Note: Descriptions are shown in the official language in which they were submitted.
CA 02375033 2001-11-27
1
Vaccines against conformation-dependent antigens and against
antigens that are not, or are not exclusively, proteins or
peptides
Description
The invention refers to vaccines against conformation-dependent
antigens and against antigens that are not, or are not
exclusively, proteins or peptides. In addition, the invention
refers to processes for their production and use as well as
human antiidiotypical antibody fragments against the MUCl
conformation epitope and amino acid sequences of mimicry
peptides against the MUC1 conformation epitope as well as
antiidiotypical antibody fragments against the TF antigen and
amino acid sequences of mimicry peptides against the TF
carbohydrate epitope.
Target structures of vaccines against pathogens of infectious
diseases and non-infectious diseases, including tumours, may be
proteins or peptides, carbohydrates or lipids as well as
combinations thereof. In the case of proteins and peptides, the
immunogenic determinant (epitope) may be determined either by
the sequence of the amino acids of a section of the molecule
(sequential epitope) or by a specific arrangement of binding
forces that do not correspond to the linear arrangement of the
amino acids (conformation epitope). Conformation epitopes occur
more frequently than sequential epitopes; mixed forms also
occur.
Conformation epitopes and antigens that are not, or not
exclusively, proteins or peptides are difficult to convert into
an effective and practicable vaccine. As a rule, conformation
epitopes only develop in native protein and not in shorter
peptides. Antigens that are not, or not exclusively, proteins
or peptides (such as glycostructures or lipids) are only
CA 02375033 2001-11-27
2
slightly immunogenic. Their synthesis is often complicated.
A particularly serious fact is that, in many cases, these
antigens are not correctly presented to the immune system.
However, an effective antigen presentation is, amongst other
things, a prerequisite for the development of cytotoxic T
lymphocytes, i.e. for an effective cellular resistance.
Finally, the very effective form of DNA vaccination is not
applicable to these antigens.
In DNA vaccination (genome vaccination), instead of a protein
or peptide antigen, the coding DNA sequence itself, or packed
in a vector, is injected as an intramuscular or intradermal
vaccine. In this way, an effective humoral response and
cellular response can be achieved (Wolff, J.A. et al., Science
247:1465, 1990; Ulmer, J.B. et al., Vaccine 12:1541, 1994; Raz,
E. et al., Cancer res. 52:1954, 1992). A particularly successful
procedure is what is known as the prime boost protocol (Keystone
Symposia: DNA Vaccines, April 12 - 17, 1999, Snowbird, Utah,
USA, conference tape) in which the intradermal, intramuscular
or intrarectal inj ection of a DNA (priming) is followed by a
booster with the corresponding antigen. A corresponding
recombinant virus-vector particle (e. g. fowlpox, constructs
derived from adeno or alpha virus) can also be used
successfully. Of course, the prime boost procedure results in
a strong cellular immune response with the activation of
specific cytotoxic T cells, which is particularly desirable in
tumour vaccines. The immune response can be significantly
strengthened through the additional administration of suitable
cytokines, also in the form of a DNA, through immunostimulating
CpG-DNA motives (non-methylated cytosine-guanine-dinucleotides)
or through suitable adjuvants (e. g. aluminium phosphates).
The object of the invention is to circumvent the aforementioned
disadvantages and develop a vaccine, and more particularly a DNA
CA 02375033 2001-11-27
3
vaccine, that can also be used in cases that, to date, were not
suitable for such a vaccination.
The invention is realized according to the claims. On the one
hand, it refers to a process by which the scope of application
of the vaccination is expanded, and more particularly the DNA
vaccination, to conformation-dependent antigens and mixed forms
(these also fall under the term conformation epitopes within the
meaning of the invention), as well as antigens whose relevant
epitopes are not, or not exclusively, proteins or peptides, e.g.
carbohydrates, combined carbohydrate-peptide epitopes, lipids,
glucolipids, thus circumventing the aforementioned
disadvantages. According to the invention, this is achieved
through the detour via a peptide that is the immunological image
of the original epitope (the antigen determinant) but whose
amino acid sequence is different (mimicry peptide). The mimicry
peptide is preferably obtained by using the well known methods
of phage display or ribosome display (Scott, J.K. and Smith,
G.P., Science, 249:386, 1990; Winter, G. et al., Annu Rev
Immunol, 12:433, 1994; Hanes, J. et al., Proc Natl Acad Sci USA,
95:14130, 1998), either as a shorter peptide from peptide gene
banks or in the form of an antiidiotypical antibody fragment
from the respective gene banks. A third, more complicated
method is to obtain antiidiotypical antibodies by means of the
hybridome method. The common objective of the above three
methods is to "rewrite" the original conformation epitope or the
epitope that is not, or not exclusively, a protein or peptide
into a corresponding immunological sequential epitope which
permits a better immunological presentation and is suitable for
a DNA vaccination. According to the invention, the vaccines,
and more particularly the DNA vaccines, can be used not only in
the form of the above example (prime boost protocol), but also
in comparable variants and in the form of DNA vaccines alone or
in mimicry structures alone and in suitable formulations.
CA 02375033 2001-11-27
4
In addition, the invention refers to vaccines against
conformation-dependent antigens according to Claim 1. In the
process according to the invention, using the phage display or
the ribosome display method the relevant conformation epitopes
are "rewritten" into a corresponding immunological sequential
epitope that mimics the conformation epitope. The primary
reagents used are molecules that specifically bind the target
antigen in its desired conformation, e.g. antibodies, antibody
fragments or receptors. Thus, from the various gene libraries,
antibody fragments (antiidiotypical antibody fragments, Ab 2)
or linear or circular peptides are obtained that specifically
bind the primary reagents and immunologically mimic the antigen.
Alternatively, antiidiotypical antibodies are obtained by means
of the hybridome method and fragments are isolated from them,
if required. These mimicry peptides are rewritten into a DNA
and used as a DNA vaccine. One process is what is known as the
prime boost protocol, in which the intradermal, intramuscular
or intrarectal injection of a DNA (priming) in the form of a
plasmid DNA, linear DNA or a plasmid replicon vector is followed
by a booster with the corresponding antigen, alone, in the form
of a chemical coupling of proteins, in the form of
bacteriophages as fusion proteins with phage coat proteins on
their surface, in the form of a fusion protein on the surface
of other viruses or attenuated biological carriers or in the
form of dendritic cells loaded with a peptide. In this case,
the DNA as well as the expressed mimicry peptide are required,
which is easy using the phage display or ribosome display
method. Alternatively, a corresponding recombinant virus-vector
particle (e. g. fowlpox, constructs derived from adeno or alpha
virus) can be used successfully. The immune response can be
significantly strengthened through the additional administration
of suitable cytokines, also in the form of a DNA, through
immunostimulating CpG-DNA motives (non-methylated cytosine-
CA 02375033 2001-11-27
guanine dinucleotides) or through suitable adjuvants (e. g.
aluminium phosphates).
Besides vaccines against conformation-dependent antigens, the
invention also refers to vaccines against antigens that are not,
or not exclusively, proteins or peptides according to Claim 3.
A target antigen type of the group of antigens that are not, or
not exclusively, proteins or peptides are glycostructures;
additional immunogenic structures are combined carbohydrate-
protein epitopes, lipids, glycolipids or synthetic structures.
