U.S. patent application number 11/028793 was filed with the patent office on 2005-09-15 for methods for inhibiting an immune response by blocking the gp39/cd40 and ctla4/cd28/b7 pathways and compositions for use therewith.
Invention is credited to Aruffo, Alejandro A., Hollenbaugh, Diane L., Larsen, Christian P., Ledbetter, Jeffrey A., Linsley, Peter S., Pearson, Thomas C..
Application Number | 20050202011 11/028793 |
Document ID | / |
Family ID | 21761563 |
Filed Date | 2005-09-15 |
United States Patent
Application |
20050202011 |
Kind Code |
A1 |
Larsen, Christian P. ; et
al. |
September 15, 2005 |
Methods for inhibiting an immune response by blocking the GP39/CD40
and CTLA4/CD28/B7 pathways and compositions for use therewith
Abstract
The present invention provides a method for inhibiting an immune
reponse and a method for inhibiting rejection of transplanted
tissues. This method comprises preventing an endogenous molecule on
a cell selected from the group consisting of gp39 and CD40 antigens
from binding its endogenous ligand and preventing an endogenous
molecule on a cell selected from the group consisting of CTLA4,
CD28, and B7 antigens from binding its endogenous ligand. The
prevention of such molecules from binding their ligand thereby
blocks two independent signal pathways and inhibits the immune
response resulting in transplanted tissue rejection.
Inventors: |
Larsen, Christian P.;
(Atlanta, GA) ; Aruffo, Alejandro A.; (Edmonds,
WA) ; Hollenbaugh, Diane L.; (Seattle, WA) ;
Linsley, Peter S.; (Seattle, WA) ; Ledbetter, Jeffrey
A.; (Seattle, WA) ; Pearson, Thomas C.;
(Atlanta, GA) |
Correspondence
Address: |
STEPHEN B. DAVIS
BRISTOL-MYERS SQUIBB COMPANY
PATENT DEPARTMENT
P O BOX 4000
PRINCETON
NJ
08543-4000
US
|
Family ID: |
21761563 |
Appl. No.: |
11/028793 |
Filed: |
January 4, 2005 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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11028793 |
Jan 4, 2005 |
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09862255 |
May 22, 2001 |
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09862255 |
May 22, 2001 |
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09172892 |
Oct 15, 1998 |
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09172892 |
Oct 15, 1998 |
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08821400 |
Mar 20, 1997 |
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5916560 |
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60013751 |
Mar 20, 1996 |
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Current U.S.
Class: |
424/144.1 |
Current CPC
Class: |
C07K 14/70532 20130101;
C07K 16/2875 20130101; C07K 14/70575 20130101; C07K 16/2827
20130101; C07K 2317/73 20130101; A61P 37/06 20180101; A61K 39/395
20130101; A61P 43/00 20180101; C07K 16/2878 20130101; C07K 14/70578
20130101; C07K 14/70521 20130101; A61K 39/395 20130101; A61K 39/395
20130101; A61K 2300/00 20130101; C07K 16/2818 20130101; A61K 38/00
20130101; A61K 38/17 20130101 |
Class at
Publication: |
424/144.1 |
International
Class: |
A61K 039/395 |
Claims
1. A method for inhibiting rejection of a transplanted tissue
comprising: a) contacting a B7-positive cell with a first soluble
ligand which recognizes and binds the B7 antigen, and b) contacting
a gp39-positive cell with a second soluble ligand which recognizes
and binds the gp39 antigen, the binding of the B7-positive cell to
the first soluble ligand thereby blocking the reaction of the B7
antigen with endogenous CTLA4 or CD28 and the binding of the gp39
antigen to the second soluble ligand thereby blocking the reaction
of gp39 antigen with endogenous CD40, the blockage thereby
inhibiting the immune response.
2. (canceled)
3. (canceled)
4. The method of claim 1, wherein the first soluble ligand is a
recombinant binding molecule having at least a portion of the
extracellular domain of CTLA4.
5. The method of claim 4, wherein the ligand is CTLA4Ig fusion
protein.
6. (canceled)
7. The method of claim 5, wherein the CTLA4Ig fusion protein is
CTLA4Ig designated ATCC 68629.
8. (canceled)
9. (canceled)
10. (canceled)
11. The method of claim 1, wherein the second soluble ligand for
the gp39 antigen is a monoclonal antibody reactive with the gp39
antigen.
12. (canceled)
13. The method of claim 4, wherein the extracellular portion of
CTLA4 is joined to a non-CTLA4 protein sequence.
14. The method of claim 13, wherein the non-CTLA4 protein sequence
is at least a portion of an immunoglobulin molecule.
15. (canceled)
16. (canceled)
17. A method for inhibiting an immune response comprising: a)
preventing an endogenous antigen on a cell selected from the group
consisting of gp39 and CD40 from binding its endogenous ligand; and
b) preventing an endogenous antigen on a cell selected from the
group consisting of CTLA4, CD28, and B7 from binding its endogenous
ligand, the prevention of such antigens from binding their ligand
thereby blocking two independent cell signals and inhibiting the
immune response.
18. The method of claim 17, wherein: a) the step of preventing the
endogenous gp39 antigen from binding its endogenous ligand
comprises contacting a gp39-positive cell with a soluble ligand
which recognizes and binds the gp39 antigen, b) the step of
preventing the endogenous CTLA4 antigen from binding its endogenous
ligand comprises contacting a B7-positive cell with a soluble
ligand which recognizes and binds the B7 antigen, the binding of
the gp39-positive cell to its soluble ligand of step (a) thereby
blocking the reaction of endogenous gp39 antigen with endogenous
CD40, the binding of the B7-positive cell to its soluble ligand of
step (b) thereby blocking the reaction of the endogenous B7 antigen
with endogenous CTLA4, the blockage thereby inhibiting the immune
response.
19. (canceled)
20. (canceled)
21. The method of claim 17, wherein: a) the step of preventing the
endogenous CD40 antigen from binding its endogenous ligand
comprises contacting a CD40-positive cell with a soluble ligand
which recognizes and binds the CD40 antigen, b) the step of
preventing the endogenous CTLA4 antigen from binding its endogenous
ligand comprises contacting a B7-positive cell with a soluble
ligand which recognizes and binds the B7 antigen, the binding of
the CD40-positive cell to its soluble ligand of step (a) thereby
blocking the reaction of endogenous CD40 antigen with endogenous
gp39, the binding of the B7-positive cell to its soluble ligand of
step (b) thereby blocking the reaction of the B7 antigen with
endogenous CTLA4, the blockage thereby inhibiting the immune
response.
22. The method of claim 21, wherein the soluble ligand of step (a)
is a monoclonal antibody directed against CD40.
23. The method of claim 21, wherein the soluble ligand of step (b)
is CTLA4Ig.
24. The method of claim 17, wherein: a) the step of preventing the
endogenous gp39 antigen from binding its endogenous ligand
comprises contacting a gp39-positive cell with a soluble ligand
which recognizes and binds the gp39 antigen, b) the step of
preventing the endogenous CD28 antigen from binding its endogenous
ligand comprises contacting a B7-positive cell with a soluble
ligand which recognizes and binds the B7 antigen, the binding of
the gp39-positive cell to its soluble ligand of step (a) thereby
blocking the reaction of gp39 antigen with endogenous CD40, the
binding of the B7-positive cell to its soluble ligand of step (b)
thereby blocking the reaction of the B7 antigen with endogenous
CD28, the blockage thereby inhibiting the immune response.
25. (canceled)
26. (canceled)
27. The method of claim 17, wherein: a) the step of preventing the
endogenous CD40 antigen from binding its endogenous ligand
comprises contacting a CD40-positive cell with a soluble ligand
which recognizes and binds the CD40 antigen, b) the step of
preventing the endogenous B7 antigen from binding its endogenous
ligand comprises contacting a CD28-positive cell with a soluble
ligand which recognizes and binds the CD28 antigen, the binding of
the CD40-positive cell to the soluble ligand of step (a) thereby
blocking the reaction of CD40 antigen with endogenous gp39, the
binding of the CD28-positive cell to the soluble ligand of step (b)
thereby blocking the reaction of the CD28 antigen with endogenous
B7, the blockage thereby inhibiting the immune response.
28. The method of claim 27, wherein the soluble ligand of step (a)
is a monoclonal antibody directed against CD40.
29. (canceled)
30. The method of claim 17, wherein: a) the step of preventing the
endogenous CD40 antigen from binding its endogenous ligand
comprises contacting a CD40-positive cell with a soluble ligand
which recognizes and binds the CD40 antigen, b) the step of
preventing the endogenous B7 antigen from binding its endogenous
ligand comprises contacting a CTLA4-positive cell with a soluble
ligand which recognizes and binds the CTLA4 antigen, the binding of
the CD40-positive cell to the soluble ligand of step (a) thereby
blocking the reaction of CD40 antigen with endogenous gp39, the
binding of the CTLA4- or CD28-positive cell to the soluble ligand
of step (b) thereby blocking the reaction of the CTLA4 antigen with
endogenous B7, the blockage thereby inhibiting the immune
response.
31. (canceled)
32. (canceled)
33. The method of claim 17, wherein: a) the step of preventing the
endogenous CD40 antigen from binding its endogenous ligand
comprises contacting a gp39-positive cell with a soluble ligand
which recognizes and binds the gp39 antigen, b) the step of
preventing the endogenous B7 antigen from binding its endogenous
ligand comprises contacting a CD28-positive cell with a soluble
ligand which recognizes and binds the CD28 antigen, the binding of
the gp40-positive cell to the soluble ligand of step (a) thereby
blocking the reaction of gp39 antigen with endogenous CD40, the
binding of the CD28-positive cell to the soluble ligand of step (b)
thereby blocking the reaction of the CD28 antigen with endogenous
B7, the blockage thereby inhibiting the immune response.
