U.S. patent application number 09/764076 was filed with the patent office on 2005-03-31 for antibodies against t cells as therapeutics.
Invention is credited to Thierfelder, Stefan.
Application Number | 20050069543 09/764076 |
Document ID | / |
Family ID | 6520949 |
Filed Date | 2005-03-31 |
United States Patent
Application |
20050069543 |
Kind Code |
A1 |
Thierfelder, Stefan |
March 31, 2005 |
Antibodies against T cells as therapeutics
Abstract
The invention relates to antibodies to T cells as therapeutics.
The object underlying the invention is to provide antibodies for a
clinical therapy for prolonging the immunosuppressive antibody
effect while avoiding the formation of antiantibodies. This object
is achieved by providing antibodies consisting of at least two
different groups which are applied at different times and in which
at least one antibody of group B differs from one antibody of group
A in the constant regions of its heavy chains and wherein group A,
which is first applied once or several times, has a T-cell
eliminating effect, while the other group B (which is applied at a
different time) has a T-cell eliminating and/or T-cell antigen
modulating effect.
Inventors: |
Thierfelder, Stefan;
(Eichenau, DE) |
Correspondence
Address: |
SUGHRUE, MION, ZINN, MACPEAK & SEAS, PLLC
2100 PENNSYLVANIA AVENUE, N.W.
WASHINGTON
DC
20037-3213
US
|
Family ID: |
6520949 |
Appl. No.: |
09/764076 |
Filed: |
January 19, 2001 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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09764076 |
Jan 19, 2001 |
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09134575 |
Aug 14, 1998 |
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6290955 |
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09134575 |
Aug 14, 1998 |
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08737798 |
May 7, 1997 |
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5830473 |
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08737798 |
May 7, 1997 |
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PCT/EP95/01898 |
May 19, 1995 |
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Current U.S.
Class: |
424/144.1 ;
530/388.22 |
Current CPC
Class: |
C07K 16/2812 20130101;
C07K 16/2815 20130101; A61P 35/00 20180101; A61K 47/6849 20170801;
Y10S 530/868 20130101; A61P 37/06 20180101 |
Class at
Publication: |
424/144.1 ;
530/388.22 |
International
Class: |
A61K 039/395; C07K
016/28 |
Foreign Application Data
Date |
Code |
Application Number |
Jun 18, 1994 |
DE |
P 44 21 391.3 |
Claims
1-5. cancelled
6. A method for eliminating tumors comprising administering, to a
tumor-bearing subject, a pharmaceutically effective amount of a
first antibody, having binding specificity for T cells and which is
capable of eliminating T cells in vivo, and thereafter
administering a pharmaceutically effective amount of a second
antibody having binding specificity for T cells and which is
capable of eliminating T cells in vivo, or is capable of modulating
the antigen effect of T cells or is both capable of eliminating T
cells in vivo and capable of modulating the antigen effect of T
cells, wherein said first antibody has a different constant region
in its heavy chains than said second antibody, and thus belongs to
a different animal species than said second antibody.
Description
FIELD OF THE INVENTION
[0001] The present invention relates to antibodies against T cells
which are useful as therapeutic agents for prolonging
immunosuppression and for tumor cell elimination.
BACKGROUND OF THE INVENTION
[0002] Heretofore, transplant rejection has been treated with
immunosuppressant agents, e.g., monoclonal, immunosuppressive
antibodies against human T lymphocytes which have been generated
from mice, rats or golden hamsters. However, the effect of these
antibodies is limited, since the patient develops an immunoreaction
to antibodies which are derived from another animal species. This
results in what are called antiantibodies, which inhibit the
immunosuppressive effect of the injected monoclonal antibodies.
Thus, at present, e.g., patients with kidney transplants that
suffer from transplant rejection crises, are usually treated with
only a single antibody therapy. If another rejection crisis occurs,
this treatment is usually not repeated because of the possible
formation of antiantibodies.
