Note: Descriptions are shown in the official language in which they were submitted.
CA 02328057 2000-11-14
Composition for the induction of a tumour-specific immune response,
methods for its production and use of the composition
for the treatment of neoplasia
The present invention relates to a composition comprising a phage or a
functionally
equivalent fragment thereof expressing, on its surface, at least one tumour
antigen
as fusion protein with a phage coat protein or a functionally equivalent
derivative
thereof and, optionally, a pharmaceutically acceptable carrier and/or a
pharmaceutically acceptable diluent. The present invention further relates to
a
method for cloning and expressing tumour antigens on phages by which a
specific
immune response is induced. Moreover, the invention relates to a composition
produced by means of said method which is meant for use as injection
preparation
for specific immune therapy of neoplasia in mammals, particularly in humans.
The immune system is the body's own defence system dealing with the fight
against
pathogens. In principle, there are two kinds of defence systems against
infectious
factors: the specific acquired (adaptive) immune response and the non-specific
inherent immune response. In the following, the adaptive immune response, in
particular, which is only triggered by an antigen is of importance. The
special features
of the adaptive immune response are adjustment, memory and specificity. They
are
the consequence of a clonal selection process triggered by antigen contact.
The immune system has a humoral and cellular organisation. With the humoral
immune response there is, amongst others, a destruction of antibody-loaded
tumour
cells by natural killer cells (NK cells) triggered by the antibodies bound to
the cell
surface of tumours contacting the NK cells. This antibody-dependant, cell-
mediated
cytotoxicity (ADCC) is a very effective mechanism for the destruction of
tumour cells.
See also RJ. Dearman et al. 1988 (Blood 72: 1985-91 ) and George AJ. et al.
1987 (J.
Immunol. 140: 1695-170). Within the frame of the cellular immune response,
too, the
CA 02328057 2000-11-14
immune system sees to the fighting of tumour cells: An activation of T-
lymphocytes,
however, presupposes, in most cases, the interaction with antigen-presenting
cells
(APCs). Therefor, the T-cell requires another co-stimulatory signal which is
delivered
by the antigen-presenting cells. The lack of these co-stimuli means that, as a
consequence, the T-cells no longer react to an activation. Due to the lack of
co-
stimulating molecules cancer cells can escape an activation of the immune
system
Some tumour cells can be recognised and destroyed by the immune system. This
function of the immune system becomes clear e.g. by the spontaneous remissions
of
tumours observed at times. With the regression of tumours T-lymphocytes,
amongst - -.
others, play a role, wherein the T-cell reaction against a tumour presupposes
the
recognition of so-called tumour-specific transplant antigens (so-called TSTA).
It is,
however, still not clear why some tumours trigger a spontaneous adaptive
immune
response and others do not. Burnet calls the ability of the immune system to
find and
destroy tumour cells as "immune surveillance" (Burnet FM. 1970, The concept of
immunological surveillance, Prog. Exp. Tumor. Res. 13, 1-27). Due to the wide
spread of cancer diseases it can be seen, however, that the immune
surveillance is
not particularly effective or that, apparently, the tumour cells have
immunological
escape mechanisms which enable them to escape the effect of the functioning
immune system.
Tumour cells can have, e.g. only little immunogenic effect since they lack MHC
molecules or co-stimulatory molecules. Another possibility to escape the
immune
system is that tumour cells expressing antigens detectable by the immune
system
lose said antigens due to a antibody-induced absorption into the cell or due
to
antigenic variation (immune selection). After all, tumours often produce
substances
such as e.g. TGF-~i which directly suppress the immune response.
In the past few years a variety of tumour-associated antigens have been
described
most of which were identified by means of monoclonal antibodies in mice
(Reisfeld
and Sell, eds.: Monoclonal Antibodies and Cancer Therapy, UCLA Symposia on
Molecular and Cellular Biology, Vol. 27, Alan R. Liss, 1985, NY, 1-609). In
spite of
most antigens having proven to be oncofetal or differentiation antigens and
their
tumour specificity constituting a quantitative but not a qualitative feature
since they
also occur on normal cells to a small extent, some antigens are also
sufficiently
specific for neoplastic cells in comparison with normal cells so that they can
potentially be used for the identification of tumour cells and for therapy.
Some of the best characterized tumour-specific antigens are the idiotype
proteins
expressed by neoplastic lymphocytes (Stevenson et al. 1977: Idiotypic
determinants
on the surface immunoglobulin of neoplastic lymphocytes: a therapeutic target.
Fed.
2
CA 02328057 2000-11-14
Proc.; 36: 2268-71 ). In this context an idiotype is the total of antigenic
epitopes
(idiotopes) in the variable region of an antibody. Lymphome cells can be
recognised
selectively via said idiotopes. Thus, it is not surprising that with
immunotherapeutic
strategies in lymphome therapy idiotype vaccination plays a central role.
Animal experiments of various kinds have shown that immunisation with tumour
antigens can lead to the defence against viable cancer cells administered
subsequently. A possible approach for this is the use of cell vaccines
consisting
either of living, killed or genetically modified cancer cells. The
effectiveness of cell
vaccination is based on the induction of cytotoxic T-cells (CTLs) which are
able to
specifically destroy tumour cells. So far, however, it seems to be the case
that the
effectiveness of cell vaccination is not particularly high since not enough
cytotoxic T-
cells can be recruited for an effective tumour therapy to be achieved. Another
approach for immunotherapy of cancer diseases is the immunisation with tumour
antigens. In this context, the destruction of tumour cells is mainly mediated
via an
antibody-dependant cell-mediated cytotoxicity (ADCC). In this context, one of
the
main problems is the weak immunogenicity of the tumour antigens. The reason
for
that is that the tumour-associated antigens representing the targets for such
an
immune response are often present in normal cells, even if only in small
amounts,
and are, thus, recognised by the immune system as "self' against which there
is
normally no immune response. Thus, it is not certain whether cancer patients
can be
treated effectively with this approach. In contrast, the idiotype protein, for
example, is
a tumour-specific antigen which is produced by lymphoma cells exclusively. As
could
be shown in mice and clinical studies, a partly very effective immune response
can
be induced against idiotype proteins. The problem with idiotype vaccination is
that
the idiotype proteins have to be produced for each patient individually and
that
idiotpye proteins themselves are only little immunogenic. Idiotype proteins,
therefore,
have to be coupled to an immunogenic carrier molecule, e.g. KLH so that an
anti-
idiotypic immune response can be induced (George AJ. et al.: Idiotypic
vaccination
as a treatment for a B cell lymphoma. J. Immunol. 1988, 141: 2168-74; Levy R.
et al.:
Therapy of lymphoma directed at idiotypes. J. Natl. Cancer Inst. Monogr. 1990;
1990:61-8). Vaccination with fragments of the idiotype proteins such as the
CDR3-
peptide is by far less effective. (Campbell MJ. et al.: Idiotype vaccination
against
murine B cell lymphoma. Humoral and cellular responses elicited by tumor-
derived
immunoglobulin M and its molecular subunits. J. Immunol. 1987; 139: 2825-33).
Furthermore, it turned out that correct folding is crucial for the
effectiveness of
immunotherapy which is essentially mediated via anti-idiotypic antibodies.
Until
recently, idiotype proteins had to be produced by means of time- and cost-
consuming
hybridoma techniques. Today, however, it is possible to produce a patient-
specific
idiotypic vaccine by recombinant techniques e.g. in form of a single-strand
fragment
3
CA 02328057 2000-11-14
(Hawkins RE. et al.: Idiotypic vaccination against human B-cell lymphoma.
Rescue of
variable region gene sequences from biopsy material for assembly as single-
chain Fv
personal vaccines. Blood 1994; 83: 3279-88). Said single-strand fragments have
meanwhile been successfully tested as DNA vaccines (Spellerberg MB et al.: NA
vaccines against lymphoma: promotion of anti-itiotypic antibody responses
induced
by single chain Fv genes by fusion to tetanus toxin fragment C., J. Immunol.
1997: .
159: 1885-92 and King CA. et al.: DNA vaccines with single-chain Fv fused to
fragment C of tetanus toxin induce protective immunity against lymphoma and
myeloma., Nat. Med. 1998; 4: 1281-6). Single chain (sc) idiotypic vaccines
could also
be produced in tobacco plants and used for vaccination successfully.
(McCormick AA _.
et al.: Rapid production of specific vaccines for lymphoma by expression of
the
tumor-derived single-chain Fv epitopes in tobacco plants. Proc. Natl. Acad.
Sci. USA
1999; 96: 703-8). With the growing importance of tumours with regard to their
absolute frequency, which is mainly due to demographic reasons, there are many
endeavours to treat the cancer effectively by appropriate measures but, at the
same
time to treat the patients with care.
Phages are particles similar to viruses which infect bacteria and can multiply
in them.
At present, A-bacteriophages are frequently used for forming cDNA libraries.
For that
purpose h-ZAP~ vectors have proved to be suitable, as described in the US-A
5,128,
256 (Huse, Sorge, Short). In this patent specification a method is disclosed
wherein
genes can be cloned with the high efficiency of the phage system and
recombinant
phagemid vectors (e.g. BIueScript~ vectors) can be produced via the step of in
vivo
excision without further subcloning processes.
Filamentous phages are oblong stretched phage particles with a length of 7 x
900 nm. They can infect Gram-negative bacteria and multiply here. They consist
of
an oblong capsid in helical order surrounding the single-stranded virus DNA.
The
virus coat consists of five phage proteins which consists of the major coat
protein
cpVlll and the four minor coat proteins cplll, cpVl, cpVll and cplX. Since the
coat
proteins cplll and cpVlll are secreted in the periplasmatic space of Gram-
negative
bacteria to be assembled here as a component of the phage surface, they are an
ideal target molecules for the fusion with idiotypes (cf. US-A 5,658,727
(Barbas,
Kang, Lerner), granted 19 August 1997). At present, filamentous phages are
widely
used within "Phage Display" technology. "Phage Display" technology means the
expression of peptides or proteins in fusion with phage coat proteins on the
phage
surface. Particularly preferred for that are the coat proteins III and VIII.
The gVlll
protein consisting of 50 amino acids is one of the main components of the
phage
coat and is present in approx. 2700 copies in the coat: The gill protein with
its 406
amino acids is essentially bigger, it is, however, only present in 3-5 copies
at the
4
CA 02328057 2000-11-14
infectious end of the phage. The gill protein plays an important role with the
infection
of bacteria. Further coat proteins occurring about as frequently as the gill
protein on
the phage coat are the gVl, gVll and gIX protein with the gVl protein serving
as
anchor of the glll protein and the gVll and gIX proteins being located at the
other end
of the phage.
At present, the "Phage Display" technology is widely used, in particular, when
identifying peptide ligands, in epitope mapping and when selecting recombinant
antibodies.
The technology may, however, also be used to produce immunogenes. Therefore, a
partly good (protective) immune response can be induced against antigenic
epitopes
expressed on phages both with and without adjuvants. See studies by de la Cruz
et
al. 1988 (JBC 263, 4318-22), Greenwood et al. 1991 {JMB 220:821-27), Minenkova
et al. 1993 (Gene 128:85-88), Meola et al. (J. Immunol. 154, 3162-72), Bastien
et al.
1997 (Virol. 234, 118-22). Moreover, DE 69124800 T2 describes genetically
modified
filamentous phages which, amongst others, express immunogenic oligopeptides of
FMDV or of the malaria pathogen Plasmodium falciparum on their surface. The
described immunogenes have in common that small antigenic oligopeptides
expressed by phages are used for vaccination. There, peptides are expressed
either
in fusion with the glll or gVlll protein. By immunisation with said phages a
specific T-
cell dependant humoral immune response can be induced with or without addition
of
an adjuvant against the antigenic peptides specifically cross-reacting with
the original
antigenes (see Willis et al., Gene 1993; 128, 97-83), wherein an immunogenic
effect
preferably was detected with "gVlll vaccines" but, at least, "gVlll vaccines"
were
more effective than "gill vaccines", possible because they exhibit a higher
antigen
density on their surface. There is no indication in the literature that bigger
proteins (or
polypeptides) expressed by phages are effective as immunogenes. On the
contrary,
a person skilled in the art would even advise not to express complex proteins
on
phages, since by doing so the load with antigens would be multiplied and,
thus, the
induction of an immune response would either not be possible at all or not be
specific
enough. By the expression of bigger proteins or domains on phages it was to be
expected - not knowing the results of the present invention - that the
biological
behaviour could be changed in an undesirable way which makes the use of phages
vaccines unsafe.