34. The method of claim 33, wherein the soluble ligand of step (a)
is a monoclonal antibody reactive with the gp39 antigen.
35. (canceled)
36. The method of claim 17, wherein: a) the step of preventing the
endogenous CD40 antigen from binding its endogenous ligand
comprises contacting a gp39-positive cell with a soluble ligand
which recognizes and binds the gp39 antigen, b) the step of
preventing the endogenous B7 antigen from binding its endogenous
ligand comprises contacting a CTLA4-positive cell with a soluble
ligand which recognizes and binds the CTLA4 antigen, the binding of
the CD40-positive cell to the soluble ligand of step (a) thereby
blocking the reaction of gp39 antigen with endogenous CD40, the
binding of the CTLA4-positive cell to the soluble ligand of step
(b) thereby blocking the reaction of the CTLA4 antigen with
endogenous B7, the blockage thereby inhibiting the immune
response.
37. The method of claim 36, wherein the soluble ligand of step (a)
is a monoclonal antibody reactive with the gp39 antigen.
38.-50. (canceled)
Description
[0001] This application is based on U.S. provisional patent
application Ser. No. 60/013,751 filed on Mar. 20, 1996.
[0002] Throughout this application various publications are
referenced. The disclosures of these publications in their
entireties are hereby incorporated by reference into this
application in order to more fully describe the state of the art to
which this invention pertains.
BACKGROUND OF THE INVENTION
[0003] CD28 is expressed on most T lineage cells and plasma cells
(June, C. H. et al., Immunol. Today 11, 211-16 (1990); Damle et
al., Proc. Natl. Acad. Sci. 78:5096-6001 (1981)). The ligand for
CD28 is B7, which is expressed on activated B cells (Linsley, P. S.
et al., Proc. Natl. Acad. Sci. USA 87, 5031-35 (1990); Linsley, P.
S. et al., J. Exp. Med. 173, 721-730 (1991).
[0004] CD40 is a member of the tumor necrosis factor receptor
(TNFR) family of type I membrane-bound signaling receptors. Though
originally identified as a B cell antigen, CD40 is expressed by all
antigen presenting cells (APC) including dendritic cells,
monocytes, and B cells.
[0005] The ligand for CD40 is gp39, which binds to CD40 and thus
can activate B cells. Gp39 is also known as CD40L, TRAP and T-BAM.
Gp39 is a type II cell surface protein with significant homology to
TNF and is transiently expressed by activated T cells. In addition
to T cells, gp39 is expressed by basophils, mast cells, and
eosinophils.
[0006] The CD28 and CD40 pathways play essential roles in the
initiation and amplification of T-dependent immune responses
(Bluestone, J. A. Immunity 2, 555-9 (1995); Banchereau J., et al.
Own. Rev. Immunol. 12, 881-922 (1994); Durie, F. H., et al. Science
261, 1328-30 (1993); Foy, T. M., et al. J Exp Med 178, 1567-75
(1993); Van den Eertwegh, A. J. M., et al. J Exp Med 178, 1555-65
(1993)).
[0007] CD28/B7 interactions provide critical "second signals"
necessary for optimal T cell activation, and IL-2 production
(Jenkins, M. K., et al. J. Immunol. 147, 2461-6 (1991); Schwartz,
R. H. Cell 71, 1065-8 (1992); Boussiotis, V. A., et al. J. Exp.
Med. 178, 1753-1763 (1993)), whereas CD40/gp39 signals provide
costimulation for B cell, macrophage, endothelial cell, and T cell
activation (Grewal, I. S., et al. Nature 378, 617-620 (1995); van
Essen, D., et al. Nature 378, 620-623 (1995); Hollenbaugh, D., et
al. J. Exp. Med. 182, 33-40 (1995); Armitage, R. J., et al. Nature
357, 80-2 (1992); Cayabyab, M., et al. J. Immunol. 152, 1523-31
(1994); Noelle, R., et al. Proc. Natl. Acad. Sci. USA 89, 6550-6554
(1992); Alderson, M. et al. J. Exp. Med. 178, 669-674 (1993)).
[0008] Host immune responses often cause rejection of transplanted
tissues and organs. Thus, inhibition of those immune responses are
critical in the success of tissue transplantation. There have been
studies aimed at blocking either of the CD28 or CD40 pathways,
however, blockade of either of these pathways alone has not been
sufficient to permit engraftment of highly immunogenic allografts
(Turka, L. A., et al. Proc. Nat'l Acad. Sci. USA 89, 11102-11105
(1992); Parker, D. C., et al. Proc. Nat'l Acad. Sci. USA 92,
9560-9564 (1995); Larsen, C. P. et al. Transplantation 61, 4-9
(1996)). The monotherapies blocking either CD28 or CD40 pathway
only resulted in at best temporary, and sometimes longer, periods
of survival of transplanted tissues. Neither blockade alone
uniformly promoted graft survival.
[0009] The vigorous immune response to xenogeneic organ transplants
has served as a powerful barrier to the application of this
technique to clinical transplantation (Platt J. L., Curr. Opin.
Imm. 8, 721-728 (1996). Previous experimental attempts to prolong
xenogeneic skin grafts have required either whole body irradiation
followed by mixed xeno/syngeneic reconstitution (Ildstad S. T.,
Sachs D. H., Nature, 307: 168-170 (1984)), or rigorous
preconditioning with thymectomy combined with depleting anti-T cell
antibodies (Pierson III R. N., Winn H. J., Russell P. S.,
Auchincloss Jr. H., J. Exp. Med., 170:991-996 (1989); and Sharabi
Y., Aksentijevich I., Sundt III T. M., Sachs D. H., Sykes M., J.
Exp. Med., 172:195-202 (1990). These strategies have recently been
used to promote skin graft acceptance across a discordant
xenogeneic barrier (Zhao Y., Swenson K., Sergio J., Arn J. S.,
Sachs D. H., Sykes M., Nat. Med., 2(11):1211-1216 (1996)). However,
the potential morbidity associated with cytoablative treatment
regimens present a significant obstacle to the introduction of
these strategies into use in clinical solid organ transplantation.
Thus the development of non-cytoablative strategies to prolong
xenograft survival would greatly facilitate the clinical
application of these techniques.
[0010] Presently, there exists a need to provide ways to effect
long-term tolerance of transplanted tissues by the host, thereby
increasing the survival rate of transplantation. To do so, it is
necessary to ensure sufficient immunologic unresponsiveness in the
transplant recipient.
[0011] We have found that the inhibition of T-dependent immune
responses resulting from blockade of either CD28 or CD40 signals is
potent, but incomplete. The data herein demonstrate that
simultaneous blockade of these pathways unexpectedly inhibits acute
and chronic rejection of transplanted tissue in vivo. Independent
blockade of these pathways using a soluble CTLA4 molecule or
antibodies which recognize and bind gp39 failed to even minimally
prolong. survival of primary skin transplanted tissue.
[0012] The invention herein involves the discovery that
simultaneous blockade of CD28 and CD40 signals promoted long-term
survival of fully allogeneic as well as xenogeneic skin grafts.
Prolongation of skin allograft survival was eliminated by
cyclosporine A (CyA), suggesting that it is an active process
requiring intact signaling via the TcR/CD3 complex and/or other CyA
sensitive pathways. Moreover, CTLA4Ig/MR1 promoted long-term
acceptance of primarily vascularized cardiac graft tissue, and
inhibited the development of chronic vascular rejection.
[0013] The effect demonstrated in the two transplantation models
herein indicates that CD28 and CD40 provide interrelated, yet
independent signaling pathways required for the generation of
effective T cell responses. This discovery provides methods which
are new and more effective strategies to manipulate immune
responses including suppressing graft rejection.
SUMMARY OF THE INVENTION
[0014] The present invention provides a method for inhibiting
rejection of a transplanted tissue. This method comprises
preventing an endogenous molecule (e.g., antigen) on a cell
selected from the group consisting of gp39 and CD40 from binding
its endogenous ligand and preventing an endogenous molecule on a
cell selected from the group consisting of CTLA4, CD28, and B7 from
binding its endogenous ligand. The prevention of such molecules
from binding their ligands thereby blocks two independent signal
pathways and inhibits the immune response responsible for
transplanted tissue resection.
[0015] Further, the invention provides a method for inhibiting an
immune response involved with transplanted tissue rejection
comprising contacting a B7-positive cell with a first soluble
ligand which recognizes and binds the B7 antigen, and contacting a
gp39-positive cell with a second soluble ligand which recognizes
and binds the gp39 antigen. The binding of the B7-positive cell to
the first soluble ligand blocks the reaction of the B7 antigen with
endogenous CTLA4 or CD28. Additionally, the binding of the gp39
antigen to the second soluble ligand blocks the reaction of gp39
antigen with endogenous CD40. This blockage of both the gp39 and B7
pathways inhibits immune responses.
[0016] Applicants' discovery includes a method for inhibiting
immune responses mediated by the gp39 and B7 pathways in a subject.
This method comprises administering to the subject a first soluble
ligand which recognizes and binds the B7 antigen and a second
soluble ligand which recognizes and binds the gp39 antigen.
[0017] The binding of both the first and second soluble ligands to
their receptors inhibits the immune response mediated by the gp39
and B7 pathways by preventing an endogenous molecule on a cell
selected from the group consisting of gp39 and CD40 antigens from
binding its endogenous ligand and preventing an endogenous molecule
on a cell selected from the group consisting of CTLA4, CD28, or B7
from binding its ligand.
