasma.
  • Non-limiting examples of compounds which may be used to supplement the culture medium are TPO, FL, KL, IL-1, IL-2, IL-3, IL-6, IL-12, JX-11, stem cell factor, G-CSF, GM- CSF, Stl factor, MCGF, LIF MJP-l ⁇ and EPO. These compounds may be used alone or in any combination, and preferred concentration ranges may be readily determined from the published art.
  • a preferred non-limiting medium includes mIL-3, mIL-6 and mSCF.
  • Other molecules can be added to the culture media, for instance, adhesion molecules, such as fibronection or RetroNectinTM (commercially produced by Takara Shuzo Co., Otsu Shigi, Japan).
  • LTCIC long-term culture initiating cell assay
  • CAFC cobblestone-area-forming cell
  • This assay gives frequency readouts that correlate with LTCIC and are predictive of engraftment in in vivo assays and patients.
  • a particularly preferred CAFC assay is described in Young, et al., Blood 88:1619 (1996).
  • Flow cytometry can be used to subset hematopoietic cells from various tissue sources by the surface antigens they express.
  • a combination of these assays may be used to test for hematopoietic cells or stem cells.
  • the invention concerns a method of identifying genetically modified mammalian cells comprising introducing a nucleic acid sequence encoding a mutated protein-tyrosine kinase receptor (PTKR) as a selective marker and operatively linked to an expression control sequence into a target cell to form a genetically modified cell; allowing expression of the mutated PTKR in the genetically modified cell; and identifying said genetically modified cell expressing the mutated PTKR.
  • the selective marker is a mutated epidermal growth factor receptor (EGFR) family member, particularly preferred is a mutated EGFRl and more specifically the sequences designated EGFRl-I and EGFRl-II.
  • a polynucleotide or nucleic acid sequence is said to "encode" a polypeptide if, in its native state or when manipulated by methods well known to those of skill in the art it can be transcribed and/or translated to reproduce a polypeptide or fragment thereof.
  • a construct or vector including a mutated PTKR of the invention may be introduced into the target population by any means of genetic transfer or modification known in the art.
  • genetic modification refers to any addition, deletion or disruption to a cells normal nucleotides and the methods of genetic modification are intended to encompass any genetic modification method of introducing nucleic acid sequences encoding the selective markers according to the invention and including but not limited to heterologous or foreign genes into mammalian target cells. These techniques are generally known.
  • introducing is broadly used herein, and includes for example inserting.
  • genetic modification includes but is not limited to transduction (viral mediated transfer of host DNA from a host or donor to a recipient, either in vivo or ex vivo), transfection (transformation of cells with isolated viral DNA genomes), liposome mediated transfer, electroporation, calcium phosphate transfection and others.
  • Methods of transduction include direct co-culture of cells with producer cells (Bregni, et al., Blood 80:1418-1422 (1992)); culturing with viral supernatant alone, with or without appropriate growth factors and polycations (Xu, et al., Exp. Hemat. 22:223-230 (1994), and spinoculation.
  • the mutated PTKRs of the invention and particularly the mutated EGFR markers are introduced into target cells by transduction with a retroviral vector as previously described.
  • the host cell range that may be infected is determined by the viral envelope protein.
  • the recombinant virus can be used to infect virtually any other cell type recognized by the env protein provided by the packaging cell, resulting in the integration of the viral genome in the transduced cell and the stable incorporation of the foreign gene product.
  • murine ecotropic env of MoMLV allows infection of rodents cells
  • amphotropic env allows infection of rodent, avian and primate cells including human cells.
  • Amphotropic packaging of cell lines for use with retroviral systems are known in the art and are commercially available.
  • Xenotropic vector systems also exist which allow infection of human cells.
  • the target cells are genetically transformed with a nucleotide sequence including the mutated PTKR as a marker, and optionally a heterologous gene, the modified cells expressing the mutated PTKR selectable marker may be identified by numerous techniques.
  • identify or “identification” used herein in reference to genetically modified cells unless otherwise indicated means to mark, to purify, to enrich, to select, to isolate, or to separate. Identification can be by a single or multiple steps.
  • the genetically modified cells expressing the selective marker according to the invention are identified by an antibody that specifically recognizes and binds to the selectable marker, and particularly an antibody that recognizes a mutated EGFR.
  • This kind of antibody has been described by ORourke, et al., Oncogene, 16:1197-1207 (1998).
  • a secondary antibody may then be used to further identify or select the antibody coated cells, if the secondary antibody is coupled to either a fluorophore or immuno-magnetic beads.
  • the marker gene expressing cells may then be selected by flow cytometry or a by using a magnet to select bead-coated cells.
  • the techniques used for the identification of genetically modified cells expressing a selective marker of the invention include those described above and other well known techniques including but are not Umited to immunose lection; nucleotide detection by northern blots wherein RNA bound to a solid support is analyzed for binding to a liquid phase DNA; nucleotide detection by southern blots, wherein genomic DNA bound to a solid support is analyzed for binding to a liquid phase DNA; PCR amplification of genomic DNA; protein detection by western blots whereby protein bound to a solid support is analyzed for binding to a liquid phase antibody; reverse transcription of mRNA and amplification with PCR; and FISH wherein chromosomes are analyzed by Fluorescence in situ hybridization with a liquid phase DNA (Lawrence, et al., Science, 249(4971 ):928 -932 (1990)).