In the patent specification DE 3703702 A1 a vaccine against melanoma is
described
which essentially comprises pure antigenic peptides or proteins belonging to
the
melanoma-associated p97 antigen. The peptides or proteins can also be
expressed
by recombinant viruses, including bacteriophages, by means of which vaccine
CA 02328057 2000-11-14
formulations on the basis of inactivated viruses or on the basis of active
viruses can
be produced. DE 3703702 A1 does, however, not contain any teaching with regard
to
a possible method for the production of recombinant bacteriophages comprising
said
peptides or proteins belonging to p97. Due to steric reasons, however, it is
not
possible to produce recombinant phages with immunogenic proteins being
expressed
on their surface. It is only possible if phages hybrids are produced. Thus,
there is
only the possibility of a conjugation to a carrier molecule giving
immunogenicity.
Accordlingly, the general concept to present antigenic structures belonging to
p97 as
fusion proteins with a coat protein of viruses or bacteriophages is not
mentioned. -
Inducement of a specific immune therapy against a tumour presupposes the
knowledge of a tumour antigen. With knowledge of such antigen the immune
therapy
can be induced artifically by addition of a specific antibody or by
immunisation with a
tumour antigen. Therapy with monoclonal antibodies has, however, the
disadvantage
that the biological half-life in the body is only short and, therefore, long-
term anti-
tumour effects are not being observed. Furthermore, the risk of tumour escape
mechanisms is high, since the immune response is only directed against an
epitope.
The advantage of active immune therapy by immunisation with a tumour antigen,
however, is the long-lasting immune response. The advantages of said active
immunisation can be seen very clearly in the example of idiotype vaccination:
Induction of a polyclonal, lymphoma-specific and long-lasting immune response.
In
hospital, however, idiotype vaccination has not been applied a lot, since
idiotype
vaccines have to be produced for the patients individually which, up to now,
has only
been possible in complex, time-consuming and expensive hybridoma techniques.
The production of idiotype vaccines as soluble sc-fragments, however, presents
the
difficulty, apart from the folding problem, to produce these in bigger amounts
and to
purify them for therapeutic use. It cannot be expected either that the
expressed
tumour antigens necessarily are immunogens. Accordingly, the technical problem
underlying the present invention was to provide a method which makes an
inexpensive and fast production of patient-specific tumour vaccines possible.
Preferably, these should also be good immunogens.
This technical problem has been solved by providing the embodiments
characterized
in the claims.
Thus, the present invention relates to a composition comprising a phage or a
functionally equivalent fragment thereof expressing at least one tumour-
specific
andlor tumour-associated antigen as fusion protein with a phage coat protein
or a
fragment or a functionally equivalent derivative thereof on its surface and,
optionally,
6
CA 02328057 2000-11-14
a pharmaceutically acceptable carrier andlor a pharmaceutically acceptable
diluent.
Within the meaning of the present invention the term "functionally equivalent
fragment" of a phage comprises fragments of phage particles produced by
physical,
chemical andlor biological treatment and which differ due to said treatments)
morphologically andlor structurally andlor functionally from native phage
particles,
however, exhibit essentially the same immunological, preferably immune-
stimulating
properties as native phage particles. A "functionally equivalent fragment" of
a phage
comprises, for example, phage particles whose genome was destroyed by chemical
treatment without changing the nucleo capsid andlor the coat, or phage
particles
whose genome andlor nucleo capsid and/or coat has been changed by e.g.
enzymatic, heat andlor ultra-sound treatment.
Within the meaning of the present invention the term "functionally equivalent
derivative" of a phage coat protein comprises a phage coat protein exhibiting
an N-
terminal and/or C-terminal andlor internal deletion andlor exhibiting an
insertion, in
addition to the naturally-occurring amino acid sequence, orland whose amino-
acid
sequence differs from the naturally-occurring amino-acid sequence by one or
more
substitutions, while at least the ability to interact with other molecules
(e.g. the viral
genome or other viral coat proteins) necessary for the virion assembly is
maintained.
Suitable "fragments" are known to the person skilled in the art.
Within the meaning of the present invention the term "tumour-specific" and
"tumour-
associated" relates to antigens which are components of cancer cells and can
derive
from the cytoplasm, the cell surface or the nucleus. Preferably, said antigens
are
expressed on the tumour cell surface as surface antigens, they can, however,
also
be detectable as multiplied intracellularly. A presentation of a tumour cell
can e.g.
also take place in the context of MHC class II molecules.
According to the invention "tumour-specific antigens"' are polypeptides which
are
exclusively formed by tumour cells. These can be tumour-specific neo-antigens
which e.g. were formed due to gene rearrangement, mutations or generally as a
consequence of a transformation in malignant cells. A typical example of a
tumour-
specific antigen is the idiotype protein expressed by lymphoma cells.
Furthermore,
tumour-specific antigens can also be proteins formed by activation of
suppressed
genes if they only occur in or on tumour cells. 'Tumour-associated antigens",
however, are, according to definition, antigens which preferably occur in or
on tumour
cells but also are detectable on non-malignant cells. They can, for example,
be
detectable as physiologically-occurring differentiation antigens or as
intermediate
products of the ontogenesis of a cell type which disappear from normal cells
with
further differentiation. Some of said antigens can also be detected
increasingly with
7
CA 02328057 2000-11-14
specific pathological processes in non-tumour cells. Examples of said antigens
are
oncofetal antigens such as the carcinoembryonic antigen (CEA) in colon
carcinomas,
the "squamous cell carcinoma" antigen (SSC) in epithelial tumours, a~ feto
protein in
prim. liver cell carcinoma, isoferritin and fetal sulfoglyco protein in
gastric and colon
carcinoma, a2-H ferro protein in early-childhood malignoma or y-feto protein
in
sarkomas, leukaemia and mammary carcinoma. Other examples according to the
invention are the Tennessee antigen (Tennagen), tissue-polypeptide antigen
TPA,
onkofetal membrane antigens (OFMA), tumour-specific transplantation antigens
(TSTA), membrane-associated antigens (MATA) as well as crypt antigens such as
the "A-like" antigen.
Furthermore, "tumour-associated antigens" within the meaning of the invention
are
polypeptides produced in low copy number on normal, non-tumourous cells and in
high copy number by tumour cells so that, in this case, the specificity for
the tumour
cell is not qualitative but quantitative. Preferably, tumour-associated
antigens are
presented in a copy number enabling a quantitative determination e.g. via flow-
cytometric methods, wherein the signal intensity ("mean fluorescence" value)
is the
same as or higher than the signal intensity in normal cells, however
preferrably at
least higher by the factor 10 or one log-level higher than in normal cells,
which can
clearly be seen by a shift of the curve to the right in the single parameter
histogram.
An example of said antigen is the onkogen Bcl-2 which is clearly overexpressed
e.g.
in lymphoma cells. Furthermore, tumour-associated antigens within the meaning
of
the present invention are further antigens making an effective tumour therapy
possible, even if said therapy is non-specific.
Within the meaning of the present invention the terms "tumour-specific
antigen" and
"tumour-associated antigen" also comprise fragments of the above-mentioned
surface antigens essentially corresponding to the relevant extracellular
domains.
Preferably, the surface antigens or fragments thereof have a length of at
least 20
amino acids, preferably 30, 40 or 50, more preferably a number of amino acids
great
enough far the length comprising at least one immunogenic domain of the tumour
antigen in their three-dimensional structure and, most preferably, the
complete
primary amino-acid sequence of a native tumour antigen. Moreover, the above-
mentioned terms within the meaning of the present invention comprise surface
antigens and fragments thereof whose amino-acid sequence deviates by one or
more substitutions from the amino-acid sequence occurring naturally, while the
amino-acid substitutions preferably represent conservative exchanges. Such
exchanges comprise, for example, the exchange of a neutral, hydrophobic amino
acid, the exchange of a neutral, polaric one, the exchange of an alcaline one
or the
exchange of an acidic amino acid by an amino acid of the respective same class
which are known to the person skilled in the art.
8
CA 02328057 2000-11-14
Preferably, the composition according to the invention is a pharmaceutical
composition.
Ln a particularly preferred embodiment the pharmaceutical composition is a
vaccine.
As mentioned above, the composition of the invention can comprise a
pharmaceutically acceptable carrier and/or a pharmaceutically acceptable
diluent.
Examples of suitable carriers are known to the person skilled in the art and
comprise,
e.g. phosphate-buffered saline solutions, water, emulsions such as oillwater J
emulsions, various types of wetting agents or detergents, sterile solutions
etc.
Compositions comprising said carriers can be produced by means- of known
conventional methods.
The compositions according to the invention can be administered to an
individual in
suitable dosage. Administration can take place orally or parenterally, e.g. in
an
intravenous, an intraperitoneal, a subcutan, perinodal, an intramuscular, a
topic, an
intradermal, intranasal or intrabronchial way or via a catheter at a place in
the artery.
The amount of the dosage is determined by the attending doctor and essentially
depends on clinical factors. These factors are known in medicine and science
and
comprise, e.g. the body size and the weight, the body surface, the age, the
sex and
the general physical condition of the patient, the specific composition to be
administered, the length of treatment, the kind of administration and the
possible
simultaneous treatment with other pharmaceutical compositions. A typical
dosage
can be within a range e.g. between 0.001 and 1000 Ng, while dosages can
possible
be below or above the range given as an example, especially under
consideration of
the above-mentioned factors. In general, the dosage should be within a range
of 1 Ng
and 10 mg units per day when the composition of the invention is administered
regularly. If the composition is administered intravenously, which is not
preferably
recommended to minimise the risk of an anaphylactic reaction, the dosage
should be
within a range of 1 Ng and 10 mg units per kilogram body weight per minute.
The composition of the invention can be administered locally or systemically.
Compositions for a parenteral administration comprise sterile aqueous or non-
aqueous diluents, suspensions and emulsions. Examples of non-aqueous solvents
are propylenglycol, polyethylenglycol, vegetable oils such as olive oil, and
organic
ester compounds such as ethyloleate, which are suitable for injection. Aqueous
carriers comprise water, alcoholic-aqueous solutions, emulsions, suspensions,
saline
solutions and buffered media. Parenteral carriers comprise sodium chloride
solutions,
Ringer's dextrose, dextrose and sodium chloride, Ringer's lactate and bound
oils.
Intravenuous carriers comprise e.g. fluid, nutrition and electrolyte
supplements (as
e.g. the ones based on Ringer's dextrose). The composition according to the
invention can further comprise preservatives and other additives, such as e.g.
9
CA 02328057 2000-11-14
antimicrobial compounds, antioxidants, complex formers and inert gases.
Furthermore, depending on the intended use, compounds such as interleukines,
growth factors, differentiation factors, interferons, chemotactic proteins or
a non-
specific immunomodulating agent can be contained.
Due to the composition according to the invention, surprisingly, a tumour-
specific
immune response is triggered, which exerts a protective immunity among
Vertebrata
against an administration of tumour cells and is essentially based on a
humoral but
also on a cellular immune response. It could be shown in vitro that dendritic
cells
pulsed with the phage vaccines of the invention induce a T-cell proliferation.
It could
further be shown in cytotoxicity assays that a specific lysis of the lymphoma
cells _-
occurs due to Bcl-2 expressing phages. The Daudi-idiotypic phages carried
along as
controls, however, could induce no cytotoxic immune response, as expected,
since
Daudi lymphoma cells express no histocompatibility antigens due to the a2-
microglobulin defect and, thus, are not receptable for cytotoxic T-cell
response.
These data suggest that via antigen-presenting cells a specific T-cell-
depending
immune response can be triggered in vitro. These data further suggest that
said
properties play an important role in vivo, too, and, thus, apart from the
expected
humoral immune response, a specific cell-mediated anti-tumour response is
induced,
by which the effect of the phage vaccine is potentially enhanced. One of the
essential
features is likely to be the preferred absorption of the phages by antigen-
presenting
cells, by which an effective loading with antigens takes place. Contrary to
general
expectations the loading with antigens did not prove to be too high
apparantly.
The composition of the vaccine of the invention allows for a fast and
inexpensive
production of tumour-specific phage vaccines, wherein, preferably, knowledge
of
immunogenic epitopes is not necessary. Equally, the ligands of the tumour
antigens
do not have to be characterized in detail either, nor do the expressed tumour
antigens have to exhibit the same functional effects. A further advantage is
the fact
that an immunisation against several epitopes of a tumour antigen follows the
use of
complex proteins, which induces a polyclonal immune response directed against
the
tumour and, thus, reduces the risk of tumour escape mechanism. This effect
can,
within the meaning of the present invention, be further enhanced by the
expression of
several different tumour antigens on phages in the form of a polyvalent
vaccine. A
further advantage is that epitopes, due to the composition according to the
invention,
can be recognised not only in their linear form, as is the case when using
oligopeptides, but also in their three-dimensional structure-dependant form.
Said
properties are particularly advantageous since they are the prerequisites e.g.
for
effective idiotypic vaccination. With the idiotypic vaccines it can further be
seen that
the presentation of the idiotypic proteins on phages has a very advantageous
effect
on immunisation, since the immune response against only weak immunogenic
CA 02328057 2000-11-14
idiotypic proteins is enhanced by the phages and thereby only becomes fully
usable.
