U.S. patent application number 13/632737 was filed with the patent office on 2013-04-18 for use of trifunctional bispecific and trispecific antibodies for the treatment of malignant ascites.
This patent application is currently assigned to TRION PHARMA GMBH. The applicant listed for this patent is TRION PHARMA GMBH. Invention is credited to Horst Lindhofer.
Application Number | 20130095106 13/632737 |
Document ID | / |
Family ID | 7654853 |
Filed Date | 2013-04-18 |
United States Patent
Application |
20130095106 |
Kind Code |
A1 |
Lindhofer; Horst |
April 18, 2013 |
USE OF TRIFUNCTIONAL BISPECIFIC AND TRISPECIFIC ANTIBODIES FOR THE
TREATMENT OF MALIGNANT ASCITES
Abstract
The invention describes the use of a pharmaceutical preparation
containing a trifunctional bispecific and/or trispecific having the
following properties: a) binding to a T cell; b) binding to at
least an antigen on a tumor cell associated with malignant ascites
and/or pleural effusion; c) binding, by its Fc portion (in the case
of bispecific antibodies) or by a third specificity (in the case of
trispecific antibodies), to Fc receptor-positive cells for the
destruction of the tumor cells in the treatment of malignant
ascites and/or pleural effusion.
Inventors: |
Lindhofer; Horst;
(Grobenzell, DE) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
TRION PHARMA GMBH; |
Muenchen |
|
DE |
|
|
Assignee: |
TRION PHARMA GMBH
Muenchen
DE
|
Family ID: |
7654853 |
Appl. No.: |
13/632737 |
Filed: |
October 1, 2012 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
10378218 |
Mar 3, 2003 |
8277806 |
|
|
13632737 |
|
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Current U.S.
Class: |
424/136.1 |
Current CPC
Class: |
A61K 39/39558 20130101;
A61K 2039/505 20130101; A61P 35/00 20180101; C07K 16/2809 20130101;
C07K 16/30 20130101; C07K 16/32 20130101; C07K 2317/31 20130101;
A61K 2039/5152 20130101 |
Class at
Publication: |
424/136.1 |
International
Class: |
A61K 39/395 20060101
A61K039/395 |
Foreign Application Data
Date |
Code |
Application Number |
Sep 4, 2000 |
DE |
100434371 |
Sep 4, 2001 |
EP |
PCT/EP01/10184 |
Claims
1. A method for inhibiting or reducing the formation of malignant
ascites, pleural effusion, or a combination thereof in a subject,
the method comprising administering to a subject having malignant
ascites and/or pleural effusion one or more doses of a
pharmaceutical preparation comprising a trifunctional bispecific
and/or trispecific antibody, wherein the trifunctional bispecific
and/or the trispecific antibody comprises: a) a first binding arm
that binds to a T cell in the subject; b) a second binding arm that
binds to an antigen on a tumor cell associated with malignant
ascites or pleural effusion in the subject; and; c) an Fc portion
that binds to an Fc receptor-positive cell in the subject, thereby
destroying the tumor cell and treating the malignant ascites, the
pleural effusion, or the combination thereof in the subject.
2. The method according to claim 1, wherein the pharmaceutical
preparation is adapted for administration in the form of a first
dose and of at least one further dose.
3. The use method according to claim 1, wherein the administering
step comprises administering at least three doses of the
pharmaceutical preparation, a first dose for testing the subject
for an allergic reaction against the trifunctional bispecific
and/or trispecific antibody, a second dose for activating and
proliferating immune cells in the subject, and a third dose, and
optionally one or more subsequent doses, each of which is for
destroying tumor cells associated with malignant ascites, pleural
effusion, or the combination thereof in the subject.
4. The use method according to claim 1, wherein said pharmaceutical
preparation is adapted for administration in the form of a first
dose to test for allergic reactions against the antibody
administered.
5. The method according to claim 3, wherein the first dose
comprises 1 to 20 .mu.g of the trifunctional bispecific and/or
trispecific antibody, the second dose comprises 20 to 100 .mu.g of
the trifunctional bispecific and/or trispecific antibody, and the
third dose, and each of the one or more subsequent doses if
present, comprises 100 to 500 .mu.g of the trifunctional bispecific
and/or trispecific antibody.
6. The method according to claim 1, wherein the administering step
comprises administering 3 to 8 doses.
7. The use method according to claim 1, wherein the pharmaceutical
preparation further comprises inactivated autologous tumor cells,
inactivated allogenic tumor cells, autologous immune cells, or
combinations thereof.
8. The method according to claim 7, wherein the autologous immune
cells comprise apheresis cells obtained from the subject.
9. The method according to claim 1, wherein the administering step
comprises a primary immunization of the pharmaceutical preparation
intraperitoneal administration, followed by one or more secondary
immunizations by intradermal, subcutaneous, or intramuscular
administration of the pharmaceutical preparation.
10. The method according to claim 7, wherein for the one or more
secondary immunizations, the pharmaceutical preparation comprises
1-10 .mu.g trifunctional bispecific and/or trispecific antibodies
and at least one of: a) 1-200.times.10.sup.6 inactivated tumor
cells, and b) 10-500.times.10.sup.6 immune cells.
11. The method according to claim 1, wherein the trifunctional
bispecific and/or trispecific antibody is an intact bispecific
and/or trispecific antibody.
12. The method according to claim 1, wherein the trifunctional
bispecific and/or trispecific antibody binds to an Fc
receptor-positive cell having an Fc.gamma. receptor I or III.
13. The method according to claim 1, wherein the trifunctional
bispecific and/or trispecific antibody binds to an Fc.gamma.
receptor I- or III-positive cell selected from the group consisting
of a monocyte, a macrophage, a dendritic cell, a "natural killer"
cell (NK cell, and an activated neutrophil.
14. The method according to claim 1, wherein the trifunctional
bispecific and/or trispecific antibody induces tumor-reactive
complement-binding antibodies and a humoral immune response in the
subject.
15. The method according to claim 1, wherein the first binding arm
binds to a molecule selected from the group consisting of CD2, CD3,
CD4, CD5, CD6, CD8, CD28, CD154, and CD44 that is present on the T
cell.