[0018] The present invention also provides a method for inhibiting
transplant rejection in a subject. This method comprises
administering to the subject an effective amount of a combination
of a first soluble ligand which recognizes and binds the B7 antigen
on B7-positive cells and a second soluble ligand which recognizes
and binds the gp39 -antigen on gp39-positive cells.
[0019] The binding of B7-positive cells with the first soluble
ligand and gp39-positive cells with the second soluble ligand
disrupts endogenous. CTLA4-, CD28-, and gp39-positive cell
interactions with B7-positive cells and gp39-positive cells so that
transplant rejection is inhibited.
BRIEF DESCRIPTION OF THE FIGURES
[0020] FIG. 1 is a bar graph showing that simultaneous blockade of
CD28 and CD40 signals ablate popliteal lymph node alloimmune
responses in vivo.
[0021] FIG. 2A is a line graph that shows CTLA4Ig/MR1 treatment
prolongs cardiac allograft survival in comparison with CTLA4Ig or
MR1 alone.
[0022] FIG. 2B is a photograph of a histologic section showing
CTLA4Ig-treated cardiac allograft at day 62 having extensive
lymphocytic infiltration, interstitial fibrosis, and severe
coronary arterial intimal thickening and fibrosis consistent with
chronic rejection (left panel 100.times. magnification; right panel
400.times. magnification).
[0023] FIG. 2C is a photograph of a histologic section showing a
MR1-treated cardiac allograft at day 62 having less lymphocytic
infiltration and interstitial fibrosis, but severe coronary
vasculopathy characteristic of chronic rejection (left panel
100.times. magnification; right panel 400.times.
magnification).
[0024] FIG. 2D is a photograph of a histologic section showing
CTLA4Ig/MR1-treated cardiac allografts at day 58, free from
lymphocytic infiltration, fibrosis, coronary arterial intimal
lesions (left panel 100.times. magnification;, right panel
400.times. magnification).
[0025] FIG. 2E is a photograph of a histologic section showing
normal untransplanted BALB/c hearts (left panel 100.times.
magnification; right panel 400.times. magnification).
[0026] FIG. 3A is a photograph of ethidium bromide stained gel
strips showing intragraft expression of immune mediator transcripts
using RT-PCR in untreated, MR1 treated, CTLA4Ig treated, and
MR1/CTLA4Ig treated cardiac allografts.
[0027] FIG. 3B is a series of bar graphs showing the mean PCR
product band intensities.+-.standard deviation.
[0028] FIG. 4A is a line graph showing data of mice treated with
MR1 alone, CTLA4Ig alone, and a combination of MR1 and CTLA4Ig.
[0029] FIG. 4B is a line graph showing data of mice treated with
CyA, CyA and CTLA4Ig, and CyA and MR1.
[0030] FIG. 4C is a line graph showing the effects of perioperative
treatment with YTS191 and MR1, alone, MR1 and CTLA4Ig, and YTS191
and CTLA4Ig on primary skin allografts.
[0031] FIG. 4D is a photograph showing healthy appearance of a
BALB/c skin graft on a CTLA4Ig/MR1 treated recipient.
[0032] FIG. 4E is a photograph showing a control allograft
undergoing rejection. FIG. 4F is a photograph of a histologic
section of a skin graft showing healthy appearance of an accepted
graft at 100 days after transplant showing well preserved epidermis
hair follicles and adnexal structures.
[0033] FIG. 4G is a photograph showing a BALB/c skin graft on an
untreated recipient eight days after transplant. The graft shows
extensive lymphocytic infiltrate.
[0034] FIG. 5A is a series of line graphs showing the effects in
vitro of using MR1 alone, CTLA4Ig alone, and a combination of
MR1/CTLA4Ig on three different cell populations.
[0035] FIG. 5B is a series of bar graphs showing the effects in
vivo of using MR1 alone, CTLA4Ig alone, and a combination of
MR1/CTLA4Ig.
[0036] FIG. 6A is a bar graph showing weight of the immunized
popliteal lymph node relative to the contralateral node in C3H mice
in response to foot pad immunization with irradiated (2000 RADS)
rat (Sprague-Dawley) splenocytes. Human IgG (stippled), CTLA4-Ig
(gray), MR1 (white), CTLA4-Ig/MR1 (black), normal unimmunized node
(hatched).
[0037] FIG. 6B is a line graph showing in vitro proliferation of
lymph node cells after harvesting the popliteal lymph at five days
after immunization. Human IgG (stippled), CTLA4-Ig (gray), MR1
(white) CTLA4-Ig/MR1 (black), normal unimmunized node
(hatched).
[0038] FIG. 6C is a bar graph showing that simultaneous blockade of
the CD40 and CD28 pathways markedly inhibits cytokine production of
IL-2. Human IgG (stippled), CTLA4-Ig (gray), MR1 (white),
CTLA4-Ig/MR1 (black), normal unimmunized node (hatched).
[0039] FIG. 6D is a bar graph showing that simultaneous blockade of
the CD40 and CD28 pathways markedly inhibits cytokine production of
INF.gamma.. Human IgG (stippled), CTLA4-Ig (gray), MR1 (white),
CTLA4-Ig/MR1 (black), normal unimmunized node (hatched).
[0040] FIG. 7A is a line graph showing that C3H recipients treated
with CTLA4-Ig (500 .mu.g) on days 0, 2, 4 and 6 combined with MR1
(500 .mu.g) on days 0, 2, 4 and 6 had prolonged survival of
Sprague-Dawley rat cardiac allografts.
[0041] FIG. 7B is a photograph of an untreated cardiac xenograft at
day 6 showing widespread tissue destruction (400.times.).
[0042] FIG. 7C is a photograph of a CTLA4-I-g treated cardiac
xenograft at day 20 showing lymphocytic infiltration, myocyte
destruction, and coronary vasculopathy (400.times.).
[0043] FIG. 7D is a photograph of a MR1 treated -cardiac xenograft
at day 20 showing lymphocytic infiltration, myocyte destruction,
and coronary vasculopathy (400.times.).
[0044] FIG. 7E is a photograph of a normal untransplanted.
Sprague-Dawley rat heart (400.times.).
[0045] FIG. 7F is a photograph of a CTLA4-Ig/MR1 treated cardiac
xenograft at day 20, essentially free from lymphocytic infiltration
and fibrosis (400.times.).
[0046] FIG. 7G is a photograph of a CTLA4-Ig/MR1 treated cardiac
xenograft at day 122, demonstrating excellent preservation of both
myocytes and vascular structures (400.times.).
[0047] FIG. 8A is a series of line graphs showing prolongation of
Sprague-Dawley rat skin xenograft survival in C3H mice treated with
MR1 and CTLA4-Ig administered together in the perioperative period
as compared with xenograft recipients treated with either MR1 alone
or CTLA4-Ig alone and untreated controls.
[0048] FIG. 8B is a series of line graphs showing no significant
change in xenograft survival following chronic treatment (beginning
after the standard 4 dose regimen) with either the CTLA4-Ig/MR1
combination or MR1.
[0049] FIG. 8C is a photograph showing the healthy appearance of a
Sprague-Dawley rat skin graft on a CTLA4-Ig/MR1 treated C3H
recipient at 100 days after transplant.
[0050] FIG. 8D is a photograph showing a control xenograft of skin
undergoing rejection at 10 days post transplant.
[0051] FIG. 8E is a photograph of a hematoxylin-eosin stained
histologic section of an accepted CTLA4-Ig/MR1 treated graft at 50
days after transplant showing well-preserved histologic
architecture (400.times.).
[0052] FIG. 8F is a photograph of a hematoxylin-eosin stained
histologic section of a Sprague-Dawley rat skin graft on an
untreated C3H recipient 8 days after transplant showing extensive
lymphocytic infiltrates (400.times.).
[0053] FIG. 9A is a scatter plot showing ablation of evoked
xenoantibody response in serum collected from C3H recipients 55
days after skin xenografts from Sprague-Dawley donors. Control mice
that had received no treatment had readily detectable IgG
xenoantibody. Either CTLA4-Ig or MR1 alone partially blocked the
xenoantibody response. The combination of CTLA4-Ig and MR1
essentially ablated the evoked xenoantibody response. Each data
point represents the analysis of an individual recipient.
[0054] FIG. 9B is a scatter plot showing ablation of evoked
xenoantibody response in serum collected from C3H recipients 20
days after heart xenografts from Sprague-Dawley donors.
DETAILED DESCRIPTION OF THE INVENTION
DEFINITIONS
[0055] All scientific and-technical terms used in this application
have meanings commonly used in the art unless otherwise specified.
As used in this application, the following words or phrases have
the meanings specified.
[0056] As used herein "monoclonal antibodies directed against gp39"
or "anti-gp39" includes MR1. Anti-gp39 is also known in the
literature as an antiCD40 ligand. Examples of MR1 include, but are
not limited to monoclonal antibodies directed against gp39 from
mouse; antibodies directed against gp39 from other species such as
monkey, sheep, human are included. Additionally, "monoclonal
antibodies directed against gp39" or "anti-gp39" includes any
antibody molecule, fragment thereof, or recombinant binding protein
that recognizes and binds gp39.
[0057] As used herein, "administering" means oral administration,
administration as a suppository, topical contact, intravenous,
intraperitoneal, intramuscular or subcutaneous administration, or
the implantation of a slow-release device such as a miniosmotic
pump, to the subject.
[0058] As used herein, "pharmaceutically acceptable carrier"
includes any material which when combined with the antibody retains
the antibody's immunogenicity and is non-reactive with the
subject's immune systems. Examples include, but are not limited to,
any of the standard pharmaceutical carriers such as a phosphate
buffered saline solution, water, emulsions such as oil/water
emulsion, and various types of wetting agents. Other carriers may
also include sterile solutions, tablets including coated tablets
and capsules.