The injection preparation according to the invention can, in principle, be
used in any
mammal and, particularly, also in humans to induce, in the fight against
neoplasia, a
polyclonal, antibody-mediated immune response directed against the tumour. The
advantage of said compositions is a long-term protection of the individual.
The
composition is, as already mentioned above, e.g. in the form of a sterile
solution,
emulsion or suspension. It can optionally contain a suitable antimicrobial
substance,
tools for isotonication as well as isohydria or euhydria. Furthermore,
substances for
the chemical stabilisation (e.g. antioxidants) and substances for the non-
specific
enhancement of the immune response, such as the aluminium hydroxide, can be .
added. The additives are to improve the immunising effect without influencing
the
tolerability in a negative way.
In a preferred embodiment the composition of the invention is an injection
preparation for the specific immune therapy of neoplasia in mammals,
particularly
preferred in humans.
In a further preferred embodiment the composition according to the invention
is an
injection preparation for the patient-specific treatment of lymphoma,
particularly non-
Hodgkin lymphoma or an injection preparation for the specific immune therapy
of
malignant melanoma, urogenital, gynecological andlor gastrointestinal
carcinoma,
thyroid carcinoma andlor bronchial carcinoma.
In ae most preferred embodiment the composition according to the invention is
designed for the subcutaneous, intradermal, perinodal or intraperitoneal
applicationladministration.
In another preferred embodiment of the composition according to the invention
the
phage or the functionally equivalent fragment thereof expresses at least one
cytokine
on its surface. Said cytokine, advantageously is an interleukin, preferably IL-
1 a, IL-
1 Vii, IL-2, IL-4, IL-6, IL-10, IL-12, IL-13; IL-15 andlor IL-18, a growth or
differentiation
factor, preferably GM-CSF, G-CSF, M-CSF andlor FIt3-Ligand, a member of the
TNF-family, preferably TNF-o, LT-a, LT-Vii, OX-40, 4-1 BB or CD40 ligand, a
chemotactic protein, preferably MCP-1, an interferon, preferably IFN-a and/or
IFN-y,
or another immunomodulating agent. Cytokines can also be viral proteins with
similar
functional effect or fragments or analogous proteins which have the same
functional
effect as the cytokine.
The at least one cytokine is expressed preferably as fusion protein with a
phage coat
protein or a functionally equivalent derivative thereof.
11
CA 02328057 2000-11-14
In another preferred embodiment of the composition of the present invention
the
phage belongs to the family of the Corticoviridae, Cystoviridae, Inoviridae,
Leviviridae, Lipothrixviridae, Microviridae, Myoviridae, Plasmaviridae,
Podoviridae,
Siphoviridae, Sulpholobus shibatae viruses (SSV) or Tectiviridae.
In a particularly preferred embodiment the phage is a lambda, PM2-, -6-
cystovirus,
Acholeplasma, MS2-, Qbeta, Thermoproteus1, X174-, Spiroplasma-, Mac-1-, T1-,
T2-, T3-, T4-, T5-, T6-, T7-, P1-, p2-, MS2-, M20- or S13-phage.
In another preferred embodiment the phage is a filamentous phage.
In an alternative embodiment, during the production of recombinant ZAP~ phages
the
tumour antigen (TA) fusion protein is transported into the periplasmatic space
of the
bacteria where it gets its native folding as to structure and is then passed
into the
medium during lysis of the bacteria, since A-bacteriophages do not carry the
cplll
proteins typical of filamentous phages on the cell surface. In other words,
during this
process step tumour-specific antigens are expressed as soluble fusion proteins
which can be purified, for example, via an affinity column. To facilitate the
procedure,
a recognition sequence may be attached to the tumour-specific antigen. In a
further
step the TA fused with cplll can be anchored on the surface of the filamentous
phages after in vivo exzision. The genes of the TA are expressed either in
fusion with
cplll andlor cpVIII. There are 3 - 5 copies of the cplll protein on the phage
surface.
TA in fusion with this molecules can be present in one or two copies. The coat
protein VIII is significantly more frequent on phages (2700-3000 copies) so
that TA
fused with cpVlll can be present in multiple copies, preferably up to 100
copies on
the phage surface.
In a particularly preferred embodiment of the composition according to the
invention
the filamentous phage is a class I phage, preferably a fd- M13-, f1-, If1-,
Ike-, ZJIZ-
and Ff-phage or a class II phage, preferably an Xf, a Pf1- and Pf3-phage.
In an additional particularly preferred embodiment the phage coat protein is
cplll
andlor cpVlll or a functionally equivalent fragment thereof. In the present
specification and in the present claims the terms "cplll" and glll" and
"cpVlll" and
"gVlll", respectivly, are used synonymously.
In a most preferred embodiment the composition of the present invention, the
at least
one tumour-specific andlor tumour-associated antigen is expressed as fusion
protein
with cpVlll or a functionally equivalent fragment thereof and the at least one
cytokine
12
CA 02328057 2000-11-14
as fusion portein with cplll or a functionally equivalent fragment thereof.
In another preferred embodiment the at least one tumour-specific andlor tumour-
associated antigen is a single-strand fragment of a lymphoma-specific
idiotypic
protein or one (or more) antigenic determinants) of the idiotypic protein. The
single-
strand fragment preferably is an scFv fragment or a (chimeric) single-chain
Fab
fragment. The latter can e.g. exhibit human VH and V~ chains and a mouse C-
part. In
a preferred embodiment the rearranged genes of the variable fragments of the
immunoglobulin are amplified and connected to a single strand via a linker
peptide,
see Hawkins et al. 1994 (Blood 83: 3279-88). These are then expressed in
fusion
with a coat protein glll or gVlll of the phages, preferably VIII. This
embodiment allows
for the expression of correctly folded, structurally intact idiotypic proteins
on the
surface of phages.
In another embodiment the present invention relates to a method for the
production
of the composition according to the invention, wherein the method comprises
the
following steps:
(a) growing of a host cell infected with a phage characterized as above, under
conditions which allow for the production of said phage:
(b) isolation of said phage from the culture; and optionally
(c) combination of the phage isolated in step (b) with a pharmaceutically
acceptable carrier andlor diluent.
The phage is isolated from the bacterial supernatant. The concentration
generally
takes place by means of PEGINaCI precipitations. By means of multiple
precipitations phages can be freed from bacterial contamination very
effectively. A
further purification step with the aim to eliminate endotoxins from the phage
preparation can be carried out by means of several methods, wherein, in
principle,
there is the possibility of purification via CsCI density gradient
centrifugation,
ultracentrifugation, polymyxin B-column chromatography or the TitronX-114 two
phase separation and the combination of the various methods. The TitronX-114
two
phase separation has proven particularly advantageous which, when carried out
several times in sequence, makes a reduction of the endotoxin contamination of
initially >300,000 EUlml to below 10 EUlml possible, see Aida, Y. et al. 1990
(J.
Immunol. Methods 132: 191-5}. Said separation presents a feature of a
preferred
embodiment of the method of the invention.
In another or preferred embodiment the invention relates to a method for the
production of the composition according to the invention which is suitable for
the
induction of a tumour-specific immune response in Vertebrate, wherein the
method
comprises the following steps:
13
CA 02328057 2000-11-14
(a) recovery of nucleic acids from an individual tumour cell, wherein the
genes of
tumour-specific antigens are amplified from the nucleic acids;
(b) cloning, preferably after gel-electrophorectic purification methods and
digestion with suitable restriction enzymes, of the genes in a vector system;
and
(c) expression of the genes in a phage expression system.
Due to the purposeful sequence of the processing steps mentioned above, the
production method according to the present invention allows cloning and
expression
of tumour antigens and antigen receptors on phages within a short period of
time. An
injection preparation produced according to this method causes, as mentioned
before, a polyclonal, antibody-mediated immune response directed against the
tumour.
The steps of the method of the present invention for the production of an
injection
preparation are discussed in detail according to a preferred embodiment in the
following:
The individual tumour cells are obtained from the tumour conglomerate
according to
standard methods as described e.g. in Sambrook et al., see below.
The nucleic acid, preferably RNA, is isolated and transfered into cDNA and can
then
be subjected to an RT-PCR-method for fast amplification to multiply the genes
of the
tumour antigens or fragments thereof.
By means of e.g. a gel-electrophoretic purification method (agarose gel
electrophoresis) the DNA fragments are separated according to their size and
purified. By means of a connection PCR reaction recognition sequences for
restriction endonucleases are introduced into the PCR products to make
subsequent
cloning possible. After purification of the gene of a tumour-specific antigen
e.g. with
Geneclean~ (Bio 101 Inc., La Jolla, CA, USA), the genes are split with
different
restriction nucleases. The purification and concentration of the DNA fragments
is
followed by ligation of said DNA fragments in a phagemid vector. This phagemid
vector is a vector as e.g. illustrated in Figure 4. In said phagemid vector
the gene is
preferably cloned N-terminally in the reading frame with the gene of a coat
protein.
Preferably, the coat proteins are glll or gVlll. In a preferred embodiment
there is a
short Gly-Ser-linker between the proteins. Contrary to traditional phage
display
vectors, in the present invention and further preferred embodiments)
preferably
vectors are used which do not carry suppressible Amber mutation or its
equivalent in
front of the coat protein since that could have a negative effect on the
presentation
density. A recognition signal can be attached N- or C-terminally to the tumour
antigen
to make its detection or purification possible. Due to the N-terminal fusion
of the
14
CA 02328057 2000-11-14
recombinant protein with a leader sequence like e.g. the OmpA or p~LB sequence
the transport into the periplasmatic space, where the virus assembly takes
place, is
made possible. By means of phagemid rescue with helper phages such as M13K07
or VCSM13, hybrid phages are produced carrying the tumour antigens as fusion
with
a coat protein on their surface. Due to the hybrid order also normal coat
proteins,
apart from recombinant coat proteins, are present phages, which makes the
virus
assembly and, thus, the expression of greater proteins on the phage surtace
possible
to start with. The amount of the tumour antigens expressed on the surface can
be
optimised by induction of the promoter so that the maximal expression is
stopped,
during which a disruption of the virus assembly has not occurred just yet.
Examples .
of said promoters can be the P~a~, the Ptet~ Pea,o Promoter, while other
promoters are
possible, too.
In an alternative embodiment a J~-ZAP~-bacteriophage system is used as vector
system, as described in the US patent 5,128,256 (Huse, Sorge, Short). After a
packaging process with commercially available packaging extracts (e.g.
GigaPack~,
Fa. Statagene) and the amplification of the bacteriophages a transformation in
filamentous phages follows by means of co-infection with M13 helper phages.
The
underlying principle thereby is that the A-bacteriophage vectors carry a
plasmid or
phagemid vector in them which can be cut out from the phage vector by means of
co-
infection with helper phages. This step is called in vivo excision and was
described
for the first time by Short et al. 1988 (Nucleic Acids Res. 16: 7583) and
Short and
Sorge 1992 (Methods Enzymol. 216: 495-508). An efficient excision of said
phagemids is possible with simultaneous co-infection of an E. coli cell with ~-
ZAP~
and filamentous helper phages. After infection of the bacteria the ssDNA of
the
filamentous phage transducted into the cell is quickly transformed into dsDNA.
Immediately afterwards the expression of phage proteins starts in the host
organism,
whereby the multiplication of the phages can begin. By means of this method it
is
possible to express specific tumour antigens for individual tumour cells so
that they
are present as soluble proteins or as fusion proteins on the phage surface.
In a further preferred embodiment of the method of the present invention the
nucleic
acids are cytosolic RNAs.
In another preferred embodiment of the method the tumour-specific antigen is
fused
with a recognition sequence.
Furthermore, the invention relates to the use of a phage characterized as
above for
the production of a pharmaceutical composition for the induction of a specific
immune
CA 02328057 2000-11-14
response, preferably for the specific immune therapy of neoplasia in a
Vertebrate.
The invention also relates to methods for the induction of a specific immune
response, preferably for the specific immune therapy of neoplasia in a
Vertebrate,
wherein a phage characterized as above or the composition according to the
invention is administered to a Vertebrate.
In a preferred embodiment the neoplasia is a hemoplastosis, preferably a
malignant
lymphoma or leukaemia, a malignant melanoma, a urogenital carcinoma,
preferably
a kidney, bladder, prostate or orchic carcinoma, a gyneological carcinoma,
preferably -
a mammary, an ovary, uterus or a cervix carcinoma, a gastrointestinal
carcinoma,
preferably an esophagus, stomach, colon, rectum, pancreas, gall bladder,
biliary duct
or hepatocellular carinoma, a malignant endocrinological tumour, preferably a
thyroid
carcinoma, a malignant lung tumour, preferably a bronchial carcinoma or
mesothelioma, a sarkoma or a ZNS tumour.
In another preferred embodiment of the use according to the invention and of
the
method according to the invention the Vertebrate is a mammal, preferably a
human.
