Polynucleotide immunogenic agents

Polynucleotide immunogenic agents capable of inducing an immune response towards the members of the Epidermal Growth Factor Receptors (EGFR) family are disclosed.

FIELD OF THE INVENTION 
The present invention refers to expression vectors coding for fragments or 
full length erbB proteins (EGF receptors) as immunogenic agents, 
particularly her-2/erbB-2/neu receptor. 
More particularly, the invention refers to the use of said expression 
vectors for the preparation of medicaments inducing a specific immune 
response in the host's cells. 
BACKGROUND OF THE INVENTION 
Different EGF receptors are known, such as EGFR1, HER-2/neu, HER-3, HER-4. 
Particularly HER-2/neu has been proposed as target for antitumor strategies 
(WO 90/14357). 
The proto-oncogene HER-2/neu codes for a 185 KDa membrane receptor protein 
(p185) (Schechter, A. et al. 1984, Nature 312/513). 
The functions of HER-2/neu are presently not well known but they seem to be 
associated to the increase of tyrosine kinase activity (Di Fiore, P. et 
al. 1990 Mol. Cell. Biol. 10:2749). 
Different ligands, which can induce both stimulatory and inhibitory signals 
depending on the ligand and/or the experimental conditions, were proposed 
for the receptor (Peles, E. et al. 1992 Cell. 69:205). 
HER-2/neu is expressed during placentation and organogenesis (Kokay. Y et 
al. 1987 Proc. Natl. Acad. Sci. U.S.A. 84:8498; Knezevic V. et al. 1994 J. 
Anat. 1985:181) and it is detectable in small amounts in a number of 
epithelial/glandular adult tissues (Press. M. et al. 1990 Oncogenes. 
5:953). 
Mutations and/or overexpression of p185 are associated to tumor 
pathogenesis. The proto-oncogene is activated by punctiform mutations 
(Bargmann, C. & Weimberg R. A. 1988 EMBO J. 7:2043). In man mutations were 
not found and it was found that in the carcinomas of glandular origin 
(e.g. breast, ovary, lung and kidney adenocarcinomas), the transforming 
activity is related to an amplification/overexpression of p185 with normal 
structure. 
The amplification of p185 has been associated with an unfavourable 
prognosis in patients with breast carcinoma (Yokota, J. et al. 1986 
Lancet. i. 765; Slamon. D. J. et al. 1987 Science. 235:1772; Yonemura. Y. 
et al. 1991. Cancer Research. 51:1034, Gustarson. B. A. et al. 1992. 
Europ. J. Cancer. 28:263). 
Recent studies also suggest a connection between the receptor 
overexpression and the phenomenon of drug resistance. 
In fact, carcinomas overexpressing HER-2/neu are insensitive to 
5-fluoruracyl treatment (Paikj S. M. et al. 1991 Proc. Am. Assoc. Cancer 
Resear. 32, 291). This was confirmed by transfection studies of HER-2/neu 
in breast carcinoma cell lines becoming resistant to Tamoxifen and 
cisplatin (Benz C. C. et al. 1992 Breast Cancer Research Treat. 24, 84). 
The patients at stage I of breast carcinoma, with good prognosis but with 
immunochemically detectable HER-2/neu, relapse after chemiotherapeutic 
treatment (Alfred D. C. et al. 1992 J. Clin. Onc. 10, 599 and Gusterson B. 
A. et al. 1992 J. Clin. Onc. 10, 1049). Moreover, anti-HER-2/neu 
antibodies increase the cisplatin and tumor necrosis factor (TNF) 
cytotoxicity against the breast and ovary carcinoma (Hancock M. C. et al. 
1991 Canc. Res. 51, 4575 and Hudziac R. M. 1989 Mol. Cell. Biol. 9, 1165). 
On the other hand, in the drug resistant cell lines, the amount of 
epidermal growth factor (EGF) receptor is increased (Meyers M. B. et al. 
1986 Proc. Natl. Acad. Sci. U.S.A. 83, 5521), and the antibodies against 
said receptors increase the cytotoxic effects of drugs (Aboud-Pirak E. et 
al. 1988 J. Natl. Canc. Inst. 80, 1605; Baselga J. et al. 1993 J. Natl. 
Canc. Inst. 85, 1327). 
