U.S. patent application number 10/203206 was filed with the patent office on 2003-07-24 for immunization of an individual against carcinoma and the preliminary stages thereof.
Invention is credited to Doeberitz, Magnus Von Knebel, Linnebacher, Michael, Rudy, Wolfgang.
Application Number | 20030139334 10/203206 |
Document ID | / |
Family ID | 7630548 |
Filed Date | 2003-07-24 |
United States Patent
Application |
20030139334 |
Kind Code |
A1 |
Doeberitz, Magnus Von Knebel ;
et al. |
July 24, 2003 |
Immunization of an individual against carcinoma and the preliminary
stages thereof
Abstract
The present invention relates to a pharmaceutical composition,
comprising a cell cycle regulatory protein and/or an expressible
nucleic acid coding for this in an amount suitable for immunization
of an individual against carcinomas and the preliminary stages
thereof and common auxiliary agents and/or to the use of a cell
cycle regulatory protein and/or an expressible nucleic acid coding
for this to immunize an individual against carcinomas and the
preliminary stages thereof.
Inventors: |
Doeberitz, Magnus Von Knebel;
(Heidelberg, DE) ; Linnebacher, Michael;
(Steinweiler, DE) ; Rudy, Wolfgang; (Bretten,
DE) |
Correspondence
Address: |
BURNS DOANE SWECKER & MATHIS L L P
POST OFFICE BOX 1404
ALEXANDRIA
VA
22313-1404
US
|
Family ID: |
7630548 |
Appl. No.: |
10/203206 |
Filed: |
December 23, 2002 |
PCT Filed: |
February 7, 2001 |
PCT NO: |
PCT/DE01/00470 |
Current U.S.
Class: |
424/185.1 ;
514/19.3; 514/44R |
Current CPC
Class: |
A61K 2039/53 20130101;
A61P 35/00 20180101; A61K 39/001149 20180801 |
Class at
Publication: |
514/12 ;
514/44 |
International
Class: |
A61K 038/17; A61K
048/00 |
Foreign Application Data
Date |
Code |
Application Number |
Feb 10, 2000 |
DE |
100 06 033.1 |
Claims
1. A pharmaceutical composition, comprising a cell cycle regulatory
protein and/or an expressible nucleic acid coding for this in an
amount suitable for immunizing an individual against carcinomas and
the preliminary stages thereof as well as common auxiliary agents,
wherein the cell cycle regulatory protein is a cyclin-dependent
kinase inhibitor and the carcinomas are those of the upper
respiratory tract or anogenital carcinomas.
2. The pharmaceutical composition according to claim 1, wherein the
cyclin-dependent kinase inhibitor is a protein p15, p16, p18 or
p19.
3. The pharmaceutical composition according to claim 1 or 2,
wherein the cell cycle regulatory protein is available in the form
of one or more fragments.
4. The pharmaceutical composition according to claim 3, wherein the
fragment or fragments contain epitopes which can be detected by
cytotoxic T cells and elicit a cytotoxic immune response.
5. The pharmaceutical composition according to any of claims 1 to
4, wherein the anogenital carcinoma is a cervical carcinoma.
6. Use of a cell cycle regulatory protein and/or an expressible
nucleic acid coding for this for immunizing an individual against
carcinomas and the preliminary stages thereof, wherein the cell
cycle regulatory protein is a cyclin-dependent kinase inhibitor and
the carcinomas are those of the upper respiratory tract and/or
anogenital carcinomas.
7. Use according to claim 6, wherein the cyclin-dependent kinase
inhibitor is a protein p15, p16, p18 or p19.
8. Use according to claim 5 or 6, wherein the cell cycle regulatory
protein is available in the form of one or more fragments.
9. Use according to claim 8, wherein the fragment or fragments
contain epitopes which can be detected by cytotoxic T cells and
elicit a cytotoxic immune response.
10. Use according to any of claims 6 to 9, the anogenital carcinoma
is a cervical carcinoma.