A process is known from DE 196 27 352 A1 with which a monoclonal
antiidiotypical antibody can be obtained using the hybridome
method, which immunologically mimics pure carbohydrate
structures. According to the invention, starting with this
antiidiotypical antibody, a vaccine (preferably a DNA vaccine
of this antibody or a suitable fragment thereof) is used for the
vaccination. Thus, the present invention expands several points
of this process from DE 196 27 352 A1. Antiidiotypical antibody
fragments can be obtained directly from the antibody gene
libraries using the phage display method or the ribosome display
method. Also with this process, human antibody fragments can
be obtained directly. In addition, combined carbohydrate-
peptide epitopes can also be used. Plus there is a process with
which short linear or circular peptides which immunologically
mimic the antigen (also known as mimicry peptides) can be
obtained from peptide gene libraries, also using the phage
display method or the ribosome display method. To this end, not
only specific idiotypical antibodies (Abl) are used as primary
reagents for the selection of these mimicking structures, but
also other substances that specifically recognize the
glycostructure, such as lectins or receptors. The process also
includes the use of the obtained structures preferably as DNA
vaccines, alone or in conjunction with the antibodies that
CA 02375033 2001-11-27
6
immunologically mimic the antigen, antibody fragments or
peptides in a suitable formulation (see above and claims), for
example in a suitable formulation of the prime boost protocol.
Furthermore, according to the invention the mimicking protein
structures can also be used alone for vaccination.
The invention also refers to vaccines (in the full scope of the
description for conformation-dependent antigens) against the
antigens glycopeptides, glycolipids, lipids, synthetic
structures or other antigens that are not, or are only
partially, proteins or peptides, the relevant epitopes having
improved immunological structures, as well as to their
production processes and their use.
The immunotherapy approach to diseases involving tumours is
based on the assumption that it is possible to strengthen or
activate the natural immune response. The rationale for
vaccination lies in combating the residual disease (metastasis
prophylaxis) according to a conventional therapy (e. g. surgical
removal of the main body of tumour cells). As the name implies,
mimicry peptides immunologically mimic the original antigen or
epitope. They do this to very high degree, but not completely.
This can be seen as positive for applications within the
framework of a vaccine (and more particularly in the case of a
tumour vaccine) in that specifically inhibiting processes, e.g.
tolerance phenomena, are circumvented.
The prerequisite for the development of defined tumour vaccines
is not only the presence of tumour-specific antigens, but also
knowledge thereof. Great progress has been achieved in this
area during the past three decades, not least through the
development of monoclonal antibodies.
One widespread cancer antigen is the epithelial mucin, MUC1,
CA 02375033 2001-11-27
7
whose immune-dominant epitope occurs multiple times on the
extracellular part of the molecule. In its native state, this
epitope forms a type I-(3 turn, but on synthetic peptides only
under certain conditions, e.g. when the theonin of the dominant
immune region is glycosylated with GalNAca1-0-Thr or Gal~il-
3GalNAca1-0-Thr (Karsten, U. et al., Cancer res 58:2541-2549,
1998). As a rule, this epitope is perceived as a typical
conformation epitope by the immune system, see Example 1.
According to the invention, using the phage display method, this
conformation epitope is mimicked by immunologically identical
(or almost identical) sequential epitopes which, in the form of
a DNA, are part of a tumour vaccine in a DNA vaccination vector
(Example 1) .
Therefore, the object of the invention is also human
antiidiotypical antibody fragments against the MUC1 conformation
epitope as well as all DNA sequences that encode these
fragments, and protein sequences or DNA or protein partial
sequences that can be derived from them and that have the
corresponding characteristics.
Primarily, this concerns the following human antiidiotypical
antibody fragments against the MUC1 conformation epitope with
the following sequence nos. 1 to 31.
Fragments that contain the desired DNA of the scFv and of the
peptides were multiplied using the PCR and subsequently
sequenced.
(The numbering, e.g. Q33, corresponds to a specific isolated
clone; the sequences of the various scFv are aligned to each
other; the complete sequence of each clone must be read
continuously throughout the different blocks)
CA 02375033 2001-11-27
g
No.1:Q33 EVQLLESGEGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSSIQRHGTWTGY
No.2:Q1.3 EVQLLESGEGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSSINYNGDATSY
No.3:Q12 EVQLLESGEGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSTINAAGAQTGY
No.4:Q4 EVQLLESGEGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSRIGQKGNKTTY
No.5:R2 EVQLLESGEGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSRITQSGTYTQY
No.6:Q15 EVQLLESGEGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSSINAFGQSTRY
No.7:R10 EVQLLESGEGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSGINASGTLTRY
No.8:Q5 EVQLLESGEGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSSISDTGSATTY
No.9:N6 EVQLLESGEGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSNISDAGCATYY
No.l0:Q32 EVQLLESGEGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSTIHSAGQETIY
No.11:R6 EVQLLESGEGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSYITTNGSTTSY
No.12:Q9.3 EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSYITTNGSTTSY
No.13:Q24 EVQLLESGEGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSSITTSGGDTAY
No.14:Q3.1 EVQLLESGEGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSYINASGASTSY
No.15:Q25 EVQLLESGEGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSTITSSGQQTFY
No.16:N2 EVQLLESGEGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSSIYSQGPVTWY
No.17:Q3.3 EVQLLESGEGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSGISTSGSYTTY
No.18:Q21 EVQLLESGEGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSTINGLGTPTAY
No.19:N4 EVQLLESGEGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSTIQTSGRDTTY
No.20:R3 EVQLLESGEGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSAITQYGGDTGY
No.21:Q2 EVQLLESGEGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSTISNLGQPTHY
No.22:Q30 EVQLLESGEGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSTISNLGQLTHY
No.23:Q16 EVQLLESGEGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSTIDPMGQSTNY
No.24:R5 EVQLLESGEGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSAITNTGQWTTY
No.25:Q26 EVQLLESGEGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSTIQSVGTYTVY
No.26:Q34 EVQLLESGEGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSTIPATGQRTFY
No.27:Q6.1 EVQLLESGEGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSSISRTGKVTDY
No.28:Q1.2 EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSAIEAGGGETTY
No.29:R4 EVQLLESGEGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSGIRPQGHPTQY
No.30:N1 EVQLLESGEGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSAIRPPGQTTQY
No.31:R7 EVQLLESGEGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSQIQENGVTTTY
Q33 ADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKRNGEFDYWGQGTLVTVSSGGGG
Q1.3 ADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKSSSTFDYWGQGTLVTVSSGGGG
Q12 ADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKTGTNFDYWGQGTLVTVSSGGGG
Q4 ADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKKSHDFDYWGQGTLVTVSSGGGG
CA 02375033 2001-11-27
9
R2 ADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKGLSRFDYWGQGTLVTVSSGGGG
Q15 ADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKYDHSFDYWGQGTLVTVSSGGGG
R10 ADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKSAKSFDYWGQGTLVTVSSGGGG
Q5 ADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKNYYDFDYRGQGTLVTVSSGGGG
N6 ADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKNSCGFDYWGQGTLVTVSSGGGG
Q32 ADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKTLLGFDYWGQGTLVTVSSGGGG
R6 ADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKDYSDFDYRGQGTLVTVSSGGGG
Q9.3 ADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKDYSDFDYRGQGTLVTVSSGGGG
Q24 ADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKNYADFDYRGQGTLVTVSSGGGG
