WO2000071578A2

WO2000071578A2 - New polypeptides and their use for the rescue of fusion defective virus or retrovirus glycoproteins

Info

Publication number: WO2000071578A2
Application number: PCT/EP2000/004534
Authority: WO
Inventors: François-Loïc Cosset; Dimitri Lavillette
Original assignee: Centre National de la Recherche Scientifique CNRS
Current assignee: Centre National de la Recherche Scientifique CNRS
Priority date: 1999-05-20
Filing date: 2000-05-19
Publication date: 2000-11-30
Anticipated expiration: 2001-11-20
Also published as: WO2000071578A3; WO2000071578A9

Abstract

The present invention relates to the use of at least one activating polypeptide comprising an amino acid sequence corresponding for instance to: the amino acid sequence of the surface subunit (SU) of an envelope glycoprotein of a first virus, or of a first retrovirus; for the rescue of the fusion property of a fusion defective envelope glycoprotein, which has the property of attachment to target cells and is inactive with respect to the fusion with said target cells, said fusion defective envelope glycoprotein belonging to a second virus or a second retrovirus of the same type as or of a different type from the above designated 'first virus or first retrovirus'.

Description

NEW POLYPEPTIDES AND THEIR USE FOR THE RESCUE OF FUSION DEFECTIVE VIRUS OR RETROVIRUS GLYCOPROTEINS

The invention relates to new polypeptides, and their use for the rescue of fusion defective virus or retrovirus glycoproteins.

Retroviruses enter cells following their attachment to specific cell surface receptors. This function is mediated by the envelope glycoproteins expressed as trimeric complexes on the viral particles (Hunter, 1997). Interaction with the receptor is thought to cause a conformational change of the glycoprotein structure necessary to expose a "fusion peptide" , buried within the native envelope complex and involved in the actual membrane fusion process (Gerlier, 1998). On the basis of both genetic (Battini et al. , 1995; Battini et al. , 1992; Morgan et al., 1993; Ott and Rein, 1992) and structural (Fass et al. , 1997; Fass et al., 1996) evidence, the attachment and fusion functions of murine leukemia virus (MLV) envelopes have been separated on the two subunits of the envelope monomer. Thus it is generally considered that the amino-terminal half of the MLV SU (surface) subunit is responsible for the binding to the receptor whereas the ectodomain of the TM

(transmembrane) subunit contains the "fusion machinery" composed of an amino-terminal fusion peptide associated to a coiled-coil structure that rearrange upon receptor interaction.

How retrovirus envelope glycoproteins convert binding to the receptor to a signal which activates the fusion machinery is currently unknown. Ample evidence from the literature indicate that functional inter-relations between different domains of the envelope complex are essential to promote fusion activation (Lavillette et al., 1998; Rein et al. , 1998; Zhao et al. , 1997; Zhao et al. , 1998).

Retroviruses can be involved in two different types of membrane fusion : - a fusion between viral and cell membranes triggered by the interaction of the virion envelope glycoproteins with specific cell surface molecules, which are the viral receptors (virus-cell fusion), with said fusion being responsible for retroviral infection, - a fusion between two cell membranes triggered by the interaction of the envelope glycoproteins expressed at the surface of a cell and the viral receptors expressed on a neighbouring cell (cell-cell fusion), the visible outcome of this latter being the formation of cell syncytia containing up to 100 nuclei (also known as polykaryocytes or multinucleated giant cells); syncytium-formation results in the death of the cells which make up the syncytium.

Furthermore, cleavage of the R peptide (inhibitory peptide on the cytoplasmic tail of the TM subunit of the envelope glycoprotein) is required to activate fully the fusogenic potential of these envelope glycoproteins and mutants lacking the R peptide show greatly enhanced activity in cell fusion assays (Rein et al, J. Virol., 1994, 68, 1773-1781; Ragheb

& Anderson, J. Virol., 1994, 68, 3220-3231; Brody et al, J. Virol., 1994, 68, 4620-4627).

Furthermore, it is known that a vector directing the expression on a eukaryotic cell surface of a syncytium-inducing polypeptide can be used in gene therapy of malignant diseases, as a result of selective elimination of unwanted cells, through syncytia formation (WO 98/40492).

The fusogenicity of envelope glycoproteins has been enhanced as described in Lavillette et al. (1998) and internal application WO 99/36561. The proline-rich region (PRR) of amphotropic MLV envelope glycoprotein was shown to be an essential determinant of membrane fusion activation and specific PRR-mutated envelope glycoproteins were obtained and were characterised by an increased cell-cell or virus-cell fusogenicity.

Studies on Mo-MLV envelope protein GP70 have shown that the first eight amino acids of the mature gp70 protein are essential for fusion and virus entry. Within this region, a particularly important residue is H8. This residue belongs to the PHQ motif which is conserved among all MLVs and some other retroviruses, such as a gibbon ape leukemia virus SEATO and feline leukemia virus types A, B, and C. The amphotropic counterpart of Del2-8, which has the first six amino acids (Met-Ala-Glu-Ser-Pro-His) deleted has been constructed. Vectors bearing the deletion mutant envelope protein could not transduce NIH 3T3 or HeLa cells (Bae et al. , Journal of Virology, March 1997, p. 2092-2099).

As the fusion mechanisms induced by interaction of retrovirus envelope glycoproteins and receptors of cell membrane are partially unknown, the regulation of the fusogenicity of retroviral or viral envelope glycoproteins was up to day not possible.

One of the aims of the invention is to enable fusion control of envelope glycoproteins with target cells.

It is an aim of the invention to provide means for the rescue of the fusion property of fusion defective viral or retroviral envelope glycoproteins. It is another aim of the mvention to provide a process for restoring the fusogenic property of retroviral or viral envelope glycoproteins, the fusogenic property of which has been inactivated and for controlling their activation by external means.

It is another aim of the invention to provide a process in which envelope glycoproteins are in a first stage inactivated as to their fusion property and then in a second stage activated in a controlled and regulated way, by means of activating polypeptides, which possibly present targeting capacities, and which are able to restore efficiently the fusogenic and cytotoxic properties of said envelope glycoproteins.

It is another aim of the invention to provide polypeptides liable to activate the cell- cell fusogenicity or the virus-cell fusogenicity.

It is another aim of the invention to provide polypeptides liable to activate the virus- cell fusogenicity and which thus can be used in strategies involving retroviral vectors comprising envelope glycoproteins, the fusion property of which can be activated.

It is another aim of the invention to provide fusogenic envelope glycoproteins presenting cytotoxic properties for use in the treatment of malignant diseases, for which the fusogenic property could be controlled by means of activating polypeptides.

It is another aim of the invention to provide cytotoxic fusogenic envelope glycoproteins having cellular specificity and for which the fusogenic property can be controlled by means of activating polypeptides. All these aspects are achieved through the present invention, which in one of its broad embodiments is the use of at least one activating polypeptide comprising an amino acid sequence corresponding to :

- the amino acid sequence of the surface subunit (SU) of an envelope glycoprotein of a first virus, or of a first retrovirus, or - an amino acid sequence derived from said surface subunit, or

- a fragment of an amino acid sequence of said surface subunit, encompassing the receptor binding domain (RBD) and the amino teπ inal part of the surface subunit (SU), for the rescue of the fusion property of a fusion defective envelope glycoprotein, which has the property of attachment to target cells and is inactive with respect to the fusion with said target cells, said fusion defective envelope glycoprotein belonging to a second virus or a second retrovirus of the same type as or of a different type from the above designated "first virus or first retrovirus" . Thus the invention lies in the possibility of desactivating the inactivation of the fusogenic property of an envelope glycoprotein, said envelope glycoprotein having previously been made inactive with respect to fusion. This restoration of the fusion property of the envelope glycoprotein is unexpectedly achieved by polypeptides whose common structure is the presence of the receptor binding domain (RBD) and the amino terminal part of SU in the environment of cells expressing fusion defective envelope glycoproteins and of cells expressing retroviral or viral receptors and the interaction of which will lead to the formation of syncytia, or in the environment of retroviruses incorporating fusion defective envelope glycoproteins and of cells expressing retroviral or viral receptors and the interaction of which will lead to infection.

The expression "polypeptide comprising an amino acid sequence corresponding to" , means that the polypeptide has the same amino sequence as that of the surface subunit, or as that of an aminoacid sequence derived from the surface subunit or of a fragment and can be prepared by recombinant technique or by chemical synthesis. The expression "polypeptide comprising an amino acid sequence derived from said surface subunit" means that the polypeptide has an aminoacid sequence which is not strictly identical to that of the sequence of the surface subunits and may contain mutations such as deletion or substitution of aminoacids of the parental sequence or insertion of aminoacid sequence of other origins. The "amino terminal part of the surface subunit" is defined as the aminoacid sequence of the first aminoacids of a length of about 10 to about 15 aminoacids of the envelope surface subunit, not including the aminoacid sequence of the signal peptide.

A fusion defective envelope glycoprotein derives from a parental envelope glycoprotein in which the fusogenic property has been inactivated, but which still presents the property of recognizing (attachment) target cells.

The expression "inactive with respect to the fusion" means that in a test for the measure of the fusogenic activity, the fusion index is either nul or significantly reduced when the fusion assay is allowed to proceed for about 20 minutes.

The rescue of the fusion property can be measured according to one of the following tests :

- test of formation of syncytia : expressing vectors encoding the envelope glycoprotein to be tested are transfected into cells on day 0. On day 2, the transfected cells are overlaid by indicator cells which express the cell surface receptors corresponding to the tested envelope glycoprotein. Detection of syncytia (i.e. fusion of envelope-expressing cells with neighbouring indicator cells) is performed at day 3 or day 4 and fusion indexes are measured as previously described (Lavillette et al, 1998) by counting the number of syncytia and the number of nuclei outside and inside the syncytia.