Finally, in an additional preferred embodiment of the use according to the
invention
or of the method according to the invention the pharmaceutical composition is
produced as injection preparation, preferably for subcutaneous, intradermal,
perinodal, intravenous, peritoneal or intramuscular application or for per os,
rectal,
intrabronchial or intranasal administration.
The subject matter of these documents is herewith incorporated into the
specification
by reference.
The figures show:
Figure 1:
Agarose gel electrophoresis of the PCR products of the idiotypic genes of the
Daudi
cell line (e.g. obtainable from the ATCC) with specific primers for the heavy
chains
(VH and JH primer). (b) Corresponding illustration of the VK and JK primers.
For size
determination of the fragments the molecular weight marker VI by Boehringer
Mannheim was used.
16
CA 02328057 2000-11-14
Figure 2:
Assembly PCR of Daudi VH and VK fragments. The purified VH and VK fragments
are
linked to each other during assembly PCR by means of a single chain Fv (scFv)
(GIy4Ser)3 linker which is complementary to the 3'-end of the JH gene and the
5'-end
of the V~ gene. The markers correspond to the ones of Figure 1.
Figure 3:
Western blot analysis of the scFvlcplll fusion proteins with an anti-kappa
antibody,
which were expressed on filamentous phages, and SDS-PAGE electrophoresis of
scFv/cplll after purification via M1 affinity columns. For the M1 affinity
column
chromatography, the soluble scFv/cplll fusion proteins which were released
into the
medium from the amplification of recombinant surfZAP~ phages were applied in
the
calcium-containing sample buffer. In the Western blot analysis with anti-kappa
antibodies and after M1 affinity chromatography the scFvlcplll fusion protein
can be
detected as a protein with a size of about 48 kD.
Figure 4:
Illustration of the phagemid vectors used in the Examples with Figure 4a
representing
the surfscript~ vector (Stratagene) and Figure 4b representing the cpVlll
script.
Figure 5:
Cytotoxicity assay: Dentritic cetls (DC) pulsed with Bcl2 paghes, Daudi-Id
phages
and wildtype control paghes were used to induce a primary T-cell response.
Unpulsed DCs were also used as control. The T-cells stimulated by DCs after 48
hrs
were added to the tumour cells at various ratios and the cytotoxicity was
measured
after 3 days. A lysis of Bcl-2 phages could clearly be seen, while Daudi Id
phages
induce no lysis.
17
CA 02328057 2000-11-14
The examples illustrate the invention
Example 1:
In this Example, the Bcl-2 antigen is cloned and expressed on the surface of
the
page in fusion with the coat proteins gill and gVlll. The phagemid vectors
used are
shown in Figure 4, with a shortened form of the glll protein being used as a
gill
protein, as has been described in the SurfScript vector (Stratagene). The
gVlll
protein consisted of the complete sequence of the protein that can be detected
in _
filamentous phages. It was fused N-terminally in both coat proteins, with the
restriction sites Notl and Spey being used for cloning. For the amplification
of Bcl-2,
the plasmid DNA of the plasmid PB4 from E. coli 79804 (ATCC) was prepared and
used in a PCR. Primers for the amplification of Bcl-2 were derived from the
Bcl-2
sequence (GenBank M13994) published by Tsujimoto (Tsujimoto and Croce, 1986).
The primers HSBCL2-FOR and HSBCL2-BACK, which were used for the
amplification of Bcl-2, correspond to the nucleic acid 1459-1478 or to the
reverse
complementary sequence 2159-2182 of the aforementioned published article. The
amplification of Bcl-2 was carried out in a 50 NI-PCR with 1 x PCR buffer (10
mM
Tris-HCL, pH 8.8, 1.5 mM MgCI, 25 Mm KCI) (Stratagene), 0.2 mM dNTPs, 2.5 pmol
primer and 2.5 U Taq-polyrnerase (Boehringer Mannheim) in an OmniGene
thermocycler (Hybaid). After 3 minutes of initial denaturation, the Bcl-2 gene
was
amplified in 30 cycles (1 min 94°C, 1 min 55°C, 1 min
72°C) and in a terminal
extension cycle (8 min 72°C). The resulting Bcl-2 PCR product could be
shown as a
0.7 kb-fragment in a 1.5%-agarose gel. In a subsequent PCR, restriction sites
were
inserted so that the product could be cloned into the gill or gVlll phagemid
vector in
frame with the coat protein. The hybrid phages expressing Bcl-2 were produced
as
described in the following section on procedures. The expression of the Bcl-2
protein
as fusion protein with the coat proteins of the filamentous phage was carried
out in a
Western blot analysis.
Examale 2:
In this Example, lymphoma-specific idiotype genes are cloned and expressed on
the
surface of the phage in fusion with cplll. First of all, cytoplasmic RNA was
isolated
from the lymphoma cell Daudi according to known methods. Then, the RNA was
transcribed into cDNA with (dT),5 primers. For one RT-PCR it was sufficient to
produce one single-stranded cDNA. The RT-PCR was carried out in an OmniGene
18
CA 02328057 2000-11-14
thermocycler (Hybaid, clo MWG-Biotech, Ebersberg) under standard conditions,
wherein family-specific human VH and VK primers (cf. Table 1 ) were used for
the
amplification. After the RT-PCR, an agarose gel electrophoresis was conducted
to
analyse the amplification products, i.e. the genes for the heavy chain (VH)
and the
light chain (V~) (cf. Figure 1 ). After extracting the corresponding VH and VK
bands
from the agarose gel, the two bands were purified with Geneclean~ (Bio 101
Inc., La
Jolla, CA, USA) and joined to a single strand (single chain Fv) in an assembly-
PCR
under conditions as described in the section on procedures. The primers used
are
listed in Tables 2 to 4. In an assembly-PCR, recognition sequences for the
restriction
endonucleases are inserted simultaneously into the scFv fragment for
subsequent w
cloning. Then, the product of the PCR was again analysed by means of an
agarose
gel electrophoresis. Subsequently, the scFv fragments were digested with the
respective restriction enzymes and ligated into the g3-script vector. As a
comparison,
a ligation into the SurfZAP vector (Stratagene) was carried out. The SurfZAP
vector
was packed in vitro according to the manufacturer's instructions (cf. section
on
procedures). After in vivo excision (cf. section on procedures), filamentous
phage
hybrids having Daudi idiotypes on the surface were generated by means of
phagemid rescue, as described in the section on procedures. The phage proteins
were separated on SDS polyacrylamide gels for the immunochemical detection of
the
expressed idiotype proteins. In the Western blot analysis, the presence of the
expressed idiotype proteins (cf. Figure 2) could be proven. It was also
possible to
purify overexpressed soluble TA fusion-proteins via M1 affinity-columns, with
the
phages being first diluted 1:1 in a buffer solution (50 mM Tris-HCI, 0.15 M
NaCI, 1
mM CaCl2, 5% Triton X-100, pH 7.0). Then, the pre-equilibrated M1 column
(Kodak)
was sequentially rinsed several times with 5 ml glycine-HCI solution (0.1 M
glycine-
HCI, pH 3.0) and finally equilibrated with 5 ml TBS. The affinity column was
loaded
with the soluble TA in the presence of 1 mM CaCl2. The column was washed three
times with 12 ml TBSICa (TBS buffer + 1 mM CaCl2) and the proteins bound to
the
column were eluated six times with 1 ml glycine-HCI buffer. Tris-HCI, pH 8.0
was
added for neutralization. After washing with TBS buffer, the eluated proteins
were
analysed on polyacrylamide gel.
Example 3:
This Example shows the lymphoma-specific idiotype genes of the cell line Daudi
expressed on the surface of the phage in fusion with cpVlll. Isolation,
amplification
and assembly-PCR of the idiotype genes were carried out as described in
Example
2. After restriction digestion, the patient-specific scFv obtained in this
manner were
19
CA 02328057 2000-11-14
cloned into the cpVlll-script phagemid vector and phages were generated via
phagemid rescue as described in the section on procedures. The Daudi idiotype
proteins expressed on the surface of phages were detected in a Western blot
analysis.
Example 4:
This Example gives in vitro data by which a cellular immune response triggered
by
the phage vaccines produced in Examples 1-3 can be detected. Dendritic cells
were
prepared as described in the section on procedures and pulsed with the phage
idiotype on the 5t" day of the re-differentiation. For this reason, first of
all dendritic
cells (DCs) were split and washed with medium. Then, resuspension was carried
out
in a DMEM/10% FCS at a concentration of 2 x 106 cells per ml of medium.
Subsequently, 500 NI (5 x 105) DCs per well were placed on a 24-well plate.
For the
Loading with antigens, they were pulsed with 50-500 NI phage suspension (1 x
10~~ -
1 x 10~~ pfulml) for several hours (at least 2 hours). The DCs stimulated in
this way
were used in proliferation assays and cytotoxicity experiments. T-cell
proliferation
assays were carried out in 96-well plates. In this case, 5 x 104 T-cells and 5
x 103
irradiated dendritic cells per well were used, which corresponds to an
effectorlstimulation ratio of 10:1. Each experimental set-up was made out in
triplicate.
The DCs loaded with antigens were adjusted to a concentration of 1 x 105
cellslml in
Iscove's medium110% FCS and irradiated with 4000 rad in order to prevent the
3H-
thymidine incorporation of the proliferating T-cells to be superimposed by
potentially
proliferating DCs. After irradiation, the DCs were diluted 1:1. Autologous T-
cells were
adjusted to a concentration of 1 x 106 cellslml. After 1:1 dilution in
Iscove's medium,
100 girl thereof were placed in the respective wells. Wells containing only
DCs or T-
cells were filled up to 200 NI with 100 NI Iscove's medium. In all wells 10 NI
ConA
solution (10 pg/ml Iscove's medium) were pipetted to achieve a submaximum
stimulation of the cells, excluding the wells which were to exhibit a maximum
stabilisation of the T-cells with PHA (50-100 ~rglml). After 48 h, 72 h and 96
h, 25 NI
[3H]-tymidine (25 NCilml) per well and the incorporation of tymidine was
determined
for 18 h. Subsequent to these 18 hours, the 96-well plate was deep-frozen at -
80°C.
The 96-well plates were harvested in a harvester and the glass fibre membranes
were then rrleasured in the Wallac 1460 scintilation counter. For both Bcl-2
phages
and Daudi idiotype phages a proliferation of the T-cells was detected. For
measuring
the cytotoxicity, 1 x 106 T-cells were activated with 5 x 104 antigen-loaded
DCs at a
ratio of 1:20 (stimulatoraarget) for 48 hours. As regards T- and stimulator
cells,
autologous cells taken from the same donor were used. Iscove's medium with
CA 02328057 2000-11-14
20 Ulml IL-2 and 5% FCS was used as medium. Then, the IL-2-activated T-cells
were used for cytotoxic experiments. T-cells and allogenic lymphoma cells
(Daudi or
the lymphoma cell HOL which is a lymphoma cell that is typical of a CBCC non-
Hodgkin lymphoma) were incubated together in different concentrations (10:1,
1:1,
0.1:1 ). Subsequently, the proportion of lysed lymphoma cells were measured
applying standard methods. The result can be seen in Fig. 5 and shows that a
specific lysis may be induced by Bcl-2 phages, whereas, due to the lacking
expression of the histocompatibility antigens on Daudi cells, there is no
lysis in the
control experiment with Daudi idiotype phages.
Methods:
The following methods used by the applicants are described in Sambrook, J.,
Fritsch,
E.F. and Maniatis, T. (ed.), 1989, Molecular Cloning, A Laboratory Manual, 2"d
edition, Cold Spring Harbor Laboratories, Cold Spring Harbor, NY: therefore,
rapid
DNA isolation, restriction digestion, ligation, agarose gel electrophoresis,
immunoblotting, bacterial cultures with different amounts media and
polyacrylamide
gel electrophoresis are not described herein in more detail.
Restriction enzymes. and ligases by Boehringer Mannheim and New England
Biolabs
were used according to the manufacturer's indications.
The E. coli strain 79804 which carries the plasmid PB4 with the Bcl-2 gene was
obtained from the American Type Culture Collection (ATCC, Rockville, MD, USA).
Both the universally available cell line Daudi and the lymphoma cell HOL were
taken
from our culture collection. The experiments can also be carried out or
repeated with
other cancer cellslcancer cell lines which have corresponding properties and
which
are available to the skilled person or which helshe can isolate without
further ado.
RNA preparation modified accordin4 to Favaloro:
Lymphoma cells of the cell line Daudi were used. 1-2 cryotubes containing
approximately 2 x 107 cells were thawed and diluted with 10m1 McCoy medium.
