WO1999034006A2

WO1999034006A2 - A method for the positive selection of cd4+ t-lymphocytes transduced with anti-hiv genes

Info

Publication number: WO1999034006A2
Application number: PCT/EP1998/008395
Authority: WO
Inventors: Claudio Bordignon
Original assignee: Genera S.P.A.
Priority date: 1997-12-23
Filing date: 1998-12-22
Publication date: 1999-07-08
Also published as: ITMI972866A1; AU2415999A; WO1999034006A3; IT1297016B1

Abstract

The present invention describes CD4+ T-lymphocytes genetically modified to express a cell surface marker and able to sustain HIV infection both in in vivo and in in vitro systems. The invention also concerns a positive selection method for CD4+ T-lymphocytes, resulting in a population fully permissible to HIV or to other retrovirus or retroviral vectors infection, for peripheral blood mononuclear cells or CD4+ T-lymphocytes transduction.

Description

A METHOD FOR THE POSITIVE SELECTION OF CD4+ T-LYMPHOCYTES TRANSDUCED WITH ANTI-HIV GENES

Background of the invention

Prevention or treatment of Human Immunodeficiency Virus (HIV-1) infection is one of the potential applications of gene therapy. Several studies have suggested the potential of intracellular immunization for the inhibition of HIV replication and spreading. In this strategy the goal is to replace the patient's HIV-susceptible cells, in a very progressive way, by genetically modified cells carrying molecules able to interfere with different steps of the HIV life cycle. This includes targeting of structural and regulatory proteins (Cell 58, 205-214, 1989 and 65, 241- 248, 1991), RNA decoys (TIG, 10, 139-144, 1994), anti-sense RNA (Science 261, 1004-1012, 1993) and ribozymes (Gene Therapy 1, 38-45, 1994; J. Virol. 65, 5531-5534, 1991). Reports on anti-HIV approaches showed variable levels of induction of resistance in trasduced cell lines. In particular, a clone of the human CEM T cell line stably expressing the transdominant mutant RevMl O developed resistance to HIV infection. Nevertheless, inhibition of viral replication was obtained in other human cell lines by expression of intracellular anti-sense RNA (Science 258, 1485-1488, 1992). Furthermore, constitutive transcription of chimeric neo-Rev Responsive Element (RRE) suppressed HIV-1 transdominant Rev protein function in CEM cells (Hum. Gen. Ther. 5, 193-201 , 1994). The expression of RRE-derived sequences (New Biol. 4, 66-74, 1992; J. Viral 68, 8254-8264, 1994) or polymeric TAR decoy (Cell 63, 601 -608, 1990; Gene Therapy 2, 377-384, 1995) inhibited HIV- 1 replication in CD4⁺ human T-cell lines. However, no methods are actually available for trasducing PBMC, or other bone marrow derived cells, so as to obtain genetically modified cell lines, still susceptible to HIV infection. Cells like these represent an ideal model to study the intracellular immunization approach in HIV infection therapy or prevention.

Conventional systems based on negative selection, such as G-418 resistance of neomycin trasduced cells, allow the recovery of a trasduced CD8⁺ T-lymphocyte population characterized by very low infectability. Moreover this selection protocol is time consuming, a condition which together with standard T-lymphocytes activation protocols, may favour HIV spreading during cell culture manipulation.

It has now been found a positive selection method that allows the recovery of a CD4⁺ enriched population that maintains unaltered susceptibility to HIV infection, as observed both in in vivo and in in vitro systems.

The method of the invention allows the recovery of a CD4⁺ T- lymphocyte PBMC derived population which can be efficiently trasduced by retroviral vectors and very quickly selected, minimizing HIV spreading in culture, thus representing an ideal model to study anti-HIV gene expression and effects in vitro.

Summary of the invention

It has now been found that it is possible to trasduce PBMC with a retrovirus encoding a cell surface molecule for positive selection, maintaining PBMC susceptible to HIV (or other retroviruses) infection. According to a first embodiment, the invention provides PBMC or

CD4⁺ T-lymphocytes genetically modified to express a cell surface marker molecule, still susceptible to HIV infection, both in in vivo and in in vitro systems.

Moreover, the invention provides a positive selection method for

CD4⁺ T-lymphocytes which can be later infected by HIV, which comprises a step of PMBC trasduction by retroviral vectors carrying the cell surface marker and their purification (or enrichment) by immunoselection with specific cell surface marker antibodies.

