AU647013B2 - Retroviral vectors expressing soluble CD4: a gene therapy for aids - Google Patents

Description

OPI DATE 23/03/90 AOJP DATE 26/04/90 APPLN. I D 41932 89 PCT NUMBER PCT/US89/03541 INTERNATIONAL APPLICATION PUBLISHED UNDER THE PATENT COOPE.RATION TREATY (PCT) (51) International Patent Classification 4 t(1 I) International Publication Number: \XO 90/018701 AO IH 1/04, A61 K39/00 Al C12N 15/00 (43) International Publication Date: 8 March 1990 (08.03.90) (21) International Application Number: PCT, US89'03541 (74) Agents: STERN, Man- in, R, et aL. Flcit, Jacobson. Cohn, Price, Holman Stern, The Jenifer Building, 400 Seventh St., (22) International Filing Date: 21 August 1989 (21,08.89) Washington, DC 20004 (US).

Priority data: (81) Designated States: AT (European patent), AU, BE (Euro- 234,646 22 August 1988 (22.08.88) us pean patent), CH (European patent), DE (European pa- 395,454 18 August 1989 (18.08.89) us tent), FR (European patent), GB (European patent), ITI (European patent), JP, LU tEuropean patentt, NL (European patent), SE (European patent).

(71) Applicant: TH4E UNITED STATES OF AMERICA, as represented by THE SECRETARY, U.S. DEPARTMENT OF COMMERCE [USA12S); 5285 Port Royal Road, Published Springfield, VA 22161 If1W: irnaonal search repor.

(72) Inventors: ANDERSON, French -,6820 Melody Lane,__ Bethesda, MD 20817 GALLO, Robert, C. 8513 Thornden Terrace, Bethesda. MD 20814 WVONG- 0U' STAAL, Flossie ;8418 Hacker Drive, Potomac, MND 6 4 73 20854 MORGAN. Richard, A. ,7851 Butterfield Drive. Elkridge. MD 21227 MUENCHAU. Dar\I ;972 1-203 Clocktower Avenue. Columbia, MD 2106!

(US).

(54)Title: RETROVIRAL VECTORS EXPRESSING SOLUBLE CD4: A GENE THERAPY FOR AID)S (57) Abstract The invention disclosed herein provides a means of blocking, retroviral infection of human cells b> in vivo production of secreted CD4 (sCD4) molecule proteins and CD4 protein analogues. These proteins block interaction betwkeen the CD4 receptor' on cells and virus, viral particles and viral products shich would other.~ise bind to the CD4 receptors.

WO 90/01870 PCT/US89/03541 1 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 amino acid residues from the amino terminal end of the intracellular region of the eIRc-.T1Tt1TE SHEET .WO 90/01870 PCT/US89/03541 2 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 owv 7i i- cr-!FFT WO 90/01870 PCT/US89/03541 3 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 oi 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., J. Exp. Med.

166:219-34 (1987).

STATEMENT OF DEPOSIT The,.plasmids..SSC and SCSX have been deposited using<'E. coli DH5 alpha as carriers in the American Type lture-Collecti'b at'RocKville, Maryland in accord with the requirements of the Budapest Treaty for 06 :2 ug LT-t cS deposit of patented organisms and biological products.

The deposits have been assigned the ATTC accession numbers: SSC Acc. No. 67760 and SCSX ACC.

No. 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.

i, SUBSTITUTE SHEET vr? IWO 90/01870 PCT/US89/03541 4 FIGURE LEGENDS Figure 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 neomycin resistance gene (NEOR), truncated CD4 gene (sCD4), and virus promoter (SV40) are as indicated. The arrow indicates the direction of transcription of the promoter. Restriction enzyme sites are dafined as follows: X Xba I, B Bst NI, E Eco RI, N Nru I, and Xh Xho I.