The
cells were centrifuged at 300 x g and washed three times with 5 ml cold PBS
solution
(120 mM NaCI, 2.7 mM KCI, 10 mM K-phosphate buffer, pH 7.4, 4°C). The
cell pellet
was put into 375 NI RNA extraction buffer (50mM Tris-HCI, pH 8.0, 100 mM NaCI,
5
mM MgCl2, 0.5% (vlv) Nonidet P-40, 1 mM DTT, 1000 Ulml RNAsin, 4°C) and
pipetted up and down until a complete lysis took place. Subsequently, it was
incubated in an ice bath, centrifuged and 4 NI 20% SDS was added to the
21
CA 02328057 2000-11-14
supernatant. Finally, the RNA was extracted twice with phenol-chloroform-
isoamyl
alcohol {25:24:1 ). By adding 0.1 vol. 3 M Na acetate, pH 5.2 and 2.5 vol. ice-
cold
ethanol, the RNA was precipitated over night at -20°C or for 30 minutes
at -80°C.
After centrifugation and one washing step, the RNA pellet was put into 50 NI
DEPC-
H20.
cDNA synthesis modified accordinct to Gubler and Hoffmann:
For a cDNA synthesis reaction 1-2 Ng cytoplasmic RNA were used which was
filled
up to a volume of 8 NI with ddH20 and incubated for 10 minutes at 65 °C
in order to
remove disturbing secondary structures. Then it was put on ice. At the same
time,
the cDNA reaction mixture was prepared containing 2 NI reaction buffer (100 mM
Tris-HCI, 500 mM KCI, pH 8.3), 4 NI 25 mM MgCl2, 2 NI 10 mM dNTP mixture, 2 NI
oligo-(dT)~5 primers (0.8 NgINI), 50U RNase inhibitor (Boehringer Mannheim)
and
20 U AMV reverse transcriptase (AMV-RT, Boehringer Mannheim) per reaction
mixture. The denatured RNA was incubated with the cDNA reaction mixture for 10
minutes at 25°C for the oligo-dT primers to hybridize to the RNA. Then,
the first
strand of cDNA was written during an incubation of 1 hour at 37°C.
After the cDNA
synthesis, the AMV-RT was deactivated for 5 minutes at 94°C. The single-
stranded
cDNA was either used immediately in the PCR or stored at -20°C until
further use.
Standard conditions for the PCR:
For a standard PCR 100 ng matrix DNA were used. Each 100 ~I reaction mixture
contained 1 x Taq buffer (10 mM Tris-HCI, 50 mM KCI, pH 8.3), 0.2-0.02 mM dNTP
mixture, 1.5 mM (1-2.5 mM) MgCl2, 20 pmol of primers and 2.5 U Taq-polymerise.
In
the standard reaction 25-30 cycles of 1 min at 94°C (denaturation), 1
min at
annealing temperature (melting temperature of the primer minus 5°C) and
1 min at
72°C (polymerization) were carried out. Instead of Taq polymerise also
a "proof-
reading" polymerise such as Pwo-polymerise can be used for amplification of
DNA
fragments. Pwo-polymerise makes distinctly fewer mistakes.
RT-PCR reaction:
5-20 NI cDNA were used in the RT-PCR reaction. With a total volume of 100 NI,
the
PCR reaction mixture contained 8 NI PCR buffer (identical with cDNA reaction
buffer),
22
CA 02328057 2000-11-14
2 NI 25 mM MgCl2, 1 NI gelatine 0.05%, 0.5 NI Taq DNA polymerise (5 Ullrl) and
1.5 NI upstream and downstream primers (20 pmollNl) per reaction. (The
sequences
of the primers used are listed in the appendix). The RT-PCR mixture was
layered
with 2 drops of mineral oil (Sigma Chemical, St. Louis, MO, USA) in order to
avoid
condensation. The PCR was carried out in an OmniGene thermocycler (Hybaid, clo
MWG-Biotech, Ebersberg) under the following amplification conditions:
Initial cycle
Denaturation 94°C 4 min -
Amplification cycles, 30 cycles
Denaturation 94°C 1 min
Annealing 50-60°C 1 min
Extension 72°C 1 min
Extension cycle
Extension 72°C 8 min
After the RT-PCR reaction, an agarose gel electrophoresis was conducted to
analyse
the amplification products. The visibility of weak bands was improved by
staining with
SYBRT~~-Green (FMC Bioproducts). In part, a proof-reading polymerise like the
Pwo-
polymerase was used instead of Taq-polymerise. Ln this case, MgS04 instead of
MgCl2 had to added to the PCR.
Standard conditions for the assembly PCR of idiotyae genes:
At least 100 ng of the idiotype PCR fragments and/or the heavy and light chain
Were
used for the assembly PCR since the starting amount of primary DNA fragments
is
crucial for the results of the assembly PCR. For removing the primers still
present,
idiotype PCR products were purified with Geneclean (Bio 101 Inc., La Jolla,
CA,
USA). 100 to 500 ng of the two DNA fragments were used in the reaction. An
about
equimolar amount of Linker primers was used. With a total volume of 50 NI, the
assembly reaction mixture contained 5 NI 10 x PCR buffer (100 mM Tris-HCI,
500 mM KCI, pH 8.3), 3 NI 25 mM MgCl2 (MgS04), 100-500 ng each amplified VH
and
VKIa., 2.5 U Taq (Pwo-) DNA polymerise and 2 girl each linker primers (25
pmoIINI) per
reaction. The sequences of the linker primers used are listed in Table II. The
23
CA 02328057 2000-11-14
assembly PCR was layered with 2 drops mineral oil. The amplification
conditions for
the assembly PCR were as follows: an initial cycle of 94°C - 4 min, ten
repeating
cycles of 94°C - 1 min, 60°C - 2 min, 72°C - 1 min.
Immediately after that, a pull-
through PCR with mega primers was carried out to amplify the variable single-
stranded fragments (scFv). Degenerated primers were used which flank the scFv
on
the outside and which additionally contain sequences for restriction
endonucieases, a
pelB-leader sequence and a flag protein. 50 NI master mixture were prepared
for the
PCR with said mega primers. This master mixture contained 5 NI 10 x PCR buffer
#5
(Stratagene, La Jolla, CA, USA), 5 NI formamide 50%, 1 NI 10 mM dNTPs, 20 pmol
each outside primers, 2.5 U Taq (Pwo) DNA polymerise. In this mixture, -the
mixture
of the assembly PCR was pipetted and layered with mineral oil. This pull-
through
PCR was carried out under the following amplification conditions:
Initial cycle
Denaturation 94°C 1 min
Amplification cycles, 5 cycles
Denaturation 94°C 1 min
Annealing 50°C 2 min
Extension 72°C 1 min
Amplification cycles, 30 cycles
Denaturation 94°C 1 min
Annealing 55°C 2 min
Extension 72°C 1 min
Extension cycle
Extension 72°C 8 min
After the PCR reaction, an agarose gel electrophoresis was conducted to
analyse the
PCR products fromed.
Packaginc~ of ~,-bacteriophaaes:
For packaging ~,-bacteriophage DNA packaging extracts by Stratagene,
Heidelberg
were used. 1 Ng ligated phage DNA was incubated briefly on ice in a freeze
thaw
24
CA 02328057 2000-11-14
extract. Then, 15 NI of the thawed Sonic extract were added. The packaging
reaction
as such was then carried out during a two-hour incubation at 22°C. The
reaction was
stopped by adding 500 NI SM buffer (100 mM NaCI, 0.2% MgS04, 50 mM Tris-HCI,
pH 7,5, 2% glv gelatine) and the proteins were precipitated by adding
chloroform.
After a brief centrifugation, the supernatant was transferred into a clean
Eppendorf
vessel and stored at 4°C until amplification. After packaging, the
titre of the produced
infected phage particles was determined.
Amplification of the SurfZAP~-~, aha4es: - -
20 ml TB medium (LB + 0.2% glv maltose + 10 mM MgS04) was over-injected with a
bacteria colony of a fresh overnight culture and incubated in a shaking
incubator until
reaching the late midlog stage. After centrifugation the bacteria were
resuspended in
mM MgS04 and an ODD of 0,5 was adjusted. 600 NI of this bacteria suspension
was infected with 5 x 104 packaged phages. For the absorption of the phages,
incubation for 10 min at 37°C was carried out. Subsequently, the
bacteria were mixed
with warm Top agar and plated onto a 150 mm-agar plate. The plated agar plates
were cultivated for 6-8 hours at 37°C and then coated with an SM
buffer. The agar
plates were incubated over night at 4°C while rotating. During this
time, the amplified
phages diffused in the buffer. It was possible to catch them by sucking them
off. The
amplified phages were centrifuged briefly and stored at 4°C adding 0.3%
chloroform.
In vivo excision and amplification of SurfScript~ phaqes in SOLR~ cells:
For the in vivo excision XL-1 Blue and SOLR cells were cultivated in TB
medium.
After the midlog stage had been reached, the bacteria were centrifuged and
resuspended in 10 mM MgS04. For XL-1 Blue cells an ODs of 5.0 and for SOLR
cells an ODD of 1.0 was adjusted, and the cells were stored at 4°C
until further use.
The XL-1 Blue cells were infected with SurfZAP~ phages. Then, the bacteria
were
super-infected with 1:1 ExAssist~ helper phages. For the absorption of the
phages,
bacteria were incubated in a shaking incubator at 37°C while shaking
gently.
Subsequently, 20 ml LB medium was added to the bacteria and the culture was
incubated in a shaking incubator at 37°C at 150 rpm for 2 to 3 hours.
After culturing,
the bacteria were incubated for 20 min at 70°C, by which the bacteria
and ~,-phages
were killed. After centrifugation, the supernatant containing the SurfScript~
phages
was transferred to a fresh tube and stored for 1-2 months at 4°C.
Amplification was
carried out with SOLR~ strains. They were infected at a ratio of 1:1 and
incubated in
CA 02328057 2000-11-14
100 ml LB medium adding 100 Nglml Carbenicillin and 50 Ng/ml Kanamycin until
the
midlog stage. The bacteria were pelleted by centrifugation and resuspended in
mM MgS04.
Production of comaetent bacteria (according to a method by Hannahan):
The E. coli strain XL-1 Blue was pre-cultivated over night on an LB-agar plate
to
obtain individual colonies. A colony was multiplied in TYM medium (2°~
Bacto
Trypton, 0.5% YEAST extract, 0.1 M NaCI and 10 mM MgS04) until an 4D~ of 0.5.
This suspension was put into 100 ml TYM and multiplied again until an ODD of
0.5.
Then, the bacteria were cooled to 0°C and centrifuged (15 min at 4000
rpm, 4°C).
The pellet was carefully resuspended in 20 ml TFB-I solution (30 mM KOH, 50
mM MnCl2, 100 mM CaCl2, 15% glycerine) and incubated for 15 min at 0°C.
After
centrifugation (3800 rpml5 minl5°C), the pellet was resuspended in 4 ml
TFB-II
solution (10 mM MOPS, pH 7, 0.75 mM CaCl2, 10 mM KCI, 15% glycerin) and
aliquotted in pre-cooled Eppendorf vessels at 100-200 NI. Then, it was frozen
in liquid
nitrogen and stored at -80°C.
Transformation of bacteria:
100 pl competent cells were thawed and 2 NI 0.5 M ~i-ME was added. Part (1-5
NI) of
a 20 NI-ligation mixture was carefully added and carefully mixed with the
cells.
Subsequently, the mixture was incubated for 30 minutes in an ice bath. Then,
the
suspension was incubated for 45 sec at 42°C in a water bath (thermo
chamber) and
again placed in an ice bath for 5 min. 0.9 ml SOB medium was added to the
suspension, then it was shaken for 60 min at 37°C. An aliquot of the
mixture (200 NI)
was plated onto a pre-warmed LB-agar plate plus Ampicillin and incubated over
night
at 37°C. Clones grown in this way were multiplied and analysed by a
standard rapid
disruption method and subsequent restriction digestion.
Phagemid Rescue:
Fiiamentous hybrid phages were generated carrying the tumour antigens on the
surface in fusion with a coat protein. TG1-clones were incubated on Minimal
agar
plates (+ 100 Nglml Ampicillin) over night at 37°C. In the morning, an
inoculating loop
containing bacteria of the plate was diluted in 50 ml 2 x n-AG medium (2x YT
26
CA 02328057 2000-11-14
medium + 100 Ng/ml Amp and 2% glucose) and cultivated until the midlog stage
or
until an OD5so nm of 0.5. When the cells were in the midlog stage (ODSSO nm of
0.5)
M13K07 or VCSM13 helper phages were added with a MOI of 1:10 to the bacteria.
The bacteria were incubated fvr 30 min at 37°C while shaking gently.
The bacteria
were centrifuged and resuspended in 500 ml 2 x YT-AK (2 x YT medium + 100
Nglml
Amp and 50 Ng/ml Kanamycin). IPTG was added at the desired concentration
(normally 0.5-2 mMlml) and the bacteria were cultivated for 18 h in a shaker
at
180 rpm at 30°C. Then, the bacteria culture was centrifuged at 4500 x
upm for 15
minutes at 4°C. The supernatant was transferred to a 1000 ml-vessel and
the phages
were precipitated over night in an ice bath adding 115 vol. (125 ml) PEGlNaCI.
