Anti-RAS intracellular binding proteins and use thereof

The present invention relates to nucleic acid sequences encoding intracellular binding proteins. More particularly, the nucleic acid comprises a gene coding for an intracellular single chain antibody specific for a ras oncogene under the control of a promoter, the antibody is functional in mammalian cells, and inhibits the transformation of cells that express a ras oncogene.

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 intra-cellular 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 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 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 and 
expressing in a target cell (or tissue) an 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 to express 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 localized at the cell surface, by bringing about a 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 responsible for the 
deregulation of the affected cells 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 in vitro 
expression and purification of heavy or light-chain variable regions of 
antibodies. Likewise, U.S. Pat. No. 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 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. Furthermore, this approach does not involve the 
development of an immune response, but acts intracellularly. 
A first subject of the invention hence lies in a nucleic acid sequence 
comprising a gene coding for an intracellular binding protein (IBP) under 
the control of regions permitting its expression in mammalian cells. 
The invention also relates to vectors containing a nucleic acid sequence as 
defined above. More especially, the vectors of the invention are of viral 
origin, such as retroviruses, adenoviruses, adeno-associated viruses, 
herpesvirus, vaccinia virus, HSV virus, and the like. 
The invention also relates to the use of these nucleic acid sequences or 
these vectors for the preparation of pharmaceutical compositions intended 
for the surgical and/or therapeutic treatment of the human or animal body. 
It also relates to any pharmaceutical composition comprising a vector, in 
particular a viral vector, or a nucleic acid sequence as are defined 
above. 
For the purpose of the present invention, the term intracellular binding 
protein (IBP) denotes any protein or protein fragment capable of 
recognizing a component of the cell in which it is expressed, and of 
interacting selectively with, and with affinity for, this component. The 
interactions may be covalent or non-covalent chemical interactions. The 
interaction with the cell component (proteins, lipids, amino acids, mRNA, 
tRNA, rRNA, DNA, and the like) makes it possible to act on a cell function 
in which the said component is involved, and thus to control (stimulate, 
slow down, inhibit) this function. 
Preferably, the IBPs according to the invention consist of molecules 
derived from antibodies or having binding properties comparable to those 
of an antibody. In particular, they are proteins having sufficient 
selectivity and 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 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 responsible 
for its secretion. The IBPs according to the invention can, in particular, 
consist of antibody fragments, and for example of Fab or F(ab)'2 fragments 
which carry the antigen binding domains. The use of this type of 
intracellular antibody involves, however, the expression of a nucleic acid 
sequence comprising several genes coding respectively for the heavy and 
light regions of these fragments, and it also involves these chains being 
correctly assembled in vivo. For this reason, an especially advantageous 
form of intracellular antibodies which are usable in the context of the 
invention consists of a 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. 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 
antibodies according to the invention is illustrated in 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 (SEQ ID NO: 13) (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 in which the 
sequences of the invention are incorporated, or alternatively synthetic or 
semi-synthetic 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 promoter 
activity, to generate inducible promoters, improve their level of 
regulation or alternatively change the nature of their regulation. Such 
modifications may be performed, for example, by in vitro mutagenesis, by 
introduction of additional control elements or of synthetic sequences or 
by deletions or substitutions of novel control elements. It can be 
especially advantageous to use tissue-specific 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. 
To prepare a vector according to the invention, in a first step, a cell 
function, for example one involved in or responsible for a pathology, on 
which it is desired to act should be identified. A suitable cell component 
involved in this function should then be identified, and the IBP which 
appears best suited to this component (antibody, derivatives, and the 
like) on the basis of its localization, its role, its nature, and the 
like, should thereafter be determined. When the IBP has been selected, a 
corresponding nucleic acid sequence may be obtained by molecular biology 
techniques (chemical synthesis, cloning, enzymatic modification, and the 
like) and inserted into a suitable vector according to the methodology 
described in the examples. 
Another subject of the invention relates to pharmaceutical compositions 
comprising at least one nucleic acid sequence or one vector as are defined 
above. 
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 DEAE-dextran (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. 
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 intra-cellular 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. 
