JP2003522525A - Cell surface selection marker gene - Google Patents

Abstract

(57) SUMMARY The present invention relates to a method for identifying genetically modified mammalian cells using a mutant (mutated) protein tyrosine kinase receptor (PTKR) as a selectable marker in mammalian cells. Particularly preferred mutant (mutated) PTKR selectable markers are mutant epidermal growth factor receptor (EGFR) family members, and mutant muscle-specific kinase receptor (MuSK-R) family members. Further, the nucleic acid sequence encoding the mutant EGFR is transduced into a mammalian cell by a retrovirus, the transduced cells are incubated with a marked antibody that specifically recognizes and binds to the mutant EGFR, and the marked transduction is performed. Identifying a cell, the method comprising immunoselection of a transduced mammalian cell.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD OF THE INVENTION

The present invention provides a mutant protein tyrosine kinase receptor (PTKR),
In particular, it relates to a method of identifying genetically modified cells using a mutant epidermal growth factor receptor (EGFR) family member or a mutant muscle-specific kinase (MuSK) family member as a cell selection marker.

[0002]

[Prior art]

The use of selectable markers to identify prokaryotic and eukaryotic cells is well known. The use of such selectable markers is very important, as often the introduction of the DNA sequence of interest into a cell does not necessarily result in a phenotype that is easily measurable. The number of selectable markers that can be used to identify eukaryotic cells, especially mammalian cells, is limited. In the past, selectable markers have been used that confer drug resistance (eg, G-418 and hygromycin). More recently, selectable markers have been used in combination with a fluorescence activated cell sorter (FACS), such as green fluorescent protein (GFP). Alternatively, an antibody that recognizes a cell surface molecule can be bound to a fluorescent source (fluorescent dye) to identify the target cell.

[0003]

[Problems to be Solved by the Invention]

Several types of cell surface molecules have been used as cell selectable markers, including CD8, CD24 and human low affinity nerve growth factor receptor (NGFR). See the following publications: WO95 / 06723; WO98 / 19540; Joly et al., Pro. Natl. Acad. Sci. 80: 477 (1983); and Lady (Reddy)
Others, Mol. Brain Res. 8: 137 (1990). Some of these cell surface molecules have mutations in their intracellular regions (domains), which allow them to circumvent molecular signals (signaling) when bound to their ligands. However, upon binding of the ligand, some of the mutated molecules in the cell may form hetero or homodimers, or trimers. If the newly introduced molecule in the cell forms a heterodimer with the endogenous (sex) receptor, a predominant negative effect may occur. The present invention provides cell surface markers that do not form heterodimers with endogenous receptors.

[0004]

[Means for Solving the Problems]

The present invention introduces into a cell a nucleic acid sequence encoding a mutant protein tyrosine kinase receptor (PTKR) functionally linked to an expression regulatory sequence to form a genetically modified cell, and the mutant PTKR is formed in the genetically modified cell. And a genetically modified cell that expresses a mutant PTKR is identified, and a method for identifying a genetically modified mammalian cell is provided. As a preferred first embodiment, the mutant PTKR is a mutant epidermal growth factor receptor (EGFR) family member, or a MuSK family member, in particular, mutant EGFR1, further mutant EGFR1-I and EGFR1.
-II is the sequence named. As a second specific example, mutant PTKR, especially mutant E
The introducing step is carried out by including the nucleic acid sequence encoding GFR in a vector and introducing the vector into cells. Furthermore, as one specific example, the mammalian cells are human cells, particularly hematopoietic cells, hepatocytes, endothelial vascular cells, and smooth muscle cells, and more preferably hematopoietic cells. In yet another embodiment, the mutant PTK
A heterologous (foreign) gene is contained in the vector or construct containing the R marker sequence. In yet another embodiment, the identifying step is performed by contacting the genetically modified cell with an antibody that recognizes and binds the mutant PTKR. In another embodiment, the genetically modified cells are separated from the non-genetically modified cells in the identifying step.

In a second aspect, the present invention includes a nucleic acid sequence encoding a mutant epidermal growth factor receptor (EGFR) family member operably linked to an expression control sequence in a vector, the vector comprising: Introduced into cells to form genetically modified cells,
There is provided a method for identifying a genetically modified mammalian cell, comprising expressing the mutant EGFR in the genetically modified cell and identifying the genetically modified cell expressing the mutant EGFR. In a preferred embodiment, the EGFR is EGFR1-I or EGFR1-I.
I, and the vector is a retroviral vector.

In a third aspect, the present invention retrovirally transduces mammalian cells with a nucleic acid sequence encoding a mutant epidermal growth factor receptor (EGFR) family member operably linked to expression control sequences. Incubating the transduced cells with a marked antibody that specifically recognizes and binds the mutant EGFR, and identifies the marked transduced cells. . In a preferred embodiment, the cells are human cells, especially hematopoietic cells. In another embodiment, the cells are Moloney murine leukemia virus (MoMLV), myeloproliferative sarcoma virus (MPSV), mouse embryonic stem cell virus (MESV), mouse stem cell virus (MSCV), and spleen focus forming virus (
SFFV) and is transduced with a retroviral vector from the group consisting of: In yet another embodiment, the cells are transduced with a lentivirus-derived vector. In yet another embodiment, the method comprises separating the identified marked transduced cells from the unmarked cells. In yet another embodiment, the step of expanding marked transduced cells is included.

As a fourth aspect, the present invention provides a nucleic acid encoding a mutant protein tyrosine kinase receptor (PTKR) functionally linked to an expression regulatory sequence and a nucleic acid encoding a target protein in a mammalian cell. A mammalian cell expressing a target protein, which comprises introducing, culturing the obtained mammalian cell, and identifying a cell expressing the mutant PTKR to obtain a cell expressing the target protein. The identification method of

Unless otherwise stated, cell biology, molecular biology, cell culture, immunology, virology, and other techniques known in the art are used in the practice of the present invention. Although these techniques are disclosed in known literature, in particular, for example, Sa
mbrook, Fritsch, Mmaniatis, eds., “MOLECULAR CLONING: A LABORATORY MANU
^{AL ", 2 nd edition (1989} ); Celis, JE" Cell Biology, A Laoratory Handb
ook ”, Academic Press, Inc. (1994); Coligan et al.,“ Current Protocol i
n Immunology ”, John Wiley and Sons (1991); and Harlow et al.,“ Antibo.
dies: A Laboratory Manual ”(1988), Biosupplynet Source Book (1999), Co
See ld Springs Harbor Laboratory.

Various publications, patents and published patent specifications are cited. These disclosures are
In order to describe the state of the art in the technical field to which the present invention is related more completely, it is incorporated into the disclosure content of the present specification by reference.

Unless otherwise stated, each term in this specification means singular and plural. For example, "stem cell" includes a plurality of stem cells.

The selectable marker of the present invention is a mutant protein tyrosine kinase receptor (PTKR)
It is a molecule. The PTKR molecule is activated by the polypeptide ligand and is closely related in its catalytic region. They are type I transmembrane proteins, their N-termini are extracellular and have a single membrane spanning region (v
an der Geero et al., Annu. Rev. Cell Biol. 10: 251 (1991)). As used herein, "PTKR" includes, but is not limited to, the following subfamilies. That is, MuSK-R (Glass, et al., Cell 85: 513 (1996)), which is believed to initiate neuromuscular junction formation in response to agrin, epidermal growth factor receptor (EGFR), and platelet-derived Growth factor receptor (PDGFR), insulin receptor (INSR), nerve growth factor receptor (NGFR), fibroblast growth factor (FGFR) and EPH. Each of these subfamilies consists of various members. PDGFR family includes PDGFR receptor α, PD
GFR receptor β, SCF receptor (c-Kit), CSF-1 receptor (c-F)
ms), Flk2 (Flt3), FLT1 (Quek1), Flk1 (KDR)
And FLT4 (Quk2). The INSF subfamily includes the insulin receptor, IGF-1 receptor, IRR, ROS and Ltk. NGF
The R subfamily includes the NGFR receptor (TrkA), TrkB and TrkC. The EGFR subfamily includes EGFR1, EGFR2, EGFR3
, And EGFR4. Other names for these members are known to those of skill in the art. For example, EGFR1 is EGFR, HER, c-ErbB and ErbB-.
Also known as 1. EGFR2 is also known as HER2, Neu and ErbB-2. EGFR3 is also known as HER3 and ErbB-3. EGFR4 is also known as HER4 and ErbB-4 (Urlli
ch, Schlessinger, Cell, 61: 203-212 (1990)).

The mutant PTKRs of the present invention include a modification in the PTKR molecule such that the receptor no longer has the corresponding unmodified (wild type) signal (signaling) activity. In describing the present invention, "wild-type PTKR" refers to natural PT.
Not only KR, but also genetically modified PTKRs that have undergone changes in the DNA sequence that do not significantly affect the protein tyrosine kinase receptor molecular properties are included. Such changes include those that do not change the encoded amino acid sequence, those that result in conservative substitutions of the amino acid sequence, and those that result in the deletion or addition of one or several amino acids. Appropriate substitutions are known to those skilled in the art. Examples of amino acid residues that can be conservatively substituted include glycine / alanine, valine / isoleucine / leucine, asparagine / glutamine, aspartic acid / glutamic acid, serine / threonine, lysine / arginine, phenylalanine / tyrosine. I can.

Modifications of the wild-type PTKR molecule include deletion, truncation and the like. More specifically, mutant PTKRs of the invention include those that have modifications in the cytoplasmic region and lack signal activity. Signal activity can be generally defined as the activity that triggers the response (pathway) to the nucleus in the cytosol that ultimately leads to transcriptional activation. Mutant PTK of the present invention
R also includes modifications in the extracellular region. However, it is preferable that the extracellular region retains the ability to bind to the antibody. Generally, the minimal peptide fragment of the extracellular region capable of binding to an antibody is about 15 amino acid residues, more preferably at least 50 amino acid residues.

In a preferred embodiment, the selectable marker PTKR is a mutated member of the EGFR family. Members of the EGFR family are similar in overall morphology, with striking amino acid sequence relatedness. Members of this family are not limited to those listed above, but share common features such as a single transmembrane region, two cysteine-rich regions consisting of approximately 100-200 amino acid residues, and a single tyrosine kinase region. It also includes new members that have. In addition, members of the EGFR family are capable of forming heterodimers with other members of the EGFR family.

A preferred mutant EGFR family member is EGFR1. It is a receptor with high affinity for many ligands including, for example, epidermal growth factor (EGF), transforming growth factor (TGFα) and amphiregulin. Many EGFR sequences have been identified and EMBL / GenBank Accession Numbers
: X00588 / X06370 (Ullich, et al., Nature 309: 418 (1984)); K01885 / K02047 (
Lin et al., Science 224: 843 (1984)); K03193 (Merlino, et al., Mol. Cell.
Biol 5: 1722 (1985)); X00663 (Xu, et al., Nature 309: 806 (1984)); AC00697
7; M38425 (Simmen, et al., Biochem. Biophys. Res. Commun. 124: 125 (1984).
) And M20386 (Lax, et al. Mol. Cell. Biol. 8: 1970 (1988)). Some EGFRs have been found in other than vertebrate species, eg EM
Mention may be made of Drosophila with BL / GenBank Accession Numbers: K03054 (Livneh, et al., Cell 40: 599 (1985).

A schematic diagram of the region structure of wild-type EGFR is shown in FIG. EGFR has a signal peptide that directs this protein to the secretory pathway. The extracellular region follows this signal peptide. This region consists of hundreds of amino acids. The extracellular region usually means a part of the receptor protruding from the cell into the extracellular environment, and has a clear Cys residue in EGFR. The transmembrane region is generally localized to the cell membrane and is linked by hydrophobic residues followed by some basic residues. The cytoplasmic region (also referred to as “intracellular region”) contains the catalytic site of the molecule and is located intracellularly. This region contains the substrate binding site and regulatory tyrosine and threonine phosphorylation sites.