Description
[0001] The present invention relates to a pharmaceutical
composition containing a cell cycle regulatory protein and to the
use of the pharmaceutical composition for immunizing an individual
against carcinomas and the preliminary stages thereof.
[0002] Several million people fall ill with, and die of, carcinomas
world-wide every year. These mortality rates have remained
unchanged for many years despite intensive therapy research. Until
now, patients suffering from carcinomas often have to undergo
carcinoma-removing surgery or chemotherapy or radiation therapy.
However, this is accompanied by very massive side-effects which
then contribute to the mortality rates of patients suffering from
carcinomas.
[0003] It is thus the object of the present invention to provide a
product by means of which therapeutic and prophylactic steps can be
taken against carcinomas, the above side-effects being avoided.
[0004] According to the invention this is achieved by the subject
matters defined in the claims.
[0005] The present invention is based on Applicant's findings that
in carcinomas or the preliminary stages thereof cell cycle
regulatory proteins are available in modified form or amount. For
example, overexpression of cyclin-dependent kinase inhibitors is
found in carcinomas (cf. Applicant's German patent 198 29 473).
Applicant also found out that individuals can be immunized against
cell cycle regulatory proteins modified as regards form or amount
so as to take therapeutic and prophylactic steps against carcinomas
and the preliminary stages thereof. Applicant showed this by way of
in vitro and in vivo experiments (cf. below example).
[0006] The present invention thus relates to a pharmaceutical
composition, comprising a cell cycle regulatory protein and/or an
expressible nucleic acid coding for this in an amount suitable for
immunization of an individual against carcinomas and the
preliminary stages thereof as well as common auxiliary agents.
[0007] The employed term "cell cycle regulatory protein" comprises
cell cycle regulatory proteins of any kind and origin. For example,
these may be cyclins. In particular, these may be cyclin-dependent
kinases, such as cdk4 and cdk6, which regulate the cyclins. More
particularly, these may be cyclin-dependent kinase inhibitors
which, in turn, regulate the cyclin-dependent kinases. Examples of
cyclin-dependent kinase inhibitors are the proteins p15, p16, p18,
p19, with p16 being preferred. The cell cycle regulatory proteins
may be available in wild-type or modified form. The latter form
comprises modifications of the amino acid sequence, such as
additions, deletions, substitutions and/or inversions of one or
more amino acids. Fragments of cell cycle regulatory proteins as
such or in combination with carriers may also be present, the
fragments being able to have a wild-type or modified amino acid
sequence. It is favorable for the carriers in the individual not to
be immunogenic. Such carriers may be the individual's own proteins
or foreign proteins or fragments thereof. Carriers, such as serum
albumin, fibrinogen or transferrin or a fragment thereof are
preferred. It is particularly favorable for the fragments of the
cell cycle regulatory proteins to contain epitopes which are
recognized by cytotoxic T cells, e.g. CD8.sup.+ T cells, and may
induce a cytotoxic immune response. Such epitopes of cell cycle
regulatory proteins can be determined by methods with which a
person skilled in the art is familiar, in particular by using an
NIH software system (NIH bioinformation service
http:/bimas.dcrt.nih.gov.cgi-bin/molbi- o/ken_parker_comboform). It
can also be advantageous for different cell cycle regulatory
proteins or fragment thereof, to which the above explanations apply
correspondingly, to be simultaneously present. For the production
of the above cell cycle regulatory proteins, reference is made e.g.
to Sambrook et al., Molecular Cloning: A Laboratory Manual,
2.sup.nd edition, Cold Spring Harbor Laboratory Press, Cold Spring
Harbor N.Y. (1989).