- test of infection of target cells : retroviral vectors carrying a lac Z transgene are being generated with the tested envelope glycoproteins. Serial dilutions of the vectors are applied on target cells at day 0. Infected cells are stained at day 2 with X-Gal to reveal the number of cell colonies having integrated the lac Z-vectors and titers are determined according to standard procedures (Lavillette at al., 1998).

It is to be noted that the activating polypeptide corresponds to the amino acid sequence of the SU of an envelope glycoprotein of a first virus or retrovirus and that the fusion defective envelope glycoprotein belongs to a second virus or retrovirus of the same type or of a type different from the first virus or retrovirus. This means that there are in fact two possibilities :

- either the first and second viruses are of the same type, which means that the activating polypeptide and the fusion defective envelope glycoprotein are homologous,

- or the first and second viruses are of a different type, which means that the activating polypeptide and the fusion defective envelope glycoprotein are heterologous. According to an advantageous embodiment the fusion defective envelope glycoprotein involved are those derived from retroviruses.

Retroviral envelope glycoproteins allow receptor binding of the viral particles onto which they have been incorporated during virion assembly. This preliminary attachment on the viral receptor activates a second function of the retroviral envelope glycoproteins which induces the fusion between the viral and the target cell membranes. While for type C mammalian retroviruses the determinants of receptor binding (RBD, receptor binding domain), are located at the amino-terminal end of the SU envelope subunit, the molecular determinants that are responsible of membrane fusion are located in the TM subunit of the glycoprotein. The molecular determinants or events which are involved in the conversion of binding to the receptor into a signal which activates the TM fusion deteπninants are not precisely known although by mutagenesis analysis, they are believed to be present in all the SU subunit, in particular in the N-terminal end of the RBD itself (Bae et al. , 1997; Lavillette et al. , 2000), in the proline-rich region (Lavillette et al. , 1998) and in the carboxy-terminal domain of the SU (Nussbaum et al., 1993; Pinter et al. , 1997). Thus the control of the activation of the fusion machinery harbored by the TM subunit might be achieved by introducing specific mutations that affect the fusion-activation determinant of the SU. This invention describes several mutations in the SU which can inhibit or partially inhibit the SU determinants which control activation of the TM fusion-machinery. It is important to understand that such mutations do not impair the fusogenic potential of the mutant envelope glycoproteins, but only impair the molecular determinants that control the activation of the fusion machinery. The latter one is still intact, despite the mutations introduced in the SU, but is not activated upon receptor binding. In addition the invention describes the use of soluble polypeptides that can alleviate the effect of these mutations and hence couple the fusion machinery to a device that is provided exogenously in substitution to the normal fusion control determinants which are disrupted in the mutant envelope glycoproteins. Thus the addition of the "activating" polypeptides can restore or rescue the fusogenicity of the mutant envelope glycoproteins.

Several mutations or modifications of the envelope glycoprotein can be used to reversibly inhibit or reduce their fusogenicity.

A first method is the introduction of the delH mutation that removes an histidine from the PHQ conserved motif located at the amino-terminal end of the SU envelope subunit of type C mammalian retroviruses such as MLV-A, MoMLV and GALV (mutations H5del, H8del or H9del, respectively). Such a mutation induces a drastic effect in the capacity of the receptor binding domain (RBD) of these envelope glycoproteins to triggers further events required to promote membrane fusion.

A second method consists in the replacement of the SU C-terminal domain (C domain) with an heterologous C domain derived from the envelope glycoprotein of another type C mammalian retrovirus. For example; the chimeric CMO amphotropic glycoprotein (hereafter defined) can be constructed by combining the amphotropic MLV RBD and PRR (proline-rich region) with the ecotropic MoMLV C-terminal domain (example 4, Fig. 29 A and 29B). For such chimeras, there is a low compatibility between their RBD (amphotropic origin) and C domain (ecotropic origin), which significantly impairs the kinetic of membrane fusion. A third method consists in the replacement of the RBD by a ligand such as a cytokine or a single chain antibody targeted to a cell surface specific receptor. In contrast to the RBD of the SU of a type C mammalian retrovirus, such ligands do not contain the determinants that allow further activation of the Env fusogenicity. Such chimeric envelope glycoproteins therefore resembles a delH envelope glycoprotein which would carry an RBD that is targeted to a specific receptor.

This invention relates to a method which can alleviate the inhibitory effect of these different mutations and thus restore the fusogenic properties of the mutant envelope glycoproteins. This method uses preparations of polypeptides which are derived from the RBD of different type C mammalian retrovirus envelope glycoproteins. For example, the addition of an RBD which is compatible with the C domain of a fusion-defective envelope glycoprotein will fully restore its fusogenic properties and will activate the kinetic of membrane fusion. Similarly such a peptide will also restore the fusogenicity of envelope glycoproteins that harbor a delH mutation or, alternatively, which carry a cell-type specific ligand in place of the RBD.

This method can be applied to the activation of infectivity of retroviral vectors whose envelope glycoproteins carry one of these fusion-defective mutations. Depending on the presence of a ligand in the fusion-deficient envelope glycoprotein or, alternatively, in the activating polypeptide, control of infection to specific target cell types will therefore be obtained. The invention can also be applied to the control of the hyper-fusogenic activity of retroviral envelope glycoproteins that can induce cell-cell fusion and elimination of unwanted cells by syncytia formation. Indeed while most retroviral envelope glycoproteins are not truly fusogenic when expressed at the surface of the cells that produce the retroviral particles, they acquire their fusion competency during or after assembly and release of the viral particles. Yet there is considerable interest to convert the retroviral envelope glycoproteins into hyperfusogenic glycoproteins that can fuse cells in the close vicinity of the cells into which they are expressed. In addition to the introduction of the above- mentioned mutations that can control activation of their fusogenicity, the fusogenicity of retroviral envelope glycoproteins can be stimulated by several ways to obtain the best effect upon activation with an exogenous activating polypeptide. Thus, additional mutations will be introduced into the retroviral envelope glycoprotein, such as the truncation of the cytoplasmic tail (Rless mutation) and/or the modification of their proline-rich regions. According to an advantageous embodiment, the activating polypeptide used in the invention recognizes substantially the same receptors of the target cells as the fusion defective envelope glycoprotein.

According to another advantageous embodiment, the activating polypeptide used in the invention recognizes receptors of the target cells which are different from the ones recognized by the fusion defective envelope glycoprotein.

It is thus quite unexpected that the activating polypeptides can restore the fusogenicity of fusion defective envelope glycoproteins by interacting with retroviral receptors which are expressed at the surface of the same target cells, and which are different from the receptors recognized by the fusion defective envelope glycoprotein.

According to another advantageous embodiment of the invention, the activating polypeptide and/or the fusion defective envelope glycoprotein is (are) linked to a ligand, which is specific with respect to some receptors of the target cells, the ligand being preferably linked to the activating polypeptide. The addition of specific ligands on the activating polypeptides and/or on the fusion defective envelope glycoprotein enables to control the fusion activity of said fusion defective envelope glycoprotein and to increase the specificity with respect to target cells. Therefore, the design of a specific system to eliminate unwanted cells, may consist in a fusion defective envelope glycoprotein associated to an activating polypeptide that is linked to a ligand that can specifically recognize a receptor expressed on the target cells, whereas the activating polypeptide recognizes the same receptor or a different receptor expressed on the target cell.

The link between the activating polypeptide and the ligand is preferably a covalent bond, whereby both the activating polypeptide and the ligand have been genetically fused and encoded by an expression vector as a single protein chain, and whereby the ligand is fused either to the amino-terminus or to the carboxy terminus of the activating polypeptide.

According to another advantageous embodiment, the ligand is chosen among molecules liable to recognize receptors of target cells, for instance receptors of tumoral cells, such as EGF, FGF, VEGF, or liable to recognize epitope of proteins expressed by the target cells, such as recombinant monoclonal antibody.

According to another advantageous embodiment, the activating polypeptide is under the form of a product as such, or is expressed by a host, such as a virus or a cell, or is expressed, in particular by the target cells, transformed by nucleotide sequences coding for said activating polypeptide.

In the use of the invention, one resorts either directly to chemical substances (which is the meaning of activating polypeptide under the form of a product as such), or to host (virus or cell) expressing said activating polypeptide.

In the following of the text, the expression "soluble activating polypeptides" will be sometimes used, meaning that said polypeptides are not integrated in the envelope proteins of a virus.

The capacity of the activating polypeptides to rescue the infectivity of fusion defective envelope glycoproteins is similar whether the target cells i) constitutively express and secrete the activating polypeptides, ii) are co-incubated with both fusion defective envelope glycoprotein and said activating polypeptides iii) are pre-incubated with said activating polypeptides before adding the fusion defective envelope glycoprotein, or iv) are pre-incubated with the fusion defective envelope glycoprotein before adding the activating polypeptides.