Q3.1 ADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARNTSDFDYRGQGTLVTVSSGGGG
Q25 ADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKRARPFDYWGQGTLVTVSSGGGG
N2 ADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARHSWPFDYWGQGTLVTVSSGGGG
Q3.3 ADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKSGTTFDYWGQGTLVTVSSGGGG
Q21 ADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKDLFGFDYRGQGTLVTVSSGGGG
N4 ADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKRSQRFDYWGQGTLVTVSSGGGG
R3 ADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKNWPYFDYWGQGTLVTVSSGGGG
Q2 ADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKLPYSFDYWGQGTLVTVSSGGGG
Q30 ADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKLPYSFDYWGQGTLVTVSSGGGG
Q16 ADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKDGREFDYWGQGTLVTVSSGGGG
R5 ADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKAGQNFDYWGQGTLVTVSSGGGG
Q26 ADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKRHNPFDYWGQGTLVTVSSGGGG
Q34 ADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKTASPFDYWGQGTLVTVSSGGGG
Q6.1 ADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKKMTSFDYWGQGTLVTVSSGGGG
Q1.2 ADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKATTTFDYWGQGTLVTVSSGGGG
R4 ADSVKGGFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKRPPPFDYWGQGTLVTVSSGGGG
N1 ADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKTASVFDYWGQGTLVTVSSGGGG
R7 ADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAIYYCAKERLQFDYWGQGTLVTVSSGGGG
Q33 SGGGGSGGGGSTDIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLI
Q1.3 SGGGGSGGGGSTDIQMTQSPSSLSASVGDGVTITCRASQSISSYLNWYQQKPGKAPKLLI
Q12 SGGGGSGGGGSTDIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLI
Q4 SGGGGSGGGGSTDIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLI
R2 SGGGGSGGGGSTDIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLI
Q15 SGGGGSGGGGSTDIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLI
R10 SGGGGSGGGGSTDIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLI
, CA 02375033 2001-11-27
1
Q5 SGGGGSGGGGSTDIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLI
N6 SGGGGSGGGGSTDIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLI
Q32 SGGGGSGGGGSTDIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLI
R6 SGGGGSGGGGSTDIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLI
Q9.3 SGGGGSGGGGSTDIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLI
Q24 SGGGGSGGGGSTDIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLI
Q3.1 SGGGGSGGGGSTDIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLI
Q25 SGGGGSGGGGSTDIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLI
N2 SGGGGSGGGGSTDIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLI
Q3.3 SGGGGSGGGGSTDIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLI
Q21 SGGGGSGGGGSTDIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLI
N4 SGGGGSGGGGSTDIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLI
R3 SGGGGSGGGGSTDIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLI
Q2 SGGGGSGGGGSTDIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLI
Q30 SGGGGSGGGGSTDIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLI
Q16 SGGGGSGGGGSTDIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLI
R5 SGGGGSGGGGSTDIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLI
Q26 SGGGGSGGGGSTDIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLI
Q39 SGGGGSGGGGSTDIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLI
Q6.1 SGGGGSGGGGSTDIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLI
Q1.2 SGGGGSGGGGSTDIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLI
R4 SGGGGSGGGGSTDIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGEAPKLLI
N1 SGGGGSGGGGSTDIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLI
R7 SGGGGSGGGGSTDIQMTQSPPSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLI
Q33 YSASTLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQNSTIPRTFGQGTKVEIKR
Q1.3 YSASTLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQTSNSPATFGQGTKVEIKR
Q12 YSASALQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQTNTDPATFGQGTKVEIKR
Q4 YRASDLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQPWDPPRMFGQGTKVEIKR
R2 YHASFLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQPWEPPRTFGQGTKVEIKR
Q15 YAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQPWLPPRTFGQGTKVEIKR
R10 YNASMLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQTLLWPLTFGQGTKVEIKR
Q5 YDASTLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQGTASPSTFGQGTKVEIKR
N6 YNASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYTGNPATFGQGTKVEIKR
Q32 YAASWLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYSGHPSTFGQGTKVEIKR
R6 YSASYLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQTSANPYTFGQGTKVEIKR
CA 02375033 2001-11-27
11
Q9.3 YSASGLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQNGATPNTFGQGTKVEIKR
Q24 YAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSSATPGTFGQGTKVEIKR
Q3.1 YSASTLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSGSAPATFGQGTKVEIKR
Q25 YAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSTPNTFGQGTKVEIKR
N2 YAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSTPNTFGQGTKVEIKR
Q3.3 YAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSTPNTFGQGTKVEIKR
Q21 YAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSTPNTFGQGTKVEIKR
N4 YAASHLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQQGQTPVTFGQGTKVEIKR
R3 YYASNLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQNSFTPYTFGQGTKVEIKR
Q2 YDASFLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQDTRPPTTFGQGTKVEIKR
Q30 YDASFLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQDTRPPITFGQGTKVEIKR
Q16 YDASKLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQDTRNPGTFGQGTKVEIKR
R5 YDASFLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQDTRGPGTFGQGTKVEIKR
Q26 YDASFLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQDTRGPGTFGQGTKVEIKR
Q34 YSASRLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQDTRQPGTFGQGTKVEIKR
Q6.1 YDASNLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQDTRQPGTFGQGTKVEIKR
Q1.2 YDASNLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQDTRPPVTFGQGTKVEIKR
R4 YDASVLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQRRTYPPTFGQGTKVEIKR
N1 YGASVLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQHLNYPLTFGQGTKVEIKR
R7 YDASNLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQFGNYPRTFGQGTKVEIKR
The object of the invention is also amino acid sequences of
mimicry peptides against the MUC1 conformation epitope as well
as all DNA sequences that encode these amino acid sequences, DNA
and peptide and partial peptide sequences that can be derived
from them and that have the same characteristics.
More particularly, this concerns the amino acid sequences of
mimicry peptides with the following sequence nos. 32 to 47.
(The numbering, e.g. S1, corresponds to a specific isolated
clone; the sequences of the various peptides are aligned to each
other) .
CA 02375033 2001-11-27
12
No.32:S1 CEYYDVPMARC
No.33:S12 CDYPSRLIDLC
No.34:Rol CGLACERPCGWVC
No.35:Ro5 CLGGCERPCMYSC
No.36:Rol3 CRGRCGEWCSRPC
No.37:Ro6 CRGRCDQRCSRPC
No.38:Rol2 CPARCGVPCAMGC
No.39:V11 CIPHRHDGC
No.40:V4 CQPHRYDKSLPC
No.4l:V10 CTTRLLNEDGSC
No.42:U7 LHGPLWD
No.43:U10 LHGPLGM
No.44:U6 LHGPLWE
No.45:U7a LHGPLWDGAAGAETVES
No.46:UlOa LHGPLGMGPLGPKLLKV
No.47:U6a LHGPLWEGPLGPKLLKV
Antigens that are not, or not exclusively, proteins or peptides
(e. g. carbohydrate antigens) are, similar to conformation
epitopes of proteins, perceived by the immune system as three-
dimensional patterns of charges and other molecular interactions
and, like them, are subject to limitations in the generation of
a cellular immune response. In these cases, too, the selection
of mimicry peptides by the phage display method according to the
invention can result in the antigen being "rewritten" into a
peptide sequence, thus permitting the DNA vaccination technique,
see Example 2.
The object of the invention is also protein sequences of
antiidiotypical antibody fragments against TF as well as amino
acid sequences of mimicry peptides against the TF carbohydrate
epitope, all DNA sequences that encode these amino acid
sequences, and DNA and protein or peptide and protein and
peptide partial sequences that can be derived from them and that
CA 02375033 2001-11-27
13
have the same characteristics.
More particularly, this concerns the following protein sequences
of antiidiotypical antibody fragments against TF with the
following sequence nos. 48 to 71.
