U.S. patent application number 10/714055 was filed with the patent office on 2004-09-23 for methods of treating autoimmune disease via ctla-4ig.
Invention is credited to June, Carl H., Thompson, Craig B..
Application Number | 20040185046 10/714055 |
Document ID | / |
Family ID | 30449654 |
Filed Date | 2004-09-23 |
United States Patent
Application |
20040185046 |
Kind Code |
A1 |
Thompson, Craig B. ; et
al. |
September 23, 2004 |
Methods of treating autoimmune disease via CTLA-4Ig
Abstract
The method of immunotherapy of the present invention involves
the regulation of the T cell immune response through the activation
or suppression/inactivation of the CD28 pathway. Induction of
activated T cell lymphokine production occurs upon stimulatory
binding of the CD28 surface receptor molecule, even in the presence
of conventional immunosuppressants. Inhibition of CD28 receptor
binding to an appropriate stimulatory ligand or inactivation of the
CD28 signal transduction pathway through other means down-regulates
CD28-pathway related T cell lymphokine production and its resulting
effects.
Inventors: |
Thompson, Craig B.;
(Chicago, IL) ; June, Carl H.; (Rockville,
MD) |
Correspondence
Address: |
LAHIVE & COCKFIELD, LLP.
28 STATE STREET
BOSTON
MA
02109
US
|
Family ID: |
30449654 |
Appl. No.: |
10/714055 |
Filed: |
November 14, 2003 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10714055 |
Nov 14, 2003 |
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08385194 |
Feb 7, 1995 |
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6685941 |
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08385194 |
Feb 7, 1995 |
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08076071 |
Jun 10, 1993 |
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08385194 |
Feb 7, 1995 |
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PCT/US93/03155 |
Apr 6, 1993 |
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PCT/US93/03155 |
Apr 6, 1993 |
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07864805 |
Apr 7, 1992 |
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PCT/US93/03155 |
Apr 6, 1993 |
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07864807 |
Apr 7, 1992 |
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PCT/US93/03155 |
Apr 6, 1993 |
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07864866 |
Apr 7, 1992 |
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07864866 |
Apr 7, 1992 |
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07275433 |
Nov 23, 1988 |
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Current U.S.
Class: |
424/144.1 ;
424/185.1 |
Current CPC
Class: |
Y10S 514/885 20130101;
A61K 39/02 20130101; C12N 2501/51 20130101; C07K 2319/00 20130101;
C07K 14/70521 20130101; C07K 16/2815 20130101; C07K 16/289
20130101; C07K 16/2818 20130101; C07K 16/2812 20130101; C07K
2319/30 20130101; C07K 16/2866 20130101; C07K 2317/74 20130101;
C07K 16/2896 20130101; A61K 2039/5158 20130101; C07K 16/2806
20130101; C07K 2317/54 20130101; C07K 16/2833 20130101; C12N
2501/515 20130101; A61K 2039/505 20130101; C12N 5/0636 20130101;
C07K 16/00 20130101; C07K 16/2809 20130101; A61K 38/13 20130101;
C07K 2317/24 20130101; A61K 38/13 20130101; A61K 2300/00
20130101 |
Class at
Publication: |
424/144.1 ;
424/185.1 |
International
Class: |
A61K 039/395; A61K
039/00 |
Goverment Interests
[0002] Work on this invention was supported in part by Naval
Medical Research and Development Command, Research Task No.
M0095.0003-1007. The Government has certain rights in the
invention.
Claims
What is claimed is:
1. A method of inhibiting CD28 pathway activation associated with
an increase in cellular production of a T.sub.HCD28 lymphokine in a
T cell population, wherein activation occurs by the binding of a
stimulatory CD28 ligand to a CD28 receptor stimulatory binding
site, the method comprising the steps of: a) selecting an
inhibitory ligand capable of binding to the stimulatory CD28
ligand; b) providing the inhibitory ligand in a biologically
compatible form; and c) administering the inhibitory ligand to the
population in an amount sufficient to bind and inhibit the
stimulatory ligand from binding the CD28 receptor stimulatory
binding site.
2. The method of claim 1, wherein the stimulatory ligand comprises
a natural CD28 ligand.
3. The method of claim 1, wherein the inhibitory ligand comprises
an antibody or fragment thereof to the stimulatory ligand.
4. The method of claim 1, wherein the inhibitory ligand comprises a
soluble form of CTLA-4.
5. The method of claim 4, wherein the ligand comprises
CTLA-4lg.
6. The method of claim 1, wherein the inhibitory ligand is of
synthetic origin.
7. The method of claim 1, wherein the inhibitory ligand comprises a
recombinant molecule.
8. The method of claim 1, further comprising the step of: d)
administering a second inhibitory ligand capable of binding but not
stimulating the CD28 receptor binding site.
9. A method of suppressing the production of a T.sub.HCD28
lymphokine by a population of T cells, the method comprising the
steps of: a) administering an inhibitory ligand which binds a
stimulatory ligand for CD28; b) providing the ligand in
biologically compatible form; and c) administering the provided
ligand in an amount sufficient to suppress production of the
lymphokine in the population.
10. The method of claim 9, wherein the inhibitory ligand comprises
a soluble form of CTLA-4.
11. The method of claim 10, wherein the inhibitory ligand comprises
CTLA-4lg.
12. The method of claim 9, wherein the T cell population is in a
patient in an autoimmune state.
13. A method of suppressing T.sub.HCD28 lymphokine production in a
patient having a population of T cells, the method comprising the
steps of: a) providing an inhibitory ligand which binds a natural
stimulatory ligand for CD28; and b) administering the inhibitory
ligand in a therapeutically effective amount to the population of T
cells.
14. The method of claim 13, wherein the administration of the
ligand to the population of T cells is in vivo.
15. The method of claim 13, wherein the administration of the
ligand to the population of T cells is in vitro, and further
comprising the step of: d) introducing the population of T cells
into the patient after administration.
16. The method of claim 15, wherein the T cell population is
removed from the patient prior to ligand administration.
17. The method of claim 13, wherein the inhibitory ligand comprises
a soluble form of CTLA-4.
18. The method of claim 17, wherein the inhibitory ligand comprises
CTLA-4lg.
19. A method of treating an autoimmune disease in a patient
comprising the steps of: a) selecting an inhibitory ligand which
binds a natural stimulatory ligand to CD28; and b) administering
atherapeutically effective amount of the ligand to the patient.
20. The method of claim 19, wherein the stimulatory ligand is B7
and the inhibitory ligand comprises a soluble form of CTLA-4.
21. The method of claim 19, wherein the inhibitory ligand comprises
CTLA-4lg.
22. The method of claim 20, wherein the administration is in
vivo.
23. The method of claim 20, wherein the administration is in vitro
to a population of cells removed from the patient, and further
comprising the step of: c) reintroducing the cells to the patient
after administration.
24. The method of claim 20, wherein the autoimune disease is
multiple sclerosis.
Description
RELATED APPLICATIONS
[0001] This is a continuation-in-part of International Application
Serial No. PCT/US93/03155, entitled "CD28 Pathway
Immunoregulation," filed Apr. 6, 1993 by Thompson et al., which is
a continuation of U.S. application Ser. No. 07/864,805, entitled
"CD28 Pathway Immunoregulation," U.S. application Ser. No.