16. The method according to claim 1, wherein binding of the
trifunctional bispecific and/or trispecific antibody to the Fc
receptor-positive cell, induces or enhances Fc receptor-positive
cell expression of at least one of CD40, CD80, CD86, ICAM-1, and
LFA-3; secretion of a cytokine; or combinations thereof.
17. The method according to claim 16, wherein the cytokine is
selected from the group consisting of IL-1, IL-2, IL-4, IL-6, IL-8,
IL-12, INF-.gamma., and TNF-.alpha..
18. The method according to claim 15, wherein the bispecific and/or
trispecific antibody is selected from the group consisting of an
anti-CD3 X anti-tumor-associated antigen bispecific and/or
trispecific antibody; an anti-CD4 X anti-tumor-associated
bispecific and/or trispecific antibodyl an anti-CD5 X
anti-tumor-associated antigen bispecific and/or trispecific
antibody; an anti-CD6 X anti-tumor-associated antigen bispecific
and/or trispecific antibody; an anti-CD8 X anti-tumor-associated
antigen bispecific and/or trispecific antibody; an anti-CD2 X
anti-tumor-associated antigen bispecific and/or trispecific
antibody; an anti-CD28 X anti-tumor-associated antigen bispecific
and/or trispecific antibody; an anti-CD40L X anti-tumor-associated
antigen bispecific and/or trispecific antibody; and an anti-CD44 X
anti-tumor-associated antigen bispecific and/or trispecific
antibody.
19. The method according to claim 1, wherein the trifunctional
bispecific and/or trispecific antibody is a heterologous
trifunctional bispecific and/or trispecific antibody.
20. The method according to claim 1, wherein said trispecific
antibody has one T cell binding arm, one tumor cell binding arm,
and a third specificity for binding to Fc receptor-positive
cells.
21. The method according to claim 1, wherein the trispecific
antibody is selected from the group consisting of an anti-CD3 X
anti-tumor-associated antigen antibody, an anti-CD4 X
anti-tumor-associated antigen antibody, an anti-CD5 X
anti-tumor-associated antigen antibody, an anti-CD6 X
anti-tumor-associated antigen antibody, an anti-CD8 X
anti-tumor-associated antigen antibody, an anti-CD2 X
anti-tumor-associated antigen antibody, an anti-CD28 X
anti-tumor-associated antigen antibody, an anti-CD40L X
anti-tumor-associated antigen antibody, and an anti-CD44 X
anti-tumor-associated antigen antibody.
22. The method according to claim 7, wherein the inactivated
autologous tumor cells or the inactivated allogenic tumor cells
have been inactivated by exposing autologous tumor cells or
allogenic tumor cells to irradiation.
23. The method according to claim 7, further comprising heating the
inactivated allogenic tumor cells or the inactivated allogenic
tumor cells prior to administration to the subject, whereby
immunogenicity of the heated inactivated autologous tumor cells or
the heated inactivated allogenic tumor cells is enhanced in the
subject.
24. The method according to claim 1, wherein the inding of the
trifunctional bispecific and/or trispecific antibody to an Fc
receptor-positive cell induces or enhances expression of a
cytokine, a co-stimulatory antigen, or combinations thereof in the
Fc-receptor-positive cell.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a divisional of U.S. patent application
Ser. No. 10/378,218, filed Mar. 3, 2002 (now pending), which itself
is a continuation of PCT International Patent Application Serial
Number PCT/EP01/10184, filed Sep. 4, 2001, which itself claims
priority to German patent application number 10043437.1, filed Sep.
4, 2000. The disclosure of each of these applications is
incorporated herein by reference in its entirety.
TECHNICAL FIELD
[0002] The invention relates to the use of a pharmaceutical
preparation containing trifunctional bispecific antibodies and/or
trispecific antibodies for the destruction of the tumor cells
associated with malignant ascites and/or pleural effusion in order
to treat malignant ascites or pleural effusion.
BACKGROUND
[0003] Malignant ascites may be caused by a plurality of primary
tumors such as e.g. breast cancer, ovarian carcinoma or the
gastrointestinal carcinomas. Although ascites is detected in a high
percentage of patients already during the first manifestation of a
tumor disease, it is an indication of a progressive disease.
[0004] The present options for a therapy of ascites include
puncture, local chemotherapy, or diuretic treatment. All these
options have dramatic disadvantages; thus, puncture only leads to a
short-term alleviation and has to be repeated after 9.5 days on
average (Mackey et al., J. Pain Symptom Manage, 19:193, 2000).
Chemotherapy, however, can only be successful in patients who have
not already developed chemotherapy-resistant tumor cells which
unfortunately is often the case. In this respect, there is an
enormous need for an improvement of the clinical treatment options
in the case of ascites.
[0005] The situation is similar in the case of tumor cell-induced
pleural effusion. Pleural effusion is an accumulation of liquid in
the pleural space which may form again after a puncture. Since one
of the causes of pleural effusion is the presence of tumor cells
within this body compartment, destruction of the tumor cells is an
important prerequisite to inhibit reformation of the pleural
effusion. This means that the problems associated with malignant
ascites and tumor cell-based pleural effusion and therefore the
respective approaches to a solution are very similar.
SUMMARY
[0006] It is an object of the invention to provide a novel means
for the treatment of malignant ascites and for the treatment of
pleural effusion which overcomes the disadvantages of the prior art
and, particularly, offers a noticeable alleviation to the
patient.
[0007] According to the invention, this object has been achieved by
a pharmaceutical preparation containing a trifunctional bispecific
and/or trispecific antibody having the following properties: [0008]
a) binding to a T cell; [0009] b) binding to at least an antigen on
a tumor cell associated with malignant ascites and/or pleural
effusion; [0010] c) binding, by its Fc portion (in the case of
bispecific antibodies) or by a third specificity (in the case of
trispecific antibodies), to Fc receptor-positive cells for the
destruction of the tumor cells in the treatment of malignant
ascites and/or pleural effusion.
[0011] Further embodiments of the present invention may be seen
from the accompanying claims as well as the following description.
It has to be pointed out that the invention is not limited to the
preferred embodiments and the Examples mentioned below. In
contrast, those skilled in the art in the frame of their technical
knowledge may modify the invention in the scope of the accompanying
claims in connection with the description without departing from
the invention.
BRIEF DESCRIPTION OF THE DRAWINGS
[0012] The accompanying Figures serve to further illustrate the
present invention.
