AU722702B2 - Intracellular binding proteins and use thereof - Google Patents

Description

AUSTRALIA

PATENTS ACT 1990 COMPLETE SPECIFICATION FOR A STANDARD PATENT

(ORIGINAL)

S

Name of Applicant: Actual Inventors: Address for Service: Invention Title: Rhone-Poulenc Rorer S.A.

Fabien SCHWEIGHOFFER AND Bruno TOCQUE DAVIES COLLISON CAVE, Patent Attorneys, 1 Little Collins Street, Melbourne, 3000 Intracellular binding proteins and use thereof The following statement is a full description of this invention, including the best method of performing it known to us: Q:\oPER\PDB\70763-94.282 9/10/99 INTRACELLULAR BINDING PROTEINS (IBPs) AND THEIR USES This is a divisional application from Australian Patent Application No: 70763/94, the entire contents of which are incorporated herein by reference.

The present invention relates to nucleic acid sequences, to vectors containing them and to their therapeutic uses, in particular in gene therapy. More especially, the present invention relates to nucleic acid sequences comprising a gene coding for an intracellular binding protein (IBP) and to their use in gene therapy, where appropriate incorporated in suitable expression vectors.

Gene therapy consists in correcting a deficiency or abnormality (mutation, aberrant :o 15 expression, and the like) by introduction of genetic information into the affected cell or organ. This genetic information may be introduced either in vitro into a cell extracted from the organ, the modified cell then being reintroduced into the body, or directly 20 in vivo into the appropriate tissue. In this connection, different techniques of transfection and of gene transfer have been described in the literature (see Roemer and Friedman, Eur. J. Biochem. 208 (1992) 211). To date, the approaches proposed in the prior art for gene therapy consist in transferring genes coding for active polypeptides involved in genetic disorders (hormones, growth factors, and the like), antisense genes, or antigenic peptides for the production of vaccines. The present invention relates to a new approach to gene therapy, consisting in transferring o! and expressing in a target cell (or tissue) an

/-T

intracellular peptide capable of interacting with cell components and thus of interfering with cell functions.

The present invention is based more especially on the demonstration that it is -possible toexpress in -vivo modified antibodies which remain in the intracellular compartment and which can control certain cell functions. The invention is also based on the demonstration that it is possible to clone DNA sequences coding for such intracellular antibodies into vectors, in particular viral vectors, for use in gene therapy.

The use of antibodies in human therapy makes it possible, in general, to target and neutralize circulating biological complexes and/or those which are 15 localized at the cell surface, by bringing about a o~oo cascade of events conducted by the immune system which leads to their removal. However, in many cases, including cancers or diseases due, for example, to viruses, this approach is fruitless since the antigen ooo.

20 responsible for the deregulation of the affected cells Soo.

is inaccessible to the injected antibodies. The present invention affords a new, especially advantageous therapeutic approach, consisting in causing antibodies or therapeutic agents whose binding to their epitope decreases and/or abolishes the deregulation to be produced continuously and intracellularly.

The possibility of recombinant expression of antibodies has already been described in the literature. Thus, Patent EP 88,994 describes the rHLh in vitro expression and purification of heavy- or 'iT light-chain variable regions of antibodies. Likewise, Cw.

Patent US 4,946,778 describes the in vitro expression of DNA sequences coding for modified antibodies composed of heavy- and light-chain variable regions of an antibody linked via a linker However-, the antibodies described in this patent are inactive, and generally synthesized in the form of insoluble inclusion bodies. The antibodies must hence be purified and then subjected to chemical treatments (denaturation, renaturations, and the like) in order to recover an activity. The present' invention demonstrates the possibility of using such DNA sequences for the expression of active intracellular antibodies directly in vivo. The present invention thus demonstrates the possibility of using such DNA sequences coding for 15 intracellular antibodies, under the control of regions permitting their expression in mammalian cells, for *gene therapy, in particular in man. This new approach hence makes it possible to target cell components which are not accessible by traditional vaccination methods.

20 Furthermore, this approach does not involve the development of an immune response, but acts intracellularly.

o" Accordingly the invention provides a defective recombinant virus comprising in its genome a nucleic acid sequence comprising a gene coding for an intracellular binding protein (IBP) under the control of a promoter which is functional in mammalian cells, wherein the IBP is a peptide directed against the expression product of a ras oncogene, against GAP protein or against p53 protein, and wherein the peptide 0" k comprises at least one peptide corresponding to the q 9* S

S

S

binding site of the light-chain variable region of an antibody linked via a peptide linker to a peptide corresponding to the binding site of the heavy-chain variable region-of an -ant-ibody, and in the case where the IBP is directed against the product of a ras oncogene the IBP is capable of inhibiting transformation of a cell by a ras oncogene.

The invention also provides an isolated nucleic acid sequence comprising a gene coding for an intracellular binding protein (IBP) under the control of a promoter which is functional in mammalian cells, wherein the IBP is capable of inhibiting transformation of a cell by a ras oncogene, and is a peptide directed against the expression product of a ras oncogene, which peptide comprises at least one peptide corresponding to the binding site of the light-chain variable region of an antibody linked via a peptide linker to a peptide corresponding to the binding site of the heavy-chain variable region of an antibody.

20 The viral vectors of the invention are typically retroviruses, adenoviruses, adeno-associated viruses, herpesvirus, vaccinia virus or HSV virus.

The invention also provides the use of these nucleic acid sequences or these vectors for the preparation of pharmaceutical compositions intended for the treatment of a cancer. Thus the invention provides a method of treating a cancer comprising administering a nucleic acid sequence or vector of the invention.

The IBPs according to the invention consist 430 of molecules derived from antibodies. In particular, they are proteins having sufficient selectivity and itV

VI

t'"'t -7 3 tiY Yi i; ~i~irc affinity to permit an in vivo interaction having a neutralizing effect on the antigen. These molecules are designated hereinafter intracellular antibodies, on account of their properties and, their-localization.