07/864,807, entitled "Immunotherapy Involving Stimulation of
T.sub.HCD28 Lymphokine Production," and U.S. application Ser.
07/864,866, entitled "Enhancement of CD28-Related Immune Response,"
all filed Apr. 7, 1992 by Thompson, et al., which are
continuations-in-part of U.S. Ser. No. 07/275,433, entitled
"Immunotherapy Involving CD28 Stimulation," filed Nov. 23, 1988 by
Thompson et al., now abandoned, and is also a continuation-in-part
of International Application Serial No. PCT/US89/05304 (Publication
No. WO 90/05541), entitled "Immunotherapy Involving CD28
Stimulation," filed Nov. 22, 1989 by Thompson et al. and U.S.
patent Ser. No. 07/902,467 entitled "Immunotherapy Involving CD28
Stimulation," filed Jun. 19,1992 by Thompson et al., all herein
incorporated by reference.
BIOLOGICAL DEPOSIT
[0003] Murine hybridoma cell line 9.3 has been deposited with the
American Type Culture Collection in Rockville, Md., in compliance
with the provisions of the Budapest Treaty, and has been assigned
ATCC Designation No. HB10271.
FIELD OF THE INVENTION
[0004] The present invention generally relates to immunotherapy.
More particularly, the present invention relates to immunotherapy
involving regulation of the CD28 T cell surface molecule.
BACKGROUND OF THE INVENTION
[0005] Thymus derived lymphocytes, referred to as T cells, are
important regulators of in vivo immune responses. T cells are
involved in cytotoxicity and delayed type hypersensitivity (DTH),
and provide helper functions for B lymphocyte antibody production.
In addition, T cells produce a variety of lymphokines which
function as immunomodulatory molecules, such as for example,
interleukin-2 (IL-2), which can facilitate the cell cycle
progression of T cells; tumor necrosis factor-.alpha. (TNF-.alpha.)
and lymphotoxin (LT), cytokines shown to be involved in the lysis
of tumor cells; interferon-.gamma. (IFN-.gamma.), which displays a
wide variety of anti-viral and anti-tumor effects; and IL-3 and
granulocyte-macrophage colony stimulating factor (GM-CSF), which
function as multilineage hematopoietic factors.
[0006] Current immunotherapeutic treatments for diseases such as
cancer, acquired immunodeficiency syndrome (AIDS) and attending
infections, involve the systemic administration of lymphokines,
such as IL-2 and IFN-.gamma., in an attempt to enhance the immune
response. However, such treatment results in non-specific
augmentation of the T cell-mediated immune response, since the
lymphokines administered are not specifically directed against
activated T cells proximate to the site of infection or the tumor.
In addition, systemic infusions of these molecules in pharmacologic
doses leads to significant toxicity. Present therapies for
immunodeficient or immunodepressed patients also involve
non-specific augmentation of the immune system using concentrated
.gamma.-globulin preparations. The stimulation of the in vivo
secretion of immunomodulatory factors has not, until now, been
considered a feasible alternative due to the failure to appreciate
the effects and/or mechanism and attending benefits of such
therapy.
[0007] It would thus be desirable to provide a method of
immunotherapy which enhances the T cell-mediated immune response
and which is directed specifically toward T cells activated by an
antigen produced by the targeted cell. It would further be
desirable to provide a method of immunotherapy which could take
advantage of the patient's natural immunospecificity. It would also
be desirable to provide a method of immunotherapy which can be used
in immunodepressed patients. It would additionally be desirable to
provide a method of immunotherapy which does not primarily rely on
the administration of immunomodulatory molecules in amounts having
significant toxic effects.
[0008] It would also be desirable to provide a method of
immunotherapy which, if so desired, could be administered directly
without removal and reintroduction of T cell populations. It would
further be desirable to provide a method of immunotherapy which
could be used not only to enhance, but to suppress T cell-mediated
immunoresponses where such immunosuppression would be advantageous,
for example, in transplant patients, in patients exhibiting shock
syndrome and in certain forms of autoimmune disease.
SUMMARY OF THE INVENTION
[0009] The present invention comprises a method of immunotherapy in
which the T cell-mediated immune response is regulated by the CD28
pathway. Binding of the CD28 receptor with anti-CD28 antibodies or
other stimulatory binding equivalents induces activated T
cell-mediated lymphokine production. Immunosuppression or
down-regulation is achieved by preventing CD28 receptor binding to
stimulatory ligands or inactivation of the CD28 signal transduction
pathway.
[0010] The method of immunotherapy of the present invention takes
advantage of the surprising and heretofore unappreciated effects of
stimulation of the CD28 surface receptor molecule of activated T
cells. By activated T cells is meant cells in which the immune
response has been initiated or "activated," generally but not
necessarily by the interaction of the T cell receptor (TCR)/CD3 T
cell surface complex with a foreign antigen or its equivalent.
While such activation results in T cell proliferation, it results
in only limited induction of T cell effector functions such as
lymphokine production.
[0011] Stimulation of the CD28 cell surface molecule with anti-CD28
antibody results in a marked increase of T cell lymphokine
production. Surprisingly, even when the stimulation of the TCR/CD3
complex is maximized, upon costimulation with anti-CD28, there is a
substantial increase in the levels of IL-2 lymphokine, although
there is no significant increase in T cell proliferation over that
induced by anti-TCR/CD3 alone. Even more surprisingly, not only are
IL-2 levels significantly increased, but the levels of an entire
set of lymphokines, hereinafter referred to as T.sub.HCD28
lymphokines, previously not associated with CD28 stimulation are
increased. Remarkably both the T cell proliferation and increased
lymphokine production attributable to CD28 stimulation also exhibit
resistance to immunosuppression by cyclosporine and
glucocorticoids.
[0012] The method of immunotherapy of the present invention thus
provides a method by which the T cell-mediated immune response can
be regulated by stimulating the CD28 T cell surface molecule to aid
the body in ridding itself of infection or cancer. The method of
the present invention can also be used not only to increase T cell
proliferation, if so desired, but to augment or boost the immune
response by increasing the levels and production of an entire set
of T cell lymphokines now known to be regulated by CD28
stimulation.
[0013] Moreover, because the effectiveness of CD28 stimulation in
enhancing the T cell immune response appears to require T cell
activation or some form of stimulation of the TCR/CD3 complex, the
method of immunotherapy of the present invention can be used to
selectively stimulate T cells preactivated by disease or treatment
to protect the body against a particular infection or cancer,
thereby avoiding the non-specific toxicities of the methods
presently used to augment immune function. In addition, the method
of immunotherapy of the present invention enhances T cell-mediated
immune functions even under immunosuppressed conditions, thus being
of particular benefit to individuals suffering from
immunodeficiencies such as AIDS.
[0014] It will also be appreciated that although the following
discussion of the principles of the present invention exemplifies
the present invention in terms of human therapy, the methods
described herein are similarly useful in veterinary
applications.
[0015] A better understanding of the present invention and its
advantages will be had from a reading of the detailed description
of the preferred embodiments taken in conjunction with the drawings
and specific examples set forth below.
BRIEF DESCRIPTION OF THE DRAWINGS
[0016] FIG. 1 is a bar graph illustrating the absence of
augmentation of the uptake of thymidine by CD28 stimulated T
cells.
[0017] FIG. 2 is a bar graph illustrating the increase in uridine
incorporation by CD28 stimulation of anti-CD3 stimulated T
cells.