Modifications of EGFR family members, in particular EGFR1 mutations, are known. WO3 / 05148 discloses three mutations of EGFR1. One of them (HE
R721A) is a point mutation at the amino acid position 721 in the cytoplasmic region of EGFR1 in which lysine is replaced with alanine. The second mutation (HERCD-533) contains the C-terminus of the molecule such that the intracellular region is deleted but the transmembrane region remains. The third mutation (HERCD-566) has a deletion of the C-terminal 566 amino acids,
Both intracellular and transmembrane regions are missing. Three truncated forms of EGFR have been detected in some malignancies. Type I (EGFRvIII) is a large extracellular region (
Amino acids 6-273) have been deleted and cannot bind to EGF (Mosca
tello, DK et al., Oncogene 13:85 (1996)). Type II has 8 extracellular domains IV
EGF with an in-frame deletion of 3 amino acids (520-603)
And does not inhibit TGFα binding (Humphrey, PA, et al., Biophys. Res. Commu
n. 178: 1413 (1991)). Type III has an in-frame deletion of 267 amino acids (29-296) in extracellular regions II and III, and these deletions inhibit ligand binding (Humphrey, PA, et al. , Proc. Natl. Acad. Sci.
USA 87: 4207 (1990)).

Although specific modifications such as, but not limited to, those described above are known for EGFR family members, examples of using such mutant EGFR family members as selectable markers in mammalian cells Has never been known. According to the invention, suitable modifications to PTKR molecules, in particular EGFR molecules, are to the cytoplasmic region, eg at least 150 of the cytoplasmic region.
, Preferably at least 250, more preferably at least 400, even more preferably at least 500 amino acids have been deleted. In a preferred embodiment the deletion consists of a truncation of the molecule. Truncation can include deletion of tyrosine phosphorylation sites and deletion of kinase catalytic sites in the range of 1-15 sites. However, the mutant EGFR useful as a selectable marker in the present invention may contain a deletion of tyrosine phosphorylation site of 1 or more and less than 15. In addition, such modifications also include in-frame deletions such as deletions of at least 50 amino acids in the cytoplasmic region. Such a deletion may include a deletion of protein tyrosine kinase activity. Particularly preferred selectable marker sequences are mutated EGFR1, EGFR2 and EGFR3 molecules in which at least 400, preferably 500 amino acids in the cytoplasmic region have been deleted from the corresponding wild type molecule.

A preferred mutant EGFR molecule in the present invention is mutant EGFR1. Particularly preferred are the modified sequences derived from EGFR1 shown in Figures 2 and 3 (SEQ ID NOS: 1 and 2). In this EGFR1 molecule, the extracellular region is nucleotides 1-19
35, the transmembrane region is encoded by nucleotides 1938-2004, and the intracellular region is encoded by nucleotides 2007-3630.
An example of a suitable mutant EGFR1 marker is designated as EGFR1-I, and amino acids 679 to 1210 in the cytoplasmic region are deleted (see FIG. 2).

A second preferred example of the selectable marker PTKR is a mutant member of the MuSK-R family. MuSK-R contains a signal sequence or leader sequence that directs the protein to the secretory pathway. Following the signal sequence is an extracellular region, which consists of hundreds of amino acids, varying in number, but typically about 500.
It is an amino acid. The extracellular region is a part of the receptor that normally projects from the cell into the extracellular environment and has a ligand binding region. The extracellular domain is one of the most distinguishing features of kinase receptors. In MuSK-R, the extracellular region contains an immunoglobulin-like (Ig-like) region. Typically, four immunoglobulin-like regions have been discovered. However, there are also some reports on MuSK-R with three immunoglobulin-like regions. In the extracellular region, "C6-b
It may contain 6 consecutive cysteine residues known as "ox". The position of this C6-box varies depending on the particular MuSK-R, but is found at approximately amino acid residues 373-382 in some MuSK-Rs. The transmembrane region is generally localized in the cell membrane, and several basic residues are bound after a series of hydrophobic residues. The intracellular region (used interchangeably with the "cytoplasmic region") contains the catalytic site of the molecule and is located intracellularly.

MuSK-R is also known in the art as a denervated muscle kinase receptor and is also referred to as DmKs (US Pat. No. 5,656,473, particularly SEQ ID NOs: 16 and 17).
reference). The MuSK-R sequence has been isolated and identified from human, rat, mouse and Xenopus. Closely related to human MuSK-R is torpedo (T
orpedo) tyrosine kinase receptor, a receptor isolated from the electric ray Torpedo California, and the ROR tyrosine kinase receptor (Jennings, et al., Proc. Natl. Acad. Sci. USA 90: 2
895 (1993); Masiakowski et al., J. Biol. Chem. 267: 26181-26190 (1992); V
alenzuela et al., Neuron, 15: 573-584 (1995); Hesser et al., FEBS Letters
, 442: 133-137 (1999)).

Other MuSK-R available from public depositary institutions such as GeneBank and ATCC
Accession number: NM005592; AF006464; A448972; AI800924; AI700028; A
I41265; AI341122; AI302067; U34985; AA448972; and ATCC75498. As mentioned above, MuSK-R is specific for the skeletal muscle junction.

As used herein and in the claims, the term “MuSK-R” refers to known MuSK-R (including DmK receptors), known MuS having a similar structure.
KR isoforms or variants (Villian), known tyrosine kinase receptors functionally similar to MuSK-R, and novel and previously unidentified screening techniques known to those of skill in the art. It is broadly defined as including the MuSK-R. Such screening techniques include the use of degenerate oligodeoxyribonucleotide primers.

Thus, when the term “MuSK-R” refers to a nucleic acid molecule,
(a) a nucleic acid sequence containing the known mammalian cell MuSK-R coding region, (b) a mammalian cell MuSK which hybridizes with the nucleic acid of (a) under stringent conditions
-R-encoding nucleic acid sequence, and (c) a polynucleotide-encoded MuSK
-Degenerate Mu with changes in the nucleic acid sequence that do not significantly affect the properties of the R protein
SK-R is included. Such changes include those that do not result in changes in the encoded amino acids, those that result in conservative substitutions of the amino acid sequence, and those that result in the deletion or addition of one or several amino acids. Appropriate substitutions are known to those skilled in the art. Non-limiting examples of amino acid residues that are conservatively substituted with another amino acid include glycine / alanine, valine / isoleucine / leucine, asparagine / glutamine, aspartic acid / glutamic acid, serine / threonine, lysine / arginine, phenylalanine / Tyrosine can be mentioned. Any conservative amino acid substitution that does not significantly affect the properties of MuSK-R is encompassed by this term. MuSK-
R includes not only natural MuSK-R but also MuSK-R obtained by genetic engineering.

Thus, when the term “MuSK-R” refers to a polypeptide,
Included are the known MuSK-R, MuSK-R isoforms or virients, and functionally equivalent receptors. A functionally equivalent receptor is a MuSK-R that competes with the known MuSK-R for binding. More specifically, functionally equivalent MuSK-R means an amino acid sequence that is at least 40%, preferably at least 60%, and more preferably at least 80% identical to the amino acid sequence represented by SEQ ID NO: 8. M, as shown in FIG. 6 for ligand or substrate binding.
Those that can compete with uSK-R.

According to the present invention, the mutant MuSK-R is used as a selectable marker to identify genetically modified cells. This marker is normally introduced onto the nucleic acid construct of target cells that do not express MuSK-R. The term "introduction" is used herein in a broad sense and is used in the sense of insertion, inclusion and the like. When the mutant MuSK-R is used as a selectable marker, the molecule no longer has signal activity.
Signal activity can be generally defined as the activity that triggers the response (pathway) to the nucleus in the cytosol that ultimately leads to transcriptional activation. The lack of signal activity is due to (a) the use of MuSK-R in tissues or cells other than muscle (Glass et al 85: 513-523 (1996)), or (b) mutant M.
By using uSK-R.

Although the modification of MuSK-R is known, no method is known to identify genetically modified cells using mutant MuSK-R as a selectable marker.

As already mentioned, MuSK-R is localized in muscle tissue and functions as a functional agrin receptor. Agrin is a factor that can induce molecular reorganization in motor end plates. Therefore, MuSK-R can be used as a selectable marker in tissues other than muscle.

In a preferred embodiment, the modification of MuSK-R comprises truncation and / or deletion of MuSK-R. Mutations can occur in the extracellular and / or intracellular regions by means well known to those of skill in the art. This mutation causes the molecule to lose signal activity.
It is preferable that the extracellular region still has antibody binding activity. Generally, the minimal peptide fragment of the extracellular region that is capable of binding an antibody is about 15 amino acid residues, more preferably at least 50 amino acid residues.

A preferred MuSK-R in the present invention is the sequence set forth in SEQ ID NO: 7 and 8, designated as “hMuSK-R”. Extracellular region is nucleotides 1-1479
, The transmembrane region is encoded by nucleotides 1480 to 1545, and the intracellular region is encoded by nucleotides 1546 to 2607. Other suitable MuSK-Rs are those closely related to the sequences set forth in SEQ ID NOs: 7 and 8. Such closely related sequences are described in US Pat.
It is the sequence described in Nos. 16 and 17.

Mutants of MuSK-R are known and are described in Ape et al., Neuron 18: 623-635 (1997).
Can be referred to. In the present invention, suitable modifications to MuSK-R are those to the cytoplasmic region, such as at least 150, preferably at least 200, more preferably at least 250, even more preferably at least 300, most preferably at least 350 amino acids of the cytoplasmic region. Is deleted. In a preferred embodiment, the deletion consists of truncation of the molecule. Deletions or truncations include deletions of tyrosine phosphorylation sites in the range of 1-19 sites, preferably in the range of 2-15 sites, more preferably in the range of 2-10 sites. Furthermore, MuS
It may include a deletion of the kinase catalytic site from KR. In one embodiment, the tyrosine phosphorylation site is approximately amino acid residue 672 of SEQ ID NO: 8.
~ 691. There is no limitation on the number of deletions or truncations in the cytoplasmic region as long as the protein is stably expressed.

A particularly preferred mutant MuSK-R used as a selectable marker of the present invention is shown in FIG.
It is a modification to the MuSK-R sequence described in SEQ ID NO: 8). In one specific example, M
At least 300 amino acids are truncated in the cytoplasmic region of uSK-R. A preferred specific example is mMuSK-RI in which the amino acid sequence of 538 to 869 is deleted.
Is called. Another preferred embodiment is what is referred to as mMuSK-RII in which the amino acid sequence of 577-869 is deleted.

In addition to modification to the cytoplasmic region, it is also possible to modify the extracellular region.
At least about 100, preferably at least about 1, for modifications in the extracellular region.
50, more preferably at least about 200, even more preferably at least about 250.
Deletions of amino acids are included. Preferably, the mutant MuSK-R used as a selectable marker of the present invention should include an antibody binding site in the extracellular region.

In all preferred embodiments, the mutant PTKR, particularly the mutant EGFR or mutant MuSK-R useful as selectable markers, includes modifications in both the cytoplasmic and extracellular regions as described above. This is expected to avoid the formation of heterodimers between the mutant PTKR and the endogenous receptor.

For modification in the extracellular region, at least about 100, preferably at least about 150, more preferably at least about 200, even more preferably at least about 25.
0 amino acids have been deleted. In one embodiment, the modification is generally about 10
It involves the removal of one cysteine-rich region characterized by 0 amino acids. Particularly preferred selectable markers are mutant EGFR1, mutant EGFR2 and mutant EGFR3, which have a truncation in the cytoplasmic region as well as a deletion of at least 200, preferably 250 amino acids in the extracellular region. As mentioned above, preferably the mutant MuSK-R used as selectable marker of the present invention should include an antibody binding site in the extracellular region.

More specifically, a preferred mutant EGFR molecule with both extracellular and cytoplasmic regions modified is the WT EGFR1-derived sequence shown in FIG. 2 (SEQ ID NOs: 1 and 2). Particularly preferred is EGFR1-II in which the amino acid sequences 25 to 313 of the extracellular region and the amino acid sequences 679 to 1210 of the cytoplasmic region in FIG. 2 (SEQ ID NO: 2) are deleted.

As used herein, “mutant PTKR”, “mutant EGFR” or “mutant MuSK”
A "-R" selectable marker means a nucleotide or protein, depending on the context. The polynucleotide of the present invention may be in the form of RNA or DNA, the DNA being cDNA
It includes NA, genomic DNA or synthetic DNA.