Similarly to antibodies also EGF increases the cytotoxic effect of 
alkylating agents such as cisplatin, of ionizing rays, of mytomicin, of 
5-fluoruracyl and of adriamycin (Christen R. D. 1990 J. Clin. Inv. 86, 
1632; Kowk T. T. et al. 1989 J. Natl. Canc. Inst. 81, 1020; Amagase H. et 
al. 1990 J. Pharm. Dyn. 13, 263; Amagase H. et al. XXX Jpn. J. Canc. Res. 
80, 670; Kowk T. T. et al. 1991 Int. J. Cancer 49, 73). 
The small amounts of p185 expressed in normal epithelia in comparison to 
the larger amounts in cancer tissues induced to believe that the receptor 
may act as target antigen for passive immunotherapeutic strategies, (e.g. 
by administering monoclonal antibodies). It is known, for instance, that 
some anti-p185 monoclonal antibodies can inhibit the growth of breast 
carcinoma cells both in vitro and in nude mice (Marx. J. 1993 Science 
259:226). 
Recently it has been demonstrated that anti-erbB-2 monoclonal antibodies 
can exert a cytostatic effect on tumor by down-regulation of the receptor 
itself (Katsumatu M. et al., 1985, Nature Medicine 1:644-648). 
Although active immunization strategies (vaccination) were proposed for 
this antigen, this hypothesis was prejudiced by the possibility that the 
induced immunity could be toxic for the epithelia physiologically 
expressing this antigen. 
On the other hand, the inoculation of recombinant vaccinia viruses 
expressing the extracellular domain of neu oncogene completely and 
specifically protected mice against tumor cells expressing this antigen 
(Bernards. R. et al. 1987 Proc. Natl. Acad. Sci. U.S.A. 84:6854). 
Since the growth factors and their receptors play a central role as 
autocrine and paracrine regulatory circuits not only during tumorigenesis 
but also during embryonic development, experimental models based on both 
these developmental processes are particularly significant for the 
purposes of this invention. 
SUMMARY OF THE INVENTION 
The present invention refers to the use of substantially pure cDNA coding 
for homologous foreign HER-2/neu or for another EGF receptor or for 
partial sequences of the said receptors, for the preparation of 
immunogenic medicaments for use in oncological immunotherapy. 
The invention refers also to expression vectors, such plasmids, containing 
one of the above mentioned cDNA. 
The invention refers also to the use of said cDNA for the isolation and 
selection of B-lymphocytes to be used in the production of specific 
monoclonal antibodies.

DETAILED DESCRIPTION OF THE INVENTION 
According to the invention, the in vivo administration of non infectious 
cDNA sequences coding for receptor of EGF, particularly of HER-2/neu, 
induces an immune response useful in antitumoral therapy. 
In fact, such therapy significantly reduces the tumoral masses and inhibits 
the growth of experimentally induced syngenic tumors. 
The effectiveness of the in vivo immunization was also shown using the 
pregnancy model. The embryonic and tumoral development share in fact, at 
least in their initial step, some relevant biological features e.g. the 
increased expression of erbB-2/neu gene. 
In fact, the immune response in pregnant mice turned out to be selective 
against the antigen so as to damage the embryonic development, with 
negligible or no cytotoxic effects on differentiated adult epithelia. Said 
selectivity, according to the observed specific cytotoxic effects, is 
associated with the production of anti p185 antibodies recognizing 
epitopes of the receptor extracellular domain, showing an unusual 
resistance to the formaldheyde treatment. Said selectivity is further 
confirmed by the ability of the antibodies to interact with NHI-3T3 
fibroblasts transformed by the pCDneuNT plasmid, and with the breast tumor 
cell line SK-BR3 overexpressing c-erbB-2, and by staining histological 
samples both from human breast infiltrating ductal carcinomas and from 
nodular mammary adenocarcinoma in neu transgenic mouse. 
Since the inhibition of two processes, tumorigenesis and pregnancy, 
following immunization is surprisingly limited to the structures 
overexpressing erbB-2/neu in developing tissues and not in differentiated 
tissues, it follows that the DNA vaccination with sequences coding for EGF 
receptors may be used as a specific and effective antitumor therapy. 
The invention has several advantages in comparison with the known 
immunotherapies. In comparison with the passive transfer of monoclonal 
antibodies, the polynucleotide vaccination has the advantage of inducing 
not only the production of antibodies but also the production and 
expansion of cytotoxic CD8+MHC class 1 T lymphocytes. 