[0018] FIG. 3 is a graph illustrating the elevated cyclosporine
resistance of T cell proliferation induced by CD28 stimulation.
[0019] FIG. 4 is a Northern blot analysis of the effects of
cyclosporine on PMA-or anti-CD3 activated T cell lymphokine
expression induced by anti-CD28.
[0020] FIG. 5 is a graph illustrating in vivo activation of T cells
in monkeys by CD28 stimulation.
[0021] FIGS. 6A and 6B are graphs representing changes in
lymphocyte levels after infusion of anti-CD28 Mab.
[0022] FIGS. 7A and 7B are graphs representing in vitro production
of TNF and IL-6 by PBLs under various conditions.
[0023] FIGS. 8A and 8B are graphs representing serum concentration
of IL-1 .beta. after single and multiple doses of anti-CD28
Mab.
[0024] FIGS. 9A and 9B are graphs representing the serum
concentration of IL-6 after single and multiple doses of anti-CD28
Mab.
[0025] FIGS. 10A and 10B are graphs representing IL-6 production of
in vitro stimulated PBLs isolated from monkeys treated with a
single bolus or multiple injections of anti-CD28 Mab.
[0026] FIG. 11 is a graph representing the inhibitory effect of
CTLA-4lg on .sup.3H-thymidine incorporation in a one-way mixed
lymphocyte culture.
[0027] FIGS. 12A and 12B are photographs of cardiac allografts to
illustrate histopathology.
[0028] FIG. 13 is a Kaplan-Meier life analysis of cardiac allograft
survival after CTLA-4lg treatment.
[0029] FIG. 14 is a bar graph illustrating CTLA-4lg and
cyclosporine as synergistic immunosuppressants.
[0030] FIG. 15 is a bar graph illustrating the effect of herbimycin
A on CD28-stimulated IL-2 production.
[0031] FIG. 16 is a bar graph illustrating activation by SEB and
anti-CD28 on purified resting T cells in the presence and absence
of a blocking Mab to HLA-DR.
[0032] FIG. 17 is a bar graph illustrating activation by SEB alone
or SEB and blocking Mab to HLA-DR in peripheral blood mononuclear
cells.
[0033] FIG. 18 is a graph showing in vitro long term growth of
CD4.sup.+ peripheral blood T cells propagated with anti-CD3 and
anti-CD28.
[0034] FIG. 19 is a Northern blot analysis of the enhancement of
MRNA for IL-2 and TNF-.alpha. after costimulation with anti-CD3 and
anti-CD28.
[0035] FIG. 20 is a Northern blot analysis of the ability of
mitogens to induce CTLA-4 mRNA expression.
[0036] FIG. 21 is a Northern blot analysis of the induction of
CTLA-4 mRNA expression by costimulation with anti-CD3 mAb and
soluble anti-CD28 mAb.
[0037] FIG. 22 is a graph illustrating the effects on disease
progression of CTLA-4lg treatment of syngeneic, MBP-sensitized
cells used to adoptively transfer the murine autoimmune disease,
Experimental Autoimmune Encephalomyelitis (EAE).
[0038] FIG. 23 is a graph illustrating the effect on disease
progression of CTLA-4lg or control IgG treatment of donor mice
and/or isolated cells used to adoptively transfer EAE.
[0039] FIG. 24 is a graph depicting the effect on disease severity
of direct administration of CTLA-4lg or control human IgG to
PLSJLFI/J mice with adoptively transferred EAE.
[0040] FIG. 25 is a graph illustrating the effect on disease
progression of direct administration of CTLA-4lg or control human
IgG to SJL/J mice with adoptively transferred EAE.
[0041] FIG. 26 is a graph depicting the effect of direct
administration of CTLA-4lg or IgG on disease severity in SJL/J mice
with adoptively transferred EAE.
[0042] FIG. 27 is a graph illustrating the effect on disease
severity of direct administration of CTLA-4lg or control IgG to
PLSJLFI/J mice directly immunized with MBP and treated with PT.
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS
[0043] In one preferred embodiment of the immunotherapeutic method
of the present invention, the CD28 molecule is stimulated to
enhance the T cell-mediated immune response of antigen or otherwise
activated T cells. CD28 is a 44 kilodalton protein expressed on the
surface of about 80% mature T cells which exhibits substantial
homology to immunoglobulin genes. See Poggi, A. et al., Eur. J.
Immunol., 17:1065-1068 (1987) and Aruffo, A. et al., PNAS (USA),
84:8573-8577 (1987), both herein incorporated by reference. Binding
of the CD28 molecule's extracellular domain with anti-CD28
antibodies in accordance with the method of the present invention
results in an increase in T cell proliferation and elevated
lymphokine levels.
[0044] In Specific Examples III-IV and VI-VIII, T cell activation
was accomplished by stimulating the T cell TCR/CD3 complex (which
mediates the specificity of the T cell immune response) with
immobilized anti-CD3 monoclonal antibodies, such as mAb G19-4, or
by chemically stimulating with PMA and ionomycin. It should also be
appreciated, however, that activation of the T cell can instead be
accomplished by routes that do not directly involve CD3
stimulation, such as the stimulation of the CD2 surface
protein.
[0045] In practice, however, an activated T cell population will be
provided by the patient's own immune system, which, barring total
immunosuppression, will have T cells activated in response to any
foreign or substantially elevated level of antigen present due to
disease, infection, inoculation or autoimmunity. The term "foreign
antigen" is used broadly herein, meaning an antigen which is either
not normally produced by the organism, or, as in carcinomas, an
antigen which is not normally produced by the cell which is
producing it. The term is also meant to include an antigen which
should normally be seen as "self," but, as occurs in autoimmune
disease states, provokes an immune response as would a foreign
antigen. By "substantially elevated" level of antigen is meant an
antigen level exceeding normal ranges and having potentially
deleterious effects to the organism due to such elevation.
[0046] In accordance with the method of the present invention,
stimulation of the CD28 molecule itself is achieved by
administration of a ligand, such as a monoclonal antibody or a
portion thereof, (e.g. F(ab').sub.2), having a binding specificity
for and stimulatory effect on CD28. Suitable antibodies include mAb
9.3, an IgG2a antibody on deposit with the ATCC which has been
widely distributed and is available (for non-commercial purposes)
upon request from Dr. Jeffrey A. Ledbetter of Oncogen Corporation,
Seattle, Wash., or Mab Kolt-2. Both these monoclonal antibodies
have been shown to have binding specificity for the extracellular
domain of CD28 as described in "Leukocyte Typing II," Ch. 12, pgs.
147-156, ed. Reinherz, E. L. et al. (1986). Monoclonal antibody 9.3
F(ab').sub.2 has also been shown to be a satisfactory ligand
capable of stimulating the CD28 receptor. It should also be
understood that the method of the present invention contemplates
the use of chimaeric antibodies as well as non-immunoglobulin,
natural and recombinant ligands which bind the CD28 surface
molecule. More recently, the natural ligand for CD28, B7/BB1, has
also been identified and can be used in accordance with the
principles of the present invention. See Linsley, P. S. et al., J.
Exp. Med. 174:561 (1991). In addition, binding homologs of a
natural ligand, whether natural or synthesized by biochemical,
immunological, recombinant or other techniques, can also be used in
accordance with the principles of the present invention. It will be
appreciated that the ligands referred to herein can be utilized in
their soluble or cell-bound forms, depending on their application.