  • antibodies may be obtained by methods well known and reference is made to Harlow, et al., "Antibodies: A Laboratory Manual: (1988), Biosupplynet Source Book, (1999) Cold Springs Harbor Laboratory. Either polyclonal antibodies that are reactive to the antigen of interest may be used or monoclonal antibody producing cell clones may be generated. According to the invention the antibody must recognize the extracellular domain of the mutated PTKR selective marker. More particularly if parts of the extracellular domain are modified, by for example deletion, the antibody should recognized an epitope of the remaining amino acid sequence of the mutated PTKR.
  • Monoclonal antibodies to EGFR are available commercially. Some sources include Calbiochem (CA), Pharmingen (CA), Becton Dickinson (CA), and the American Type Culture Collection (ATCC) (Virginia).
  • CA Calbiochem
  • CA Pharmingen
  • CA Becton Dickinson
  • ATCC American Type Culture Collection
  • the antibody may be identified and assayed in vitro by a range of methods including gel diffusion, immunoassay, immunoelectrophoresis and immunofluorescence. Once genetically modified cells are labeled they can be incubated with an antibody against the mutated receptor.
  • the genetically modified cells may be physically separated by the use of antibodies, flow cytometry including fluorescence activated cell sorting (FACS), and bead selection. (U.S. Pat. No. 5,011,912).
  • FACS fluorescence activated cell sorting
  • an antibody that recognizes the mutated PTKR may be used to identify the cells that express the PTKR.
  • This primary antibody can be conjugated to a fluorophore, such as fluorescein isothiocyanate (FITC), phycoerythrin (PE), cy-chrome (CyC), allophycocyanine (APC), tricolor (TC) or Texas Red (TX).
  • FITC fluorescein isothiocyanate
  • PE phycoerythrin
  • CaC cy-chrome
  • APC allophycocyanine
  • TX Texas Red
  • a secondary antibody that is conjugated to a fluorophore may be introduced into the cell sample containing cells which have expressed the mutated PTKR and which are recognized by the primary antibody.
  • the primary antibody is attached to the mutant PTKR.
  • the secondary antibody binds to the primary antibody and cells having the secondary antibody can be induced to fluoresce. Separation may be achieved by the fluorescence activate
  • the genetically modified target cells expressing a mutated PTKR, and optionally a second nucleic acid sequence encoding a protein of interest may be expanded, either prior to or after identification and selection, by culturing the cells for days or weeks in appropriate culture media, with or without supplements by means well known in the art. (Freshney supra, Celis supra, and Coligan et al., supra).
  • the genetically modified marked cells obtained according to the methods of the invention may further be used in an autologous or allogeneic setting wherein the genetically modified target cells, preferably hematopoietic cells are expanded and then used in gene therapy for example in bone marrow transplantation, graft facilitation, or immune reconstitution.
  • the expanded cells expressing the mutated cell surface marker may be infused into a subject. Samples may be taken and then tested for the selectable marker by FACS analysis, PCR, or FISH, as referenced above, to determine the persistence of the marked cells and further to assess efficiency of transduction.
  • EGFR cDNA is isolated by PCR from cDNA that has been generated from colorectal adenocarcinoma: SW480 cell line (Marathon cDNA, Clontech (CA); Leibowitz, et al., Cancer Research 36:4562-4569 (1976).
  • the following primers are used in the described methods and were obtained from Life Technologies (MD):
  • the primers EGFRl and EGFR2220R are used to generate an intracellular deletion mutant of EGFR ( Figures 2 and 3) from the SW480 adenocarcinoma cell line cDNA. Amino acid residues 679 - 1210 of the sequence in Figure 2 (SEQ ID NO. 2) are deleted, and this mutant EGFR is designated EGFRl -I.
  • the 5' primer EGFRl covers the start codon of the EGFR.
  • the 3 '-primer EGFR2220R contains a stop codon in place of amino acid 679 of the SEQ ID NO. 2 ( Figure 2).
  • Using primers EGFRl and EGFR2220R results in the amplification of the EGFR sequence that has a deletion of amino acids 679 to 1210.
  • the following PCR reaction is performed: Marathon cDNA ( ⁇ 0.5 ng) is mixed with
  • PCR is performed as follows: Cycle 1 : 95°C for 5 min, Cycle 2-15: 95°C for 1 min, 60°C for 1 min, 68°C for 4 min, and Cycle 16: 68°C for 10 min.
  • the reaction is then cooled to 4°C in the PCR machine and subsequently the amplified cDNA is ethanol precipitated with 0.3 M sodium acetate.
  • the pellet is washed once with 70% ethanol, dried and resuspended in 50 ⁇ l H 2 O.
  • the reaction is cooled to 4°C in the PCR machine and the amplified cDNA is ethanol precipitated with 0.3 M sodium acetate. The pellet is washed once with 70% ethanol, dried and resuspended in 20 ⁇ l H 2 O.
  • the PCR reaction is loaded on a lxTAE gel. A band with the size of approximately 2200 bp is isolated from the gel and cloned into the Srfl restriction site of pPCR-Script Amp vector (Stratagene; CA) according to the manufacturer's protocol. The resulting vector is called pPCR-Script EGFRl-I. The correctness of the subcloned PCR product is confirmed by restriction analysis and sequencing according to well known methods.
  • EGFRl -II contains the same deletion in the intracellular domain as EGFRl-I (a stop codon in position 679).