In an advantageous use of the invention the fusion defective envelope glycoprotein is expressed by a host, such as a virus incorporating in its envelope said fusion defective envelope glycoprotein, or such as a cell, transformed by nucleotide sequences coding for said fusion defective envelope glycoprotein. According to the invention, the activating polypeptide advantageously corresponds to a fragment of about 200 to about 250 amino acids of said surface subunit comprising the receptor binding domain (RBD) and comprising the first amino acids of the amino terminal part of the surface subunit, the number of said first aminoacids varying from about 10 to about 15, in particular comprising the RBD and the PHQ peptide, in particular the fragments corresponding to amino acid sequences derived from type C mammalian retroviruses such as: - Murine leukemia virus such as :

. amphotropic . ecotropic . xenotropic

. polytropic . 10 Al MLV - or GALV type retroviruses and related retrovirus such as SSAV, FELV-A, FELV-B, FELV-C in particular wherein the activating polypeptide corresponds to one of the following amino acid sequences :

A-RBD

E-RBD

GALV-RBD

34-1-A-RBD 34-1-E-RBD

34-1 -GALV-RBD

EGF-A-RBD

EGF-E-RBD

EGF-GALV-RBD - A-RBD corresponds to the activating polypeptide comprising the RBD domain and the

PHQV motif of the amphotropic MLV surface subunit.

- E-RBD corresponds to the activating polypeptide comprising the RBD domain and the PHQV motif of the ecotropic MLV surface subunit.

- GALV-RBD corresponds to the activating polypeptide comprising the RBD domain and the PHQP motif of the GALV surface subunit.

- 34.1-A-RBD corresponds to the activating polypeptide A-RBD above defined, linked to a ligand which is a single chain antibody directed against MHCI.

- 34.1 -E-RBD corresponds to the activating polypeptide E-RBD above defined, linked to a ligand which is a single chain antibody directed against MHCI. - 34.1 GALV-RBD corresponds to the activating polypeptide GALV-RBD above defined, linked to a ligand which is a single chain antibody directed against MHCI.

- EGF-A-RBD corresponds to the activating polypeptide A-RBD above defined, linked to a ligand which is the epidermal growth factor.

- EFG-E-RBD corresponds to the activating polypeptide E-RBD above defined, linked to a ligand which is the epidermal growth factor.

- EGF-GALV-RBD corresponds to the activating polypeptide GALV-RBD above defined, linked to a ligand which is the epidermal growth factor. According to the invention, the fusion defective envelope glycoprotein advantageously corresponds to the amino acid sequence of a mutated form of an envelope glycoprotein of a virus or a retrovirus which, in its non mutated form, presents attachment and fusion properties with respect to target cells, said fusion defective envelope glycoprotein comprising one or several mutations in the amino terminal part of its surface subunit, particularly in the first amino acids of the amino terminal part of the surface subunit, the number of said first aminoacids varying from about 10 to about 15, said mutations inactivating the fusion property without substantially affecting the attachment property, in particular comprises one or several mutations within the PHQ amino sequence, wherein in particular the fusion defective envelope glycoprotein is derived from type C mammalian retroviruses such as:

- Murine leukemia virus such as :

. amphotropic . ecotropic . xenotropic

. polytropic . 10 Al MLV

- or GALV type retroviruses and related retrovirus such as SSAV, FELV-A, FELV-B, FELV-C, in particular wherein the fusion defective envelope glycoprotein corresponds to one of the following amino acid sequences :

- MO del H

- AdelH

- PROMOdelH - GALVdelH

- C2delH

- MO del H R less

- AdelHRless

- PROMOdelHRless - GALVdelHRless

- C2delHRless - C MO - TM MO

- C TM MO

- C MOdelH

- TM MOdelH - C TM MOdelh - 34.1-A

* MO del H corresponds to the ecotropic MLV envelope glycoprotein, in which the histidine residue of the PHQV motif is deleted (i.e. the 8th residue of the SU subunit is removed).

* AdelH corresponds to the amphotropic MLV envelope glycoprotein, in which the histidine residue of the PHQV motif is deleted (i.e. the 5th residue of the SU subunit is removed).

* PROMOdelH corresponds to the amphotropic MLV envelope glycoprotein, in which the proline rich region has been replaced by the proline rich region of the ecotropic MLV envelope and in which the histidine residue of the PHQV motif is deleted.

* GALVdelH corresponds to the GALV envelope glycoprotein, (gibbon ape leukemia virus envelope glycoprotein), in which the histidine residue of the PHQP motif is deleted (i.e. the 9th residue of the SU subunit is removed).

* C2delH corresponds to the amphotropic MLV envelope glycoprotein, in which the proline rich region has been modified by a small deletion at its carboxy terminus and in which the histidine residue of the PHQV motif is deleted (i.e. the 5th residue of the SU subunit is removed) .

* MO del H R less corresponds to the ecotropic MLV envelope glycoprotein, in which the histidine residue of the PHQV motif is deleted and in which the R peptide of the TM subunit has been deleted.

* AdelHRless corresponds to the amphotropic MLV envelope glycoprotein in which the histidine residue of the PHQV motif is deleted and in which the R peptide of the TM subunit has been deleted.

* PROMOdelHRless corresponds to the amphotropic MLV envelope glycoprotein, in which the proline rich region has been replaced by the proline rich region of the ecotropic MLV envelope, in which the histidine residue of the PHQV motif is deleted and in which the R peptide of the TM subunit has been deleted. * GALVdelHRless corresponds to the GALV envelope glycoprotein, in which the histidine residue of the PHQP motif is deleted and in which the R peptide of the TM subunit has been deleted.

* C2delHRless corresponds to the amphotropic MLV envelope glycoprotein, in which the proline rich region has been modified by a small deletion at its carboxy terminus and in which the histidine residue of the PHQV motif is deleted (i.e. the 5th residue of the SU subunit is removed) and in which the R peptide of the TM subunit has been deleted.

* CMO envelope corresponds to the amphotropic MLV envelope glycoprotein, in which the SU carboxy-terminal domain, C domain, has been replaced by the C domain derived from the ecotropic MoMLV envelope glycoprotein.

* CMOdelH envelope corresponds to the amphotropic MLV envelope glycoprotein, in which the SU carboxy-terminal domain, C domain, has been replaced by the C domain derived from the ecotropic MoMLV envelope glycoprotein and in which the histidine residue of the PHQV motif is deleted (i.e. the 5th residue of the SU subunit is removed). * TMMO envelope corresponds to the amphotropic MLV envelope glycoprotein, in which the ectodomain of the TM subunit has been replaced by the TM ectodomain derived from the ecotropic MoMLV envelope glycoprotein.

* TMMOdelH envelope corresponds to the amphotropic MLV envelope glycoprotein, in which the ectodomain of the TM subunit has been replaced by the TM ectodomain derived from the ecotropic MoMLV envelope glycoprotein and in which the histidine residue of the PHQV motif is deleted (i.e. the 5th residue of the SU subunit is removed).

* CTMMO envelope corresponds to the amphotropic MLV envelope glycoprotein, in which both the SU carboxy-terminal domain, C domain, and the ectodomain of the TM subunit have been replaced by the C domain and TM ectodomain derived from the ecotropic MoMLV envelope glycoprotein.

* CTMMOdelH envelope corresponds to the amphotropic MLV envelope glycoprotein, in which both the SU carboxy-terminal domain, C domain, and the ectodomain of the TM subunit have been replaced by the C domain and TM ectodomain derived from the ecotropic MoMLV envelope glycoprotein and in which the histidine residue of the PHQV motif is deleted (i.e. the 5th residue of the SU subunit is removed). * 34.1-A envelope corresponds to the amphotropic MLV envelope glycoprotein, in which the receptor binding domain has been removed and replaced by an single chain antibody against human class I MHC molecules.

The invention also relates to the use of at least one activating polypeptide and of at least one fusion defective envelope glycoprotein for the preparation of a drug useful for the treatment of cancer and infectious diseases.

The invention also relates to products containing an activating polypeptide and a fusion defective envelope glycoprotein as defined above, as a combined preparation for simultaneous, separate, or sequential use for the rescue of the fusion defective property of said fusion defective envelope glycoprotein.

The invention also relates to products containing an activating polypeptide and a fusion defective envelope glycoprotein as defined above, as a combined preparation for simultaneous, separate or sequential use for the treatment of cancer pathologies. The invention also relates, as new products, to activating polypeptides corresponding to a fragment of about 200 to about 250 amino acids of said surface subunit comprising the RBD and the PHQV peptide, in particular the fragments corresponding to amino acid sequences derived from type C mammalian retroviruses such as: - Murine leukemia virus such as : . amphotropic

. ecotropic . xenotropic . polytropic . 10 Al MLV - or GALV type retroviruses and related retrovirus such as SSAV, FELV-B in particular wherein the activating polypeptide corresponds to one of the following amino acid sequences : A-RBD E-RBD GALV-RBD 34-1-A-RBD 34-1-E-RBD 34-1 -GALV-RBD EGF-A-RBD EGF-E-RBD EGF-GALV-RBD The invention also relates, as new products, to fusion defective envelope glycoprotein to the amino sequence of an envelope glycoprotein of a virus or a retrovirus which in its native state presents attachment and fusion properties with respect to target cells, and comprises one or several mutations in the .imino terminal part of its surface subunit, inactivating the fusion property without substantially affecting the attachment property, in particular comprises one or several mutations within the PHQ amino sequence of the amino terminal part of its surface subunit, wherein in particular the fusion defective envelope glycoprotein is derived from type C mammalian retroviruses such as:

- Murine leukemia virus such as :

. amphotropic . ecotropic

. xenotropic . polytropic . 10 Al MLV

- or GALV type retroviruses and related retrovirus such as SSAV, FELV-A, FELV-B or FELV-C in particular wherein the fusion defective envelope glycoprotein corresponds to one of the following amino acid sequences :

- AdelH

- PROMOdelH - GALVdelH

- C2delH

- AdelHRless

- PROMOdelHRless

- GALVdelHRless - C2delHRless

- C MO - TM MO - C TM MO

- C MOdelH

- TM MOdelH

- C TM MOdelh - 34.1-A with the proviso that they are different from the ecotropic MLV sequence, wherein the amino acids in position 2 to 8 are either substituted or deleted.