[0003] So far, there is no clinical therapy of choice for
prolonging the immunosuppressive effect of antibodies while
avoiding the formation of antiantibodies. A repeated treatment with
another monoclonal antibody can lead to an accelerated formation of
antiantibodies (Chatenoud, Transpl. Proc., 25. 2(Suppl. 1):68
(1993)). In addition, patients have developed antiantibodies even
against immunosuppressive antibodies that had been humanized using
genetic engineering methods, i.e., where the antibodies have been
substantially adapted to the patient's species, i.e., "primate
species" or "species-adapted" antibodies (Isaacs et al, Lancet.,
340:748 (1992)).
[0004] Experimentally, a clear prolongation of the survival time of
skin transplants has been found in a mouse model, which was
considered a tolerance induction. Such prolongation was observed
after the injection of high doses of a rat antibody directed to
mouse T(L3T4+Lyt-2) cells, followed by injection of a second
antibody of the same species and the same cell binding specificity,
which, however, differed from the first antibody by its low
elimination of T cells from the blood circulation of the mouse (a
"non-depleting", i.e., eliminating antibody). Unlike the present
invention, the described principle of action therein was not based
on a combined therapy of at least two antibodies with
species-different Fc regions (Cobbold et al, Eur. J. Immunol.,
20:2747 (1990)).
[0005] Prolonged survival time of skin transplants and lack of
formation of antiantibodies, were also found after the injection of
a rat anti-mouse T(L3T4=CD4+lymphocyte subpopulation)-cell
antibody, followed by injection of (Fab').sub.2 fragments and
unfragmented monoclonal hamster anti-mouse T(CD3) antibodies
(Hirsch et al, Transplantation, 47:853 (1989)). Here, too, the
described principle of action is not based on a combined therapy of
two antibodies having species--different Fc regions that are
directed to all T cells, as in the present invention, but, rather,
on the suppression of the CD4+T lymphocyte subpopulation (Hirsch et
al, J. Immunol., 140:3766 (1988)) achieved by means of the first
antibody, which is, however, not sufficient.
[0006] Permanent tolerance of skin transplants can be achieved in
irradiated mice after transplantation of bone marrow of the donor
of the skin transplant, while protecting anti-T cell antibodies
(Thierfelder et al, Blood, 68:818 (1986)). However, this technique
involves risks.
[0007] So far, there is no therapy of choice for definitely
preventing the formation of antiantibodies in a patient in the case
of conventional poly- or monoclonal immunosuppressive antibodies.
The first clinical experiences with antibodies that have recently
been humanized by means of genetic engineering show that
antiantibodies may be formed (Isaacs et al, supra) similarly to
what was seen with murine immunosuppressive anti-mouse T cell
antibodies (Kremmer et al, Eur. J. Immunol., 23:1017 (1993)).
[0008] Also, a combination of immunosuppressive antibody treatment
with chemotherapy, e.g., cyclophosphamide or busulfan, involves the
risk of side-effects, particularly on hematopoiesis, and, also, on
the transplanted tissue, due to lack of cell specificity of the
chemotherapeutic agents (Cobbold et al, supra; and Leong et al,
Eur. J. Immunol., 22:2825 (1992)).
SUMMARY OF THE INVENTION
[0009] An object of the present invention is to provide antibodies
for clinical therapy for prolonging the immunosuppressive antibody
effect, while avoiding the formation of antiantibodies.
[0010] The above-described object of the present invention has been
met by the use of antibodies against T cells as a therapeutic for
prolonged immunosuppression and tumor cell elimination, wherein the
antibodies consist of at least two different groups A, B, which are
administered at different times and in which at least one antibody
type of group B differs from at least one antibody type of group A
in the constant regions of their heavy chains, and wherein group A,
which is first applied once or several times, has a T-cell
eliminating effect, whereas the other group B (which is applied at
a different time) has a T-cell eliminating and/or T-cell antigen
modulating effect.