[0013] The Figures show:
[0014] FIG. 1: Elimination of EpCAM+ tumor cells and proliferation
of immune cells in the ascites under bsab therapy;
[0015] FIG. 2: FACS analysis of tumor cells for surface
antigens;
[0016] FIG. 3: Destruction of residual tumor cells after
CD3.times.EpCAM treatment by means of RT PCR;
[0017] FIG. 4: Detection of EpCAM-positive tumor cells from the
ascites liquid in flow cytometry during an antibody therapy;
[0018] FIG. 5: Decrease of ascites liquid formed under therapy
using the antibodies REMOVAB.RTM. (anti EpCAM.times.anti CD3) and
REXOMAB (anti Her2/neu.times.anti CD3).
DETAILED DESCRIPTION
[0019] Trifunctional bispecific and trispecific antibodies and
different fields of use of these antibodies are known from the
prior art. Reference is made in this respect to DE 198 44 157.6 as
well as DE 199 25 586. Although these state of the art documents
describe the use of bispecific and trispecific antibodies having
the three features a, b, and c for anti-tumor immunization, a
direct treatment of the tumors by destruction of the tumor cells
which goes beyond induction of an immunity is not described in
these documents. These documents in particular do not disclose that
a very specific partial area of the extraordinarily broad spectrum
of tumor diseases, namely malignant ascites but also pleural
effusion, may be treated by trifunctional bispecific and
trispecific antibodies having the features listed above in a highly
efficient and unexpected manner. Furthermore, a successful
treatment has already been achieved in two clinical patients
suffering form a final tumor disease (cf. Examples 1 and 2).
[0020] The pharmaceutical preparation has been designed to achieve
at least a primary immunization by destruction of the tumor cells.
For this purpose, the pharmaceutical preparation containing the
trifunctional antibodies described in more detail below is
administered in several doses. The first dose is selected to test a
reaction to foreign proteins of the patient in response to the
antibodies administered. The second dose is preferably selected to
achieve activation and proliferation of the patient's immune cells.
A third dose and further doses are selected to particularly achieve
destruction of the tumor cells.
[0021] Administration of the first dose is carried out in an amount
of 1 to 20 .mu.g, preferably 5 to 10 .mu.g of antibody to test for
allergic reactions. Administration of the second dose is performed
in an amount of 20 to 100 .mu.g, preferably 80 .mu.g, further
preferred 30 to 60 .mu.g of antibody. The administration of the
third dose and of any further dose is performed in an amount of
more than 80 .mu.g, preferably 100 to 500 .mu.g, further preferred
100 to 300 .mu.g of antibody. It should be understood by those
skilled in the art that these amount are only guide numbers which
may vary between patients and antibodies. It is within the skill of
those working in the medical field, however, to appropriately
select the amount of antibody to be employed in individual
cases.
[0022] The number of applications preferably is an application of
one dose 3 to 8 times, preferably 3 to 6 times. The administration
is carried out in certain intervals, and preferably intervals of 48
to 72 hours are selected. Preferably, between individual
applications an interval of 2-3 days plus/minus several hours is
selected. The applications are preferably continued over a period
of 12-14 days. The period is selected in a way that no further
antibody doses will be administered from the point when first
immune reactions against the antibody protein are observed. If a
mouse antibody is used then for example the HAMA (human anti-mouse
antibody) reaction will be determined which generally is initiated
after 16 to 21 days, in some patients earlier, in others later or
not at all.
[0023] As detailed above, malignant ascites is particularly
frequent in a plurality of primary tumors such as breast cancer,
ovarian carcinoma and gastrointestinal carcinomas. Such tumor
diseases are treated by first resecting the tumor tissue by surgery
and subsequent chemotherapy. In a preferred embodiment of the
invention the patient's immune cells are obtained by apheresis.
This apheresis product is an accumulation of blood cells which
particularly does not contain erythrocytes. Contained are
especially T cells, macrophages, monocytes, NK cells, dendritic
cells, granulocytes, and B cells. The preparation of the apheresis
product must be particularly carried out prior to the first
chemotherapy to ensure integrity of the immune cells which are
almost always affected by the chemotherapeutics.
[0024] In one embodiment of the invention, the patient is not only
treated by the trifunctional bispecific and/or trispecific
antibodies but, in the frame of a secondary immunization, also
receives in addition inactivated autologous and/or allogenic tumor
cells and autologous immune cells. The autologous immune cells
preferably are T lymphocytes and accessory cells, for example
monocytes, macrophages and dendritic cells. These cells are for
example contained in PBMC (peripheral blood mononuclear cells)
which may be obtained for example from heparinized blood via Ficoll
density gradient centrifugation. It is also possible, however, to
employ subfractions, e.g. only T lymphocytes and dendritic cells or
macrophages, respectively, etc. In a preferred embodiment the
autologous immune cells are the apheresis cells which are employed
in an admixture together with the antibody and the inactivated
autologous or allogenic tumor cells.
[0025] For successful immunization it is further advantageous to
select the amount of tumor cells administered in a suitable manner.
Thus, it has been shown in experiments with the murine tumor model
that too low a number of tumor cells does not bring about the
desired immunization success. Too high a number of tumor material,
however, may have an disadvantageous effect since for example
tolerance phenomena may arise. If these results are applied to the
situation in the patient, this would mean that it is advantageous
for a successful immunization to administer a defined amount of
tumor material in the correct spatial context together with amount
of antibodies which has also been defined. Although successful
immunization will also be achieved if one of these parameters has
not been optimized, particularly good results are achieved,
however, if the amounts of the antibody and the amount of tumor
material as well as the spatial context have been matched and
optimized.
[0026] Considering what has been explained above, in the case of
too low a number of tumor cells only an insufficient immune
protection can be established. Therefore, it is required for a
complete success of immunization to immunize with a defined number
of inactivated tumor cells and a defined amount of bispecific
and/or trispecific antibodies. The respective numbers may be
determined by those skilled in the art by way of experiments.
[0027] The tumor cells are preferably administered in an amount of
10.sup.5-10.sup.8 cells per application while a cell number of
about 10.sup.7 has been found to be preferable. Prior to their
reapplication, the tumor cells have been treated as detailed above
so that their survival after reapplication is inhibited while they
optionally may additionally be subjected to heat pretreatment.