Even in comparison with the use of peptides with selected sequences 
representing epitopes of the protein against which the immune response has 
to be raised, the advantages of the present invention are remarkable: in 
fact the latter is not limited to selected aplotypes, since potentially T 
cell repertoire for cryptic and subdominant epitopes can be activated by 
homologous foreing antigen in order to obtain Th responses spontaneously 
selected in each subject on the basis of his own aplotype. 
Moreover, as high affinity antibodies are produced by the conventional 
immunization when the antigen concentration becomes limitant, the 
expression of low concentration antigens, as observed with the 
polynucleotide immunization, may induce the development of high affinity 
antibodies already in the first immunization steps. 
In comparison to the immunotherapies with viral vectors, the administration 
of naked DNA is remarkably safer since: 1) DNA is not infective; 2) there 
is no integration of the antigenic DNA in the host chromosome (the 
expression vector remains in episomal form for about one week). 
The cytotoxic effects induced by the viral immunization may decrease the 
number of host cell expressing viral antigens. 
Moreover, many viruses negatively regulate the host's transcriptional 
factors, which may on one side decrease the expression of MHC I class 
molecules on the cell surface and on the other side impair the normal 
processing of the intracellular proteins also in this way decreasing the 
presence of epitopes on MHC molecules. Further advantages of the invention 
are provided by the easy availability and control of the antigens DNA 
sequences. 
According to the invention, the immunogenic agent may be prepared by known 
methods. The DNA sequences of EGFR or HER-2/neu are known and can be 
prepared by conventional methods such as PCR, recombinant DNA etc. It is 
optionally possible to use also partial sequences of genes or chimeric 
structures, providing that the resulting expression product has suitable 
immunogenic properties. For instance, the agent can be prepared by linking 
the EGFR DNA to one or more sequences (promoter and replication origin) 
allowing the expression of said DNA in tissues and in microorganisms for 
the preparation process. The control sequences may be obtained from 
plasmids or viruses such as SV40 or cytomegalovirus (CMV) or Rous sarcoma 
virus (WO 94/21707 of 29.09.94). 
The agent may be administered according to known methods (Donnelly J. J. et 
al. 1994. The immunologist 2:1). 
The effects of intramuscular injections of DNA were considered as a new 
vaccination tool for the treatment of infectious diseases. In addition to 
the intramuscular route, other administration routes of DNA are possible, 
e.g. the parenteral and mucosal route (Proc. Natl. Acad. Sci. U.S.A. 1986 
83, 9551; WO 90/11092 of 4.10.90). DNA may also be adsorbed on gold 
microparticles for the transcutaneous administration by means of balistic 
apparatuses (Johnston, 1992 Nature. 356, 152). 
Moreover, in view of the previous arguments, the immunization has 
remarkable advantages in the preparation of the monoclonal antibodies 
having in vivo biological activity. In fact, the DNA vaccination with said 
oncogene or related proto-oncogene (neuNT, neu) allows the selection, 
directly from the animal which responded to the treatment, the lymphocyte 
lines isolated from the spleen to be used for the preparation of said 
monoclonal antibodies. Similarly said vaccine may be used for the 
selection of human lymphocyte lines from peripheral blood for the 
production of monoclonal-antibodies for use as adjuvants, optionally 
conjugated with cytotoxic molecules to be used in passive immunotherapy. 
EXAMPLE 
1.1 Cloning of pCDneuNT expression vector. 
All the enzymatic manipulations of the DNA, cloning and characterisation of 
the recombinant constructs have been carried out with strict adherence to 
the methods described by Maniatis ("Gene Cloning"). The plasmid pSV2 neuNT 
which contains the cDNA encoding the entire neuNT oncoprotein (ref. 
Weinberg) was digested with the enzymes HindIII, Sal I and EcoRI. 
Digestion with EcoRI generates two fragments of the original plasmid 
(PSV2neo). The combined digestion with HindIII/SalI generates the 
full-length cDNA (about 4600 bp). The HindIII/SalI gene segment has then 
been cloned into the intermediate vector pSP64, previously digested with 
the enzymes HindIII/SalI. The recombinant construct pSP64 neuNT (Amersham) 
has been cloned and characterised as described previously. pSP64 neuNT was 
digested with HindIII/EcoRI generating two fragments: 1) 3000 bp (pSP64); 
2) about 4600 bp (neuNT). Fragment 2 was cloned in pCDNA3 neo (), 
previously prepared and digested with the enzymes HindIII/EcoRI. The final 
recombinant vector was utilised for the immunization procedures. 