[0059] Typically such carriers contain excipients such as starch,
milk, sugar, certain types of clay, gelatin, stearic acid or salts
thereof, magnesium or calcium stearate, talc, vegetable fats or
oils, gums, glycols, or other known excipients. Such carriers may
also include flavor and color additives or other ingredients.
Compositions comprising such carriers are formulated by well known
conventional methods.
[0060] As used herein, "transplanted tissue" includes autografts,
isografts, allografts, and xenografts. Examples of transplanted
tissue include, but are not limited to, solid organ transplants
such as heart, liver or kidney, skin grafts, pancreatic islet
cells, bone marrow grafts or cell suspensions.
[0061] As used herein, "B7" includes: B7-1 (also called CD80), B7-2
(also called CD86), B7-3, and the B7 family, e.g., a combination of
B7-1, B7-2 and/or B7-3.
[0062] In order that the invention herein described may be more
fully understood, the following description is set forth.
[0063] The discovery herein is related to a method for inhibiting
rejection of a transplanted tissue. In one embodiment, the method
comprises preventing an endogenous molecule on a cell selected from
the group consisting of gp39 and CD40 from binding its endogenous
ligand. The method provide's preventing an endogenous molecule on a
cell selected from the group consisting of CTLA4, CD28, and B7 from
binding its endogenous ligand. The prevention of these molecules
from binding their endogenous ligands blocks two independent signal
pathways. The blockage of these two independent signal pathways
inhibits the immune responses that cause transplanted tissue
rejection.
[0064] In one example of the invention, endogenous gp39 antigen is
prevented from binding its endogenous ligand. This example
comprises the step of contacting a gp39-positive cell with a
soluble ligand which recognizes and binds the gp39 antigen (e.g.,
by using soluble ligands such as MR1 or other antibodies which bind
gp39, and soluble CD40 molecules).
[0065] This example comprises the additional step of preventing the
endogenous CTLA4 antigen from binding its endogenous ligand. This
comprises the step of contacting a B7-positive cell with a soluble
ligand which recognizes and binds the B7 antigen such as CTLA4Ig
(U.S. Pat. No. 5,434,131, issued Jul. 18, 1995), the BB-1
monoclonal antibody or other antibodies directed against B7.
[0066] The binding of the gp39-positive cell to its soluble ligand
blocks the reaction of endogenous gp39 antigen with endogenous
CD40. The binding of the B7-positive cell to its soluble ligand
blocks the reaction of the endogenous B7 antigen with endogenous
CTLA4 and CD28. This combined blockage inhibits the immune
response.
[0067] In another example, endogenous CD40 antigen is prevented
from binding its endogenous ligand. This example comprises the step
of contacting a CD40-positive cell with a soluble ligand which
recognizes and binds the CD40 antigen. Suitable ligands include
antibodies directed against CD40 or soluble gp39 (sgp39).
[0068] This example comprises the additional step of preventing the
endogenous CTLA4 antigen from binding its endogenous ligand. This
step comprises contacting a B7-positive cell with a soluble ligand
which recognizes and binds the B7 antigen. Examples of this soluble
ligand include CTLA4Ig, soluble CD28 molecules, and antibodies
directed against B7.
[0069] The binding of the CD40-positive cell to its soluble ligand
blocks the reaction of endogenous CD40 antigen with endogenous
gp39. The binding of the B7-positive cell to its soluble ligand
blocks the reaction of the B7 antigen with endogenous CTLA4. The
combined blockage inhibits the immune response.
[0070] In yet another example, endogenous gp39 antigen is prevented
from binding its endogenous ligand as described above. The example
comprises the additional step of preventing the endogenous CD28
antigen-from binding its endogenous ligand. This step comprises
contacting a B7-positive cell with a soluble ligand which
recognizes and binds the B7 antigen. Examples include CTLA4Ig,
soluble CD28 molecules, and antibodies directed against B7 such as
BB-1.
[0071] The binding of the gp39-positive cell to its soluble ligand
blocks the reaction of gp39 antigen-with endogenous CD40. The
binding of the B7-positive cell to its soluble ligand blocks the
reaction of the B7 antigen with endogenous CD28. This combined
blockage inhibits the immune response.
[0072] In another example, endogenous CD40 antigen is prevented
from binding its endogenous ligand as described above. The example
provides the additional step of preventing the endogenous B7
antigen from binding its endogenous ligand. This comprises
contacting a CD28-positive cell with a soluble ligand which
recognizes and binds the CD28 antigen. Examples of such soluble
ligands include soluble B7 molecules and antibodies directed
against CD28.
[0073] The binding of the CD40-positive cell to the soluble ligand
blocks the reaction of CD40 antigen with endogenous gp39. The
binding of the CD28-positive cell to the soluble ligand blocks the
reaction of the B7 antigen with endogenous CD28. This combined
blockage inhibits the immune response.
[0074] In yet another example, endogenous CD40 antigen is prevented
from binding its ligand as described above. This example provides
the additional step of preventing the endogenous B7 antigen from
binding its endogenous ligand which comprises contacting a
CTLA4-positive cell with a soluble ligand which recognizes and
binds the CTLA4 antigen. Examples of such soluble ligands include
soluble B7 molecules and antibodies directed against CTLA4.
[0075] The binding of the CD40-positive cell to the soluble ligand
blocks the reaction of CD40 antigen with endogenous gp39.
Additionally, the binding of the CTLA4- or CD28-positive cell to
the soluble ligand blocks the reaction of the CTLA4 antigen with
endogenous B7. This combined blockage inhibits the immune
response.
[0076] In a further example, endogenous CD40 antigen is prevented
from binding its ligand as described above. This example provides
the additional step of preventing the endogenous B7 antigen from
binding its endogenous ligand which comprises contacting a
CD28-positive cell with a soluble ligand which recognizes and binds
the CD28 antigen. Examples of such soluble ligands include soluble
B7 molecules and antibodies directed against CD28.
[0077] The binding of the CD40-positive cell to the soluble ligand
blocks the reaction of gp39 antigen with endogenous CD40. Further,
the binding of the CD28-positive cell to the soluble ligand blocks
the reaction of the CD28 antigen with endogenous B7. This combined
blockage inhibits the immune response.
[0078] Also, in another example, endogenous CD40 antigen is
prevented from binding its ligand as described above. This example
provides the additional step of preventing the endogenous B7
antigen from binding its endogenous ligand which comprises
contacting a CTLA4-positive cell with a soluble ligand which
recognizes and binds the CTLA4 antigen.
[0079] The binding of the CD40-positive cell to the soluble ligand
blocks the reaction of gp39 antigen with endogenous CD40.
Additionally, the binding of the CTLA4-positive cell to the soluble
ligand blocks the reaction of the CTLA4 antigen with endogenous B7.
This combined blockage inhibits the immune response.
[0080] Additionally, the present invention provides another
embodiment for a method for inhibiting an immune response resulting
in graft rejection. This embodiment comprises contacting a
B7-positive cell with a first soluble ligand which recognizes and
binds the B7 antigen, and contacting a gp39-positive cell with a
second soluble ligand which recognizes and binds the gp39
antigen.
[0081] The binding of the B7-positive cell to the first soluble
ligand blocks the reaction of the B7 antigen with endogenous CTLA4
or CD28. Further, the binding of the gp39 antigen to the second
soluble ligand blocks the reaction of gp39 antigen with endogenous
CD40. The combination of this blockage inhibits the immune
response.
[0082] Additionally, the invention provides a method for inhibiting
an immune response mediated by the CTLA4/CD28/B7 and gp39/CD40
pathways in a subject. In accordance with the practice of the
invention, the subject may be an animal subject such as a human, a
dog, a cat, a sheep, a horse, a mouse, a pig, or a cow.
[0083] The method comprises administering to the subject a first
soluble ligand which recognizes and binds the B7 antigen (e.g.
soluble CTLA4 or CD28 molecules) and a second soluble ligand which
recognizes and binds the gp39 antigen (e.g., monoclonal antibodies
directed against gp39 (MR1) or soluble CD40 molecules). The binding
of the first and second ligands to their receptor inhibits the
immune response mediated by CTLA4-, CD28-, and gp39-cell
interactions with B7- and CD40-positive cells.
[0084] Also, the invention provides a method for inhibiting
transplant rejection in a subject. This method comprises
administering to the subject an effective amount of a combination
of a first soluble ligand which recognizes and binds the B7 antigen
on B7-positive cells and a second soluble ligand which recognizes
and binds the gp39 antigen on gp39-positive cells. The binding of
B7-positive cells with the first soluble ligand and gp39-positive
cells with the second soluble ligand disrupts endogenous CTLA4-,
CD28-, and gp39-cell interactions with B7-positive cells and
gp39-positive cells so that transplant rejection is inhibited.
[0085] In accordance with the practice of the invention, the first
soluble ligand may be a recombinant binding molecule having at
least a portion of the extracellular domain of CTLA4. In accordance
with the practice of the invention, the extracellular portion of
CTLA4 is joined to a non-CTLA4 protein sequence. The non-CTLA4
protein sequence may be at least a portion of an immunoglobulin
molecule.
[0086] In one specific example of the invention, the ligand is
CTLA4Ig fusion protein, e.g., the CTLA4Ig fusion protein deposited
with the American Type Culture Collection (ATCC) in Rockville, Md.,
under the provisions of the Budapest Treaty on May 31, 1991 and
accorded ATCC accession number: 68629. Alternatively, the ligand
may be a CD28Ig/CTLA4Ig fusion protein hybrid (U.S. Pat. No.