Fiqure 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, SSCtransduced PA317; 3, SV-T2; 4, SCSX-transduced SV-T2; 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 SIIBSTITUTE SHEET WO 90/01870 PCT/US89/03541 producer cell lines was immunoprecipitated 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 or coprecipitation with no HIV gpl20 (lane 0.2 ig of partially purified HIV gpl20 (lane or gg partially purified HIV gpl20 (lane 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 (5il) 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 METIIHODS Construction of sCD4 retroviral vectors A soluble form of the gene was constructed by first tr erring the original T4B cDNA clone as co RI plus Barn HI fragment, into the Bluesc p' vector (Stratagene) to produce pCDW. The 3 gene was excised w.ith Hind III plucs Xba I and c^, f-u r,.TlrEIr7r LzrF-r WO 90/01870 PCT/US89/03541' 6 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 to derive the SCSX vector. The SSC vector was produced from the SCSX vector by first digesting with Bst NI followed by endfilling 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, 50pg of SSC or SCSX vector DNA was used to transfect (via calcium phosphate coprecipitation) the ecotropic packaging cell line 2 and 48hr post transfection, virus ICf I IIn C 1? 1 *r 19 ft IIF- 5a Figure 5. Production of sCD4 by retroviral vector producer cell line. Plasmid DNA for the SSC retroviral vector was introduced into the PA317 packaging cell line by calcium phosphate transfection, followed by selection for resistance to the neomycin analog G418. The producer cell population (labeled SSC) and a control packaging cell lines (labeled PA317) were radiolabeled with and cell-free culture medium was immunoprecipitated with CD4 monoclonal antibody OKT4a.

Immune complexes were isolated and subjected to polyacrylamide gel electrophoresis and autoradiography.

Size markers of known MW (in Kd) were run in parallel lanes. The 'sCD4 protein is indicated by the arrow (CD4S).

Figure 6. Production of sCD4 by transduced fibroblasts. Retroviral vector containing cell culture medium from an SCSX produced cell line was used to infect the mouse fibroblast cell line SV-T2. Infected (transduced) cells were selected for the presence of the vector by growth in G418 containing culture medium.

Following selection, cells (infected inf., and uninfected controls, uninf.) were radiolabeled with and subjected to immunoprecipitation with the OKT4a monoclonal antibody. Radiolabel immune complexes were isolated and subjected to polyacrylamide gel electrophoresis and autoradiography. Size markers of known MW (in Kd) were run in parallel lanes. The sCD4 protein is indicated by the arrow (cD4S).

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 as an Eco RI plus Bam HI fragment, into the Bluescript vector (Stratagene) to produce pCDW. The CD4 gene was excised with Hing III plus Xba I and

'S

WO 90/01870 PCT/US89/03541 7 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 Ag/ml).

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 fetal bovine serum). Cell free supernants were collected by filtration through 0.224m 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 (300pg/ml) for 14 days.

Immunoprecipitation Cells were grown to approximately confluence on 100mm tissue culture dishes and prior to radiolabeling incubated for 1-2 hr in cysteine-free MEM labeling medium (GIBCO) containing 2% dialyzed fetal calf serum. Labeling was initiated by removal/replacement of the labeling medium with fresh cystein-free medium containing 100-200 MCi/ml of 35 S]cysteine (approximately 1000Ci/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 i rf v- 1- 1 -r r-LY r r-q WO 90/01870 PCT/US89/03541 8 adding 1/10 vol of 10X buffer (200mM Tris HCi pH 1.2M NaC1, 5% NP.40, 2% NaDeoxycholate, 2mM EGTA, 2mM NaF, 50 g/ml aprotinin, 2mM phenyl methyl sulphonyl 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 Ag 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 lX buffer. and resuspended in 50 pl of sample loading buffer (10mM Tris HC1 pH 8.0, 2% SDS, 5% 2-mercaptoethanol, 10% glycerol). Samples wer'.

heated for 30 min at 700C before electrophoresis on 12% SDS-PAGE (18).