[0028] The tumor cells are obtained from the tumor material of the
patient to be treated. For example, the tumor material will be
worked up to yield a single cell suspension by enzymatic treatment,
preferably by collagenase treatment. It is important that the tumor
cells are as undamaged as possible. Tumor cell lysates have been
found unsuitable. Prior to their application the tumor cells are
treated to exclude survival of the tumor cells in the patient.
Therefore, the tumor cells are treated in a manner known per se,
e.g. by irradiation or treatment with chemical agents. By this
treatment, especially the outer structure of the tumor cell should
remain unaffected to retain the recognition pattern for the
antibodies.
[0029] Preferably, .gamma.-irradiation is used for irradiation,
which preferably is carried out at a dose of 20 to 200 Gy. For a
chemical treatment, mitomycin C has been found particularly
successful.
[0030] A further improvement of the immunogenicity of the tumor
cells may be achieved by subjecting them to a heat pretreatment
prior to infusion. The preferred temperature is in the range of 41
to 45.degree. C. while a range of 41 to 42.degree. C. is preferred.
The optimum may be determined by experimentation. Preferred results
are achieved particularly at a temperature of about 41.8.degree. C.
The period for the heat pretreatment generally is 1 to 6 hours,
preferably about 3 hours. The period as well as the temperature
which may be optimally employed are dependent on the type of tumor
to be treated. The respective optimal values may be determined by
those skilled in the art by way of experiments.
[0031] Administration of the single doses generally is performed in
a way that an intraperitoneal application form is chosen wherein
the antibodies are infused into the patient to achieve a primary
immunization, i.e. destruction of the tumor cells. Administration
of the doses for secondary immunization generally is carried out by
local application, for example intradermally, subcutaneously or
intramuscularly. It may also be performed intraperitoneally or
intravenously.
[0032] If together with the trifunctional antibodies also the
inactivated autologous and/or allogenic tumor cells and the
autologous immune cells are administered to achieve a secondary
immunity the following amounts are preferably used: [0033] a)
1-200.times.10.sup.6 inactivated tumor cells, preferably
1-100.times.10.sup.6 inactivated tumor cells [0034] b)
10-500.times.10.sup.6 immune cells, preferably
50-200.times.10.sup.6 immune cells [0035] c) 1-10 .mu.g
trifunctional bispecific antibodies or trispecific antibodies,
preferably 2-5 .mu.g antibodies.
[0036] As already detailed above, however, those skilled in the art
will choose the respective amount and the site of application to
ensure an optimal therapeutic success.
[0037] The pharmaceutical preparation provided according to the
invention is for example present in an isotonic saline. Other
components may be for example stabilizing agents such as Tween 80
or buffer solutions.
[0038] For treatment, preferably intact trifunctional bispecific
and/or trispecific antibodies are used. The treatment not only
achieves the surprising direct destruction of the tumor cells
described above but an immunity is also induced which is directed
against the tumor.
[0039] By "intact antibodies" there are meant those antibodies
having a functional Fc portion. Preferably those are heterologous
antibodies, i.e. they are combined of heavy immune globulin chains
of different subclasses (combinations, also fragments) and/or
different origin (species).
[0040] Besides the features a, b, and c described above, in
preferred embodiments of the invention the antibodies employed also
show the additional features d and e: [0041] d) activates the Fc
receptor-positive cell by its binding to the Fc receptor-positive
cell whereby the expression of cytokines and/or of co-stimulatory
antigens is initiated or increased; [0042] e) the co-stimulatory
antigens and/or cytokines transfer to the T cell at least one
2.sup.nd activation signal which is required for physiological
activation of the T cell, this activation being indicated by an
up-regulation of activation markers, a destruction of the tumor
cell and/or a proliferation of the T cell.
[0043] In the following, the invention will be described
particularly using bispecific antibodies as an example. The
results, however, can also be achieved with trispecific
antibodies.
[0044] The antibodies useful according to the present invention are
capable of activating the Fc receptor-positive cell whereby the
expression of cytokines and/or co-stimulatory antigens is initiated
or increased.
[0045] In the case of the trispecific antibodies, binding to the Fc
receptor-positive cells preferably occurs for example via the Fc
receptor of Fc receptor-positive cells or alternatively via other
antigens on Fc receptor-positive cells (antigen-presenting cells)
such as the mannose receptor.
[0046] By means of the combination and the way of application of
the intact, preferably heterologous bispecific and/or trispecific
antibodies of the present invention, besides a treatment of the
ascites and the pleural effusion by direct destruction of the tumor
cells also an anti-tumor immunity, preferably a long-lasting
anti-tumor immunity is developed in the patient. The administration
is preferably performed in a patient after treatment of the primary
tumor, preferably in patients in a minimal residual disease (MRD)
situation. In patients with a low amount of residual tumor cells in
whom, however, the risk of a recurrency may be high, the
application of the pharmaceutical preparation described according
to the present invention is especially successful.
[0047] The heterologous bispecific and/or trispecific antibodies
which may be used according to the present invention are known per
se. For example they have been described in Lindhofer et al.,
Blood, 88:4651, 1996; or Lindhofer et al., J. Immunology, 155:219,
1995.
[0048] On the tumor cell, an up-regulation of MHC I as well as an
activation of the intracellular processing machinery (proteasome
complex) occurs due to the release of cytokines (such as
INF-.gamma. and TNF-.alpha.) in the direct vicinity of the tumor
cell. The cytokines are released due to bispecific
antibody-mediated activation of T cells and accessory cells. This
means that due to the intact bsab not only tumor cells are
destroyed or phagocytized but indirectly also the anti-tumor
immunogenicity is increased.
[0049] Activation of the Fc receptor-positive cell by the bsab is
dependent on the subclass or the subclass combination,
respectively, of the bsab. As demonstrated in in vitro experiments,
for example, bsabs of the subclass combination
mouse-IgG2a/rat-IgG2b are capable of binding to and simultaneously
activating Fc receptor-positive cells resulting in an up-regulation
or new formation (expression), respectively, of co-stimulatory
antigens such as CD40, CD80, or CD86 on the cell surface of these
cells. (Zeidler et al., J. Immunol., 163:1246, 1999). In contrast,
bsabs of the subclass combination mouse-IgG1/rat-IgG2b are able to
bind to Fc receptor-positive cells (Haagen et al., J. Immunology,
1995, 154: 1852-1860) but obviously are unable to activate these
cells to a comparable extent (Gast et al., Cancer Immunol.