General methods for making mutations in nucleic acids and proteins are known. These methods can be used to make mutant PTKRs, particularly mutant EGFR family members, useful as selectable markers of the invention. Those skilled in the art can obtain PTKR molecules containing EGFR family members from sources such as various sequence databases such as GenBank. Both random and site-directed mutagenesis are effective in producing mutations in wild-type PTKR. The random method involves altering sequences within restriction endonuclease sites, inserting oligonucleotide linkers into the plasmid, using chemical agents to damage the plasmid, and incorporating the wrong nucleotide during in vitro DNA synthesis. . However, site-directed mutagenesis may be a more beneficial tool. Particularly suitable site-directed mutagenesis methods are oligonucleotide-directed mutagenesis and polymerase chain reaction (PCR) amplification oligonucleotide-directed mutagenesis. These methods are known to those of skill in the art and are described in Wu, et al., Methods in Enzymol.
ogy, Vol. 154: Recombinant DNA, Part E, Academic NY (1987); Landt et al.
, Gene 96: 125-128 (1990); Kirchhoff et all., Methods Mol. Biol. 57: 323-3.
33 (1995); Herlitze, et al., Gene 91: 143-147 (1990); Sambrook, et al., M
olecular Cloning, A Laboratory Manual (above) and Current Protocols in
Molecular Bioloby, Greene Publishing Associates, John Wiley & Sons, Inc.
See Canada (1998)).

The utility of mutant PTKRs as selectable markers is related to their ability to identify mammalian cells that have been genetically modified in vitro (in vitro), in vivo (in vivo), and ex vivo (in vitro). The mutated PTKR sequence is introduced into the target cell as part of a nucleic acid construct operably linked to an expression control sequence, in a preferred embodiment,
The construct containing the mutated PTKR sequence is placed in a vector and introduced into target cells. As used herein, "operably linked" means that the elements are arranged such that each element assumes a structure that serves its normal function.
The regulatory elements need not be contiguous with the coding region.

Vectors having both a cloning site and a promoter to which a polynucleotide is operably linked are well known to those of skill in the art. Such vectors are capable of transcribing RNA in vitro or in vivo and are capable of producing Stratagene (LaJolla, CA) and Pr.
Commercially available from omega Biotech (Madison, WI). Examples of such vectors include viruses such as baculovirus, retroviruses, adenoviruses, adeno-associated viruses and herpes simplex viruses, bacteriophages, cosmids, plasmid vectors, fungal vectors, synthetic vectors and others typical in the art. Recombinant vehicles that have been used in the past can be mentioned. These vectors have been described for expression in a variety of prokaryotic and eukaryotic hosts and can be used to express proteins.

In a preferred embodiment, the vector comprises a nucleic acid sequence encoding a selectable marker of the invention operably linked to an expression control sequence. Appropriate regulatory sequences can be appropriately selected by those skilled in the art based on the target cells. Examples of the expression regulatory sequence (regulatory sequence) include a promoter, an enhancer, a polyadenylation signal, an RNA polymerase binding sequence, a sequence inducing transcription, and a scaffold attachment region (SAR).
Other expression control elements, such as

The promoter may be either a prokaryotic or a eukaryotic promoter. Furthermore,
The promoter can be a tissue-specific promoter, an inducible promoter, a synthetic promoter, or a fusion (hybrid) promoter. Non-limiting examples of promoters include phage lambda (PL) promoter, SV40 early promoter, adenovirus promoters such as adenovirus major late promoter (AdMLP), herpes simplex virus (HSV) promoter, human cytomegalovirus (CMV). Cytomegalovirus promoter such as immediate early promoter, long terminal repeat (LTR) promoter such as MoMLV LTR, U3 region promoter of Moloney murine sarcoma virus (MoMSV), granzyme A promoter, metallothionein gene regulatory sequence, CD34 promoter, CD8 promoter , Thymidine kinase (TK) promoter, B19
The Pablo virus promoter and the Rous sarcoma virus (RSV) promoter can be mentioned. Additionally, promoter elements from yeast and other fungi such as the Gal4 promoter and the alcohol dehydrogenase (ADH) promoter can be used. These promoters are based on the Stratagene (Str
commercially available from various sources such as atagene, La Jolla, CA). The scope of the invention is not limited to a particular promoter.

The vector may further include a polyadenylation signal located 3 ′ to the carboxyl terminal amino acid. Vectors containing both a polynucleotide and a promoter operably linked to a cloning site are known to those of skill in the art. Such vectors are capable of transcribing RNA in vitro or in vivo and are commercially available. Non-limiting examples include Stratagene (La Jolla,
Mention may be made of pSG, pSV2CAT and pXt1 commercially available from CA) and pMSG, pSVL, pBPV and pSVK3 commercially available from Pharmacia. Other vectors include pCMV mammalian expression vectors such as pCMV6b and pCMV6c (Chiron, CA), pSFFV-Neo and pB.
luesscript-SK + can be mentioned. To optimize expression and / or in vitro transcription, the 5'and / or 3'untranslated portions of the polynucleotide may be removed, added or modified to allow for extra and inappropriate alternative translation initiation codons. It may be necessary to exclude sequences, or other sequences that interfere with or reduce expression at the transcriptional or translational level. Alternatively, a consensus ribosome binding site can be inserted immediately 5'to the start codon to enhance expression.

A particularly preferred vector is a retroviral vector, as described by Coffin et al., “Retro
viruses ”, (1997) Chapter 9, pp.437-473 Cold Springs Harbor Laboratory P
ress). Retroviral vectors useful in the present invention can be prepared recombinantly by procedures already taught in the art.
WO94 / 29438, WO97 / 21824 and WO97 / 21825 describe retroviral packaging plasmids and packaging cell lines. Common retroviral vectors are retroviruses of mouse, bird or primate origin. The most common retroviral vectors are Moloney murine leukemia virus (MoMLV) and mouse stem cell virus (MSCV). For MoMLV-derived virus, LM
ily, LINGFER, MINGFER, MND 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 US Pat. No. 5,707,865). Furthermore, non-limiting examples of vectors include Gibbonsal leukemia virus (GALV), Moloney mouse sarcoma virus (MoMSV), myeloproliferative sarcoma virus (MPSV), mouse embryonic stem cell virus (MESV), spleen focus forming virus (SFFV), And the human immunodeficiency virus (HIV-
1 and HIV-2). Host range,
New vector systems are constantly being developed to take advantage of specific properties of parental retroviruses, such as altered cell surface receptors (C. Baum et al., Chapter 4, Gene Therapy.
of Cancer Cells eds., Lattime & Gerson (1998)). The present invention is not limited to any particular retroviral vector and includes all types of retroviral vectors. A particularly preferred vector is two long terminal repeats (LT
DNA from mouse virus corresponding to R) and packaging signal. As one specific example, the vector is a MoMLV- or MSCV-derived vector. In another preferred embodiment, the vector is MND.

In the production of retroviral vector constructs, viral gag, poll and env sequences are usually removed from the virus, providing a place for insertion of foreign DNA sequences. This gene, encoded by an exogenous DNA sequence, is usually expressed under the control of a strong promoter within the long terminal repeat (LTR). The LTR promoter is preferred, but various promoters such as those mentioned above are known.

Such constructs can be efficiently encapsulated within viral particles if the gag, poll and env functions are conferred in trans by the packaging cell line. That is, the vector construct is introduced into packaging cells and the gag, pol and env proteins produced by the cells are combined with vector RNA to produce infectious virions which are secreted into the culture medium. The virus thus produced infects the target cell and integrates into its DNA,
It does not produce infectious viral particles due to the lack of essential packaging sequences. Most of the packaging cell lines currently available are transfected with separate plasmids, each containing one of the required coding regions, so that the replication component virus cannot be produced. Multiple recombination events are required. Alternatively, the packaging cell line has the provirus already integrated (a DNA form of reverse transcribed RNA, incorporated into the genome of the infected cell). In this case, the cells produce all the proteins necessary to make up the infectious virus, but because they are crippled,
It cannot package its own RNA into a virus. Instead, the RNA produced by the recombinant virus is packaged. Therefore, the virus stock released from the packaging cells contains only recombinant virus. As non-limiting examples, PA12, PA317, PE501, PG13, ΨCRI
P, RD114, GP7C-tTA-G10, ProPak-A (PPA-6)
, And PT67 (Miller et al., Mol. Cell. Biol. 6: 2.
895 (1986); Miller et al. Biotechniques 7: 980 (1989); Danos et al. Proc.
Natl. Acad. Sci. USA 85: 6460 (1988); Pear et al., Proc. Natl. Acad. Sci
USA 90: 8392 (1993); Rigg, et al., Virology 218: 290-295 (1996); Finer e
t al., Blood 83:43 (1994)). Retroviral vector DNA is introduced into packaging cells by stable or transient transfection to produce vector particles.

Further preferred vectors include adenovirus vectors (Frey et al., Blood 9
1: 2781 (1998); WO95 / 27071) and adeno-associated virus vector (AAV) (Chat
terjee et al., Current Topics in Microbiol. And Immunol. 218: 61 (1996)
Can be mentioned. Shenk, Chapter 6, 161-78, Breakefield et al., Cha
pter 8 201-235; Kroner-Lux et al., Chapter 9, 235-256 in Stem Cell Biolo
gy and Gene Therapy, eds., Quesenberry et al., John Wiley & Sons, 1998
See also US Pat. No. 5,693,531, US Pat. No. 5,691,176. The use of adenovirus-derived vectors is advantageous under certain conditions because they can infect non-dividing cells, and, unlike retroviral DNA, adenovirus DNA is used within the genome of target cells. Will not be incorporated into. Moreover, the ability of adenovirus vectors to carry foreign DNA is considerably higher than that of retrovirus vectors. Adeno-associated virus is another useful delivery system. The DNA of these viruses has been integrated into non-dividing cells and has been successfully used to introduce many polynucleotides into different cell types using adeno-associated virus vectors. The vector is capable of transducing different types of cells including hematopoietic cells and endothelial cells.

Vectors include WO96 / 13598 and WO99 / 47691 (University of Pennsylvania), WO98 / 2134
5 (General Hospital), U.S. Patent No. 5965441 (General Hospital),
Alternatively, a hybrid vector of an adenovirus vector and an adeno-associated virus vector, as described in WO99 / 58700 (allied gene therapy), is also included, and the disclosures of these documents are incorporated herein. ..

In one embodiment, the construct or vector comprises a mutated PTK as a selectable marker.
It contains not only the nucleic acid sequence encoding R or the mutant EGFR, but also the nucleic acid sequence encoding the heterologous gene to be transferred into the target cell. In a preferred embodiment, the nucleic acid is DNA
Is. The term "heterologous or exogenous" when used in reference to nucleic acid sequences such as gene sequences is not normally related to the particular cell or vector, and is appropriately inserted into the construct or vector under the control of the promoter. Means a sequence capable of being expressed in the target cell to be genetically modified. The heterogeneous gene can be located 5'or 3'of the selectable marker of the present invention.

Non-limiting preferred examples of vector constructs have the general structure shown below in 5'or 3 '. (A) LTR-X-IM-LTR; (b) LTR-M-LTR; (c) LTR-M- (I) -X-LTR; (d) LTR-X-pM-LTR; and ( e) LTR-X-IM-SAR-LTR. Here, "LTR" is a long terminal repeat sequence, "X" is an isomer gene for a desired protein, "M" is a selectable marker of the present invention, "I" is an internal ribosome entry site of the present invention, and "SAR" is a scaffold. The attachment region, and "p", is a second promoter, preferably the CMV or PGK promoter.

A gene, coding sequence, or sequence encoding 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 that one desires to express. Non-limiting examples of heterologous genes can include therapeutic proteins, structural genes, ribozymes, antisense sequences. The heterologous gene may be the complete protein or a functionally active fragment thereof. Such proteins include, for example, those that control cell differentiation and therapeutic genes that can compensate for patient disease resulting from endogenous genetic defects. Further, therapeutic proteins or genes may antagonize or neutralize the production or function of infectious agents, antagonize pathological processes, improve the genetic make-up of the host, or engraft. Includes facilitators.

Specific examples of therapeutic genes or gene sequences include the following therapeutically effective genes or gene sequences: Adenosine deaminase deficiency (ADA).
, Sickle cell anemia, recombinase deficiency, recombinase regulatory gene deficiency, HIV such as antisense or transdominant REV genes, or herpes simplex thymidine kinase gene (HSV-tk). The heterologous gene encodes a novel antigen or drug resistance gene, or a toxin or an apoptosis inducer effective in specifically killing tumor cells, or a suicide gene specific for hematopoietic cells. It can also be included. Further, the heterologous gene may be a non-human therapeutic gene such as a yeast gene (Seo et al., Proc. Natl.
Acad. Sci. 95: 9167 (1998)).