The invention also relates to the use of at least one activating polypeptide to the rescue of the fusion property of a fusion defective envelope glycoprotein wherein the fusion defective envelope glycoprotein corresponds to the amino acid sequence of a mutated form of an envelope glycoprotein of a virus or a retrovirus which, in its non mutated form, presents attachment and fusion properties with respect to target cells, said fusion defective envelope glycoprotein being advantagously derived from type C mammalian retroviruses such as: - Murine leukemia virus such as :

. amphotropic . ecotropic . xenotropic . polytropic . 10 Al MLV

- or GALV type retroviruses and related retrovirus such as SSAV, FELV-A, FELV- B, FELV-C and comprising :

* one or several mutations in the amino terminal part of its surface subunit, particulary in the first amino acids of the amino terminal part of the surface subunit, the number of said first aminoacids varying from about 10 to about 15, said mutations inactivating the fusion property without substantially affecting the attachment property, in particular comprises one or several mutations within the PHQ ammo-terminal sequence, wherein in particular the fusion defective envelope glycoprotein corresponds to one of the following amino acid sequences :

- MO R less del H

- MO del H - A R less del H

- AdelH

- PROMO R less del H

- PROMOdelH - GALV R less del H

- GALVdelH

- C2 R less del H

- C2delH

- C MOdelH - TM MOdelH

- C TM MOdelh

* or the replacement of the amino-terminal domain of its surface subunit (RBD) by a ligand such as a growth factor, or a cytokine or a single chain antibody, the extent of said mutations inactivating the fusion property and the attachement property to the normal viral receptor, in particular wherein the fusion defective envelope glycoprotein corresponds to the following amino-acid sequence :

- 34.1-A

* or the replacement of its SU carboxy-terminal domain and / or its TM ectodomain, and preferably its SU carboxy-terminal domain, by the corresponding SU carboxy-terminal domain and / or the TM ectodomain of another "second" glycoprotein derived from type C mammalian retroviruses, with the proviso that said fusion defective glycoprotein and said second glycoprotein are not derived from the same type C mammalian retroviruses, in particular wherein the fusion defective envelope glycoprotein corresponds to one of the following amino-acid sequences : - CMO - TM MO

- C TM MO FIGURE LEGENDS

Fig 1A, IB and 1C represent titres of AdelH retroviruses in the presence of soluble envelope fragments of the invention. Retroviral vectors carrying VSV-G or/and wild-type amphotropic, A, envelope glycoproteins were used as controls. Titres expressed as lacZ infectious units (i.u.) per ml of viral supernatants. In figures 1A, IB and 1C, the titres are on the y-axis.

Fig 1A. Different target cell types (XC, NIH3T3, CHO-PIT-2, TE 671, from left to right) were incubated with both lacZ retroviruses and conditioned media containing, or not (control), the indicated polypeptides during the 3 hrs of infection. The black columns correspond to the control, the grey columns correspond to A-RBD and the hatched columns correspond to SU polypeptide.

Fig IB. XC target cells were incubated with dilutions of A-RBD (x axis) and infected with LacZ retroviruses carrying the AdelH envelope glycoproteins (curve with black circles), A envelope glycoproteins (curve with black squares) or VSV-G envelope glycoproteins (curve with hollow squares).

Fig 1C. Retroviral vectors carrying A envelope glycoproteins (curves with black squares) or AdelH envelope glycoproteins (curves with black circles) were incubated with XC target cells for 1 hr at 4°C to allow virion binding while preventing cell entry. After PBS washing to remove unbound retroviruses, cells were incubated at 37 °C with undiluted A-RBD- containing supernatants for the indicated times of dilution represented on the x axis, the titres being expressed on the y axis. A-RBD was then eliminated from the cell supernatant by washing the cells four times with 1 ml PBS (resulting in dilution of unbound A-RBD by more than 100,000 times). Cells were then grown in regular medium for 2 days before X- Gal staining.

Fig 2. represents the titers of A-RBD-mediated activation of AdelH retroviruses. A-

RBD (used undiluted) was bound to XC target cells at 37 °C for 30 min. Unbound A-RBD was removed and cells were washed two times with PBS and incubated at 37°C. At the indicated times, cells were further washed two times with PBS and incubated at 37 °C for 3 hrs with retroviruses carrying A envelope glycoproteins or AdelH envelope glycoproteins. Titers, determined 2 days later by X-Gal staining, are expressed as lacZ i.u./ml (y-axis), as a function of time before infection (x-axis). The curve with black circles corresponds to retrovirus carrying A envelope glycoproteins and the curve with black squares corresponds to retrovirus carrying AdelH envelope glycoproteins.

Fig 3. represents titers of AdelH retroviruses after ultrafiltration. Titers expressed on the y-axis (as lacZ i.uJml) of retroviruses carrying wild-type, A, or AdelH envelope glycoproteins before (1) or after incubation with A-RBD (2), and following A-RBD elution on two successive 700 KD-cut off ultrafiltration cartridges (3). After elution AdelH retroviruses were subjected to re-stimulation with fresh A-RBD (4).

Fig. 4 and 4B represent infection assays of AdelH and MOdelH retroviruses in the presence of RBD or RBDdelH polypeptides.

Fig. 4A. Retroviruses carrying A (upper left) or AdelH (upper right) envelope glycoproteins were mixed with A-RBD (hatched column) or A-RBDdelH (grey column) during infection of XC cells. Retroviruses carrying MO (lower left) or MOdelH (lower right) envelope glycoproteins were mixed with E-RBD (hatched column) or E-RBDdelH

(grey column) during infection of XC cells. Titers are expressed as lacZ i.uJml on the y- axis. In Figure 4A, the black column corresponds to control.

Fig. 4B. represents binding assays of A-RBD (solid lines) and A-RBDdelH fragments (broken lines) on Cearl3 (top), and binding assays of E-RBD (solid lines) and E-RBDdelH fragments (broken lines) on Cearl3 cells (bottom). The background of fluorescence (filled histograms) was provided by incubating the cells with supernatants devoid of envelope fragments.

Fig 5A and 5B represent the cross-activation of AdelH and MOdelH retroviruses by MLV RBDs.

Fig. 5A. Titers of retroviruses carrying AdelH or MOdelH envelope glycoproteins in the presence of the indicated RBDs. Titers are determined (y-axis) on XC target cells as lacZ i.uJml. On figure 5A, the black column corresponds to control, the hatched column corresponds to A-RBD and the grey column corresponds to E-RBD. Fig. 5B. Titers of AdelH retroviruses (y-axis) on CHO cells expressing, or not (control),

PiT-2 and/or mCAT-1 MLV receptors in the presence of the indicated RBDs. On figure 5B, the hatched column corresponds to E-RBD, the grey column corresponds to A-RBD and the black column corresponds to control.

Fig. 6 represents the A-RBD sequence

Fig. 7 represents the E-RBD sequence Fig. 8 represents the GALV-RBD sequence

Fig. 9 represents the EGF-A-RBD sequence

Fig. 10 represents the EGF-E-RBD sequence

Fig. 11 represents the EGF-GALV-RBD sequence

Fig. 12 represents the 34.1-A-RBD sequence Fig. 13 represents the 34.1-E-RBD sequence

Fig. 14 represents the 34.1-GALV-RBD sequence

Fig. 15 represents the AdelH sequence

Fig. 16 represents the PROMOdelH sequence

Fig. 17 represents the GALVdelH sequence Fig. 18 represents the C2delH sequence

Fig. 19 represents the AdelHRless sequence

Fig. 20 represents the PROMOdelHRless sequence

Fig. 21 represents the GALVdelHRless sequence

Fig. 22 represents the C2delHRless sequence Fig. 23 represents the C MO sequence

Fig. 24 represents the C MO delH sequence

Fig. 25 represents the TM MO sequence

Fig. 26 represents the TM MOdelH sequence

Fig. 27 represents the C TM MO sequence Fig. 28 represents the C TM MO delH sequence

Fig. 29A represents the schematic structure of chimeric amphotropic envelope glycoproteins in which the C domain (CMO chimera), the TM ectodomain (TMMO chimera) or both C and TM domains (CTMMO chimera) are replaced by the ecotropic counterpart derived from MoMLV envelope glycoprotein. The CMOdelH, TMMOdelH and CTMMOdelH chimeric envelope glycoproteins harbor the H5del mutation. Figure 29B represents the kinetic of infection of retroviral vectors carrying the wild-type amphotropic envelope glycoprotein (A) or the CTMMO amphotropic chimera on XC cells in the presence, or not, of soluble polypeptides encompassing the amphotropic RBD (A- RBD) or the ecotropic RBD (E-RBD), as indicated. The curve with points and black circles corresponds CTMMO on XC cells + E-RBD The curve with points and hollow circles corresponds to A on XC cells + E-RBD The curve with continuous line and hollow circles corresponds to A on XC cells The curve with dotted line and hollow circles corresponds to A on XC cells + A-RBD The curve with continuous line and black circles corresponds to CTMMO on XC cells The curve with dotted line and black circles corresponds to CTMMO on XC cells + A- RBD

The X axis corresponds to the time expressed in seconds. The Y axis corresponds to the number of infected cells. Fig. 30 represents the 34.1-A Sequence

Example 1:

MATERIALS AND METHODS

Cell lines. The TELCeBό cell line (Cosset et al., 1995b) has been derived from the TELac2 line after transfection and clonal selection of a Moloney murine leukemia virus (MoMLV)- based expression plasmid to produce Gag and Pol proteins. The TELac2 cells were originally derived from the TE671 human rhabdomyosarcoma cells (ATCC CRL8805) to express the nlsLacZ reporter retroviral vector (Takeuchi et al., 1994). Production of infectious retroviral particles by TELCeBό cells depends on newly introduced envelope expression vectors.