Immunother., 1995, 40: 390).
[0050] While the intact bsab simultaneously binds to and activates
the T cell with one binding arm (e.g. to CD3 or CD2),
co-stimulatory signals from the Fc receptor-positive cell bound to
the Fc portion of the bsab may be transferred to the T cell. This
means that only the combination of T cell activation via one
binding arm of the bsab and simultaneous transfer of co-stimulatory
signals from the Fc receptor-positive cell to the T cell results in
an efficient T cell activation. Also, tumor-specific T cells which
have been insufficiently activated at the tumor cell and therefore
are anergic may be reactivated by the treatment with intact
bispecific antibodies or trispecific antibodies according to the
present invention.
[0051] Another important aspect in the induction of an anti-tumor
immunity is possible phagocytosis, processing and presentation of
tumor components by the accessory cells (monocytes/macrophages, or
dendritic cells) which have been recruited and activated by the
bsab. By this classical mechanism of presentation of antigens both
tumor-specific CD4- as well as CD8-positive cells may be generated.
Moreover, tumor-specific CD4 cells play an important role in the
induction of a humoral immune response in the context of T/B cell
cooperation.
[0052] Bispecific and trispecific antibodies are able to bind to
the T cell receptor complex of the T cell with one binding arm and
to tumor-associated antigens on the tumor cell with the second
binding arm. Thereby, they activate T cells which destroy the tumor
cells by releasing cytokines or by apoptosis-mediating mechanisms.
Moreover, there seems to be the possibility that in the frame of
activation by bispecific antibodies T cells recognize
tumor-specific antigens via their receptor and thereby a
long-lasting immunization is initiated. Of particular importance in
this respect is the intact Fc portion of the bispecific or
trispecific antibody mediating the binding to accessory cells such
as monocytes/macrophages and dendritic cells and causing them to
become cytotoxic themselves and/or to concomitantly transfer
important co-stimulatory signals to the T cell. Obviously, in this
manner under certain circumstances a T cell response may be induced
against so far unknown tumor-specific peptides.
[0053] By redirection of possibly anergized tumor-specific T cells
to tumor cells by means of bispecific and/or trispecific antibodies
and simultaneous co-stimulation of such T cells by accessory cells
binding to the Fc portion of the bispecific or trispecific antibody
the anergy of cytotoxic T cells (CTLs) could be abolished. This
means that a T cell tolerance against the tumor existing in the
patient may be abolished by means of intact heterologous bispecific
and/or trispecific antibodies and, thus, a long-term anti-tumor
immunity may be induced.
[0054] This last issue is supported by initial in vivo data from
experiments with mice indicating a long-term anti-tumor immunity of
this kind after treatment with a syngeneic tumor and intact bsabs.
In these experiments, a total of 14 out of 18 animals which could
be successfully treated with bsabs following a first tumor
injection survived a second tumor injection carried out 144 days
after the first injection--without an additional bsab
administration.
[0055] Preferably, the antibodies employed according to the present
invention are capable of reactivating tumor-specific antigens being
in the anergic state. Furthermore, they are capable of inducing
tumor-reactive complement-binding antibodies and therefore inducing
a humoral immune response.
[0056] Binding to the T cell preferably takes place via CD3, CD2,
CD4, CD5, CD6, CD8, CD28, CD40L and/or CD44. The Fc
receptor-positive cells at least have one Fc.gamma. receptor type I
or III.
[0057] Antibodies which may be employed according to the present
invention are able to bind to monocytes, macrophages, dendritic
cells, "natural killer" cells (NK cells) and/or activated
neutrophils being Fc.gamma. receptor I and/or III-positive cells
(Zeidler, 1999, Zeidler 2000 loc. cit.)
[0058] The antibodies which may be employed according to the
invention have the effect that the expression of CD40, CD80, CD86,
ICAM-1, and/or LFA-3 as co-stimulatory antigens, or/and the
secretion of cytokines by the Fc receptor-positive cell is
initiated or increased. Preferably, the cytokines are IL-1, IL-2,
IL-4, IL-6, IL-8, IL-12, INF-.gamma. and/or TNF-.alpha..
[0059] The bispecific antibodies which may be employed according to
the present invention are for example: [0060] an anti-CD3 X
anti-tumor-associated antigen antibody and/or anti-CD4 X
anti-tumor-associated antigen antibody and/or anti-CD5 X
anti-tumor-associated antigen antibody and/or anti-CD6 X
anti-tumor-associated antigen antibody and/or anti-CD8 X
anti-tumor-associated antigen antibody and/or anti-CD2 X
anti-tumor-associated antigen antibody and/or anti-CD28 X
anti-tumor-associated antigen antibody and/or anti-CD40L X
anti-tumor-associated antigen antibody and/or anti-CD44 X
anti-tumor-associated antigen antibody.
[0061] The trispecific antibodies which may be employed according
to the present invention preferably are: [0062] an anti-CD3 X
anti-tumor-associated antigen antibody and/or anti-CD4 X
anti-tumor-associated antigen antibody and/or anti-CD5 X
anti-tumor-associated antigen antibody and/or anti-CD6 X
anti-tumor-associated antigen antibody and/or anti-CD8 X
anti-tumor-associated antigen antibody and/or anti-CD2 X
anti-tumor-associated antigen antibody and/or anti-CD28 X
anti-tumor-associated antigen antibody and/or anti-CD40L X
anti-tumor-associated antigen antibody and/or anti-CD44 X
anti-tumor-associated antigen antibody.
[0063] The trispecific antibodies which may be used according to
the present invention at least have one T cell binding arm, one
tumor cell binding arm as well as an arm binding to Fc
receptor-positive cells. This last binding arm may be an anti-Fc
receptor binding arm or a mannose receptor binding arm.
[0064] Preferably, the bispecific antibody is a heterologous intact
rat/mouse bispecific antibody.