In a preferred embodiment, the invention hence relates to nucleic acid 
sequences as defined above incorporated in a viral vector. The invention 
also relates to any recombinant virus comprising, inserted into its 
genome, at least one nucleic acid sequence coding for an IBP. 
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. 
Advantageously, the recombinant virus according to the 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. 2 (1990) 739; Miyanohara et al., New Biol. 
4 (1992) 238; WO91/18,088; 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 somewhat, but which are not pathogenic in man, 
and in particular non-immunosuppressed subjects. Moreover, 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 5). 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 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 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 
(i) 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 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 Ad5 
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 present invention hence also relates to a pharmaceutical composition 
comprising at least one defective recombinant virus as defined above. 
The pharmaceutical compositions of the invention may be formulated for the 
purpose of topical, oral, parenteral, intranasal, intravenous, 
intramuscular, subcutaneous, intraocular, and the like, administration. 
Preferably, the pharmaceutical compositions 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 accordance with different parameters, and in particular 
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 viruses according to 
the invention, these are formulated and administered in the form of doses 
of between 10.sup.4 and 10.sup.14 pfu/ml, and preferably 10.sup.6 to 
10.sup.10 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 used for the 
treatment of many pathologies. 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 pathology 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 sequences or vectors of the invention are advantageously 
used for the production in man or animals, in vivo and intracellularly, of 
proteins capable of acting specifically on various cell functions such as 
cell proliferation, synthesis of metabolites, protein synthesis, DNA 
replication and/or transcription, and the like. The present invention thus 
makes it possible to treat specifically, locally and effectively many cell 
dysfunctions at the origin of or resulting from different pathologies, and 
especially cancers, viral or bacterial diseases or, more generally, any 
pathology in which a cellular mediator can be identified. 
Use for the treatment of pathologies linked to cell proliferation 
In an especially advantageous embodiment, the nucleic acid sequences of the 
invention comprise genes coding for IBPs capable of interacting and 
interfering with the activity of factors involved in cell proliferation. 
Cell proliferation involves a multitude of factors such as membrane 
receptors (G proteins), oncogenes, enzymes (protein kinases, farnesyl 
transferases, phospholipases, and the like), nucleosides (ATP, AMP, GDP, 
GTP, and the like), activation factors [guanosine exchange factors (GRF, 
GAP, and the like), transcriptional factors, and the like], and the like. 
The intracellular expression of IBPs according to the invention capable of 
binding and neutralizing such factors enables the process of cell 
proliferation to be controlled. This is especially advantageous in 
situations in which cell proliferation eludes the natural regulatory 
mechanisms, leading, for example, to the appearance of tumours. Many 
factors (products of oncogenic genes 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. 
In an especially advantageous embodiment, the present invention relates to 
vectors containing nucleic acid sequences comprising a gene coding for an 
intracellular antibody capable of interacting with the expression product 
of an oncogene or with a factor participating in the pathway of signalling 
of an oncogene. 
Among target oncogenes, the ras, fos, jun, myc, myb and erb oncogenes may 
be mentioned for the purpose of the invention. 
Among factors participating in the pathway of signalling of an oncogene, 
membrane receptors, which may be targeted at the level of their 
intracellular domains [G proteins, kinases (for example tyrosine kinase), 
phosphorylases, farnesyl transferases], nucleoside exchange factors (GAP, 
GRF, and the like, factors), and the like, may be mentioned in particular. 
More preferably, the present invention relates to vectors, in particular of 
viral origin, containing a nucleic acid sequence coding for an 
intra-cellular antibody directed against an oncogene or a factor 
participating in the pathway of signalling of an oncogene, consisting of a 
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. 
More especially, the invention relates to defective recombinant viruses 
expressing an intra-cellular antibody directed against a factor of the 
ras-dependent signalling pathway. 
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. 
Use for the treatment of viral pathologies 
The nucleic acid sequences according to the invention can also be sequences 
coding for intra-cellular antibodies capable of interacting with the 
infectious cycle of a pathogenic virus (HIV, papillomavirus, and the 
like). 