The
phages were then transferred to 250 ml-Corning centrifuge tubes and
centrifuged at
4000 rpm for 15 min at 4°C. The phage pellet was resuspended in 30 ml 1
x PBS and
transferred to Oak-Ridge centrifuge tubes (Nalgene 3115-0050) and centrifuged
for
min at 15,000 rpm at 4°C in an SS34 rotor. Thereby, the remaining cell
components of the bacteria were pelleted. The phages were then precipitated
once
more adding 115 vol. PEGINaCI and resuspended in 5 ml PBS. For pasteurization,
the phages were filtered through a 0.45 pm-sterile filter and stored at
4°C until use or
aliquots of it were deep-frozen at -20°C.
Endotoxin purification of phases by means of Triton X-114:
The phage suspension was sterile filtered and precipitated with 1l5 vol.
PEG800011.5M NaCI. The phages were resuspended in 1 ml PBS. 1 % Triton X-114
was added. 5 min-incubation on ice until the suspension appears to be
homogenous.
The sample was incubated for 5 min at 37°C and briefly centrifuged at
max. speed in
a standard Eppendorf centrifuge. As a result, 2 phases were formed, the top
aqueous
phase containing the phages. This phase is carefully sucked off without
contaminating the bottom phase. This Triton X-114 two-phase separation is
repeated
three times with the aqueous phase. Finally, the phages are again precipitated
with
PEG/NaCI and the phages were resuspended in PBS. The endotoxin content was
determined using the LAL method, with the endotoxin content normally being in
a
range of 0.1 to 10 EU/ml. The Triton X-114 two-phase separation is to be
repeated
until the desired degree of purity has been reached.
27
CA 02328057 2000-11-14
Isolation of leukocytes from the aeripheral blood:
After a blood sample from the vein was taken, 5 ml each of heparin blood was
transferred to a 50 ml-Falcon~ tube and layered with 40 ml of cold Ery-lysis
buffer
(155 mM ammonium chloride, 10 mM potassium hydrogen carbonate, 0.2 mM
EDTA). During the lysis, the solution was incubated for 10 min at 4°C.
Then, the
suspension was centrifuged (1280 rpm, 10 min, 4°C) and washed three
times with
PBS Dulbecco's (GibcoIBRL, Grand Island, NY, USA). The pellet was resuspended
in medium and a concentration of 1 x 107 cellslml was adjusted. Until further
use, the
cells were stored in liquid nitrogen adding 20% FCS and 10% DMSO, or they were
used immediately.
Isolation of mononucleic cells by means of density 4radient centrifu4ation:
Heparin blood was diluted 1:1 in PBS. 20 ml Ficoll separating solution
(Seromed,
Biochrom AG, Berlin), which had a temperature of 20°C was put in a 50
ml-Falcon~
tube and carefully layered with 20 ml PBS blood. After centrifugation (2000
rpm,
20 min, room temperature, without brake), the inter-phase (containing
monocytes,
lymphocytes and blasts) was carefully sucked off with a Pasteur pipette. The
mononuclear cells were washed twice in ice-cold PBS and either processed
immediately or deep-frozen in freezing medium (450 pl RPMI, 450 NI McCOY, 10%
DMSO).
Magnetic cell separation:
For the magnetic cell separation, the cells were incubated with a sufficient
amount of
specific antibodies as regards separation (10-20 NII1 x 107 cells) in 200 NI
PBSIBSA
at 4-8°C for 30 min. For removing the surplus antibodies, the cells
were washed
three times with PBSIBSA (2000 rpm, 4 min, RT). Then, the cells stained with
the
primary antibody were put in 100 NI PBSIBSA and incubated with a second
ferrochrome-containing secondary antibody (25 NII1 x 107 cells) for 30 min at
4°C.
After washing again, the cells treated in this way were applied to a pre-
equilibrated
RS+ column hanging in magnetic field (Milteyni-Biotech) so that the labelled
cells
were magnetically fixed to the column. The cells fixed in that way were washed
in a
sufficient amount of PBSIBSA while the unfixed cells were collected in a
different
28
CA 02328057 2000-11-14
tube for further use. After removing the magnet, the magnetically fixed cells
could be
eluted from the column by washing with PBSIBSA.
Generation of dendritic cells:
For generating dendritic cells, monocytes from a huffy coat or from periphery
blood
were used. After density gradient centrifugation with Ficoll solution (see
above), the
mononucleic cells were adjusted to a concentration of 5-10 x 10g cellslml in
Iscove's
medium (without serum}. 15 ml cells were then pipetted in cell culture flasks
and -
cultivated in an incubator for one hour in order to separate monocytes from
lymphocytes. During this time, the monocytes adhere to the bottom of the
culture
flasks. Subsequently, the lymphocytes that do not adhere to the bottom of the
culture
flasks were sucked off and used for preparing the T-cells. The adhering
monocytes
were washed several times with warm medium and incubated again in an incubator
for one hour. After washing again, the adhering cells were put in RPMI 1640
medium
containing 5°~ FCS or 1 % autologous serum. Normally, the monocytes
were
enriched to about 60% in this way. For obtaining a more pure CD14+ population,
the
adhering monocytes were purified via antibodies coupled with a magnet by means
of
MACS (Miltenyi-Biotech, Bergisch Gladbach) to a degree of more than 95%.
For the redifferentiation into dendritic cells, the monocytes were cultivated
in RPMI
1640 medium adding PenIStrep and L-glutamine at 87°C, 5% C02. 800 Ulml
GM-
CSF and 500 Ulml IL-4 (both cytokines by Genzyme) was added to the medium.
Every 2-4 days, the medium and the cytokines were renewed. The purity of the
preparation and the steps of the redifferentiation into dendritic cells were
controlled
by FACS analyses.
Further methods which are not described in more detail herein, bacteria
strains,
phage strains, and other material are of conventional origin or can be bought.
29
CA 02328057 2000-11-14
Table I: Primer for the polymerase chain reaction (PCR)
A. Primer for the amplification of the variable fragment of the heavy chain:
1. VH primer:
HSVH IA 5'- CDR RVG CAR CTK GTG CAR TCT GG -3'
HSVH 2A 5'- CAG GTN CAR CTG SWG NAG TCK GG -3'
HSVH 3A 5'- SAG GTN CAG STG GTG SAG TCN G -3'
HSVH 4A 5'- CAG GTG CAG CTG CAG SAG TCG -3'
HSVH 5A 5'- SAR STG CAG CTG KTG CAG TCV G -3'
HSVH 6A 5'- CAG GTA CAG CTG CAG CAG TCA -3'
2. CN primer:
HSIGM-CH 5'- AAA GGG TTG GGG CGG ATG CAC TCC -3'
3. JH primer:
HSJH-12FOR 5'- TGA GGA GAC GGT GAC CAG GGT GCC -3'
HSJH-3FOR 5'- TGA AGA GAC GGT GAC CAT TGT CCC -3'
HSJH-45FOR 5'- TGA GGA GAC GGT GAC CAG GGT TCC -3'
HSJH-6FOR 5'- TGA GGA GAC GGT GAC CGT GGT CCC -3'
B. Primer for the amplification of the variable fragment of the K-light chain:
1. VK Primer:
HSVK IA 5'- RHC ATC VRG ATG ACC CAG TCT CC -3'
HSVK 2A 5'- GAW RTT GTG WTG ACN CAG WCT CCA -3'
HSVK 3A 5'- GAA ATW GTR WTG ACR CAG TCT CCA -3'
HSVK 4A 5'- GAC ATC GTG ATG ACC CAG TCT CCA -3'
HSVK 5A 5'- GAA ACG ACA CTC ACG CAG TCT CCA -3'
HSVK 6A 5'- GAW RTT GTG MTG ACW CAG TCT CCA -3'
HSVK 7A 5'- GAC ATT GTG CTG ACC CAG TCT CCA -3'
CA 02328057 2000-11-14
2. JK Primer:
HSJK 124FOR 5'- ACG TTT GAT CTC CAC CTT GGT CCC -3'
HSJK 3FOR 5'- ACG TTT GAT ATC CAC TTT GGT CCC -3'
HSJK SFOR 5'- ACG TTT AAT CTC CAG TCG TGT CCC -3'
C. Primer for the amplification of the variable fragment of the ~,-light
chain:
1. Va, primer:
HSVL 1A 5'- CAG TCT GTG TTG ACG CAG CCG CCC TC -3'
HSVL 2A 5'- CAG TCT GCC CTG ACT CAG CCT GCC TC -3'
HSVL 3A 5'- TCC TAT GAG CTG ACT CAG CCA CVC TC -3'
HSVL 4A 5'- CAC GTT ATA CTG ACT CAA CCG CCC TC -3'
2. Ja, primer:
HSJL 1 FOR 5'- ACC TAG GAC GGT GAC CTT GGT CCC -3'
HSJL 23FOR 5'- ACC TAG GAC GGT CAG CTT GGT CCC -3'
HSJL 7FOR 5'- ACC GAG GAC GGT CAG CTG GGT GCC -3'
31
CA 02328057 2000-11-14
Table II: Primers for assembling light and heavy chains to form a variable
single-
strand fragment (scFv)
(GIy4Ser)3 linker sequence:
GGT GGC GGT GGC TCG GGC GGT GGT GGG TCG GGT GGC GGC GGA TCT
reverse (GIy4Ser)3 linker: --
AGA TCC GCC GCC ACC CGA CCC ACC ACC GCC CGA GCC ACC GCC ACC
scJH linker:
scHSJH-12FOR 5'- CGA CCC ACC ACC GCC CGA GCC ACC GCC ACC
TGA GGA GAC GGT GAC CAG GGT GCC -3'
scHSJH-3FOR 5'- CGA CCC ACC ACC GCC CGA GCC ACC GCC ACC
TGA AGA GAC GGT GAC CAT TGT CCC -3'
scHSJH-45FOR 5'- CGA CCC ACC ACC GCC CGA GCC ACC GCC ACC
TGA GGA GAC GGT GAC CAG GGT TCC -3'
scHSJH-6FOR 5'- CGA CCC ACC ACC GCC CGA GCC ACC GCC ACC
TGA GGA GAC GGT GAC CGT GGT CCC -3'
scVL linker:
scHSVK-1 A 5'- GGC GGT GGT GGG TCG GGT GGC GGC GGA TCT
RHC ATC VRG ATG ACC CAG TCT CC -3'
scHSVK-2A 5'- GGC GGT GGT GGG TCG GGT GGC GGC GGA TCT
GAW RTT GTG WTG ACN CAG WCT CCA -3'
scHSVK-3A 5'- GGC GGT GGT GGG TCG GGT GGC GGC GGA TCT
GAA ATW GTR WTG ACR CAG TCT CCA -3'
scHSVK-4A 5'- GGC GGT GGT GGG TCG GGT GGC GGC GGA TCT
GAC ATC GTG ATG ACC CAG TCT CCA -3'
scHSVK-5A 5'- GGC GGT GGT GGG TCG GGT GGC GGC GGA TCT
GAA ACG ACA CTC ACG CAG TCT CCA -3'
scHSVK-6A 5'- GGC GGT GGT GGG TCG GGT GGC GGC GGA TCT
GAW RTT GTG MTG ACW CAG TCT CCA -3'
scHSVK-7A 5'- GGC GGT GGT GGG TCG GGT GGC GGC GGA TCT
GAC ATT GTG CTG ACC CAG TCT CCA -3'
32
CA 02328057 2000-11-14
scHSVL-1A 5'- GGC GGT GGT GGG TCG GGT GGC GGC GGA TCT
CAG TCT GTG TTG ACG CAG CCG CCC TC -3'
scHSVL-2A 5'- GGC GGT GGT GGG TCG GGT GGC GGC GGA TCT
CAG TCT GCC CTG ACT CAG CCT GCC TC -3'
scHSVL-3A 5'- GGC GGT GGT GGG TCG GGT GGC GGC GGA TCT
TCC TAT GAG CTG ACT CAG CCA CVC TC -3'
scHSVL-4A 5'- GGC GGT GGT GGG TCG GGT GGC GGC GGA TCT
CAC GTT ATA CTG ACT CAA CCG CCC TC -3'
33
CA 02328057 2000-11-14
Table III: Degenerated primers with pelB-leader and FLAG~ Taq recognition
sequence
SZ-pelB-FLAG-HSVH-1 primer:
CTA TTG CCT ACG GCG GCC GCA GGT CTC CTC CTC TTA GCA GCA CAA CCA
GCA ATG GCC GAC TAC AAA GAC GAT CDR RVG CAR CTK GTG CAR TCT GG
SZ-pelB-FLAG-HSVH-2 primer:
CTA TTG CCT ACG GCG GCC GCA GGT CTC CTC CTC TTA GCA GCA CAA CCA
GCA ATG GCC GAC TAC AAA GAC GAT CAG GTN CAR CTG SWG NAG TCK GG
SZ-pelB-FLAG-HSVH-3 primer:
CTA TTG CCT ACG GCG GCC GCA GGT CTC CTC CTC TTA GCA GCA CAA CCA
GCA ATG GCC GAC TAC AAA GAC GAT SAG GTN CAG STG GTG SAG TCN G
SZ-pelB-FLAG-HSVH-4 primer:
CTA TTG CCT ACG GCG GCC GCA GGT CTC CTC CTC TTA GCA GCA CAA CCA
GCA ATG GCC GAC TAC AAA GAC GAT CAG GTG CAG CTG CAG SAG TCG
SZ-pelB-FLAG-HSVH-5 primer:
CTA TTG CCT ACG GCG GCC GCA GGT CTC CTC CTC TTA GCA GCA CAA CCA
GCA ATG GCC GAC TAC AAA GAC GAT SAR STG CAG CTG KTG CAG TCV G
SZ-pelB-FLAG-HSVH-6 primer:
CTA TTG CCT ACG GCG GCC GCA GGT CTC CTC CTC TTA GCA GCA CAA CCA
GCA ATG GCC GAC TAC AAA GAC GAT CAG GTA CAG CTG CAG CAG TCA
34
CA 02328057 2000-11-14
Table IV: Primers with Spel cleavage site
SZ-FX-HSJK/L primer:
AGCATCACTAGT - (CTA)-HSJKIL primer
HSJK 124FOR 5'- AGCATCACTAGT - (CTA) ACG TTT GAT CTC CAC CTT
GGT CCC -3'
HSJK 3FOR 5'- AGCATCACTAGT - (CTA) ACG TTT GAT ATC CAC TTT
GGT CCC -3'
HSJK 5FOR 5'- AGCATCACTAGT - (CTA) ACG TTT AAT CTC CAG TCG
TGT CCC -3'
HSJL 1 FOR 5'- AGCATCACTAGT - (CTA) ACC TAG GAC GGT GAC CTT
GGT CCC -3'
HSJL 23FOR 5'- AGCATCACTAGT - (CTA) ACC TAG GAC GGT CAG CTT
GGT CCC -3'
HSJL 7FOR 5'- AGCATCACTAGT - (CTA) ACC GAG GAC GGT CAG CTG
GGT GCC -3'
CA 02328057 2000-11-14
Claims
1. A composition comprising a bacteriophage or a functionally equivalent
fragment thereof expressing at least one tumour-specific andlor tumour-
associated antigen as fusion protein with a phage coat protein or a fragment
or
a functionally equivalent derivative thereof on its surface, and optionally
containing a pharmaceutically acceptable carrier andlor a pharmaceutically
acceptable diluent.