1.2 Immunization of mice and rats. 
Female outbred Swiss, CD1 and Balb-c and inbred FVBN and Balb-c 
H.sub.2.sup.D mice were anaesthetised with a nembutal-Equithesinain 
solution at a dose of 0.3 ml/100 g body weight. The injections with an 
insulin syringe (1 ml) delivered 100 .mu.l of a DNA solution in saline (1 
mg/ml). The animals were immunised with three inoculations carried out at 
biweekly intervals. The same immunization schedule was used on Wistar 
rats. 
1.3 In vivo expression of the antigen. 
To verify the capability of the pCDneuNT plasmid of expressing the neuNT 
antigen in vivo, the animals were sacrificed 48 hours from the injections 
and the quadriceps femoral muscles were removed in one piece. The muscular 
tissue was homogenised in the presence of protease inhibitors and of 
non-ionic detergent. The total extracted proteins were then assayed in dot 
blots for the presence of the p185 antigen using monospecific rabbit 
polyclonal anti-neu IgG antibodies (K15) SC-07 (Santa Cruz Biotechnology, 
USA), following a standard procedure (both the dot-blotting and every 
other immunochemical technique reported from here onwards have been 
carried out according to the procedures indicated in: "Antibodies: a 
laboratory manual" 1988 Cold Spring Harbor Laboratory Ed. Harlow/David 
Lane). 
The results have evidenced that only the proteins derived from the muscles 
in which pCDneuNT had been injected, and not those treated with the 
control plasmid, were able to bind to anti-p185 antibodies, from which one 
can conclude that the pCDneuNT plasmid is able to direct the synthesis of 
the p185 molecule inside the muscular fibres. 
1.4 Immunoreactivity in the host. 
i) mouse. 
It is known that the capability of responding to exogenous antigens varies 
greatly according to the animal strain used. Also with respect to the 
immunogenicity of neu rat in the mouse, it is known from the literature 
(Bernards R. et al. 1987 Proc. Natl. Acad. Sci. USA 89: 6854-6858) that 
antibody production against the extracellular domain (ECD) of the oncogene 
by immunization with a vaccine virus is restricted to hosts that belong to 
heterozygous populations. It was therefore necessary to assay the 
anti-p185 immunoreactivity of different strains following the immunization 
with the gene. 
Outbred Balb-c, CD1 or Swiss or inbred Balb-c H.sub.2 D or FVBN mice of 
eight weeks were inoculated either with 100 .mu.g control plasmid or with 
an equal dose of pCDneuNT according to the previously described schedule. 
As controls, the preimmune sera from the same animals were used. 
The production and specificity of the anti-p185 antibodies was evaluated 
through the Western blotting technique, using as antigen source cellular 
lysates from 3T3 cells transformed with pSVneuNT (Bernards, R. et al., 
1987, Proc. Natl. Acad. Sci. USA 84: 6854-6858). The results demonstrate 
that both the homo- and heterozygotic mice were able to recognise the rat 
protein, following the immunization with pCDneuNT. 
The formation of antigen antibody complexes was further evidenced through 
the immunochemical analysis of murine mammary tumors in transgenic MMTV, 
neuN mice characterised by the overproduction of the receptor (C. T. Guy 
et al. 1992 PNAS 89: 10578-582). 
Sections of mammary carcinomas of MMTV, neuN transgenic mice (3-5 u) were 
prepared following the same procedures reported above and incubated with 
antisera derived from mice immunised either with pCDneuNT or with control 
plasmids (dilution of the antiserum 1/30) followed by the addition of goat 
anti-mouse antibodies conjugated with peroxidase (1/100 dilution). It has 
been found that only the pCDneuNT antisera were able to develop a clear 
staining on the membrane of the tumor cells. 
ii) rat: 
The immunization with pCDneuNT in mice represents a classical xenogenic 
immunization given that the procedure presents an immunogenic protein of a 
species (rat) in a host of a different species (mouse). To verify if the 
anti-p185 immunity could be generated through immunization of the gene in 
an entirely syngeneic system, Wistar rats (outbred) have been inoculated 
with the pCDneuNT plasmid using the same immunization scheme as employed 
in the mouse. No anti-p185 seropositivity was evidenced in the treated 
animals even increasing the quantity inoculated in the muscle up to 0.8 
mg. This result is in accordance with that reported by Bernards R. et al. 