Cerd9 and Cearl3 cells (Kozak et al. , 1995) and CHO-PiT-2 cells (Rodrigues and Heard, 1999) were derived from CHO (Chinese hamster ovary) cells (ATCC CCL-61) and express ecotropic MLV receptors alone or both ecotropic and amphotropic receptors or amphotropic MLV receptors alone, respectively. XC-A-RBD cells were derived from XC rat sarcoma cells (ATCC CCL-165) after transfection with the pA-ST plasmid expressing the amino terminal receptor binding domain of the amphotropic envelope glycoprotein (Battini et al., 1996). TE671 and TELCeBό cells were grown in DMEM (Life-Technologies) supplemented with 10% fetal bovine serum. NIH-3T3 mouse fibroblasts were grown in DMEM supplemented with 10% new-born calf serum. Cerd9, Cearl3, CHO-PiT-2 and CHO cells were grown in DMEM supplemented with 10% fetal bovine serum and with proline (Life-Technologies).

Construction of envelope expression vectors. Plasmids FBASALF and FBMOSALF carrying a phleomycin-resistance gene and encoding the MLV-4070A amphotropic (noted as A) and MoMLV ecotropic (noted as MO) envelope glycoproteins, respectively, have been described elsewhere (Cosset et al. , 1995a) and were used as backbones for construction and expression of envelope mutants. All constructs were generated by PCR-mediated and ohgonucleotide site-directed mutagenesis (details and sequences available upon request) . The FBASALF plasmid was modified to produce a cell entry-defective form of the amphotropic glycoprotein, designated AdelH envelope, by deleting the 36th codon of the 4070A env gene (Ott et al. , 1990). The resulting mutant envelope glycoprotein, in which the 5th residue of the SU envelope subunit was removed, was named AdelH. The expression plasmid encoding the fusion-defective MOdelH envelope glycoproteins, harboring a deletion of the 8th residue of the SU envelope subunit corresponding to the 41th codon of the MoMLV env gene (Shinnick et al., 1981), was derived from FBMOSALF. Plasmids encoding soluble receptor binding domains (RBD) were derived from FBASALF and FBMOSALF expression vectors. The boundaries of either amphotropic (A-RBD) and ecotropic (E-RBD) RBDs, defined as A32244 and A34269, were fused in frame with the carboxy-terminal ends of either envelope glycoproteins by fusion to aminoacids R623 or R634, respectively. Residues are numbered starting from the initiation methionine deduced from the aminoacid sequences of the 4070A amphotropic MLV (Ott et al. , 1990) or the Moloney MLV (Shinnick et al. , 1981). Expression vectors encoding either A-RBDdelH or E-RBDdelH were generated similarly by using the plasmids expressing the AdelH or MOdelH envelope glycoproteins. In plasmids encoding A-RBD, E-RBD, A-RBDdelH or E- RBDdelH polypeptides, the 11 aminoacid-long VSV tag (YTDIEMNRLGK) (Kreis and Lodish, 1986) was inserted after either RBDs.

Transfections, production of RBDs and infection assays. Envelope glycoproteins expression plasmids were transfected into TELCeBό as reported elsewhere (Cosset et al. , 1995a). RBD expression vectors were transfected by calcium phosphate precipitation in NIH-3T3, XC or TE671 cells. Transfected cells were selected with phleomycin (50 μg/ml) and phleomycin-resistant colonies were pooled.

RBD-containing supernatants were collected after an overnight production from confluent RBD-transfected cells, filtrated trough 0.45 micron pore-sized membranes and stored at 4°C. Virus-containing supernatants were collected after an overnight production from freshly confluent env-transfected TELCeBό cells in regular medium.

The supernatant were filtered trough 0.45 micron pore-sized membranes and diluted in DMEM for titration assays. Target cells were seeded in 24 multi-well plates at a density of 5xl0⁴ cells per well and incubated overnight at 37°C. Unless otherwise indicated in figure legends, 200 μl of conditioned cell culture media containing the RBDs were added to the cells after removing their supernatants. Then 200 μl of viral supernatant dilutions containing 5 μg/ml polybrene were added and cells were incubated for 3 hrs at 37 °C. Cell supernatants were then removed and cells were incubated in regular medium for 48 hrs. X-Gal staining and viral titer determination were performed as previously described (Cosset et al. , 1995a) and expressed as lacZ i.u. (infectious units)/ml.

Antibodies. Anti-SU: a rat monoclonal antibody 83A25 (Evans et al. , 1990) cell culture supernatant against MLV SU used undiluted for FACS analysis. Anti- VSV tag: a purified mouse monoclonal antibody P5D4 (SIGMA-Aldrich) used diluted 1/100 for FACS analysis.

Binding assays and FACS analysis. For binding assays, target cells were washed in PBS and detached by a 10 min incubation at 37°C with versene 0.02% in PBS. Cells were washed in PBA (PBS with 2% FCS and 0.1 % sodium azide). 5xl0⁵ cells were incubated with 1 ml of conditioned supernatants containing the VSV tagged RBDs for 45 min at 37 °C. Cells were then washed with PBA and were incubated with the P5D4 antibody, for 45 min at 4°C. Cells were washed twice with PBA and incubated with anti-mouse FITC-conjugated antibodies (DAKO, U.K.), respectively. 5 min before the two final washes in PBA, cells were counter-stained with 20 μg/ml propidium iodide. Fluorescence of living cells was analysed with a fluorescent-activated cell sorter (FACSCalibur, Beckton Dickinson). RESULTS

H5del phenotype can be efficiently compensated by soluble A-RBD. Viral particles were generated with either wild-type amphotropic A or AdelH envelope glycoproteins and used to infect target cells in the presence or in the absence of soluble SU or soluble A-RBD, an envelope fragment encompassing the amphotropic receptor binding domain. When SU or A- RBD were provided during infection the infectivity of retroviruses carrying wild-type amphotropic envelope glycoproteins was decreased by approximately 5-100 times (Fig. 1A), most likely owing to the partial blocking of PiT-2 receptors on the target cell surface. In striking contrast, while in the absence of envelope fragments retroviruses generated with AdelH envelopes were very poorly infectious, with titers in the range of lO'-lO² lacZ i.u. (infectious units)/ml, the presence of SU or of A-RBD in the medium of infected cells could strongly stimulate the infectivity of the former retroviruses by up to 30,000 fold (Fig. 1A). Stimulation of AdelH infection with either soluble envelope fragments was detected on a wide range of target cell types including rat XC cells, 3T3 murine fibroblasts, TE671 human cells and PiT-2-transfected CHO hamster cells, though with different efficacies (Fig. 1A). Significant differences were detected when soluble SU or A-RBD were used to activate the AdelH retroviruses. Comparatively to soluble SU, A-RBD was 100 to 1,000 fold more potent to activate AdelH retroviruses (Fig. 1A). These results indicated that the determinants of activation were located in the first half of the SU protein. Therefore to facilitate the characterization of this activation pathway, the subsequent experiments were performed by using the soluble RBD. A-RBD could fully activate AdelH retroviruses when diluted 10 fold and retained 90% of activity at a 1:100 dilution, thus suggesting that it was effective at very low doses (Fig. IB). In contrast the infectivity of control retroviruses pseudotyped with either wild-type amphotropic envelopes or with VSV-G glycoproteins was weakly increased or unchanged, respectively, when A-RBD was diluted (Fig. IB). Additionally the origin of the cell types used to produce the envelope fragments did not influence the stimulation of infectivity since conditioned media harvested from 3T3, TE671 or XC cells that were engineered to express the envelope fragments could similarly activate the infectivity of AdelH retroviruses (data not shown). Finally activation of AdelH retroviruses was found to be the result of a specific interaction of A-RBD with the PiT-2 amphotropic receptor. Indeed while A-RBD could efficiently rescue the infectivity of AdelH retroviruses on PiT-2-transfected CHO cells, no infection could be detected when using parental CHO cells as target cells (see below and Fig. 5A). Altogether these data demonstrated that activation of AdelH retroviruses by A-RBD was specific. The capacity of the soluble envelope fragments to rescue the infectivity of AdelH retroviruses was similar whether the target cells i) constitutively expressed and secreted the envelope fragments, ii) were co-incubated with both AdelH retroviruses and A-RBD, iii) were pre-incubated with A-RBD prior to infection, or iv) were pre-incubated with the AdelH retroviruses before adding A-RBD (Table 1). Using this latter experimental condition, the kinetic of activation of infection was found very rapid. Indeed a brief incubation of target cells with A-RBD, by no more than 30 sec, was found sufficient to fully stimulate the infectivity of pre-bound AdelH retroviruses (Fig. 1C).

A-RBD stimulates the target cell membrane rather than the AdelH envelope. In theory, it is possible that the envelope fragments might rescue the infectivity of AdelH retroviruses either by interacting with the fusion-defective viral particles or by activating the target cell membrane upon receptor binding. Indeed within the envelope complex, the SU subunits are not tightly attached to either the TM subunits or to the other SU units and can easily shed off the viral particles (Hunter and Swanstrom, 1990). However the reassociation of shed SU with the viral particle is unlikely since they are incorporated on virions by virtue of their association with the TM subunit that results from their synthesis as a common SU-TM polypeptide precursor (Hunter and Swanstrom, 1990). Therefore it is unlikely that soluble SU or A-RBD mixed with AdelH retroviruses may interact with the envelope complex of the latter retroviruses and thus rescue their fusion defect. Nevertheless the following experiments were performed to address this possibility.