5,434,131, issued Jul. 18, 1995).
[0087] In an alternative embodiment, the first soluble ligand may
be a monoclonal antibody reactive with B7. antigen, e.g., the
antibody may be anti-BB1 monoclonal antibody (Clark et al., Human
Immunol. 16:100-113 (1986); Yokochi et al., J. Immunol. 128:823
(1981)); Freeman et al. (J. Immunol. 143(8):2714-2722 (1989); and
Freedman et al., J. Immunol. 139:3260 (1987)).
[0088] In another embodiment, the ligand may be a CD28Ig/CTLA4Ig
fusion protein hybrid having a first amino acid sequence
corresponding to a portion of the extracellular domain of CD28
receptor fused to a second amino acid sequence corresponding to a
portion of the extracellular domain of CTLA4 receptor and a third
amino acid sequence corresponding to the hinge, CH2 and CH3 regions
of human immunoglobulin C.gamma.1.
[0089] In one embodiment of the invention, the second soluble
ligand for the gp39 antigen may be a monoclonal antibody reactive
with the gp39 antigen, e.g., the MR1 anti-murine monoclonal
antibody or the anti-human gp39 antibody (U.S. Pat. No. 5,474,771,
issued Dec. 12, 1995).
[0090] In another embodiment of the invention, the method comprises
administering to the subject a soluble fusion protein, the soluble
fusion protein comprising a first binding domain and a second
binding domain.
[0091] In one example, the first binding domain is a ligand which
recognizes and binds the gp39 antigen. Examples include CD40 and
monoclonal antibodies directed against gp39. In another example,
the first binding domain is a ligand which recognizes and binds the
CD40 antigen. Examples include gp39 and monoclonal antibodies
directed against CD40.
[0092] In one example, the second binding domain is a ligand which
recognizes and binds CTLA4. Examples include B7 and monoclonal
antibodies directed against CTLA4. In another example the second
binding domain is a ligand which recognizes and binds the CD28
antigen. Examples include B7 and monoclonal antibodies directed
against CD28. In another example, the second binding domain is a
ligand which recognizes and binds the B7 antigen. Examples include
CTLA4, CD28 and monoclonal antibodies directed against B7.
[0093] Soluble ligands may be administered during transplant,
before transplant, or after transplant. Soluble ligands may be
administered by oral means, transdermal means, intravenous means,
intramuscular means, intraperitoneal, or by subcutaneous
administration.
[0094] The most effective mode of administration and dosage regimen
for the molecules of the present invention depends upon the
location of the tissue or disease being treated, the severity and
course of the medical disorder, the subject's health and response
to treatment and the judgment of the treating physician.
Accordingly, the dosages of the molecules should be titrated to the
individual subject.
[0095] By way of example, the interrelationship of dosages for
animals of various sizes and species and humans based on mg/m.sup.2
of surface area is described by Freireich, E. J., et al. Cancer
Chemother., Rep. 50 (4): 219-244 (1966). Adjustments in the dosage
regimen may be made to optimize suppression of the immune response
resulting in graft rejection, e.g., doses may be divided and
administered on a dally basis or the dose reduced proportionally
depending upon the situation (e.g., several divided doses may be
administered daily or proportionally reduced depending on the
specific therapeutic situation).
[0096] It would be clear that the dose of the composition of the
invention required to achieve an appropriate clinical outcome may
be further reduced with schedule optimization.
[0097] The present invention also provides pharmaceutical
compositions useful in inhibiting graft rejection or in inhibiting
an immune response. In one embodiment, these compositions comprise
an effective amount of a combination of (a) soluble ligands which
recognize and bind any one of CTLA4, CD28, and B7 antigens,
together with (b) soluble ligands which recognize and bind any one
of gp39 and CD40 antigens and an acceptable carrier. In another
embodiment, these compositions comprise an effective amount of a
soluble fusion protein comprising a first binding domain and a
second binding domain, wherein the first binding domain is a ligand
which recognizes and binds any one of gp39 or CD40 antigens and the
second binding domain is a ligand which recognizes and binds any
one of CTLA4, CD28, and B7 antigens.
[0098] ADVANTAGES OF THE INVENTION: Despite the many, advances in
clinical immunosuppression, chronic vascular rejection remains the
major source of transplant failure for which there remains no
effective therapy. The experiments described herein show that
blocking the CD28/CTLA4/B7 and gp39/CD40 pathways inhibits the
development of chronic transplant vasculopathy in transplanted
tissues. These data show that immune responses to allogeneic and
xenogeneic grafts can be inhibited without cytoablation. When
compared to the use of soluble CTLA4 molecules alone, the use of
soluble CTLA4 molecules together with a soluble ligand that
recognizes and binds gp39 provides dramatically prolonged
immunosuppression.
[0099] The following examples are presented to illustrate the
present invention and to assist one of ordinary skill in making and
using the same. The examples are not intended in any way to
otherwise limit the scope of the invention.
EXAMPLE 1
[0100] The data in this example show that simultaneous blockade of
CD28 and CD40 signals ablates popliteal lymph node alloimmune
responses in vivo.
[0101] Method
[0102] Male C3H/HeJ (The Jackson Laboratory, Bar Harbor, Me.) mice
were subcutaneously immunized with 2.times.10.sup.6 BALB/c
splenocytes in 50 .mu.l of sterile normal saline in the left foot
pad and 50 .mu.l of sterile normal saline in the right foot pad on
day 0 and then treated intraperitoneally with MR1 (250 .mu.g),
CTLA4Ig. (250 .mu.g), or both reagents on days 0, 2 and 4.
[0103] The mice were sacrificed on day 5, the popliteal lymph nodes
were harvested using an operating microscope (20.times.
magnification) and the fresh weight of each node was determined to
the nearest 0.1 mg with an analytical balance (Model A-160, Denver
Instrument Company, Arvada, Colo.).
[0104] Discussion
[0105] Five days after subcutaneous immunization with allogeneic
splenocytes, the draining popliteal lymph nodes on the side of
antigen challenge underwent a >5 fold increase in weight
relative to the contralateral node in untreated control mice.
Treatment with either CTLA4Ig or MR1 resulted in a 50-60%
inhibition of the response, whereas concomitant administration of
CTLA4Ig and MR1 ablated lymph node expansion in response to antigen
challenge. The results represent the mean.+-.standard deviation for
3 individual mice in each group. Similar results were obtained in
three independent experiments.
[0106] Control mice demonstrated a 4-6 fold increase in the weight
of the node draining the immunized foot relative to the node
draining the contralateral foot injected with sterile saline (FIG.
1). This increase in weight was accompanied by a dramatic expansion
of the lymphocyte-rich paracortical (T cell) and cortical (B cell)
regions. When administered alone, CTLA4Ig and MR1 each produced
partial inhibition of this response (57% and 56% inhibition,
respectively). The combination of CTLA4Ig/MR1 ablated lymph node
expansion (98% inhibition, FIG. 1) and prevented expansion of the
paracortical and lymphoid follicles.
EXAMPLE 2
[0107] This example shows prolongation of cardiac allograft
survival and inhibition of vasculopathy associated with chronic
rejection.
[0108] Method
[0109] Male C3H/HeJ mice were transplanted with primarily
vascularized BALB/c heart allografts at 8-12 weeks of age using
microsurgical techniques (Corry, R. J., Winn, H. J. & Russell,
P. S. Transplantation 16, 343-350 (1973)).
[0110] Rejection was defined by the loss of palpable cardiac
contractions with confirmation at laparotomy by direct
visualization. At specified times after transplant, the
transplanted hearts were excised, formalin fixed and embedded in
paraffin. Tissue sections (5 .mu.m) were stained with Masson's
Trichrome or hematoxylin-eosin. Each histologic specimen was
reviewed by a cardiac transplant pathologist (KJW) blinded to the
treatment modality.
[0111] Discussion
[0112] In FIG. 2A C3H/HeJ recipients were treated with CTLA4Ig (200
.mu.g/dose) on days 0, 2, 4 and 6 combined with MR1 (250
.mu.g/dose) on days 0, 2 and 4, and had long term survival of
BALB/c cardiac allografts (Median Survival Time (MST) >70 days,
n=7). The control groups included recipients treated with: CTLA4Ig
alone (MST=50 days, n=12); MR1 alone (MST=70 days, n=12); and no
treatment (MST=12 days, n=7).
[0113] All recipients were followed for 70 days with the exception
of three mice with surviving transplants in each experimental group
which were sacrificed for histologic analysis at 58-63 days post
transplant.
[0114] In FIG. 2B, CTLA4Ig-treated cardiac allograft at day 62
shows extensive lymphocytic infiltration, interstitial fibrosis,
and severe coronary arterial intimal thickening and fibrosis
consistent with chronic rejection.
[0115] In FIG. 2C, MR1-treated cardiac allograft at day 62
demonstrates less lymphocytic infiltration and interstitial
fibrosis, but severe coronary vasculopathy characteristic of
chronic rejection.
[0116] In FIG. 2D, CTLA4Ig/MR1-treated cardiac allografts at day
58, in marked contrast, were remarkably free from lymphocytic
infiltration, fibrosis, and most significantly, coronary arterial
intimal lesions. The parenchyma and blood vessels of these grafts
were virtually indistinguishable from normal untransplanted BAL-B/c
hearts.
[0117] In FIG. 2E, normal untransplanted BALB/c hearts are
shown.
[0118] Similar histologic results were obtained from three
allografts in each experimental group. C3H/HeJ (H-2.sup.k)
recipients treated with CTLA4Ig alone, MR1 alone, or CTLA4Ig/MR1,
all showed prolonged survival of BALB/c (H-2.sup.d) cardiac
allografts when compared to untreated controls (FIG. 2A). However,
when examined histologically at 58-62 days post-transplant marked
differences were apparent.