Monoclonal antibody 9.3 and B7 are currently preferred stimulatory
ligands.
[0047] The extracellular domain of CD28, which was sequenced by
Aruffo, A. et al., PNAS (USA), 84:8573-8577 (1987), generally
comprises the following amino acid sequence:
1 MetLeuArgLeuLeuLeuAlaLeuAsnLeuPheProSerIleGln
ValThrGlyAsnLysIleLeuValLysGlnSerProMetLeuVal
AlaTyrAspAsnAlaValAsnLeuSerCysLysTyrSerTyrAsn
LeuPheSerArgGluPheArgAlaSerLeuHisLysGlyLeuAsp
SerAlaValGluValCysValValTyrGlyAsnTyrSerGlnGln
LeuGlnValTyrSerLysThrGlyPheAsnCysAspGlyLysLeu
GlyAsnGluSerValThrPheTyrLeuGlnAsnLeuTyrValAsn
GlnThrAspIleTyrPheCysLysIleGluValMetTyrProPro
ProTyrLeuAspAsnGluLysSerAsnGlyThrIleIleHisVal
LysGlyLysHisLeuCysProSerProLeuPheProGlyProSer LysPro
[0048] By the term "extracellular domain" as used hereinafter in
the specification and claims, is meant the amino acid sequence set
forth above, any substantial portion thereof, or any sequence
having substantial homology thereto.
[0049] As shown by the data of Specific Examples III-V, substantial
augmentation of the T cell-mediated immunoresponse by CD28
stimulation appears specific for activated T cells. Such
specificity is of particular clinical importance and is one of the
significant advantages of the method of immunotherapy of the
present invention. Administration of anti-CD28 antibodies or other
CD28 ligands will specifically augment the response of T cells
which are already activated and engaged in the immune response or
those in the process of activation. It should, however, also be
appreciated that CD28 stimulation may be effective even where the T
cells are activated after the binding of the CD28-specific ligand
of the present invention to CD28 receptor. Thus, the T cells at or
near the tumor site or site of infection, which are being activated
by the antigens produced or present at those sites, will be
selectively "boosted" by the CD28 stimulation.
[0050] Boosting of the immune response can also be beneficial to
healthy individuals, for example, in augmenting their response to
antigens presented in vaccines (see Specific Example IX). CD28
stimulation coupled with antigen administration in accordance with
the present invention can result in more effective immunization,
not only with conventional vaccines, but in situations where an
adequate immune response is difficult to elicit, e.g. with human
retroviruses such as HIV and some herpes viruses. Examples where
CD28 stimulation of the present invention can be used to augment
the immune response include, but are not limited to viral vaccines
against measles, influenza, polio, herpes viruses (i.e. HCMV,
Epstein Barr Virus, Herpes Simplex Type I and 11); bacterial
vaccines against whooping cough (Bordatella pertussis), tetanus
(Clostridium tetanus), pneumonia (Streptococcus pneumoniae),
meningitis and gonorrhea (Neisseria) and against enteropathic
bacteria such as Salmonella, E. coli and Shigella. The principles
of the present invention are also applicable in inoculations
against parasitic infection, including those caused by protozoal
parasites, e.g. malaria, trypanosomiasis, leishmaniasis, amebiasis,
toxoplasmosis, pneumocystis carinni and babesiosis, by cestodes
(e.g. tapeworm) and by other parasitic organisms. It will also be
appreciated that immunization for a humoral response through
injection of cDNA for intracellular antigenic production, as
described in Nature 356:152 (1992), costimulated with anti-CD28 is
also contemplated as within the scope of the present invention.
[0051] Indeed, recently CD28 engagement and signal transduction
have been used to identify IL-13 (Minty, A. et al., Nature 362:
(6417):248-50 (1993)), a lymphokine involved in the inflammatory
and immune response. Punnonev, J. et al., PNAS (USA) 90(8): 3730-4
(1993). When TCR/CD3 stimulation is maximized, although T cell
proliferation is not markedly increased, the levels of certain
lymphokines are substantially increased by CD28 activation,
indicating an increase in cellular production of these lymphokines.
Thus, in patients undergoing natural maximal TCR/CD3 stimulation or
its equivalent T cell activation in vivo due to disease or
infection, the administration of anti-CD28 antibody or other CD28
ligand to stimulate CD28 in accordance with the method of the
present invention will result in substantially elevated lymphokine
production.
[0052] The increase in lymphokine production achieved by
administration of a CD28 stimulator in accordance with the method
of the present invention, as particularly shown in Specific Example
III, surprisingly results in the increased production of an entire
set of lymphokines, indicating that these lymphokines are under
some form of CD28 regulation. Part of this set of lymphokines,
which includes IL-2, TNF-.alpha., LT, IFN-.gamma., and IL-3 as
later determined, is somewhat analogous to the T.sub.H1 cell
lymphokines present in the mouse which were described by Mosmann,
T. R. et al., Immunol. Today, 8:223-227 (1987). Although it was
originally believed that human IL-4 production was not increased by
CD28 stimulation, more recent assays as set forth in Specific
Example III have now also shown an increase in the production of
other lymphokines, including IL-4 and IL-5 and the increased
production of IL-6 and IL-1 was also confirmed in Specific Example
IX. It will be appreciated, however, that the term "T.sub.H1
lymphokines" was originally used for ease of reference and was
expressly not limited to the lymphokines listed above, but was
meant to include all lymphokines whose production is affected or
regulated by the binding or stimulation of the CD28 T cell surface
molecule. Thus the group of lymphokines affected by CD28 will
hereinafter be referred to as T.sub.HCD28 lymphokines, it will
again be appreciated that the term T.sub.HCD28 lymphokine is not
intended to be limiting to the specific lymphokines listed herein.
Furthermore, it will be appreciated that the principles of the
present invention can be used in veterinary applications to
increase the T cell-mediated immune response and lymphokine
production in animals. The term T.sub.HCD28 lymphokines, as used
herein, is thus also meant to include analogous animal lymphokines.
Thus, by administration of anti-CD28 antibodies or other CD28
ligands in accordance with the method of the present invention, the
production and levels of an entire set of lymphokines can be
significantly increased.
[0053] The method of immunotherapy of the present invention can
also be used to facilitate the T cell-mediated immune response in
immunodepressed patients, such as those suffering from AIDS. As
shown in Specific Examples VI-VII, T cell proliferation and the
increased levels or production of CD28-regulated lymphokines
continue to function even in the presence of immunosuppression such
as that caused by cyclosporine or dexamethasone. Thus
administration of CD28 stimulators such as mAb 9.3 or other CD28
ligands can be used to treat immunodepressed patients to increase
their in vivo lymphokine levels.
[0054] In addition, a variety of syndromes including septic shock
and tumor-induced cachexia may involve activation of the CD28
pathway and augmented production of potentially toxic levels of
lymphokines. The immune response can also be deleterious in other
situations such as in organ transplant recipients or in autoimmune
disease. Thus down-regulation or inactivation of the CD28 pathway,
as discussed more fully below and in Specific Examples X and XI,
can also provide immunotherapy for those and other clinical
conditions.
[0055] It should be appreciated that administration of an anti-CD28
antibody has not heretofore been seriously contemplated as a
potential immunotherapeutic method for the substantial increase of
lymphokine levels at the sites of activated T cells. For example,
the addition of mAb 9.3 has been thought only to somewhat augment T
cell proliferation, not to induce substantial increases in
T.sub.HCD28 lymphokine production.