[0065] By means of the bispecific and trispecific antibodies which
may be used according to the present invention, T cells are
activated and redirected against the tumor cells. Preferably used
heterologous intact bispecific antibodies are selected from one or
more of the following isotype combinations:
[0066] rat-IgG2b/mouse-IgG2a,
[0067] rat-IgG2b/mouse-IgG2b,
[0068] rat-IgG2b/mouse-IgG3;
[0069] rat-IgG2b/human-IgG1,
[0070] rat-IgG2b/human-IgG2,
[0071] rat-IgG2b/human-IgG3[oriental allotype G3m(st)=binding to
protein A],
[0072] rat-IgG2b/human-IgG4;
[0073] rat-IgG2b/rat-IgG2c;
[0074] mouse-IgG2a/human-IgG3[caucasian allotypes G3m(b+g) =no
binding to protein A, in the following indicated as *]
[0075]
mouse-IgG2a/mouse-[VH--CH1,VL-CL]-human-IgG1-[hinge]-human-IgG3*-[C-
H2--CH3]
[0076]
mouse-IgG2a/rat-[VH--CH1,VL-CL]-human-IgG1-[hinge]-human-IgG3*-[CH2-
--CH3]
[0077]
mouse-IgG2a/human-[VH--CH1,VL-CL]-human-IgG1-[hinge]-human-IgG3*-[C-
H2--CH3]
[0078]
mouse-[VH--CH1,VL-CL]-human-IgG1/rat-[VH--CH1,VL-CL]-human-IgG1-[hi-
nge]-human-IgG3*-[CH2--CH3]
[0079]
mouse-[VH--CH1,VL-CL]-human-IgG4/rat-[VH--CH1,VL-CL]-human-IgG4-[hi-
nge]-human-IgG4[N-terminal region of CH2]-human-IgG3*[C-terminal
region of CH2: >aa position 251]-human-IgG3[CH3]
[0080]
rat-IgG2b/mouse-[VH--CH1,VL-CL]-human-IgG1-[hinge-CH2--CH3]
[0081]
rat-IgG2b/mouse-[VH--CH1,VL-CL]-human-IgG2-[hinge-CH2--CH3]
[0082] rat-IgG2b/mouse-[VH--CH1,VL-CL]-human-IgG3-[hinge-CH2--CH3,
oriental allotype]
[0083]
rat-IgG2b/mouse-[VH--CH1,VL-CL]-human-IgG4-[hinge-CH2--CH3]
[0084]
human-IgG1/human-[VH--CH1,VL-CL]-human-IgG1-[hinge]-human-IgG3*-[CH-
2--CH3]
[0085]
human-IgG1/rat-NH--CH1,VL-CL]-human-IgG1-[hinge]-human-IgG4[N-termi-
nal region of CH2]-human-IgG31C-terminal region of CH2: >aa
position 251]-human-IgG3[CH3]
[0086]
human-IgG1/mouse-NH--CH1,VL-CL]-human-IgG1-[hinge]-human-IgG4[N-ter-
minal region of CH2]-human-IgG31C-terminal region of CH2: >aa
position 251]-human-IgG3*[CH3]
[0087]
human-IgG1/rat-[VH--CH1,VL-CL]-human-IgG1-[hinge]-human-IgG2[N-term-
inal region of CH2]-human-IgG31C-terminal region of CH2: >aa
position 251]-human-IgG3*[CH3]
[0088]
human-IgG1/mouse-[VH--CH1,VL-CL]-human-IgG1-[hinge]-human-IgG2[N-te-
rminal region of CH2]-human-IgG31C-terminal region of CH2: >aa
position 251]-human-IgG3[CH3]
[0089]
human-IgG1/rat-[VH--CH1,VL-CL]-human-IgG1-[hinge]-human-IgG3*-[CH2--
-CH3]
[0090]
human-IgG1/mouse-[VH--CH1,VL-CL]-human-IgG1-[hinge]-human-IgG3*-[CH-
2--CH3]
[0091]
human-IgG2/human-[VH-CH1,VL-CL]-human-IgG2-[hinge]-human-IgG3*-[CH2-
--CH3]
[0092]
human-IgG4/human-[VH--CH1,VL-CL]-human-IgG4-[hinge]-human-IgG3*-[CH-
2--CH3]
[0093]
human-IgG4/human-[VH--CH1,VL-CL]-human-IgG4-[hinge]-human-IgG4[N-te-
rminal region of CH2]-human-IgG31C-terminal region of CH2: >aa
position 251]-human-IgG3[CH3]
[0094]
mouse-IgG2b/rat-[VH--CH1,VL-CL]-human-IgG1-[hinge]-human-IgG3*-[CH2-
--CH3]
[0095]
mouse-IgG2b/human-[VH--CH1,VL-CL]-human-IgG1-[hinge]-human-IgG3*-[C-
H2--CH3]
[0096]
mouse-IgG2b/mouse-[VH--CH1,VL-CL]-human-IgG1-[hinge]-human-IgG3*-[C-
H2--CH3]
[0097]
mouse-[VH--CH1,VL-CL]-human-IgG4/rat-[VH--CH1,VL-CL]-human-IgG4-[hi-
nge]-human-IgG4-[CH2]-human-IgG3*-[CH3]
[0098]
human-IgG1/rat-[VH--CH1,VL-CL]-human-IgG1-[hinge]-human-IgG4-[CH2]--
human-IgG3*-[CH3]
[0099]
human-IgG1/mouse-[VH-CH1,VL-CL]-human-IgG1-[hinge]-human-IgG4-[CH2]-
-human-IgG3*-[CH3]
[0100]
human-IgG4/human-[VH--CH1,VL-CL]-human-IgG4-[hinge]-human-IgG4-[CH2-
]-human-IgG3*-[CH3]
[0101] The antibodies which may be used according to the present
invention preferably are monoclonal, chimeric, recombinant,
synthetic, semi-synthetic, or chemically modified intact antibodies
having for example Fv, Fab, scFv, or F(ab).sub.2 fragments.
[0102] Preferably, antibodies or derivatives or fragments of humans
are used, or those which have been modified to be suitable for the
use in humans (so-called "humanized antibodies") (see for example
Shalaby et al., J. Exp. Med. 175 (1992), 217; Mocikat et al.,
Transplantation 57 (1994), 405).
[0103] The preparation of the different types of antibodies and
antibody fragments mentioned above is well-known to the skilled
artisan. The preparation of monoclonal antibodies, preferably of
those originating in mammals, e.g. human, rat, mouse, rabbit, or
goat, can be performed using conventional methods as those
described for example in Kohler and Milstein (Nature 256 (1975),
495), in Harlow and Lane (Antibodies, A Laboratory Manual (1988),
Cold Spring Harbor) or in Galfre (Meth. Enzymol. 73 (1981), 3) or
in DE 195 31 346.