A-RBD was bound to target cells at 37°C for 30 min. After removal of unbound fragments by washing target cells, binding of A-RBD was verified by FACS analysis. Cells were then incubated at 37 °C for 0 (TO) to 28 hrs (T28) to allow internalisation of receptor/A-RBD complexes (Rodrigues and Heard, 1999) and A-RBD disappearance from the cell surface, as checked by FACS analysis (Data not shown). Cells were then washed again to remove A- RBD that may have been released by the cells and the AdelH retroviruses were then added at the different time points. Comparatively to infection performed at TO, no significant decrease of infectivity could be detected when the AdelH retroviruses were added until 8 hrs after the initial binding with A-RBD. Likewise the infectivity of AdelH retroviruses added at T20 was at least 50 fold higher than before pre-stimulation of the cells with A- RBD, thus indicating that stimulation of infectivity was durable (Fig. 2). Since A-RBD was efficiently internalised and removed from the cell surface after 4 hrs of incubation (Data not shown) , these results indicated that activation of AdelH retroviruses most likely occurred by activation of the cell surface rather than by interaction with the AdelH envelope complex itself.

Further evidence indicated that A-RBD could not associate with the AdelH retroviruses. Indeed retroviruses generated with either A or AdelH envelopes and mixed or co-expressed with A-RBD were not found to incorporate A-RBD as shown by western-blotting of purified viral particles with antibodies recognising A-RBD (data not shown). However these experiments could not exclude that undetected quantities of A-RBD might be associated with the virions. Therefore to rule out this possibility, a mixture of A-RBD and retroviruses carrying either AdelH or wild-type A envelope glycoproteins were separated on two consecutive 700 KD-pore sized ultrafiltration columns in order to elute out A-RBD. No decrease of infectivity could be detected for retroviruses carrying wild-type amphotropic envelopes after the two subsequent ultrafiltrations (Fig. 3), demonstrating that this process did not affect the viability of the retroviruses. However while before ultrafiltration the AdelH viral particles mixed with A-RBD could efficiently infect XC target cells, as expected, only a residual infectivity could be detected after A-RBD elution (Fig. 3), indicating that stimulation of AdelH retroviruses was lost after separation from A-RBD. As control, the AdelH retroviruses processed through the two columns retained their full capacity to be stimulated by newly added A-RBD (Fig. 3). Similar experiments were conducted after separation of AdelH retroviruses from A-RBD by sephacryl chromatography and led to the same conclusions (data not shown). Altogether these data indicated that stimulation of infectivity of the AdelH retroviruses proceeded via activation of the target cell membrane, most likely as a consequence of interaction of A-RBD with the PiT-2 amphotropic receptor, rather than via interaction with the AdelH envelope incorporated on the retrovirus itself. Thus A-RBD could activate the target cell membrane which in turn became competent to allow entry of retroviruses carrying the fusion-defective AdelH envelope glycoproteins. The SU amino-terminal end activates a cell entry pathway common to type C mammalian retroviruses. The histidine residue deleted in the H5del mutant glycoprotein belongs to a peptide motif, PHQ, found at the amino-termini of the SUs of all type C mammalian retroviruses, suggesting a conserved function of this motif. Thus the H8del mutant ecotropic envelope glycoprotein (MOdelH), harboring the deletion of the histidine in the PHQV peptide motif of the MoMLV SU, is impaired for both cell-cell and virus-cell fusion (Bae et al. , 1997). Similarly the H9del mutation of the GALV envelope glycoprotein GAdelH mutant results in impairement of both cell-cell and virus-cell fusion. Further experiments were performed to demonstrate that MOdelH, AdelH and GAdelH (delH) envelopes were phenotypically similar. Indeed the E-RBD fragment, containing the MoMLV ecotropic receptor binding domain, could rescue the infectivity of MOdelH retroviruses by up to 1 ,000 fold (Fig. 4A). Similar results were obtained for GAdelH retrovirus, whose fusogenicity could be efficiently rescued by soluble envelope fragment encompassing the GALV envelope glycoprotein receptor-binding domain. This result indicated that fusion activation by soluble envelope fragments was not particular to AdelH envelopes and suggested that the MLV SU amino-terminus may convey a signal common to other type C mammalian retrovirus which may be required to achieve early post-binding events.

To address this hypothesis the delH mutations, H5del, H8del, were introduced in the A- RBD or in the E-RBD fragments, respectively. The resultant A-RBDdelH and E-RBDdelH could bind their respective cell surface receptors as efficiently as the parental envelope fragments (Fig. 4B). They could also decrease the infectivity of retroviruses bearing wild- type amphotropic or ecotropic glycoproteins, respectively, by up to 10-fold (Fig. 4A). This effect was most likely a consequence of partial receptor blocking. However in contrast to A- RBD or to E-RBD, neither the A-RBDdelH nor the E-RBDdelH fragments could rescue the infectivity of AdelH or MOdelH retroviruses, respectively (Fig. 4A). These data therefore demonstrated that the integrity of the amino-terminal ends of either RBD fragments was absolutely required to activate postbinding functions and suggested that an essential determinant of cell membrane activation may reside at the amino-terminal end of the MLV RBD. Thus the so-called "receptor binding domain" located in the amino-terminal half of the MLV SU may in fact be composed of two different entities, one involved in specific receptor binding and a second, likely to be non functional in AdelH or MOdelH envelopes, involved in transmission of a signal that may activate cell entry upon receptor binding.

Therefore to test whether the binding function and the activating function of the MLV receptor binding domain could be uncoupled, cells "infected" with AdelH or MOdelH retroviruses were cross-incubated with either E-RBD or A-RBD polypeptides, respectively. Interestingly the infectivity of MOdelH retroviruses could be activated by either A-RBD or E-RBD fragments (Fig. 5A). This cross-activation was reciprocal since either fragments could activate, with similar efficacies, the infectivity of AdelH retroviruses (Fig. 5A). The cross-reactivity of either RBDs was dependent on the presence of both mCAT-1 and PiT-2 receptors on the cell surface since neither PiT-2-transfected CHO cells nor mCAT-1- transfected CHO cells could be infected when E-RBD was used to activate the infectivity of AdelH retroviruses (Fig. 5B) and vice-versa (data not shown). The specificity of activation of AdelH/MOdelH retroviruses by ecotropic envelope fragments was further demonstrated by using the MOD84K-ST envelope fragment, harboring the D84K point mutation that inactivates MoMLV envelope binding (MacKrell et al., 1996). This fragment could activate neither AdelH nor MOdelH retroviruses despite the presence of both amphotropic and ecotropic receptors on target cells (Fig. 5A). Cross-activation of AdelH retroviruses could also be obtained by using soluble envelope glycoproteins from Gibbon Ape leukemia virus (data not shown), thus indicating that infection by delH retroviruses could be activated by a pathway common to type C mammalian retrovirus.

Table 1. Activation of AdelH retroviruses by A-RBD

Cells Pre-binding^a binding and infection^b Titre^c

XC - lacZ(AdelH) 5X10¹

XC - lacZ(AdelH)^d + A-RBD^d 3.1xl0⁵

XC-A-RBD^e - lacZ(AdelH) 9xl0⁴

XC A-RBD lacZ(AdelH) l. lxlO⁵

XC lacZ(AdelH) A-RBD 2.7xl0⁵

a: Target cells were pre-incubated for one hr at 4°C with conditioned supernatants containing A-RBD (used undiluted) or with lacZ retroviral vectors carrying AdelH envelope glycoproteins, as indicated. b: Target cells were incubated for 3 hrs at 37 °C. c: Titres as lacZ i.uJml determined 48 hrs post-infection. d: Both A-RBD (used undiluted) and AdelH retroviruses were added at the same time. e: XC cells constitutively expressing A-RBD polypeptides.