[0056] Although it is not the intent herein to be bound by any
particular mechanism by which CD28 binding regulates the T
cell-mediated immune response, a model for the mechanism of
stimulation has been postulated and supported with experimental
data, some of which is shown in Specific Example VIII. It has
previously been shown that a number of lymphokine genes undergo
more rapid degradation in the cytoplasm than mRNAs from
constitutively expressed housekeeping genes, leading to the
hypothesis that the instability of these inducible mRNAs has been
selected to allow for rapid regulation of gene expression. It is
believed that the mechanism of CD28 regulation herein described and
claimed is related to the stabilization of rapidly degradable mRNAs
for the set of T.sub.HCD28 lymphokines set forth above. To date, it
appears no other mechanism in any eukaryotic cell system has been
described to demonstrate that a cell surface activation pathway can
alter gene expression by inducing specific alteration in mRNA
degradation. (A more in-depth analysis of possible models of CD28
activation is presented later herein.)
[0057] As shown in Specific Example IV, costimulation of CD28 and
TCR/CD3 caused an increase in mRNA of the T.sub.HCD28 lymphokines
which was not the result of a generalized increase in a steady
state mRNA expression of all T cell activation-associated genes.
The increase was disproportionate and thus could not be accounted
for by the increase in percentage of proliferating cells in
culture. These data, in addition to further studies not detailed
herein, demonstrate that activation of the CD28 surface molecule of
activated T cells functions to specifically stabilize lymphokine
mRNAs. Increased mRNA stability, i.e. slower degradation thereof,
results in increased translation of the mRNA, in turn resulting in
increased lymphokine production per cell. An increase in per T cell
production of lymphokines that allows the T cell to influence the
response of other inflammatory and hematopoietic cells is referred
to as paracrine production. In contrast, an increase in lymphokine
levels merely due to increased cell proliferation, such as that
shown in Martin, P. J. et al., J. Immunol. 136:3282-3287 (1986), is
commonly referred to as autocrine production. For ease of
reference, paracrine production is also herein referred to as
"cellular" production of lymphokines.
[0058] Thus, in accordance with the principles of the present
invention, ligands with binding specificity for the CD28 molecule
are administered in a biologically compatible form suitable for
administration in vivo to stimulate the CD28 pathway. By
"stimulation of the CD28 pathway" is meant the stimulation of the
CD28 molecule resulting in increased T cell production of
T.sub.HCD28 lymphokines. By "biologically compatible form suitable
for administration in vivo" is meant a form of the ligand to be
administered in which the toxic effects, if any, are outweighed by
the therapeutic effects of the ligand. Administration of the CD28
ligand can be in any suitable pharmacological form, including but
not limited to intravenous injection of the ligand in solution.
[0059] It should be understood that, although the models for CD28
regulation of lymphokine production are described with respect to
stimulation and enhancement of lymphokine levels, as noted above,
down-regulation or inhibition of the CD28 pathway is also in
accordance with the principles of the present invention.
Down-regulation or suppression of the immune response is of
particular clinical interest for a variety of conditions, including
septic shock, tumor-induced cachexia, autoimmune diseases and for
patients receiving heart, lung, kidney, pancreas, liver and other
organ transplants. One preferred approach to down-regulation is the
blocking of the CD28 receptor stimulatory binding site on its
natural ligand. For example, CTLA-4 (discussed in more detail
below), which shares 32% amino acid homology with CD28 and appears
to have greater binding affinity for B7 than CD28, can be used to
bind B7 and prevent CD28 binding and activation thereby. See
Linsley, P. S. et al., J. Exp. Med., 174:561 (1991). Such
regulation has been accomplished in vivo as described in Specific
Example X. In this Example, acute rejection of fully MHC-mismatched
cardiac allografts was prevented by blocking B7-dependent T cell
activation, i.e. CD28 binding, with the soluble recombinant fusion
protein CTLA-4lg. The immunosuppression observed with CTLA-4lg did
not result in significant alterations in circulating T cell
subsets. It will be appreciated that other B7-binding ligands such
as a monoclonal antibody to B7 can be similarly employed. In
addition, CTLA-4lg treatment can be efficacious in the treatment of
autoimmune diseases, as shown in the murine model for multiple
sclerosis (i.e. Experimental Autoimmune Encephalomyelitis (EAE)).
CTLA-4lg treatment of T cells isolated from a mouse immunized with
Myelin Basic Protein (MBP) results in reduced disease severity when
the treated cells are introduced into a syngeneic animal. Likewise,
when mice immunized with MBP and injected with pertussis toxin (PT)
are treated directly with CTLA-4lg, disease severity is reduced.
These findings confirm the in vivo immunosuppressive effects of
CTLA-4lg treatment. Thus, administration of CTLA-4 can provide an
effective therapeutic strategy for the treatment of autoimmune
diseases. Those skilled in the art will also appreciate that the
cell lines and animal models used to exemplify the present
invention are recognized predictors of efficacy in humans.
[0060] It will be appreciated that down-regulation can also be
accomplished by blocking CD28 receptor binding to B7 by occupying
the CD28 binding site with nonstimulatory ligands which may mimic
stimulatory ligands but do not result in activation of the CD28
pathway, e.g. Fabs, modified natural, synthetic, recombinant or
other ligands which do not crosslink or otherwise do not activate
receptors.
[0061] As discussed above and shown in the Specific Examples, the
blockade of stimulatory ligands which bind to CD28 and activate the
CD28 pathway (e.g. B7) or the blocking of the CD28 binding site can
reduce the increased lymphokine expression which occurs upon CD28
activation. Thus, just as manipulation of the CD28 pathway can be
used to enhance T cell immune responses, it can also be used to
suppress such responses. Since unregulated lymphokine production
has been implicated in the aetiology of autoimmunity, CD28-mediated
immunosuppression can be exploited to treat various autoimmune
diseases. Methods of suppressing the CD28 pathway in accordance
with the present invention are desirable since this pathway is
resistant to the effects of cyclosporine, which is commonly used as
an immunosuppressive agent in the treatment of autoimmune diseases.
Immunosuppression via the CD28 pathway can restore immunoregulation
and thus reduce the pathologic effects of such autoimmune diseases
as systemic lupus erythematosis, rheumatoid arthritis, hemolytic
anemia, myasthenia gravis, schleroderma, Sjogren's syndrome,
ulcerative colitis, multiple sclerosis, and a host of other
systemic as well as organ-specific autoimmune diseases.
[0062] Administration of stimulatory ligands (e.g. mAb 9.3, Kolt,
B7) or inhibitory ligands (e.g. CTLA-4lg, Fab fragments of mAb 9.3,
and the like) can be in any suitable pharmacological form,
including parenterally or topically. Pharmaceutical compositions
may also take the form of ointments, gels, pastes, creams, sprays,
lotions, suspensions, solutions and emulsions of the active
ingredient in aqueous or nonaqueous diluents, syrups, granulates or
powders. In addition to a ligand of the present invention, the
pharmaceutical compositions can also contain other pharmaceutically
active compounds or a plurality of compounds of the invention.