[0008] The employed term "expressible nucleic acid coding for a
cell cycle regulatory protein" comprises any nucleic acid, e.g. RNA
or DNA, expressible in an individual and coding for a cell cycle
regulatory protein, to which the above explanations apply
correspondingly. The nucleic acid can be present as such, i.e.
together with elements suitable for the expression thereof, or in
combination with a vector. Examples of such elements are promoters
and enhancers, such as CMV, SV40, RSV metallothionein I and
polyhedrin promoter or CMV and SV40 enhancers. Further sequences
suitable for expression are disclosed in Goeddel: Gene Expression
Technology: Methods in Enzymology 185, Academic Press, San Diego,
Calif. (1990). Moreover, any vectors suitable for expression in
mammalian cells can be used as vectors. These are e.g. pcDNA3,
pMSX, pKCR, pEFBOS, cDM8 and pCEV4 as well as vectors derived from
pcDNAI/amp, pcDNAI/neo, pRc/CMV, pSV2gpt, pSV2neo, pSV2-dhfr, pTk2,
pRSVneo, pMSG, pSVT7, pko-neo and pHyg. Recombinant viruses, e.g.
adenovirus, vaccinia virus or adeno-associated virus, can also be
used as vectors. As regards the production of the above nucleic
acids, in particular vectors containing such nucleic acids,
reference is made to Sambrook et al., supra, for example.
[0009] The employed term "carcinomas and the preliminary stages
thereof" comprises carcinomas of any kind and origin and
preliminary stages thereof. For example, these may be carcinomas of
the upper respiratory tract or anogenital carcinomas, in particular
the cervical carcinoma and the preliminary stages thereof, such as
cervical intraepithelial neoplasia (CIN I-III), carcinoma in situ
(CIS), etc. Likewise benign modifications such as papillomas,
adenomas, hyperplasias or similar proliferations of epithelial,
mesenchymal or hematopoietic proliferations are also to be counted
thereamong.
[0010] The employed term "individual" comprises an individual of
any kind and origin having cell cycle regulatory proteins and being
able to fall ill with carcinomas and/or their preliminary stages.
Examples of such individuals are humans and animals as well as
cells thereof.
[0011] The employed term "amount suitable for immunization of an
individual" comprises any amount of a cell cycle regulatory
protein, to which the above explanations apply correspondingly, or
an expressible nucleic acid coding for this, to which the above
explanations apply correspondingly, and with which an individual
can be immunized. The amount depends on whether a cell cycle
regulatory protein or an expressible nucleic acid coding for this
is used. The amount also depends on whether immunization of the
individual rather aims at an induction of antibodies directed
against modified cell cycle regulatory proteins or a stimulation of
cytotoxic T cells, e.g. CD8.sup.+ T cells, directed against
modified cell cycle regulatory proteins. Both possibilities of
immunization can be achieved by the present invention. Furthermore,
the amount depends on whether immunization is intended as a
prophylactic or therapeutic treatment. In addition, the
individual's age, sex and weight play a role for determining the
amount. It is favorable to give the individual 100 .mu.g-1 g of a
cell cycle regulatory protein or 10.sup.6-10.sup.12 MOI of a
recombinant virus containing an expressible nucleic acid coding for
a cell cycle regulatory protein by means of injection. The
injection may be made at various sites of the individual
intramuscularly, subcutaneously, intradermally or in any other form
of application. It may also be favorable to carry out one or more
"booster injections" having about equal amount. In this case, it
may be particularly favorable to use different fragments of the
respective cell cycle regulatory proteins for the individual
injections.
[0012] The employed term "common auxiliary agents" comprises any
auxiliary agents suitable for a pharmaceutical composition to
immunize an individual. Such auxiliary agents are e.g. immunization
adjuvants, such as GM-CSF or Freund's adjuvant, buffered common
salt solutions, water, emulsions, such as oil/water emulsions,
wetting agents, sterile solutions, etc.
[0013] By means of the present invention it is possible to immunize
individuals, in particular humans and animals, against modified
cell cycle regulatory proteins. Immunization takes place by both
induction of antibodies and stimulation of CD8.sup.+ T cells,
directed against modified cell cycle regulatory proteins. Thus, it
is possible to take prophylactic and therapeutic steps against
carcinomas and the preliminary stages thereof.