2. The composition according to claim 1, wherein the phage or the-functionally
--.
equivalent fragment thereof co-expresses at least one cytokine as fusion
protein with a phage coat protein or a functionally equivalent derivative
thereof
on its surface.
3. The composition according to claim 1 or 2, wherein the bacteriophage
belongs
to the family of the Corticoviridae, Cystoviridae, Inoviridae, Leviviridae,
Lipothrixviridae, Microviridae, Myoviridae, Plasma-viridae, Podoviridae,
Siphoviridae, Sulpholobus shibatae viruses (SSV) and Tectiviridae.
4. The composition according to claim 3, wherein the phage is a Lambda, PM2-,
-6-cystovirus, Acholeplasma, MS2-, Qbeta, Thermoproteus1, X174-,
Sprioplasma-, Mac-1-, T1-, T2-, T3-, T4-, T5-, T6-, T7-, P 1-, N2-, MS2-, M20-
or S 13-phage.
5. The composition according to claim 1 or 2, wherein the bacteriophage is a
filamentous phage.
6. The composition according to claim 5, wherein the filamentous phage is a
class I phage, preferably a fd-, M13-, f1-, If1-, Ike-, ZJIZ- or Ff-phage or a
class
II phage, preferably an Xf, Pf1 or Pf3 phage.
7. The composition according to claim 6, wherein the phage coat protein is
cplll
and/or cpVlll or a functionally equivalent fragment thereof.
8. The composition according to claim 7, wherein the at least one tumour-
specific
and/or tumour-associated antigen as fusion protein with cpVlll or a
functionally
equivalent fragment thereof and the at least one cytokine as fusion protein
with cplll or a functionally equivalent fragment thereof is expressed.
36
CA 02328057 2000-11-14
9. The composition according to any one of claims 1 to 8, wherein the at least
one tumour-specific andlor tumour-associated antigen is a single-strand
fragment of a lymphoma-specific idiotype protein or an antigenic determinant
thereof.
10. The composition according to any one of claims 1 to 9 which is a
pharmaceutical composition.
11. The composition according to claim 10 which is a vaccine.
12. A method for the production of a composition according to any one of
claims 1
to 11, wherein the method comprises the following steps:
(a) cultivation of a host cell infected with a phage as defined in any one of
claims 1 to 11 under conditions allowing for the production of said
phage;
(b) isolation of said phage from the culture; and optionally
(c) combination of the phage isolated in step (b) with a pharmaceutically
acceptable carrier andlor diluent.
13. A method, preferably the one according to claim 12, for the production of
a
composition, preferably the one according to any one of claims 1 to 11,
suitable for the induction of a tumour-specific immune response in vertebrata,
wherein said method comprises the following steps:
(a) recovery of nucleic acids from an individual tumour cell, wherein the
genes of tumour-specific antigens are amplified from said nucleic acids;
(b) cloning, preferably after gel-electrophoretic purification methods and
digestion with suitable restriction enzymes of the genes in a vector
system; and
(c) expression of the genes as phage fusion proteins.
14. The method according to claim 13, wherein the nucleic acids are DNAs or
RNAs.
15. The method according to claim 14, wherein the RNAs are cytosolic RNAs.
16. A method according to claim 13 or 14, wherein the tumour-specific antigen
is
fused with a recognition sequence.
17. Use of a phage as defined in any one of claims 1 to 9 for the production
of a
37
CA 02328057 2000-11-14
pharmaceutical composition for the induction of a specific immune response,
preferably for the specific immune therapy of neoplasia in a vertebrata.
18. Use according to claim 17, wherein the neoplasia is hemoplastosis,
preferably
a malignant lymphoma or leukaemia, a malignant melanoma, a urogenital
carcinoma, preferably a kidney, bladder, prostate or orchic carcinoma, a
gyneological carcinoma, preferably a mammary, an ovary, uterus or a cervix
carcinoma, a gastrointestinal carcinoma, preferably an esophagus, stomach,
colon, rectum, pancreas, gall bladder, biliary duct or hepatocellular
carinoma,
a malignant endocrinological tumour, preferably a thyroid carcinoma, a
malignant lung tumour, preferably a bronchial carcinoma or mesothelioma, a
sarkoma or a ZNS tumour.
18. Use according to claims 17 or 18, wherein the vertebrate is a mammal,
preferably a human.
19. Use according to any one of claims 17 to 19, wherein the pharmaceutical
composition is produced as injection preparation for parenteral, preferably
for
subcutane, intradermal, intramuscular, perinodal, intravenous or
intraperitoneal application, or for per os, preferably oral, rectal,
intrabronchial
or intranasal administration.
38
CA 02328057 2000-11-14
' VOSSIUS & PARTNER
PATENTAlVINALTE
SEQUENZPROTOKOLL $ ~ s 7~g~ ~ ~ C ~ ~ N
<110> RdHNISCH, Tim
PACHMANN, Katharina
<120> Zusammensetzung zur Induktion einer tumorspezifischen
Immunantwort, Verfahren zu deren Herstellung Bowie
Verwendung der Zusammensetzung zur Behandlung von
Neoplasien
<130> D1221PCT/a
<140> PCT/EP99/03380
<141> 1999-05-17
<150> DE 198 21 925.3
<151> 1998-05-15
<150> PCT/EP99/03353
<151> 1999-05-14
<160> 57
<170> Pater~tIn Ver. 2.1
<210> 1
<211> 23
<212> DNA
<213> Kiinstliche Sequenz
<220>
<223> Beschreibung der kiinstlichen Sequenz: synthetisch,
kein natiirlicher Ursprung
<400> 1
cdrrvgcarc tkgtgcartc tgg 23
<210> 2
<211> 23
<212> DNA
<213> Kiinstliche Sequenz
<220>
<223> Beschreibung der ktznstlichen Sequenz: synthetisch,
kein natiirlicher Ursprung
<400> 2
1
CA 02328057 2000-11-14
caggtncarc tgswgnagtc kgg _y 23
<210> 3
<211> 22
<212> DNA
<213> Kunstliche Sequenz
<220>
<223> Beschreibung der kunstlichen Sequenz: synthetisch,
kein naturlicher Ursprung _
<400> 3
saggtncags tggtgsagtc ng
22
<210> 4
<211> 21
<212> DNA
<213> Kunstliche Sequenz
<220>
<223> Beschreibung der kunstlichen Sequenz: synthetisch,
kein naturlicher Ursprung
<400> 4
caggtgcagc tgcagsagtc g 21
<210> 5
<211> 22
<212> DNA
<213> Kunstliche Sequenz
<220>
<223> Beschreibung der kunstlichen Sequenz: synthetisch,
kein naturlicher Ursprung
<400> 5
sarstgcagc tgktgcagtc vg 22
<210> 6
<211> 21
<212> DNA
<213> Kunstliche Sequenz
<220>
2
CA 02328057 2000-11-14
<223> Beschreibung der kiinstlichen Sequenz: synthetisch,
kein natiirlicher Ursprung
<400> 6
caggtacagc tgcagcagtc a 21
<210> 7
<211> 24
<212> DNA
<213> Kiinstliche Sequenz
<220>
<223> Beschreibung der kiinstlichen Sequenz: synthetisch,
kein natiiriicher Ursprung
<400> 7
aaagggttgg ggcggatgca ctcc 24
<210> 8
<211> 24
<212> DNA
<213> Kiinstliche Sequer~z
<220>
<223> Beschreibung der kiinstlichen Sequenz: synthetisch,
kein natiirlicher Ursprung
<400> 8
tgaggagacg gtgaccaggg tgcc 24
<210> 9
<211> 24
<212> DNA
<213> Kiinstliche Sequenz
<220>
<223> Beschreibung der kiinstlichen Sequenz: synthetisch,
kein natiirlicher Ursprung
<400> 9
tgaagagacg gtgaccattg tccc 24
<210> 10
<211> 24
3
CA 02328057 2000-11-14
<212> DNA _
<213> Kiinstliche Sequenz
<220>
<223> Beschreibung der kiinstlichen Sequenz: synthetisch,
kein natiirlicher Ursprung
<400> 10
tgaggagacg gtgaccaggg ttcc
24
<210> 11
<211> 24
<212> DNA
<213> Kiznstliche Sequenz
<220>
<223> Beschreibung der kilnstlichen Sequenz: synthetisch,
kein natiirlicher Ursprung
<400> 11
tgaggagacg gtgaccgtgg tccc
24
<210> 12
<211> 23
<212> DNA
<213> Kiinstliche Sequenz
<220>
<223> Beschreibung der kiinstlichen Sequenz: synthetisch,
kein natiirlicher Ursprung
<400> 12
rhcatcvrga tgacccagtc tcc 23
<210> 13
<211> 24
<212> DNA
<213> Kiinstliche Sequenz
<220>
<223> Beschreibung der kiinstlichen Sequenz: synthetisch,
kein natiirlicher Ursprung
<400> 13
gawrttgtgw tgacncagwc tcca 24
4
CA 02328057 2000-11-14
<210> 14
<211> 24
<212> DNA
<213> Kiinstliche Sequenz
<220>
<223> Beschreibung der kiinstlichen Sequenz: synthetisch,
kein natilrlicher Ursprung
<400> 14
gaaatwgtrw tgacrcagtc tcca ~ 24
<210> 15
<211> 24
<212> DNA
<213> Kiinstliche Sequenz
<220>
<223> Beschreibung der kunstlichen Sequenz: synthetisch,
kein natiirlicher Ursprung
<400> 15
gacatcgtga tgacccagtc tcca 24
<210> 16
<211> 24
<212> DNA
<213> Kiinstliche Sequenz
<220>
<223> Beschreibung der kitnstlichen Sequenz: synthetisch,
kein natiirlicher Ursprung
<400> 16
gaaacgacac tcacgcagtc tcca 24
<210> 17
<211> 24
<212> DNA
<213> Kiinstliche Sequenz
<220>
<223> Beschreibung der kunstlichen Sequenz: synth.etisch,
CA 02328057 2000-11-14
kein natiirlicher Ursprung
<400> 17
gawrttgtgm tgacwcagtc tcca 24
<210> 18
<211> 24
<212> DNA
<213> Kiinstliche Sequenz
<220>
<223> Beschreibung der kiinstlichen Sequenz: synthetisch, -
kein natiirlicher Ursprung
<400> 18
gacattgtgc tgacccagtc tcca 24
<210> 19
<211> 24