No. 48 - >H16
EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSMIDGSGSQTYYADSVKGRFTISRDN
SKNTLYLQMNSLRAEDTAVYYCAKSDLDFDYWGQGTLVTVSSGGGGSGGGGSGGGGSTDIQMTQSPSSLSASVG
DRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQ
SYSTPNTFGQGTKVEIKR
No. 49 - >P3
EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSSISYSGATTNYADSVKGRFTISRDN
SKNTLYLQMNSLRAEDTAVYYCAKSDASFDYWGQGTLVTVSSGGGGSGGGGSGGGGSTDIQMTQSPSSLSASVG
DRVTITCRASQSISSYLNWYQQKPGKAPKLLIYDASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQ
DYGGPTTFGQGTKVEIKR
No. 50 -'>P8
EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSTISATGGSTYYADSVKGRFTISRDN
SKNTLYLQMNSLRAVDTAVYYCAKSSDGFDYWGQGTLVTVSSGGGGSGGGGSGGGGSTDIQMTQSPSSLSASVG
DRVTITCRASQSISSYLNWYQQKPGKAPKLLIYSASNLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQ
ASSAPATFGQGTKVEIKR
No. 51 - >H6
EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSTISAQGLTTTYADSVKGRFTISRDN
SKNTLYLQMNSLRAEDTAVYYCAKGRSSFDYWGQGTLVTVSSGGGGSGGGGSGGGGSTDIQMTQSPSSLSASVG
DRVTITCRASQSISSYLNWYQQKPGKAPKLLIYGASGLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQ
RKLLPWTFGQGTKVEIKR
No. 52 - >H1
EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSSITELGRSTQYADSVKGRFTISRDN
SKNTLYLQMNSLRAEDTAVYYCAKPWPHFDYWGQGTLVTVSSGGGGSGGGGSGGGGSTDIQMTQSPSSLSASVG
DRVTITCRASQSISSYLNWYQQKPGKAPKLLIYGASGLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQ
AARRPTTFGQGTKVEIKR
, CA 02375033 2001-11-27
14
No. 53 - >H13
EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSKISELGRNTSYADSVKGRFTISRDN
SKNTLYLQMNSLRAEDTAVYYCAKDITAFDYWGQGTLVTVSSGGGGSGGGGSGGGGSTDIQMTQSPSSLSASVG
DRVTITCRASQSISSYLNWYQQKPGKAPKLLIYGASGLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQ
SMRMPPTFGQGTKVEIKR
No. 54 - >K3
EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSAIQWSGESTWYADSVKGRFTISRDN
SKNTLYLQMNSLRAEDTAVYYCAKSTSSFDYWGQGTLVTVSSGGGGSGGGGSGGGGSTDIQMTQSPSSLSASVG
DRVTITCRASQSISSYLNWYQQKPGKAPKLLIYDASLLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQ
RRHTPTTFGQGTKVEIKR
No. 55 - >K3
EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSAIQWSGESTWYADSVKGRFTISRDN
SKNTLYLQMNSLRAEDTAVYYCAKSTSSFDYWGQGTLVTVSSGGGGSGGGGSGGGGSTDIQMTQSPSSLSASVG
DRVTITCRASQSISSYLNWYQQKPGKAPKLLIYDASLLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQ
RRHTPTTFGQGTKVEIKR
No. 56 - >K4
EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSGIQFSGQGTRYADSVKGRFTISRDN
SKNTLYLQMNSLRAEDTAVYYCAKTLSTFDYWGQGTLVTVSSGGGGSGGGGSGGGGSTDIQITQSPSSLSASVG
DRVTITCRASQSISSYLNWYQQKPGKAPKLLIYRASHLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQ
GYRQPTTFGQGTKVEIKR
No. 57 - >K2
EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSSIRPLGSATQYADSVKGRFTISRDN
SKNTLYLQMNSLRAEDTAVYYCAKSNMAFDYWGQGTLVTVSSGGGGSGGGGSGGGGSTDIQMTQSPSSLSASVG
DRVTITCRASQSISSYLNWYQQKPGKAPKLLIYGASGLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQ
TTRPPTTFGQGTKVEIKR
No. 58 - >J6
EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSDISEQGARTMYADSVKGRFTISRDN
SKNTLYLQMNSLRAEDTAVYYCAKSTPAFDYWGQGTLVTVSSGGGGSGGGGSGGGGSTDIQMTQSPSSLSASVG
DRVTITCRASQSISSYLNWYQQKPGKAPKLLIYGASGLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQ
MNNKPNTFGQGTKVEIKR
, CA 02375033 2001-11-27
IS
No. 59 - >E3
EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSQITGLGSQTRYADSVKGRFTISRDN
SKNTLYLQMNSLRAEDTAVYYCAKGETAFDYWGQGTLVTVSSGGGGSGDIQMTQSPSSLSASVGDRVTITCRAS
QSISSYLNWYQQKPGKAPKLLIYGASGLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQRQQRPSTFGQ
GTKVEIKR
No. 60 - >K1
EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSNITQMGMTTAYADSVKGRFTISRDN
SKNTLYLQMNSLRAEDTAVYYCAKGEQTFDYWGQGTLVTVSSGGGGSGGGGSGGGGSTDIQMTQSPSSLSASVG
DRVTITCRASQSISSYLNWYQQKPGKAPKLLIYGASGLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQ
RRTHPQTFGQGTKVEIKR
No. 61 - >E5
EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSAISQTGTRTKYADSVKGRFTISRDN
SKNTLYLQMNSLRAEDTAVYYCAKGSASFDYWGQGTLVTVSSGGGGSGGGGSGGGGSTDIQMTQSPSSLSASVG
DRVTITCRASQSISSYLNWYQQKPGKAPKLLIYGASGLQSGVPTRFSGSGSGTDFTLTISSLQPEDFATYYCQQ
VTTHPNTFGQGTKVEIKR
No. 62 - >K2+
EVQLVESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSAISGSGGSTYYADSVKGRFTISRDN
SKNTLYLQMNSLRAEDTAVYYCARQVKSWTRWGQGTLVTVSSGGGGSGGGGSGGSALSSELTQDPAVSVALGQT
VRITCRGDSLRSYYASWYQQKPGQAPVLVIYGKNNRPSGIPDRFSGSSSGNTASLTITGAQAEDEADYYCNSRD
SSGNHYVFGGGTKLTVLG
No. 63 - >K4+
EVQLVESGGGLVQPGGSLRLSCAASGFTFSSYSMNWVRQAPGKGLEWVSAISGSGGSTYYADSVKGRFTISRDN
SKNTLYLQMDSLRAEDTAVYYCARGRRKQDKSTRWGQGTLVTVSSGEGGSGGGGSGGSALSSELTQDPAVSVAL
GQTVRITCQGSLRSYYASWYQQKPGQAPVLVIYGKNNRPSGIPDRFSGSSSGNTASLTITGAQAEDEADYYCNS
RDSSGSSSVFGGGTKLTVLG
No. 64 - >K4-
EVQLVESGGGLVQPGGSLRLSCAASGFTFSSYSMNWVRQAPGKGLEWVSAISGSGGSTYYADSVKGRFTISRDN
SKNTLYLQMDSLRAEDTAVYYCARGRRKQDKSTRWGQGTLVTVSGSGGGGSGGSALSSELTQDPAVSVALGQTV
RITCQGDSLRSYYASWYQQKPGQAPVLVIYGKNNRPSGIPDRFSGSSSGNTASLTITGAQAEDEADYYCNSRDS
SGSSSVFGGGTKLTVLG
, CA 02375033 2001-11-27
16
No. 65 - >K9+
EVQLVESGGGLVQPGGSLRLSCAASGFTFSSYEMNWVRQAPGKGLEWVSYISSSGSTIYYADSVKGRFTISRDN
AKNSLYLQMNSLRAEDTAVYYCARDPFHPWGQGTLVTVSSGGGGSGGGGSGGSALSSELIQDPAVSVALGQTVR
ITCQGDSLRSYYASWYQQKPGQAPVLVIYGKNNRPSGIPDRFSGSSSGNTASLTITGAQAEDEADYYCNSRDSS
GTVFGGGTKLTVLG
No. 66 - >K1+