The vector or construct can contain a second heterologous gene in addition to the first heterologous gene encoding the protein of interest. The treatment of certain diseases requires more than one gene. Alternatively, one or more genes can coexist (compat
It is also possible to carry using an (ible) vector. Depending on the genetic defect, the therapeutic gene can include regulatory and untranslated sequences. For human patients, the therapeutic gene is generally of human origin, but genes of closely related species that exhibit high homology and biologically identical or equivalent function in humans also have the same gene in the recipient. It can be used as long as it does not cause an adverse immune reaction.

The nucleotide sequence of the heterologous gene of interest is known in the art or G
It is available from various sequence databases such as enBank. Those skilled in the art will readily recognize that any structural gene can be cleaved into compatible restriction enzyme fragments and incorporated into the vector in a manner that allows the structural gene to be appropriately expressed in hematopoietic cells.

The target cells of the present invention are mammalian cells, non-limiting examples of which include livestock such as humans, mice, monkeys, chimpanzees, cows, sheep, pigs, goats, horses, sports animals, dogs, Examples thereof include pets such as cats, and experimental rodents and animals such as rats, mice, and guinea pigs. Preferably, the target cells are human cells. Suitable human cells include hepatocytes, hematopoietic cells, smooth muscle cells, nerves, endothelial vascular cells, tumor cells, and epithelial cells. Hematopoietic cells are particularly suitable, and include hematopoietic stem cells, erythrocytes, neutrophils, monocytes, platelets, mast cells, eosinophils, basophils, B and T lymphocytes, NK cells, and these. Included are precursor cells of each cell lineage. Hematopoietic stem cells and T cells are particularly preferred. Hematopoietic stem cells (HSC) can be defined as a hematopoietic cell population having a long-term multilineage reconstitution (proliferation) capacity. T cells are one type of lymphocyte and are considered to have developed from hematopoietic stem cells. There are many types of T cells, including cytotoxic T cells, helper T cells, induced T cells and suppressor T cells.

Methods for obtaining target cells, particularly hematopoietic cells, are well known to those of skill in the art and are not specifically repeated here. Non-limiting sources of hematopoietic cells can include hematopoietic stem cells, bone marrow, embryonic yolk sac, fetal liver tissue, adult spleen, and blood such as adult peripheral blood and cord blood (To et al., Blood 89: 2233 (1997)). Bone marrow cells can be obtained from the ilium, sternum, tibia, femur, spine, and other bone cavities.

The method of separating target cells from other cells is not critical to the invention. Various procedures can be used, such as physical separations, magnetic separations with antibody-coated magnetic beads, affinity chromatography, and cytotoxic agents for use with or attached to monoclonal antibodies. Also included in the procedure is a fluorescence activated cell sorter (FACS) that separates cells according to the level of staining of a particular antigen. These techniques are known to those skilled in the art and are described in various documents, for example US Pat.
061,620; 5,4098213; 5,677,136; 5,750,397 and Yau et al., Exp. Hematol.
18: 219-222 (1990).

[0058]   The order of cell separation is not critical to the invention. Mutant PTKs are specific cell types
Separation can be done either before or after genetic modification by R. Preferably
, Cells are first roughly separated and then positively and / or negatively selected. Human
In, the surface antigen expression profile of the enriched hematopoietic stem cell population was ⁺ Thy-1⁺Lin⁻Identified by. Non-limiting examples of other enriched phenotypes
As CD2⁻, CD3⁻, CD4⁻, CD7⁻, CD8⁻, CD10⁻, C
D14⁻, CD15⁻, CD19⁻, CD20⁻, CD33⁻, CD34⁻, C
D38^{lo /-}, CD45, CD59^+/-, CD71⁻, CDW109⁺, Gu
Lycophorin⁻, AC133⁺, HLA-DR^+/-, And EM⁺To list
You can Lin⁻The cells have at least one lineage-specific marker (CD2,
CD3, CD4 and CD15, CD56, etc.) are selected based on their lack of expression.
Combination of expression markers used for separation and identification of enriched HSC cell population
Varies depending on various factors, and other expression markers are available.
It can change.

Mouse HSCs with properties similar to human CD34 ⁺ Thy-1 ⁺ Lin ⁻ can be identified as kit ⁺ Thy-1.1 ^lo Lin ^{− / lo} Sca-1 ⁺ (KTLS). Other phenotypes are well known. Combining CD34 expression with Thy-1 selection can result in compositions containing cells with less than about 5% lineage committed cells (US Pat. No. 5,061,620).

CD3 is expressed on most T cells and such cells express the cell surface antigen CD2.
, CD4 and CD8 have been shown to be expressible. CD45 is also a useful T cell marker. The best known T cell marker is the T cell antigen receptor (T
CR). Currently, there are two types of TCR, α, β-TCR, and γ, δ-TCR.
Is defined. B cells can be selected by, for example, expression of CD19 and CD20. Bone marrow cells can be selected by, for example, expression of CD14, CD15 and CD16. NK cells can be selected by the expression of CD56 and CD16. Erythrocytes can be selected by the expression of glycophorin. Neurons can be identified by NCAM and LNGFR (Baldwin et al., J. Cell Biochem. 15: 502 (1996)). VE endothelial cells
GFR2, CD34, P-selectin, VCAM-1, ELAM-1 and ICA
It can be identified by M-1 (Horvathova et al., Biol. Trace. Elem.
Res., 69: 15-26 (1999)). One of ordinary skill in the art will know of other useful markers for identifying other surface fluid cells.

Once the population containing the target cells has been recovered, the target cells, in particular the hematopoietic cells, are separated and cultured in a suitable medium containing a combination of growth factors sufficient to maintain growth. . Methods for culturing target cells are well known to those of skill in the art and will only be described briefly here. Any suitable medium can be used and these are readily available from commercial vendors. The seeding level is not critical and depends on the cell type used, but generally the seeding level for hematopoietic cells is that the cells are CD34
Is at least 10 per ml, more usually about 100 and usually less than 10 ⁶ .

Various types of media (solid or liquid) can be used. As a non-limiting example, DMEM, Iscove's modified Dulbecco's medium (IMDM), X-vivo15 (
Serum-free), and RPMI-1640. These are commercially available, for example, from various vendors. Various types of nutrients, growth factors, cytokines can be added to the preparation. The medium may or may not contain an appropriate amount of serum such as fetal bovine serum, autologous serum or plasma. However, when cells or cell populations are used in humans, the medium is preferably serum-free or autologous serum or plasma (Lansdorp, et al., J. Exp. Med. 175: 1501 (1992) and Petzer,
et al., PNAS 93: 1470 (1996)). In addition, Freshney, RI, “Culture of Animal
See Cells, A Manual of Basic Techniques ”, Wiley-Liss, Inc. (1994).

Non-limiting examples of other chemical compounds that can also be added to the medium include TPO
, FL, KL, IL-1, IL-2, IL-3, IL-6, IL-12, IL-
11, stem cell factor, G-CSF, GM-CSF, Stl factor, MCGF, LIF
, MIP-1α and EPO. These compounds can be used alone or in any combination, and a suitable concentration range can be easily determined from published literature. When culturing mouse stem cells, suitable non-limiting media include mIL-3, mIL-6, and mSCF. Other molecules can also be added to the medium, for example adhesion molecules such as fibronectin and / or RetroNectin ^™ (Takara Shuzo, Otsu, Shiga, Japan).

In vitro assays for mammalian stem cell activity include long-term culture initiation assays (L
TCIC) and Coblestone region formation assay (CAFC) (Pettenge
ll, et al., Blood 84: 3653 (1994); Breens, et al., Leukemia 8: 1095 (1994);
Reading, et al., Exp. Hem. 22: 786 (Abst # 406) (1994); Ploemacher, et al.
, Blood 74: 2755 (1989)). In CAFC, sparsely populated cell populations simply expressed in terms of their ability to form distinct clonal outgrowths on stroma cell monolayers over a period of time. To be tested. This assay provides frequency readouts associated with LTCIC and can predict engraftment in in vivo assays and patients. A particularly suitable CAFC assay is Young, et al., Blood 88: 1619 (1
996). Flow cytometry can be used to obtain (subset) a subpopulation of hematopoietic cells from various tissues based on the surface antigens expressed by the hematopoietic cells. These assays can also be combined to test hematopoietic cells or stem cells.

In one preferred embodiment of the present invention, a nucleic acid sequence encoding a mutant protein tyrosine kinase receptor (PTKR) as a selectable marker which is operably linked to an expression control sequence is introduced into a cell to form a genetically modified cell. Mutated PTKR forming and genetically modified in cells
And a genetically modified cell that expresses a mutant PTKR is identified. In a preferred embodiment, the selectable marker is a mutant epidermal growth factor receptor (EGFR) family member, particularly mutant EGFR1 and more preferably EGFR1-I and EGFR1-II. A polynucleotide or nucleic acid "encodes" a polypeptide and is transcribed and / or translated to produce a polypeptide or fragment thereof, either in nature or when manipulated by methods well known to those of skill in the art. .. The construct or vector containing the mutant PTKR of the present invention can be introduced into a target population by genetic modification or genetic transfer.

As used herein, the term “genetic modification” means an addition, deletion, or disruption to a cell's normal nucleotides. This method of genetic modification includes any method of genetic modification in which a nucleic acid sequence encoding the selectable marker of the present invention and containing, for example, a heterologous or foreign gene is introduced into a mammalian target cell. These methods are generally known. The term "introduce" is used broadly and includes, for example, insertion. Non-limiting examples of "genetic modification" include transduction (in vivo or ex vivo, virus-mediated transfer of host DNA from host or donor to recipient), transfection (by isolated viral DNA). Transformation of cells), liposome-mediated transfer, electroporation, calcium phosphate transfection and other means well known in the art. For transduction, direct co-culture of cells and producer cells (Bregni, et al., (1992)
Blood 80: 1418-1422), a method of culturing with appropriate growth factors and polycations or virus supernatant alone (Xu, et al., Exp. Hemat. 22: 223-230) and spinoculation. is there.

In a preferred embodiment, the mutant PTKR of the invention, in particular the mutant EGFR marker, comprises
Target cells are transduced with a retroviral vector as described above. The range of infected hosts is determined by the viral envelope protein. Recombinant virus can infect virtually any type of cell recognized by the env protein provided by the packaging cells, integrates the viral genome into transduced cells, and stably produces foreign gene products. To do. In general, MoMLV mouse ecotropic env can infect rodent cells, but
v can infect several primate cells, including rodents, avian cells, and human cells. Amphotropic packaging cell lines that can be used in retroviral systems are known in the art and are commercially available. As a non-limiting example, PA12, P
A317, ΨCRIP, and FLYA13 can be mentioned (Miller et al.
Mol. Cell. Biol. 5: 431-437 (1985); Miller et al. Mol. Cell. Biol. 6: 289.
5-2902 (1986); Danos et al. Proc. Natl. Acad. Sci. USA 85: 6460-6464 (1988).
). Recently, vesicular stomatitis virus G-glycoprotein (VSV-G)
Was replaced with the MoMLV env protein (Burns, et al., Proc. Natl. Cad.
Sci. USA 90: 8033-8037 (1993); and WO92 / 14829). There are also heterologous vector systems capable of infecting human cells.

When the target cells are transformed with a nucleotide sequence containing the selectable marker mutant PTKR and, optionally, a heterologous gene, the modified cells expressing the selectable marker PTKR can be identified in a variety of ways. As used herein, the term "identifying" with respect to modified cells means labeling, purifying, concentrating, isolating or separating, unless otherwise specified. Identification can be done in single or multiple steps.

In a preferred embodiment, genetically modified cells expressing a selectable marker of the invention can be identified by an antibody that specifically recognizes and binds the selectable marker, particularly an antibody that recognizes a mutant EGFR. . This type of antibody is described by O'Rourke, et al., Oncog
ene, 16: 197-1207 (1998). Furthermore, if the secondary antibody is bound to a fluorescent source or immunomagnetic beads, this secondary antibody can be used to further identify or select antibody-coated cells. Genetically modified cells expressing the selectable marker are then selected using flow cytometry, or magnetically coated cells are selected using a magnet.