Antibodies, molecules of the immunoglobulin superfamily, are synthesized naturally (essentially by B lymphocytes) in the form of secreted proteins. They are hence released into the extracellular compartments (circulatory system) where they exert their activity (recognition of and binding to non-self antigens). It has now been shown that it is possible to express in vivo modified genes coding for intracellular antibodies, without affecting the specificity and affinity properties of the antibodies. The nucleic acid 15 sequences according to the invention, which code for intracellular antibodies, hence comprise an antibody gene modified so that the antibody is not secreted. In particular, the gene for the antibody is generally modified by deletion or mutation of the sequences 20 responsible for its secretion.

The form of intracellular antibodies which are used in the context of the invention consists of a peptide corresponding to the binding site of the lightchain variable region of an antibody linked via a peptide linker to a peptide corresponding to the binding site of the heavy-chain variable region of an antibody. The use of this type of intracellular antibody, designated ScFv, is advantageous since they are expressed by a single gene. The construction of nucleic acid sequences coding for such modified 3\ antibodies according to the invention is illustrated in \f it Aj 59 the examples.

Moreover, the nucleic acid sequences coding for the intracellular antibodies according to the invention can also be-modified-chemically, enzymatically or genetically for the purpose of generating stabilized and/or multifunctional intracellular antibodies, and/or which are of reduced size, and/or with the aim of promoting their localization in one or other intracellular compartment.

Thus, the nucleic acid-sequences of the invention can comprise sequences coding for nuclear localization peptides (NLS). In particular, it is possible to fuse the sequences of the invention with the sequence coding for the NLS of SV40 virus, the peptide sequence of which is as follows: MPKKKRK (Kalderon et al., Cell 39 (1984) 499).

As mentioned above, the nucleic acid sequences according to the invention comprise sequences permitting expression of the gene or genes coding for IBPs in mammalian cells. Generally, the IBP genes are hence placed under the control of transcription and translation promoter regions which are functional in the mammalian cell in which expression is sought. These can be sequences which are homologous with respect to the said cell, that is to say sequences naturally responsible for gene expression in the said cell. They can also be sequences of different origin, that is to say sequences responsible for the expression of proteins in other cell types, sequences responsible for antibody expression under natural conditions, viral expression sequences, for example present in a vector arTin which the sequences of the invention are incorporated, or alternatively synthetic or semisynthetic sequences.

As regards-use for man, many functional promoters have been described in the literature, such as, for example, the CMV, SV40, Ela, MLP, LTR, and the like, viral promoters. Cellular promoters such as, for example, the promoter of the villin gene are useful since they permit a specific tissue expression (limited to the intestine-in the case of villin).

Moreover, the expression sequences can also be modified, for example, in order to adapt them to expression in a particular type of vector or of cell, to reduce their size, to increase their transcription 15 promoter activity, to generate inducible promoters, improve their level of regulation or alternatively eo change the nature of their regulation. Such .modifications may be performed, for example, by in vitro mutagenesis, by introduction of additional 20 control elements or of synthetic sequences or by deletions or substitutions of novel control elements.

It can be especially advantageous to use tissuespecific promoters in order to targets the expression of the IBP in one type of tissue only.

Moreover, when the nucleic acid sequence does not contain an expression sequence, the latter may be incorporated in an expression vector, downstream of such a sequence.

Another subject of the invention relates to pharmaceutical compositions comprising at least one it nucleic acid sequence or one vector as are defined

S

above and a pharmaceutically acceptable vehicle.

The sequences of the invention may be used as they are, for example after injection into a human or animal, to induce the intracellular expression of an IBP for the purpose of affecting a particular cell function. In particular, they may be injected in the form of naked DNA according to the technique described in Application WO 90/11,092. They may also be administered in complexed form, for example with DEAEdextran (Pagano-et al., J. Virol. 1 (1967) 891), with nuclear proteins (Kaneda et al., Science 243 (1989) 375), with lipids (Felgner et al., PNAS 84 (1987) 7413), in the form of liposomes (Fraley et al., J. Biol. Chem. 255 (1980) 10431), and the like.

15 In a preferred embodiment of the invention, the nucleic acid sequences as defined above are incorporated in a vector. The use of such vectors makes it possible, in effect, to promote entry into cells, to enhance resistance to enzymes and to increase intracellular stability and expression levels. The vectors of the invention can equally well be plasmid vectors or viral vectors. However, it is preferable to use a viral vector.

As mentioned above, different viruses are capable of being used as vectors for the in vivo transfer and expression of genes according to the invention. By way of example, retroviruses (RSV, HMS, MMS, and the like), HSV virus, adeno-associated viruses, adenoviruses, vaccinia virus, and the like, may be mentioned.

The recombinant virus according to the i 3

C-

S

.q

S

S. S

S

invention is a defective virus. The term "defective virus" denotes a virus incapable of replicating in the target cell. In general, the genome of the defective viruses used in the context of the present invention hence lacks at least the sequences needed for replication of the said virus in the infected cell.

These regions may be either removed (completely or partially), or rendered non-functional, or substituted by other sequences and in particular by the nucleic acid sequences of the invention. Preferably, the defective virus nevertheless retains the sequences of its genome which are needed for encapsidation of the viral particles.

Defective recombinant viruses derived from retroviruses, from adeno-associated viruses, from HSV virus (herpes simplex virus) or from adenoviruses have already been described in the literature [Roemer and Friedmann, Eur. J. Biochem. 208 (1992) 211; Dobson et al., Neuron 5 (1990) 353; Chiocca et al., New Biol.

20 2 (1990) 739; Miyanohara et al., New Biol. 4 (1992) 238; W091/18,088; Akli et al., Nature Genetics 3 (1993) 224; Stratford-Perricaudet et al., Human Gene Therapy 1 (1990) 241; EP 185,573, Levrero et al., Gene 101 (1991) 195; EP 243,204)].

It is especially advantageous to use the nucleic acid sequences of the invention in a form incorporated in a defective recombinant adenovirus.

There are, in effect, different serotypes of adenovirus, the structure and properties of which vary 30 somewhat, but which are not pathogenic in man, and in Sparticular non-immunosuppressed subjects. Moreover, i T r

Y

4 s these viruses do not integrate in the genome of the cells they infect, and can incorporate large fragments of exogenous DNA. Among the different serotypes, it is preferable to use, in-the context-of the present invention, adenoviruses type 2 or 5 (Ad 2 or Ad In the case of Ad 5 adenoviruses, the sequences needed for replication are the E1A and E1B regions.