Example 2: Control of syncytia formation by RBD polypeptides

Ideally, the transfer of therapeutic genes into tumour cells should lead both to direct local cell destruction and to activation of anti-tumour immunity to clear tumour deposits to which the genes cannot be delivered. The most commonly used genes for the control of local tumour growth have been the suicide gene/prodrug systems such as Herpes Simplex Virus thymidine kinase (HSVtk)/Ganciclovir (GCV) or cytosine deaminase (CD)/5FC systems. Importantly, these systems not only kill gene-transduced target cells but also close neighbours via local bystander effects. These effects depend upon cell-cell communication or on the diffusion of toxic metabolites, but generally act only over short ranges. Nonetheless, the existence of such bystander effects are crucial to compensate for the relatively poor efficiencies of gene transfer that are currently possible. However, results from early clinical trials have demonstrated that both better delivery systems and more effective genes are clearly still required to improve the efficiency of both local tumour control and of anti tumour immune responses. Hence, therapeutic genes with greater local killing activity would represent a significant advance for gene transfer strategies for cancer. It has been recently demonstrated that the well-known ability of several viral fusogenic membrane glycoproteins (FMG) to fuse target cells could be exploited therapeutically to kill tumour cells. These previous results introduced the FMG as a novel class of therapeutic genes which are more potent than existing suicide gene systems in killing tumour cells and do so by mechanisms which are highly immunostimulatory. Table 2 shows the results of four FMGs derived from the non fusogenic ecotropic or amphotropic murine leukemia virus glycoproteins, whose fusogenicity were increased by deleting the p2R peptide (naturally removed during retrovirus egress) for the ARless and MoRless FMG and also by modifying the proline-riche region (Lavillette et al., 1998) for the C2Rless and PROMORless FMGs. The four resulting FMGs, Arless, C2Rless and PROMORless and MORless induced syncytia formation in most of the cell types that express amphotropic receptors and/or ecotropic receptors . However for selective eliminations of cells as required in cancer gene therapy protocols, the design of a mechanism that can control the formation of syncyia is required. We therefore introduced in all four FMGs the H5del or the H8del mutation (delH mutation) which removes the 5th or the 8^th residue of the amphotropic or the ecotropic SU, respectively, an histidine belonging to the PHQV conserved motif. This mutation could inactivate an early event of the fusion process after receptor binding (Example 1 and Lavillette et al. , 2000). As previously demonstrated the virus-cell fusion activity of delH MLV envelopes bearing the delH mutation can be efficiently rescued by using polypeptides encompassing the first 213 amino-acids of amphotropic MLV SU (A-RBD) or the first 236 amino-acids of ecotropic MoMLV SU (E-RBD) (example 1 and Lavillette et al. , 2000). Consistently with Example 1, the data in Table 2 demonstrated that the cell-cell fusion activity of the four FMGs engineered to harbor the delH mutation, AdelRless, C2delHRless, and PROMOdelHRless and MOdelHRless, could be rescued by providing either the A-RBD or E-RBD polypeptides thus leading to syncytia formation and elimination of target cells that express the relevant viral receptors. Interestingly formation of syncytia by delH FMGs could be achieved by using an heterologous polypeptide (E-RBD) that interacted with a receptor different of that recognised by the delH FMGs.

Table 2. Modulation of cell-cell fusion by soluble envelope fragments

Envelope RBD Syncytia delH envelope RBD Syncytia

A

ARless - + + ΛueixiiS-ie-SS

A-RBD +/-

E-RBD +

C2Rless - + + + C2delHRless - -

A-RBD +/-

E-RBD + + PROMORless - + + + + PROMOdelHRless - +/-

A-RBD + +

E-RBD + + + +

MORless - + + + + MOdelHRless - -

A-RBD + + + +

E-RBD + + + +

fusion indexes (calculated according to Lavillette et al. , 1998) -: less than 100, +/-: 100-304, + : 300-1000, + + : 1000-10 000, + + + : 10 000-15 000, + + + + : > 15000 Of note, similar results of fusion activation with RBD polypeptides were obtained with the GALVdelHRless envelope glycoprotein, indicating that the delH mutation of the potent GALVRless FMG (Bateman et al. , 2000) can be controlled by adding a soluble Env- derived polypeptide (Lavillette and Cosset, unpublished results). These data indicate that it is possible to exploit a well-known phenomenon - the fusion of virally infected cells with uninfected cells as a novel, potent cytotoxic therapy using gene transfer. There are several options to exploit the use of delH FMGs in cell elimination stategies. Firstly viral vectors such as adenovirus vectors can be used to transduce both the delH FMG and the gene encoding the activating RBD polypeptide in cancer cells in vivo. Secondly cells transduced in vitro or ex vivo by a vector expressing a delH fusion-defective FMG can be induced to form syncytia after inoculation of an RBD presented as a protein preparation. In addition as well as using direct gene transfer, delH FMG and RBD can be effectively delivered through cell-mediated mechanisms, which will also be useful as an option for in vivo delivery of FMG into tumours for both local tumour eradication as well as enhancement of anti tumour vaccination strategies.

Example 3: Addition of ligands on RBD polypeptides

To further control the fusion activation of FMGs by RBD polypeptides, a ligand was inserted at the amino-terminal end of the RBD. The anti-MHC-I 34.1 scFv was chosen as we previously demonstrated that it could redirect the host-range of retroviral vectors carrying 34.1 -ecotropic MLV chimeric envelope glycoproteins and allow specific infection of human cells (Marin et al. 1996). Therefore the 34.1 scFV was fused to A-RBD or to E- RBD and preparation of 34.1-A-RBD or 34.1-E-RBD polypeptides were obtained. Retroviral vectors carrying wild-type envelope glycoproteins, A, or AdelH fusion-defective amphotropic envelope glycoproteins were used to infect target cells in the presence of A- RBD, E-RBD, 34.1-E-RBD or 34.1-A-RBD polypeptides (Table 3). While either polypeptides could only weakly decrease the infectivity of parental retroviruses, they could strongly stimulate the infectivity of AdelH retrovirus. No significant differences could be detected when comparing the A-RBD, the E-RBD, the 34.1-E-RBD or the 34.1-A-RBD polypeptides in activation of AdelH retrovirus infectivity (Table 3). Table 3. Modulation of virus-cell fusion by ligand-displaying envelope fragments

Envelope RBD fragment Titers

A 8.1x10°

A-RBD 5.6xl0⁵ E-RBD 8xl0⁶ 34.1-A-RBD 1.35xl0⁶ 34.1-E-RBD 3xl0⁶

AdelH 2x10*

A-RBD 8.5xl0⁴ E-RBD 8xl0⁵ 34.1-A-RBD 7.3xl0⁴ 34.1-E-RBD 7xl0⁵

These data therefore suggest that it is possible to control the fusion activity of FMGs by adding specific ligands on the activating polypeptides. Therefore the design of a specific system to eliminate unwanted cells may consist in a delH FMG associated to an RBD polypeptide that displays a ligand that can specifically recognise a receptor expressed on the target cells. Table 4 shows that 34.1-A-RBD and 34.1-E-RBD could activate the cell-cell fusion activity of the PROMOdelHRless FMG with an efficiency similar to that of A-RBD or E-RBD.

Altogether these results indicate that since the 34.1-E-RBD or 34.1-A-RBD polypeptides could cross-activate the fusogenicity of AdelH, AdelHRless, or PROMOdelHRless envelope glycoproteins on cells that express the targeted antigen (the MHC class I molecules) but no receptor corresponding to either the chimeric RBD or the delH retrovirus (Tables 3 & 4), efficient retargeted infection or cell-type specific syncytia formation could be obtained. Table 4. Modulation of cell-cell fusion by ligand-displaying envelope fragments

Envelope RBD Syncytia

A - 18

ARless - 4464

ARlessdelH - 14

RBD 1014

PROMORless - 14796

PROMORlessdelH - 304

A-RBD 18920

E-RBD 17000

34.1-A-RBD 19000

34.1-E-RBD 20000

Example 4: Activation of Env fusogenicity by RBD and C/TM domains interaction

The H5del mutation was introduced into the CMO, the TMO and the CTMMO MLV amphotropic envelope glycoprotein (Lavillette et al., in preparation), derived from the MLV 4070A envelope glycoprotein by substitution of the SU carboxy-terminal domain (C domain) and/or TM ectodomain with those derived from the MLV ecotropic envelope glycoprotein (Figure 29A). Recent evidence from the literature suggest that the C domain of MLV envelope glycoproteins is involved in activation and/or control of the fusion machinery contained in the TM subunit of the envelope glycoprotein (Nussbaum et al., 1993; Pinter et al. , 1997). Retroviruses carrying either the parental or the delH-mutated CMOdelH, TMOdelH and CTMMOdelH chimeric envelope glycoproteins were prepared and their infectivity was assayed in the presence or the absence of A-RBD or E-RBD soluble polypeptides (Table 5). While the infectivity of AdelH control retroviruses could easily be rescued when either A-RBD or E-RBD polypeptides were present in the infection mixture (Lavillette et al. , 2000), the infectivity of CMOdelH, TMMOdelH and CTMMOdelH retroviruses could not be rescued with the A-RBD polypeptide although it was efficiently rescued with the E-RBD polypeptide (Table 5). These results indicated that the fusogenicity of the CMOdelH, TMMOdelH and CTMMOdelH envelope glycoproteins could only be activated by soluble RBD polypeptides derived from the same envelope glycoprotein from which the C and/or TM domain were inserted in the delH chimera. Further evidence for the requirement of a compatibility between the RBD and the C and/or TM domains were provided by the finding that the CTMMO retroviruses displayed a significantly slower kinetic of infection comparatively to that of retroviruses carrying wild- type MLV amphotropic envelope glycoproteins (Figure 29B). In the absence of exogenous RBD in the infection mixture, the replication retroviruses carrying the CTMMO envelope glycoproteins was delayed, most likely owing to the unability of the MLV 4070A-derived RBD of the latter to activate their MoMLV-derived C and/or TM domains. Yet the addition in the infection mixture of exogenous RBD polypeptides derived from the ecotropic MoMLV envelope glycoprotein (E-RBD), matching the C and/or TM domains of the CTMMO chimera could restore the infection kinetic of CTMMO retroviruses to a level similar if not identical to that of wild-type amphotropic retroviruses (Figure 29B). In contrast the kinetic of infection of retroviruses carrying wild-type amphotropic envelope glycoproteins was not increased in the presence of the E-RBD polypeptide. Additionally, infectivity with retroviruses carrying either wild-type amphotropic or CTMMO envelope glycoproteins was strongly decreased or inhibited, respectively, when infection was performed in the presence of A-RBD (Figure 29B). This inhibitory effect was most likely due to A-RBD-mediated partial blocking of amphotropic receptors in the case of wild-type amphotropic envelope glycoproteins and both receptor interference and incompatibility between this MLV amphotropic Env-derived fragment and the ecotropic C domain in the case of CTMMO chimera.