Techniques for identifying genetically modified cells that express the selectable marker of the present invention include, in addition to the above, non-limiting examples of immunoselection, solid support bound RNA.
Nucleotide analysis by Northern blot to analyze the binding between DNA and liquid phase DNA, nucleotide analysis by Southern blot to analyze the binding between genomic DNA bound to solid support and liquid phase DNA, PCR amplification of genomic DNA, on solid support Western blot protein analysis to analyze the binding between bound protein and liquid phase antibody, reverse transcription of mRNA and amplification by PCR, and chromosome by in situ hybridization by fluorescence with liquid phase DNA FISH (Lawrence, et al., Sciene, 249 (4971): 928-932 (1990)) which is an analysis of the above.

Each of the above methods is not described in detail herein. They are well known to those skilled in the art. Briefly, antibodies can be obtained by well known methods or Harl
ow, et al., “Antibodies: A Laboratory Manual: (1988), Biosupplynet Sour
See Ce Book, (1999) Cold Springs Harbor Laboratory.
Either polyclonal or monoclonal antibody-producing cells that react with the antigen of interest can be produced. According to the invention, the antibody must recognize the extracellular region of the mutant PTKR selectable marker. More specifically, if a portion of the extracellular region has been modified, eg, a deletion, the antibody must recognize an epitope on the remaining amino acid sequence of the mutant PTKR.

Monoclonal antibodies against EGFR are commercially available. For example, Calibiochem (CA), Pharmingen (CA), Becton Dixon (CA), and American Type Culture Collection (ATCC) (Virginia). The antibody is
For example, assayed and identified in vitro by gel diffusion, immunoassay, immunoelectrophoresis, and immunofluorescence. Once the genetically modified cells are labeled, they are incubated with antibodies to the mutant receptor.

Genetically modified cells are physically separated using antibodies, flow cytometry such as fluorescence activated cell sorter (FACS) or bead selection (US Pat. No. 5,011,912).

In FACS, an antibody that recognizes a mutant PTKR is used to identify cells that express PTKR. This primary antibody is conjugated to a fluorescent source such as fluorescein isothiocyanate (FITC), phycoerythrin (PE), cy-chromium (CyC), allophycocyanin (APC), tricolor (TC), or Texas red (TX). You can When the primary antibody is not bound to the fluorescent source, the secondary antibody binding to the fluorescent source is introduced into a cell sample containing cells expressing the mutant PTKR and recognized by the primary antibody. The primary antibody attaches to the mutant PTKR. The secondary antibody binds to the primary antibody and cells carrying this secondary antibody fluoresce. Separation can be done with a fluorescence activated cell sorter.

Genetically modified target cells expressing a mutant PTKR, and optionally a second nucleic acid sequence encoding a protein of interest, may be added to the cells prior to or after identification and selection, with or without additives by methods well known to those of skill in the art. However, it can be grown in an appropriate medium for a few days or several weeks (Freshney, Celis, and Coligan, supra).

The genetically modified cells obtained by the method of the invention can further be used in an autologous or allogenic setting. There, genetically modified target cells, preferably hematopoietic cells, are expanded and used for genetic therapy such as, for example, bone marrow transplantation, facilitation of engraftment, or immune reconstitution. Proliferated cells expressing the mutant cell surface antigen are loaded into the patient. Samples can be taken and tested for selectable markers by FACS analysis, PCR or FISH, above, to determine the viability of labeled cells and to further assess transduction efficiency.

The invention as generally described above is explained in more detail with reference to the following examples. These examples are included solely for the purpose of describing certain embodiments of the invention and are not intended to limit the invention in any way.

[0078]

[Example 1]A. Isolation of human EGFR cDNA   Human EGFR cDNA is obtained from colon adenocarcinoma SW480 cell line (Mar
athon cDNA Clontech (CA); Leibowitz, et al., CancerResearch 36: 4562-4569
 (1976)) and isolated by PCR from the cDNA. The following primers
Software from Life Technologies (MD) and use it as described.

[0079]

[Table 1]

[0080]B. Manufacture of EGFR1-I   SW480 colon adenocarcinoma cell line cDNA to EGFR intracellular
To produce deletion mutants (FIGS. 2 and 3), primers EGFR1 and EGF
R2220R is used. Amino acid residues 679-1210 of the sequence of Figure 2 were deleted.
This EGFR mutant is designated as EGFR1-I.

The 5 ′ primer EGFR1 covers the start codon of EGFR. The 3'primer EGFR2220R contains a stop codon in place of amino acid 679 of SEQ ID NO: 2 (Figure 2). By using the primers EGFR1 and EGFR2220R,
An EGFR sequence deleted of amino acid residues 679-1210 can be amplified.

The following PCR reaction was performed. Marathon cDNA (~ 0.5 ng) Advantage cDNA buffer (10 mM Tris-HCl (pH = 7.5, 42 ° C), 50)
mM KCl, 2.5 mM MgCl ₂ , 0.001% Gelatin, 2.5 μmol dATP, 2.5 μmol dCTP, 2
.5 μmol dGTP, 2.5 μmol dTTP), 0.01 OD260 Primer EGFR1 and 0.01 OD
260EGFR2220R, 1 μl Advantage cDNA polymerase (Life Tech
nologies; MD), 5U Pfu Turbo polymerase (from Pyrococcus furiosus) (Promega; WI) and water (final volume 50 μl). PCR is performed as follows. Cycle 1: 95 ° C for 5 minutes; Cycle 2-15: 95 ° C for 1 minute,
60 ° C. for 1 minute, 68 ° C. for 4 minutes; and cycle 16: 68 ° C. for 10 minutes.

The reaction is cooled to 4 ° C. in a PCR machine and then the amplified cDNA is ethanol precipitated in 0.3 M sodium acetate. Wash the pellet with 70% ethanol,
Dry and resuspend in 50 μl water.

1 μl of the above PCR reaction was reamplified and in addition to 1 μl of the above PCR reaction, the reaction mixture contained the following for the second round of amplification: Pfu buffer (20 mM Tris-HCl (p
H = 8.8), 2 mM MgSO4 , 10 mM KCl, 10 mM (NH 4) 2 SO 4, 0.1% Triton X-100, 0.
1 mg / ml BSA), 2.5 μmol dATP, 2.5 μmol dCTP, 2.5 μmol dGTP, 2.5 μmol
dTTP, 0.01 OD260 primer EGFR1 and 0.01 OD260EGFR2220R, 5
U Pfu Turbo Polymerase (from Pyrococcus furiosus) (Promega; WI)
And water (final volume 50 μl). PCR is performed as follows. Cycle 17: 9
5 ° C. for 5 minutes; Cycle 18-48: 95 ° C. for 1 minute, 60 ° C. for 1 minute, 72 ° C.
For 4 minutes; and cycle 49: 72 ° C. for 10 minutes.

The reaction is cooled to 4 ° C. in a PCR machine, then the amplified cDNA is ethanol precipitated in 0.3 M sodium acetate. Wash the pellet with 70% ethanol,
Dry and resuspend in 20 μl water. The PCR reaction is loaded on a 1 × TAE gel. A band of approximately 2200 bp was isolated from the gel and the SrfI restriction site pPCR script Amp vector (Strateg
ene; CA). The resulting vector was designated as pPCR-script (Sc
ript) EGFR1-I. The accuracy of the subcloned PCR product is confirmed by restriction enzyme analysis and well known sequencing methods.

[0086]C. Manufacture of EGFR1-II   EGFR1-II has the same deletion in the intracellular region as EGFR1-I (instead of 679).
Stop codon). Furthermore, the extracellular region of EGFR shown in FIG. 2 (SEQ ID NO: 2)
Amino acids 25-312 are deleted in the region. As a result, extracellular EGFR
Amino acid 1-24, which is a signal peptide in the region, is fused with amino acid 313
is doing. In order to delete the extracellular region of EGFR, a protocol described in detail below
Use call. White, ed. Mehods in Molecular Biology, Chapter 25 (1993)
 See also This method uses "gene splicing by overlap extension.
It is known to those skilled in the art as "Gene SOEing" (Fig. 4).

Primer pair EGFR1 / EGFR3 and EGFR2 / EGFR2220R
To amplify two PCR products (a) and (b). Primer EGFR3
Encodes amino acids 18-24 fused to the nucleotide sequence of amino acids 313-319. Using the EGFR1 / EGFR3 primers, amino acids 313-
A PCR product encoding amino acids 1-24 fused to 319 is prepared.

The primer EGFR2 encodes the same amino acid as the primer EGFR3 in the opposite direction. Primer EGFR2220R contains a stop codon instead of amino acid 679. This deletes the intracellular region after amino acid 678, as described in Section B above.

Using these two primer pairs, amino acids 18-24 and amino acid 3
Two PCR products (a) and (b) are obtained which overlap the sequence coding 13-319 (Fig. 4-first PCR reaction).

In the second PCR reaction, primers EGFR1 and EGFR2220
R is used with the intermediate PCR products (a) and (b). When these intermediate PCR products are mixed, denatured, and annealed, one of the strands of the two PCR products overlaps at their 3'ends and functions as the other primer, producing a mutant product. Then, the mutated PCR product (c) was used as primers EGFR1 and EG
Amplify using FR2220R. The resulting PCR product has a stop codon of 679
Amino acid 313-678 of wild type EGFR and amino acid 1 of wild type EGFR
-24 encodes fused EGFR1-II (FIG. 4).

The PCR reaction is as follows. First PCR reaction: a) 0.01 OD260 primer EGFR1 and 0.01 OD260 EGFR3 and b) 0.01 OD260 primer EGF
R2 and 0.01 OD260EGFR2220R were cloned into Marathon cDNA (~ 0
.4 ng), 1x Advantage cDNA Polymerase Buffer (B above), 1 μl Advantage cDNA Polymerase, 2.5 μmol dATP, 2.5 μmol dCTP, 2.5 μmo
l dGTP, 2.5 μmol dTTP, 5U Pfu polymerase (Promega) and water (final volume 50
μl). PCR is performed as follows. Cycle 1: 94 ° C for 5 minutes; Cycle 2-16: 94 ° C for 0.5 minutes, 60 ° C for 1 minute, 68 ° C for 7 minutes;
And cycle 17: 68 ° C. for 10 minutes.

The reaction is cooled to 4 ° C. in a PCR machine, then the amplified cDNA is ethanol precipitated in 0.3 M sodium acetate. Wash the pellet with 70% ethanol,
Dry and resuspend in 50 μl water.

5 μl each of the above PCR reactions (a) and (b) are reamplified. In addition to each PCR reaction, the reaction mixture contains for the second round of amplification: a) 0.01 OD260 primer EGFR1 and 0.01 OD260 EGFR3 and b) 0.01 OD260 primer E
GFR2 and 0.01 OD260EGFR2220R1 x Pfu buffer, 2.5 μmol dATP, 2
.5 μmol dCTP, 2.5 μmol dGTP, 2.5 μmol dTTP, 5U Pfu polymerase (Prome
ga; WI) and water (final volume 50 μl). PCR is performed as follows. Cycle 1
8: 94 ° C for 5 minutes; cycle 19-49: 94 ° C for 0.5 minutes, 60 ° C for 1 minute, 72 ° C for 6 minutes; and cycle 50: 72 ° C for 10 minutes.

The reaction is cooled to 4 ° C. in a PCR machine, then the amplified PCR product is phenol / chloroform extracted and ethanol precipitated in 0.3 M sodium acetate.
The pellet is washed with 70% ethanol, dried and resuspended in 20 μl water.

In the second PCR reaction, PCR reactants (a) and (b) were mixed in equal amounts,
Pfu turbopolymerase using primers EGFR1 and EGFR2220R
Re-amplify with (Stragene). PCR mix is the same as in cycles 18-50. P
CR will be conducted as follows. Cycle 1: 95 ° C for 5 minutes; Cycle 2-32:
95 ° C for 1 minute, 60 ° C for 1 minute, 72 ° C for 4 minutes; and cycle 33: 72 ° C.
For 10 minutes. The PCR reaction is loaded on the gel. Bands up to 1200 bp are isolated from the gel. Clone in SrfI restriction enzyme site pPCR script Amp vector (Strategene; CA) according to the manufacturer's protocol. The resulting vector is called pPCR-Script EGFR1-II. The accuracy of the subcloned PCR product is confirmed by restriction enzyme analysis and well known sequencing methods.