BRIEF DESCRIPTION OF THE DRAWINGS
[0011] FIG. 1 is a graph showing prolonged immunosuppression due to
sequential treatment with two anti-T cell antibodies, the Fc
regions of which are species-different.
[0012] FIG. 2 is a graph showing immunosuppression using anti-T
cell antibodies with mouse or rat Fc regions.
[0013] FIG. 3 is a table showing suppression of antiantibodies by
Fc-region-incompatible monoclonal antibody therapy.
DETAILED DESCRIPTION OF THE INVENTION
[0014] As discussed above, the above-described objects of the
present invention have been met by the use of antibodies against T
cells as a therapeutic for prolonged immunosuppression and tumor
cell elimination, wherein the antibodies consist of at least two
different groups A, B, which are administered at different times
and in which at least one antibody type of group B differs from at
least one antibody type of group A in the constant regions of their
heavy chains, and wherein group A, which is first applied once or
several times, has a T-cell eliminating effect, whereas the other
group B (which is applied at a different time) has a T-cell
eliminating and/or T-cell antigen modulating effect.
[0015] Preferably, antibody B is a bispecific antibody.
[0016] Also, preferably, antibody B has a different constant region
in its heavy chains than antibody A, and antibody A or antibody B
is obtained by genetic engineering of the region encoding the
constant region of its heavy chain.
[0017] Also, preferably, antibody A or antibody B is a humanized
antibody.
[0018] Also, preferably, antibody A or antibody B has a hapten
covalently linked to the constant region of its heavy chain. The
hapten is preferably DNP or TNP.
[0019] Each of the two groups of antibodies, i.e., antibodies A and
antibodies B, may also consist of only one antibody type or several
kinds of antibodies.
[0020] In addition, the two groups of antibodies, i.e., antibodies
A and antibodies B, may also be monoclonal or polyclonal.
[0021] The sequential treatment with anti-T cell antibodies that
are partially or fully humanized using molecular biological means,
and non-humanized antibodies, or with at least two anti-T cell
antibodies generated from different species, as described herein,
leads to prolonged immunosuppression and tumor cell elimination.
This treatment principle has been experimentally tested on animals,
as shown below, and has not heretofore been described in the
art.
[0022] The novel therapy principle of the present invention, which
has not heretofore been described in the art, is not obvious at all
in terms of immunology. Commonly, immunobiologists and experts in
the field of medicine, search for a reduction of the immunogenicity
of anti-T cell antibodies (which causes the formation of
antiantibodies) by adaptation thereof, as much as possible, to the
patient's antibody immunoglobulin structures so that the patient is
more likely to tolerate the antibodies, e.g., by humanization by
means of genetic engineering of monoclonal immunosuppressive
antibodies derived from mice. However, principally this adaptation
cannot possibly be complete, and is the cause for the formation of
antiantibodies, because the T cell binding (V) region of the
immunosuppressive antibody is so variable that the patient's
immunoapparatus can still form antiantibodies thereto.
[0023] The present invention is based on a contrasting experience,
i.e., on the suppression of antiantibodies by creating a high
species difference between the anti-T cell antibodies, wherein one
or both, applied alone, may be potentially immunogenic in the
recipient of the antibodies.
[0024] It was found in the present invention that:
[0025] (a) the survival time of skin transplants was basically
prolonged, not by applying two different mouse anti-mouse T cell
antibodies (or two different rat anti-mouse T cell antibodies) one
after the other, but by using two antibodies which are
species-different to one another, but not necessarily to the
recipient of the antibodies, and a clear prolonged
immunosuppression was achieved; and
[0026] (b) two antibodies are effective even when they are as
different from one another as human and mouse.