[0104] It is further possible to prepare the antibodies described
by means of recombinant DNA technology according to techniques
known to those skilled in the art (see Kurucz et al., J. Immunol.
154 (1995), 4576; Hollinger et al., Proc. Natl. Acad. Sci. USA 90
(1993), 6444).
[0105] The preparation of antibodies having two different
specificities, the so-called bispecific antibodies, can be
performed on the one hand using recombinant DNA technology but on
the other hand also by the so-called hybrid hybridoma fusion
technique (see for example Milstein et al., Nature 305 (1983),
537). For this purpose, hybridoma cell lines each producing
antibodies having one of the desired specificities are fused and
cellular clones (quadromas) producing antibodies having both
specificities are identified and isolated.
[0106] The problem underlying the present invention can be solved
using both bispecific and trispecific antibodies which preferably
exhibit the properties and effects characterized. In the following,
the preparation of antibodies showing two and three specificities
is described in more detail. To provide such bispecific and
trispecific antibodies is known from the prior art, and references
describing such techniques of preparation are incorporated herein
by reference in their entirety.
[0107] The preparation of antibodies exhibiting three
specificities, so-called trispecific antibodies, by which the
problem underlying the present invention can also be solved may be
for example carried out by coupling to one of the IgG heavy chains
of a bispecific antibody a third antigen binding site having an
additional specificity, e.g. in the form of a "single chain
variable fragment" (scFv). The scFv may be coupled for example
using a
--S--S(G.sub.4S).sub.nD-I-linker
to one of the heavy chains (S=serine, G=glycine, D=aspartate,
I=isoleucine).
[0108] In an analogous manner trispecific F(ab).sub.2 constructs
may be prepared by replacing the CH2--CH3 regions of the heavy
chain of one specificity of a bispecific antibody by an scFv having
a third specificity while the CH2--CH3 regions of the heavy chain
having the other specificity are removed for example by insertion
of a stop codon (at the end of the "hinge" region) into the coding
gene, e.g. by homologous recombination.
[0109] It is also possible to prepare trispecific scFv constructs.
For this purpose three VH-VL regions representing three different
specificities are arranged behind each other in series.
[0110] According to the present invention there are for example
used intact bispecific antibodies. Intact bispecific antibodies are
composed of two antibody semi-molecules (each having a H and a L
immunoglobulin chain) each of which represents one specificity
which additionally like normal antibodies have an Fc portion
performing the well-known effector functions. They are preferably
prepared using the quadroma technology. This method of preparation
is described exemplarily in DE-A-44 19 399. For complete disclosure
this document is incorporated by reference in its entirety also
with respect to a definition of bispecific antibodies. It should be
understood, however, that also other methods of preparation may be
employed if they lead to the intact bispecific antibodies according
to the above definition which are required by the present
invention.
[0111] For example, intact bispecific antibodies may be produced in
a sufficient amount using a newly developed method of preparation
(Lindhofer et al., J. Immunology 1995, 155: 219). The combination
of 2 bispecific antibodies directed against 2 different
tumor-associated antigens (e.g. c-erb-B2, and Ep-CAM) on the
mammary carcinoma cells minimizes the risk that tumor cells
expressing only one antigen would remain unrecognized.
[0112] Further advantages of intact bsabs having the ability of
redirecting T cells over the above-mentioned bsF(ab')2 fragments
may be detailed as follows: [0113] 1. It is possible for Fc
receptor-positive cells to bind to intact bsabs and to contribute
on the one hand directly to tumor destruction via ADCC
(antibody-dependent cell-mediated cytotoxicity) and on the other
hand to T cell activation as explained in more detail above. [0114]
2. By intact T cell-redirecting bsabs also anergized tumor-specific
T cells are recruited to the tumor cell which according to the
invention may be reactivated directly at the tumor site. This may
not be achieved by an bsF(ab')2 fragment having the specificities
anti-CD64 X anti-tumor-associated antigen.
[0115] Binding of the bsab to Fc.gamma.-RI has two essential
advantages with regard to an optimal anti-tumor effectiveness:
[0116] (1) Fc.gamma.-RI-positive cells have the ability to
eliminate tumor cells by ADCC and, thus, are able to contribute
synergistically to the anti-tumor effect of the cytotoxic T cells
recruited to the tumor cell by the bsab. [0117] (2)
Fc.gamma.RI-positive cells (such as monocytes/macrophages/dendritic
cells) are able to provide important co-stimulatory signals similar
to those involved in antigen presentation to the T cell and,
thereby, to prevent anergizing of the T cell. Furthermore, because
of the intact bsab-mediated interaction of T cell with accessory
cell and tumor cell there can be stimulated as a desired by-product
even T cells the T cell receptor of which recognizes tumor-specific
peptides (presented via MHC antigens on the tumor cell). In this
constellation, the co-stimuli necessary for correct activation of
the T cell would be provided by the accessory cell (such as the
monocyte). Thus, besides the direct, T cell receptor-independent
bsab-mediated tumor destruction the antibody of the present
invention should also be able to activate and generate
tumor-specific T cells which after degradation of the bsab continue
to patrol in the patient. This means that similar to
gene-therapeutical approaches (e.g. by incorporation of
co-stimulatory antigens such as B-7 into the tumor cell) the tumor
tolerance in the patient may be abolished by means of intact
bsabs.
[0118] As could be surprisingly demonstrated in two patients, the
immune therapy of tumors by means of trifunctional bispecific
antibodies can also be therapeutically used in the treatment of
ascites.
[0119] In one patient suffering from ovarian carcinoma, the new
formation of ascites following treatment with bsab could be
inhibited for a period of 6 months. In this case also the complete
destruction of the tumor cells present in the ascites liquid could
be confirmed.
EXAMPLE 1
[0120] A patient with ovarian carcinoma first underwent a
peritoneum puncture, then ascites liquid (700 ml) was removed and
the cells contained therein were analyzed. As shown in FIG. 1 the
tumor cells could be unambiguously identified already by visual
inspection due to their morphology. In addition, anti-EpCAM
peroxidase staining was performed.
[0121] Counting of the cells in the ascites prior to treatment gave
a ratio of about 50:50 of tumor cells to immune cells.