More especially, in the case of HIV virus, the antiviral agents at present 
available or in protocols of advanced clinical phases do not enable the 
virus to be blocked in its multiplication, but merely make it possible to 
slow down the progression of the disease. One of the main reasons for this 
lies in the appearance of strains resistant to these antiviral agents. The 
development of an effective vaccination also runs into many obstacles: the 
genetic variability of HIV makes it impossible to define a stable 
antigenic structure to give rise to a humoral or cellular immunological 
response against the different existing strains. As in the case of 
antiviral agents where the virus withstands a selection pressure by the 
expedient of point mutations, in the vaccination trials attempted to date, 
HIV appears to elude the immune system. 
The present invention constitutes a new approach for the treatment of HIV 
infection, consisting in blocking the virus during its replicative cycle 
by expression of IBPs, and in particular of intracellular antibodies. The 
present invention makes it possible, in particular, to circumvent the 
problem of genetic variability of the virus, in contrast to the vaccinal 
and antiviral approaches. In the case of traditional vaccination, the 
immune responses detected mainly take place against variable regions. In 
contrast, the use of intracellular antibodies according to the invention 
enables an epitope to be chosen which is not only conserved but also 
essential to the function of a viral protein. Preferably, the 
intracellular antibody is directed against an epitope of sufficient size 
to prevent the virus withstanding and adapting by the expedient of point 
mutations. Moreover, the small size of the gene coding for the 
intracellular antibody according to the invention advantageously enables 
several regions (of only one or of several proteins) to be targeted from 
the same gene therapy vector. 
The two main regulatory proteins of the virus, tat and rev, are targets of 
primary importance for implementing the present invention. In effect, 
these two proteins are essential for viral replication. Furthermore, the 
expression of antisense messenger RNAs or of ribozymes targeting the tat 
or rev messenger RNA prevents viral replication. The mechanism of action 
of these proteins is relatively well documented: tat is a transcription 
transactivation factor, while rev provides for transition between the 
early and late phases of the replicative cycle. These two proteins exert 
their activity by binding to the viral messenger RNAs, and the peptide 
region needed for this binding, which is essential for their function, is 
relatively well delimited. In addition, it has been shown that 
overexpression of their site of binding to RNA (TAR region for tat and RRE 
region for rev) inhibits their function regarding viral expression. 
Under these conditions, an especially advantageous embodiment of the 
invention lies in the use of a DNA sequence coding for an intracellular 
antibody directed against the tat or rev proteins and capable of 
neutralizing their activity. Advantageously, such antibodies are directed 
against the region of tat or rev responsible for their binding to RNA. 
Intracellular antibodies according to the invention directed against these 
epitopes may be prepared according to the methodology described in the 
examples. In particular, as regards anti-tat antibody, this may be 
obtained by genetic modification from T-B monoclonal antibody (12) 
(Hybridolab). 
Another important target protein in HIV replication whose function may be 
readily inhibited by an intracellular antibody is the nucleocapsid protein 
NCP7. In effect, this protein plays an important part in reverse 
transcription (early phase), in integration and in a late but nevertheless 
important phase: encapsidation. This multifunctionality is explained by 
the fact that it has an enzymatically active structural role. It has been 
described as a hybridase which is considered to permit the complexing of 
viral nucleic acids: RNA/DNA and DNA/DNA during reverse transcription, as 
well as RNA/RNA and RNA/tRNA-lys3 during encapsidation. NCP7 appears to be 
essential for the production of infectious viruses. 
The cytoplasmic expression by gene therapy of intracellular antibodies 
directed against NCP7, inhibiting its function in the dimerization of the 
viral RNAs and in encapsidation, constitutes another particular embodiment 
of the invention. 
Intracellular antibodies according to the invention directed against 
neutralizing epitopes of this protein may be prepared according to 
methodology described in the examples. 
Other viral components can also form the subject of a gene therapy 
according to the invention, and in particular: the CD4 molecule binding 
site (for example the envelope glycoprotein (gpl20/41)), the envelope 
multimerization region, the gpl20/gp41 cleavage site, the protease, the 
integrase and, more generally, any viral protein. These domains are not 
generally accessible when the envelope is in native form. For this reason, 
they are very feebly immunogenic and a vaccination employing them is 
impossible. The present invention enables these viral components to be 
targeted, and thus possesses much greater therapeutic potential. In 
addition, as mentioned above, the small size of the genes coding for the 
intracellular antibody according to the invention advantageously makes it 
possible to express simultaneously, in the same vector, several 
intracellular antibodies directed against different regions of one or more 
of these targets. 