[0096]D. Production of retroviral vector containing mutant EGFR and viral supernatant   pPCR-script EGFR1-I and pPCR-script EGFR1-
EGFR1-I and E excised from II with restriction sites NotI and XhoI, respectively.
Murine leukemia virus (MFR) cleaved with GFR1-II sequence by NotI and XhoI
oMLV-based retrovirus vector pGla (GTI, Maryland)? (pGla
 Mutant sequence-IRES-Nerve growth factor receptor (NGFR)
Learn. These retrovectors are pGlaEGFR1-I and pGla.
Name it EGFR1-II.

The construct is an envelope construct pC expressing the vesicular stomatitis virus G-protein (VSV-G envelope) under the control of cytomegalovirus (CMV).
Human embryonic kidney cells 293T (293T cells) are co-transfected with iGL. In addition, the packaging construct pCiGP (encoding MoMLVgag-pol under the control of the CMV promoter) was added to 293T cells by the CaCl ₂ method (Clontech; CA).
)) (WO97 / 21825 and Rigg et al., Virology
21: 290-295 (1996)).

Viral supernatants were harvested 24, 48 and 72 hours after transfection and centrifuged at 1200 rpm in a Beckman GS-6KR centrifuge to remove particulate matter and then immediately used to transform cells. Or store in a dry ice / methanol bath. Viral supernatant packaging cell line (cell line) Pro
Used to transduce Pak-A-6 (PPA-6) (Systemix, Inc.). The PPA-6 cell line is a cell line derived from 293T cells that express the amphotropic MLV envelope and MLV gag-pol under the control of the CMV promoter. PP
A-6 cells are sorted by immunomagnetic bead selection or cell expressed EGF
If R is small, cells are sorted by FAC Sorting as described below. Supernatants from PPA-6 cells are harvested 2, 3 and 4 days after transduction and treated as for 293T cells. The supernatant from the PPA-6 cells produced contains recombinant viral particles that have an ambiotropic envelope and can be used for transduction of human primary cells and cell lines.

[0099]E. Tissue culture and cell lines:   The following saline and primary cells are used: (a) Human T cell line, CEMS
S (Frederico, et al., J. Biol. Regul. Homeost Agents, 7: 41-49 (1993)),
(b) Human embryonic kidney cells 293T (293T) (Pear, et al., Proc. Acad. Natl.
Sci. USA 90: 8392-8396 (1993)), and (c) PPA-6 (Pear, et al., Supra).
Human primary T cells were collected from human blood using a Ficoll gradient to separate the mononuclear cell fraction.
Get away (Noble and Cutts, Can. Vet. J. 8: 110-111 (1967), and Boyle
And Chow, Transfusion 9: 151-155 (1969)). Enter from Stanford Blood Bank
Human blood (Buffy coat Pietersz, et al., Vox Sang 49: 81-85 (198
5) Piertersz, et al., Blut. 54: 201-206 (1987)) in phosphate buffered saline (
Dilute 1: 1 with PBS). In a 50 ml tube, add 15 ml blood to 21 ml
Placed on Ficoll (Isoprep; Robbins Scientific, CA)
It 30 minutes at 1700ppm in a Beckman GS-6KR centrifuge at room temperature
Apply a slope between and stop without using the brakes. Peripheral blood mononuclear cells (PBM
C) is recovered from the border of Lymphoprep.

The resulting PBMCs are purified on a second Ficoll gradient to remove residual red blood cells. PBMC with the following medium for PBMC in a tissue culture flask 37
Incubate at 5 ° C., 5% CO ₂ for 1 hour to attach adherent cells such as macrophages to the tissue culture flask. C using non-adherent cells (T / B / NK cells)
Purify D ⁴⁺ T cells. Cells (2x10 ⁸ ) are incubated with 300 μl of anti-CD4 antibody for 1 hour at 4 ° C. Cells are washed 3 times with PBS and incubated with 1 × 10 ⁸ anti-mouse IgG conjugated immunomagnetic beads (Dynal. Oslo) for 1 hour at 4 ° C. CD4 ⁺ cells bound to anti-CD4 antibody and secondary antibody-conjugated beads are positively selected by incubating the cells with the Dynal magnet for 10 minutes. After removing unbound cells, the remaining cells are removed from the magnet and placed in culture (below). Usually the beads remain on the cells for up to 10 days.

CD34 ⁺ cells were isolated from G-CSF mobile peripheral blood (MPB) on Isolex 300SA.
Alternatively, it is isolated using 300I (Baxter, IL) (Systemix, CA). Cells are about 80-90
% Purity.

The cells were inoculated with a Steri-Cult 200 incubator (Forma-Scientif).
ic), culture with 5% CO ₂ . Culture medium (DMEM, Iscove medium, RPM
I), PBS, and sodium pyruvate are obtained from JRH BioSciences (CA). FBS is Hyclone, (Utah), L-glutamine, trypsin, and MEM vitamins are Life Technology (Maryland), ITS (insulin, transferrin and sodium selenate), phytohemagglutinin (PHA), and interleukin 2 (IL-2). Is from Sigma, (Missouri).

Cells were treated with DMEM, 10% FBS, 1% sodium pyruvate, and 1% L-glutamine (293T, -6); RPMI, 10% FBS, 1% sodium pyruvate, and 1% L-glutamine (CEMSS). ); Iscove's medium, 10% FBS,
And 1% L-glutamine, 1% ITS (0.5 mg / ml stock), 1% MEM vitamin (human PBMC). CD4 ⁺ T cells 1-2 every 10-12 days
Stimulate with μg / ml PHA, irradiated feeder cells and 40 U / ml IL-2.

Irradiated feeder cells are prepared as follows. PBMCs are isolated as above and irradiated at 3500 rad. The cells are mixed with B cell lymphoma JY irradiated at 6000 rad at a ratio of 10: 1 (Barbosa, et al., Proc. Natl. Acad.
Sci. 81: 7549-7553 (1984)).

To obtain quiescent T cells, T cells were injected with 20 U / ml IL 4-5 days after stimulation.
Transfer to fresh medium containing -2. CD34 ⁺ cells were 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.). Let To passage adherent cells, cells are washed once with PBS, trypsinized for 5 minutes and then split into new tissue culture flasks (VWR, New Jersy).

[0106]F. Traits of PPA-6, human T cell line, primary T cells, and CD34 ⁺ cells Introduction:   Viral supernatants produced from either 293T cells or PPA-6 cells 1-
Using 3 ml, 10 from step (E)⁶8 μg / ml sulfate
Spinoculation with Rotamine (Sigma, Missouri)
To transduce. Spinoculation for 3 hours at 37 ° C, 3000 rp
m (CD34⁺Cells, PPA-6), or 2750 rpm (human T cell line,
CEMSS). PPA-6 cells are transduced in 6-well plates. Non-contact
Adherent cells are transduced in 6 ml test tubes (VWR). Human T cells and CD34⁺Fine
Cells were PHA / IL-2 or TPO / FL / SF 2 days before transduction, respectively.
Activate with.

[0107]G. FACS analysis of cells expressing EGFR1-I and EGFR1-II, FA Selection by CSsorting and immuno-magnetic beads:   Stain using the following antibodies and reagents. CD4-FITC (Caltag, CA),
Anti-CD34-APC (Becton Dickinson, NJ), Thy-1-PE (Becton Dic
kinson, NJ), propidium iodide (PI, Sigma), unconjugated
-EGFR antibody (GR01) (Calbiochem; CA), goat anti-mouse IgG-PE /
FITC (Caltag, CA), goat anti-mouse IgG2a-PE / FITC (Caltag, CA)
, Anti-mouse IgG conjugated magnetic beads (Dynal, Oslo). Antibodies and immunobeads are manufacturers
Use according to the protocol. 1x10⁶50 μl PBS / 2% F
Stain in BS for 20-60 minutes at 4 ° C. When using a secondary antibody,
Wash once with 2 ml PBS / 2% FBS and once again 50 μl PBS / 2% FBS
Incubate in and add secondary antibody. Before FACS analysis,
The cells were further washed once with PBS / 2% FBS, centrifuged, and 1 μg / ml PI was added.
Resuspend in 500 μl PBS / 2% FCS containing. FACS analysis is manufacturer
FACSscan (Becton Dickinson Immunocytometry Group; CA
).

The cells expressing the marker genes EGFR1-I and EGFR1-II are sorted as follows. Anti-E was added to cells in 2 × 10 ⁷ / ml PBS / 2% FBS.
Stain with GFR antibody (at least 1 μl antibody / 10 ⁷ cells; GR01). P cells
Fluorescence source (FITC) that recognizes anti-EGFR antibody after washing twice with BS / 2% FBS
, PE) conjugated rat anti-mouse IgG antibody (5 μl / 10 ⁶ cells). The cells are further stained with PI to distinguish between dead and viable cells. Sort for EGFR-positive and PI-negative cells per 1x10 ⁶ cells / ml. This is a FACSstar Plus (Becton Dickinson Immun) according to the manufacturer's instructions.
ocytometry Group (CA).

To isolate cells by bead selection, cells are stained with anti-EGFR antibody. Incubate 10 ⁷ / ml with 10 μl anti-EGFR antibody (GR01) in PBS / 2% FBS for 1 hour on ice with occasional rocking. Cells with PBS / 2% FCS 3
Washed twice, anti-IgG antibody-bound magnetic beads (Dynal, Oslo) that recognize anti-EGFR antibody
) Is added (~ 5 beads per positive cell). The cells are again incubated on ice for 1 hour. Cells expressing EGFR are positively selected by Dynal magnetism in 10 minutes. Unbound cells are removed and cells expressing EGFR are provided in culture (step (E)).

FIG. 5 shows EGFR1-I after transduction with supernatant prepared from PPA-6 cells.
Expression of ICD34 ⁺ cells is shown. Primary human T cells were transduced with PPA-6 supernatant, selected with immunomagnetic beads and sorted by FACSstar Plus (
Without data). Cells are enriched from 16% to 92%.

[0111]

Example 2A. Isolation of human MuSK-R cDNA   Human MuSK-R is derived from fetal smooth muscle cDNA (Marathon cDNA, Invitron)
By PCR using primers linked to the 5'and 3'of MuSK-R cDNA.
To isolate. The following primers were obtained from Operon Technologies, Inc.
Used for amplification of uSK-R cDNA.

[0112]

[Table 2]

The 5 ′ primer MuSK21FN covers the 25 nucleotides before the start codon of MuSK-R. The second 5'primer MuSK34FN is MuSK-
It covers the R start codon (aa1) and its surrounding sequences. 3'primer Mu
SK2666RN covers the stop codon of MuSK-R and its surrounding sequences.
By using the primers MuSK21FN, MuSK34FN and MuSK2666RN, a ~ 2600 bp DNA fragment encoding wild-type MuSK-R can be amplified.

The following PCR reaction was performed. Marathon cDNA (~ 2 ng) Advantage cDNA buffer (10 mM Tris-HCl (pH = 7.5, 42 ° C), 50)
mM KCl, 2.5 mM MgCl ₂ , 0.001% Gelatin, 2.5 μmol dATP, 2.5 μmol dCTP, 2
.5 μmol dGTP, 2.5 μmol dTTP), 1 μg Primer MuSK21FN, 1 μg
Mix with primer MuSK2666RN, 1 μl Advantage cDNA polymerase, and water (final volume 50 μl). PCR is performed as follows. Cycle 1: 94 ° C for 5 minutes; Cycle 2-11: 94 ° C for 0.5 minutes, 63 ° C for 1 minute
Min, 68 ° C for 6 minutes; and cycle 12: 68 ° C for 10 minutes.

The reaction is cooled to 4 ° C. in a PCR machine and then the amplified cDNA is ethanol precipitated in 0.3 M sodium acetate. Wash the pellet with 70% ethanol,
Dry and resuspend in 100 μl water.

10 μl of the above PCR reaction is reamplified. In addition to 10 μl of the above PCR reaction, the reaction mixture contains for the second round of amplification: Pfu buffer (20 mM Tris-
HCl (pH = 8.8), 2 mM MgSO ₄ , 10 mM KCl, 10 mM (NH ₄ ) ₂ SO ₄ , 0.1% Triton X-10
0, 0.1 mg / ml BSA), 2.5 μmol dATP, 2.5 μmol dCTP, 2.5 μmol dGTP, 2.5
μmol dTTP, 1 μg primer MuSK21FN, 1 μg primer MuSK2
666RN, 5U Pfu Turbo Polymerase (from Pyrococcus furiosus)
And water (final volume 50 μl). PCR is performed as follows. Cycle 13: 9
4 ° C. for 5 minutes; Cycle 14-43: 94 ° C. for 0.5 minutes, 62 ° C. for 1 minute, 7
2 ° C. for 6 minutes; and cycle 44: 72 ° C. for 10 minutes.