Moreover, the small size of the genes coding for the intracellular antibodies according to the invention makes it possible advantageously to incorporate simultaneously, in the same vector, several genes coding for intracellular antibodies directed against different regions of one or more targeted cell components. A particular embodiment of the invention 15 hence consists of a vector, in particular a viral vector, comprising at least two nucleic acid sequences coding for intracellular binding proteins directed against different epitopes.

The defective recombinant viruses of the 20 invention may be prepared by homologous recombination between a defective virus and a plasmid carrying, inter alia, the nucleic acid sequence as defined above (Levrero et al., Gene 101 (1991) 195; Graham, EMBO J.

3(12) (1984) 2917). Homologous recombination takes place after cotransfection of the said virus and said plasmid into a suitable cell line. The cell line used should preferably be transformable by the said elements, and (ii) contain the sequences capable of complementing the portion of the defective virus genome, preferably in integrated form in order to avoid risks of recombination. By way of example of a line

C

which is usable for the preparation of defective recombinant adenoviruses, the human embryonic kidney line 293 (Graham et al., J. Gen. Virol. 36 (1977) 59), which contains, in particular, integrated in its genome, the left-hand portion of the genome of an adenovirus (12 may be mentioned. By way of example of a line which is usable for the preparation of defective recombinant retroviruses, the CRIP line (Danos and Mulligan, PNAS 85 (1988) 6460) may be mentioned.

The viruses which have multiplied are then recovered and purified according to traditional molecular biology techniques.

The pharmaceutical compositions of the 15 invention may be formulated for the purpose of topical, oral, parenteral, intranasal, intravenous, intramuscular, subcutaneous, intraocular, and the like, administration.

Preferably, the pharmaceutical compositions ego 20 contain pharmaceutically acceptable vehicles for an injectable formulation. These can be, in particular, sterile, isotonic saline solutions (monosodium or disodium phosphate, sodium, potassium, calcium or magnesium chloride, and the like, or mixtures of such salts), or dry, in particular lyophilized, compositions which, on addition, as appropriate, of sterilized water or of physiological saline, enable injectable solutions to be formed.

The doses of nucleic acids (sequence or vector) used for the administration can be adjusted in .Nl accordance with different parameters, and in particular

S.

S

S

I

K

4 in accordance with the mode of administration used, the pathology in question, the gene to be expressed or the desired treatment period. Generally speaking, in the case of the recombinant virusesaccording-to the invention, these are formulated and administered in the form of doses of between 10' and 10" pfu/ml, and preferably 106 to 1010 pfu/ml. The term pfu (plaque forming unit) corresponds to the infectious power of a solution of virus, and is determined by infection of a suitable cell culture and measurement, generally after 48 hours, of the number of infected cell plaques. The techniques of determination of the pfu titre of a viral solution are well documented in the literature.

The subject of the invention is also any recombinant cell comprising a nucleic acid sequence as defined above.

The sequences of the invention, where appropriate incorporated in vectors, and the pharmaceutical compositions containing them, may be 20 used for the treatment of cancer. They may hence be used for the transfer and expression of genes in vivo in any type of tissue. The treatment can, moreover, be targeted in accordance with the cancer to be treated (transfer to a particular tissue can, in particular, be determined by the choice of a vector, and expression by the choice of a particular promoter).

The intracellular expression of IBPs according to the invention is capable of binding and neutralizing factors involved in cell proliferation, enabling the process of cell proliferation to be S controlled. Many factors (products of oncogenic genes 0* a.

a a. a a a.

13 and factors involved in the signalling of the effect of these products) have, in effect, been associated with these phenomena of deregulation of cell proliferation.

Thus, 90 of adenocarcinomas of the pancreas possess a Ki-ras oncogene mutated on the twelfth codon (Almoguera et al., Cell 53 (1988) 549). Likewise, the presence of a mutated ras gene has been detected in adenocarcinomas of the colon and cancers of the thyroid (50 or in carcinomas of the lung and myeloid leukaemias (30 Bos, J.L. Cancer Res. 49 (1989) 4682). Many other oncogenes have now been identified (myc, fos, jun, ras, myb, erb, and the like), mutated forms of which appear to be responsible for a deregulation of cell proliferation.

The expression of IBPs capable of binding these cell factors (preferably their oncogenic form), and hence of slowing down or inhibiting their effects, affords the possibility of a new therapeutic approach to cancer.

20 As shown in the examples, the expression of intracellular anti-p21 (expression product of the ras gene), anti-GAP or anti-p53 antibodies enables the transforming phenotype of a cancer cell to be reverted.

The present invention will be described more completely by means of the examples which follow, which are to be considered to be illustrative and nonlimiting.

Legend to the figures Figure 1: Restriction map of ScFv-ras. (B) Restriction map of ScFv-Gap. Neutralizing effect of the transient expression of an intracellular antibody 3 t( A. @3 S 3*A*

A

A

AS

AR

S

of the invention on the transforming power of a ras or Her2 oncogene. Py control cells; ScFv cells transected with plasmid psv2.ScFv.ras alone, VAL cells transected with the vector expressing Ha-ras Vall2 oncogenic ras; VAL+ScFv cells cotransected with plasmid psv2.ScFv.ras and Ha-ras Vall2; HER2 cells transected with the vector expressing oncogenic Her2; HER2+ScFv cells cotransected with plasmid psv2.ScFv.ras and with Her2.

General molecular biology techniques The methods traditionally used in molecular biology, such as preparative extractions of plasmid DNA, centrifugation of plasmid DNA in a caesium chloride gradient, agarose or acrylamide gel electrophoresis, purification of DNA fragments by electroelution, protein extraction with phenol or phenol/chloroform, ethanol or isopropanol precipitation of DNA in a saline medium, transformation in Escherichia coli, and the like, are well known to a person skilled in the art and are amply described in the literature [Maniatis T. et al., "Molecular Cloning, a Laboratory Manual", Cold Spring Harbor Laboratory, Cold Spring Harbor, 1982; Ausubel F.M. et al.

(eds), "Current Protocols in Molecular Biology", John Wiley Sons, New York, 1987].