[0122] The flow cytometric analysis revealed a strong positivity of
the tumor cells for the EpCAM (epithelial cell adhesion molecule,
Balzar et al., Mol. Cell. Biol., 18:4833, 1998) surface antigen as
well as a weaker expression of the tumor-associated surface antigen
Her2/neu (FIG. 2).
[0123] To exclude the possibility of a reaction to foreign protein,
first 10 .mu.g of TPBS03 REXOMAB bsab (anti-Her2/neu X anti-CD3;
isotype combination mouse IgG2a X rat IgG2b) were infused
intraperitoneally by means of a perfusor syringe over 8 h. The
precise application is presented in Table 1.
TABLE-US-00001 TABLE 1 Dose regimen Day Bispecific Antibody Dose 1
CD3xHer2/neu 10 .mu.g 4 CD3xEpCAM 10 .mu.g 7 CD3xEpCAM 40 .mu.g 9
CD3xEpCAM 80 .mu.g 11 CD3xEpCAM 160 .mu.g 15 CD3xEpCAM + 200 .mu.g
+ CD3xHer2/new 50 .mu.g
[0124] A first change could be observed 1 day after the 40 .mu.g
infusion. At this point a peritoneal lavage was performed and the
cells contained therein were analyzed revealing that the ratio of
immune cells to tumor cells had improved to a value of 3:1.
[0125] Surprisingly, on day 11 immediately prior to the 160 pg
infusion only occasional tumor cells could be found. The ratio of
immune cells to tumor cells had changed dramatically to a value of
330:1.
[0126] In the third peritoneal lavage on day 15 after the
administration of 160 .mu.g and immediately prior to the 250 .mu.g
infusion no more tumor cells could be detected (FIG. 1). This
result could be confirmed using a particularly sensitive nested
EpCAM RT PCR (FIG. 3).
[0127] It should be pointed out that during treatment no severe
side effects occurred. Only after the highest dose of 250 .mu.g
bsab a short-term fever (38.6.degree. C.) was observed.
[0128] In the patient, no new formation of the ascites liquid was
observed over a period of 6 months and no further puncture had to
be performed. This is remarkable because the disease stage of this
patient at the beginning of immune therapy was very advanced and
liver metastases already existed.
EXAMPLE 2
[0129] In another patient suffering from mammary carcinoma and
ascites formation also a peritoneal puncture was performed, ascites
liquid (3 liters) was collected and the cells contained therein
were analyzed by flow cytometry. For this purpose, detection
antibodies against the tumor-associated antigen EpCAM and CD14 were
used to be able to distinguish unspecific binding to Fc
receptor-positive cells (of the detection antibodies) from a
binding to tumor cells. As shown in FIG. 4 at the point of day 0
prior to therapy more than 70% of the cells which were detectable
in the ascites liquid were EpCAM-positive tumor cells which,
however, under therapy were reduced to 0.7%.
[0130] On day 0 after removal of the ascites liquid the patient
received 10 pg of the anti-EpCAM.times.anti-CD3 REMOVAB.RTM.
antibody (isotype combination: mouse IgG2a X rat IgG2b) as well as
on the following days 2 and 5 40 .mu.g and 100 .mu.g, respectively,
of REMOVAB.RTM. intraperitoneally over a period of about 6 hours.
On day 8 a combination of the Rexomab (anti-Her3/neu X anti-CD3;
100 .mu.g) and REMOVAB.RTM. (50 .mu.g) antibodies were applied
intraperitoneally to the patient because in the flow cytometry also
the tumor-associated antigen Her2/neu could be detected on the
tumor cells (not shown).
[0131] As depicted in FIG. 5 the new formation of liquid of the
ascites continuously decreased under therapy and was completely
abolished by the end of therapy.
[0132] The treatment was well tolerated. After the highest dose,
however, a transient increase in liver values and a short-term
fever occurred.
Conclusion:
[0133] From these results it can be concluded that already a dose
of 40 .mu.g bsab has a certain anti-tumor effect and an activation
and proliferation of immune effector cells is achieved. After an
administration of 160 .mu.g bsab no indication as to the presence
of residual tumor cells in the peritoneal lavage of the first
patient could be found even with very sensitive detection methods,
and thus an effective destruction of the autologous tumor cells in
the ascites liquid can be assumed. A similar effect could also be
observed in the second patient after administration of 100 .mu.g
REMOVAB.RTM.. The tolerability of the doses administered was good,
and only in one of the patients an increase in liver values or
other negative side effects were observed. After the 250 .mu.g dose
(patient 1) and the 100 .mu.g dose (patient 2) short-term fever
(38.6.degree. C.) occurred.
[0134] In this view, for future treatments for the destruction of
tumor cells in the ascites and for inhibiting the new formation of
ascites the following application of trifunctional bispecific
antibodies results: [0135] Day 0: 10 .mu.g (starting dose to test
for a hypersensitivity reaction against the foreign protein) [0136]
Day 2: 40 .mu.g (activation and proliferation of immune cells)
[0137] Day 4: 200 .mu.g (main dose for the destruction of the tumor
cells)
[0138] Since at present there is no satisfactory treatment of
malignant ascites which accompanies tumor diseases, the immune
therapy by means of bsab is a novel and promising method.
[0139] The intraperitoneal immunotherapeutic treatment of ascites
by means of trifunctional bsabs may also serve as a primary
immunization for inducing a long-term anti-tumor immunity.
[0140] The abdominal cavity contains a number of immunological
organs such as spleen, Peyer's plaques and a plurality of lymph
nodes. Due to their interactions with tumor cells, T cells, and
accessory cells (Zeidler et al., J. Immunol. 163:1246, 1999;
Zeidler et al., British J. Cancer 83:261, 2000), the trifunctional
bsabs are capable of directing tumor material to the
antigen-presenting system and to induce an infiltration of
activated dendritic cells into the immunological organs.
[0141] These events are essential prerequisites for the induction
of an immune response against the autologous tumor. To establish a
long-term anti-tumor immune response and particularly to generate a
functional polyclonal humoral and cellular immune response a
secondary immunization is necessary. The secondary immunization can
be performed subcutaneously or intradermally. As the autologous
tumor material in the case of malignant ascites the tumor cells
present therein may be prepared simply by means of puncture and may
be used.
* * * * *