QVQLQESGPGLVKPSETLSLTCWSGGSISSSNWWSWVRQPPGKGLEWIGEIYHSGSPNYSPSLKSRATISVDK
SKNQFSLKLSSVTAADTAVYYCARQDMTQQTSWGQGTLVTVSSGGGGSGGGGSGGSALQSVLTQPPSASGTPGQ
RVTISCSGSSSNIGSNYVYWYQQLPGTAPKLLIYRNNQRPSGVPDRFSGSKSGTSASLAISGLRSEDEADYYCA
AWDDSLRNLVFGEGTKLTVLG
No. 67 - >K3+
QVQLQESGPGLVKPSETLSLTCVVSGGSISSSNWWSWVRQPPGKGLEWIGEIYHSGSPNYSPSLKSRATISVDK
SKNQFSLKLSSVTAADTAVYYCARQDMTQQTSWGQGTLVTVSSGEGGSGEGGSGGSALQSVLTQPPSASGTPGQ
RVTISCSGSSSNIGSNYVYWYQQLPGTAPKLLIYRNNQRPSGVPDRFSGSKSGTSASLAISGLRSEDEADYYCA
AWDDSLRNLVFGEGTKLTVL
No. 68 - >ZA4
QVQLQESGPGLVKPSGTLSLTCAVSGGSISSSNWWSWVRQP
PGKGLEWIGEIYHSGSTNYNPSLKSRVTISVDKSKNQFSLKLSSVTAADTAVYYCARDDK
GGWGQGTLVTVSSGGGGSGGGGSGGSALQSVLTQPPSASGTPGQRVTISCSGSSSNIGSN
TVNWYQQLPGTAPKLLIYSNNQRPSGVPDRFSGSKSGTSASLAISGLQSEDEADYYCAAW
DDSLRSLVFGGGTKLTVLG
No. 69 - >ZA36
QVQLQESGPGLVKPSGTLSLTCAVSGGSISSSNWWSWVRQPPGKGLEWIGEIYH
SGSTNYNPSLKSRVTISVDKSKNQFSLKLSSVTAADTAVYYCARPSSIWGQGTLVTVSSG
GGGSGGGGSGGSALQSVLTQPPSASGTPGQRVTISCSGSSSNIGSNYVYWYQQLPGTAPK
LLIYRNNQRPSGVPDRFSGSKSGTSASLAISGLRSEDEADYYCAAWDDSLRSLVFGGGTK
LTVLG
, CA 02375033 2001-11-27
17
No. 70 - >ZA14
QVQLQESGPGLVKPSGTLSLTCAVSGGSISSSNWWSWVRQPPGKGLEWIGEIYHS
GSTNYNPSLKSRVTISVXKSKNQFSLKLSSVTAXDTAVYYCARPSHHAGTHTWGQGTLVT
VSSGGGGSGGGGSGGSALQSVLTQPPSASGTPGQRVTISCSGSSSNIGSNTVNWYQQLPG
TAPKLLIYSNNQRPSGVPDRFSGSKSGTSASLAISGLQSEDEADYYCAAWDDSLRALVFG
GGTKLTVLG
No. 71 - >Z9
QVQLQESGAGLLKPSETLSLTCAVYGGSFSGYYWSWIRQPPGKGLEWIGEINHSGS
TNYNPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCARKGLNFGPWGQGTLVTVSSG
GGGSGGGGSGGSALQSVLTQPPSASGTPGQRVTISCSGSSSNVGSNTVNWYQQLPGTAPK
LLIYSNNQRPSGVPDRFSGSKSGTSASLAISGLQSEDEADYYCAAWDDSLRSYVFGGGTK
LTVLG
Furthermore, this concerns the amino acid sequences of mimicry
peptides against the TF carbohydrate epitope with the following
sequence nos. 72 to 96.
(The numbering, e.g. S1, corresponds to a specific isolated
clone)
No.72:T1 CLREGHFASFC
No.73:T14 CGMLTPAWIKC
No.74:T4 CETFSNLAFLC
No.75:T7 CEGPEIPAFVC
No.76:T3 CESMVEPAWVC
No.77:T15 CTNDIMPPWVC
No.78:T2 CDGLLLPIWAC
No.79:T11 CAGEFVPVWAC
No.80:T16 CDLGLKPAWLC
No.8l:X3 CGPMCSGSCVPQC
No.82:X9 CDAGCNFFCPWRC
No.83:X2 CGPMCSGSCXPQC
CA 02375033 2001-11-27
Ig
No.84:Y8 VWWWQWS
No.85:Y1 MWRPFWL
No.86:Y4 PPWVXHL
No.87:Y9 LIPQWIV
No.88:W4 CTPADMSGC
No.89:W3 CTPADMSGC
No.90:W16 CPSVWMLDLGPC
No.9l:W15 CHGGLTPLC
No.92:W8 CGPMMLWHW
No.93:W5 CTRHIHWGNAHW
No.94:W14 CTPADMSGW
No.95:A1 CFRGGPWWSLC
No.96:A2 CAVRTWVISEC
The invention is explained in more detail by examples of
embodiments, however, it is not limited to these.
Examples of embodiments
Example 1
Production of hybridome cell line A76-A/C7 and of antibodies
After treatment with Neuraminidase (V. cholerae), Balb/c mice
were immunized i.p. with a suspension of live human
mammacarcinoma cells of the cell line T-47D (Keydar, I. et al.,
Eur J Cancer, 15:659, 1979) without an adjuvant. The fusion cell
line was X63-Ag8.653 (Kearney, J.F. et al., J Immunol 123:1548,
1979). The hybridome method itself was carried out according
to standard methods (e. g. Peters, H.H., et al., "Monoklonale
Antikorper, Herstellung and Charakterisierung" [Monoclonal
Antibodies, Production and Characterization], Berlin 1985;
Friemel, H. "Immunologische Arbeitsmethoden" [Immunological
Working Methods], 4th edition, Jena 1991). The specificity
CA 02375033 2001-11-27
19
analysis of the monoclonal antibodies (mAK) produced by the
hybridome cell lines was based on enzyme immunoassays with
natural glycoproteins and synthetic peptides and glycopeptides,
immunofluorescence analyses with diverse cell lines as well as
immune histochemical studies of tissue sections. For the
monoclonal antibody A76-A/C7, the epithelial mucin, MUC1, was
unequivocally determined as the specific antigen. IgGl,k was
determined as the isotope, with a small portion of IgM of the
same specificity using a commercial isotyping kit (Pharmingen,
San Diego, USA). An epitope mapping within the framework of the
ISOBM TD-4 International Workshop on Monoclonal Antibodies
against MUC1 (Tumor Biol. 19, Suppl. 1, 1998) defined the
epitope as APDTRPAP. Additional studies using synthetic,
glycosylated and non-glycosylated peptides showed that the
epitope of the monoclonal antibody A76-A/C7 is, to great extent,
determined by its conformation:
- The antibody binds only insignificantly to a single unit
(a repeat), despite its containing the epitope sequence.
- The binding to non-glycosylated peptides depends on the
length of the peptide or, more specifically, on the number
of linked repeats (Fig. la). It is known from the
literature that the native conformation of the PDTRP
motive only develops with a peptide length of more than 3
repeats (Fontenot, J.D., et al., J Biomol Struct Dyn
13:245, 1995).
- The binding of the monoclonal antibody A76-A/C7 to a
single MUCl unit (1 repeat) is greatly increased if it is
glycosylated with GalNAc-3GalNAc or Galal-3GalNAc in the
area of the epitope at the Thr (Fig. lb~ see also Karsten,
U., et al., Cancer res, 58:2541, 1998).
The antibody was cleaned by ammonium sulphate precipitation
followed by an affinity chromatography on protein A-Sepharose.