  • amino acids 25 to 312 are deleted in the extracellular domain of EGFR depicted in Figure 2 (SEQ ID NO. 2).
  • the sequence of the signal peptide, amino acid 1 to 24, is fused to amino acid 313 of the EGFR extracellular domain.
  • a protocol is used that is described in further detail below. (Also see White, ed. Methods in Molecular Biology, Vol 15. PCR Protocols, Chapter 25 (1993)). This method is known in the art as "gene splicing by overlap extension (gene SOEing)".
  • Primer pairs EGFR1/EGFR3 and EGFR2 EGFR2220R are used to amplify 2 PCR products (a) and (b).
  • Primer EGFRl encodes amino acid 1 to 7
  • Primer EGFR3 encodes amino acid 18 to 24 fused to the nucleotide sequence of amino acid 313 to 319.
  • a PCR product is generated that encodes amino acid 1 to 24 which is fused to amino acid 313 to 319.
  • Primer EGFR2 encodes the same amino acid acids as Primer EGFR3 only in the reverse orientation.
  • Primer EGFR2220R encodes a stop codon in place of amino acid 679. As described in section B above, this results in the deletion of the intracellular domain after amino acid 678.
  • primers EGFRl and EGFR2220R are used with the intermediate PCR products (a) and (b).
  • these intermediate PCR products are mixed, denatured and reannealed, one of the strands of the two PCR products can overlap at their 3' ends, and act as primers on one another to make the mutant product.
  • the mutant PCR product (c) is ampUfied with the primers EGFRl and EGFR2220R.
  • the resulting PCR product encodes EGFRl-II amino acid 1 to 24 of the wt EGFR fused to amino acids 313 to 678 of the wt EGFR with a stop codon in position 679 ( Figure 4).
  • the PCR reactions are as follows: The first PCR reaction in: a.) 0.01 OD 26 o of EGFRl and 0.01 OD ⁇ o of EGFR3 and in b.) 0.01 OD ⁇ o EGFR2 and 0.01 OD 260 EGFR2220R are mixed with -0.4 ng Marathon cDNA, lx Advantage cDNA Polymerase buffer (as described in B), 1 ⁇ l Advantage cDNA polymerase, 2.5 ⁇ mol of each dNTP, 5U Pfu Polymerase (Promega) and H 2 O in a final volume of 50 ⁇ l.
  • the PCR is performed as follows: Cycle 1 : 94°C for 5 min, Cycle 2-16: 94°C for 0.5 min, 60°C for 1 min, 68°C for 7 min, and Cycle 17: 68°C for 10 min.
  • the reactions are cooled to 4°C in the PCR machine and the amplified PCR products are ethanol precipitated with 0.3 M sodium acetate.
  • the pellets are washed once with 70% ethanol, dried and resuspended in 50 ⁇ l H 2 O.
  • each PCR reaction (a) and (b) are then further amplified.
  • the reaction mix contains for the second round of amplification, in addition to each PCR reaction (a) 0.01 OD 260 of primers EGFRl and EGFR3 or (b) 0.01 OD 260 of primers EGFR2 and EGFR2220R: 1 x Pfu buffer, 2.5 ⁇ mol of each dNTP, and 5 U Pfu Polymerase (Promega; WI) and H 2 O in a final volume of 50 ⁇ l.
  • the PCR is performed as follows: Cycle 18: 94°C for 5 min, Cycle 19-49: 94°C for 0.5 min, 60°C for 1 min, 72°C for 6 min and Cycle 50: 72°C for 10 min.
  • the reactions are then cooled to 4°C in the PCR machine and the amplified PCR products are phenol/chloroform extracted and ethanol precipitated with 0.3 M sodium acetate.
  • the pellet is washed once with 70% ethanol, dried and resuspended in 20 ⁇ l H 2 O.
  • PCR product (a) and (b) are mixed in equimolar amounts and reamplified with Pfu Turbo Polymerase (Stratagene) using the primer pair EGFRl and EGFR2220R.
  • the PCR mix is the same as described for cycles 18-50.
  • the PCR is performed as follows: Cycle 1: 95°C for 5 min, Cycle 2 - 32: 95°C for 1 min, 60°C for 1 min, 72°C for 4 min, and Cycle 33: 72°C for 10 min.
  • the PCR is then loaded on a gel (as described above) and a band of -1200 bp is isolated.
  • This PCR product is cloned into the Srfl site pPCR-Script (Stratagene; CA) according to the manufacturer's protocol.
  • the resulting vector is called pPCR- Script EGFR1-JJ.
  • the correctness of the subcloned PCR product (c) is confirmed by restriction analysis and sequencing according to well known methods.
  • EGFRl-I and EGFRl-II sequences are excised from pPCR-Script EGFR-I and pPCR-Script EGFR-II using the restriction sites Notl and Xhol and are cloned into the multiple cloning site of the Murine Leukemia Virus (MoMLV) based retroviral vector pGla (GTI, Maryland) - (pGla-mutated sequence - IRES- nerve growth factor receptor (NGFR)), which has been cut with Notl and Xhol.
  • the retroviral vectors are designated pGlaEGFRl-I and pGlaEGFRl-H.