The reaction is cooled to 4 ° C. in a PCR machine, then the amplified cDNA is ethanol precipitated in 0.3 M sodium acetate. Wash the pellet with 70% ethanol,
Dry and resuspend in 20 μl water. The PCR reaction is loaded on a 1 × TAE gel. Bands up to 2600 bp were isolated from the gel and SrfI restriction site pPCR script Amp vector (Strateg) according to the manufacturer's protocol.
ene; CA). The resulting vector was pPCR-script MuS.
It is called KR-wt. The accuracy of the subcloned PCR product is confirmed by restriction enzyme analysis and well known sequencing methods (nucleotide sequence shown in SEQ ID NO: 7).

[0118]B. Generation of mutations in the intracellular region of MuSK-R by PCR:   Using primers MuSK1380F, MuSK1657R and 1747R
, Plasmid pPCR-script MuSK-R to MuSK-R intracellular region
Create a mutant. The primers are as follows, where "p" means phosphorylation
To do.

[0119]

[Table 3]

The use of primers MuSK1380F and MuSK1657R resulted in the deletion of amino acid residues 538-879 of MuSK-R, resulting in primer Mu
By using SK1380F and MuSK1747R, MuSK-R
Resulting in a deletion of amino acid residues 577-879. These two MuSK-R mutants were designated as MuSK-RΔ538-879 (MuSK-RI) and MuSK-RΔ57.
7-879 (MuSK-RII). MuSK-RI and MuSK-RI
Both I and I lacked most of the intracellular region of MuSK-R (see Figure 6).
Although not limiting the present invention in any way, it is considered that these two cleavages delete most of the substrate binding motif and the kinase region of wild-type MuSK-R shown in FIG.

The 5'primer MuSK1380F is the nucleotide 1333-of MuSK-R.
1410 is covered. The 3'primers MuSK1657R and 1747R are
A stop codon is included in place of amino acids 538 and 577 of MuSK-R. Using nucleotides 1333-1614 of MuSK-R having a stop codon at amino acid 538 or nucleotides 1333-1728 of MuSK-R having a stop codon at amino acid 577, respectively, using primer MuSK1380F and primer MuSK1657R or 1747R. Can be done. The PCR reaction was -10 μgh MuSK-R-wtDNA, 1 × Pfu buffer, 2.5 μmol dAT.
P, 2.5 μmol dCTP, 2.5 μmol dGTP, 2.5 μmol dTTP, 1 μg Primer Mu
SK1380F, 1 μg primer MuSK1657R or 1747R, 5U Pfu
Turbo polymerase and water (final volume 50 μl). PCR is performed as follows. Cycle 1: 95 ° C for 5 minutes; Cycle 2-31: 95 ° C for 0.5 minutes, 60 ° C for 1 minute, 72 ° C for 4 minutes; and cycle 32: 72 ° C for 10 minutes.
The reaction is cooled to 4 ° C. in a PCR machine and the PCR reaction is loaded on a 1 × TAE gel.

Two PCR products, MuSK-RI (nt 1380-1614) and MuSK-.
RII (nt 1380-1728) with SrfI according to the manufacturer's protocol.
Clone in the restriction enzyme site pPCR script Amp vector (Strategene; CA). These constructs contained the MuSK 5'coding region (nt1-137).
Due to the lack of 9), this sequence is excised from pPCR-script MuSK-R-wt using the restriction enzyme sites NaeI and AatII. Modified MuSK sequence nt1-1
PPCR-script with 614 and nt1-1728, respectively,
Called R-script MuSK-RI and pPCR-script MuSK-RII. The correctness of the vector is confirmed by restriction enzyme analysis and sequencing methods well known in the art.

[0123]C. Production of retrovirus vector containing mutant MuSK-R and virus supernatant
  Wild-type MuSK-R and mutant MuSK-R were cloned into pPCR-script MuSK.
-R-wt, pPCR-script MuSK-RI, and pPCR-script
It was excised from MuSK-RII using the NotI and XhoI sites, NotI and Xh
Retrovirus vector based on murine leukemia virus (MoMLV) cleaved with oI
Clone into the multiple cloning site of pGla. These retro vectors
Are pGlaMuSK-R, pGlaMuSK-RI, and pGlaMuSK.
-Name it RII. These constructs were used as described in Example 1 to
Produce Irus supernatant.

[0124]D. Tissue culture and cell lines:   The experiment is carried out in the same way as described in Example 1.

[0125]E. Transduction of PPA-6 and human T cell lines:   The experiment is carried out in the same way as described in Example 1.

[0126]F. FACS analysis of cells expressing MUSK-RI and MUSK-RII, and , Immuno-selection by magnetic beads:   In addition to the antibodies listed in Example 1, anti-MuSK-R polyclonal serum, anti-
Flow using MuSK-R hybridoma supernatant (see Section G below)
Perform cytometry and immunomagnetic bead selection.

To isolate cells by bead selection, cells are stained with anti-MuSK-R antibody. To this end, 10 ⁷ cells / ml were added to 1-3 ml anti-M in PBS / 2% FBS.
Incubate with the uSK-R hybridoma supernatant for 1 hour with occasional shaking on ice. The cells were washed 3 times with PBS / 2% FCS, and anti-I that recognizes anti-MuSK-R antibody was detected.
Add gG antibody-coupled magnetic beads (~ 5 beads per positive cell). again,
Incubate cells on ice for 1 hour. The cells expressing MuSK-R are Dyn
It is positively selected in 10 minutes by al magnetism. Unbound cells are removed and MuSK-R
The cells expressing the are provided in culture (step (D)).

[0128]G. Production of a monoclonal antibody against the extracellular region of MuSK-R:   To produce a monoclonal antibody against the extracellular region (XC) of MuSK-R
In addition, MuSK-R XC was amplified by PCR, and the expression construct pSecTag2b (I
nvirtogen). Plasmid pSe of XC region of MuSK-R
CMV by cloning into the multivalent cloning site (MCS) of cTag2b
XC is expressed under the control of the promoter. In addition, the plasmid is
Myc and (His) after the site₆Since it has a -tag sequence,
Protein (MuSK-RXC) and myc and (His)₆-Tag fusion is alive
Jijiru The signal peptide of MuSK-R is replaced by the Igκ leader (Fig.
3). The extracellular region of MuSK-R without signal peptide is
Amplified by the PCR used, the primers are as follows, and "p" is
Means phosphorylation.

[0129]

[Table 4]

Primer MuSK116FPC starts the sequence after the signal peptide (nt 69-93). Primer MuSK1532RC contains the sequence before the start of the transmembrane region and the first two amino acids of the transmembrane region (nucleotide 146 of SEQ ID NO: 7).
(Corresponding to 2-1586). PCR reaction is -10 μgh MuSK-R
-WtDNA, 1xPfu buffer, 2.5 μmol dATP, 2.5 μmol dCTP, 2.5 μmol
dGTP, 2.5 μ? mol dTTP, 1 μg primer MuSK116FPC, 1 μg primer MuSK1532RC, 5U Pfu Turbo polymerase and water (final volume 50 μl). PCR is performed as follows. Cycle 1: 95 ° C for 5 minutes; Cycle 2-7: 96 ° C for 0.5 minutes, 60 ° C for 1 minute, 72 ° C for 6 minutes; Cycle 8-27: 95 ° C for 0.58 minutes, 58 ° C. 1 minute, 72 ° C for 6 minutes; and cycle 28: 72 ° C for 10 minutes. The reaction is cooled to 4 ° C in a PCR machine and
The CR reaction is gel purified. The PCR fragment is cloned into the SecTag2b EcoRV site. Thus, MuSK-R XC is colonized in frame with the N-terminal Igκ leader and the C-terminal myc- and (His) ₆ -tag. The resulting plasmid is called pSecTag-hMUSK-R.

To express MuSK-R XC, the plasmid pSecTag-hMUS was expressed.
KR is transfected into 293T cells by the CaCl ₂ method (described in Section C). 24 hours after transfection, the medium was replaced with fresh DMEM / 1.
Replace with 0% FBS or serum-free X-Vivo15. Supernatants of transfected cells are harvested after 48 and 72 hours. A total of 400 ml of supernatant is collected and frozen at -80 ° C until purification.

MuSK-R XC is purified from tissue culture supernatant by immobilized metal affinity chromatography. The metal ion is 0.1M NiCl ₂ . The column is a 1 or 5 ml Farcia Metal HiTrap Chelating Sepharose column. The equilibration buffer (buffer A) consists of 20 mM Na ₂ HPO ₄ , pH 7.4, 1 M guanidine hydrochloride, and 1 M NaCl and is passed through a 0.2 μM cellulose acetate filter. The elution buffer (buffer B) was 20 mM Na ₂ HPO ₄ , p.
H7.4 consisting of 1M guanidine hydrochloride, and 1M NaCl, and 0.5M imidazole and passed through a 0.2 μM cellulose acetate filter. A Pharmacia FPLC chromatography system is used to operate a column equipped with FPLC operating program software and a Pharmacia P50 pump. Purification is 4 ℃
Will be carried out.

Before starting the loading, inject buffer A into the pump. Prior to loading the carau, the load is pumped so that the pink color of tissue culture is visible at the junction to prevent the column from being washed in non-equilibrium conditions.

Tissue culture supernatant was 0.85 M NaCl, 1 M guanidine chloride, and 40 mM
Adjust to contain imidazole and adjust pH to 7.4. 8 columns
Equilibrate with% buffer B. Load the sample and place the column in column 7 under the above conditions.
Wash with volume. MuSK-R is 30% buffer B (150 mM imidazole)
Is eluted in 8 volumes or more. Fractions are collected from the start of operation.

Each fraction is tested by Dot Blot Assay (below). Selected positive fractions are tested by Western blot assay (Elisa: below). The positive fractions are collected and dialyzed against PBS with 10,000 MWCO membrane (Pierce Snakeskin). After dialysis, the optical density measured at _{OD 280.} Samples are passed through a 0.2 μM filter and concentrated on a Centricon Centriprep 30 instrument in a refrigerator Sorvall RT6000D according to the manufacturer's protocol.

Dot Blot Assay To test positive samples, pipet 10 μl of each fraction onto nitrocellulose. The membrane was dried, blocked with superblock, probed for MuSK-R protein, and
Western: Color is developed as described below.

Western blotting is performed by methods well known to those skilled in the art. Materials (sample buffer, running buffer and gel) are obtained from Novex.

For Western blot analysis, use 25-35 μl per fraction. The guanidine-containing fractions are precipitated in ice-cold ethanol and stored on ice for 15 minutes or 4 ° C overnight. Samples in a refrigerated microcentrifuge (TOMY) at 14,000 rpm
Centrifuge for 10 minutes. Discard the supernatant, add ice cold acetone and centrifuge as above. Pellets containing SDS sample buffer containing 5% β-mertacaptoethanol (Novex)
Resuspend (final volume: 50-70 μl). Samples are denatured above 90 ° C. for 5 minutes, briefly centrifuged and 25-35 μl loaded on a 4-20% gradient gel. Bi
The orad wet transfer blotting cassette was replaced with Tris-glycine-methanol transfer buffer (25 mM Trizma base, 192 mM glycine, 20 mM).
% Methanol) and run on 0.45 μM nitrocellulose for 1.4 h. After blotting the gel, the blot is blocked in the Pierce TBS superblock for 10 minutes with gentle agitation. Blots were blotted to TBST (50 mM Tris, pH 7.5, 150 mM NaCl, 0.05% Tween on a rotating platform.
20) Wash twice for 5 minutes each. The Pierce India ™ His-HRP probe is diluted 1: 5,000 in TBST and the blot is incubated with this probe for 1 hour at room temperature and washed 3 times with TBST. The horseradish peroxidase (HRP) reagent (Sigma Fast HRP insoluble substrate D4418) is then added to the blot to develop the blot. Alternatively, mouse anti-c-myc antibody (Sant
a Cruz Biochemistry; CA) is used to detect recombinant MuSK-R protein. This antibody is diluted in superblock to a final concentration of 1 μg / ml. Blots are washed 3 times with TBST and goat anti-mouse IgG-HRP antibody (Sigma) diluted 1: 5,000 in superblock is added. The blot is incubated at room temperature with gentle agitation for 1 hour and developed with Sigma Fast HRP insoluble substrate as described above. The recognized protein migrates on SDS-PAGE at approximately 85 kD and is believed to be 19 kD heavy due to glycosylation.