Plasmids of the pBR322 and pUC type and phages of the M13 series are of commercial origin (Bethesda Research Laboratories).

For ligation, the DNA fragments may be separated according to their size by agarose or t acrylamide gel electrophoresis, extracted with phenol .j 4 i~V i C: or with a phenol/chloroform mixture, precipitated with ethanol and then incubated in the presence of phage T4 DNA ligase (Biolabs) according to the supplier's recommendations.

The filling in of 5' protruding ends may be performed with the Klenow fragment of E. coli DNA polymerase I (Biolabs) according to the supplier's specifications. The destruction of 3' protruding ends is performed in the presence of phage T4 DNA polymerase (Biolabs) used according to the manufacturer's recommendations. The destruction of 5' protruding ends is performed by a controlled treatment with Sl nuclease.

Mutagenesis directed in vitro by synthetic 15 oligodeoxynucleotides may be performed according to the method developed by Taylor et al. [Nucleic Acids Res.

13 (1985) 8749-8764] using the kit distributed by Amersham.

u.

S

S

I

0O*4

S

0 0 6 0* 6

S

S

Sq 6 S S

S..

The enzymatic amplification of DNA fragments 20 by the so-called PCR [Polymerase-catalyzed Chain Reaction, Saiki R.K. et al., Science 230 (1985) 1350- 1354; Mullis K.B. and Faloona Meth. Enzym. 155 (1987) 335-350] technique may be performed using a "DNA thermal cycler" (Perkin Elmer Cetus) according to the manufacturer's specifications.

Verification of the nucleotide sequences may be performed by the method developed by Sanger et al.

[Proc. Natl. Acad. Sci. USA, 74 (1977) 5463-5467] using the kit distributed by Amersham.

Example 1: Cloning and expression of a DNA sequence codinc for an intracellular anti-ras antibody

C

r i:' ~t This example describes the cloning and expression of a nucleic acid sequence coding for an intracellular binding protein reproducing the properties of Y13-259 monoclonal antibody. Y13-259,.

antibody is directed against Ras proteins (ref. ATCC CRL 1742) Virol. 43, 294-304, 1982), and is a neutralizing antibody against the function of the oncogenic Ras proteins when injected into cells [Smith et al., (1986) Nature, 320, 540-543; Kung et al., Exp.

Cell. Res. (1986) 162, 363-371].

1.1. Preparation of the DNA sequence A DNA sequence coding for an intracellular antibody (ScFv fragment) was prepared according to the technique described in Patent US 4,946,778. This 15 sequence was then placed under the control of a promoter which is functional in mammalian cells.

Poly(A) RNAs are isolated from a cell culture of the hybridoma which secretes Y13-259 antibody according to the technique described by Chirguin S.H.

et al. [Biochemistry 18, 5294 (1979)]. These RNAs are used for a reverse transcription reaction using primers o composed of random hexanucleotides. The cDNAs obtained serve as a template for two PCR reactions: one intended for amplifying the heavychain variable fragment (VH) of Y13-259 with primers specific for murine VH regions, the second enabling the VL fragment to be obtained using a mixture of 10 primers derived from murine sequences.

30 Two fragments of 340 bp and 325 bp are S- thereby obtained and then assembled by means of a \S s H

/"U

17 linker which permits a correct positioning of the VH cDNA at the 5' end of that of VL. This linker codes for amino acids composed of three repeats of the unit (Gly) 4 Ser [Orlandi, R. et al., Proc. Natl. Acad..--Sci.

USA, 86, 3833-3837 (1979)]. The sequence of the intracellular antibody is presented in SEQ ID No. 1 (residues 28 to 270). This sequence endows the VH-VL fusion with enough degrees of freedom to permit their assembly in a parallel orientation and to provide for a correct affinity for the antigen.

The VH-linker-VL fused nucleic acid sequence is then inserted into a phagemid which permits expression of the intracellular antibody (ScFv fragment) at the surface of an M13 phage (Figure 1A).

15 This expression readily permits identification and Sselection of the intracellular antibodies which correctly recognize the antigen.

1.2. Functional evaluation of the modified intracellular antibody 20 The DNA sequence which codes for the modified o intracellular anti-Ras antibody (VH-linker-VL) is isolated from the phagemid by restriction, and then inserted into the vector sv2 under the control of the I enhancer-early promoter system of SV40 (Schweighoffer et al., Science, 256, 825-827, 1992) in order to test its capacity to antagonize the effects of an oncogenic Ras. The plasmid thereby obtained is designated psv2.ScFv.ras. The functional evaluation was performed according to several tests: a) Transient transfection in mammalian cells 18 For evaluation by transient transfection in mammalian cells, plasmid psv2.ScFv.ras was cotransfected into NIH 3T3 cells according to the protocol described in Schweighoffer et al. [Science, 256, 825-827 (1992)] with a vector which permits the expression of a Ha-ras Val 12 gene. The activation state of the signalling pathway under study was recorded by measuring the enzymatic activity obtained from the chloramphenicol acetyltransferase (CAT) reporter gene placed under the control of a-promoter containing nucleotide elements responding in trans to the action of Ras (RRE), which were also cotransfected: these RRE elements consist of a polyoma TK hybrid promoter-enhancer (Wasylyk et al., EMBO, J. 7, 2475, 15 1988).

The results obtained are presented in Figure 1C. The analysis of the CAT activities obtained demonstrates the capacity of the modified intracellular o* antibody, prepared from Y13-259 antibody, to antagonize 20 the activity of the oncogenic Ras.

Plasmid psv2.ScFv.ras cotransfected with a plasmid permitting the expression of an oncogene endowed with tyrosine kinase activity, the HER2 (human epidermal growth factor type II) oncogene, also blocks its activity on the test "CAT" plasmid (Figure 1).

b) Formation of foci of transformed cells: Cancer cells have the property of forming foci of transformation, and in particular NIH 3T3 fibroblasts expressing an oncogenic Ras (Barlat et al., Oncogene (1993), B, 215-218).