  • the constructs are cotransfected into human embryonic kidney cells 293T (293T cells) (Gary Nolan, Stanford) with an envelope construct pCiGL that permits expression of the Vesicular Stomatitis Virus G-Protein (VSV-G envelope) under the control of the cytomegalovirus (CMV). Also cotransfected into 293T cells is the packaging construct pCiGP (encoding MoMLV gag-pol under the control of the CMV promoter ) using the CaCl 2 technique (Clontech; CA). (WO 97/21825 and Rigg, et al., Virology 218:290-295 (1996))
  • Viral supernatants are collected 24, 48, and 72 hours after transfection, centrifuged at 1200 rpm in a Beckman GS-6KR centrifuge to remove particulate material, and either used immediately to transduce ceUs or frozen in a dry ice/methanol bath.
  • the viral supernatants are used to transduce the packaging cell line ProPak-A-6 (PPA-6) (Systemix, Inc.).
  • PPA-6 cell line is a derivative of 293T cells expressing the MLV amphotropic envelope and MLV gag/pol under the control of the CMV promoter (Rigg et al. supra).
  • the PPA-6 cells are sorted by immuno-magnetic bead selection.
  • the cells are sorted by FACSorting as described in detail below.
  • Supernatants from PPA-6 cells are collected on day 2, 3 and 4 after transduction and treated as described for 293T cells.
  • the generated supernatants of PPA-6 cells contain recombinant viral particles that have the amphotropic envelope and are used to transduce human primary cells and cell lines.
  • Tissue culture and cell lines The following cell lines and primary cells are used: (a) human T cell Une, CEMSS
  • Human primary T cells are obtained by isolating the mononuclear cell fraction from human blood using Ficoll-gradient centrifugation (Noble and Cutts, Can. Vet. J. 8: 110 - 111 (1967) and Boyle and Chow, Transfusion 9:151 - 155 (1969).
  • PBMCs Peripheral blood mononuclear cells
  • the resulting PBMCs are further purified on a second Ficoll gradient to remove remaining blood cells.
  • the PBMCs are incubated for 1 hour in a tissue culture flask in tissue culture media for PBMCs (see below) at 37°C in 5% CO 2 allowing adherent cells such as macrophages to attach to the tissue culture flask.
  • Non-adherent cells T/B/NK cells
  • Cells (2 x 10 8 ) are incubated with 300 ⁇ l anti-CD4 antibody for 1 hour at 4°C.
  • the cells are washed 3 times with PBS and incubated with 1 x 10 8 anti-mouse IgG coupled immuno-magnetic beads (Dynal, Oslo) for 1 hour at 4°C.
  • the CD4 + cells that bound the CD4-antibodies and the secondary antibody coupled magnetic beads are positively selected by incubating the cells for 10 minutes on a Dynal magnet. After removing the unbound cells, the remaining cells are taken off the magnet and put into culture (see below). Usually the beads remain for up to 10 days on the cells.
  • CD34 + cells are isolated from G-CSF mobihzed peripheral blood (MPB) using Isolex 300SA or 3001 (Baxter, EL) (Systemix, CA). The cells are approximately 80 - 90% pure.
  • DMEM fetal calf serum
  • PBS fetal bovine serum
  • sodium pyruvate obtained from JRH Biosciences (California), FBS from Hyclone (Utah), L-glutamine, Trypsin and MEM vitamins from Life Technologies (Maryland), ITS (insulin/transferrin/sodium selenite), PHA (phythemaglutinin), Interleukin-2 (11-2) from Sigma (Missouri).
  • the cells are cultured in DMEM, 10% FBS, 1% sodium pyruvate, 1% L-glutamine (293 T, PPA-6); RPMI, 10% FBS, 1% sodium pyruvate, 1% L-glutamine (CEMSS), Iscove's medium 10% FBS, 1% L-glutamine, 1% ITS (insulin/transferrin/sodium selenite, 0.5 mg/ml stock), 1 % MEM vitamins (human PBMCs).
  • CD4 + T cells are stimulated every 10-12 days with 1-2 ⁇ g/ml PHA, irradiated feeder cells and 40 U/ml E-2.
  • Irradiated feeder cells are generated as follows: PBMCs are isolated as described above and irradiated with 3500 rads. The cells are mixed at a 10:1 ratio with the B cell lymphoma JY (Barbosa, et al., Proc. Natl. Acad. Sci. 81:7549-7553 (1984)) that had been irradiated with 6000 rads.
  • B cell lymphoma JY Barbosa, et al., Proc. Natl. Acad. Sci. 81:7549-7553 (1984)
  • T cells are transferred into fresh medium that contained 20 U/ml E-2.
  • CD34 + cells are grown in X vivo 15, 50 ng/ml thrombopoietin) (TPO; R & D Systems, MN), 100 ng/ml flt-3 ligand (FL), 100 ng/ml steel factor (SF). (SyStemix, Inc.)
  • TPO thrombopoietin
  • FL 100 ng/ml flt-3 ligand
  • SF steel factor
  • the cells are washed once with PBS, then trypsinized for 5 min and subsequently split into new tissue culture flasks (NWR, New Jersey).
  • PPA-6 human T cell lines, primary T cells and CD34 + cells
  • 10 6 cells/ml from step (E) are transduced with 1-3 ml of viral supernatant, that has been either generated from 293 T cells or PPA-6 cells, by spinoculation with 8 ⁇ g/ml protamine sulfate (Sigma, Missouri). Spinoculation is done at 37°C for 3 hrs at 3000 rpm (CD34 + cells, PPA-6) or at 2750 rpm (human T cells, CEMSS).