The nucleic acid sequences according to the invention can also be sequences 
coding for IBPs capable of interacting and interfering with the activity 
of factors involved in the synthesis of metabolites, in protein synthesis 
or alternatively in DNA replication and/or transcription. 
The present invention will be described more completely by means of the 
examples which follow, which are to be considered to be illustrative and 
non-limiting.

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, N.Y., 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 acrylamide gel electrophoresis, extracted with phenol 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 S1 nuclease. 
Mutagenesis directed in vitro by synthetic 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. 
The enzymatic amplification of DNA fragments by the so-called PCR 
[Polymerase-catalyzed Chain Reaction, Saiki R. K. et al., Science 230 
(1985) 1350-1354; Mullis K. B. and Faloona F. A., 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 Coding for an Intracellular 
Anti-ras Antibody 
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) (J. 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 U.S. Pat. No. 4,946,778. 
This 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 composed of random 
hexanucleotides. The cDNAs obtained serve as a template for two PCR 
reactions: 
one intended for amplifying the heavy-chain 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. 
Two fragments of 340 bp and 325 bp are thereby obtained and then assembled 
by means of a linker which permits a correct positioning of the VH cDNA at 
the 5' end of that of VL. This linker codes for 15 amino acids composed of 
three repeats of the unit (Gly).sub.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 Nos. 1 & 2 (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 (FIG. 1A). This expression 
readily permits identification and selection of the intracellular 
antibodies which correctly recognize the antigen. 
1.2. Functional evaluation of the modified intracellular antibody 
The DNA sequence which codes for the modified 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 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 
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, 
1988). 
The results obtained are presented in FIG. 1C. The analysis of the CAT 
activities obtained demonstrates the capacity of the modified 
intracellular antibody, prepared from Y13-259 antibody, to antagonize 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 (FIG. 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 test in Dulbecco's modified 
Eagle medium (DMEM) containing 10% of foetal calf serum, at 37.degree. C. 
in a humid environment containing 5% of CO.sub.2. These cells are then 
cotransfected with an oncogenic Ras: Ha-ras Val12, plasmid psv2.ScFv.ras 
(see a) above) and a 10-fold 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 transfection, the 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 foci of transformation obtained per 
.mu.g 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. 
______________________________________ 
Number of foci per .mu.g of 
Transfected plasmids 
transfected DNA 
______________________________________ 
Ha-Ras Val12 110 
psv2.ScFv.ras 2 
Ha-Ras Val12 + psv2.ScFv.ras 
30 
______________________________________ 
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 a), 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 intracellular antibody from the antibody, its mRNA or hybridoma 
obtained. Other components involved in processes of cell transformation 
may thus be targeted. 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 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, 259, 
525-528, 1993). 
According to the methodology described in 1.1., it is possible to 
synthesize a DNA sequence coding for an intracellular antibody (ScFv 
fragment) corresponding to this antibody (SEQ ID Nos. (3 and 4 ) residues 
11 to 250, FIG. 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 
decapeptide every other amino acid. 
Synthesis of overlapping peptides 
35 peptides covering the whole of the fragment of FIG. 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 (SEQ ID NO: 5) 
EISF (SEQ ID NO: 6) 
EDGWM (SEQ ID NO: 7) 
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 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: (SEQ ID NO: 8) QYRLKKISKEEKTPGCVKIKKCIIM 
Peptide 2B: (SEQ ID NO: 9) 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 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 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 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 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 intra-cellular 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 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; Munger et al., J. Virol. 63 
(1989) 4417). Particular regions of this protein may be identified by 
"epitope scanning" (see Example 2), 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 after in vivo expression, by measuring: 
the increase in the level of wild-type p53 in cells expressing E6, 
the morphological reversion of HPV-transformed 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. 
EXAMPLE 6 
Preparation of a Nucleic Acid Sequence Coding for an Intracellular Anti-HIV 
Antibody from mRNAs Extracted from Spleens of Mice Immunized with Peptides 
Derived from Active Regions of the tat, rev and NCP7 Proteins 
This example describes the preparation of nucleic acid sequences coding for 
intracellular antibodies according to the invention (such as ScFv 
fragments), 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 further 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 has been described 
in the prior art. 