Table 5

Infectivity in the presence of :

Example 5: Retargeted gene delivery using RBD fragments.

We have therefore demonstrated that the RBD of a retroviral envelope glycoprotein is not only a domain responsible of attaching the viral particle to a cell surface receptor but is also a domain that is responsible, upon receptor binding, of fusion activation most likely via an interaction with the carboxy-terminal domain of the SU Env subunit (C domain). The RBD determinant responsible of this activation appears to be located at its amino-terminal end and can be disrupted by the delH mutation. Thus receptor binding of RBD most probably promotes a conformational change of this amino-terminal end which, in turn, is rendered competent for inducing further conformational changes in the Env C domain that control the fusion machinery located in the TM Env subunit. We surmised that the replacement of the RBD of a retroviral envelope glycoprotein by a cell type specific ligand may redirect the attachment of viral particles carrying such targeting envelope glycoproteins, yet specific attachment of these virions would not promote fusion activation due to the absence in the novel ligand of a molecular device responsible of fusion activation. Such an activation, leading to infection, may therefore be restored by providing in trans a soluble RBD in the infection mixture.

Data in Table 6 demonstrate that retroviral vectors carrying a chimeric envelope glycoprotein in which the wild-type RBD is replaced by a single-chain antibody against human class I MHC molecules cannot infect human cells although they can efficiently bind to class I MHC molecules expressed at the surface of the target cells. The addition in trans of RBD polypeptides during infection can promote the events necessary to allow infection of the target cells, demonstrating that retargeted binding of chimeric viral particles can give rise to productive infection and/or gene delivery when the fusion domains of the targeting envelope are activated by an exogenous polypeptide.

Table 6. RBD-mediated activation of the fusion activity of retargeted envelope glycoproteins

Enva - + RBDb + RBD & competitor'

A ΪO6 Ϊ05 105

34.1-A < 101 105 102

a: LacZ retroviral vectors were generated with the indicated envelope glycoproteins. A, wild-type amphotropic Env; 34.1-A, a chimeric envelope glycoprotein derived from the MLV amphotropic Env in which the receptor binding domain was replaced by a single chain antibody (scFv) against human class I MHC molecule (Marin et al, 1995). The vectors were used to infect class I positive human cells. Titers as lacZ i.uJml. b: A preparation of A-RBD polypeptide was added during infection. Similar results were obtained when the A-RBD polypeptide was added before or after infection with the 34.1-A retroviruses . c: Anti-MHC-I IgGs, corresponding to the Mab which was used to derive the anti-MHC-I scFv inserted into the 34.1-A chimera, were used as competitor in this infection assay. The anti-MHC-I IgGs were used to block the targeted antigens before infection with the 34.1-A retroviruses .

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Claims

1. Use of at least one activating polypeptide comprising an amino acid sequence corresponding to :

- the amino acid sequence of the surface subunit (SU) of an envelope glycoprotein of a first virus, or of a first retrovirus, or

- an amino acid sequence derived from said surface subunit, or - a fragment of an amino acid sequence of said surface subunit, encompassing the receptor binding domain (RBD) and the amino terminal part of the surface subunit, for the rescue of the fusion property of a fusion defective envelope glycoprotein, which has the property of attachment to target cells and is inactive with respect to the fusion with said target cells, said fusion defective envelope glycoprotein belonging to a second virus or a second retrovirus of the same type as or of a different type from the above designated "first virus or first retrovirus" .

2. Use of an activating polypeptide according to claim 1, wherein the activating polypeptide recognizes substantially the same receptors of the target cells as the fusion defective envelope glycoprotein or wherein the activating polypeptide recognizes receptors of the target cells which are different from the ones recognized by the fusion defective envelope glycoprotein.

3. Use according to any one of the claims 1 or 2, wherein the activating polypeptide and/or the fusion defective envelope glycoprotein is (are) linked to a ligand, which is specific with respect to some receptors of the target cells, the ligand being preferably linked to the activating polypeptide.

4. Use according to claim 3, wherein the ligand is chosen among molecules liable to recognize receptors of target cells, for instance receptors of tumoral cells, such as EGF, FGF, VEGF.

5. Use according to any one of claims 1 to 4, wherein the activating polypeptide is under the form of a product as such, or is expressed by a host, such as a virus or a cell, or is expressed, in particular by the target cells, transformed by nucleotide sequences coding for said activating polypeptide.

6. Use according to any one of claims 1 to 5, wherein the fusion defective envelope glycoprotein is expressed by a host, such as a virus incorporating in its envelope said fusion defective envelope glycoprotein, or such as a cell, transformed by nucleotide sequences coding for said fusion defective envelope glycoprotein.

7. Use according to any one of claims 1 to 6, wherein the activating polypeptide corresponds to a fragment of about 200 to about 250 amino acids of said surface subunit comprising the receptor binding domain (RBD) and comprising the first amino acids of the amino terminal part of the surface subunit, the number of said first aminoacids varying from about 10 to about 15, in particular comprising the RBD and the PHQ peptide, in particular the fragments corresponding to amino acid sequences derived from type C mammalian retroviruses such as: - Murine leukemia virus such as : . amphotropic

. ecotropic . xenotropic . polytropic . 10 Al MLV - or GALV type retroviruses and related retrovirus such as SSAV, FELV-A,

FELV-B, FELV-C in particular wherein the activating polypeptide corresponds to one of the following amino acid sequences : A-RBD E-RBD

GALV-RBD 34-1-A-RBD 34-1-E-RBD 34-1 -GALV-RBD EGF-A-RBD EGF-E-RBD EGF-GALV-RBD

8. Use according to any one of claims 1 to 7, wherein the fusion defective envelope glycoprotein corresponds to the amino acid sequence of a mutated form of an envelope glycoprotein of a virus or a retrovirus which, in its non mutated form, presents attachment and fusion properties with respect to target cells, said fusion defective envelope glycoprotein being advantagously derived from type C mammalian retroviruses such as:

- Murine leukemia virus such as :

. amphotropic . ecotropic

. xenotropic . polytropic . 10 Al MLV

- or GALV type retroviruses and related retrovirus such as SSAV, FELV-A, FELV-B, FELV-C and comprising :

* one or several mutations in the amino terminal part of its surface subunit, particulary in the first amino acids of the amino terminal part of the surface subunit, the number of said first aminoacids varying from about 10 to about 15, said mutations inactivating the fusion property without substantially affecting the attachment property, in particular comprises one or several mutations within the PHQ amino- terminal sequence, wherein in particular the fusion defective envelope glycoprotein corresponds to one of the following amino acid sequences :

- MO R less del H - MO del H

- A R less del H

- AdelH - PROMO R less del H

- PROMOdelH

- GALV R less del H

- GALVdelH

- C2 R less del H

- C2delH

- C MOdelH

- TM MOdelH

- C TM MOdelh

- 34.1-A

* or the replacement of its SU carboxy-terminal domain and / or its TM ectodomain, and preferably its SU carboxy-terminal domain by the corresponding SU carboxy-terminal domain and / or the TM ectodomain of another "second" glycoprotein derived from type C mammalian retroviruses, with the proviso that said fusion defective glycoprotein and said second glycoprotein are not derived from the same type C mammalian retroviruses, in particular wherein the fusion defective envelope glycoprotein corresponds to one of the following amino-acid sequences : - CMO

- TM MO

- C TM MO

9. Use of at least one activating polypeptide and of at least one fusion defective envelope glycoprotein for the preparation of a drug useful for the treatment of cancer and infectious diseases.

10. Products containing an activating polypeptide and a fusion defective envelope glycoprotein as defined in any one of claims 1 to 9, as a combined preparation for simultaneous, separate, or sequential use for the rescue of the fusion defective property of said fusion defective envelope glycoprotein.

11. Products containing an activating polypeptide and a fusion defective envelope glycoprotein as defined in any one of claims 1 to 9, as a combined preparation for simultaneous, separate or sequential use for the treatment of cancer pathologies.

12. Activating polypeptides corresponding to a fragment of about 200 to about

250 amino acids of said surface subunit comprising the RBD and the PHQ peptide, in particular the fragments corresponding to amino acid sequences derived from type C mammalian retroviruses such as:

- Murine leukemia virus such as : . amphotropic

GALV-RBD 34-1-A-RBD 34-1-E-RBD 34-1 -GALV-RBD EGF-A-RBD

EGF-E-RBD EGF-GALV-RBD

13. Fusion defective envelope glycoprotein to the amino sequence of an envelope glycoprotem of a virus or a retrovirus which in its native state presents attachment and fusion properties with respect to target cells, and comprises one or several mutations in the amino terminal part of its surface subunit, inactivating the fusion property without substantially affecting the attachment property, in particular comprises one or several mutations within the PHQ amino sequence of the amino terminal part of its surface subunit, wherein in particular the fusion defective envelope glycoprotein is derived from type C mammalian retroviruses such as: - Murine leukemia virus such as :

. amphotropic . ecotropic . xenotropic . polytropic . 10 Al MLV

- or GALV type retroviruses and related retrovirus such as SSAV, FELV-A, FELV-B or FELV-C in particular wherein the fusion defective envelope glycoprotein corresponds to one of the following amino acid sequences : - AdelH

- PROMOdelH

- GALVdelH

- C2delH

- A R less del H - PROMO R less del H

- GALV R less del H

- C2 R less del H - C MO - TM MO - C TM MO

- C MOdelH

- TM MOdelH - C TM MOdelh

- 34.1-A with the proviso that they are different from the ecotropic MLV sequence, wherein the amino acids in position 2 to 8 are either substituted or deleted.