Proc. Natl. Acad. Sci. USA 84: 6854-6858 (1987) and reinforces the concept 
that the murine model presents an elevated tolerance (with respect for 
example to the human) towards the self antigen (Houghton A. N. J. Exp. 
Med. 180:1-4 (1994)). It is still possible in any case that the 
simultaneous inoculation of the vector and of drugs through the same or 
other routes could reduce or even avoid the phenomenon of tolerance 
towards self p185. 
1.5 Antibodies specificity 
i) Anti-rat P185 IgGs as autoantibodies. 
Given that the injection of rat pCDneuNT is able to induce the production 
of antibodies reactive against the rat receptor (reactivity against 
neuNT), it was reasonable to expect, for simple philogenetic reasons, that 
the same mouse antibodies should be able to recognise both the mouse 
receptor itself (self antigen) and the homologous human variant erbB-2. 
The immunoreactivity of the circulating anti-p85 antibodies in the mouse 
towards the self antigen was evidenced by histochemical means. 
In the mouse as in the adult rat, the neu proto-oncogene is physiologically 
expressed in epithelial/endocrine tissues such as the lung, liver, kidney, 
gut and in the basal layer of the epidermis cells (Knerewik V. et al. 1994 
J. Anat. 181:1985). In the experiments, the animals were sacrificed with 
an overdose of ether vapours and sections (3-5 u) were prepared from 
tissues that constitutively express erbB-2/neu, such as the kidney, from 
material prepared with the method by Carnoix, and prepared for the 
histological analysis according to a standard procedure. The samples were 
then incubated with the anti-p185 antisera or with control sera at a 1:60 
dilution and successively with goat anti-mouse antibodies conjugated with 
alkaline phosphatase (dilution 1/100). The results clearly prove: a) the 
reactivity of the circulating p185 antibodies towards the kidney cells of 
the same animal and b) the absence of recognition of the same cells by 
antibodies induced following immunization with the control pCDNA-3. The 
identity of murine p185 as the target antigen was then verified by Western 
blotting using mouse kidney homogenate extracts incubated with immune 
mouse serum or with the rabbit anti neu K15 SC-07 antibodies previously 
described. 
ii) Anti-rat p185 IgGs cross-react with human receptor. 
The possibility that the anti-p185 antibodies produced by the mice 
immunised with pCDneuNT could also be able to recognise the human p185 
homologous variant erbB-2, has resulted from studies of immunofluorescence 
in confocal microscopy using SKBR3 cells derived from a human mammary 
carcinoma cell line, that overexpress p185.sup.erbB-2 (10.sup.6 receptor 
molecules/cell; Kraus M. H. et al. 1987 EMBO J. 6: 605-610. 
After the preparation of slides of SKBR3 cells, grown on cover-slip slides, 
fixed, either with a mixture of cold methanol-acetone (1:1 V/V) 
(permeabilised cells) or with 2% paraformaldehyde (non permeabilised 
cells), the cellular samples were incubated with anti-p185 antisera at a 
1/30 final dilution, and the antigen-antibody reaction was evidenced using 
sheep anti-mouse IgG antibodies conjugated with fluorescein (FITC) 
(Boehringer) (dilution 1:60). The mounted slides were examined under a 
laser confocal microscope (BioRad MRC800K) with a triple Argon laser and a 
60.times. oil immersion objective. 
The reactivity of both the non permeabilised cells that showed a strong 
fluorescence distributed in a diffuse way on the extracellular side of the 
plasma membrane, and of the permeabilised ones, that presented a well 
defined fluorescence in the perinuclear region, indicated that the 
anti-p185 antibodies were composed of a mixed population of 
immunoglobulins capable of recognising both the extracellular and 
intracellular domain of the antigen. 
The confirmation that the murine anti-p185 antibodies were able to 
recognise the human variant of the antigen was obtained by western 
blotting using cell lysates of SKBR3, T23.1 (3T3 NIH fibroblasts 
transfected with retroviral erbB-2 DNA), and control 3T3 cells. The 
results indicated clearly the presence, in the sera of mice immunised with 
pCDneuNT, of anti-p185 antibodies, detected with biotinylated goat 
anti-mouse IgG antibodies (Santa Cruz Biotechnology Inc) reacted with 
peroxidase (horseradish) conjugated with streptavidin (1:200). 