The method of the invention allows the production of trasduced T- lymphocytes from seropositive, infected individuals, in conditions that minimize HIV spreading from infected to still uninfected CD4+ cells in culture, thanks to the fast selection procedure.

The invention further comprises retroviral vectors which encode anti-HIV DNA sequences and a cell surface marker.

The invention comprises, in a further embodiment, the use of T- lymphocytes, trasduced by retroviral vectors and selected by immunoaffϊnity, as experimental models for intracellular immunization in the prevention and treatment of HIV infection and spreading.

Moreover the invention provides the use of T-lymphocytes trasduced by retroviral vectors (carrying a cell surface marker and anti- HIV genes), selected by immunoaffmity and susceptible to HIV infection, as an experimental model for anti-HIV therapy. Description of the Figures

Fig 1 : Acute infection of G418-selected or ΔLNGFR-selected PHA-stimulated T-lymphocytes with NL4-3-derived HIV virus.

After trasduction and G418-selection or positive selection for ΔLNGFR expression PBMCs and CD8+-depleted T lymphocytes were plated in 24-well plates at the concentration of 2.5X10⁵ cells/well and infected with NL4-3 derived virus (m.o.i. = 0.5). Virus replication was measured by RT activity released into the culture supernatants.

Fig 2: Acute infection of G-418-selected or ΔLNGFR-selected PHA stimulated T-lymphocytes with primary isolate CSL-1 of HIV.

After trasduction and G-418 selection or positive selection for ΔLNGFR expression PBMCs and CD8+-depleted T lymphocytes were plated in 24-well plates at the concentration of 2.5X10⁵ cells/well and infected with the CSL-1 virus isolate (m.o.i. = 0.5). Viral replication was measured by RT activity released into the culture supernatants. Detailed disclosure of the invention Positive selection of trasduced PBMC or CD4⁺ T-lymphocytes can be performed by using modified retroviral vectors containing genes which encode for cell surface proteins. Growth factor receptors may represent useful cell surface molecules.

In this particular application, the sequence encoding the low affinity Nerve Growth Factor, LNGFR (Blood 83, 1988-1997, 1994), deleted in the intracellular domain to avoid activation of intracellular signaling, has been used (ΔLNGFR).

Retroviral plasmid DNA encoding for ΔLNGFR and anti-HIV genes has been obtained by conventional techniques (Sambrook, J., E. F. Fritsch, e T. Maniatis. 1989. Molecular cloning: a laboratory manual. 2nd ed., Cold Spring Harbor Laboratory Press, New York), and as already described (see WO 95/06723). Trasfection of the DNA in conventional amphotropic AM 12 or the ecotropic E86 packaging cell lines to reconstitute infectious virions, has been performed either by electroporation or Calcium Phosphate coprecipitation. The E86 supernatants containing the recombinant retroviral progeny has been harvested and used to infect the amphotropic packaging cell line AM 12 to obtain helper-free amphotropic recombinant retroviruses and a producer cell line for PBMC trasduction.

Since LNGFR is not expressed either in haemopoietic cells or in PBMC, trasduced cells could be characterized and isolated for ΔLNGFR surface expression. ΔLNGFR expression levels are determined by FACS analysis with a monoclonal antibody (MoAb) to LNGFR, using an indirect fluorescence method. Selection of trasduced cells is performed (with antibodies specific for the extracellular domain of LNGFR) as already described in WO 95/06723. Anti-HIV genes are defined as sequences able to interfere with HIV replication, to decrease its replication efficiency or to interfere with virion assembly such as: 1) decoy sequences for HIV transcription factors, such as tat binding region (TAR) or Rev Responsive Element (RRE); 2) ribozymes for viral RNA, such as naturally occurring (anti-tat and anti-Rev hammerhead ribozyme) or synthetic catalytic RNA molecules targeted to essential components of HIV genome (i.e.

Reverse Transcriptase or Protease genes);

3) negative dominant HIV proteins, such as C-terminal Rev mutants or gag mutants;

4) HIV mutants able to interfere with wild type HIV replication or packaging, such as the F-12 mutant, as described in WO 97/13861 ;

5) anti-sense nucleic acids targeted to different regions of the HIV genome (i.e. Reverse Transcriptase, Protease or other essential genes);

6) combinations of the above genes.