NIH 3T3 cells are cultured as in the previous A f 19 test in Dulbecco's modified Eagle medium (DMEM) containing 10 of foetal calf serum, at 37 0 C in a humid environment containing 5 of CO,. These cells are then cotransfected with an oncogenic Ras: Ha-ras Vall2, plasmid psv2.ScFv.ras (see a) above) and a excess of the neomycin resistance gene, by the cationic lipid transfection technique (Schweighoffer et al., Science, 256, 825-827, 1992). The same total amount of DNA is transfected for each dish.

24 hours after transfectionr, th e transfected cells originating from each 100 mm Petri dish are divided in a ratio of 1 to 10 and cultured in the same medium but in the presence of G418 (GIBCO/BRL) at a concentration of 0.4 mg per ml of medium. The number of 15 foci of transformation obtained per pg of transfected DNA is counted after 14 days of culture.

°The results obtained are presented in the table below. They represent the mean of four independent tests.

20

TABLE

t Transformation of NIH 3T3 cells with Ha-ras Vall2 in the presence of intracellular anti-ras antibody •gee 30

_F

-s t r Transfected plasmids Number of foci per pg of transfected DNA Ha-Ras Vall2 110 psv2.ScFv.ras 2 Ha-Ras Vall2 psv2.ScFv.ras The results obtained show clearly that the intracellular anti-ras antibody very greatly decreases the transforming power of an oncogenic ras gene.

Moreover, in the light of the results obtained in expression of this ScFv fragment of Y13-159 antibody should also prevent transformation by other oncogenes such as HER1, HER2 facilitating the activation of cellular Ras proteins....- It is understood that a person skilled in the art can, on the basis of the results described in the present application, reproduce the invention with nucleic acid sequences coding for intracellular antibodies (such as ScFv fragments) directed against other cell components. These may be prepared either from known antibodies directed against cell components, or by identification of an antigen to be neutralized, immunization by means of this antigen or of a preferred epitope of the latter, and then preparation of the 15 intracellular antibody from the antibody, its mRNA or hybridoma obtained. Other components involved in processes of cell transformation may thus be targeted.

So For example, other DNA sequences coding for intracellular anti-ras binding antibodies may be prepared according to the same methodology from M38, M8, M70, M90 and M30 (ATCC HB 9158) hybridomas, the antibodies of which are directed, respectively, against residues 1 to 23, 24 to 69, 90 to 106, 107 to 130 and 131 to 152 of the Ha-Ras protein. Furthermore, as mentioned above, vectors simultaneously carrying several sequences coding for different intracellular antibodies may advantageously be prepared in order to confer a superior neutralizing activity.

Example 2: Cloning and expression of a DNA sequence coding for an intracellular anti-GAP antibody This example describes the cloning and expression of a nucleic acid sequence coding for an intracellular binding protein reproducing the properties of a monoclonal antibody directed against GAP protein GAP protein (for GTPase Activating Protein) is involved in the ras-dependent signalling pathway. It interacts catalytically with ras proteins and multiplies 100- to 200-fold the rate of hydrolysis of GTP, measured in vitro for the normal p21 protein.

Various studies have shown that-the catalytic domain of this protein of approximately 1044 amino acids is located in the carboxy-terminal region (residues 702-1044), and that this region is reponsible for the interaction of GAP protein with the ras proteins (see 15 W091/02,749).

It has now been shown that a monoclonal antibody directed against the so-called "SH3" domain of GAP protein neutralizes the functions of oncogenic Ras proteins in the Xenopus egg (Duchesne et al., Science, 20 259, 525-528, 1993).

According to the methodology described in it is possible to synthesize a DNA sequence coding for an intracellular antibody (ScFv fragment) corresponding to this antibody (SEQ ID No. 2, residues 11 to 250, Figure 1B). Such a sequence, incorporated in a vector, can enable the transforming power of an oncogenic ras gene in tumour cells to be inhibited.

Moreover, the Applicant has also identified more precisely the epitopes recognized by this antibody. These epitopes were then synthesized artificially, and may be used to generate new neutralizing antibodies capable of being used for carrying out the invention.

a) More precise identification: The identification was-carried out by the "epitope scanning" technique. This technique is based on the principle that a given antibody can react with peptides of 5 to 15 amino acids. As a result, the identification of sequential epitopes may be obtained by preparing a complete set of overlapping peptides, of 5-15 amino acids, corresponding to the complete sequence of the antigen in question. This technique was used to determine the functional epitopes of the "SH3" domain of GAP. To this end, the whole of this domain was explored by sequential overlaps, by synthesis of a 15 decapeptide every other amino acid.

Synthesis of overlapping peptides 35 peptides covering the whole of the o fragment of Figure 1 were synthesized chemically. The synthesis was performed in duplicate, on 2 independent supports, by the Fmoc/t-butyl solid-phase method (Cambridge Research Biochemicals kit).

Detection of functional epitopes The functional epitopes recognized by Ac200 antibody were visualized in an ELISA test with a peroxidase-coupled rabbit anti-mouse antibody. The chromogenic substrate used for the enzyme is aminobis(3-ethylbenzothiazodinesulphonate)

(ABTS).

The results obtained show that the epitopes recognized by this antibody are as follows:

PVEDRRRVRAI

EISF

1

EDGWM

These epitopes may be used according to the traditional techniques of a person skilled in the art to generate antibodies neutralizing the effects of ras.

These antibodies or hybridomas producing them are then used to generate nucleic acid sequences and vectors of the invention, according to the methodology described above.

Example 3: Preparation of a nucleic acid sequence coding for an intracellular anti-Ki-ras antibody from mRNAs extracted from spleens of mice immunized with peptides derived from the hypervariable portions of Ki- Ras (2A and 2B) This example describes the preparation of 15 nucleic acid sequences coding for intracellular antibodies (such as ScFv fragments) according to the invention, by identification of an antigen to be neutralized, immunization by means of this antigen or of a preferred epitope of the latter, and then preparation of the nucleic acid sequence from the antibody, its mRNA or hybridoma obtained.

This example demonstrates the possibility of applying the present invention to any desired antigen or epitope, even when no monoclonal antibody directed against the said antigen or epitope is available.