[0027] These thoughts, results and antibody combinations define a
therapy model offering, inter alia, the advantage that it can
immediately be tested clinically, and does not expose the patients
to any additional treatment risks. A prolongation of
immunosuppression should not only be a more successful therapy for
rejection crises of organ transplants and immune complications with
bone marrow transplantations, but should help prevent them
altogether by prophylactic treatment. In addition, autoimmune
diseases, chronic diseases of all kinds of rheumatism, and also
individual tumor conditions might face new therapeutic
perspectives. For instance, in the mouse model studies carried out
by the inventors on the suppression of murine or human T cell
leukemias transplanted into mice, a prolonged survival time due to
antibody injection was observed. Upon T cell depletion, foreign
immunocompetent cells can be introduced into chimeric mice, i.e.,
mice transplanted with bone marrow and suffering from leukemia,
which foreign immunocompetent cells attack the neoplastic cells in
the recipient. Furthermore, the tolerance induction, vis--vis
heterologous serum proteins makes possible passive vaccination with
antibodies of a different species that is free of hypersensitive
reactions, e.g., for tetanus.
[0028] In the murine skin transplant model it could be shown that a
monoclonal, immunosuppressive antibody, that was humanized by
genetic engineering methods achieves a survival time of transplants
against murine T lymphocytes, i.e., prolonged by a multitude, when
its application was preceded by one or more injections of a
monoclonal immunosuppressive mouse antibody. The preceding antibody
injections as such did not have to be immunosuppressive in the
sense of transplant prolongation. It turned out that this antibody
therapy induced a complete tolerance towards heterologous, human
serum protein, which still remained five months after the end of
the immunosuppressive therapy. The unexpected prolongation of the
immunosuppressive effect was thus, accompanied by a lack of a
formation of antiantibodies in the treated mice due to their
tolerance of the heterologous antibody immunoglobulin. The
principle of action underlying this phenomenon is analyzed
particularly with regard to species-related differences in the Fc
region of the combined antibodies. It also proved effective when
using anti-T cell antibodies that had not been modified by
molecular biology, if they were species-different from one
another.
[0029] Antibodies have what is called a variable region that
includes the antibody binding site and what is called a constant Fc
region that mediates antibody effector functions (e.g., elimination
from the system of body cells occupied by antibodies), which is
located on what are called the constant regions of the heavy chains
of the antibody. In this way, two antibodies can be similar with
regard to their specificity to bind, e.g., human T lymphocytes.
Such antibodies with the same cell binding specificity, however,
may differ in their Fc region due to the fact that they are derived
from different normal or molecular biology-manipulated animal
species. They can also be modified in vitro in the Fc region using
methods of molecular biology on generating antibody-secretory cells
(e.g., hybridomas or hybrid hybridomas) so that there is the degree
of difference obtained as found between humans and rodents, and as
described in the present invention.
[0030] In the following examples, the present invention is
described in more detail.
EXAMPLE 1
[0031] Combination antibody treatment was carried out by first
injecting a mouse IgG.sub.2, anti-mouse-Thy-1.2 antibody (MmT1
antibody; (Kremmer et al, supra)) on day 3, followed by injecting a
chimeric antibody having a MmT1 idiotype (V region) and human Fc
IgG.sub.1 region (T23 antibody) on day 0 and twice a week. The T23
antibody differs from the MmT1 antibody by an exchange of the
murine IgG.sub.1 Fc region for a human IgG.sub.1 Fc region, which
was achieved by means of genetic engineering. The results are shown
in FIG. 1.
[0032] As shown in FIG. 1, a single dose of MmT1 did not prolong
the (average) skin survival time. T23 alone, applied twice a week,
did prolong it by nine days from 16 to 24. MmT1 (first dose)
followed by T23 (applied twice a week) prolonged it to more than 90
days. Thus, FIG. 1 shows immunosuppression that was prolonged
approximately ten-fold, as measured in a rodent skin
transplantation model of maximum histoincompatibilty, by the
combined FC-region incompatible antibody treatment of the present
invention.