[0119] Allografts from CTLA4Ig-treated recipients showed extensive
lymphocytic infiltration, interstitial fibrosis, and severe
coronary arterial intimal thickening and fibrosis consistent with
chronic rejection (FIG. 2B). While the MR1-treated allograft
demonstrated less lymphocytic infiltration and interstitial
fibrosis, these grafts also had severe coronary vasculopathy
characteristic of chronic rejection (FIG. 2C).
[0120] In marked contrast, the allograft from CTLA4Iq/MR1 treated
recipients were remarkably free from lymphocytic infiltration,
fibrosis, and most significantly, coronary arterial intimal lesions
(FIG. 2D). In fact, the parenchyma and blood vessels of these
grafts were virtually indistinguishable from those found in normal
BALB/c hearts (FIG. 2E).
EXAMPLE 3
[0121] This example shows blockade of T cell cytokine and
costimulatory molecule transcript expression.
[0122] Method
[0123] At 8 days after transplantation, the cardiac grafts were
removed and total RNA was prepared from tissues using TRIzol
Reagent (GIBCO BRL, Gaithersburg, Md.). cDNA was synthesized using
5 .mu.g of total RNA template with a Superscript Preamplification
System (GIBCO BRL, Gaithersburg, Md.) in a final volume of 20
.mu.l. PCR reactions were carried out. PCR products were visualized
on ethidium bromide stained 1% agarose (BIO-RAD, Hercules, Calif.),
2% NuSieve GTG agarose (FMC BioProducts, Rockland, Me.) gels. Gel
images were stored using a UVP Gel Documentation System 5000. Band
intensity was quantified using Gelreader analysis software
(National Center for Supercomputing Applications, Urbana,
Ill.).
[0124] In FIG. 3A, intragraft expression of immune mediator
transcripts was assessed using RT-PCR in untreated, MR1-treated,
CTLA4Ig treated, and MR1/CTLA4Ig treated cardiac allografts.
[0125] Three allografts from each treatment group and the control
group were analyzed at 8 days post-transplant. Normal heart tissue
(N) and a syngeneic heart graft (S) at 8 days after transplantation
were included for comparison.
[0126] In FIG. 3B, graphical representation of the mean PCR product
band intensities.+-.standard deviation are shown.
[0127] Discussion
[0128] No consistent differences in the expression of T cell
cytokine transcripts for IL-2, IL-4, IL-10, and IFN.gamma. or
costimulatory molecule transcripts (B7-1, and B7-2) were detectable
between the control allografts (FIG. 3A, untreated) and MR1-treated
allografts, (FIG. 3A), whereas CTLA4Ig partially inhibited
expression of IL-4 transcripts.
[0129] Allografts from CTLA4Ig/MR1-treated recipients showed a
striking decrease in the expression of both Th1 cytokine (IL-2 and
IFN.gamma.) and Th2 cytokine (IL-4, and IL-10) transcripts.
However, intragraft B7-1 and B7-2 costimulatory molecule
transcripts were only modestly reduced in recipients treated with
CTLA4Ig/MR1.
[0130] PCR reactions using template prepared without reverse
transcriptase yielded no products, even for the intron-less GADPH
gene (FIG. 3A, GADPH, NO RT), confirming the absence of
contaminating genomic DNA.
[0131] Intragraft B7-1 and B7-2 costimulatory molecule transcripts
were only modestly reduced in recipients treated with CTLA4Ig/MR1,
(FIG. 3) suggesting that CD28/B7-independent or
CD40/gp39-independent factors, such as GMCSF (Larsen, C. P., et al.
J Immunol 152, 5208-5219 (1994)), may be important regulators of
intragraft B7 expression. Thus, MR1-mediated blockade of the CD40
pathway not only inhibits T cell cognate help for effector APC's,
but enhances the ability of CTLA4Ig to inhibit T cell activation
transcript expression within allografts. These data are consistent
with those from our studies in vitro which indicate that while MR1
alone has only a modest negative effect on cellular proliferation
in allogeneic mixed leukocyte reactions, it potentiates the
inhibitory effects of suboptimal concentrations of CTLA4Ig.
EXAMPLE 4
[0132] This example demonstrates prolongation of murine skin
allograft survival using C3H/HeJ mice which received full thickness
skin allografts from BALB/c mice.
[0133] Method
[0134] Segments of either full thickness tail or ear skin of
approximately 1 cm square were grafted on to the posterior-lateral
thoracic wall of recipient mice and secured in place with a
circumferential Bandaid.RTM.. The grafts were then followed by
daily visual inspection. Rejection was defined as the complete loss
of visible epidermal graft tissue. Treatment protocols for MR1 and
CTLA4Ig were as detailed for heart transplant recipients in FIG. 1.
CyA (Sandoz, East Hanover, N.J.) at a concentration of 50 mg/ml was
administered at a rate of 0.5 .mu.l/hr (.about.20 mg/kg/day) for 14
days via an osmotic pump (Alzet Model No. 2002, Alza, Palto Alto,
Calif.) which was implanted subcutaneously in the dorsal region of
the recipient at the time of-skin grafting and removed at 21 days
after transplant (Pereira, G. M., Miller, J. F. & Shevach, E.
M. J Immunol 144, 2109-2116 (1990)). After sacrifice, the skin
graft was excised, formalin fixed and embedded in paraffin. Tissue
sections (5 .mu.m) were stained with hematoxylin-eosin.
[0135] In FIG. 4A, C3H/HeJ recipients treated with either MR1 alone
(MST=13 days, n=5) or CTLA4Ig alone (MST=12 days, n=7) rejected
fully MHC-disparate BALB/c skin grafts at the same rate as an
untreated control group (MST=13 days, n=5). In contrast, when MR1
and CTLA4Ig were administered together in the perioperative period,
the allografts enjoyed markedly prolonged survival (MST>50,
n=15).
[0136] In FIG. 4B, mice treated with CyA alone (MST=30 days, n=4),
CyA plus CTLA4Ig (MST=30 days, n=5), or CyA and MR1 (MST=32 days,
n=4) all displayed similar modestly prolonged skin graft survival.
Surprisingly, the salutary effect of CTLA4Ig/MR1 on skin graft
survival was abolished by concomitant cyclosporine administration
(MST=34 days, n=4).
[0137] In FIG. 4C, C3H recipients of BALB/c skin grafts-were not
treated (MST 10d, n=3), or treated with MR1 (MST 13d, n=3),
YTS191.1 (MST 14d, n=6), YTS191.1 and MR1 (MST 16d, n=6), YTS191.1
and CTLA4Ig (MST 19d, n=5), or CTLA4Ig and MR1 (MST>50d, n=22).
Thus far, >53 mice have been treated with CTLA4Ig/MR1. Of these,
2 died on days 13 and 21. All others have remained healthy
throughout the experiments without signs of weight loss, infection,
or malignancy.
[0138] Healthy appearance of a BALB/c skin graft on an CTLA4Ig/MR1
treated C3H/HeJ recipient at 50 days after transplant (FIG. 4D),
contrasts sharply with a control allograft undergoing rejection
(FIG. 4E). On hematoxylin-eosin stained sections the accepted graft
at 100 days after transplant demonstrated well preserved epidermis,
hair follicles and adnexal structures (FIG. 4F), which is in
contrast to a BALB/c skin graft on an untreated C3H/HeJ recipient 8
days after transplant which shows an extensive lymphocytic
infiltrate (FIG. 4G).
[0139] Discussion
[0140] The effects of CTLA4Ig and. MR1, alone and in combination on
primary skin allograft survival in mice were tested (FIG. 4). For
comparison, recipients were also treated with CyA alone or CyA
combined with either CTLA4Ig or MR1. C3H/HeJ recipients treated
with either MR1 alone or CTLA4Ig alone rejected fully MHC-disparate
BALB/c skin grafts at the same rate as untreated controls (FIG.
4A).
[0141] Mice treated with CyA alone, CyA plus CTLA4Ig, or CyA and
MR1 all displayed modestly prolonged skin graft survival (FIG. 4B).
However, all of these allografts were ultimately rejected with no
apparent effects between either drug and CyA.
[0142] As a rigorous test of the ability of CD40/CD28 blockade to
interrupt alloimmune responses, we studied the effects of
perioperative treatment with CTLA4Ig and MR1, alone and in
combination on primary skin allograft survival in mice. For
comparison, recipients were also treated with CyA, an anti-CD4 mAb
YTS191.1, or with either of these agents combined with CTLA4I-g or
MR1 (FIGS. 4B and 4C). C3H/HeJ recipients treated with either MR1,
CTLA4Ig, or YTS191.1 alone rejected fully MHC-disparate BALB/c skin
grafts at essentially the same rate as untreated controls (FIGS. 4B
and 4C). Mice treated with YTS191.1 and MR1, YTS191.1 and CTLA4Ig,
CyA alone, CyA plus CTLA4Ig, or CyA and MR1 displayed modestly
prolonged skin graft survival (FIGS. 4B and 4C). However, all of
these allografts were ultimately rejected.
[0143] In contrast, on recipients treated with both MR1 and CTLA4Ig
in the perioperative period, the skin allografts demonstrated
markedly prolonged survival. Visual examination of these allografts
at 50 days after transplantation showed the grafts to be healthy in
appearance, well vascularized, supple, and bearing short white hair
(FIG. 4D). Histologically, the accepted grafts demonstrated well
preserved epidermis, hair follicles and adnexal structures (FIG.
4F). Surprisingly, the salutary effect of CTLA4Ig/MR1 on skin
graft'survival was abolished by concomitant cyclosporine
administration (FIG. 4B).