[0063] More particularly, a ligand of the present invention (also
referred to herein as the active ingredient) may be administered by
any suitable route, including parenteral (including subcutaneous,
intramuscular, intravenous and intradermal), topical (including
transdermal, buccal and sublingual), rectal, vaginal, nasal and
pulmonary. Although the preferred route of administration is
currently parenteral, it will be appreciated that the delivery
route of choice will vary with the condition and age of the
recipient and the nature of the disease or condition being
treated.
[0064] While it is possible for the active ingredient to be
administered alone, it is preferable to present it as a
pharmaceutical formulation comprising at least one active
ingredient, as defined above, together with one or more
pharmaceutically acceptable carriers therefor and optionally other
therapeutic agents. Each carrier must be "acceptable" in the sense
of being compatible with other ingredients of the formulation and
not injurious to the patient.
[0065] Formulations suitable for parenteral administration include
aqueous and non-aqueous isotonic sterile injection solutions which
may contain anti-oxidants, buffers, bacteriostats and solutes which
render the formulation isotonic with the blood of the intended
recipient; and aqueous and non-aqueous sterile suspensions which
may include suspending agents and thickening agents, and liposomes
or other microparticulate systems which are designed to target the
compound to blood components or one or more organs. The
formulations may be presented in unit-dose or multi-dose sealed
containers, for example, ampoules and vials, and may be stored in a
freeze-dried (lyophilized) condition requiring only the addition of
the sterile liquid carrier, for example water for injections,
immediately prior to use. Extemporaneous injection solutions and
suspensions may be prepared form sterile powders, granules and
tablets of the kind previously described.
[0066] Formulations include those suitable for parenteral
(including subcutaneous, intramuscular, intravenous and
intradermal), topical (including transdermal, buccal and
sublingual), rectal, vaginal, nasal and pulmonary administration.
The formulations may conveniently be presented in unit dosage form
and may be prepared by any methods well known in the art of
pharmacy. Such methods include the step of bringing into
association the active ingredient with the carrier which
constitutes one or more accessory ingredients. In general, the
formulations are prepared by uniformly and intimately bringing into
associated the active ingredient with liquid carriers or finely
divided solid carriers or both, and then if necessary shaping the
product.
[0067] Pharmaceutical compositions for topical administration
according to the present invention may be formulated as an
ointment, cream, suspension, lotion, solution, paste, gel, spray
aerosol or oil. Alternatively, a formulation may comprise a patch
or a dressing such as a bandage or adhesive plaster impregnated
with active ingredients and optionally one or more excipients or
diluents. Formulations suitable for topical administration in the
mouth include mouthwashes comprising the active ingredient in a
suitable liquid carrier. It will also be appreciated that in a
carrier suitable to preserve efficacy of the ligand, oral
administration is also contemplated.
[0068] For conditions of the eye or other external tissues, e.g.
mouth and skin, the formulations are preferably applied as a
topical ointment or cream containing the active ingredient. When
formulated in an ointment, the active ingredient may be employed
with either a paraffinic or a water-miscible ointment base.
Alternatively, the active ingredients may be formulated in a cream
with an oil-in-water cream base. The topical formulations may
desirably include a compound which enhances absorption or
penetration of the active ingredient through the skin or other
affected areas. Examples of such dermal penetration enhancers
include dimethylsulphoxide and related analogues. Formulations
suitable for topical administration to the eye also include eye
drops wherein the active ingredient is dissolved or suspended in a
suitable carrier, especially an aqueous solvent for the active
ingredient.
[0069] Formulations for rectal administration may be presented as a
suppository with a suitable base. Formulations for vaginal
administration may be presented as pessaries, tampons, creams,
gels, pastes, foams or spray formulations containing in addition to
the active ingredient, such carriers as are known in the art to be
appropriate. Formulations suitable for nasal administration,
include formulations wherein the carrier is a liquid for
administrations, for example, a nasal spray or a nasal drops,
include aqueous or oily solutions of the active ingredient.
[0070] Preferred unit dosage formulations are those containing a
daily dose or unit, daily subdose, or an appropriate fraction
thereof, of the active ingredient. In any event, in practicing the
present invention, the amount of active ingredient to be used or
administered, alone or in combination with other agents, will vary
with the patient being treated and will be monitored on a
patient-by-patient basis by the physician. Generally, a
therapeutically effective amount of the vaccine will be
administered for a therapeutically effective duration. By
"therapeutically effective amount" and "therapeutically effective
duration" are meant an amount and duration to achieve the result
desired in accordance with the present invention without undue
adverse or with medically acceptable physiological effects, which
effects can be determined by those skilled in the medical arts. It
will also be appreciated that, particularly when a natural ligand
is used in the practice of the invention, its isolation or
production should render it substantially free of undesirable
contaminants such as other proteins (i.e. "substantially pure")
which may adversely impact on its efficacy or use. Acceptable
levels of purity can be determined by those skilled in the
pharmaceutical and medical arts and can depend on the specific
ligand and composition and its intended use.
[0071] It should also be understood that in addition to the
ingredients particularly mentioned above, the formulations of this
invention may include other agents conventional in the art having
regard to the type of formulation in question. In accordance with
the present invention, ligands may also be presented for the use in
the form of veterinary formulations, which may be prepared, by
methods conventional in the art.
[0072] Accumulating evidence suggests that in addition to T cell
receptor occupancy, other costimulatory signals are required to
induce a complete T cell-mediated immune response. The CD28
receptor expressed on T cells serves as a surface component of a
novel signal transduction pathway that can induce paracrine levels
of cellular production of lymphokines. Interaction of CD28 with its
natural ligand B7 which is expressed on the surface of activated B
cells macrophages or dendritic cells can act as a costimulus to
induce high level lymphokine production in antigen
receptor-activated T cells. Thus, another approach to
down-regulation is to inhibit the activation of the CD28 signal
transduction pathway as described below.
[0073] Although the CD28 signal transduction pathway is not
entirely understood, Specific Example XI demonstrates that binding
of CD28 induces protein tyrosine phosphorylation distinct from T
cell receptor (TCR)-induced tyrosine phosphorylation. For example,
TCR-induced tyrosine phosphorylation occurs in both resting and
activated T cells, while CD28-induced tyrosine phosphorylation
occurs primarily in previously activated T cells. Most striking
were the results after CD28 receptor ligation by cell-bound B7,
where phosphorylation was consistently detectable on only a single
substrate. Experiments using the Jurkat E6-1 T cell line indicated
an absolute requirement for PMA pretreatment in order to observe
CD28-induced tyrosine phosphorylation. In contrast, there was no
requirement for cellular preactivation in the Jurkat J32 line,
while, as noted above, there was a relative requirement for PMA or
TCR prestimulation of normal T cells in order to induce CD28
responsiveness. Studies with Jurkat mutants further indicated that
CD28-induced tyrosine phosphorylation and biologic function can
occur in the absence of the TCR. In this respect, CD28 appears to
be unique in that other accessory molecules involved in T cell
activation, such as CD2, Ly-6, Thy-1, and CD5 appear to require the
presence of the TCR. Thus, specific tyrosine phosphorylation
appears to occur directly as a result of CD28 ligand binding and is
involved in transducing the signal delivered through CD28 by
accessory cells that express the B7/BB1 receptor.