Recombinant MuSK-R protein is injected into 3 Balb / c mice. To this end, 25-50 μg is 2.25 mg alhydrogel.
And 100 μg MDP (Muranyl dipeptide: Pierce) are mixed (final volume 200 μl) and injected subcutaneously 5 times every 14 days. After the 3rd and 5th injections, 3 sera are tested for reactivity to MuSK-R by FACS analysis and Elisa. For FACS analysis, 5 × 10 ⁵ cell lines CEMSS and CEMSSMuSK-R are used. 1: 100 both pre-serum and serum,
Dilute 1: 300, 1: 900 and 1: 2700. Rat anti-mouse IgG-
PE antibody is used as the secondary antibody at a dilution of 1: 200. For Elisa, 96-well plates are coated with 50 μl of 10 μg / ml anti-mouse IgG Fc (Jackson; Maine) antibody. Dilute plates variously (1: 100 to 1: 218700)
Incubated with the MuSK-R protein and then 1:10
Incubate with 00 dilution of nickel activated horseradish peroxidase (HRP, Pierce). Nickel-activated HRP was added to recombinant MuSK-R protein (
His) _6- tag. To develop this, TMP the plate
Incubate with peroxidase substrate (Zymed; CA). In both of these assays, one mouse shows the highest reactivity to native and recombinant MuSK-R. The mice are boosted with a sixth injection of MuSK-R (200 μg) in PBS. Injections are subcutaneous and intravenous. One week later, the spleen was removed, lymphocytes were isolated with lympholite M (Accurate Chemicals), and the myeloma cell line P3X63AG was isolated using 50% polyethylene glycol.
Fusion with 8.0653 by standard procedure. The resulting hybridoma is cultured in HAT bulk form. Viable cells are harvested with lympholite M and cultured in HAT medium containing cloning factor (Igen). When the hybridomas have grown for an additional week, one of the cell batches is HAT containing 10% DMSO.
Store frozen in medium. The other cell batch is split into each clone by FACSsorting using a single cell deposit unit. PI-negative cells are sorted by forward and side scatter. Cells are cultured in HAT medium for 2 weeks. Regarding monoclonal antibodies capable of recognizing native and recombinant MuSK-R proteins,
The supernatant is tested by Elisa and FACS. IgG1, 2a, 2b, 3, IgM
The isotype of the antibody is identified by an Elisa assay using a secondary antibody (Caltag, CA) that reacts with κ, κ and λ.

Three monoclonal antibodies H1, H2 and H4 have been identified. All three are capable of reacting with MuSK-R expressed on the CEMSS-MuSK-R cell line in a FACS assay. H1 is IgG1, κ, H
2 is IgG1, kappa and H4 is an IgM antibody.

[Sequence list]

[Brief description of drawings]

FIG. 1A is a schematic representation of a wild-type (WT) EGFR molecule and two mutant EGFRs: EGFR1-I with amino acid deletions from the cytoplasmic region and EGFR1-II with amino acid deletions from both the cytoplasmic and extracellular regions. Express B shows wild type and mutant MuSK.

FIG. 2 corresponds to the wild type (WT) EGFR1 molecule and SEQ ID NO: 2. The signal peptide is amino acid residues 1-24. Extracellular region contains amino acid residues 25-645
, The transmembrane region contains amino acid residues 646-668, the cytoplasmic region contains amino acid residues 669-1210, and the threonine phosphorylation site contains amino acid residues 678.
Is.

FIG. 3A is a WT EGFR1 nucleotide sequence encoding the amino acid sequence of FIG. 2 and corresponds to SEQ ID NO: 1.

FIG. 3B is a nucleotide sequence of wild-type (WT) EGFR1 encoding the amino acid sequence of FIG. 2 and corresponds to SEQ ID NO: 1.

FIG. 3C is a WT EGFR1 nucleotide sequence encoding the amino acid sequence of FIG. 2 and corresponds to SEQ ID NO: 1.

FIG. 4 shows a general scheme for creating deletions in the extracellular and intracellular regions of the EGFR sequences shown in FIGS.

FIG. 5 shows EGFR1-II expression on primary T cells and CD34 ⁺ cells after being transduced with supernatant obtained from PPA-6 cells. Panel A corresponds to non-transduced cells, panel B to post-transduction expression, and panel C to immunomagnetic bead selection for mutant EGFR expression on T cells. Panel D corresponds to untransduced, and panel E corresponds to the expression of CD34 ⁺ cells.

FIG. 6 represents MuSK-R designated hMuSK-R, corresponding to the nucleotide sequence shown in SEQ ID NO: 7 and the amino acid sequence shown in SEQ ID NO: 8.

─────────────────────────────────────────────────── ─── Continuation of front page (51) Int.Cl. ⁷ Identification code FI theme code (reference) G01N 33/53 G01N 33/566 33/566 C12N 15/00 ZNAA (31) Priority claim number 09/539, 248 (32) Priority date March 30, 2000 (March 30, 2000) (33) Priority claiming country United States (US) (81) Designated country EP (AT, BE, CH, CY, DE, DK, ES, FI, FR, GB, GR, IE, IT, LU, MC, NL, PT, SE, TR), OA (BF, BJ, CF, CG, CI, CM, GA, GN, GW, ML, MR, NE, SN, TD, TG), AP (GH, G M, KE, LS, MW, MZ, SD, SL, SZ, TZ, UG, ZW), EA (AM, AZ, BY, KG, KZ) , M , RU, TJ, TM), AE, AG, AL, AM, AT, AU, AZ, BA, BB, BG, BR, BY, BZ, CA, CH, CN, CR, CU, CZ, DE, DK , DM, DZ, EE, ES, FI, GB, GD, GE, GH, GM, HR, HU, ID, IL, IN, IS, JP, KE, KG, KP, KR, KZ, LC, LK, LR, LS, LT, LU, LV, MA, MD, MG, MK, MN, MW, MX, MZ, NO, NZ, PL, PT, RO, RU, SD, SE, SG, SI, SK, SL , TJ, TM, TR, TT, TZ, UA, UG, US, UZ, VN, YU, ZA, ZW F terms (reference) 2G045 AA34 AA35 AA40 BA11 BB50 CB01 DA12 DA13 DA14 DA36 FB02 FB03 4B024 AA15 BA63 CA04 DA03 EA02 GA11 HA14 HA15 4B063 QA05 QA18 QQ08 QQ79 QR55 QS33

Claims (30)

[Claims] 1. The following steps: (a) Introducing into a cell a nucleic acid sequence encoding a mutant protein tyrosine kinase receptor (PTKR) operably linked to an expression regulatory sequence to form a genetically modified cell ( Here, the mutant PTKR contains a modification in the intracellular region and the extracellular region, or
A gene other than any nerve growth factor receptor (NGFR) and containing modifications in the intracellular region), (b) a genetically modified cell that expresses a mutant PTKR, and (c) a genetically modified cell that expresses a mutant PTKR. A method for identifying a genetically modified mammalian cell, which comprises: 2. The method according to claim 1, wherein the mutant PTKR is selected from a mutant epidermal growth factor receptor (EGFR) family member and a mutant muscle-specific kinase receptor (MuSK-R) family member. 3. A mutant EGFR family member comprising EGFR1-I and EG
The method according to claim 2, which is a mutant EGFR1 appropriately selected from the sequence named FR1-II. 4. The mutant MuSK-R comprises the mMuSK-RI and mMuSK-RI.
The method of claim 2, wherein the method is selected from the sequence designated I. 5. The method according to any one of the preceding claims, wherein the intracellular region of the mutant PTKR is excised, and the extracellular region thereof is also excised appropriately. 6. The method according to claim 2, wherein the introducing step is carried out by incorporating a nucleic acid sequence encoding a mutant EGFR or a mutant MuSK-R into a vector and introducing the vector into cells. 7. The method according to claim 6, wherein the vector is a retrovirus vector. 8. The method of claim 1, wherein the identifying step is performed by contacting the genetically modified cell with an antibody that recognizes and binds the mutant PTKR. 9. The method according to claim 1, wherein the genetically modified cells are separated from the non-genetically modified cells in the identifying step. 10. The method of claim 1, further comprising the step of isolating cells that express the identified mutant PTKR. 11. The method of claim 1, wherein the cells are human cells. 12. The method of claim 11, wherein the cells are selected from the group consisting of hematopoietic cells, hepatocytes, endothelial cells, and smooth muscle cells. 13. The method according to claim 11, wherein the cells are hematopoietic cells. 14. The method according to claim 13, wherein the hematopoietic cells are stem cells or T cells. 15. The method according to claim 6, wherein the vector further contains a heterologous gene. 16. The following steps: (a) A nucleic acid sequence encoding a mutant protein tyrosine kinase receptor (PTKR) family member is contained in a vector (wherein the PTKR is incorporated into an intracellular region and an extracellular region). Modified, or any nerve growth factor receptor (NG
FR) and including modifications in the intracellular region), introducing the vector into mammalian cells to form genetically modified cells, (b) expressing the mutant PTKR in the genetically modified cells, and (C) identifying a genetically modified cell that expresses a mutant PTKR, which comprises: 17. The mutant PTKR has EGFR (preferably EGFR1-I or EGFR1-II) and MuSK-R (preferably mMuSK-RI or mMu).
The method according to claim 16, which is selected from SK-RII). 18. The method according to claim 16, wherein the vector is a retroviral vector. 19. The method according to claim 16, wherein the vector further contains a heterologous gene. 20. The following steps: (a) a protein tyrosine kinase receptor (P) operably linked to an expression regulatory sequence.
A nucleic acid sequence encoding a TKR family member is retrovirally transduced into mammalian cells, wherein the PTKR contains modifications in the intracellular and extracellular regions, or any nerve growth factor receptor. (Other than NGFR) and containing modifications in the intracellular region), (b) incubating the transduced cells with a marked antibody that specifically recognizes and binds the mutant PTKR, and (c) the marked transduction A method of immunoselection of transduced mammalian cells comprising identifying the cells. 21. The method of claim 20, wherein the cells are human cells or hematopoietic cells. 22. The mutant PTKR has EGFR (preferably EGFR1-I or EGFR1-II) and MuSK-R (preferably mMuSK-RI or mMu).
21. The method according to claim 20, selected from SK-RII). 23. Moloney murine leukemia virus (MoMLV), myeloproliferative sarcoma virus (MPSV), mouse embryonic stem cell virus (MESV), mouse stem cell virus (MSCV), and spleen focus forming virus (cells).
21. The method of claim 20, transduced by a retroviral vector from the group consisting of SFFV). 24. The method of claim 20, wherein the cells are transduced with a vector derived from a lentivirus. 25. The method of claim 20, further comprising the step of separating the identified marked transduced cells from the unmarked cells. 26. The method of claim 20, further comprising expanding marked transduced cells. 27. The following steps: (a) DNA encoding a mutant protein tyrosine kinase receptor (PTKR) family member operably linked to an expression regulatory sequence and a protein of interest.
A nucleic acid encoding a nucleic acid containing a sequence is introduced into a mammalian cell (where the mutant PT
KR contains modifications in the intracellular domain and extracellular domain, or is any other than nerve growth factor receptor (NGFR) and contains modifications in the intracellular domain), (b) the obtained mammalian cells are A method for identifying a mammalian cell that expresses a target protein, which comprises culturing, and (c) identifying a cell that expresses a mutant PTKR to obtain a cell that expresses the target protein. 28. The mutant PTKR has EGFR (preferably EGFR1-I or EGFR1-II) and MuSK-R (preferably mMuSK-RI or mMu).
28. The method according to claim 27, selected from SK-RII). 29. The method according to claim 27, wherein the nucleic acid encoding the mutant PTKR and the nucleic acid encoding the protein of interest are introduced into a retroviral vector in step (a). 30. A mutant muscle-specific kinase receptor (MuSK-R) family member having a truncated cytoplasmic region, which is preferably mMuSK-RI or mMuSK-RII.