IL147557A - Method for the immunoselection and immunoseparation of transfected eukaryotic cells - Google Patents

Abstract

PCT No. PCT/EP94/02687 Sec. 371 Date Apr. 4, 1996 Sec. 102(e) Date Apr. 4, 1996 PCT Filed Aug. 11, 1994 PCT Pub. No. WO95/06723 PCT Pub. Date Mar. 9, 1995A method of marking a eukaryotic (mammalian) cell by expressing in this cell a nucleic acid, said nucleic acid encoding a cell surface receptor, and by subsequently presenting said receptor at the cell surface, characterized by using a nucleic acid in which the region encoding the intracellular domain of the receptor has been completely or party deleted or modified in such a way that the receptor presented at the surface cannot, after binding to its binding partner, effect any signal transduction, is effective and usable in gene therapy.

Description

13255/bc/OlD METHOD FOR THE IMMUNOSELECTION AND IMMUNOSEPARATION OF TRANSFECTED EUKARYOTIC CELLS The present application is a divisional patent application from Israel Patent Application No. 110815 (hereafter "the parent application", "the parent invention").

The present invention relates to a method for the immunoselection and immunoseparation of transfected eukaryotic cells.

The parent invention is related to a method of marking eukaryotic (mammalian) cells by the use of a cell surface receptor with a modified intracellular domain as a selectable marker.

The identification of cells into which a DNA sequence has been successfully introduced is an essential step in recombinant DNA technology. As the DNA sequence to be introduced will not necessarily lead to a phenotype which may be selected in a simple manner, there is usually introduced an additional DNA sequence coding for a selectable phenotype. There are still only a very limited number of such selectable marker genes for use in recombinant DNA technology of eukaryotic cells. Most of these available marker genes, such as, for example, the Herpes simplex virus type I thymidine kinase (Wigler et al., Cell 1 (1977) 223) or hypoxanthine phosphoribosyl transferase (Jolly et al., Proc. Natl. Acad. Sci. 80 (1983) 477) correspond to genes, or are highly related to genes, which are constitutively expressed in the majority of normal cells or are identical with such genes.

Therefore, these marker genes can only be used in special mutant cells, which do not express the corresponding gene. A preferred marker gene on the other hand should not be expressed in the majority of mammalian cells or at least not be expressed in certain tissues and cell types, so that they can be used as selectable marker genes in recombinant DNA technology involving these cells.

Pawlink et al. have described the use of the CD24 cell surface antigen as a dominant selectable marker in retroviral mediated gene transfer (Journal of Cellular Biochemistry, Supplement 17 E, page 203, abstract S210). With this marker NIH-3T3 fibroblasts, BAF-3 pre-B cells as well as murine bone marrow cells could be labelled by retroviral infection and the labelled cells were detected by fluorescence activated cell sorting analysis. However, as the CD24 cell surface antigen is normally expressed on some mammalian cells, the use of this marker will be limited by the difficulty to differentiate between cells normally expressing the CD24 cell surface antigen and cells labelled with this marker.

Furthermore, it has been found that the CD24 cell surface antigen, following the heterologous expression, is presented at the cell surface to a little extent only.

It was therefore the object of the parent invention to provide a process for marking eukaryotic cells, with the use of cell surface receptors, by means of which process the cell surface receptors are presented to a large extent at the cell surface, whereby the sensitivity of the selection and of the detection of marked cells can be increased.

This object is accomplished by a process of marking a eukaryotic (mammalian) cell by expressing in this cell a nucleic acid, the nucleic acid encoding a cell surface receptor, and by presenting the receptor at the cell surface, the process being characterized by the use of a nucleic acid in which the region encoding the intracellular domain of the receptor is completely or partly deleted, or modified so that the receptor presented at the surface cannot effect any signal transduction after binding to its binding partner.

Preferably, the intracellular domain is completely deleted, or individual nucleotides are modified.

Preferably, it is used the nucleic acid encoding the NGF-, CD24- or the LDL receptor.

The sequence of the human NGF receptor, which will be referred to hereinafter as "low affinity nerve growth factor receptor, LNGFR", is described in D. Johnson, Cell 47 (1986) 545-554. Preferably, there is used a nucleic acid in which the DNA region encoding the amino acids 245 (beginning with nucleotide 930) to the C-terminus has been deleted (cf. SEQ ID NO. 1).

In SEQ ID NO. 1 the extracellular domain is coded by the nucleotides 114 (ATG) - 863, the transmembrane domain is coded by the nucleotides 864 - 929, and the intracellular domain is coded by the nucleotides 930 - 1394 (Reddy et al., Molecular Brain Research 8 (1990) 137-141).

For the deletion of the intracellular domain it is possible to delete the complete domain or only a functional part so that the receptor presented at the surface cannot effect any signal transduction after binding to its binding partner. For example, it is possible to delete the nucleotides 943 - 1512 by restriction with PvuIII and Sstl.

The nucleic acid can be introduced in the target cell, for example, via a viral vector (preferably, retrovirus) by means of lipofection or electroporation.

A further subject matter of the parent invention is a DNA, which encodes a modified LNGFR and which is illustrated in SEQ ID NO. 1, as well as an eukaryotic cell containing such a nucleic acid.

A nucleic acid of this type can be used to express a so modified cell surface receptor, and to present the receptor at the cell surface in such a way that the receptor can be used as a selectable marker for transfected eukaryotic cells.

The DNA encoding receptor or a derivative thereof will be introduced into an eukaryotic expression vector by methods known in the art. Suitable expression vectors are known to a person skilled in the art, retroviral vectors are preferably used. Also other viral vectors according to the state of the art can be used for the transfer of the receptor DNA (e.g. herpes viruses, adenoviruses, vaccinia viruses). With a certain type of retrovirus, so-called double copy vectors (Yu et al., Proc. Natl. Acad. Sci. 83 (1986) 3194-3198), a high frequency of rearranged provirus and a significantly lower virus titre have been observed. XSN vector viruses (Bio-Techniques 7 (1989) 980-990) are preferred.

The recombinant eukaryotic expression vectors are then introduced into a eukaryotic host cell, either alone for a gene marking protocol, or in combination with a further DNA to be transfected, but which has no selectable phenotype. The introduction of the DNA into the host cell is accomplished by methods for viral infections known in the art. In addition, the transfer of the receptor gene can also be achieved by utilizing non- viral transfer methods (e.g. electroporation, liposomal transfer, or calcium precipitation). Cells containing the exogenous DNA can then be recognized by their ability to produce a receptor at high levels. Recombinant eukaryotic cells controlling a DNA according to the parent invention can even be more easily detected by their ability to produce a derivative of the receptor.

A subject-matter of the present invention is the easy selection and separation of the receptor-expressing cells described above by using antibodies that bind to the receptor. Such antibodies are known in the art and described, for example, by A. Ross et al. (Proc. Natl. Acad. Sci. 81 (1984) 6681-6685, EP-A 0 202 055). Furthermore, antibodies to the receptors can be obtained by methods known in the art by immunizing an animal with a protein encoded by DNA according to the parent invention and isolating the antibodies from the serum of the immunized animals, or by fusing spleen cells of the immunized animals with immortal cells, such as, for instance, a murine myeloma cell line, to obtain monoclonal antibodies. These antibodies can be labelled by methods known in the art (such as are described, for example, in J. H. Peters, Monoklonale Antikorper, Springer Verlag, 2nd edition, 1988, pages 285 to 315). Suitable labels will be enzymes, fluorescent or chemiluminescent dyes.

It is possible to isolate the marked cells by immunoselection. To this end, the transfected cells are identified, selected with a marked antibody binding specifically to the receptor employed according to the parent invention, and isolated by being split off the antibody/cell complex.

By using an immobilized antibody instead of a marked (labelled) antibody, transfected cells can be selected. Therefore, the solid phase with the immobilized antibody against receptor is used as a matrix in affinity chromatography. After separation of non-transfected cells not expressing receptor, bound cells are, for example, cultivated and then expanded on Petri dishes overnight.

The processes as described above will be especially important as diagnostic tools for the identification of cells introduced into a mammalian organism by gene therapy protocols. For some applications, such as, for example, bone marrow transplantation, it will be of diagnostic value to differentiate between cells originating from bone marrow transplantation and cells derived from residual cells of the host. In this case, bone marrow cells containing DNA encoding receptor are transplanted to provide these cells with a cell surface label.

Preferably, progenitor or stem cells which were enriched according to the state of the art (e.g. immobilized anti-CD34 antibodies) containing the gene according to the parent invention will be transplanted. After transplantation, cells, especially eventually arising tumor cells, can be differentiated as being derived from the transplanted cells or residual cells of the host organism.

One embodiment of the present invention is therefore the use of the process for immunoselection of transfected cells according to the parent invention for the diagnostic identification of cells, which had been labelled by introduction of a DNA encoding receptor, especially of a DNA according to the parent invention.

The main application of the marking of blood cells/bone marrow cells with the described modified receptors will be the monitoring of cells from autologous and also from allogenic transplants. In the treatment of leukemias and lymphomas, it is often necessary to treat the patients with sublethal doses of cytostatic pharmaceuticals or with comparable doses of irradiation or with combinations thereof. To restore bone marrow function either autologous bone marrow, which is explanted before treatment, will be reimplanted into the patients or allogenic marrow from a HLA matching donor will be implanted.

In the case of autologous bone marrow transplantation contamination of tumor cells in the reimplanted material might lead to a tumor relapse. Purging methods to purify the marrow from tumor cells (clonogenic tumor cells) are employed but these techniques are not reliable at this time. In the case of a relapse it is of course not possible to distinguish between these potentially contaminating tumor cells and a relapse caused by residual cells after treatment. In the case of allogenic bone marrow transplantation the rejection of the allogenic marrow is the main problem. Purging techniques are described in Areman E.M. et al: Bone Marrow and Stem Cell Processing: A Manual of Current Techniques, F.A. Davies Co., Philadelphia (1992), which is incorporated herein by reference.

The parent invention further comprises a method for the preparation of a hematopoietic cell preparation (i.e. bone marrow or mobilized peripheral blood progenitor cells and/or stem cells), wherein said cells are marked with a cell surface receptor by transfecting said cells with a nucleic acid which encodes a cell surface receptor and, preferably, in which the region encoding the intracellular domain has been completely or partly deleted or modified in such a way that the domain can essentially no longer effect any signal transduction. In the present invention, said cell preparation will be substantially freed from clonogenic tumor cells by purging the marked cells through the use of cytotoxic agents or irradiation, and separating said transfected cells from non-transfected cells. The separation is preferably accomplished by incubating said cells with an antibody against the receptor immobilized prior to or after incubation, separating the cells immobilized by the antibody from unbound cells, and isolating the hematopoietic cell preparation by cleaving the antibody-receptor binding.

If after autologous transplantation the transplanted cells are marked with a gene and consequently with the gene product derived thereof which is not common to these cells it is possible to trace these cells directly after transplantation and in the case of relapse to differentiate between tumor cells derived from the transplanted material and residual tumor cells. This knowledge will lead to a very early definition of the further treatment based on the source of relapse. Besides this diagnostic purpose, gene marking in autologous transplantation will also allow to monitor and compare the efficacy of different purging methods in relatively small patient groups.

For autologous and, preferably, allogenic transplantation the main field of application will be the monitoring of rejection of transplanted cells.

The clinical protocol will contain the following steps: a) Explantation of patient cells. b) Purging of explanted cells according to the known art. c) Transfection of the patient cells with a vector containing the modified receptor gene according to the parent invention. d) Facultatively expansion of the patient cells under selective conditions, e.g. with G418 utilizing the expression of the neomycin resistance gene coded on the vector containing the modified receptor gene. e) Immunoselection of the marked cells which express the modified gene and present the receptor on the surface of the cells. f) Reimplantation of the enriched and marked cells into the patient. g) Monitoring blood/marrow samples of the patient for the marking gene by FACS analysis or ELISA techniques.

Schematic Flow Diagram for the Gene Marking Protocol Explantation of the Bone Marrow Cells Ψ Freezing of aliquots Purging of cells Treatment of patient Ψ Transfection of cells with LNGFR vector Ψ Expansion under selective conditions Immunoselection for LNGFR positive cells Ψ Reimplantation of marked cells Ψ Monitoring of blood marrow cells Ψ No relapse Relapse Ψ Marked tumor cells Tumor cells not marked Ψ Relapse caused by Relapse caused by contaminating tumor cells residual tumor cells in the transplanted cell material The present invention further comprises a method for the immunoselection of cells transfected according to the parent invention, by introducing into said cells a nucleic acid which codes for a cell surface receptor in which the region encoding the intracellular domain of the receptor has been completely or partly deleted or modified in such a way that the receptor presented at the surface cannot, after binding to its binding partner, effect any signal transduction, identifying the transfected cells by incubation of the cells with a marked antibody which binds specifically to said receptor, and recovering the cells by cleavage from the antibody/cell complex.

The invention further comprises a method for the immunoseparation of cells transfected according to the parent invention, by introducing into said cells a DNA which codes for a cell surface receptor in which the region encoding the intracellular domain of the receptor has been completely or partly deleted or modified in such a way that the receptor presented at the surface cannot, after binding to its binding partner, effect any signal transduction, incubating the cells with an antibody against the receptor immobilized prior to or after incubation, separating the cells immobilized by the antibody from unbound cells, and isolating the cells by cleaving the antibody-receptor binding.

A large number of studies have demonstrated that retroviral vectors are an efficient tool for transfer of exogenous DNA into somatic cells!. In the mouse hematopoietic system, efficient gene transfer and expression has been achieved into both progenitors and pluripotent stem cells, in vitro as well as in vivo. Lower levels of gene transfer have been obtained in canine and human hematopoietic progenitors in culture, in which significant levels of expression of the transfected genes have been demonstrated (reviewed in ¾. Retroviral-mediated gene transfer is currently utilized in gene therapy protocols for treatment of inherited and acquired diseases, such as adenosine deaminase-deficient (ADA") severe combined immunodeficiency (SCID) and advanced cancer (reviewed in Although significant effort is being devoted to optimize procedures for purification of human hematopoietic stem cells and finding efficient conditions for gene transfer and expression, peripheral blood lymphocytes (PBLs) are still considered the safest cellular delivery vehicle for human gene therapy.

A number of different retroviral vectors have been engineered and used for gene transfer into human hematopoietic cells, all based on the Moline murine leukemia virus (MoMLV) backbone. Usage of different promoters driving expression of the gene of interest, and the position of these sequences with respect to the viral transcription unit, were some of the parameters taken into account in generating alternative vector designs^ 5 6 Some of these vectors were used to transfect human lymphoid cells under different conditions. Human tumor-infiltrating lymphocytes (TILs) were transfected with the N2 retroviral vector, carrying a neomycin phosphotransferase (Neo) gene, and retained normal phenotypic and functional characteristics in vitro 7 β. Transfected cells were recovered from tumor sites up to 64 days after in vivo cell administration to melanoma patients^. Gene transfer was obtained in both CD4+ and CD8+ human T-cell subsets derived from TILs and PBLslO. A retroviral vector for expression of a functional CD 18 gene, driven by viral LTR promoter, successfully transfected lymphocytes from patients affected by leukocyte adhesion deficiency (LAD), and led to correction of LAD in vitro. Expression of human ADA driven by an ADA promoter into a double-copy vector^ in PBLs obtained from patients affected by ADA" SCID also led to correction of the enzyme deficiency, allowing reconstitution of immune functions^ 13 Finally, retroviral constructs for overexpression of HIV structural RNA sequences (transacting responsive element, TAR, and Rev-responsive element, RRE), driven by a RNA polymerase III promoter, were successfully introduced into a T-lymphoid cell line, inducing partial intracellular immunization against HIV14 15 Although in all these cases retroviral vectors were shown to transfect human lymphoid cells and express the transferred genes, no attempt has yet been made to directly compare the different vector designs for stability, efficiency of gene transfer, and expression of the transfected gene.

The human low affinity nerve growth factor receptor (LNGFR) is not expressed on the majority of human hematopoietic cells, thus allowing quantitative analysis of the transfected gene expression for each vector and each cell target by immunofluorescence analysis, even at single cell level. Different human hematopoietic cell lines of myeloid and lymphoid origin, as well as normal peripheral blood mononuclear cells (PBMC), were transfected with four vector constructs and analyzed for stability and number of viral integrations and LNGFR expression, at both RNA and protein level. FACS analysis of transfected T-cell lines and clones for coexpression of LNGFR and cell surface markers was carried out to study gene expression into specific T-cell subpopulation. Under appropriate, high efficiency infection conditions, all retroviral vectors could infect a T-cell population of the normal immune repertoire. Therefore, according to the parent invention, LNGFR (in its truncated form without the intracellular domain, or in its (essentially) not modified form) is a preferred marker for cell marking, by transfecting the cells with a nucleic acid coding for LNGFR. Vectors such as retroviral vectors or also naked DNA, e.g. with liposomes as transfecting agent, are used for transfection. LNGFR marker is preferably used for marking cells of the hematopoietic system, especially hematopoietic stem cells. Especially preferred in the parent invention, is the use for marking in gene therapy, and in the present invention, is the use for purging methods.

Efficient retroviral vector gene transfer into hematopoietic stem cells still remains the prime goal of gene therapy for many congenital and acquired disorders. As vehicles, retroviral vectors are currently the safest and most effective tools for transfer and expression of exogenous DNA into human hemato-lymphopoietic cells. Actually, all the approved clinical protocols for gene transfer in marrow or blood cells rely on retroviral vectors!^. Although the ideal target cell is represented by the pluripotent stem cell, there is no conclusive evidence about the ability of retroviral vectors to infect such cells at adequate efficiency, and maintain stable gene expression in their progeny. The results of in progress clinical protocols based on bone marrow gene transfer may clarify these problems 1,3,30 Clearly, gene transfer can be more easily achieved into peripheral blood lymphocytes, a potential alternative to bone marrow cells, at least for congenital and acquired disorders of the immune system. However, for this purpose, it is necessary to define what proportion of PBLs needs to be transfected in order to represent the whole immunorepertoire, and whether this can be stably maintained in vivo, both necessary prerequisites for a gene transfer procedure to be of therapeutic relevance. For this purpose, the efficiency of gene transfer and vector design, which affects both persistence and levels of gene expression, are crucial factors.

The overall efficiency of gene transfer into human PBLs was strictly dependent upon the infection protocol. Limited cell expansion by short-term activation of lymphocytes in vitro, followed by gene transfer into PBLs by exposure to producer cell supernatants resulted in a limited proportion of G418-resistant cells (approximately 1% per infection cycle), with additional variability introduced by the titre of vector supernatants. Efficiency of gene transfer can be increased by more extended in vitro expansion of target cells.

The analysis of the repertoire of VB usage in the population of PBLs, after three cycles of PBLs infection by vector-containing supernatants, followed by selection of transfected cells by G418, produced an intact repertoire as compared to the original control population of untransfected/unselected lymphocytes. These data show that limited expansion of target cells and utilization of vector-containing supernatants produce adequate gene transfer, independently of the relatively low frequency of gene transfer, at least when the vector supernatants have good viral titre (> 5xl05). However, low viral titre in vector supernatants produced limited νβ repertoires. This was confirmed by Southern blot analysis of the TCR-β chain rearrangement, which showed oligoclonal pattern in the transfected/selected population. This limitation was circumvented by co-culture of PBLs with irradiated vector-producing cells. Independently of viral titre, this gene transfer procedure is significantly more effective, by at least one order of magnitude. Taking advantage of the high efficiency of gene transfer by co-cultivation and the expression LNGFR on transfected lymphocytes, a simple protocol was devised involving co-cultivation and immunoselection that allows production of homogeneously transfected cells.

Since co-culture protocols are still considered unsafe for clinical use, co-culture conditions were designed in the parent invention in which vector-producing cells and PBLs were kept separated by a porous membrane, which allows virus passage while avoiding cell-to-cell contact. This approach offers some advantage when compared to supernatant infection.

Double fluorescence analysis of G418-selected T-cells for co-expression of LNGFR and different T-cell surface markers showed similar susceptibility of all the tested subpopulations with apparently equal efficiency of gene transfer. Furthermore, we obtained sporadic, though conclusive evidence of gene transfer into immature, CD4+/CD8+ cells. We have previously shown retroviral vector-mediated gene transfer into T-cell progenitors preceding TCR rearrangement^. The data show that retroviral infection of PBMCs allows gene transfer into circulating progenitors at low but detectable frequency. This may be an additional advantage of gene transfer protocols involving short term in vitro culture and limited expansion of target lymphocytes.

Further, the impact of vector construct on efficiency of gene transfer and expression is analyzed. In the vector constructs utilized in the parent invention, the reporter gene was alternatively placed within the retroviral transcription unit under the control of internal SV40 or HSV-TK promoters (NSV-N and NTK-N vectors, respectively), under the MoMLV viral LTR (LNSN/SFCM), or outside the retroviral transcription unit under the control of the human ADA promoter (the "double-copy" vector DCN). The use of a receptor according to the parent invention as a reporter gene allowed the study of gene expression levels in different PBL subpopulations, with single cell resolution.

The viral titers obtained for different vectors in the selected producer clones in the parent invention were comparable, with the exception of the DCN producer cells which consistently yielded titers up to five -fold lower than those for the other three vectors, suggesting that the extra sequence in the viral LTR may reduce the transduction efficiency, probably by interfering with the reverse transcription/integration process. This is also reflected by the relatively high tendency of DCN to integrate as a rearranged provirus, which further reduces the overall gene transfer efficiency that can be obtained with this type of vector. Rearrangement of integrated proviruses was observed at negligible frequencies for the other three vectors.

Gene expression levels showed that vectors based on internal transcription units were the least efficient in terms of both mRNA accumulation and protein expression. The only exception was the very high level of transcripts generated by the SV-40 promoter within two different vectors (NSV-N and LNSN/SFCM) in the EBV-infected B-cell line RIM. This may be due to interaction of EBV trans-activating proteins with the SV-40 enhancer. On the contrary, the vectors based on the MoMLV LTR and the human ADA promoter for expression of the reported gene of the parent invention worked very efficiently in all hematopoietic lines. Analysis of T-cell lines and clones gave essentially the same results. Both LNSN and DCN vectors were very efficient in directing high levels of gene expression in all T-cell subpopulations, including immature progenitors, and maintained these levels unaltered throughout a high number of passages.

In conclusion, the LTR-based retroviral vector is probably the most reliable and efficient for gene transfer and expression into human PBLs, whereas the DC design, although very good as far as gene expression is concerned, results in generally low gene transfer efficiency due to both the lower titer and the tendency to genomic rearrangement. This type of construct, however, could be the vector of choice when tissue-specific, inducible, or otherwise LTR-independent gene expression is a mandatory requirement, or else in tissues in which LTR inactivation might occur. Although we did not address this issue in the parent invention, human hematopoietic stem cells could be among these tissues, by analogy to what observed in the murine hematopoietic system.

In the parent invention, the utilization of a gene coding for the cell surface molecule LNGFR or coding for a cell surface molecule in which the intracellular domain of the receptor has been completely or partly deleted or modified in such a way that the receptor presented on the surface cannot, after binding to its binding partner, effect any signal transduction, for retroviral vector-mediated cell marking clearly represents an important advantage over vectors utilized in previous gene marking experiments that utilize a marker gene, usually the NeoR gene, not expressed on the cell membrane.

In the present invention, cells marked with this type of vector can be recognized by the antibody, and represent a potential advantage for in vivo preclinical and clinical studies of immunoselection and immunoseparation of the marked cells.

Material which is outside the scope of the claims does not constitute a part of the claimed invention.

Brief Description of the Drawings Figure 1: Retroviral vectors for LNGFR expression (A,C,E,G).

Schematic maps of integrated NSV-N (A), NTK-N (C), LNSN (E) and DCN (G) proviral genomes are shown, indicating the SV40 (SV), HSV-TK (TK) and human ADA (ADAp) internal promoters. Black boxes denote LTR sequences. Xba I and Hind III (H) restriction sites are indicated.

The RNA species originating from each vector are represented by numbered arrows on the maps.

Figure 2: Southern blot analysis of integration of the four retroviral vectors (A-D) in Xba I digest of DNA of K562 (1), KG 1 (2), Raji (3), Daudi (4), RIM (5), MOLT-4 (6) and J.M. (7) cell lines, hybridized to the Neo probe. Bands corresponding to intact, integrated proviruses are indicated, together with their molecular weight (in kb). Rearrangement bands are indicated by arrows.

Figures 3 and 4: Different vector constructs. (L: leader sequence; EXTR: extracellular domain; T: transmembrane domain; INTR: intracellular domain; black boxes: LTR, ψ: packaging signal) Examples Example 1 Retroviral vectors Different retroviral vectors for expression of LNGFR as a reporter gene were generated, using the full-coding 1.5 Kb Sst I fragment of the human LNGFR CDNA16 or a truncated LNGFR cDNA without an intracellular domain (e.g. by restriction with PvuII and Sstl). The NSV-N and NTK-N vectors were obtained by cloning the LNGFR cDNA into the unique Hind III and Bgl II sites of the NSV and NTK vectors, respectively. The NSV vector was derived from the original N2 vector 17 by insertion of the 0.4 kb Kpn I/Hind III fragment containing the SV40 early promoter enhancer and origin of replication into the unique Xho I cloning site. The NTK vector was also derived from N2 by insertion of the 852 bp Herpes Simplex Virus (HSV) thymidine kinase (TK) promoter.

The LNSN vector was constructed by insertion of the LNGFR cDNA into the Hpa I site of the LXSN vector 5.

In the DCN vector (double copy LNGFR) the LNGFR cDNA was cloned under the control of the 0.8 kb Ssp I/Nco I fragment of the human adenosine deaminase (ADA) promoter18 into the Bgl II/Sna BI sites of the N2A retroviral vector/poly linker^. \ Quite similarly, it is possible to use in the NSV-N, NTK-N, LNSN and DCN vectors instead of the LNGFR cDNA a truncated LNGFR cDNA (ALNGFR).

Different vector constructs according to the invention are shown in Figs. 3 and 4.

Vector DNAs were converted to corresponding viruses by the transinfection protocol. Briefly, vector DNA was transfected into the ψ2 ecotropic packaging cell line 19 by standard calcium -phosphate eo-precipitation^O 48 hrs after transfection, ψ2 supernatants were harvested and used to infect the amphotropic packaging cell lines PA31721 for 16 hrs in the presence of 8 μg/ml polybrene. Infected PA317 cells were selected in DMEM (GIBCO, Grand Island, NY) supplemented with 10% FCS (Hyclone, Logan, UT) and containing 0.8 mg/ml G418 (GIBCO), and then used to generate helper-free, virus-containing supernatants with titers ranging from 10^ to 5x10^ cfu/ml. All vectors contain the gene NeoR gene coding for neomycin phosphotransferase, that confers in vitro resistance to the neomycin analogue G418.

Infection of hematopoietic cell lines All cell lines described in this study, with the exception of RIM and J.M., were obtained from ATCC, and grown in RPMI 1640 (GIBCO) supplemented with 10% FCS. K562 and KGl are myeloid cell lines, derived from chronic and acute myelogenous leukemias, respectively; Raji and Daudi are derived from two Burkitt's lymphomas; MOLT-4 is considered a stable T-cell leukemia (CD8+); RIM is an EBV-transformed lymphoblastoid cell line; J.M. is a CD4+/CD8+ T-lymphoblastoid cell line; A875 is a human melanoma cell line expressing ~ 106 LNGFRs/cell. 5x10^ target cells were infected for 16 hrs with undiluted viral supernatants containing 8 μg ml polybrene, grown for additional 24 hrs in complete medium and then selected in the presence of the previously determined dose of G418 (0.5 to 1.5 mg/ml). Further analyses were carried out on bulk cultures of G418-selected cells.

Infection of human PBMC Peripheral blood mononuclear cells (PBMC) were obtained from healthy donors by Ficoll-Hypaque (Pharmacia, Uppsala, Sweden) gradient separation and grown for 72 hrs under phytohemagglutinin (PHA) and human recombinant interleukin2 (hu-rIL2) stimulation (2 μ /ml of purified PHA, Wellcome, Labs., Dartford, UK; 100 U/ml of hu-rIL2, Roche, Nutley, NJ). Viral infection was carried out by exposure of stimulated PBLs to a cell-free viral stock for 6 hrs in the presence of polybrene (8 μg ml). 48 hrs after infection, PBLs were selected in RPMI 1640 supplemented with 2 mM L-glutamine, 1% non-essential aminoacids, 1% Na pyruvate, 5% human serum (HS) and 100 U/ml hu-rIL2 (complete medium), containing 0.4 mg/ml G418. Cell density was maintained constant (5x10^ cells/ml) during 2 wks of G418 selection. Retroviral-infected human T-lymphocytes were also cloned in Terasaki plates at different cell concentration (1-103 cells/well) in complete medium containing 0.4 mg/ml G418, in the presence of irradiated human PBLs as feeder cells.

In order to improve the retroviral infection efficiency, human PBLs were co-cultivated with virus-producing cell for 48-72 hrs in complete medium. Co-cultivation was also carried out in Transwell plates (Costar, Cambridge, MA) to prevent cell-to-cell contact. 3x10^ producer cells were seeded in the cluster plate wells of 6-well dishes and incubated at 37°C O/N. 5x10^ stimulated PBLs were added into the Transwells and grown for 48-72 hrs in the presence of 8 μ /ml polybrene.

Retroviral-infected cells were analyzed by flow cytometry for receptor expression and expanded for further analyses.

DNA analysis High molecular weight DNA was obtained from cells by standard phenol/chloroform extraction^, digested to completion in 5 μg aliquots with appropriate restriction enzymes (GIBCO-BRL), electrophoresed in 0.8% agarose gels at 1.5 V/cm in tris-acetate-EDTA buffer, transferred to a nylon membrane (Hybond-N, Amersham, Buckinghamshire, UK) by Southern capillary blotting^O and hybridized to 10^ dpm of [32p]-labeled probe.

DNA analysis - Southern blot Probes: 1) fragment of pSV2-neo and 2) fragment containing TCR-B-constant region DNA probes were a 1.2 Kb Hind III/Sma I fragment of pSV2-neo 22 an(j the Hinc II 3' fragment of the YTJ-2 cDNA clone, containing the human TCR-13 constant region^S. Filters were washed under high stringency condition and exposed to Kodak X-AR 5 films at -70°C.

RNA analysis - Northern blot Probes: 1) fragment ov pSV2 neo and 2) fragment of LNGFR cDNA Total cellular RNA was extracted by the guanidine-isothiocyanate technique^ and selected for poly(A)+ by oligo (dT)-cellulose chromatography^. 5 μ 0f poly(A)+ RNA were size-fractionated on 1% agarose-formaldehyde gel, transferred onto nylon membrane by Northern capillary blotting^ and hybridized, washed and exposed as described for Southern blots. DNA probes were a 1.2 Kb Hind III/Sma I fragment of pSV2-neo and the 1.5 Kb Sst I fragment of the LNGFR cDNA16.

Cell surface phenotype Expression of LNGFR monitored by flow cytometry using 1) antihuman LNGFR with fluorescein label and 2) PE conjugated anti-human MoAbs.

Cell surface expression of LNGFR was monitored by flow cytometry utilizing the murine anti-human LNGFR monoclonal antibody 20.4 (ATCC) with an indirect fluorescence labelling method. Cell surface phenotype of T-lymphocytic lines and clones was determined by flow cytometry using PE-conjugated anti-human-CD4 (T4), CD8 (T8), CD5, B4, CD25R, Leu7, CD34 monoclonal antibodies (MoAb) (Coulter Immunology, Hialeah, FL). Briefly, 5x10^ cells were stained with 100 μΐ of diluted antibodies at 4°C for 30 min., washed twice in medium without FCS and resuspended in 0.5 ml of PBS for FACS analysis or in 100 μΐ of diluted FITC-conjugated secondary antibody. Double-staining analysis was performed by sequential incubation of FITC- and PE-conjugated antibodies.

Analysis of TCR VB-chain usage via PCR Total RNA was extracted from PBL and T cell lines using GTC, 26 reverse transcribed into cDNA using oligo dT and dG tailed, 27 one twentieth of the obtained DNA was subjected to PCR by the use of VB-CB specific oligonucleotides, 28 and to anchor PCR utilizing a CB-specific oligonucleotide (5'-TGCTGACCCCACTGTCGACCTCCTTCCCATT-3 (SEQ ID NO:2), as described in ref. 27. PCR amplification cycles were performed at: 94°C for 45", 57°C for 45", and 72°C for 1'. The amplified product was gel purified and cloned into bluescript plasmid vector. CB positive colonies were replicated onto different plates and transferred to nitrocellulose filters and hybridized to VB-specific oligonucleotides^, 29 under the following conditions: 6 x SSC, 1% blotto, 0.1% SDS and 5 mM EDTA at 42°C for 3-6 hrs, washed for 1 hr at the same temperature in 6 x SSC and exposed for 6-12 hrs to an X-ray film at room temperature.

Results Generation of recombinant retroviruses for expression of the LNGFR Different retroviral vector constructs for LNGFR expression were developed and utilized for generation of virus-producing cell lines. The NSV-N (Fig. 1A) and NTK-N (Fig. 1C) constructs are based on internal promoters, driving the LNGFR cDNA, i.e. the SV40 early promoter and HSV-TK promoter, respectively. In the LNSN vector (Fig. IE), the LNGFR gene is expressed from the viral LTR. In the DCN construct (Fig. 1G), the LNGFR cDNA is under the control of the human ADA promoter, within the U3 region of the 3' LTR. The SFCM constructs contain a truncated LNGFR gene, wherein the intracellular domain is deleted (Δ LNGFR). After infection of target cells, the transfected gene is duplicated and transferred to the 5' LTR^, thus generating a provirus containing two copies of the ADA- LNGFR minigene. All vectors carried the NeoR gene, under the control of either the viral LTR (NSV-N, NTK-N and DCN) or the SV40 early promoter (LNSN and SFCM).

The vectors were utilized to transfect the LNGFR gene (and the truncated LNGFR gene respectively) into human hematopoietic cell lines of different lineages and into human PBLs, and they are all able to 1) transfect human target cells, 2) integrate and form intact proviruses, and 3) express the reporter gene.

The viral titer of the amphotropic producer cell lines ranged from lxlO^ to 5x10^ cfu/ml for NSV-N, NTK-N, LNSN and SFCM vectors, and from 5xl03 to lxlO4 cfu/ml for DCN.

Molecular analysis of viral integration in human hematopoietic cell lines For the initial screening of the different vectors, a number of tumor cell lines of the hemato-lymphopoietic origin were utilized as target cells for gene transfer. Two myeloid cell lines (K562 and KG1), two Burkitt's lymphomas (Raji and Daudi), one EBV-transformed lymphoblastoid cell line (RIM), and two T-lymphoblastoid cell lines (MOLT-4 and J.M.) were infected with the four retroviral vectors and selected in the presence of G418. Molecular analysis of retroviral integrations was carried out by Southern blotting, to detect the size of integrated virus and the copy number (about 1 to 5 copies/cell). Genomic DNAs were digested with Xba I, which cuts in both 5' and 3' LTRs of all vectors (Fig. 1), allowing detection of the size of integrated proviruses, and with Bgl II, which cuts only in genomic DNAs, allowing estimation of the number of integration sites. XBa I- and Bgl II-digested DNAs were hybridized to NeoR and LNGFR genes specific probes sequentially.

Integration of the NSV-N vector generated a single Xba I band of the expected 5.1 kb size, corresponding to an intact provirus, in all the G418-selected cell lines (Fig. 2A). In the Raji cells an additional band of smaller size was observed, indicating integration of a rearranged provirus (Fig. 2A, lane 3).

Similarly, Xba I digestion generated a single band of the expected 5.4 kb size in the DNAs from all the NTK-N-infected cell lines (Fig. 2B). An additional band, probably due to the presence of a rearranged provirus, was detected only in the RIM sample (Fig. 2B, lane 5). A single band of 4.3 kb was seen in all samples transfected with the LNSN and SFCM vector with no evidence of viral rearrangements (Fig. 2C). On the contrary, the pattern of viral integrations in DNAs from cells infected with DCN was characterized by frequent rearrangements (Fig. 2D). All the DCN-infected cell lines showed the presence of a 5.4 kb band, corresponding to the intact provirus, but an additional band of 3.2 kb was detected in all but one (KGl) cell lines. The relative proportion of the two bands was variable in the analyzed samples, ranging from predominance of the intact provirus (MOLT4, Fig. 2D, lane 6) to prevalence of the rearranged one (Raji, Fig. 2D, lane 3). Repeated infections with the same retroviral stock indicated that the proportion between intact and rearranged provirus is a random, non-cell-specific event.

As observed for the cell lines transfected by all other vectors, the Bgl II pattern showed that DCN infection generated polyclonal integration in all cell lines.

In order to investigate the nature and the mechanism responsible for the generation of rearranged provirus, we carried out additional digestions of genomic DNAs with Hind III, which cuts twice in the ADA-LNGFR minigene, followed by hybridization with the LNGFR probe. The comparative analysis of Xba I and Xba I/Hind III restriction patterns with both probes indicated that the 3.2 kb band corresponded to a rearranged provirus lacking the ADA-LNGFR minigene from both LTRs. Defective proviruses were also detected at high frequency during selection of the DCN producer cell lines. The clone used in all our experiments harbored only the intact provirus, indicating that generation of defective proviruses occurs during integration in the target cells, and is not due to defects of the producer cell line.

In summary, infection of all cell lines with NSV-N, NTK-N, LNSN and SFCM gave rise to G418-resistant populations carrying low copy numbers of mostly unrearranged proviruses, whereas infection with DCN vector generated a common rearranged provirus at high frequency, most likely derived by the loss of the ADA-LNGFR minigene from the 3' LTR of the retroviral vector during the integration process. This problem may be due to the size and nature of the inserted gene in the viral LTR, similar limitations with other "double-copy" vector constructs were not observed 12-14 Detection of vector-mediated LNGFR expression in human hematopoietic cell lines Expression of LNGFR introduced by the different retroviral vectors was evaluated at both protein and mRNA levels. Cell surface expression of LNGFR in the G418-selected cell lines was quantitatively analyzed by flow cytometry with an anti-LNGFR MoAb. The results are summarized in Table I. Most of the NSV-N- and NTK-N-transfected cell lines showed a low-medium level of surface LNGFR, expressed as relative mean fluorescence (< 100 arbitrary units). High expression of LNGFR (202 a.u.) was observed only in the EBV-infected RIM cell line transfected by NSV-N. LNGFR expression was not detected in the NTK-N-transfected MOLT-4 cell line, although no rearrangement of the proviral genome was detected in Xba I-digested genomic DNA (Fig. 2B, lane 6). Repeated gene transfer by this vector in the same cell line gave consistently negative results.

All cell lines infected with LNSN and SFCM retroviral vectors expressed LNGFR at medium-high levels (50-200 a.u.) (table I).

Table 1 FACS analysis of LNGFR expression in human hematopoietic cell lines infected by the four retroviral vectors a Quantitative expression of LNGFR was scored as: -, 0 arbitrary units (a.u.) of relative mean fluorescence intensity, +, 0-50 a.u., ++, 50-100 a.u., +++, 100- 200 a.u. The relative mean value of A875 melanoma cell line, expressing ~ 106 receptors/cell, was 190 a.u. b FACS profiles of K562, Daudi, RIM, and J.M. were biphasic, and the score refers only to the positive peak (see results) c similar data for SFCM vectors Most of the DCN-infected cell lines expressed medium or high level of LNGFR (50-200 a.u.). The FACS profiles of DCN-infected cell lines correlated with results obtained from Southern analysis, in that cell lines harboring a rearranged provirus in addition to an intact one showed biphasic curves, with a negative and a positive peak proportional to the relative intensity of the bands corresponding to intact and rearranged proviruses, respectively.

Expression of vector-specific RNAs in transfected cells (K562) was determined by Northern blotting of poly(A)+ RNA isolated from G418-selected cell lines, hybridized to LNGFR and Neo probes. The patterns obtained were consistent with the expected transcription profiles of each retroviral vector. In the NSV-N- and NTK-N-transfected cells, an unspliced and a spliced RNA species hybridized to both the LNGFR and Neo probe. The SV-40- and TK-derived subgenomic transcripts containing the LNGFR-specific mRNA were observed only after hybridization to the LNGFR probe. LNSN-or SFCM-transfected cells expressed only the unspliced RNA form, hybridizing to both NeoR and LNGFR probe. This is consistent with the reported inactivation of the splice donor site in the LXSN vector, from which LNSN and SFCM were derived^. A shorter transcript, corresponding to the SV40-derived NeoR mRNA hybridized only to the NeoR probe.

The DCN vector generated two LTR-derived genomic transcripts, an unspliced and a spliced form, respectively. A third RNA species was transcribed from the ADA promoter, and most likely used as the major mRNA template for LNGFR synthesis. The relative contribution of the ADA promoter in the 5' or in the 3' LTR to the LNGFR transcript cannot be assessed by this technique. A slow-migrating RNA form might represent a read-through transcript initiated from the ADA promoter in the 5' LTR and terminated at the poly(A) addition site in the 3' LTR. Two additional, unaccounted RNA species hybridizing only to the NeoR probe were also detected. These transcripts, present in all DCN-infected cell lines, were the only RNA species detected in Raji cells, which did not express LNGFR and carried only rearranged proviruses, as demonstrated by Southern blot analysis. It is therefore likely that these transcripts are specific for the rearranged provirus, lacking the ADAp-LNGFR minigene.

The RNA expression pattern observed in the other cell lines was comparable with that obtained from K562, with the only exception of the EBV-infected RIM cell line, which consistently showed high levels of SV40-derived transcripts, from NSV-N, LNSN and SFCM.

Efficiency of vector-mediated gene transfer into human PBLs Human PBLs were infected by retroviral vectors under PHA and IL-2 stimulation. In order to estimate infection frequencies, 48 hrs after infection human T-cells were cultured under limiting dilution conditions (1 to 10^ cells/well). Infected and uninfected control cells were cultured in the presence or absence of 0.4 mg/ml of G418. Cell growth was evaluated 14 days after plating, when no surviving cells could be detected in wells containing uninfected cells cultured in the presence of G418. The overall infection frequency ranged from less than 1% to 5.1%, depending upon viral titer of vector supernatants. However, significant variability was observed between different donors. Efficiency of gene transfer can be increased by multiple infection cycles or by co-cultivation. Co-cultivation of PBLs with irradiated virus-producing cell lines for 48 hrs consistently provides good gene transfer efficiency (10-15%). Double fluorescence analysis of LNGFR and T-cell markers ruled out the possibility of a contamination by vector-producing cells in the LNGFR positive population. In the experiment, the frequency of LNGFR expression was entirely due to expression of the transfected gene by infected PBLs since all cells positive in FACS analysis were co-expressing the human CD3. Independently of initial efficiency of gene transfer, transfected PBLs can be selected to homogeneity either by negative selection with G418 or by positive immunoselection with magnetic beads coupled to a monoclonal antibody directed against the LNGFR. In the representative experiment, a single round of immunoselection was sufficient for separating the population of infected cells from uninfected PBLs. Once the magnetic beads were removed, a homogeneous population of infected lymphocytes was obtained without significant loss of infected cells.

In order to evaluate the efficiency of viral vectors in infecting human PBLs, a number of PBL bulk cultures were independently infected by exposure to supernatants containing the different retroviral vectors and selected by G418 for vector presence and expression. Ten of the vector-infected cultures (2 by NSV-N, 3 by NTK-N, 2 by LNSN/SFCM and 3 by DCN) were analysed for proviral integration by Southern blotting. Hybridisation to the NeoR probe of representative Xba I, which cuts in both 5' and 3' LTR of all vectors (Fig. 1), allows detection of the size of integrated proviruses, and with Bgl II, which cuts only in genomic DNA, allows estimation of the number of integration sites. In all the lymphocyte cultures infected by NSV-N, NTK-N, and LNSN/SFCM, only bands corresponding to the intact integrated proviruses were detected, whereas an additional band corresponding to a rearranged provirus was observed in all DCN-infected cultures.

Finally, analysis of the proviral integrations revealed that NSV-N-, NTK-N- and LNSN-/SFCM-infected T cell cultures were largely polyclonal, whereas DCN-infected cultures were essentially oligoclonal, as indicated by the presence of a predominant band and only a few minor ones. As previously suggested, this is probably due to lower viral titre and could be circumvented by different gene transfer protocols including co-cultivation of target cells with vector-producing cell lines.

Detection of vector-mediated expression of the LNGFR on human T-lymphocytes Cell surface expression of LNGFR was detected by FACS analysis in a total of 23 retroviral vector-infected T-lymphocyte cultures (5 NSV-N-, 8 NTK-N-, 5 LNSN-and 5 DCN-infected cell lines). LNGFR expression was low in all NSV-N-infected cells, and in six out of eight NTK-N-infected cell cultures. The remaining two NTK-N-infected T-cell lines showed intermediate levels of LNGFR expression. Heterogeneous expression levels were observed in LNSN-SFCM cell cultures, with two lines expressing low levels, two intermediate levels, and one high levels of LNGFR. In DCN-infected cells, four out of five lines expressed low levels and one intermediate levels of LNGFR.

In order to characterise the infected cells, we tested 13 transfected cell lines for expression of cell surface lymphocyte differentiation antigens, using monoclonal antibodies anti-CD4, CD8, CD5, B4, Leu7, CD25R and CD34. All tested cell lines scored positively for CD5 and CD25R expression and negatively for Leu7, B4 and CD34 expression (data not shown). Eight out of 13 analysed cultures were LNGFR+/CD8+, one was LNGFR+/CD4+ and four were positive for LNGFR and contained different percentages of cells co-expressing CD8 and CD4. Predominance of the CD8+ phenotype in the PHA and IL-2 stimulated cultures was also observed in the uninfected control cell lines, and probably represent a constant bias of the T-cell culture procedure. In general, we did not observe preferential infection of particular cellular subsets by any of the four retroviral vectors.

T-cell clones were obtained from vector-transfected T-cell cultures by plating cells in limiting dilution (1000, 100 and 10 cells/well), taking into account an average infection frequency of approximately 1%. 72% of all the examined clones were LNGFR+/CD4+ and 28% LNGFR+/CD8+. Predominance of the CD4+ phenotype was also observed in control, uninfected T cells cloned from the same donors, and is considered a standard finding under our cloning conditions. Furthermore, retroviral infection and LNGFR expression was achieved in double-positive, CD4+/CD8+ clones. These results demonstrate that retroviral infection might occur not only in mature CD4+ and CD8+ cells, but also in immature CD4~I7CD8+ double positive peripheral blood lymphocytes.

Analysis of T-cell repertoire in vector-transfected T-lymphocytes As shown above, Southern blot analysis of viral integration demonstrated the polyclonal nature of the vector-infected T-cell cultures. This was further confirmed by molecular analysis of T-cell receptor (TCR) 13 chain rearrangements on Xba I digested DNA samples hybridised to a probe specific for the TCRB chain constant region. A polyclonal pattern, with no predominant bands in addition to the germline pattern, was detected in T-cell cultures infected with NSV, NTK-N and LNSN/SFCM. The presence of limited numbers of predominant rearrangement bands was observed in DCN-infected cultures, as for oligoclonal cell populations.

Further confirmation to these observations was obtained by systematic analysis of the TCR VB-chain usage in transfected lymphocytes. After a single cycle of PBLs infection by vector-containing supernatants, followed by selection of infected cells by G418, the total RNA of the selected lymphocyte population was reverse transcribed and the obtained DNA was subjected to PCR by the use of VB-CB specific oligonucleotides, and to anchor PCR utilising a CB specific oligonculeotide as described in the methods section. Analysis of the amplified products by VB-specific oligonucleotides showed an identical repertoire as compared to the original control population of uninfected/unselected lymphocytes. Figure 9 shows the substantially normal repertoire of VB usage in a polyclonal population of LNSN-/SFCM-infected lymphocytes. Similar to the results of the Southern blot analysis of TCR-B chain rearrangements, low viral titre in vector supernatants produced limited VB repertoire (data not shown). This limitation of low titre vectors could be circumvented by multiple infection cycles or by co- cultivation of PBLs with the producer cell line.

Material which is outside the scope of the claims does not constitute a part of the claimed invention.

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Immunol. 22:541, 1992 ) Bordignon C: A clinical protocol for transfer of the ADA gene into bone marrow cells and peripheral blood lymphocytes for the treatment of patients affected by ADA deficient SCID. Human Gene Therapy (in press). ) Areman E.M. et al: Bone Marrow and Stem Cell Processing: A Manual of Current Techniques, F.A. Davies Co., Philadelphia (1992) ) Reddy et al., Molecular Brain Research 8 (1990) 137 - 141 SEQUENCE LISTING (1) GENERAL INFORMATION: (i) APPLICANT: (A) NAME: BOEHRINGER MANNHEIM GMBH (B) STREET: Sandhofer Str. 116 (C) CITY: Mannheim (D) STATE: BW (E) COUNTRY: Germany (F) POSTAL CODE (ZIP): D-68305 (G) TELEPHONE: 08856/60-3446 (H) TELEFAX: 08856/60-3451 (ii) TITLE OF INVENTION: Method of marking eukaryotic cells (iii) NUMBER OF SEQUENCES: 2 (iv) COMPUTER READABLE FORM: (A) MEDIUM TYPE: Floppy disk (B) COMPUTER: IBM PC compatible (C) OPERATING SYSTEM: PC-DOS/MS-DOS (D) SOFTWARE: Patentln Release #1.0, Version #1.25 (EPO) (vi) PRIOR APPLICATION DATA: (A) APPLICATION NUMBER: IT RM93/A000587 (B) FILING DATE: 01-SEP-1993 (2) INFORMATION FOR SEQ ID NO: 1: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 1600 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: double (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (ix) FEATURE: (A) NAME/KEY: misc_feature (B) LOCATION: one-of(943) (D) OTHER INFORMATION: /function cleavage site" (ix) FEATURE: (A) NAME/KEY: misc_feature (B) LOCATION: one-of(1512) (D) OTHER INFORMATION: /function cleavage site" (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 1: GCCGCGGCCA GCTCCGGCGG GCAGGGGGGG CGCTGGAGCG CAGCGCAGCG CAGCCCCATC 60 AGTCCGCAAA GCGGACCGAG CTGGAAGTCG AGCGCTGCCG CGGGAGGCGG GCGATGGGGG 120 CAGGTGCCAC CGGCCGCGCC ATGGACGGGC CGCGCCTGCT GCTGTTGCTG CTTCTGGGGG 180 TGTCCCTTGG AGGTGCCAAG GAGGCATGCC CCACAGGCCT GTACACACAC AGCGGTGAGT 240 GCTGCAAAGC CTGCAACCTG GGCGAGGGTG TGGCCCAGCC TTGTGGAGCC AACCAGACCG 300 TGTGTGAGCC CTGCCTGGAC AGCGTGACGT TCTCCGACGT GGTGAGCGCG ACCGAGCCGT 360 GCAAGCCGTG CACCGAGTGC GTGGGGCTCC AGAGCATGTC GGCGCCGTGC GTGGAGGCCG 420 ACGACGCCGT GTGCCGCTGC GCCTACGGCT ACTACCAGGA TGAGACGACT GGGCGCTGCG 480 AGGCGTGCCG CGTGTGCGAG GCGGGCTCGG GCCTCGTGTT CTCCTGCCAG GACAAGCAGA 540 ACACCGTGTG CGAGGAGTGC CCCGACGGCA CGTATTCCGA CGAGGCCAAC CACGTGGACC 600 CGTGCCTGCC CTGCACCGTG TGCGAGGACA CCGAGCGCCA GCTCCGCGAG TGCACACGCT 660 GGGCCGACGC CGAGTGCGAG GAGATCCCTG GCCGTTGGAT TACACGGTCC ACACCCCCAG 720 AGGGCTCGGA CAGCACAGCC CCCAGCACCC AGGAGCCTGA GGCACCTCCA GAACAAGACC 780 TCATAGCCAG CACGGTGGCA GGTGTGGTGA CCACAGTGAT GGGCAGCTCC CAGCCCGTGG 840 TGACCCGAGG CACCACCGAC AACCTCATCC CTGTCTATTG CTCCATCCTG GCTGCTGTGG 900 TTGTGGGCCT TGTGGCCTAC ATAGCCTTCA AGAGGTGGAA CAGCTGCAAG CAGAACAAGC 960 AAGGAGCCAA CAGCCGGCCA GTGAACCAGA CGCCCCCACC AGAGGGAGAA AAACTCCACA 1020 GCGACAGTGG CATCTCCGTG GACAGCCAGA GCCTGCATGA CCAGCAGCCC CACACGCAGA 1080 CAGCCTCGGG CCAGGCCCTC AAGGGTGACG GAGGCCTCTA CAGCAGCCTG CCCCCAGCCA 1140 AGCGGGAGGA GGTGGAGAAG CTTCTCAACG GCTCTGCGGG GGACACCTGG CGGCACCTGG 1200 CGGGCGAGCT GGGCTACCAG CCCGAGCACA TAGACTCCTT TACCCATGAG GCCTGCCCCG 1260 TTCGCGCCCT GCTTGCAAGC TGGGCCACCC AGGACAGCGC CACACTGGAC GCCCTCCTGG 1320 CCGCCCTGCG CCGCATCCAG CGAGCCGACC TCGTGGAGAG TCTGTGCAGT GAGTCCACTG 1380 CCACATCCCC GGTGTGAGCC CAACCGGGGA GCCCCCGCCC CGCCCCACAT TCCGACAACC 1440 GATGCTCCAG CCAACCCCTG TGGAGCCCGC ACCCCCACCC TTTGGGGGGG GCCCGCCTGG 1500 CAGAACTGAG CTCCTCTGGG CAGGACCTCA GAGTCCAGGC CCCAAAACCA CAGCCCTGTC 1560 AGTGCAGCCC GTGTGGCCCC TTCACTTCTG ACCACACTTC 1600 (2) INFORMATION FOR SEQ ID NO: 2: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 31 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 2: TGCTGACCCC ACTGTCGACC TCCTTCCCAT T 31

Claims (8)

33 147557/3 Claims

1. A method for the immunoselection or immunoseparation of transfected cells, wherein said cells have been transfected with a nucleic acid that encodes a cell surface receptor in which the region encoding the intracellular domain of the receptor has been completely or partly deleted or modified in such a way that the receptor presented at the surface cannot, after binding to its binding partner, effect any signal transduction, said method comprising incubating the cells with an antibody that specifically binds said cell surface receptor, and recovering the cells by cleaving the antibody-receptor binding.

2. The method of claim 1, wherein said method is for the selection of the transfected cells, and said antibody is marked.

3. The method of claim 1, wherein said method is for the separation of the transfected cells, whereby said antibody is immobilized prior to or after incubation with said cells, and said cells are immobilized through binding to the immobilized antibody, and are thus separated from any unbound cells.

4. The method of any one of claims 1 and 2, wherein said cells are marked with a cell surface receptor in which the region encodin the intracellular domain of the receptor has been completely or partly deleted or modified in such a way that the receptor presented at the surface cannot, after binding to its binding partner, effect any signal transduction, and whereby said cell preparation is substantially free of clonogenic tumor cells, by purging using cytotoxic agents or irradiation, and separating said transfected cells from non-transfected cells by immunoselection using an antibody that specifically binds said cell surface receptor. 34 147557/3

5. The method of any one of claims 1-4, wherein said cell surface receptor bound by said antibody is the NGF-, the CD24-, or the LDL receptor.

6. The method of claim 5, wherein said antibody is monoclonal or polyclonal.

7. The method of claim 6, wherein said antibody has been labeled with an enzyme.

8. The method of claim 7, wherein said antibody has been labeled with fluorescent or chemiluminescent dyes.