HIV-1 titration Virus strains used (20.23) were HTLV-III (designation, HIV-1/NIH/USA/1984/MN), and HTLV-III, (designation, HIV-l/NIH/USA/1983/RF). HTLV-IIIR 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 C11nC:_r~rV11r= CUrC*_ WO 90/01870 PCT/I'S89/03541 9 2.2 to 3.1 x 1011 particles/mi. 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 (TCIDs 5 The preparations of HTLV-III, and HTLV-III, used had 1.6 x 104 and 4.0 x 103 TCIDs,/ml in the H9 assay, respectively.

To determine the anti-viral effects of cocultivation with transduced cells lines, a different culture system was required. Mouse NIH/3T3-derived fibroblasts (cither N2 or SSC transduced PA317 cells) were seeded into 24 well.culture plates (2 x 10 5 /wells) 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 twofold dilution of HIV-1 virus preparations (RF or MN) were made in 20 ti media in a separate 96 well U-bottom microtiter 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 murine 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 TCIDs 5 values were performed by the method of Reed and Muench (26).

e1PlM1TTI1T' rWcL crr 10 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 which constitute combined in vitro host range of both xenotropic and ecotropic viruses, mouse, cat, dog and human cells were used to transduce cells which were shown to express the secreted CD4 (sCD4) gene product. Ecotropic viruses are viruses that will grow in cells of species from which they were isolated, a mouse virus propagates best in mouse cells and to a limited or undetectable level in cells of other species. It shows a low efficiency of infection in heterologous host cells. In contrast, xenotropic viruses are those that are endogenous to one species but cannot replicate well in that species, generally because of a receptor block, a mouse virus propagates in cat, dog, or human cells but not in mouse cells. Amphotropic viruses have the characteristics of both ecotropic and xenotropic viruses, viruses that will grow in cell of species from which they were isolated and also grow in other host cells. The sCD4 produced by the viral vectors is immunoprecipitated by monoclone% antibodies against CD4. Physical interaction of the vector-produced sCD4 and HIV-1 gpl20 was demonstrated by coprecipitation of sCD4/gpl20 with antiserum directed against HIV 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.

C, 1 10a 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 the SCSX is in the opposite C^p WO 90/01870 PCT/US89/03541 11 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 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 The data demonstrated that bot6 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 O 90/01870 PCT/US89/03541 12 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 Leu3a (lane and MT151 (lane but not by a monoclonal antibody which does not interfere with HIV infection, OKT4 (lane 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 The HIV gpl20 was then immunoprecipitated with goat antiserum to gpl20 and the immune-complexes resolved by SDS-Page. Without no sCD4 was immunoprecipitated (Figure 4, lane 2).

However, upon addition of gpl20, sCD4 was observed (lanes 3 and Hence, it is demonstrated that there was binding of the sCD4 to HIV 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 SUBSTITUTE SHEET WO 90/01870 PCT/US89/03541 13 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 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).

l 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 SUBSTITUTE SHEZT WO 90/01870 PCT/US89/03541 14 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 .ql]RF',TITITIr cztrc,, .WO 90/01870 PCT/US89/03541 embodiment, the expression vehicle includes a retroviral vector 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 TCIDs,/ml and standard errors of the geometric mean.

Sup-TI cells were co-cultivated with either N2transduced PA317 cells (vector control) or SSCtransduced PA317 (sCD4 producing). Independent determinations were performed in quadruplicate as described.

HIV Co-cultivation Infectivity Virus Cell Type (TCIDs 5 /ml SEM) HTLV-IIIR N2 transduced 1979 283 HTLV-IIIR SSC- transduced 200 0 HTLV-IIIm N2 transduced 2828 566 HTLV-IIIm SSC- transduced 707 141 Bone marrow cells are transfected at a multiplicity of infection (MOI) of from 10 to 20 with F:UBSTITUTE SHEET WO 90/01870 PCT/US89/03541 16 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-O8 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. Phys., 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 genecontaining 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 ,-,i1Ftc;1T[1TF !;FFT WO 90/01870 PCT/US89/03541 17 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 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, SUBSTITUTE SHEET WO 90/01870 PCT/'S89/03541 18 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) CTAGCTTAGAGTGAGTT 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 SUBSTITUTE SHEET WO 90/01870 PCT/US89/03541 19 England Biolabs). The linker reaction was terminated by heating at 65 C for 15 min. followed by subjection to digestion with Xho I restriction endonucl.ase (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 Phosphatase (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 cysteine for 12hr after which time ,ziiRc;T1TUTE SHEET WO 90/01870 PCT/US89/03541 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 polyactylamide 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 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 ,iR~~C-r~7(lr~C ~=I-lrFIP WO 90/01870 PCT/US89/03541 21 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.