Accordingly to the preferred protocol, PBMC from healthy, HIV negative donors, were depleted for CD8⁺ cells by incubation with magnetic microbeads conjugated with anti-human CD8 MoAbs.

The resulting CD4⁺ enriched population was activated by IL-2 alone or in combination with PHA. T-lymphocytes trasduction was obtained by co-cultivating activated cells with the recombinant retroviruses producing cell line, for 2-6 days, preferably 4. Immunoselection of ΔLNGFR⁺ cells was then performed on magnetic microbeads conjugated with anti LNGFR MoAbs. ΔLNGFR⁺ cells were then infected with two different HIV strains (See figure 1 : infection by NL4-3 derived HIV and figure 2: infection by a primary isolate, CSL-1).

The selected T-lymphocyte population represents a more physiological model for the gene-transfer approach in selected T- lymphocytes population. Moreover, the method allows the selection of a trasduced T-lymphocyte population from seropositive, infected individuals, in conditions that minimize HIV spreading from infected to still uninfected CD4⁺ cells.

The invention will be described in more details in the following examples:

Example 1 ; Positive selection based on ΔLNGFR expression PMBC from healthy HIV-1 negative donors have been isolated on

Ficoll-Hypaque (Pharmacia, Uppsala, Sweden) density gradient. Purified lymphocytes have been seeded in 24 well trays (Falcon Lincoln Park, NJ) and cultured for 72 hrs in the presence of phytohemagglutinin (2 μg/ml; PHA-L, Boehringer Mannheim, Mannheim, Germany) and rhIL-2 (100- U/ml; Cetus, Emeryville, CA) RPMI 1640 medium (GIBCO, Grand Island, NY) with ImM glutamine, 100 U/ml antibiotics and 5% Human Serum (HS). In alternative, PBMC stimulation was performed with IL-2 alone for 6-8 days and cells cultured as above. Stimulated cells were then washed twice and resuspended in medium containing rhIL-2 (100 U/ml). Gene transfer was performed by cocultivation of PHA-stimulated PBMCs with the recombinant ecotropic packaging cell line E86 (recombinant retrovirus producer cell line). The retrovirus producer cell line was previously obtained by standard Ca₃(PO₄)₂ coprecipitation with the LXSN vector in which the truncated form of the LNGFR cDNA had been inserted. 8X10⁶ producer cells were irradiated at 10.000 rad for 1 h and cultured 12 hrs in standard conditions, before the addition of PHA- stimulated PBMC (1X10⁶ cells/ml), in the presence of lOOU/ml rhIL-2 and 8 μg/ml polybrene for additional 48-72 hrs.

ΔLNGFR⁺ PBMCs were enriched by positive immunoselection with magnetic beads coated with goat anti-mouse IgG (Dynal A.S., Oslo, Norway). 4X10⁶ cells/ml were incubated with anti-ΔLNGFR MoAb at 4°C for 30 min, washed twice in medium containing 2% FCS and resuspended in medium with magnetic beads at 4°C for 30'. Magnetic selection was performed and the beads were detached from cells after overnight incubation at 37°C, 5% CO₂. Additionally, in order to enrich for CD4⁺-cells, PBMCs were incubated, before trasduction with magnetic microbeads conjugated with anti-human CD8 MoAbs (Miltenyi Biotech., Bergisch, Germany) for 20 min at 4°C and depleted of CD8⁺ cells by magnetic separation.

Example 2: Cell surface phenotvping

Cell surface expression of ΔLNGFR was monitored by flow cytometry using the murine anti-human ΔLNGFR monoclonal antibody

(MoAb) 20.4 (ATCC, Rockville, MD) by an indirect fluorescence labeling method. The cell surface phenotype of T-lymphocytes was determined by flow cytometry using fluorescein isothiocyanate (FITC)- or rhodamine conjugated goat anti-human CD4 (T4) or CD8 (T8) MoAbs (Coulter Immunology, Hialeah, FL). 3X10⁵ cultured cells were stained with 100 μl of 1 : 100 diluted MoAb at 4°C for 30', washed twice in medium containing 2% FCS and resuspended in 0.2 ml medium without FCS for

FACS analysis, or in 100 μl of diluted FITC-conjugated secondary MoAb (Coulter).