The targeted antigens in this example are the tat, rev and NCP7 proteins of 
HIV virus. More precisely, the antigens used for the immunization are the 
following peptides of 6, 9 and 16 amino acids, corresponding to the 
regions of these proteins responsible for their interaction with mRNAs (in 
the case of tat and rev) or for the dimerization of RNAs (in the case of 
NCP7): 
tat peptide: (SEQ ID NO: 10) RKKRRQRRR 
rev peptide: (SEQ ID NO: 11) RQARRNRRRRWRERQR 
NCP7 peptide: (SEQ ID NO: 12) RAPRKK 
After immunization of mice with these peptides according to conventional 
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 tat, rev and 
NCP7 peptides 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 functionally for their capacity to 
block the replicative cycle of HIV virus. 
__________________________________________________________________________ 
# SEQUENCE LISTING 
- (1) GENERAL INFORMATION: 
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- (2) INFORMATION FOR SEQ ID NO:1: 
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48 
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624 
Asn Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gl - #y Lys Ser Pro Gln Leu 
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145 1 - #50 1 - #55 1 - 
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48 
Leu Leu Leu Ala Ala Gln Pro Ala Met Ala Gl - #n Val Gln Leu Gln Glu 
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288 
Lys Asp Asn Ser Lys Ser Gln Val Phe Phe Ly - #s Leu Asn Ser Leu Gln 
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432 
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528 
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624 
Ala Thr Lys Pro Gly Asn Gly Val Pro Ser Ar - #g Phe Ser Gly Ser Gly 
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672 
Ser Gly Thr Gln Phe Ser Leu Lys Ile Asn Se - #r Leu Gln Pro Glu Asp 
495 5 - #00 5 - #05 5 - 
#10 
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720 
Leu Gly Asn Tyr Tyr Cys Leu His Phe Tyr Gl - #y Thr Pro Tyr Arg Phe 
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768 
Gly Gly Gly Thr Lys Leu Glu Thr Lys Arg Al - #a Ala Ala Glu Gln Lys 
# 540 
# 798 AG GAT CTG AAT TAA TAA 
#*u Ile Ser Glu Glu Asp Leu Asn * 
# 550 
- (2) INFORMATION FOR SEQ ID NO:4: 
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#acids (A) LENGTH: 264 amino 
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(D) TOPOLOGY: linear 
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- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4: 
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# 15 
- Ser Gly Pro Gly Leu Gly Gln Pro Ala Gln Se - #r Ile Ser Ile Thr Cys 
# 30 
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145 1 - #50 1 - #55 1 - 
#60 
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# 190 
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# 205 
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# 220 
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225 2 - #30 2 - #35 2 - 
#40 
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# 255 
- Leu Ile Ser Glu Glu Asp Leu Asn 
260 
- (2) INFORMATION FOR SEQ ID NO:5: 
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(B) TYPE: amino acid 
(C) STRANDEDNESS: 
(D) TOPOLOGY: linear 
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(B) TYPE: amino acid 
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(D) TOPOLOGY: linear 
- (ii) MOLECULE TYPE: peptide 
- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6: 