SUBSTITUTE SHEET

Claims (21)

1. A retroviral vector, comprising: nucleic acid that encodes a soluble CD4 receptor protein, a portion or an analog thereof, wherein, upon introduction of said vector into a mnammalian host cell, said soluble CD4 receptor, portion or analog thereof is produced in and secreted by said host cell and wherein said portion or analog thereof binds a human immunodeficiency virus (HIV) with substantially the same affinity as said soluble CD4 receptor protein binds said HIV.

2. A retroviral vector of claim 1, which is designated SSC.

3. A retrovirai vector of claim 1, which is designated SSCX.

4. The retroviral vector of claim 1, wherein said nucleic acid is RNA. The retroviral vector of claim 1, wherein said nucleic acid is DNA.

6. An amphotropically packaged retroviral particle, comprising the retroviral vector of claim 4.

7. A method for inhibiting infection by human immunodeficiency virus (HIV) of susceptible cells comprising: introducing the retroviral vector of claim 6 into said susceptible cells under conditions whereby said soluble CD4 receptor protein, portion or analog thereof is produced and secreted by said cells.

8. Transduced mammalian cells, comprising the retroviral particles of claim 6.

9. The transduced cells of claim 8, selected from the group consisting of fibroblast cells, epithelial cells, wherein said cells are fibroblast cells, epithelial cells, bone marrow cells, cultured cells derived from normal tissues, immortal cell lines that grow in suspension 23 culture or in monolayer, and transformed cell lines that grow in suspension culture or in monolayer. The cells of claim 9, which are derived from humans.

11. The cells of claim 9, wherein said cells are bone marrow cells.

12. The cells of claim 9, wherein said cells are fibroblast cells.

13. A method for producing soluble CD4 receptor protein, a portion or an analog thereof in a mammalian host, comprising: selecting transduced cells of claim 9 that are immunologically matched to said mammalian host; and introducing said transduced cells into said host, whereby said soluble CD4 receptor protein, a portion or an analog thereof is expressed and secreted by said transduced cells in said host.

14. The method of claim 13, wherein said transduced cells are obtained from said host. The method of claim 13, wherein said transduced cells are bone marrow cells.

16. The method of claim 13, wherein said transduced cells are fibroblast cells.

17. The method of claim 16, wherein said fibroblast cells are introduced as part of a patch of skin.

18. The method of claim 13, wherein said cells are introduced intravenously.

19. The method of claim 13, wherein said cells are seeded onto a solid support and implanted into said host. A method for treating an individual who is infected with a human immunodeficiency virus (HIV), comprising: selecting transduced cells of claim 9 that are immunologically matched to said individual; and introducing an effective number of said transduced cells into said individual, whereby a sufficient amount of said soluble CD4 receptor protein, a portion or an analog r( i 24 thereof is expressed and secreted by said transduced cells in said host to inhibit the progress of the infection of said individual by HIV.

21. The method of claim 20 wherein said transduced cells are obtained from said individual.

22. The method of claim 20, wherein said transduced cells are bone marrow cells.

23. The method of claim 20, wherein said transduced cells are fibroblast cells.

24. The method of claim 20, wherein said fibroblast cells are introduced as part of a patch of skin. The method of claim 20, wherein said cells are introduced intravenously.

26. The method of claim 20, wherein said cells are seeded onto a solid support and implanted into said host. DATED this 17 day of August 1992 THE UNITED STATES OF AMERICA, AS REPRESENTED BY THE SECRETARY, U.S. DEPARTMENT OF COMMERCE Patent Attorneys for the Applicant: F.B. RICE CO.