The antigen targeted in this example is the Ki-ras protein. More precisely, the antigens used for the immunization are the peptides of 25 and 24 amino acids corresponding to the following terminal ends of the Ki-Ras 2A and 2B proteins: Peptide 2A: QYRLKKISKEEKTPGCVKIKKCIIM Peptide 2B: KYREKNSKDGKKKKKKSKTKCIIM After immunization of mice with these peptides according to traditional techniques of immunology, the spleens are extracted and cDNAs are prepared from the mRNAs. The cDNAs coding for the variable regions are then cloned, leading to the formation of a library of phages expressing the ScFv fragments corresponding to the whole of the repertoire of the mice used. The intracellular antibodies (ScFv fragments) recognizing the peptides 2A and 2B are then identified and isolated by successive steps of selection on the basis of column and microtitration plate affinity tests.

These ScFv fragments are then tested 15 functionally according to the protocol described in Example 1.

The strategy developed in this example makes it possible advantageously to select intracellular 'antibodies specific for the Ki-Ras oncogenes, which will hence not affect the other Ras proto-oncogenes.

The selectivity of such tools is hence not only cellular (as a result of the uncoupling of the transduction pathways in Ras-transformed cells), but 2. also molecular.

Example 4: Preparation of a nucleic acid sequence coding for an intracellular anti-(mutated p53) antibody This example describes the preparation of nucleic acid sequences coding for intracellular antibodies (such as ScFv fragments) directed against mutated p53 proteins. These intracellular antibodies are obtained from different monoclonal antibodies r *4

S

S

S

SI

S

S

directed against the said mutated p53 proteins.

The gene coding for the p53 protein is altered in a very large number of tumour cells (Caron de Fromentel and Soussi, Genes, 4, 1-15, 1992). The mutated p53 protein does not have the same conformation as wild-type p53 (Lane and Benchimol, Genes Dev. 4, 1- 8, 1990). This change in conformation may be detected by monoclonal antibodies (Milner and Cook, Virology, 154, 21-30, 1986; Milner, Nature, 310, 143-145, 1984).

pAB 240 antibodies recognize the mutated forms of the p53 proteins.

The intracellular expression of an ScFv fragment of antibodies specific for mutated p53 or of proteins interacting specifically with these mutated 15 p53 proteins should induce a beneficial effect in tumours possessing a mutated p53.

Example 5: Cloning and expression of a DNA sequence coding for an intracellular anti-papillomavirus antibody This example describes the cloning and expression of a DNA sequence coding for an intracellular antibody (ScFv fragment) directed against a protein of human papillomavirus

(HPV).

The targeted viral protein is the E6 protein.

This protein is produced by HPV 16 and 18 viruses, which are responsible for 90 of cancers of the cervix in women and have been identified in precancerous epithelial lesions (Riou et al., Lancet 335 (1990) 117). The E6 gene product leads to the formation of tumours by strongly decreasing the amount of wild-type p53, an anti-oncogene, in HPV-positive cells (Wrede

-I

et al., Mol. Carcinog. 4 (1991) 171). In other tumours, p53 is inhibited by different mechanisms: mutation (see Example 4) or combination with proteins such as MDM2.

The sequence of the E6 protein has been described in the literature (Hawley-Nelson et al., EMBO J. 8 (1989) 3905; Minger et al., J. Virol. 63 (1989) 4417). Particular regions of this protein may be identified by "epitope scanning" (see Example and then used to immunize mice according to the protocol described in Example 3. The DNA sequence coding for the intracellular antibody (ScFv fragment) directed against the E6 protein of human papillomavirus (HPV) is then prepared according to the methodology described above.

The functionality of this sequence is demonstrated 15 after in vivo expression, by measuring: the increase in the level of wild-type p53 in cells expressing E6, the morphological reversion of HPVtransformed cells, the blocking of the effects of E6 on the transactivation of p53, and the inhibition of the transformation by E6 of human keratinocytes and fibroblasts.

Throughout this specification and the claims which follow, unless the context requires otherwise, the word "comprise", and variations such as "comprises" and "comprising", will be understood to imply the inclusion of a stated integer or step or group of integers or steps but not the exclusion of any other integer or step or group of integers or steps.

SEQUENCE LISTING GENERAL INFORMATION:

APPLICANT:

NAME: RHONE-POULENC RORER S.A.

STREET: 20, avenue R. ARON CITY: ANTONY COUNTRY: FRANCE POSTAL CODE: 920165 (ii) TITLE OF INVENTION: Nucleic acid sequences, vectors containing them, pharmaceutical compositions and therapeutic uses.

0* (iii) NUMBER OF SEQUENCES: 2 (iv) COMPUTER READABLE FORM: MEDIUM TYPE: Tape COMPUTER: IBM PC compatible SOFTWARE: PatentIn Release S(2) INFORMATION FOR SEQ ID NO: 1: SEQUENCE CHARACTERISTICS: LENGTH: 870 base pairs TYPE: nucleic acid STRANDEDNESS: double TOPOLOGY: linear REPLACEMENT SHEET (RULE 26) (ii) MOLECULE TYPE: cDNA (iii) HYPOTHETICAL: NO (iii) ANTI-SENSE: NO (ix) FEATURE: NAME/KEY: CDS LOCATION: 870 (ix) FEATURE: NAME/KEY: misc-feature LOCATION: 442. .486 OTHER INFORMATION: /product ="Linker" (ix) FEATURE: NAME/KEY: misc feature LOCATION: 82. .810 15 OTHER INFORMATION: /product= "ScFv anti- Ras" see* (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 1: be 6 0 2 0 5101 TTG TTA TTA CTC C GCC CAG CCG GCC ATG OCT C-AG GTO AAA CTG CAG 96 Leu Leu Leu Leu Ala Ala Gln Pro Ala Met Ala Gin Val Lys Leu Gin REPLACEMENT SHEET (RULE 26) 25 CAG TCA GSA GSA GGC TTA GTG CAS CCT GGA ASS Gin Ser Sly Gly Gly Leu Val Sin Pro Sly Arg 40 TGT GTA GTC TCT GSA TTC ACT TTC AGT AAC TAT TCC CTG AAA CTC TCC Ser Leu Lys Leu Ser GGA ATS Asn Tyr Gly Met AAC TGS ATT Asn Trp Ile 192 Cys Val Val Ser Gly CSC CAS ACT CCA GGG Arg Gin Thr Pro Sly 65 Phe Thr Phe Ser GSA CTG SAG TSS STT Sly Leu Slu Trp Val 75 GCA TAC ATT ACT ACT Ala Tyr Ile Ser Ser 240 GST ASC ACT Sly Ser Ser CTC TAC TAT Leu Tyr Tyr SCA SAA ACS STS Ala Glu Thr Val AAS GGC CGA TTC Lys Gly Arg Phe 288 ATC TCC AGA SAC AAT GCC A AAC ACC CTS TAC CTG CAA 15 Ile Ser Arg Asp Asn Ala Lys Asfl Thr Leu Tyr Leu Gin 100 105 ATS ACC ACT Met Thr Ser 110 6.