  • PPA-6 cells are transduced in 6 well plates, non adherent cells in 6 ml tubes (NWR). Human T cells and CD34 + cells are activated for 2 days prior to the transduction procedure with PHA/IL-2 or TPO/FL/SF, respectively.
  • FACS analysis FACSsorting and immuno-magnetic bead selection of cells expressing EGFRl-I and EGFRl-II:
  • CD4-FTf C (Caltag; CA), anti-CD34-APC (Becton Dickinson; ⁇ J), Thy-l-PE (Becton Dickinson), propidium iodide (PI) (Sigma), unconjugated anti-EGFR antibody (GR01) (Calbiochem; CA), goat anti-mouse IgG- PE/FTTC (Caltag), goat anti-mouse IgG2 a -PE/FTTC (Caltag), and anti-mouse IgG coupled magnetic beads (Dynal; Oslo).
  • the anti-bodies and immuno-magnetic beads were used according to manufacturer's protocol.
  • lxlO 6 cells are stained in 50 ⁇ l of PBS/2% FBS for 20 to 60 minutes at 4°C.
  • a secondary antibody When a secondary antibody is used the cells are washed once with 2 ml of PBS/2%FBS, then incubated again in 50 ⁇ l PBS/2%FBS and the secondary antibody is added. Before FACS analysis, the cells are again washed once with PBS 2%FBS, centrifuged and then resuspended in 500 ⁇ l PBS/2 %FCS containing 1 ⁇ g/ml PI.
  • FACS analysis is performed on a FACSscan (Becton-Dickinson Immunocytometry Group; CA) according to manufacturer's instructions.
  • Cells that express the marker genes, EGFRl-I and EGFRl-II are sorted as follows: 2 x 10 7 /ml PBS/2%FBS cells are stained with an anti-EGFR antibody (at least with l ⁇ l antibody/10 6 cells; GR01). The cells are washed twice with PBS/2%FBS and subsequently incubated with a fluorophore (FTTC, PE) conjugated rat anti-mouse IgG antibody (5 ⁇ l/10 6 cells) that recognizes the anti-EGFR antibody. The cells are also stained with PI to distinguish between dead and live cells, lx 10 6 cells/ml are sorted for EGFR-positive and Pl-negative cells. This is done on a FACSstar Plus (Becton-Dickinson Immunocytometry Group) according to manufacturer's protocol.
  • FTTC, PE fluorophore conjugated rat anti-mouse IgG antibody
  • the cells are stained with an anti-EGFR antibody.
  • the 10 7 cells/ml are incubated with lO ⁇ l ml anti-EGFR antibody (GR01) in PBS/2%FBS for 1 hr on ice with occasional shaking.
  • the cells are washed 3 times with PBS/2%FCS and then anti-IgG antibody coupled magnetic beads (Dynal; Oslo) that can recognize the anti-EGFR antibody are added ( ⁇ 5 beads per positive cell). Again the cells are incubated for 1 hr on ice.
  • the cells that express EGFR are selected by positive selection with a Dynal magnet (Dynal; Oslo) for 10 min. The unbound cells are removed and the EGFR expressing cells are put into culture (see section E.).
  • Figure 5 illustrates the expression of EGFRl-II CD34 + cells after transduction of these cells with supernatants that are generated from PPA-6 cells.
  • Primary human T cells are transduced with PPA-6 supernatants and are selected by immuno-magnetic beadselection then sorted using a FACStar Plus (data not shown). The cells are enriched from 16 % to 92 %.
  • MuSK-R is isolated by PCR from fetal skeletal muscle cDNA (Marathon cDNA, Invitrogen) using primers flanking the 5' and 3' of the MuSK-R cDNA.
  • MuSK21FN CGT CCT GCG TGA GCC TGG ATT AAT C SEQ ID NO: 9
  • MuSK34FN GCC TGG ATT AAT CAT GAG AGA GCT C SEQ ID NO: 10 MuSK2666RN: CGA GGC CTG TCT TCA ACC TTA G AC
  • the 5' primer MuSK21FN covers 25nucleotide (nt) before the start codon
  • the second 5' primer MuSK34FN covers the start codon (aa 1) of MuSK-R and surrounding sequence.
  • the 3 '-primer MuSK2666RN covers the stop codon of MuSK-R and surrounding sequence.
  • the PCR is performed as follows: Cycle 1 : 94°C for 5 min, Cycle 2-11 : 94°C for 0.5 min, 63°C for 1 min, 68°C for 6 min, and Cycle 12: 68°C for 10 min.
  • the reaction is cooled to 4°C in the PCR machine, and the amplified cDNA is ethanol precipitated with 0.3 M sodium acetate.
  • the pellet is washed once with 70% ethanol, dried and resuspended in 100 ⁇ l H 2 O. 10 ⁇ l of the above PCR reaction is then reamplified.
  • the reaction mix contains for the second round of ampUfication step in addition to 10 ⁇ l of the above PCR reaction: Pfu buffer (20mM Tris-HCl (pH8.8), 2 mM MgSO 4 , 10 mM KC1, 10 mM (NH 4 ) 2 SO 4 , 0.1 % Triton X-100, 0.1 mg/ml BSA), 2.5 ⁇ mol of each dNTP (dATP, dCTP, dGTP, dTTP), 1 ⁇ g primer MuSK34FN, 1 ⁇ g primer MuSK2666RN, 5 U Pfu Turbo Polymerase (from Pyrococcus fiiriosus) and water in a final volume of 50 ⁇ l.