CA 02375033 2001-11-27
Obtaining human recombinant antibody fragments that mimic the
conformation-dependent epitope of the MUC1 from antibody gene
libraries using the phage display method
Two different synthetic antibody gene libraries were used which
represent human single-chain antibody fragments (scFv). One
antibody gene library (Griffin 1 Library; http://www.mrc-
cpe.cam.ac.uk/-phage/) comprises more than 109 phages with
various combinations of the variable regions of the heavy and
light chains of human antibodies with, in part, randomized
hypervariable regions that are connected by a peptide piece
(linker) and are covalently bound to a phage coat protein
(pIII). It is derived from a different antibody gene library
(Griffiths, A. et al., 1994, EMBO I., 13:3245-3260). The second,
smaller gene library comprises scFv with the same framework
(single framework library) which were preselected for active
folding of the antibody fragments by binding to protein L and
protein A (I. Tomlinson., 9th anniversary conference: "Antibody
engineering", IBC Conferences, San Diego 1998; I. Tomlinson,
10th anniversary conference: "Antibody engineering" IBC
Conferences, San Diego 1999; Speaker abstract). The first
library originates from the Dr. G. Winter Laboratory and the
second from the Dr. I. Tomlinson Laboratory (both MRC Centre for
Protein Engineering, Cambridge, U.K.). The specific phages were
selected in 2-3 rounds (phage panning) using the proteolytic
selection method with the helper phage KM13 (Kristensen, P. and
Winter, G. Folding & Design, 3:321, 1998). The cleaned
monoclonal antibody A76-A/C7 (35 u/ml in 4 ml) was used as the
antigen which was immobilized overnight in a test tube
(Immunotube, Nunc, Wiesbaden) in PBS at 4 degrees Celsius.
Alternatively, A76-A/C7 was incubated with the phages; the
phages bound to the antibodies were obtained through magnet
beads with immobilized anti-IgG antibodies (Deutsche Dynal,
CA 02375033 2001-11-27
21
Hamburg) . After stringent washing steps (up to 20 times PBS/
O.lo Tween20 followed by 20 times PBS), the phages specifically
bound during the selection rounds (3 h at RT) were eluted
through the tandem repeat (100 ug/ ml; Biosynthan, Berlin-Buch)
that was glycosylated with GalNAc in the PDTR, and subsequently
treated with Trypsin (proteolytic selection method). Between
the selection rounds, the eluted phages were multiplied with
helper phages in the bacteria and selected once again.
Obtaining mimicry peptides that mimic the conformation-dependent
epitope of the MUCI from peptide gene libraries using the page
display method
Analogous to the example for generating antiidiotypical
antibodies, in multiple selection rounds specifically binding
peptides were obtained from a peptide gene library (gene library
of Dr. H. Gollasch; Oligino, L. et al., J Biol Chem 272:29046,
1997) that has 107 different short peptides coupled to the phage
coat protein pIII. The expressed peptides are randomized
nonapeptides that are flanked , and thus circularized, by two
cysteins (CX9C), thus increasing the stability and the affinity.
The selection and testing were carried out as described in the
generation of the antiidiotypical antibodies. Similarly,
additional linear and circular mimicry peptides were obtained
with other peptide libraries. These are peptide libraries that
were created in the same way as the aforementioned peptide
library. The expressed peptides are linear peptides with 7
amino acids and circular peptides with 7 randomized amino acids
flanked by two cysteins (CX10C), circular peptides with 10
randomized amino acids flanked by two cysteins (CX10C), and
circular peptides with a total of 9 randomized amino acids with
two internal and two flanking cysteins (CX3CX3CX3C).
CA 02375033 2001-11-27
22
Specificity tests of the mimicry peptides and the
antiidiotypical antibody fragments
The selected peptides and antibody fragments were tested for
their binding to the monoclonal antibody A76-A/C7 in ELISA tests
as well as in the form of a negative control with other IgG and
IgM monoclonal antibodies. Furthermore, in ELISA tests they
were tested for their binding to a series of well characterized
MUC1-specific antibodies that differ in their fine specificity.
For the ELISA tests, the form of peptides and antibody fragments
coupled to phages was used. The antiidiotypical svFr and the
mimicry peptides can be categorized in groups that:
- bind exclusively to A76-A/C7;
- bind to A76-A/C7 and other MUC1-specific antibodies
that bind only to either the conformation epitope (in the
PDTR region glycosylated MUC1 tandem repeat) (type A) or
whose binding is greatly increased through the PDTR
glycosylation of the MUC1 tandem repeat (conformation
induction) (type B)
- bind to MUCl-specific antibodies that, besides type
A and type B, also bind MUCl-specific antibodies which bind
glycosylated and unglycosylated MUC1 tandem repeats to the
same extent (type D)
- bind strongly to MUC1-specific antibodies and that,
with regard to the glycosylation of the PDTR region of the
MUC1 repeat to A76-A/C7, behave quite the opposite and do not
bind the glycosylated MUC1 peptide or bind much less than
the non-glycosylated MUC1 peptide (type C). These mimicry
peptides or antiidiotypical scFv can also bind to other types
of the MUC1-specific antibody.
CA 02375033 2001-11-27
23
The mimicry peptides and antiidiotypical antibody fragments were
also tested in ELISA inhibition tests to see whether they, in
the form of the syntheticized peptides or cleaned scFv (alone
or coupled to phages), specifically and concentration-
dependently inhibit the binding of A76-A/C7 to the glycosylated
MUC1 peptide (in the epitope PDTR with GalNAc glycosylated
tandem repeat) and non-glycosylated oligomers of the 20-mer
tandem repeats. These tests were carried out with streptavidin-
coated micro test plates (BioTeZ, Berlin-Buch) and biotinylated
MUC1 peptides (Biosynthan, Berlin-Buch; Fig. lc) as well as with
normal ELISA test plates on which the MUC1 peptides were
immobilized by drying.
Inbred mice of the Balb/c line were immunized intraperitoneally
with mimicry peptides and antiidiotypical antibody fragments in
the form of the synthesized peptides or cleaned scFv alone,
respectively coupled to the protein KLH or to bacteriophages in
PBS, mixed with an incomplete Freund's adjuvant, whereby
mixtures of antiidiotypical scFv phages or mimicry peptide
phages from the respective groups (see above) were used. Three
weeks later a booster of the same preparation, but without the
adjuvant, was administered. The booster was repeated after
three weeks and ten days later blood samples were taken from the
mice. In ELISA tests the serum was tested for antibodies that
specifically recognize the conformation-dependent epitope of the
MUC1 (test setup as above). The compositions of the
antiidiotypical scFv as well as the mimicry peptides trigger a
strong reaction against the conformation-dependent epitope of
the MUCl.
CA 02375033 2001-11-27
24
Design of the DNA vaccines and testing on mice
The antiidiotypical scFv were directionally cloned into a DNA
vaccination vector, whereby the scFv were cut out of the phage
vector by means of Sfil and NotI and directionally cloned into
various DNA vaccination vectors that had previously been split
with the same enzymes. A suitable vector for this is the vector
pVAC2 (I. Harmer et al., Keystone Symposium "DNA Vaccines",
Snowbird, USA, 1999; poster and poster abstract) which, after
insertion of the scFv into the DNA vaccination vector, encodes
a fusion protein from the antiidiotypical scFv with a tetanus
toxoid. The tetanus toxoid has the characteristic of an adjuvant
and strengthens the immune response against the fused protein
portion (C. King et al., 1998, Nat Medicine 4:1281-86).