The fact that the anti-p185 antibodies could interact with human erbB-2, 
and recognise the extracellular domain of the protein even after strong 
fixation procedures, suggested the possibility of analysing, with these 
antibodies, the overexpression of erbB-2/neu that can be observed in some 
human mammary adenocarcinomas. 
Thus, 21 samples corresponding to the ductal infiltrating carcinoma 
histotype obtained from surgical reseption and taken from the Pathological 
Anatomy Service of the "Ospedale Civile" of Macerata, were examined by 
histochemistry using the same procedures of fixation, inclusion into 
paraffin blocks, staining of the tissue sections followed for the murine 
tissues. 
The anti-p185 positivity of the sera from female mice immunised with 
pCDneuNT was observed only in the 8 out of 21 cases that had resulted 
positive for erbB-2 when stained with commercially available antibodies in 
an absolutely superimposable manner. 
In order to verify if the murine anti-erbB-2/neu antibodies could react 
with other members of the EGFR family members, such as for example EGFR-1, 
the murine antibodies were incubated with cells of the human A431 
epidermoid carcinoma line known to express a very high number of EGFR-1 
receptor molecules (2-3.times.10.sup.6 /cell) on their surface. The 
immunoreactivity of the anti-p185neu antibodies with respect to the 
p170.sup.EGFR-1 was analysed by immunofluorescence with the FACS scann. 
Controls for both the antigen and the antibodies were prepared using 
respectively the lymphoid cells CEM (human cells derived from a patient 
with acute lymphoblastic leukaemia, that do not express EGFR-1) as 
negative control, and monoclonal antibodies W600 (Dr. P. G. Natali, 
Istituto di ricerca sperimentale Regina Elena, Roma) specific for the ECD 
of erbB-2/neu and that do not cross react with the EGFR-1 antigen, as a 
positive control. The cells, after incubation with the second anti-mouse 
FIT conjugated antibody, were analysed using a Bekton Dickinson FACS 
(argon laser light of excitation, 488 nm. Measure of the emitted light at 
525 nm). The results obtained indicate clearly that the anti-p185 
immunoglobulins present in the antisera of the immunised mice behave 
exactly like the W600 MAb, that is they do not express reactivity towards 
p170.sup.EGFR-1. 
1.6 In vitro activity of the p185 antisera. 
Inhibition of the growth of SKBR3 cells by the anti p185 antisera. 
SKBR3 cells were plated in 35 mm culture plates at the concentration of 
10.sup.3 cells/plate. 30 .mu.l (about 300 .mu.g of total IgG) of anti-p185 
antisera and control antisera were added, in duplicate, at day 0. After 
one week, the cells were removed from the plate and their number was 
determined with a haemocytometer. The immunoglobulins from pCDneuNT 
immunised mice, of the IgG isotype, were able to inhibit the growth of 
SKBR3 cells. 
1.7 Biological effects of the immunization. 
i) Toxicity of the treatment. 
The non production of circulating anti-DNA antibodies following the 
administration of the DNA vaccine was demonstrated using a commercially 
available ELISA kit (INOV Diagnostics Inc; San Diego, Calif.) that uses 
highly purified calf thymus double helix DNA as antigen. Variations in the 
IgM and IgG content were not specifically noted. 
Several assays of haematologic and serum parameters, such as haemochrome, 
VES leukocyte formula, proteinaemia etc, have been carried out, all 
resulting within the normal range. 
ii) Histopathological examination of tissues. 
The fact that the immunised animals possess circulating antibodies that can 
interact with the neu receptor of their own tissues, leads to the 
possibility that pathological phenomena of an autoimmune type could arise 
through mechanisms mediated by for example the complement system 
(Complement Dependent Cytotoxicity, CDC) and/or natural killer cells 
(Antibody Dependent Cellular Cytotoxicity, ADCC) (Stribling et al. Proc. 
Natl. Acad. Sci. USA 89: 11277-11281 (1992)). 
In order to verify such a hypothesis, the animals have been sacrificed with 
an overdose of ether vapours and the tissues that constitutively express 
erbB-2/neu, the kidney, gut, ovary, placenta and the mammary gland were 
fixed using conventional methods, the inclusion in paraffin blocks, 
cutting of the sections (3-5 .mu.) and deparaffination of the same have 
been carried out using standard procedures described previously. The 
samples treated in such a way were stained with haematoxilin and eosin. 