- Glu Ile Ser Phe 
- (2) INFORMATION FOR SEQ ID NO:7: 
- (i) SEQUENCE CHARACTERISTICS: 
#acids (A) LENGTH: 5 amino 
(B) TYPE: amino acid 
(C) STRANDEDNESS: 
(D) TOPOLOGY: linear 
- (ii) MOLECULE TYPE: peptide 
- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7: 
- Glu Asp Gly Trp Met 
1 5 
- (2) INFORMATION FOR SEQ ID NO:8: 
- (i) SEQUENCE CHARACTERISTICS: 
#acids (A) LENGTH: 25 amino 
(B) TYPE: amino acid 
(C) STRANDEDNESS: 
(D) TOPOLOGY: linear 
- (ii) MOLECULE TYPE: peptide 
- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8: 
- Gln Tyr Arg Leu Lys Lys Ile Ser Lys Glu Gl - #u Lys Thr Pro Gly Cys 
# 15 
- Val Lys Ile Lys Lys Cys Ile Ile Met 
# 25 
- (2) INFORMATION FOR SEQ ID NO:9: 
- (i) SEQUENCE CHARACTERISTICS: 
#acids (A) LENGTH: 24 amino 
(B) TYPE: amino acid 
(C) STRANDEDNESS: 
(D) TOPOLOGY: linear 
- (ii) MOLECULE TYPE: peptide 
- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:9: 
- Lys Tyr Arg Glu Lys Asn Ser Lys Asp Gly Ly - #s Lys Lys Lys Lys Lys 
# 15 
- Ser Lys Thr Lys Cys Ile Ile Met 
20 
- (2) INFORMATION FOR SEQ ID NO:10: 
- (i) SEQUENCE CHARACTERISTICS: 
#acids (A) LENGTH: 9 amino 
(B) TYPE: amino acid 
(C) STRANDEDNESS: 
(D) TOPOLOGY: linear 
- (ii) MOLECULE TYPE: peptide 
- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:10: 
- Arg Lys Lys Arg Arg Gln Arg Arg Arg 
1 5 
- (2) INFORMATION FOR SEQ ID NO:11: 
- (i) SEQUENCE CHARACTERISTICS: 
#acids (A) LENGTH: 16 amino 
(B) TYPE: amino acid 
(C) STRANDEDNESS: 
(D) TOPOLOGY: linear 
- (ii) MOLECULE TYPE: peptide 
- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:11: 
- Arg Gln Ala Arg Arg Asn Arg Arg Arg Arg Tr - #p Arg Glu Arg Gln Arg 
# 15 
- (2) INFORMATION FOR SEQ ID NO:12: 
- (i) SEQUENCE CHARACTERISTICS: 
#acids (A) LENGTH: 6 amino 
(B) TYPE: amino acid 
(C) STRANDEDNESS: 
(D) TOPOLOGY: linear 
- (ii) MOLECULE TYPE: peptide 
- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:12: 
- Arg Ala Pro Arg Lys Lys 
1 5 
- (2) INFORMATION FOR SEQ ID NO:13: 
- (i) SEQUENCE CHARACTERISTICS: 
#acids (A) LENGTH: 7 amino 
(B) TYPE: amino acid 
(C) STRANDEDNESS: 
(D) TOPOLOGY: linear 
- (ii) MOLECULE TYPE: peptide 
- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:13: 
- Met Pro Lys Lys Lys Arg Lys 
1 5 
- (2) INFORMATION FOR SEQ ID NO:14: 
- (i) SEQUENCE CHARACTERISTICS: 
#pairs (A) LENGTH: 12 base 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
- (ii) MOLECULE TYPE: cDNA 
- (iii) HYPOTHETICAL: NO 
- (iv) ANTI-SENSE: NO 
- (ix) FEATURE: 
(A) NAME/KEY: CDS 
(B) LOCATION: 1..12 
- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:14: 
# 12 
Glu Phe Thr Gly 
270 
- (2) INFORMATION FOR SEQ ID NO:15: 
- (i) SEQUENCE CHARACTERISTICS: 
#acids (A) LENGTH: 4 amino 
(B) TYPE: amino acid 
(D) TOPOLOGY: linear 
- (ii) MOLECULE TYPE: protein 
- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:15: 
- Glu Phe Thr Gly 
1 
- (2) INFORMATION FOR SEQ ID NO:16: 
- (i) SEQUENCE CHARACTERISTICS: 
#pairs (A) LENGTH: 12 base 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
- (ii) MOLECULE TYPE: cDNA 
- (iii) HYPOTHETICAL: NO 
- (iv) ANTI-SENSE: NO 
- (ix) FEATURE: 
(A) NAME/KEY: CDS 
(B) LOCATION: 1..12 
- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:16: 
# 12 
Glu Phe Thr Gly 
5 
- (2) INFORMATION FOR SEQ ID NO:17: 
- (i) SEQUENCE CHARACTERISTICS: 
#acids (A) LENGTH: 4 amino 
(B) TYPE: amino acid 
(D) TOPOLOGY: linear 
- (ii) MOLECULE TYPE: protein 
- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:17: 
- Glu Phe Thr Gly 
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