  • the PCR is performed as follows: Cycle 13: 94°C for 5 min, Cycle 14-43: 94°C for 0.5 min, 62°C for 1 min, 72°C 6 min, and Cycle 44: 72°C for 10 min.
  • the reaction is cooled to 4°C in the PCR machine and the amplified cDNA is ethanol precipitated with 0.3 M sodium acetate. The pellet is washed once with 70% ethanol, dried and resuspended in 20 ⁇ l H 2 O.
  • the PCR reaction is loaded on a IxTAE gel. A band with the size of ⁇ 2600 bp is isolated from the gel and cloned into the Srfl restriction site of pPCR-Script Amp vector (Stratagene, CA) according to the manufacturer's protocol. The resulting vector is called pPCR-Script MuSK-R-wt. The correctness or the subcloned PCR product is confirmed by restriction analysis and sequencing by methods well known in the art. (The nucleotide sequence is illustrated in SEQ TD NO.7)
  • the primers MuSK1380F, MuSK1657R, and 1747R are used to generate intracellular deletion mutants of MuSK-R from the plasmid pPCRScriptMuSK-R.
  • the primer sequences are as follows wherein p means phosphorylated: Primer 1380F: 5' pCG GCC TGT GCC AGA CTG CCA CAT CTA G (SEQ ID NO: 12); Primer 1657R: 5' pCG TCT AGG TGA GGG TTA CTG CTG
  • MuSK1380F and 1657R results in the deletion of amino acid residues 538 - 879 of MuSK-R
  • primer pair MuSK1380F and 1747R results in the deletion of amino acid residues 577 - 879.
  • the two mutant forms of MuSK-R are designated MuSK- R ⁇ 538-879 (MuSK-RI) and MuSK-R ⁇ 577-879 (MuSK-RII).
  • MuSK-RI and MuSK-RII most of the intracellular domain of MuSK-R as shown in Figure 6 is deleted. While not meant to limit the invention in any manner, it is believed that both truncations result in a deletion of the kinase domain and most of the substrate binding motifs of the wt MuSK-R illustrated in Figure 6.
  • the 5' primer MuSK1380F covers the nucleotide sequence 1333-1410 of the MuSK-R.
  • the 3'-primers MuSK1657R and 1747R contain stop codons in place of amino acid 538 and 577 of MuSK-R.
  • Using primer MuSK1380F with MuSK1657R or MuSK1747R results in the amplification of MuSK-R nucleotide sequence 1333 to 1614 that has a stop codon in the position of amino acid 538 or nucleotide sequence 1333-1728 that has a stop codon in the position of amino acid 577, respectively.
  • the PCR reaction includes -10 ng hMuSK-R wt DNA, 1 x Pfu buffer, 1 ⁇ g of primer MuSK1380F and either 1 ⁇ g primer MuSK1657R or MuSK1747R, 2.5 ⁇ mol of each dNTP, 5 U Pfu polymerase and H 2 0 in a final volume of 50 ⁇ l.
  • the PCR reaction is performed as follows: Cycle 1: 95°C for 5 min, Cycle 2-31 : 95°C 0.5 min, 60°C for 1 min, 72°C for 4 min, Cycle 32: 72°C for 10 min.
  • the PCR reaction is cooled to 4°C in the PCR machine and then loaded on a 1 x TAE gel.
  • Wild-type and mutant MuSK-R are excised from pPCRScriptMuSK-Rwt, pPCRScriptMuSK-RI and pPCRScriptMuSK-RII using the Notl and Xhol site and are cloned into the multiple cloning site of the Moloney Murine Leukemia Virus (MoMLV) based retroviral vector pGla (GTI, Maryland) which is cut with Notl and Xhol.
  • the retroviral vectors are designated pGlaMuSK-R, pGlaMuSK-RI and pGlaMuSK-RII.
  • the constructs pGlaMuSK-R, pGlaMuSK-RI and pGlaMuSK-RII were used to generate viral supernatants as described in Example 1.
  • anti-MuSK-R polyclonal serum, and anti-MuSK-R hybridoma supernatant were used for flow cytometry and immuno-magnetic bead selection.
  • the cells are stained with an an ti -MUSK-R antibody.
  • the 10 7 cells/ml are incubated with 1-3 ml anti-MuSK- R hybridoma supernantant in PBS/2%FBS for 1 hr on ice with occasional shaking.
  • the cells are washed 3 times with PBS/2%FCS and then anti-IgG antibody coupled magnetic beads, that can recognize anti-MuSK-R antibodies, are added ( ⁇ 5 beads per positive cell).
  • the cells are incubated for 1 hr on ice.
  • Cells that express MuSK-R are selected by positive selection with a Dynal magnet (Dynal, Oslo) for 10 min. The unbound cells are removed and the MuSK-R expressing cells are put into culture as described in section D.
  • MuSK-R XC is amplified by PCR and cloned into the expression construct pSecTag2b (Invitrogen). Cloning the XC domain of MuSK-R into the multiple cloning site (MCS) of the plasmid pSecTag2b allows for the expression of the XC under the control of the CMV promoter.