[0144] The remarkable potency of this effect was most clearly
evident in the primary skin allograft model. Neither CTLA4Ig or MR1
alone or with CyA significantly prolonged skin allograft survival.
Only the combination of CTLA4Ig and MR1 produced >50 day
survival of fully-MHC mismatched skin allografts. Similar
prolongation in this stringent test of inhibition of the alloimmune
response has previously only been observed using vigorous
cytoablative and/or hematopoietic chimerism-based strategies
(Mayumi, H. & Good, R. A. J Exp Med 169, 213-238 (1990);
Ildstad, S. T. & Sachs, D. H., Nature 307, 168-170 (1984);
Ilstad, S. T., et al. J Exp Med 162, 231-44 (1985); Cobbold, S. P.,
Martin, G., et al. Nature 323, 164-166 (1986); Qin, S., et al.
Science 259, 974-977 (1993)).
EXAMPLE 5
[0145] To explore the effect of blockade of the CD28 and CD40
pathways on T cell proliferation, we studied the primary allogeneic
mixed leukocyte reaction using T cells from both
Ie.sup.k-restricted pigeon cytochrome c-reactive (pcc-TCRTg) and
I.sup.d-alloreactive (2C) T cell receptor transgenic mice (REF HED
and LOH). CTLA4Ig, a fusion protein which binds to the ligands for
CD28 and its homologue CTLA4, effectively inhibited proliferation
of all three T cell populations (FIG. 5A).
[0146] In contrast, blockade of the CD40 pathway with the hamster
anti-gp39 mAb, MR1, modestly (.about.50%) inhibited the
proliferation of C3H/HeJ T cells responding to BALB/c dendritic
cells, dramatically inhibited (.gtoreq.85%) pcc-TCRTg T cells to
reacting to cytochrome c, but had negligible effects on the
proliferation of 2C T cells responding to L.sup.d-bearing BALB/c
dendritic cells (FIG. 5A).
[0147] Furthermore, simultaneous blockade with these agents
cooperatively inhibited T cell proliferation in allogeneic mixed
leukocyte reactions and pcc-TCRTg T cells, whereas MR-1 had no
effect or slightly augmented the proliferation of 2C T cells when
combined with CTLA4Ig (FIG. 5A). These results indicate that not
all T cells are dependent on CD40 signals for clonal expansion and
may explain the inability of CD40 blockade to completely inhibit
allograft rejection.
[0148] The effect of CD28 and CD40 blockade on T cell responses in
vivo was assessed in C3H/HeJ (H-2.sup.k, MMTV-7.sup.-) immunized
with DBA/2(H-2.sup.d, MMTV-7.sup.+) splenocytes in their foot pads.
Five days after immunization the draining popliteal lymph nodes in
control mice treated with human IgG demonstrated a 4-6 fold
increase in weight (FIG. 5B). This was accompanied by a 30 fold
expansion in the number of MMTV-7 superantigen-reactive
V.beta..sup.+CD.sup.+4 T cells and a >90 fold increase in the
number of V.beta..sup.+CD.sup.+T cell blasts within the popliteal
lymph node. Alone, CTLA4Ig or MR1 partially inhibited these
responses. In contrast, the combination of CTLA4Ig/MR1 essentially
ablated the increase in lymph node size and the expansion and
blastogenesis of V.beta..sup.+CD4.sup.+T cells (FIG. 5B)
[0149] These data show that simultaneous blockade of the CD28 and
CD40 pathways inhibit complex T-dependent immune responses in vitro
and in vivo.
EXAMPLE 6
[0150] This example shows, that simultaneous blockade of the CD28
and CD40 pathways produces marked inhibition of both the cellular
and antibody response to xenoantigen and long-term acceptance of
xenogeneic (rat to mouse) cardiac and skin grafts without the need
for a cytoablative conditioning therapy.
[0151] Methods
[0152] LYMPH NODE ASSAY. Male C3H (Jackson Laboratory, Bar Harbor,
Me.) mice were immunized with 2.times.10.sup.6 male Sprague-Dawley
(Harlan, Indianapolis, Ind.) irradiated (2000 RADS) rat splenocytes
in 50 .mu.l of sterile normal saline in the left foot pad and 50
.mu.l of sterile normal saline in the right foot pad and then
treated intraperitoneally (i.p.) with MR1 (500 .mu.g), CTLA4-Ig
(500 .mu.g) or both reagents (500 .mu.g each) on days 0, 2 and 4.
On day 5, the popliteal lymph nodes were removed, weighed and then
teased apart, washed, before resuspension in 600 .mu.l of RPMI 1640
with 10% FBS (Mediatech, Herndon, Va.). Each resuspended node was
then divided into four equal aliquots (150 .mu.l each). Three of
the aliquots were plated into a 96 well plate. .sup.3H-thymidine (1
.mu.Ci/well) (Amersham, Arlington Heights, Ill.) incorporation was
measured after 24 hours incubation at 37.degree. C. The results for
each individual animal therefore represent the mean of the 3 wells
per node. The fourth aliquot was incubated for 24 hours at
37.degree. C. and served as the source of supernatant for cytokine
analysis with ELISA. Each point on all of the graphs represents the
mean.+-.standard deviation of 5 mice per group. The experiment was
repeated with similar results.
[0153] CYTOKINE ELISA. Sandwich ELISA was performed using paired
antibodies {anti-IL-2, anti-IFN-gamma, anti-IL-2 biotin, anti-IL-4
biotin, anti-IFN-gamma biotin (Pharmingen, San Diego, Calif.), anti
IL4 (kind gift from Peter Jensen)} and streptavidin-HRP (Pierce,
Rockford, Ill.). Colorimetric detection was assayed using TMB
substrate (Pierce). Data were collected using a SpectraMax plate
reader and plotted as absorbance (370 nm).+-.sem. Standard curves
for each cytokine were generated using recombinant cytokine (rIL2,
Boehringer Mannheim, Indianapolis, Ind.; rIL4, R&D Systems,
Minneapolis, Minn.; and rIFN-gamma, Biosource International,
Camarillo, Calif.).
[0154] MICE. Male C3H/HeJ (H-2.sup.k) and DBA/2 (H-2.sup.d) mice
and Sprague-Dawley rats were purchased from The Jackson Laboratory
(Bar Harbor, Me.) and used at 8-12 weeks of age.
[0155] CARDIAC TRANSPLANTATION. C3H/HeJ or DBA mice were
transplanted with primarily vascularized Sprague-Dawley rat heart
xenografts and monitored for rejection as described in Larsen C.
P., Alexander D. Z., Hollenbaugh D., et al., Transplantation, 61
(1):4-9 (1996) and Corry R. J., Winn H. J., Russell P. S.,
Transplantation, 16(4):343-350 (1973). Recipients were treated with
500 mg CTLA4-Ig combined with 500 mg MR1 on days 0, 2, 4 and 6.
Control groups included recipients treated with CTLA4-Ig alone, MR1
alone or Human Ig. Paraffin embedded tissue sections (5 .mu.m) were
stained with Masson's Trichrome or hematoxylin-eosin. Histologic
specimens were reviewed by a cardiac transplant pathologist (KJW)
blinded to the treatment modality.
[0156] SKIN TRANSPLANTATION. Full thickness skin grafts (.about.1
cm.sup.2) from Sprague-Dawley rats were transplanted on the dorsal
thorax of C3H recipient mice and survival followed by daily visual
inspection. Rejection was defined as the complete loss of visible
epidermal graft tissue. Control groups included recipients treated
with: CTLA4-Ig alone; MR1 alone; and Human Ig. Two additional mice
in each experimental group were sacrificed at 20 days post
transplant for histologic analysis.
[0157] XENOANTIBODY ASSAY. Serum was collected via tail bleed from
anesthetized animals. Single cell suspensions from lymph nodes of a
Sprague-Dawley rat were used as target cells, and incubated with
recipient mouse serum for 20 minutes at 4.degree. C. The cells were
washed and IgG xenoantibodies were detected with donkey anti-mouse
IgG Biotin (Jackson ImmunoResearch, West Grove, Pa.) followed by
streptavidin-PE (Southern Biotech, Birmingham, Ala.). Cells were
analyzed on a Becton-Dickinson FACscan using Cellquest
Software.
[0158] Results
[0159] As an initial approach to determine the effects of CD28 and
CD40 blockade on responses to xenoantigenic challenge in vivo, we
used the popliteal lymph node assay as described in Larsen C. P.,
Elwood E. T., Alexander D. Z., et al., Nature, 381:434-438 (1996).
C3H/HeJ (H-2.sup.k) mice were injected with irradiated
Sprague-Dawley (SD) rat splenocytes. Five days after foot pad
immunization, the draining popliteal lymph nodes in control mice
treated with human IgG demonstrated a 5.2 fold increase in weight
relative to the contralateral node which was inoculated with
sterile saline (FIG. 6A). CTLA4-Ig partially inhibited nodal
expansion. Similarly MR1 partially inhibited this response. In
contrast, the combination of CTLA4-Ig/MR1 essentially ablated
xenoantigen-induced lymph node expansion (FIG. 6A).
[0160] We then compared the ex vivo proliferation of lymph node
cells from the different groups of xenoantigen-primed mice. While
either treatment alone only partially blocked proliferation (FIG.
6B), the combination of CTLA4-Ig/MR1 essentially ablated the
proliferative response (302.+-.235 CPM for the combination versus
143.+-.145 for a normal unstimulated node). Furthermore, the
combination therapy markedly suppressed Th1 cytokines (IL-2 and
IFNg) to the level of normal unstimulated cells (FIGS. 6C and 6D).