The mimicry peptides were also cloned into various DNA vaccine
vectors. The cloning occurred according to the well known method
of PCR cloning in which, using synthetic primers, the sequences
that encode for the mimicry peptides were inserted into the DNA
vaccination vectors. Thus DNA vaccination vectors were also
produced on the basis of the pVAC2 which were encoded with the
tetanus toxoid for a fusion protein of the mimicry peptide.
The DNA of the vaccination vectors was increased according to
well known methods, cleaned and then injected into mice.
Mixtures of DNA vaccination vectors that encode antiidiotypical
scFv or mimicry peptides as the fusion protein using the tetanus
toxoid and which respectively originate from the various groups
with different binding patterns for MUC1-specific antibodies
(see above) were used for the immunization. The dose was 50 ug
or 200 ug, respectively, of total DNA and administration was
intramuscular. Four weeks later a booster of the same
preparation was administered and then repeated again after 4
CA 02375033 2001-11-27
weeks. 10 days later blood samples were taken from the mice. In
ELISA tests the serum was tested for antibodies that
specifically recognize the conformation-dependent epitope of the
MUC1 (test setup as above).
The immunization with the mixtures of DNA vaccine vectors showed
a strong humoral immune response against the conformation-
dependent epitope of the MUCl and a strong response against the
tetanus toxoid in the antiidiotypical scFv as well as the
mimicry peptides of the coding DNA vectors.
Vaccines in the tumour challenge model
In the mouse tumour challenge model, different mouse tumour
cells (3T3 and P815) were used that stably transfect with the
cDNA of the transmembrane form of the human MUC1. The MUC1-
positive mouse cell lines express the conformation epitope of
the MUC1 which was tested in immune binding studies with the
A76-A/C7. Several mouse lines were used for the studies (Balb/c,
DBA/2 and C57BL/6). After vaccinating the mice according to the
prime boost protocol described below, the mice were
subcutaneously injected near the peritoneum with 106 to 10'
tumour cells in 200 a PBS and the tumour growth (tumour size
in mm) was measured over 20 - 30 days.
Prime boost vaccination pattern:
For the immunizations (priming), a combination of DNA
vaccination vectors (encoding for scFv tetanus toxoid or mimicry
peptide tetanus toxoid fusion protein) was used with two
candidates from each of the 4 different groups of
antiidiotypical scFv or mimicry peptides. However, for the
booster, the same combinations of antiidiotypical scFv or
mimicry peptides were used in incomplete Freund's adjuvants, but
CA 02375033 2001-11-27
26
in their protein form. To this end, the scFv were cleaned by
nickel chelate chromatography according to well known procedures
and the mimicry peptides were coupled to KLH according to well
known procedures. For the immunization, 50 - 200 ug of total DNA
were administered intramuscularly and for the scFv and the
mimicry peptides, 10 - 200 ug were administered
intraperitoneally at three week intervals and boosters were
administered 2 - 3 times.
As a control, the DNA vaccination vectors were used for a scFv
with a specificity against an irrelevant bacterial protein or
for an irrelevant peptide (SSGSSSSGS), or their cleaned scFv or
the peptide KLH complex. 5 - 10 animals were studied for the
different test preparations.
The test showed that a vaccination according to the prime boost
protocol prevents the growth of injected MUCl-positive mouse
tumour cells or reduces them to a minimal size (0 - 20 mm2 after
20 days). In a subsequent injection with the same tumour cells
without transfected MUC1, the same vaccination achieves an
average tumour size of over 200 mm2 (after 20 days). The
injection of MUC1-positive mouse tumour cell lines into mice
without prior vaccination results in strong tumour growth (>200
mm2 after 20 days). An immunization and booster with the
proteins of the antiidiotypical scFv or the mimicry peptides
coupled to KLH without DNA vaccination vectors results in an
immune response against the MUC1 tumour cells, however, the
tumour protection is much less than with the prime boost
protocol with the DNA vaccination vectors.
The results show that a vaccination with DNA vaccination vectors
that encode for antiidiotypical scFv or mimicry peptides
provides excellent protection against tumours. This reaction is
MUCl-specific. It is far better or even possible compared to
CA 02375033 2001-11-27
27
vaccination studies with the proteins of the antiidiotypical
scFv or mimicry peptides without prior immunization with the
corresponding DNA vaccination vectors.
This shows that the vaccines against conformation-dependent
antigens according to the invention using DNA vaccination
vectors of mimicry structures is a successful form of fighting
tumours that carry these conformation-dependent antigens.
Example 2
Production of hybridome cell lines A78-G/A7 and of antibodies
In the case of A78-G/A7 (see also Karsten, U. et al., Hybridoma
14:37, 1995), Balb/c mice were immunized intraperitoneally with
100 ug of Asialoglycophorin (Sigma, Diesenhofen) in PBS mixed
with Freund's adjuvant. After 24 h, 100 ug of cyclophosphamid
in PBS per kg body weight was administered i.p. A booster of 100
ug Asialoglycophorin was administered two weeks later. Each time
the fusion cell line was X63-Ag8.653 (Kearney, J.F. et al., J
Immunol 123:1548, 1979). The hybridome method was carried out
according to standard methods (e. g. Peter, H.H. et al.,
"Monoklonale Antikorper, Herstellung and Charakterisierung"
[Monoclonal Antibodies, Production and Characterization], Berlin
1985; Friemel, H. "Immunologische Arbeitsmethoden"
[Immunological Working Methods], 4th edition, Jena 1991). The
specificity analysis of the monoclonal antibodies produced by
the hybridome cell lines was based on enzyme immunoassays with
natural glycoproteins, synthetic peptides and glycopeptides,
glycolipids and neoglycolipids and synthetic polyacrylamide-
carbohydrate conjugates, absorption analyses of synthetic
carbohydrate conjugates (Synsorb, Chembiomed, Edmonton, Canada),
immunofluorescence analyses with diverse cell lines as well as
immune histochemical studies of tissue sections. For the A78-
G/A7, the carbohydrate epitope Thomsen-Friedenreich (TF) that
is associated with tumours was unequivocally determined as a
CA 02375033 2001-11-27
28
specific antigen:
- A78-G/A7 binds exclusively to the disaccharide TF in the
a-anomer configuration (TFa; Gall-3GalNAca1-0-Ser/Thr) on
natural and synthetic structures as occur naturally only on
glycoproteins in the form of a direct 0-glycoside binding to
serines or threonines. However TFa, which can occur at the
end of glycan chains of glycolipids, as well as other
carbohydrate structures, portions of peptides or lipids, are
not bound.
- A78-G/A7 binds highly specifically to various cancer cell
lines in immunofluorescence studies and to various cancers in
histochemical studies. (Cao, Y. et al., Histochem Cell Biol
106:197, 1996; Cao, Y. et al., Cancer 76:1700, 1995; Cao, Y.
et al., Virchows Arch 431:159, 1997; Karsten, U. et al.,
Hybridoma 14:37, 1995.
- For A78-G/A7, IgM, k, was determined as the isotype using a
commercial isotyping kit (Pharmingen, San Diego, USA).
A78-G/A7 was cleaned using an ammonium sulphate precipitation
followed by an affinity chromatography on a proteinG affinity
matrix for cleaning undesirable IgG antibodies from the calf
serum and, finally, with an affinity chromatography using a
goat-anti-mouse-Ig affinity matrix (Perzellulose, BioTeZ,
Berlin-Buch) (Dr. G. Butschak).