  • the plasmid contains the sequence of a myc and (His) 6 -tag after the multiple cloning site, which allows to fuse the protein of interest (MuSK-RXC) to the myc and (His) 6 -tag.
  • the signal peptide of MuSK-R is replaced by the Ig ⁇ leader. ( Figure 3).
  • the extracellular domain of MuSK-R without the signal peptide is amplified by PCR using the following primers wherein p means phosphorylated:
  • MuSK 116FPC 5' pCT TCC AAA AGC TCC TGT CAT CAC C SEQ ID NO: 15
  • MuSK 1532RC 5'pCC AGT CAT GGA GTA TGT AGG TGA GAC SEQ ID NO: 16
  • Primer MuSK116FPC starts with the sequence after the signal peptide (nt 69 - 93).
  • Primer MuSK1532RC covers the sequence before the transmembrane domain starts and the first 2 amino acids of the transmembrane domain corresponding to nucleotide sequence 1462-1586 of SEQ I NO:7.
  • ng hMuSK-R wt DNA are mixed with 1 x Pfu buffer, 1 ⁇ g of primer MuSK116FPC and 1 ⁇ g primer MuSK1532RC, 2.5 ⁇ mol of each dNTP, 5 U Pfu polymerase and H 2 0 in a final volume of 50 ⁇ l.
  • the PCR reaction is performed as follows: Cycle 1: 95°C for 5 min, Cycle 2-7: 96°C for 0.5 min, 60°C for 1 min, 72°C for 6 min, and Cycle 8 - 27: 95°C for 0.58 min, 58°C for 1 min, 72°C for 6 min; Cycle 28: 72°C for 10 min.
  • the PCR reaction is cooled to 4°C in the PCR machine and then gel-purified.
  • the PCR fragment is cloned into to the EcoRV restriction site of pSecTag2b. In this way MuSK-R XC is cloned in frame with the Igk leader at the N-terminus and the myc- and (His) 6 -tag at the C- terminus.
  • the resulting plasmid is called pSecTag-hMuSK-R.
  • the plasmid pSecTag-hMuSK-R is transfected into 293T cells by the CaCl 2 technique (as described in section C). 24 hrs after the transfection, the media is replaced with either fresh DMEM/10%FBS or serum-free X-Vivo 15. Supernatants of the transfected cells are collected after 48 and 72 hrs. A total of 400 ml supernatants are collected and are frozen at -80°C until the supernatants are purified.
  • the MuSK-R XC is purified from tissue culture supernatants by immobilized metal affinity chromatography.
  • the metal ion is 0.1 M NiCl 2 .
  • the column is a 1 or 5 ml Pharmacia metal HiTrap chelating sepharose column.
  • the equilibration buffer (Buffer A) consisted of 20 mM Na 2 HPO pH 7.4, 1M guanidine hydrochloride, 1M NaCl, filtered through 0.2 ⁇ M cellulose acetate filter.
  • the elution buffer (Buffer B) is 20 mM Na 2 HPO 4 , pH 7.4, 1 M guanidine hydrochloride, 1M NaCl, 0.5 M imidazole, filtered through 0.2 ⁇ M cellulose acetate filter.
  • the Pharmacia FPLC chromatography system is used to run columns, with FPLC director program software and a Pharmacia P50 pump. The purification is performed at 4°C.
  • the pump is primed with buffer A before the load is started. Before the column the column is attached, the load is pumped through until the pink color of the tissue culture media is seen at the connection so that the column is not washed with non-equilibration conditions.
  • tissue culture supernatants are adjusted to contain 0.85 M NaCl, 1M guanidinium chloride and 40 mM imidazole and the pH is adjusted to 7.4.
  • the column is equilibrated with 8% buffer B. The sample is loaded and the column then washed in above conditions for seven column volumes. MuSK-R is eluted at 30% Buffer B (150 mM imidazole) over eight column volumes. Fractions are collected from start of the run.
  • each fraction is tested in a Dot Blot Assay (see below). Selected positive fractions are tested in Western Blot assays and Elisa (see below). Positive fractions are pooled and dialyzed in 10,000 MWCO membrane (Pierce Snakeskin) against PBS. After dialysis, the optical density is determined at OD 280 . The samples are filtered through 0.2 ⁇ M filters and then concentrated in Centricon Centriprep 30 devices in a refrigerated Sorvall RT6000D according to manufacturer's protocol. To test for positive samples in the dot blot assay, 10 ⁇ l of each fraction is pipetted on nitrocellulose. The membrane is dried, blocked with superblock and then probed for MuSK-R protein and developed as described for the India Western (see below).
  • Western Blots are performed by methods well known in the art.
  • the materials (sample buffer, running buffer and gels) are obtained from Novex.
  • For western blot analysis 25 to 35 ⁇ l/fraction are used. Guanidine containing fractions are precipitated in ice-cold ethanol and are stored on ice for 15 minutes or overnight at 4°C.
  • the samples are centrifuged in refrigerated microcentrifuge (TOMY) at 14,000 rpm for 10 min. The supernatant is discarded, ice-cold acetone is added and centrifuged as before.
  • the pellet is resuspended in SDS sample buffer (Novex) with 5% ⁇ -mercaptoethanol in a final volume of 50- 70 ⁇ l.
  • the samples are denatured at > 90°C for 5 minutes, briefly centrifuged, and 25 - 35 ⁇ l loaded on a 4-20% gradient gel.