<212> DNA
<213> Kiinstliche Sequenz
<220>
<223> Beschreibung der kiinstlichen Sequenz: synthetisch,
kein natiirlicher Ursprung
<400> 19
acgtttgatc tccaccttgg tccc 24
<210> 20
<211> 24
<212> DNA
<213> Kiinstliche Sequenz
<220>
<223> Beschreibung der kiinstlichen Sequenz: synthetisch,
kein natiirlicher Ursprung
<400> 20
acgtttgata tccactttgg tccc 24
<210> 21
<211> 24
<212> DNA
6
CA 02328057 2000-11-14
<213> Kiinstliche Sequenz
<220>
<223> Beschreibung der kiinstlichen Sequenz: synthetisch,
kein natiirlicher Ursprung
<400> 21
acgtttaatc tccagtcgtg tccc 24
<210> 22
<211> 26
<212> DNA _
<213> Kiinstliche Sequenz
<220>
<223> Beschreibung der kiinstlichen Sequenz: synthetisch,
kein natiirlicher Ursprung
<400> 22
cagtctgtgt tgacgcagcc gccctc 26
<210> 23
<211> 26
<212> DNA
<213> Kiinstliche Sequenz
<220>
<223> Beschreibung der kiznstlichen Sequenz: synthetisch,
kein natiirlicher Ursprung
<400> 23
cagtctgccc tgactcagcc tgcctc 26
<210> 24
<211> 26
<212> DNA
<213> Kiinstliche Sequenz
<220>
<223> Beschreibung der kiinstlichen Sequenz: synthetisch,
kein natiirlicher Ursprung
<400> 24
tcctatgagc tgactcagcc acvctc 26
7
i
CA 02328057 2000-11-14
<210> 25
<211> 26
<212> DNA
<213> Kiinstliche Sequenz
<220>
<223> Beschreibung der kiinstlichen Sequenz: synthetisch,
kein natiirlicher Ursprung
<400> 25
cacgttatac tgactcaacc gccctc 26
<210> 26
<211> 24
<212> DNA
<213> Kiinstliche Sequenz
<220>
<223> Beschreibung der kiinstlichen Sequenz: synthetisch,
kein nattzrlicher Ursprung
<400> 26
acctaggacg gtgaccttgg tccc 24
<210> 27
<211> 24
<212> DNA
<213> Kiinstliche Sequenz
<220>
<223> Beschreibur_g der kiinstlichen Sequenz: synthetisch,
kein natiirlicher Ursprung
<400> 27
acctaggacg gtcagcttgg tccc
24
<210> 28
<211> 24
<212> DNA
<213> Kiinstliche Sequenz
<220>
<223> Beschreibung der kiinstlichen Sequenz: synthetisch,
kein natiirlicher Ursprung
8
CA 02328057 2000-11-14
<400> 28
accgaggacg gtcagctggg tgcc 24
<210> 29
<211> 45
<212> DNA
<213> Kiinstliche Sequenz
<220>
<223> Beschreibung der kiinstlichen Sequenz: synthetisch,
kein natiirlicher Ursprung
<400> 29
ggtggcggtg gctcgggcgg tggtgggtcg ggtggcggcg gatct 45
<210> 30
<211> 45
<212> DNA
<213> Kiinstliche Sequenz
<220>
<223> Beschreibung der kiinstlichen Sequenz: synthetisch,
kein naturlicher Ursprung
<400> 30
agatccgccg ccacccgacc caccaccgcc cgagccaccg ccacc 45
<210> 31
<211> 54
<212> DNA
<213> Kiznstliche Sequenz
<220>
<223> Beschreibung der kiinstlichen Sequenz: synthetisch,
kein natiirlicher Ursprung
<400> 31
cgacccacca ccgcccgagc caccgccacc tgaggagacg gtgaccaggg tgcc 54
<210> 32
<211> 54
<212> DNA
<213> Kiinstliche Sequenz
9
i!
CA 02328057 2000-11-14
<220>
<223> Beschreibung der kilnstlichen Sequenz: synthetisch,
kein natiirlicher Ursprung
<400> 32
cgacccacca ccgcccgagc caccgccacc tgaagagacg gtgaccattg tccc 54
<210> 33
<211> 54
<212> DNA
<213> Kiinstliche Sequenz
<220>
<223> Beschreibung der kiinstlichen Sequenz: synthetisch,
kein natiirlicher Ursprung
<400> 33
cgacccacca ccgcccgagc caccgccacc tgaggagacg gtgaccaggg ttcc 54
<210> 34
<211> 54
<212> DNA
<213> Kiinstliche Sequenz
<220>
<223> Beschreibung der kiinst_ichen Sequenz: synthetisch,
kein natiirlicher Ursprung
<400> 34
cgacccacca ccgcccgagc caccgccacc tgaggagacg gtgaccgtgg tccc 54
<210> 35
<211> 53
<212> DNA
<213> Kiinstliche Sequenz
<220>
<223> Beschreibung der kiznstlichen Sequenz: synthetisch,
kein natiirlicher Ursprung
<400> 35
ggcggtggtg ggtcgggtgg cggcggatct rhcatcvrga tgacccagtc tcc 53
CA 02328057 2000-11-14
<210> 36
<211> 54
<212> DNA
<213> Kunstliche Sequenz
<220>
<223> Beschreibung der kunstlichen Sequenz: synthetisch,
kein naturlicher Ursprung
<400> 36
ggcggtggtg ggtcgggtgg cggcggatct gawrttgtgw tgacncagwc tcca 54
<210> 37
<211> 54
<212> DNA
<213> Kunstliche Sequenz
<220>
<223> Beschreibung der kunstlichen Sequenz: synthetisch,
kein naturlicher Ursprung
<400> 37
ggcggtggtg ggtcgggtgg cggcggatct gaaatwgtrw tgacrcagtc tcca 54
<210> 38
<211> 54
<212> DNA
<213> Kunstliche Sequenz
<220>
<223> Beschreibung der kunstlichen Sequenz: synthetisch,
kein naturlicher Ursprung
<400> 38
ggcggtggtg ggtcgggtgg cggcggatct gacatcgtga tgacccagtc tcca 54
<210> 39
<211> 54
<212> DNA
<213> Kunstliche Sequenz
<220>
<223> Beschreibung der kunstlichen Sequenz: synthetisch,
kein naturlicher Ursprung
11
CA 02328057 2000-11-14
<400> 39
ggcggtggtg ggtcgggtgg cggcggatct gaaacgacac tcacgcagtc tcca 54
<210> 40
<211> 54
<212> DNA
<213> Kunstliche Sequenz
<220>
<223> Beschreibung der kunstlichen Sequenz: synthetisch,
kein naturlicher Ursprung
<400> 40
ggcggtggtg ggtcgggtgg cggcggatct gawrttgtgm tgacwcagtc tcca 54
<210> 41
<211> 54
<212> DNA
<213> Kunstliche Sequenz
<220>
<223> Beschreibung der kunstlichen Sequenz: synthetisch,
kein naturlicher Ursprung
<400> 41
ggcggtggtg ggtcgggtgg cggcggatct gacattgtgc tgacccagtc tcca 54
<220> 42
<211> 56
<212> DNA
<213> K~nstliche Sequenz
<220>
<223> Beschreibung der kunstlichen Sequenz: synthetisch,
kein naturlicher Ursprung
<400> 42
ggcggtggtg ggtcgggtgg cggcggatct cagtctgtgt tgacgcagcc gccctc 56
<210> 43
<211> 56
<212> DNA
<213> Kunstliche Sequenz
12
CA 02328057 2000-11-14
<220>
<223> Beschreibung der kunstlichen Sequenz: synthetisch,
kein naturlicher Ursprung
<400> 43
ggcggtggtg ggtcgggtgg cggcggatct cagtctgccc tgactcagcc tgcctc 56
<210> 44
<211> 56
<212> DNA
<213> KUnstliche Sequenz
<220>
<223> Beschreibung der kunstlichen Sequenz: synthetisch,
kein naturlicher Ursprung
<400> 44
ggcggtggtg ggtcgggtgg cggcggatct tcctatgagc tgactcagcc acvctc 56
<210> 45
<211> 56
<212> DNA
<213> Kunstliche Sequenz
<220>
<223> Beschreibung der kunstlichen Sequenz: synthetisch,
kein nat~rlicher Ursprung
<400> 45
ggcggtggtg ggtcgggtgg cggcggatct cacgttatac tgactcaacc gccctc 56
<210> 46
<211> 95
<212> DNA
<213> Kunstliche Sequenz
<220>
<223> Beschreibung der kunstlichen Sequenz: synthetisch,
kein nat~rlicher Ursprung
<400> 46
ctattgccta cggcggccgc aggtctcctc ctcttagcag cacaaccagc aatggccgac 60
tacaaagacg atcdrrvgca rctkgtgcar tctgg 95
13
CA 02328057 2000-11-14
<210> 47
<211> 95
<212> DNA
<213> Kunstliche Sequenz
<220>
<223> Beschreibung der kiinstlichen Sequenz: synthetisch,
kein natiirlicher Ursprung
<400> 47
ctattgccta cggcggccgc aggtctcctc ctcttagcag cacaaccagc aatggccgac 60 _
tacaaagacg atcaggtnca rctgswgnag tckgg 95
<210> 48
<211> 94
<212> DNA
<213> Kiznstliche Sequenz
<220>
<223> Beschreibung der kunstlichen Sequenz: synthetisch,
kein natiirlicher Ursprung
<400> 48
ctattgccta cggcggccgc aggtctcctc ctcttagcag cacaaccagc aatggccgac 60
tacaaagacg atsaggtnca gstggtgsag tcng g4
<210> 49
<211> 93
<212> DNA
<213> Kiznstliche Sequenz
<220>
<223> Besc:~reibung der kiinstlichen Sequenz: synthetisch,
kein natiirlicher Ursprung
<400> 49
ctattgccta cggcggccgc aggtctcctc ctcttagcag cacaaccagc aatggccgac 60
tacaaagacg atcaggtgca gctgcagsag tcg g3
<210> 50
<211> 94
<212> DNA
<213> Kiinstliche Sequenz
<220>
14
CA 02328057 2000-11-14
<223> Beschreibung der kiinstlichen Se~uer~z: synthetisch,
kein natiirlicher Ursprung
<400> 50
ctattgccta cggcggccgc aggtctcctc ctcttagcag cacaaccagc aatggccgac 60
tacaaagacg atsarstgca gctgktgcag tcvg 94
<210> 51
<211> 93
<212> DNA
<213> Kiinstliche Sequenz
<220>
<223> Beschreibung der kiinstlichen Sequenz: synthetisch,
kein natiirlicher Ursprung
<400> 51
ctattgccta cggcggccgc aggtctcctc ctcttagcag cacaaccagc aatggccgac 60
tacaaagacg atcaggtaca gctgcagcag tca g3
<210> 52
<211> 39
<212> DNA
<213> Kiinstliche Sequenz
<220>
<223> Beschreibung der kiinstlichen Sequenz: synthetisch,
kein natiirlicher Ursprung
<400> 52
agcatcacta gtctaacgtt tgatctccac cttggtccc 39
<210> 53
<211> 39
<212> DNA
<213> Kiinstliche Sequenz
<220>
<223> Beschreibung der kiinstlichen Sequenz: synthetisch,
kein natiirlicher Ursprung
<400> 53
agcatcacta gtctaacgtt tgatatccac tttggtccc 39
CA 02328057 2000-11-14
<210> 54 _
<211> 39
<212> DNA
<213> Kiinstliche Sequenz
<220>
<223> Beschreibung der kiinstlichen Sequenz: syntzetisch,
kein natiirlicher Ursprung
<400> 54
agcatcacta gtctaacgtt taatctccag tcgtgtccc
39 -
<210> 55
<211> 39
<212> DNA
<213> Kiinstliche Sequenz
<220>
<223> Beschreibung der kiinstlichen Sequenz: synthetisch,
kein natiirlicher Ursprung
<400> 55
agcatcacta gtctaaccta ggacggtgac cttggtccc 39
<210> 56
<211> 39
<212> DNA
<213> Kiinstliche Sequenz
<220>
<223> Beschreibung der kiinstlichen Sequenz: synthetisch,
kein natizrlicher Ursprung
<400> 56
agcatcacta gtctaaccta ggacggtcag cttggtccc 39
<210> 57
<211> 39
<212> DNA
<213> Kiinstliche Sequenz
<220>
<223> Beschreibung der kiinstlichen Sequenz: synthetisch,
kein natiirlicher Ursprung
16
CA 02328057 2000-11-14
<400> 57
agcatcacta gtctaaccga ggacggtcag ctgggtgcc 3g
I7