Repeated histological examinations have constantly excluded the presence, 
in the immune mouse, of even minimal traces of lesions (evidenced for 
example by necrotic areas, lymphocyte infiltrations or other) in the 
pcDNAneuNT immunised animals. One can therefore state that the DNA 
vaccination is deprived of apparent toxicity both at the general and 
tissue level, indeed, despite the fact that the animal possesses 
antibodies against its own receptor, no sign of systemic or organ specific 
autoimmune pathology was observed. 
To summarise: i) the animals feed and drink normally even one year after 
the vaccination; ii) they show an excellent fur quality; no alteration in 
their individual or social behaviour is noted; iii) several haematological 
parameters remain within the normal range; iv) the tissues in which neu is 
expressed do not present any lesion upon histological examination; v) the 
vaccination does not produce a detectable increase in circulating anti-DNA 
IgM and/or IgG antibodies. 
iii) Effect of the vaccination with pCDneuNT on pregnancy in mice. 
83% of female Swiss mice immunised (n=16) developed circulating anti-p185 
antibodies. 10 seropositive animals were caged with males and mating was 
confirmed the day after (day 1 of gestation) by the presence of sperm 
cells in the vaginal smears. The body weights of the pregnants were 
recorded every two days. Seven out of ten female mice showed a reduced 
number of offspring per litter (5.+-.2 compared to 13.+-.3 in the 
controls) while the hysterectomy of the remaining three demonstrated full 
fetus reabsorption. 
More marked was the effect of said vaccination on the inbred strains such 
as Balb/c H.sub.2.sup.D that all showed a marked development of anti-p185 
antibodies. 14 out of 19 vaccinated female mice, did not give birth at 
all, the others gave birth with a limited number of offsprings (1-2) 
compared with the control ones (15 animals: 8.+-.3 offsprings). 
On the contrary, no significant difference in the fecundation capacity was 
observed between the groups of non-treated mice and of mice immunised with 
the control plasmid or of rats immunised with pCDneuNT (syngeneic 
immunization). 
iv) Histopathological examination of the Placenta and of the mammary 
tissue. 
Seven p185 serum positive pregnant mice were sacrificed at day 18 of 
gestation for the hystological examination of placenta and mammary glands. 
Sections (3-5 .mu.) of formalin fixed tissues were stained as previously 
described. All specimens were found to be devoid of hystopathological 
elements. 
v) Effect on mouse tumors. 
In order to verify the immunotherapeutic effect of the vaccination against 
erbB-2/neu with respect to tumors which overexpress this proto-oncogene, 
tumors were induced in outbred mice according to the following procedure: 
clones of NIH3T3 fibroblasts transfected with CMVneuNT and which express 
substantial levels of the receptor on the cell surface, were inoculated in 
Swiss CD1 mice two weeks after the end of the immunization cycle. The 
growth of the neoplastic masses at the site of injection was measured with 
time through a calibration method. In the non immunised mice or in those 
immunised with the control plasmid, the cells grew progressively for about 
three weeks (See Table). A radically different result was obtained when 
the transformed fibroblasts were inoculated in mice immunised with 
pCDneuNT in which no development of neoplastic masses was observed at the 
site of injection. 
These results demonstrate therefore that the immunization with the CMVneuNT 
plasmid in the form of an injectable physiological solution of naked DNA 
is able to drastically and specifically inhibit the attachment and 
dissemination of the tumor cells that overexpress the neu oncogene. 
TABLE 
______________________________________ 
Tumor area (mm.sup.2) 
1W 2W 3W 
______________________________________ 
A 150 .+-. 60 380 .+-. 50 
580 .+-. 70 
B 150 .+-. 60 410 .+-. 65 
550 .+-. 40 
C nd nd nd 
______________________________________ 
Inhibition of tumor growth. 10 weeks old Swiss CD1 (Charles River) female 
mice (12 per group) were immunized with plasmid DNA encoding both for 
neuNT (C) and for irrelevant gene (p24-FIV) (B) as described in the test. 
Mice in group A received saline injections. After two weeks the mice were 
inoculated with 10.sup.7 3T3 NIH cells transfected with neuNT. W, week 
after challenge. The data refer to mean square of the tumor (.+-.SD). 
nd, tumor not detectable.