  • the gel is blotted onto 0.45 ⁇ M nitrocellulose for 1.4 hours at 100 volts, using the Biorad wet transfer blotting cassette with tris-glycine-methanol transfer buffer (25 mM trizma Base, 192 mM glycine, 20% methanol). After blotting the gel, the blot is blocked in Pierce TBS superblock for 10 minutes with mild agitation.
  • the blot is washed twice in TBST (50 mM Tris, pH 7.5, 150 mM NaCl, 0.05% Tween 20) for five min per wash on a rotating platform.
  • the Pierce IndiaTM His-HRP Probe is diluted to 1 :5000 in TBST and the blot is incubated with the probe for 1 hour at room temperature and washed 3 times in TBST.
  • horseradish peroxidase reagent Sigma Fast HRP Insoluble Substrate D4418
  • the blot is washed in three changes of water to stop development.
  • an mouse anti-c-myc antibody (Santa Cruz Biotechnology; CA) is used to detect recombinant MuSK-R protein.
  • This antibody is diluted in superblock to 1 ⁇ g/ml final concentration.
  • the blot is washed three times with TBST and then a goat anti-mouse IgG- HRP antibody (Sigma) is added at 1 :5000 dilution in superblock.
  • the blot is incubated for 1 hour at room temperature, with gentle agitation and developed as described above with Fast HRP insoluble substrate (Sigma).
  • the recognized protein traveled at about 85 kD on the SDS PAGE, and it is considered to be 19 kD heavier due to glycosylation.
  • the recombinant MuSK-R protein is injected into 3 different Balb/c mice.
  • 25-50 ⁇ g are mixed with 2.25 mg alhydrogel and 100 ⁇ g MDP (muranyl dipeptide; Pierce) in a final volume of 200 ⁇ l and injected 5 times every 14 days subcutaneously.
  • MDP muranyl dipeptide
  • the 5x10 5 cells of cell lines CEMSS and CEMSSMuSK- R are used. Both the presera and sera are diluted 1 :100. 1:300, 1:900 and 1:2700.
  • a rat anti- mouse IgG-PE antibody is used as a secondary reagent at a 1 :20 dilution.
  • 96 well plates are coated with 50 ⁇ l of 10 ⁇ g/ml anti-mouse IgGF c (Jackson; Maine) The plates are incubated with various dilutions of sera (1 :100 to 1:218700), subsequently with MuSK-R protein and with Nickel activated horse radish peroxidase at a 1 :1000 dilution (HRP, Pierce). Nickel activated HRP is binding to the recombinant MuSK-R protein via the (His) 6 tag.
  • the plate is incubated with TMP peroxidase substrate (Zymed; CA).
  • TMP peroxidase substrate Zymed; CA
  • one mouse shows the highest reactivity against native and recombinant MuSK-R.
  • This mouse is boosted with a 6 th injection of 200 ⁇ g MuSK-R protein in PBS. The injection is done subcutaneously and intravenously. 1 week later the spleen is removed, lymphocytes isolated with lympholite M (Accurate Chemicals) and fused, using 50% polyethylene glycol to the myeloma cell line P3X63AG8.0653 using standard procedures. The resulting hybridomas are grown in bulk in HAT media for one week.
  • Viable cells are recovered using lympholite M and cultured in HAT media plus cloning factor (Igen). After the hybridoma are grown for another week, a batch of the cells are cryopreserved in HAT media plus 10% DMSO. Another batch of the cells is subdivided into individual clones by FACSsorting using the single cell deposit unit. The cells are sorted by forward and side scatter and for PI negative cells. The cells are grown up in HT media for two weeks. The supernatants are tested by Elisa and FACS (as described above) for monoclonal antibodies that can recognize native and recombinant MuSK-R protein. The antibodies are isotyped in an Elisa assay by using secondary antibodies that react with IgGl, 2a, 2b, 3, IgM, K, and ⁇ (Caltag, CA).
  • HI is an IgGl, K
  • H2 is IgGl
  • K is IgM antibody.

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Abstract

L'invention concerne un procédé permettant d'identifier des cellules de mammifère modifiées génétiquement à l'aide d'un récepteur de protéine-tyrosine kinase mutante (PTKR) comme marqueur sélectionnable de cellules de mammifère. Les membres de la famille des récepteurs du facteur de croissance épidermique (EGFR), ainsi que les membres de la famille des récepteurs de tyrosine kinase spécifique de muscle (MuSKR) sont des marqueurs sélectifs de PTKR mutant particulièrement préférés. L'invention concerne en outre un procédé d'immunosélection de cellules de mammifère transduites, qui comporte les étapes consistant à : mettre en oeuvre une transduction rétrovirale de cellules de mammifère à l'aide d'une séquence d'acides nucléiques codant pour un EGFR mutant ; incuber les cellules transduites avec un anticorps marqué qui reconnaît le PTKR mutant et se lie spécifiquement à celui-ci ; et identifier les cellules marquées transduites.
EP00988723A 1999-11-19 2000-11-17 Genes marqueurs de surface selectionnables Withdrawn EP1278773A2 (fr)

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US53924800A 2000-03-30 2000-03-30
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DK2496698T3 (en) * 2009-11-03 2019-04-15 Hope City TRUNCATED EPIDERIMAL GROWTH FACTOR RECEPTOR (EGFRt) FOR TRUNCATED T-CELL SELECTION

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