[0074] Studies with an inhibitor of the src family of tyrosine
kinases, herbimycin A, and with tyrosine phosphatase, as described
in Specific Example XI, further show that the functional effects of
CD28 stimulation on lymphokine gene expression require the
above-described protein tyrosine phosphorylation. The data on
tyrosine phosphorylation inhibitors thus demonstrate that
inactivation of CD28-mediated signal transduction can also be used
to down-regulate lymphokine production in accordance with the
principles of the present invention.
[0075] Immunotherapy through CD28 stimulation in accordance with
the present invention also has clinical applicability in the
treatment of bone marrow transplant recipients. The success of
autologous and allogeneic bone marrow transplantation (BMT) has
generally been limited by recurrent malignancy, graft-vs-host
disease (GVHD), and the life-threatening immune deficiency that
occurs after BMT. One approach to overcoming these problems has
been the adoptive transfer of lymphocytes in combination with
lymphokine infusions, to accelerate immune reconstitution or
mediate cytotoxicity directed at malignant cells. The side effects
attending such transfer with lymphokine infusions are, however,
quite significant.
[0076] Thus, T cell proliferation and lymphokine synthesis in the
absence of exogenously added IL-2 in response to CD3 and CD28
costimulation, as described in Specific Example XIII, provides a
unique opportunity for clinical use of adoptive transfer of
activated T cells to repair T cell defect in vivo without exogenous
lymphokine infusions. The studies detailed in Specific Example XIV
show that defective in vitro proliferative responses to anti-CD3
(OKT3 or G19-4) can be repaired by adding mAb 9.3 to the cultures.
See Joshi, I. et al., Blood Suppl. (Abstract) (1991). Costimulation
of T cells with OKT3/9.3 repaired proliferative responses as a
result of increasing the levels of MRNA expression for
cytokines/lymphokines such as IL-2, GM-CSF, and TNF.alpha.. See
Joshi, supra; Perrin, P. J. et al, Blood Suppl. (Abstract) (1991).
Purified normal CD4.sup.+ cells can thus be costimulated with
OKT3/9.3 to expand and secrete lymphokines for long periods of
time. Preclinical studies using mAb 9.3 stimulation have shown no
untoward effects in monkeys. Although CD3-stimulated cells (CTC)
provide helper factors to normal B cells, CD3-CD28 costimulated
cells appear even more effective in producing helper factors than
CD3-stimulated T cells. Thus, costimulation may also enhance the
growth of helper cells and cytotoxic T cells for adoptive
immunotherapy after BMT. The administration in vivo of T cells that
have been expanded in vitro, will provide two prominent benefits in
marrow transplantation. The ability of CD28-treated cells to
produce many lymphokines which have a strong positive effect on
hematopoiesis, such as GM-CSF, IL-3, and IL-6, should accelerate
engraftment after marrow transplantation. In addition, the ability
of anti-CD28 to trigger cytotoxicity and to cause the production of
lymphokines such as TNF is a novel form of adoptive immunotherapy
that should augment the anti-neoplastic efficacy of bone marrow
transplantation.
[0077] The possible role of CD28 in anergy was also examined.
Generally, the activation of a quiescent T cell is initiated
through stimulation of the T cell antigen receptor. This activation
can occur either through engagement of an antigenic peptide
presented in the antigen binding groove of a self-encoded MHC
molecule or by engagement of a foreign MHC molecule. However, while
this receptor-mediated activation event is required for the
initiation of a T cell response in a quiescent cell, recent studies
have demonstrated that signals transduced by the antigen receptor
alone are not sufficient to lead to an effective T cell-mediated
immune response. Several in vitro models suggest that, in fact, T
cell receptor (TCR)/CD3 activation alone of a quiescent T cell
leads to the induction of a state in which the T cell becomes
anergic to further stimulations through its antigen-specific
receptor. This state is relatively long-lived and, for at least
several weeks, renders that cell incapable of further response upon
antigenic stimulation. It is hypothesized that this isolated
activation of the TCR/CD3 complex alone in the absence of
additional T cell costimulatory molecules plays an important role
in regulating a peripheral immune response by preventing T cells
from responding to self antigens in the periphery.
[0078] Thus, for T cells to mount a proliferative response and
initiate a cell-mediated immune response, a quiescent T cell
normally requires stimulation not only through its antigen-specific
T cell receptor but also through a second receptor which provides
additional costimulatory signals to the cell. The data set forth
herein, e.g. in Specific Example XII, demonstrates that CD28
provides an essential costimulatory signal for T cell responses in
vitro and in vivo. Thus, the CD28 receptor's ability to augment T
cell lymphokine production not only results in the initiation of a
cell-mediated immune response, but also prevents the induction of
anergy in a quiescent T cell.
[0079] The role of CD28 in the prevention of programmed cell death
has also been tested. The induction of cell death has a major role
in the elimination of self-reactive or non-reactive T cells in the
thymus. In the thymus, it is thought that T cell receptor signals
are able to induce programmed cell death, in a selective and
specific fashion involving cells that express T cell receptors
specific for self-antigens. As described in Turka, L. A. et al., J.
Immunol., 144:1646-53 (1990), CD28 is expressed in developing T
cells in the thymus, and the binding of mAb 9.3 prevents thymocyte
cell death. Programmed cell death is also thought to occur in
mature T cells in peripheral lymphoid organs. Signals delivered
through the T cell receptor can induce cell death (see Newell, M.
K., et al., Nature, 347:286-8 (1990)). It has also been proposed
that cell death may have a role in certain forms of
immunopathology. For example, in HIV-1 infection it has been
proposed that the progressive immunodeficiency may be the result of
immunologically-mediated cell death, rather than a direct
consequence of viral-induced cytopathic effects. See Ameisen, J. C.
et al., Immunol. Today, 4:102 (1991); also see Groux H., et al., J.
Exp. Med. 175:331-340 (1992). The results in Specific Example XIII
show that CD28 can prevent cell death in mature T cells. Thus,
abnormal expression or activation of CD28 may have a role in
immunopathology of certain autoimmune disorders such as systemic
lupus erythematosus, a disorder characterized by abnormally
self-reactive T cells that have failed to undergo elimination in
the thymus or escape from anergic states in the peripheral lymphoid
system. Similarly, the ability to induce CD28 activation may be
beneficial in disorders characterized by progressive T cell
depletion such as HIV-1 infection.
[0080] It was also demonstrated in Specific Example XII that
superantigens SEA and SEB, which do not require traditional
processing for binding to MHC, can directly activate purified T
cells in the absence of accessory cells as determined by a
transition from G.sub.0 to G.sub.1 and induction of IL-2 receptor
expression. However, neither SEA nor SEB alone was sufficient to
result in T cell proliferation. The induction of T cell
proliferation by SEB or SEA required the addition of a second
costimulatory signal. This could be provided by either accessory
cells or monoclonal antibody stimulation of CD28. As previously
reported, T cell proliferation induced by enterotoxin in the
presence of accessory cells was partially inhibited by a blocking
antibody (HLA-DR) against class II MHC. In contrast, in purified T
cells when costimulation was provided through CD28, proliferation
was not inhibited by class II antibody and HLA-DR expression was
not detectable. In addition, costimulation through CD28 was
partially resistant to the effects of cyclosporine. These results
demonstrate that CD28 costimulation is sufficient to induce
lymphokine production and subsequent proliferation of
enterotoxin-activated T cells, and that this effect is independent
of class II MHC expression. This prevention of in vivo CD28
activation of superantigen-activated cells such as those occurring
in toxic shock syndrome and rheumatoid or lyme arthritis, may
substantially decrease disease morbidity.