a a a .a a CTG ASS Leu Arg SAA SAC ACT GCC TTS TAT TAC TST SCA AGA CAT SAG GGT Slu Asp Thr Ala Leu Tyr Tyr Cys Ala Arg His Glu Sly 120 125 ACS SST Thr Sly 130 STC TCC Val Ser 25 145 ACC SAC TTC TTT SAT TAC TGS SSC CAA GGG ACC ACS GTC ACC Thr Asp Phe Phe Asp Tyr Trp Sly Sin Gly Thr Thr Val Thr 135 140 TCA SST GSA Ser Sly Sly SSC SST TCA SSC GSA SST GGC TCT GSC GGT GSC Sly Sly Ser Gly Giy Sly Sly Ser Sly Sly Sly 150 155 160 GSA TCS SAC STT GAS CTC ACC CAS TCT CCA CAT TCC CTG TCT Sly Ser Asp Val Slu Leu Thr Sin Ser Pro His Ser Leu Ser 1c.; 170 SCA TCT Ala Ser 175 528 CTS GSA SAA ACT STC 3 0 Leu Sly Siu Thr Val TCC ATC GAA TST CTA SCA ACT SAG GSC ATT TCC Ser Ile GlU CyS Leu Ala Ser Glu Sly Ile Ser REPLACEMENT SHEET (RULE 26) 576 180 18S 190 A.AT TAT TTA GCG TGG Ann Tr Lou Ala Trp 195 CTG ATC TAT TAT CCA Lou Ile Tyr Tyr Ala 210 CAG AAG CCA Glu Lys Pro 200 GGG AAA TCT Gly Lys Ser 205 CCT CAG CTC Pro Gln Lou 624 TTG CAG GAT GGG GTC CCA TCA CGG TTC Lou Gln Asp Gly Val Pro Ser Arg Phe 220 AGT GGC AGT GGA TCT Ser Gly Ser Gly Ser 225 ACA CAG TTT TCT Thr Gln Phe Ser CTC AAG ATC Lou Lys Ile 235 AGC AAC ATG Ser Ann Met 240 720 230 CAA CCT GAA Gln Pro lu GAT GAA GGG GTT Asp Glu Gly Val 245 TAT TAC TGT Tyr Tyr Cys 250 CAA CAG GCT TAC Gln Gln Ala Tyr AAG TAT Lys Tyr 255 CCT TCC ACG Pro Ser Thr TTT GGA Phe Gly 260 GCT GGC ACC Ala Gly Thr CTG GAA ATA AAA Lou Glu Ile Lys CGG GCG GCCC Arg Ala Ala 270 816 GCA GAAP CAA AAAJ CTC ATC Ala Glu Gln Lys Lou Ile 275 GAG GCAT CTG AAT TAA TAA GAA TTC Glu Asp Lou Ann Glu Phe 285 ACT GGC Thr Gly 290 INFORMATION FOR SEQ ID NO: 2: SEQUENCE CHARACTERISTICS: LENGTH: 810 base pairs TYPE: nucleic acid STRANDEDNESS: double CONFIGURATION: linear REPLACEMENT SHEET (RULE 26) ;ti (ii) MOLECULE TYPE: cDNA (iii) HYPOTHETICAL: NO (iii) ANTI-SENSE: NO (ix) FEATURE: NAME/KEY: CDS LOCATION: 1..810 (ix) FEATURE: NAME/KEY: misc_feature LOCATION: 382..426 OTHER INFORMATION: /product= "Linker" (ix) FEATURE: NAME/KEY: misc feature LOCATION: 31..753 OTHER INFORMATION: /product= "ScFv antia a a a.

a a.

a.

GAP"