Production of human recombinant antibody fragments against the
Thomsen-Friedenreich antigen from antibody libraries using the
phage display method
Two different synthetic antibody gene libraries were used which
represent human single-chain antibody fragments (scFv). One
antibody gene library comprises more than 101° phages with
various combinations of the variable regions of the heavy and
CA 02375033 2001-11-27
29
light chains of human antibodies with, in part, randomized
hypervariable regions that are connected by a peptide piece
(linker) and are covalently bound to a phage coat protein
(pIII). It is derived from a different antibody gene library
(Griffiths, A. et al., 1994, EMBO I., 13:3245-3260). The second,
smaller gene library comprises scFv which were preselected for
active folding of the antibody fragments. The first library
originates from the Dr. G. Winter Laboratory and the second from
the Dr. I. Tomlinson Laboratory (both MRC Centre for Protein
Engineering, Cambridge, U.K.). The specific phages were
selected in 2-3 rounds (phage panning) using the proteolytic
selection method with the helper phage KM13 (Kristensen, P. and
Winter, G. Folding and Design, 3:321, 1998). The cleaned
monoclonal antibody A78-G/A7 (35 u/ml in 4 ml) was used as the
antigen which was immobilized overnight in a test tube
(Immunotube, Nunc, Wiesbaden) in PBS at 4 degrees Celsius.
Alternatively, the cleaned antibody was incubated with the
phages; the phages bound to the antibodies were obtained through
magnet beads with immobilized anti-IgM antibodies (Deutsche
Dynal, Hamburg). After stringent washing steps (up to 20 times
PBS/ O.lo Tween20 followed by 20 times PBS), the phages
specifically bound during the selection rounds (3 h at RT) were
specifically eluted through the TFa-carrying glycoprotein
asialglycophorin (100 - 165 ug/ ml) and, in part, subsequently
treated with Trypsin (proteolytic selection method). Between the
selection rounds, the eluted phages were multiplied with helper
phages in the bacteria and selected once again. 2 to 3 selection
rounds were carried out.
Identification of peptides using a peptide gene library that
specifical3y mimics the Thomsen-Friedenreich antigen
Analogous to the example for generating antiidiotypical
antibodies, in multiple selection rounds specifically binding
CA 02375033 2001-11-27
peptides were obtained from a peptide gene library (Oligino, L.
et al., J Biol Chem 272:29046, 1997) that has 10' different
short peptides coupled to the phage coat protein pIII (in
cooperation with Dr. H. Gollasch Robert-Rossle-Klinik, Berlin-
Buch). The expressed peptides are randomized nonapeptides that
are flanked, and thus circularized, thereby increasing the
stability and the affinity. The selection and testing were
carried out as described in the generation of the
antiidiotypical antibodies.
Specificity testing of the mimicry peptides and antiidiotypical
antibody fragments
The selected peptides and antibody fragments were tested in
ELISA tests for their binding to the TF-specific antibodies and
to the plant lectin PNA (peanut agglutinin, Arachis hypogaea
lectin; Sigma) which also , if not exclusively, binds the
Thomsen-Friedenreich antigen, as well as for comparison with
other IgM and IgG antibodies. To this end, the form of peptides
and antibody fragments that are coupled to phages were used
which were cleaned beforehand through a polyethylene glycol
precipitation in 96-well plates. The potential mimicry peptides
and antiidiotypical antibody fragments were then studied in
ELISA inhibition tests as to whether they specifically inhibit
the binding of A78-G/A7 and/or other TF-recognizing antibodies
and lectins to the disaccharide TFa. To this end, the TFa-
carrying glycoprotein asialglycophorin was immobilized on ELISA
plates by drying, and the binding of the monoclonal antibodies
and lectins was concentration-dependently inhibited by the
mimicry peptides or antiidiotypical antibody fragments in the
form of the synthetic peptides or cleaned scFv alone or coupled
to phages (Fig. 2).
~
CA 02375033 2001-11-27
31
Inbred mice of the Balb/c and NMRI lines were immunized
intraperitoneally with mimicry peptides and antiidiotypical
antibody fragments in the form of the synthesized peptides or
cleaned scFv alone, respectively coupled to the protein KLH or
to bacteriophages in PBS, mixed with a complete Freund's
adjuvant. Three weeks later a booster of the same preparation,
but without the adjuvant, was administered. The booster was
repeated after three weeks and ten days later blood samples were
taken taken from the mice. In ELISA tests the serum was tested
for antibody bindings against the Thomsen-Friedenreich antigen.
Vaccination with TF-mimicking peptides in the mouse tumour model
Cell culture: The mouse colon cancer cell line C-26 was kept in
the medium RPMI 1640 with the addition of loo foetal calf serum.
Tumour model: 105 cells of the syngene colon cancer cell line C-
26 were transplanted subcutaneously into mice of the Balb/c line
in two variants: a) untreated and b) pretreated (TF-positive)
with neuraminidase from V. cholerae (Serva, Heidelberg). At
weekly intervals the tumour size was determined externally.
After 3 weeks the animals were killed and the livers removed in
order to determine the number of visible metastases on the
surface of the liver.
Vaccination: The vaccination of the mice was begun 6 weeks prior
to the tumour transplant. The phage preparation or the cleaned
scFv (as well as corresponding controls) were emulgated 1:1 with
an incomplete Freund adj uvants and inj ected i . p . Four weeks
later a booster was administered (without adjuvant). After two
more weeks, the tumour transplant (tumour challenge) was carried
out with untreated and C-26 cells treated with neuraminidase.
CA 02375033 2001-11-27
32
Results: The present results with three of the aforementioned
antiidiotypical scFv showed that the initial rate of tumours in
the C-26 cells treated with neuraminidase can be significantly
reduced through vaccination (to 3 - 16% of the control; control:
1000 initial rate). Furthermore, the number of liver metastases
in the vaccinated animals approximately corresponded to that of
the animals that were transplanted with untreated (TF-negative)
C-26 cells (ca. 2 per liver), whereas the unvaccinated control
animals with TF-positive C-26 cells had 5 - 9 metastases per
liver.
Legends for the Figures:
Fig. lc:
Inhibition of the A76-A/C7 binding to the MUC1 glycopeptide
(Biotin-Ahx-APPAHGVTSAPD-Thr(a-D-GalNAc)-RPAPGSTAPPAHGVTSA)
through scFv phages. The MUC1 glycopeptide was immobilised on
the streptavidin ELISA plate (5 ng/well) and subsequently
blocked with 30% FKS in RPMI. The culture supernatant of the
A76-A/C7 (diluted 1:80 ) was preincubated for one hour with the
scFv phages that were cleaned through a polyethylene glycol
precipitation in the indicated concentrations (percent by volume
of adjusted phage solutions in PBS) and subsequently placed on
the MUC1 glycopeptide plate for 2 hours. Verification occurred
via an anti-mouse-POD antibody (Dako). The scFv phages Q6, Q7
and Q8 are examples of antiidiotypical scFv, whereas Q4 and Q10
are examples of control scFv that bind the A78-A/C7 but are not,
however, antiidiotypical scFv.
CA 02375033 2001-11-27
33
Figure 2:
Inhibition of the A78-G/A7 binding to Asialoglycophorin through
scFv phages. The Asialoglycophorin (A-GP) was immobilized on the
ELISA plate by drying (25 ng/well) and subsequently blocked with
30% FKS in RPMI. The culture supernatant of the A78-G/A7
(diluted 1:20 ) was preincubated for one hour with the scFv
phages that were cleaned through a polyethylene glycol
precipitation in the indicated concentrations (percent by volume
of adjusted phage solutions in PBS) and subsequently placed on
the A-GP plate for 2 hours. Verification occurred via an anti-
mouse-POD antibody (Dako). The scFv phages P9, P13, P16, P3 and
K3 are examples of antiidiotypical scFv, whereas P8 and Ql are
examples of control scFv, of which P8 binds the A78-G/A7 but is
not, however, an antiidiotypical scFv, and Ql is a phage that
does not bind A78-G/A7.