Example 3: HIV-1 acute infection of trasduced PBMC

PMBC obtained as described in the previous examples were plated in 24-well trays and incubated with the HIV-1 isolate from the NL4-3 molecular clone or with the primary HIV CSL-1 isolate at a multiplicity of infection comprised between 0.01 and 4. Viral titers were determined on PHA-stimulated PBMCs from seronegative donors, by the ID50 quantitation and the Reed-Muench accumulative method (Koup, R.A., Ho, D.D., and Poli, G. (1993). Isolation and quantitation of HIV in peripheral blood. In: Currents protocols in Immunology. (Greene Publishing associates and Wiley-Interscience, NIH-Bethesda MD) pp. 12.2.1-11). By these criteria the NL4-3 virus was estimated to contain 10⁵ IU (Infectious Unit)/ml, whereas the primary CSL-1 isolate contained 10⁸ IU/ml. Cells were maintained in RPMI 1640 with 10% FCS and rhIL-2

(100 U/ml) throughout the infection. Supernatants were harvested every three days and stocked at -80°C until their RT activity was evaluated as described in J. Virol. 62 139-147, 1988 5 μl of each culture supernatant were added to 25 μl of a mixture containing Oligo (dT) (Pharmacia; Uppsala, Sweden) used as primer for poly (A) (Pharm) template, MgCl₂ and ³²P-labelled dTTP (Amersham International pic, Amersham, UK). Six μl of the mixture reaction was spotted onto DE81 paper and air dried for 2 hrs at 37°C. The filter was washed five times in IXSSC and twice with 95% ethanol; the paper was then dried and the radioactivity content measured by Top Count scintillation counter (Canberra-Packard, Meriden, CT). RT activity of positively selected, CD8⁺ depleted PBMCs, expressed as cpm/μl of culture supernatant, is reported on the graph (see figure 1 and 2) and compared to the one measured after HIV- 1 infection of PBMCs, CD8⁺ depleted, trasduced instead, with the Neo gene and negatively selected by G-418.

The results clearly showed that infection efficiencies, both by the NL4-3 clone and the primary HIV-1 CSL-1 isolate, are higher in PBMCs which were positively selected by the method of the invention, than in G- 418-selected PBMCs. These efficiencies were optimized by CD4⁺ cells enrichment, according to the method of the invention.

The selection procedure for ΔLNGFR expression by immunoaffinity, was completed on 30' as compared to 2-3 weeks in the alternative procedure by G418. This short time minimizes HIV-1 spreading in culture.

Moreover the results have been confirmed in PBMCs stimulated with IL-2 alone. This is particularly useful for gene therapy experiments, since the use of mitogens such as PHA, should be avoidable.

Results have also shown that two different HIV isolates efficiently infected the lymphocyte populations (PBMCs and CD8⁺ depleted PBMCs), which had been previously trasduced to express the cell surface marker for positive selection, indicating that the trasduction "per se" does not alter productive replication of HIV.

Claims

1. CD4⁺-T-lymphocytes genetically engineered to express a cell surface marker and susceptible to HIV infection in vivo and/or in vitro.

2. CD4⁺-T-lymphocytes according to claim 1 obtained by trasduction with retroviral vector carrying a cell surface marker and anti-HIV genes.

3. CD4⁺-T-lymphocytes according to claim 1 and 2 in which the cell- surface marker is the truncated form, devoid of the intracellular domain of the Low Affinity Nerve Growth Factor Receptor.

4. Use of the CD4⁺ T-lymphocytes according to claims 1-3 as experimental models for intracellular immunization or therapy against HIV.

5. Method for the positive selection of CD4⁺ lymphocytes, which maintains their susceptibility to HIV infection and requires a shorter time than in conventional methods, comprising the steps of: a) trasduction of peripheral blood mononuclear cells by retroviral vectors encoding cell surface molecules, b) immunoaffinity selection with antibodies against said cell surface molecules.

6. Retroviral vectors for peripheral blood mononuclear cell and CD4⁺ T-lymphocytes trasduction encoding an anti-HIV gene and a cell surface molecule.

7. Vectors according to claim 6 in which the DNA for a cell surface molecule is the DNA encoding for the Low Affinity Nerve Growth Factor Receptor, devoid of the intracellular domain.