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 2: TTA TTA CTC GCG GCC CAG CCG GCC ATG GCC CAG GTC CAA CTG CAG GAG Leu Leu Leu Ala Ala Gin Pro Ala Met Ala Gln Val Gin Leu Gln Glu TCA GGA CCT GGC CTA GGG CAG CCC GCA CAG AGC ATT TCC ATA ACC TGC Ser Gly Pro Gly Leu Gly Gin Pro Ala Gin Ser Ile Ser Ile Thr Cys "i ic- Pi REPLACEMENT SHEET (RULE 26) ACA GTC TCT GGT TTC TCA TTA AGT AGC TAT GGT GTA CAC TGG GTT CC Thr Val Ser Gly Phe Ser Leu Ser Ser Tyr Gly Val His Trp Val Arg CAG TCT CCA GGA AAG GGT CTG GAG Gin Ser Pro Gly Lys Gly Leu Glu 55 GGA GGO ACA GAC TAC AAT GCA GCC Gly Gly Thr Asp Tyr Asn Ala Ala TGG CTG GGA GTG ATA TGG AGA GGT Trp, Leu Gly Val Ile Trp Arg Gly TTC ATG TCC AdA CTG AGC ATO ACC Phe Met Ser Arg Leu Ser Ile Thr 192 AAG GAC AAC TOO AAG AGC CAA GTT TTO TTT AAA TTG AAC AGT OTG CAA Lys Asp Asn Ser Lys Ser Gin Val Phe Phe Lys Leu Asn Ser Leu Gin COT CAT GAC ACT GCC ATG TAO TAC Pro Asp Asp Thr Ala Met Tyr Tyr 100 CCC AAA AGG GGT Ala Lys Arg Gly GGC OCG GGG 336 Gly Pro Gly 110 9* TAT TTC Tyr Phe GGA GGC 20 Giy Gly 130 GAG CTC Giu Leu 145 GTC TGG GGC Val Trp Gly OAA GGG Gin Gly 120 ACC AOG GTO ACC Thr Thr Val Thr GTO TOO TOA GGT Val Ser Ser Gly 125 GGT TOA GGC GGA GGT GGC TCT AGO GGT GGC GGA TCG GAO ATT Gly Ser Gly Gly Gly Gly Set Ser Gly Gly Gly Ser Asp Ile 432 ACC CAG TOT Thr Gin Ser CO TOO OTA TOT Ala Ser Leu Ser 140 TOT GTG GGA GAA ACT Ser Val Gly Clu Thr 160 480 25 GTO ACC ATG ACA TGT OGA GCA ACT GAG AAT ATT Val Thr Met Thr Cys Arg Ala Ser Giu Asn Ile 165 170 AGT AAT TTA GCA 528 Ser Asn Leu Ala 175 TGG TAT 0kG CAG AAA CAd GGA AAC TOT COT CAG OTO CTG GTO TAT GOT Trp, Tyr Gin Gin Lye Gin Gly Lye Ser Pro Gin Leu Leu Val Tyr Ala 180 185 190 REPLACEMENT SHEET (RULE 26) OCA ACA AAA OCA GGA AAT GOT OTG CCA TOA AGG TTC AOT GGC ACT OGA Ala Thr Lys Pro Oly Asn Gly Val Pro Ser Arg Phe Ser Oly Ser Gly 195 200 205 TCA GGC ACA OAA TTT TOT CTG AAG AbC AAO AGC CTG CAG CCT OAA OAT Ser Oly bhr Gin Phe Ser Leu Lys Ile Aen Ser Leu Gin Pro Glu Asp 210 215 220 672 OTT GGG AAO TAT Len Gly Asn Tyr 225 iO GGC GGG GGC ACC Gly Gly Gly Thr TAO TOT OTA OAT TTT TAT GGG ACT COG TAT AGO TTO Tyr Cys Len His Phe Tyr Cly bhr Pro Tyr Arg Phe 230 235 240 AAG CTG GAA ACO AAA COG GCG GCC GCA GA-A CAA AAA Lye Len Glu Thr Lys Arg Ala Ala Ala Glu Gin Lye 245 250 255 eTC ATC TCA GAA GAG GAT CTG AAT TAA TAA GAA TTC ACT GGC Leu Ile Ser Olu Glu Asp Leu Asn Glu Phe Thr Oiy 260 265 270

S

9 9

S

9.

S S 9 0

S.

99 9

S

S

S 9 59 9 9 REPLACEMENT SHEET (RULE 26)

Claims (17)

1. A defective recombinant virus comprising in its genome a nucleic acid sequence comprising a gene coding for an intracellular binding protein (IBP) under the control of a promoter which is functional in mammalian cells, wherein the IBP is a peptide directed against the expression product of a ras oncogene, against GAP protein or against p53 protein, and wherein the peptide comprises at least one peptide corresponding to the binding site of the light-chain variable region of an antibody linked via a peptide linker to a peptide corresponding to the binding site of the heavy-chain variable region of an antibody, and in the case where the IBP is directed against the 15 product of a ras oncogene the IBP is capable of inhibiting transformation of a cell by a ras oncogene. Virus according to claim 1, wherein the promoter which is functional in mammalian cells is chosen from viral, cellular or artificial promoters.

3. Virus according to claim 1 or 2 wherein the nucleic acid sequence comprises all or part of the sequence SED ID NO. 1.

4. Virus according to claim 1 or 2 wherein the nucleic acid sequence comprises all or part of the sequence SED ID NO. 2. Virus according to claim 1 or 2 wherein the nucleic acid sequence codes for a peptide directed against a mutant p53 protein.

6. Virus according to any one of the 36 preceding claims which is a retrovirus, adenovirus, f adeno-associated virus, vaccinia virus or HSV virus.

7. Virus according to any one of the proceeding claims comprising at least two nucleic acid sequences as defined in claim 1, coding for intracellular binding proteins directed against different epitopes of one or more antigens.

8. An isolated nucleic acid sequence comprising a gene coding for an intracellular binding protein (IBP) under the control of a promoter which is functional in mammalian cells, wherein the IBP is capable of inhibiting transformation of a cell by a ras oncogene, and is a peptide directed against the expression product of a ras oncogene, which peptide comprises at least one peptide corresponding to the binding site of the light-chain variable region of an antibody linked via a peptide linker to a peptide corresponding to the binding site of the heavy-chain variable region of an antibody.

9. Nucleic acid sequence according to claim 8, characterised in that it is incorporated in a vector.

10. Nucleic acid sequence according to claim 9, which is incorporated in a viral vector.

11. Vector comprising at least two nucleic acid sequences as defined in claim 8 coding for -I 25 intracellular binding proteins directed against different epitopes of one or more antigens.

12. Vector according to claim 11 which is a viral vector.

13. Pharmaceutical composition comprising at least one nucleic acid sequence according to any one of Sclaims 8 to 10 or a virus according to any one of r claims 1 to 7 and a pharmaceutically acceptable vehicle.

14. Pharmaceutical composition according to claim 13, which comprises at least one nucleic acid sequence according to any one of claims 8 to 10, in the form of liposomes or of a complex with nuclear proteins, lipids or dextran, or in untreated form. Use of a nucleic acid sequence according to claim 8, 9 or 10, for the preparation of a pharmaceutical composition intended for the treatment of a cancer.

16. Method of treating a cancer comprising administering a nucleic acid sequence according to claim 8, 9 or 15

17. Virus according to claim 1 substantially as hereinbefore described in any one of the Examples.

18. Nucleic acid sequence according to claim 8 substantially as hereinbefore described in any one of o* the Examples. 3: 20

19. Vector according to claim 11 g substantially as hereinbefore described in any one of the Examples.

20. Pharmaceutical composition according to claim 13 substantially as hereinbefore described in any one of the Examples. DATED this 2 4 th day of May, 2000 Rhone-Poulenc Rorer S.A. by DAVIES COLLISON CAVE Patent Attorneys for the Applicant(s) k c