[0014] The invention is explained by the below example.
EXAMPLE
Stimulation of CD8.sup.+ T Cells Against the Cyclin-Dependent
Kinase Inhibitor p16 and Lysis of p16-Overexpressing Carcinoma
Cells.
(A) Stimulation of CD8.sup.+ T cells against p16.
[0015] Peripheral mononuclear cells are obtained from a healthy
donor and subjected to what is called ELISPOT analysis. It is the
principle of this experiment to stimulate lymphocytes in culture
vessels with specific antigens. Whenever the lymphocytes are
activated as they recognize the antigen, the activated lymphocytes
release cytokines which, in turn, bind to specific antibodies
immobilized on the bottom surface of the culture vessels. Having
washed out the lymphocytes, the bound cytokines can be detected in
the culture vessels by means of a second antibody made visible in a
subsequent color reaction.
[0016] Peripheral blood lymphocytes (PBL) from an
HLA-A0201-positive healthy proband are purified by density
centrifugation via a Ficoll Paque.RTM. gradient. T-lymphocytes are
obtained by separating the B-lymphocytes or the monocytes using
antibody-coupled magnetobeads (CD11, CD16, CD19, CD36 and CD56)
(Pant T cell isolation Kit.RTM., Milteny, Bergisch Gladbach,
Germany). About 2.times.10.sup.7 T cells are obtained from 30 ml
blood.
[0017] HLA-A0201-restricted peptides of p16 are identified by means
of an NIH software system (NIH bioinformation service
[http:/bimas.dcrt.nih.gov- .cgi-bin/molbio/ken_parker_comboform).
These are the below peptides:
1 9mer peptides: 10mer peptides: score 1: VMMMGSARV score 1:
MMGSARVAEL score 2: VLHRAGARL score 2: LLLHGAEPNC score 3:
TLTRPVHDA score 3: GVMMMGSARV score 4: LLHGAEPNC score 5:
SMEPSADML
[0018] The isolated T cells are incubated with T2 cells, which (a)
have been loaded with a mixture of the above 9mer peptides (10
.mu.g/peptide) and (b) with a mixture of the above 10mer peptides
(10 .mu.g/peptide). The T cells are restimulated once a week for a
period of 6 weeks. 107 T cells each are cocultured with
2.times.10.sup.6 peptide-loaded T2 cells in 24-well plates.
[0019] The reactivity over the peptide-loaded T2 cells is
determined once a week, starting on day 0 of the experiment, by
carrying out IFN-.gamma. elispot analysis. On day 28, a reactivity
is observed by the mixture of (a) (400 specific cells per million
cells). The main reactivity is in this case directed against the
peptide VMMMGSARV (1,000 specific cells/1,000,000 cells) (FIG. 1).
A less intensive activity is observed against the mixture of (b)
(150 specific cells/1,000,000 cells). Here, the peptide MMGSARVAEL
shows maximum reactivity (600 specific cells/1,000,000 cells).
[0020] Hence it is evident that it is possible to stimulate CD8+T
cells activated against p16.
[0021] (B) Lysis of p16-Overexpressing Carcinoma Cells
[0022] Following another restimulation, the activated CD8.sup.+ T
cells are incubated with the HLA A0201+ cervical carcinoma cells
Caski, which overexpress p16. The colon carcinoma cells SW480 which
do not overexpress p16 are used as controls. 10.sup.6 Caski cells
are labeled with .sup.52Cr (100 .mu.Ci) at 37.degree. C. for 1 h
and cocultured with increasing numbers of activated CD8.sup.+ T
cells for 3 hours. Specific lysis of the Caski cells is determined
by the amount of released radioactivity in the supernatant.
[0023] It turns out that Caski cells are lyzed by the activating
CD8.sup.+ T cells but not by the control cells SW480 (FIG. 2).
* * * * *
References