[0081] Although as noted previously the present invention is not
intended to be restricted to specific mechanisms of activation, the
role of CTLA-4 in the CD28 activation pathway has been examined and
models consistent with the data presented herein have been
postulated. (See e.g. Specific Example XV.) Recent work in our
laboratory has shown that the CTLA-4 gene lies immediately adjacent
to CD28 on chromosome 2, with a similar genomic organization and
32% amino acid homology. Based on their chromosomal localization
and sequence and organizational similarities, CD28 and CTLA-4
likely represent evolutionary gene duplication. By standard
nomenclature they might thus more appropriately be named
CD28.alpha. and CD28.beta., although the terms CD28 and CTLA-4 are
retained herein.
[0082] Although CTLA-4 is not expressed on quiescent lymphoid
cells, its expression at the RNA level can be rapidly induced upon
T cell activation. Two potential mechanisms by which CTLA-4 might
function are postulated as follows. First, since CD28 and CTLA-4
contain an unpaired cysteine in their extracellular domain, this
cysteine residue may be used to form crosslinked dimeric receptors
on the surface. If this were the case, it may suggest that CTLA-4
is normally expressed on the surface as a heterodimer with CD28.
Under such conditions, the higher affinity of CTLA-4 for the
natural ligand B7 might in the dimeric state lead to a higher
affinity receptor with enhanced signaling capabilities. This might
allow for an enhanced signal transduction capability through the
CD28-CTLA-4 heterodimer in an antigen-activated cell. In addition,
if CD28 and CTLA-4 are found primarily in activated cells in a
heterodimeric state, this might account for observations that
CD28-containing receptors have enhanced signaling capabilities in
activated cells.
[0083] On the other hand, the data presented herein are also
compatible with a model in which CTLA-4 is induced upon T cell
activation as a competitive inhibitor of CD28 and is used to
down-modulate an ongoing immune response by inhibiting further
interactions between B7 and CD28 on the surface. In addition, it is
quite possible that the CTLA-4 expressed on the surface is also
expressed in a shed form, and this shed form of the receptor acts
as a soluble competitive inhibitor of an ongoing B7-CD28
interaction, thereby preventing the antigen-presenting cell from
activating additional T cells in its environment. Thus, the ability
of T cells to produce an additional isoform of CD28, i.e. CTLA-4,
suggests that the interplay of expression of CD28 and CTLA-4 has
profound effects on the ability of T cells to be activated through
a CD28-containing receptor.
[0084] CD28 pathway activation and inhibition studies indicate that
the ability of the CD28 natural ligand B7 to activate a T cell to
augment lymphokine production is entirely mediated through a
CD28-containing receptor, either a CD28 homodimer or a CD28-CTLA-4
heterodimer. Thus, the data suggest that a CTLA-4 homodimer is not
critical in T cell activation, but may play an important role in
down-modulation of T cell lymphokine production, while a
CD28-CTLA-4 heterodimer may account for the enhanced signaling
properties of CD28-containing receptors upon T cell activation.
[0085] The role of CTLA-4 in CD28-mediated signal transduction
event may explain why the novel and profound effects of CD28 on
normal T cell activation encountered and described herein were not
previously observed in human T cell lines. Earlier work on the CD28
pathway occurred in cell lines such as Jurkat human T cells.
Extensive attempts to stimulate these cells to express CTLA-4 have
been entirely negative (see FIG. 20). In contrast, standard
activation events that lead to cell cycle progression of normal T
cells either through chemical mitogens such as phytohemagglutinin
(PHA) and phorbol myristate acetate (PMA) leads to rapid induction
of CTLA-4 expression as does crosslinking of the TCR/CD3 complex.
Therefore, the inability of previous investigators to appreciate or
harness the CD28 activation pathway to enhance cellular production
of lymphokines was likely due to the lack of expression of the
CTLA-4 isoform of CD28 in these cell lines. Interestingly,
costimulation of resting T cells with anti-CD28 monoclonal
antibodies enhances the expression of the CTLA-4 gene (see FIG.
21). Thus, the CD28 activation pathway in normal cells may in fact
involve a positive feedback loop in which initial CD28 stimulation
through the CD28 homodimer enhances the expression of CTLA-4 thus
leading to enhanced heterodimer expression and signal transduction.
Alternatively, the enhanced CTLA-4 may lead to the production of a
receptor which competes for CD28 signal transduction thus leading
to the ultimate termination of lymphokine production and acts as a
negative feedback loop to down modulate an ongoing CD28-mediated
lymphokine production.
SPECIFIC EXAMPLE I
Preparation of CD28 Stimulator Monoclonal Antibody 9.3
[0086] The monoclonal antibody (mAb) 9.3, an IgG2a monoclonal
antibody which binds to the extracellular domain of the CD28
molecule, was produced by a hybrid cell line originally derived as
described by Hansen et al., Immunogen., 10:247-260 (1980). Ascites
fluid containing high titer monoclonal antibody 9.3 was prepared by
intraperitoneal inoculation of 5-10.times.10.sup.6 hybrid cells
into a Balb/C.times.C57BL/6 F.sub.1 mice which had been primed
intraperitoneally with 0.5 ml of Pristane (Aldrich Chemical Co.,
Milwaukee, Wis.). The monoclonal antibody 9.3 was purified from
ascites fluid on a staphylococcal protein-A sepharose column as
described by Hardy, R., "Handbook of Experimental Immunology," Ch.
13 (1986).
[0087] Prior to use in functional assays, purified mAb 9.3 was
dialyzed extensively against phosphate buffered saline (KCl 0.2
grams/liter dH.sub.2O; KH.sub.2PO.sub.4 0.2 grams/liter dH.sub.2O;
NaCl 8.0 grams/liter dH2O; Na.sub.2HPO.sub.4.7H.sub.2O 2.16
grams/liter dH.sub.2O) and then filtered through a 0.22 cubic
micron sterile filter (Acrodisc, Gelman Sciences, Ann Arbor,
Mich.). The mAb 9.3 preparation was cleared of aggregates by
centrifugation at 100,000 .times.g for 45 m at 20.degree. C. The
resulting purified mAb 9.3 was resuspended in phosphate buffered
saline to a final concentration of 200 .mu.g/ml as determined by
OD.sub.280 analysis and stored at 4.degree. C. prior to use.
SPECIFIC EXAMPLE II
Isolation of CD28.sup.+ T Cells
[0088] Buffy coats were obtained by leukopheresis of healthy donors
21 to 31 years of age. Peripheral blood lymphocytes (PBL),
approximately 2.5.times.10.sup.9, were isolated from the buffy coat
by Lymphocyte Separation Medium (Litton Bionetics, Kensington, Md.)
density gradient centrifugation. The CD28.sup.+ subset of T cells
was then isolated from the PBL by negative selection using
immunoabsorption, taking advantage of the reciprocal and
non-overlapping distribution of the CD11 and CD28 surface antigens
as described by Yamada et al., Eur. J. Immunol., 15:1164-1688
(1985). PBL were suspended at approximately 20.times.10.sup.6/ml in
RPMI 1640 medium (GIBCO Laboratories, Grand Island, N.Y.)
containing 20 mM HEPES buffer (pH 7.4) (GIBCO Laboratories, Grand
Island, N.Y.), 5 mM EDTA (SIGMA Chemical Co., St. Louis, