WO1990001870A1

WO1990001870A1 - Retroviral vectors expressing soluble cd4; a gene therapy for aids

Info

Publication number: WO1990001870A1
Application number: PCT/US1989/003541
Authority: WO
Inventors: W. French Anderson; Robert C. Gallo; Flossie Wong-Staal; Richard A. Morgan; Daryl Muenchau
Original assignee: The United States Of America, As Represented By The Secretary, U.S. Department Of Commerce
Priority date: 1988-08-22
Filing date: 1989-08-21
Publication date: 1990-03-08
Also published as: JPH03505039A; EP0431045A4; EP0431045A1; AU4193289A; AU647013B2

Abstract

The invention disclosed herein provides a means of blocking retroviral infection of human cells by in vivo production of secreted CD4 (sCD4) molecule proteins and CD4 protein analogues. These proteins block interaction between the CD4 receptors on cells and virus, viral particles and viral products which would otherwise bind to the CD4 receptors.

Description

RETROVIRAL VECTORS EXPRESSING SOLUBLE CD4: A GENE THERAPY FOR AIDS The invention disclosed herein provides a means of blocking retroviral infection of human cells by in vivo production of secreted CD4 (sCD4) molecule proteins and CD4 protein analogues. These proteins block interaction between the CD4 receptors on cells and virus, viral particles and viral products which would otherwise bind to the CD4 receptors. BACKGROUND OF THE INVENTION The ability of human immunodeficiency virus (HIV) to infect and destroy cells having CD4 receptors results in depletion of T4 cells which express the CD4 protein. It has been shown by several investigators that the envelope protein of HIV binds to the CD4 molecule. (See, for example, Dalglish, et al. , Nature (London) 312:763 (1985); McDougal, et al. Science 231;382 (1986)). It has now been shown that HIV infectivity can be blocked in vitro by recombinant soluble CD4 receptor protein (Fisher, et al.l. Nature 331:76 (1988)). Soluble CD4 protein which binds the HIV envelope protein gpl20 with affinity comparable to affinity of the intact CD4 receptor was shown to block HIV infectivity (Smith, et al.. Science, 238: 1704 (1987) ) . A soluble recombinant polypeptide comprising a fragment of about 180 a ino acid residues from the amino terminal end of the intracellular region of the CD4 was produced by expression vectors in vaccinia recombinants by Berger, et al. Proc. Natl. Acad. Sci USA. 85.:2357 (1988)). Attempts have been made previously to produce CD4 protein or fragments thereof as therapeutic agents for patients infected with HIV. BRIEF DESCRIPTION OF THE INVENTION It is the purpose of this invention to provide a continuously maintained level of sCD4 by biological engineering of cells of an AIDS afflicted individual using gene transfer methods.

Based on the results of studies showing helper/inducer T -cell antigen CD4 is an essential component in the entry of HIV into permissive cells and that soluble forms of CD4 antigen can bind to and inhibit HIV infection of CD4 positive cells the treatment of AIDS by administration of soluble CD4 (sCD4) is seen as useful. Biological engineering of the cells of an AIDS patient to provide a continuous production of sCD4 in vivo is an alternative to administration of exogenously administered CD4.

The most effective means of gene transfer currently available is through the use of retroviral vectors. Retroviral vectors have been produced which can promote the expression of a wide variety of genes, and these genes can be expressed in vivo following gene transfer. Disclosed herein is construction of retroviral vectors which, when transduced into target cells, produce the sCD4 gene product. The sCD4 so produced inhibits HIV infection of the CD4 positive target cells. The vectors are transduced into human cell (preferably the patient's own cells) . These cells are then administered to the patient in need of the sCD4- to block ligand binding at the CD4 binding sites on the cells. The compositions and method of the invention can also be used to treat patients suffering from other disease conditions which result from deficiency of CD4 receptor protein.

While the most immediate value of the invention is production of CD4 receptor proteins to bind HIV virus, particles, or proteins, the invention is not limited to the specific examples and applications disclosed herein. Cells which may be transduced by the method of the invention may include, for example, human bone marrow cells or fibroblast cells. Bone marrow cells may transformed by the procedure described in Kantoff, et al. , . Exp. Med. l :219-34 (1987).

STATEMENT OF DEPOSIT The plasmids SSC and SCSX have been deposited using E. coli DH5 alpha as carriers in the American

Type Culture Collection at Rockville, Maryland in accord with the requirements of the Budapest Treaty for deposit of patented organisms and biological products. The deposits have been assigned the ATTC accession numbers: SSC Ace. No. 67760 and SCSX ACC.

No. _t 67761

Upon issuance of a patent, the deposited organism will be made available upon request. A viable deposit will be maintained for at least 30 years consistent with provisions of applicable laws.

SUBST5TUTESHEET FIGURE LEGENDS Ficfure 1. Diagram of soluble CD4 producing retroviral vectors. Retroviral vectors SCSX and SSC were produced by inserting an SV40 promoted sCD4 cassette into the N2 (15) retroviral vector. The viral promoter (LTR) , packaging signal (-φ) , neomycin resistance gene (NEO^R) , truncated CD4 gene (sCD4) , and SV40 virus promoter (SV40) are as indicated. The arrow indicates the direction of transcription of the SV40 promoter. Restriction enzyme sites are defined as follows: X = Xba I, B = Bst NI, E = Eco RI, N = Nru I, and Xh = Xho I.

Figure 2. Soluble CD4 production by viral producer and transduced cells. Shown are the resulting autoradiograms (lanes 1-4 and 5,6 were exposed for 3 days, and 7 days respectively) of immunoprecipitated sCD4 from retroviral vector transduced cell lines (resolved on 12% acrylamide SDS-PAGE gels as described in methods) . Samples were conditioned medium from approximately one half of a near confluent 100mm tissue culture dish of the following: 1, PA317; 2, SSC- transduced PA317; 3, SV-T2; 4, SCSX-transduced SV-T2; 5, HeLa; and 6, SCSX-transduced HeLa. The sizes (in kilodaltons) of coelectrophoresed marker proteins are as indicated. Arrows point to the soluble CD4 gene product.

Figure 3. Soluble CD4 produced by retroviral vectors contains the proper antigenic determinants. Radiolabeled cell medium (approximately 1/2 of a 100mm dish per assay) from a pool of SSC and SCSX PA317 producer cell lines was i munoprecipitated with anti.CD4 monoclonal antibodies as described in Figure 2. sCD4 is immunoprecipitated by monoclonal antibodies which inhibit HIV infection: OKT4a, lane 1; Leu 3a, lane 3; and MT151, lane 4; but not by nonblocking antibody OKT4, lane 2. Size markers of 30kd and 17kd are indicated. The arrow points to the sCD4 gene product.

Figure 4. Coprecipitation of vector-produced sCD4 with HIV gpl20. Radiolabeled cell medium (prepared from the SSC-transduced PA317 cells as described in Fig. 2) was subjected to immunoprecipitation with monoclonal antibody OKT4a (lane 1) , or coprecipitation with no HIV gpl20 (lane 2), 0.2 μg of partially purified HIV gpl20 (lane 3), or 2.0 μg partially purified HIV gpl20 (lane 4) . For coprecipitation assays the stated amounts of partially purified HIV gpl20 were added and incubated for 4hr followed by the addition of goat antiserum to HIV gpl20 (5μl) plus 0.1 ml of protein A sepharose, followed by overnight incubation. Size markers of 30kd and 17kd are indicated. The arrow points to the sCD4 gene product.

MATERIALS AND METHODS Construction of sCD4 retroviral vectors

A soluble form of the CD4 gene was constructed by first transferring the original T4B cDNA clone (14) , as an Eco RI plus Bam HI fragment, into the Bluescript vector (Stratagene) to produce pCDW. The CD4 gene was excised with Hind III plus Xba I and inserted into Hind II plus Xba I cut vector pSVPLX, to yield pSVCDW. The expression plasmid pSVPLX was derived from pSV2neo by inserting an Xho I linker into the Pvu II site, and removal/replacement of the neoR gene by the polylinker sequence from pUC-13. A soluble form of CD4 was produced from pSVCDW by inserting a synthetic adaptor molecule (CTAGCTTGAGTGAGTT plus AACTCACTCAAG) into the Nhe I site of the cDNA. This procedure regenerates the Nhe I site, immediately introduces a stop codon after amino acid 202, and produces a Hpa I site adjacent to the stop codon. Ligated molecules were cleaved with Hpa I, Xho I linkers were attached (New England Biolabs) , and the SV40-SCD4 fragment was removed by Xho I digestion. This fragment was then inserted into the Xho I site of the N2 retroviral vector (15) , to derive the SCSX vector. The SSC vector was produced from the SCSX vector by first digesting with Bst NI followed by end- filling with T4 polymerase and subsequent digestion with Xho I.to release an SV40-SCD4 fragment which was then directionally cloned into N2 cut with Nru I plus Xho I.

Production of recombinant virus and the transduction protocol Replication-defective, amphotropically packaged, retroviral vector particles were generated by the transinfection procedure. In brief, 50μg of SSC or SCSX vector DNA was used to transfect (via calcium phosphate coprecipitation) the ecotropic packaging cell line 2 (16) , and 48hr post transfection, virus containing cell medium was removed and used to infect the amphotropic packaging cell line PA317 (17) . Transduced PA317 cells were selected for by growth in the presence of the neomycin analog G418 (500 μg/ l) . Following selection, recombinant virus was harvested from confluent 100mm tissue culture dishes following 24hr incubation in 10ml fresh culture medium (Dulbecco's modified Eagle's medium containing 10% fetal bovine serum) . Cell free supernants were collected by filtration through 0.22μm filter units and stored at -70"C until use. Transduction of mouse SV-T2 and human HeLa cells lines was conducted by incubation with recombinant viral supernate containing 8ug/ml polybrene at 37°C for 2hr, followed by removal of virus-containing medium and replacement with fresh culture medium. Transduced cell populations were selected by growth in G418 (300μg/ml) for 14 days. Immunoprecinitation

Cells were grown to approximately 75% confluence on 100mm tissue culture dishes and prior to radiolabeling incubated for 1-2 hr in cyεteine-free MEM labeling medium (GIBCO) containing 2% dialyzed fetal calf serum. Labeling was initiated by removal/replacement of the labeling medium with 5ml fresh cystein-free medium containing 100-200 μCi/ml of [³⁵S]cysteine (approximately lOOOCi/mmol, Amersham) . Conditioned medium was collected 14-16 hr later, and cell debris was removed by centrifugation (1,000 x g, 10 min) . Before immunoprecipitation was conducted the conditioned medium was precleared by adding 1/10 vol of 10X buffer (200mM Tris HCi pH = 7.5, 1.2M NaCl, 5% NP.40, 2% NaDeoxycholate, 2mM EGTA, 2mM NaF, 50 μg/ml aprotinin, 2mM phenyl methyl εulphonyl fluoride) plus 0.2 ml of protein A sepharose (Pharmacia) and incubating at 4oC for 4 hr followed by removal of the protein A sepharose by low speed centrifugation. One μg of the monoclonal antibody (OKT4a, Ortho; Leu 3a, Becton Dickinson; or MT151, Boehringer Mannheim) plus 0.1 ml of protein A sepharose was then added and the samples incubated overnight at 4oC on a rotating wheel. The absorbed immune-complexes were pelleted by low speed centrifugation, washed three times with IX buffer, and resuspended in 50 μl of sample loading buffer (lOmM Tris HCI pH = 8.0, 2% SDS, 5% 2-mercaptoethanol, 10% glycerol) . Samples were heated for 30 min at 70oC before electrophoresis on 12% SDS-PAGE (18) . HIV-1 titration

Virus strains used (20.23) were HTLV-III_HH (designation, HIV-l/NIH/USA/1984/MN) , and HTLV-III_RJ. (designation, HIV-l/NIH/USA/1983/RF) . HTLV-III_KF was isolated from a Haitian AIDS patient in 1983 from peripheral blood lymphocytes and in H4 cells, and HTLV/IIIMN was isolated from a child with AIDS (whose mother was an intravenous drug user) in 1984 in peripheral blood lymphocytes and JM cells. Virus preparations used were 1000-fold concentrates - prepared by direct centrifugation from infected H9 culture supernatants, and direct centrifugation from infected H9 culture supernatants, and contained from 2.2 to 3.1 x 10¹¹ particles/ml. Virus preparations were pre-titered in H9 cells in a two-week assay (24) using detection of infected cells by immunofluorescent microscopy for HIV-1 p24 to determine 50% infectious endpoints (TCID₅₀) . The preparations of HTLV-III_RJ. and HTLV-IIIrø used had 1.6 x 10 and 4.0 x 10³ TCID₅₀/ml in the H9 assay, respectively.

To determine the anti-viral effects of co- cultivation with transduced cells lines, a different culture system was required. Mouse NIH/3T3-derived fibroblasts (either N2 or SSC transduced PA317 cells) were seeded into 24 well culture plates (2 x 10⁵/^we_.l_^s) and cultured overnight. The following day, 1 x 10⁶ Sup Tl CD4+ cells (25) were added to each well of duplicate plates. After overnight co-cultivation, serial two¬ fold dilution of HIV-1 virus preparations (RF or MN) were made in 20 μl media in a separate 96 well U-bottom icrotiter plate in quadruplicate, and added to corresponding wells of the 24 well plates. After one week of incubation, cells from each well were pelleted and dotted onto slides, air-dried, and fixed in 1:1 acetone-methanol. Indirect immunofluorescent staining for HIV-1 p24 was performed using BT3 urine monoclonal antibody and sheep anti-mouse FITC conjugate. A well (and hence that virus dilution) was scored as positive if at least 2 characteristic fluorescene cells were^" seen. Calculation of TCID₅₀ values were performed by the method of Reed and Muench (26) . DETAILED DESCRIPTION OF THE INVENTION As described in the examples which follow, the sCD4 is produced by recombinant means. Retroviral vectors were developed which produce a secreted form of the CD4. Amphotropically packaged vectors were used to transduce cells which were shown to express the secreted CD4 (sCD4) gene product. The sCD4 produced by the viral vectors is immunoprecipitated by monoclonal antibodies against CD4. Physical interaction of the vector -produced sCD4 and HIV-1 gpl20 was demonstrated by coprecipitation of sCD4/gpl20 with an iserum directed against HIV gpl20.

A preferred embodiment of the invention provides means of transducing human cells (preferably the patient's own cells) with retroviral vectors which enable cells to produce secreted CD4 protein. Use of such biologically engineered cells of the afflicted individual to produce sCD4 is believed to provide benefits superior to benefit from exogenous administration of sCD4.

Two retroviral vectors which express a soluble form of the helper T-cell antigen Cd4 were constructed by truncating the Cd4 gene at the Nhe I site, inserting this fragment into an SV40-based expression plasmid, and then transferring the SV40-sCD4 into the N2 retroviral vector. The two vectors produced differ mainly in the orientation of the SV40 promoted sCD4 transcription unit relative to that of the vector LTR (SSC is in the same transcriptional orientation as the LTR, and SCSX is in the opposite orientation. (Figure 1) .

SSC and SCSX were introduced into the amphotropic packaging cell line PA317 by the method of Miller, et al. (Mol. Cell. Biol. 6: 2895-2902 (1986)) and, following selection for resistance to the neomycin analog G418, assayed for sCD4 production. An appropriately-sized secreted protein was specifically immunoprecipitated with anti-CD4 monoclonal antibody OKT4a in the transduced PA317 cell population, but not in the transduced cells (lane 1,Figure 2) . The SCSX transduced PA317 cells produce a similar secreted protein. To demonstrate the ability of our sCD4 retroviral vectors to transfer the sCD4 phenotype to other cells, a viral supernate from the PA317 SCSX producer cells was used to infect both a mouse fibroblast line (SV-T2) and a human epithelial cell line (HeLa). Following selection with G418, both lines were assayed for sCD4 production by immunoprecipitation (lines 3-6, Figure 2) . The data demonstrated that both of these transduced lines secreted the truncated CD4 gene product into the cell medium. The transduced HeLa line has yielded a consistently higher autoradiographic signal than the SV-T2 line (compare lanes 4 and 6) . Both lines have continued to produce sCD4 in vitro for up to three months, indicating stable integration and expression of the SCSX vector.

There exist several monoclonal antibodies to CD4 which are able to block HIV infection of CD4 positive T-cells, presumably by inhibiting HIV binding. Experimental data (Figure 3) demonstrated that the sCD4 produced by the retroviral vectors is recognized by three blocking antibodies, but not by a non -blocking control antibody. The secreted sCD4 is specifically immunoprecipitated by blocking antibodies OKT4a (lane 1), Leu3a (lane 3), and MT151 (lane 4), but not by a monoclonal antibody which does not interfere with HIV infection, OKT4 (lane 2) . The data demonstrates that the sCD4 produced by retroviral vectors that the sCD4 produced by retroviral vectors retains the proper antigenic determinants for interaction with the HIV gpl20 envelope.

To determine if the sCD4 produced by the sCD4 vectors could interact directly with the HIV envelope, labeled cell medium was incubated with partially purified HIV gpl20 (Figure 4) . The HIV gpl20 was then immunoprecipitated with goat antiserum to gpl20 and the immune-complexes resolved by SDS-Page. Without pgl20 no sCD4 was immunoprecipitated (Figure 4, lane 2) . However, upon addition of gpl20, sCD4 was observed (lanes 3 and 4) . Hence, it is demonstrated that there was binding of the sCD4 to HIV gpl20.

In order to be of potential therapeutic benefit, sCD4 must inhibit HIV infection of CD4 positive cells. This potential was tested in an experiment where HIV susceptible Sup-Tl cells (which grow in suspension) were added to dishes of adherent cells producing the sCD4 gene product. As a source of sCD4, the SSC-transduced PA317 producer cell line was chosen based on the observation that his line consistently yielded the most sCD4 in the immunoprecipitation assay used at this time. Because these cells were also producing the amphotropically packaged SSC virus, a PA317 cell line producing the parent N2 vector is used as a control. After an overnight co-cultivation period, both cell mixtures were challenged with two independent isolates of HIV-1 and the cultures were left to expand for one week. When Sup-TI cells were assayed for virus replication by scoring for p21 production (see Table 1) . Cells exposed to the sCD4 producing cell line were significantly protected from HIV-1 infection when compared with the control transduced cell line, providing evidence of lower infectivity of the tested HIV-1 preparations. (p=0.026, Mamm-Whitney Rank Sum). If the transient presence of sCD4 on the surface of transduced cells afforded the potential for these cells to be infected by HIV, this would seriously limit the potential use of this anti-HIV system. To address this potential complication, both the SSC and SCSX vectors were used to transduce the human K562 line (aCD4 negative line of myeloid origin) . These transduced cell populations were challenged with HIV. In neither case could HIV infection be documented (as scored by p24 production) in the transduced K562 cells. As an alternative to bone marrow transduction and transplantation, cell implants could be used as^" in vivo factories for the continuous production of sCD4 in accord with the teachings of Thompson, et al. , Science 241:58-62 (1988). Such implants could be maintained successfully in vivo following neovascularization of collagen sponges treated with angiogenesis factors. The trans-production of sCD4 by cell implants would not only be expected to protect HIV susceptible cells, but could also mitigate the cytotoxic effects of soluble HIV gpl20. To this end, the sCD4 vectors have been used successfully to transduce both primary fibroblast and endothelial cells.

Although the procedure has been described with respect to a 202 bp fragment of the CD4 protein, it is to be understood that other fragments may be employed which include the CD4 receptor region. The selection of an appropriate fragment is deemed to be within the skill of one experienced in the art in view of the teachings therein. References pertinent to soluble CD4 fragment are Smith, et al, Science 238:1704-40 and the articles appearing in Nature 331:76-86 (January 17, 1988) . Reference is also made to Tefson, et al. , Science 241:712-16 (1988) which provides information about the CD4 receptor site region. (All of the above cited references are incorporated herein by reference.)

As would be expected by one of ordinary skill in the art, the DNA or RNA sequence included in an expression vehicle need not be identical to all or any particular portion of the DNA or RNA sequence for CD4 protein, provided that the sequence included in the expression vehicle encodes for a protein which functions as a CD4 receptor. (Although, in accordance with a preferred embodiment, the expression vehicle includes a retroviral vector (i.e., the retroviral vector is packaged in retroviral particles) , the present invention is not limited to such an expression vehicle. However, this particular expression vehicle is very effective for transfecting animal cells, particularly human cells, by procedures known in the art (See Kantoff, et al., cited previously).

Table 1. Effect of co-cultivation of susceptible CD4⁺ lymphoblastic cells with sCD4 producing and control transduced cell lines upon HIV-1 infectivity. The infectivity of two preparations of RF or MN HIV-1 viruses are expressed below as average TCID₅₀/ml and standard errors of the geometric mean. Sup-TI cells were co-cultivated with either N2- transduced PA317 cells (vector control) or SSC- transduced PA317 (sCD4 producing) . Independent determinations were performed in quadruplicate as described.

Bone marrow cells are transfected at a multiplicity of infection (MOI) of from 10 to 20 with the expression vehicle, as described above, in an effective amount and are administered intravenously in a physiologically acceptable carrier, such as saline solution. The selection of a suitable carrier is deemed to be within the scope of those skilled in the art. A preferred dosage range is 10^6"108 treated cells per kilogram of patient body weight. Though only a portion of the cells which have been treated with a retroviral vector as described herein may be transduced with the functioning gene for the protein which functions as a CD4 receptor, the transduced cells are sufficiently numerous to effect the desired CD4 receptor protein level. Similarly, the patient's fibroblasts may be transduced and administered by the procedure described by Palmer, et al.. PNAS, 84:1055-59 (9187) ; Graver, et al.. PNAS 84:1050-54 (1987) and Graver et al. , Trans, Assoc. Am. Phvs. , Vol. C, 10-20.

EXAMPLES Example 1: In order to initiate the construction of a retroviral vector which would express a soluble form of the human CD4 protein, both the available CD4 gene- containing plasmid and a common SV40 virus based expression vector had to be modified. The CD4 containing plasmid (pTAB, a gift of Richard Axel of the College of Physicians and Surgeons, Columbia University, New York, New York) was digested with the restriction endonucleases Eco RI and Bam HI (New England Biolabs, Beverly, MA) to release the CD4 gene which was isolated by agarose gel electrophoresis followed by purification via binding/elution to glass beads (using the GeneClean product, BIO 101, La Jolla CA in the manner recommended by the manufacturer) . The CD4 fragment was ligated (using T4 DNA ligase as recommended by the supplier. New England Biolabs) into Eco RI plus Bam HI cut Bluescript cloning vector (Stratagene Co. , La Jolla, CA) . The product of the ligation was then transformed into competent DH5 alpha bacteria (Bethesda Research Labs, Gaithersburg, MD) and white colonies were isolated and screened for proper insert size to yield the plasmid pCDW.

To produce a suitable plasmid-based expression vector for the CD4 gene the plasmid SVlneo (obtained from American Type Culture Collection, Rockville, MD) was digested in Hind 3 plus Hpa I, and a synthetic polylinker sequence from the pUC-13 vector (Pharmacia, Piscataway, NJ) was inserted (via T4 DNA ligase) in place of the neoR gene of pSV2neo. This new vector, pSVPL, was transformed into DH5 alpha bacteria (Bethesda Research Labs) and colonies were screened for the presence of restriction enzyme sites unique to the polylinker.

The pSVPL expression vector was further modified by the insertion of an Xho I linker (conditions and reagents supplied by New England

Biolabs) into the Pvu II site on the 5' side of the" SV40 early region promoter to produce pSVPLX.

All references cited herein are incorporated herein by reference. In the examples, unless otherwise specified, restriction enzyme digests, ligations, transformations, etc., are performed as described in Molecular Cloning, A Laboratory Manual by Maniatis, et al.

Example 2: The pCDW and pSVPLX plasmids, for example, were digested with enzymes Hind 3 plus Xba I (New England Biolabs) and their DNAs isolated (using the GeneClean product) following agarose gel electrophoresis. Ligation of the CD4 fragment into the pSVPLX vector was performed using T4 DNA ligase and colonies were screened by color and analyzed for size as in Example 1 to yield pSVCDW in which the SV40 virus early region promoter is used to drive the expression of the complete CD4 gene product. Example 3:

The production of a soluble form of CD4 was accomplished by the use of a specially designed oligonucleotide adaptor to produce a mutant form of the CD4 gene. This adaptor has the unique property that when inserted into the Nhe I site of the CD4 gene it produces the precise premature termination of the CD4 protein amino acid sequence while regenerating the Nhe I site and creating a new Hpa I site. The oligonucleotide adaptor used (synthesized by Midland Certified Reagent Co.) was produced by annealing two phosphorylated oligonucleotides; 1) 5' CTAGCTTAGAGTGAGTT 3', 2) AACTCACTCAAG. This product was then ligated using T4 DNA ligase into the Nhe I site of pSVCDW. The ligation reaction was then cleaved with Hpa I followed by addition of Xho I linkers (New England Biolabs) . The linker reaction was terminated by heating at 65 C for 15 min. followed by subjection to digestion with Xho I restriction endonuclease (New England Biolabs) . This reaction product was then subjected to agarose gel electrophoresis and the fragment containing the SV40-CD4 adaptor isolated (GeneClean) . The retroviral vector N2 was prepared to accept the SV40-CD.,- adaptor fragment by digestion with Xho I and treatment with Calf Intestinal Phoεphatase (Boehringer Mannhein, Indianapolis IN) . The ligation of the CD4 expression cassette was performed using a ratio of insert fragment to vector of 5:1. The CD4 cassette was then transformed into DH5 alpha competent bacteria (Bethesda Research Labs) . Constructs were analyzed by restriction endonuclease digestion to screen for orientation and then grown up in large scale. The construct where the SV40 virus promoter is in the same orientation as the viral LTR promoters is known as SSC, while the construction in the reverse orientation is called SCSX.

Example 4:

The SSC vector is packaged into PA317 cell line (the cell line is described by Miller et al. , cited previously) to provide PA317 cells capable of producing soluble CD4 protein.

The vector-producing cell line was tested^" to ensure that it was making both retroviral particles containing the SSC vector as well as the final product itself, namely, soluble CD4. The transfected PA317 vector-producing cell line was radiolabeled with 35- cysteine for 12hr after which time the cell medium was collected and subjected to immunoprecipitation with an anti-CD4 monoclonal antibody (OKT4a) . Immune complexes were isolated (via protein A sepharose beads) and subjected to denaturing polyacrylamide gel electrophoresis. Figure 5 is an autoradiogram with protein MW size markers as indicated. The SSC cell line is producing large quantities of soluble CD4 protein (CD4S) . The PA317 cells are employed for transforming monkey bone marrow cells by the procedures described in Kantoff et al. , cited previously. Such bone marrow cells are capable of expressing CD4 protein. The same procedure may be used for transforming human bone marrow cells. Example 5:

PA317 cells transformed with SCSX, as hereinabove described, are employed for transfecting mouse fibroblast cells (SV-T2 line) as described by Eglitis et al. Science 230:1395-98 (1985) . Cells capable of growing in G418 (and, therefore, containing a functioning neoR gene) were then radiolabeled and the cell medium was subjected to immunoprecipitation to test for the presence of a functioning soluble CD4 gene, as described with reference to testing of the vector-containing PA317 cells above (Example 4) .

Figure 6 is a resultant autoradiogram from this assay (uninf. : uninfected SV-T2 cells; inf.: SCSX infected, selected SV-T2 cells) . The infected fibroblasts are secreting soluble CD4 protein (CD4S) into the medium. Human fibroblasts are obtained from skin biopsy and are transfected by known means with SCSX vector. The cells, after transfection, are delivered by cellulose pads surgically inserted subcutaneously (Thompson et al., Science, in press, 1988). Dosage of 10⁶ fibroblasts/kg of patient body weight is appropriate when administered by this method.

Claims

What is Claimed:

1. An expression vector containing a promoter sequence and a DNA (RNA) sequence insert which encodes a soluble CD4 receptor protein, a fragment thereof, or a CD4 receptor protein analogue which, when transfected into a mammalian cell, will cause said cell to express soluble CD4 receptor protein, a fragment thereof, or a CD4 receptor protein analogue.

2. An expression vector of claim 1 wherein the DNA (RNA) insert encodes soluble CD4 receptor protein or a fragment thereof.

3. A vector of claim 1 which is designated

SSC.

A vector of claim 1 which is designated SCSX.

5. A composition of matter comprising a vector of claim 1 in a mammalian cell.

6. A composition of matter comprising a vector of claim 1 in a carrier organism. 7. A composition of claim 6 wherein the carrier organism is an E. coli.

8. A method of preventing a ligand from binding with a CD4 receptor on a cell comprising of the steps of 1) Transfecting a vector of claim 1 into a mammalian cell;

2) Administering the transfected mammalian cells prepared in step 1 to a mammal of the same species as that from which the cells (which have been transfected) were obtained: an amount of transfected cells sufficient to produce adequate soluble CD4 to block binding of a ligand to CD4 receptor sites on host cells.

9. A composition of matter comprising cells of claim 5 and a pharmaceutically acceptable carrier.

10. A composition of claim 9 wherein the carrier is a solid support.

11. A composition of claim 10 wherein the support is a pad appropriate for subcutaneous insertion.

12. A composition of claim 9 wherein the carrier is a patch of skin fibroblasts.

13. A method of claim 8 wherein the mammalian cells are human cells. 14. A method of claim 12 wherein the ligand is an HIV virus, viral particle or viral product.

15. A method of claim 8 wherein the donor cells used in step 1 have been obtained from the recipient of the transfected cells.