[0033] Also, as shown in FIG. 1, a similarly increased
immunosuppression was achieved after replacement of antibody MmT1
with antibody MmT5 (Kremmer et al, supra), which does not differ
from antibody MmT1 in its T-cell specificity, but, rather, differs
in the microstructure of the antibody binding site (idiotype).
[0034] The results demonstrate that in the combined Fc-region
incompatible antibody therapy of the present invention, the
likeness or difference of the antibody binding site is not a
prerequisite for the principle of action, but, rather, the
species-dependent difference of its heavy chains incorporating the
Fc regions is a prerequisite for the principle of action.
EXAMPLE 2
[0035] MmT1/RmCD4+CD8 combination therapy was carried out by
injecting MmT1 on day 3, followed by injecting RmCD4+RmCD8 antibody
(a rat anti-mouse CD4+CD8 lymphocyte antibody) on day 0 and twice a
week, and vice versa. The results are shown in FIG. 2.
[0036] FIG. 2 shows that rat anti-mouse T cell antibodies having
Thy-1 specificity (Kummer et al, J. Immunol., 138:4069 (1987)), and
also particularly clinically-relevant antibody specificities, such
as anti-CD4 and anti-CD8 (two T cell subpopulations, which together
bind all T cells) also prolong the average survival time of skin
transplants. Furthermore, as shown therein, the reversal of the
combined antibody treatment in the RmCD4+CD8/MmT1 combination also
leads to a prolonged immunosuppression since here, too, the
prerequisite of the species-dependent difference of the Fc region
is fulfilled. Since anti-CD4+CD8 antibodies are active as first
antibodies, this excludes an effect of the preinjected first
antibody restricted to MmT1. On the contrary, the prerequisite for
the synergistic antibody action of their species-dependent Fc
region differences (apart from the application of at least two
antibodies at different times) applies again and again. Survival of
skin transplants using a combination antibody therapy was permanent
if further T cell depleting and/or T cell receptor modulating
(anti-CD3) antibodies were added to the second antibody.
EXAMPLE 3
[0037] Groups of 4 to 6 C57BL/6 mice were injected with 400 .mu.g
of the first antibody (shown in FIG. 3) and 500 .mu.g of the second
antibody (shown in FIG. 3). Tail blood was drawn 6 to 10 days after
the last injection in order to determine the antiantibody level.
The results are shown in FIG. 3.
[0038] As shown in FIG. 3, the combined rat/mouse or mouse/rat
Fc-region incompatible antibody treatment leads to a high
suppression or complete lack of the formation of antiantibodies,
i.e., antiantibody levels were extremely low or zero where there
was a species difference (rat/mouse or mouse/rat) between the first
and the second antibody. The same applies to treatment when carried
out as per Example 1.
[0039] Antiantibodies also occur when treating with polyclonal
antibodies that arise after immunization of, e.g., rabbit, rat or
horse lymphocytes. Here, too, it can be seen that a species
difference (e.g., rabbit/rat) of polyclonal antibodies leads to a
prolonged immunosuppression in mice, as well as with what are
called bispecific antibodies, i.e., antibodies having two different
binding sites, or with anti-T cell antibodies that were chemically
modified by introduction of a low-molecular compound (e.g., DNP,
TNP haptenes) or by genetic engineering, e.g., antibodies and
antibody fragments prepared in bacteria. Here, too, sequentially
injected anti-T cell antibodies may neutralize the formation of
antiantibodies to species-different polyclonal or bispecific or
chemically or molecular biology-modified antibodies. A prerequisite
is always a strong difference in the sequentially applied
antibodies or antibody groups, which either results from species
difference or from the introduction (conjugation) of chemical
compounds.
[0040] Finally, undesired immunoreactions may also occur in the
case of passive immunization with antibodies in
protein-oversensitive or presensitized patients. A treatment using
combined Fc-region incompatible antibody therapy would prevent the
formation of antiantibodies.
* * * * *