Levels of the Th2 cytokine IL4 were below the level of detection in
all samples.
[0161] It is important to note that this potent immune modulation
is not the result of cellular deletion. Flow cytometric analysis of
the peripheral blood of treated mice showed no depletion of CD4+ or
CD8+ T cells, B cells, or NK cells. These data are the result of an
individual analysis of 3 mice per group treated with either
CTLA4-Ig or MR1 alone or the combination of these agents on days 0,
2, 4, and 6 as described in the methods section. Peripheral blood
was collected by tail bleed on days 6 and 20.
[0162] The results of the lymph node assays suggested that
simultaneous blockade of the B7/CD28 and CD40/gp39 pathways would
inhibit xenograft rejection. To explore this hypothesis we studied
a vascularized cardiac xenograft model using Sprague-Dawley rats as
donors and C3H/HeJ mice as recipients. Treatment with either
CTLA4-Ig (MST=33 days) or MR1 (MST=51 days) alone prolonged
xenograft survival when compared to untreated controls (MST=6 days)
(FIG. 7A). However, CTLA4-Ig/MR1 in combination markedly prolonged
survival (MST=104.5 days).
[0163] When examined histologically at 20 days post-transplant,
xenografts treated with either CTLA4-Ig alone (FIG. 7C) or MR1
alone (FIG. 7D) showed heavy lymphocytic infiltration with evidence
of myocyte destruction and vasculopathy consistent with moderate to
severe cellular rejection. In sharp contrast, the xenografts from
CTLA4-Ig/MR1 treated recipients were essentially free from
lymphocytic infiltration, interstitial fibrosis, and coronary
arterial intimal lesions (FIG. 7F). CTLA4-Ig/MR1-treated cardiac
xenografts demonstrated excellent preservation of both myocytes and
vascular structures at day 122 (FIG. 7G). Untreated xenografts
showed widespread tissue destruction at day 6 (FIG. 7B). A normal
untransplanted Sprague-Dawley rat heart is shown in FIG. 7E.
[0164] As a more stringent test of the ability of CD40/CD28
blockade to inhibit xenogeneic immune responses, we studied the
effects of short term CD28 and/or CD40 blockade, on primary skin
xenograft survival in mice.
[0165] C3H recipients treated with either MR1 (MST=11.5 days n=4)
or CTLA4-Ig (MST=12 days n=4) alone rejected full thickness skin
grafts from Sprague-Dawley rats at the same rate as untreated
controls (untreated controls MST=11.5 days n=8) (FIG. 8A). In
contrast, the skin xenografts on recipients treated with
simultaneous. MR1 and CTLA4-Ig in the perioperative period,
demonstrated markedly prolonged survival (MST=53 days n=25) (FIG.
8A). A total of 25 mice received xenografts and treatment with
CTLA4-Ig/MR1. With the exception of one mouse that died on day 4,
all others have remained healthy throughout the experiments without
signs of weight loss, infection, or malignancy.
[0166] Chronic treatment (beginning after the standard 4 dose
regimen) with either the CTLA4-Ig/MR1 combination (500 .mu.g of
both agents weekly until day 100 or rejection, whichever came
first) or MR1 (500 .mu.g of MR1 weekly until day 100 or rejection)
resulted in no significant change in xenograft survival (FIG.
8B).
[0167] Xenograft survival after simultaneous MR1/CTLA4-Ig therapy
was 52% and 21% at 50 and 100 days respectively. The xenografts
surviving at 50 (FIG. 8E) and 100 (FIG. 8C) days after
transplantation were healthy in appearance and demonstrated well
preserved histologic architecture. In untreated controls without
the combination therapy, rejection was prompt and these xenografts
showed marked inflammatory infiltrates (FIGS. 8D and 8F).
[0168] Similar prolongation of Sprague-Dawley skin xenografts was
also observed in DBA [H-2.sup.d]) recipients (untreated controls
MST=14 days (n=5) versus CTLA4-Ig/MR1 MST=86 days n=5), suggesting
that the potent effect of the combination treatment is not limited
to a single recipient mouse strain.
[0169] The progressive loss of skin xenografts between 25 and 75
days post-transplant suggested that late graft failure might be due
to subtherapeutic concentrations of CTLA4-Ig and/or MR1. To address
this possibility, after the standard four-dose combination regimen
mice were treated weekly with either the CTLA4-Ig/MR1 combination
or MR1 alone for 100 days or until graft loss occurred. Neither of
these chronic therapy strategies appreciably improved skin
xenograft survival, suggesting that graft failure in CTLA4-Ig/MR1
treated mice results from factors other than inadequate drug titers
and that alternate pathways not completely inhibited by
CTLA4-Ig/MR1 may be important in sub-acute xenograft loss.
[0170] In addition to cell-mediated effector mechanisms, evoked
xenoantibody responses may play an important role in accelerated
vascular rejection of concordant xenografts, Aksentijevich I.,
Sachs D. H., Sykes M., Transplantation, 53(5):1108-14 (1992). To
test the effect of simultaneous blockade on evoked xenoantibody
responses, serum samples from C3H/HeJ mice were analyzed for
anti-rat antibodies at 55 days after receiving a Sprague-Dawley rat
skin graft (FIG. 9A) and at 20 days after receiving a
Sprague-Dawley rat heart graft (FIG. 9B). Treatment with either
CTLA4-Ig or MR1 alone decreased the IgG antibody response, whereas
the simultaneous combination CD28/CD40 blockade essentially
eliminated-the evoked antibody response to rat xenoantigens. Thus,
inhibition of both T cell activation and antibody production could
be functionally important in xenograft survival after simultaneous
blockade of the B7/CD28 and CD40/gp39 pathways.
[0171] Discussion
[0172] Combined blockade of the CD28 and CD40 pathways markedly
inhibits the immune response to xenoantigen. The potency of this
combination therapy was particularly demonstrated in the primary
skin xenograft model. Neither agent alone, prolonged skin xenograft
survival, while, in contrast, the simultaneous combination of
CTLA4-Ig and MR1 cooperatively inhibited xenograft rejection. The
uniqueness of the findings resides in the stringency of the skin
graft model, as CTLA4-Ig alone has previously been shown to prolong
the survival of xenogeneic pancreatic islets in a mouse model,
Lenschow D., Zeng Y., Thistlethwaite J., et al., Science,
257:789-792 (1992). Long-term survival of xenogenic skin grafts
have previously only been observed using vigorous cytoablative
and/or hematopoeitic chimerism-based strategies, Ildstad S. T.,
Sachs D. H., Nature, 307:168-170 (1984), Zhao Y., Swenson K.,
Sergio J., Arn J. S., Sachs D. H., Sykes M., Nat. Med.,
2(11):1211-1216 (1996), Mayumi H., Good R. A., J. Exp. Med.,
169(1):213-238 (1990), Cobbold S. P., Martin G., Zin S., Waldman
H., Nature, 323:164-166 (1986), Qin S., Cobbold S., Benjamin R.,
Waldmann H., J. Exp. Med., 169:779-794 (1989)).
[0173] The observation that simultaneous CD28/CD40 blockade can
dramatically prolong xenograft survival suggests that both the
antibody and cell mediated mechanisms for destruction of the
xenograft may be effectively inhibited by this strategy. While the
etiology of acute vacular xenograft rejection remains to be
completely defined, there is evidence that it is caused, at least
in part, by the development of xenoreactive antibodies (Cotterell
A. H., Collins B. H., Parker W., Harland R. C., Platt J. L,
Transplantation, 60(8):861-868 (1995)). The rapid destruction of
untreated control cardiac xenografts in our model, in the absence
of a cellular infiltrate, suggests a role for antibody mediated
rejection. This observation and those of others (Aksentijevich I.,
Sachs D. H., Sykes M., Transplantation, 53(5):1108-14 (1992)),
combined with the documented dramatic inhibition of the evoked
xenoantibody response after blockade of the CD28 and CD40 pathways
(FIGS. 9A and 9B), supports the hypothesis that xenoresponses may
be sufficiently controlled by inhibition of these pathways to
permit the development of non-cytoablative strategies for
xenotransplantation in discordant species combinations.
[0174] While combined blockade of the CD28 and CD40 pathways
markedly inhibited the xenograft rejection response, this blockade
did not achieve uniform indefinite cardiac or skin graft survival
in our experimental system. The observation that prolonged
treatment did not improve graft survival was surprising. This
suggests that inadequate blockade of these pathways is not the
cause for "late" graft failure and raises the possibility that
alternate pathways such as NK cells or other cells, which do not
require CD28/CD40 co-stimulation, may promote late xenograft
rejection. Suggestive evidence for the contribution of NK cells to
xenograft rejection support this possibility, Zhao Y., Swenson K.,
Sergio J., Arn J. S., Sachs D. H., Sykes M., Nat. Med.,
2(11):1211-1216 (1996), Malyguine A. M., Saadi S., Platt J. L.,
Dawson J. R., Transplantation, 61(1):161-164 (1996). In addition,
we have shown that the inhibition of the rejection of concordant
heart and skin xenografts by the simultaneous blockade of the CD40
and CD28 pathways is associated with the prevention of the "late"
development of the evoked xenoreactive antibody responses (FIGS. 9A
and 9B), we have not excluded the possibility that the development
of an antibody response may be associated with delayed graft
loss.
[0175] The ability of combined CTLA4-Ig/MR1 treatment to block the
development of transplant vasculopathy in cardiac xenografts and
prolong skin xenografts is of significant clinical relevance. The
refinement of techniques to inhibit the effect of natural preformed
xenoreactive antibodies combined with further study of CD28 and
CD40 pathway blockade promises the possibility of effective new
strategies to facilitate clinical xenograft transplantation.
* * * * *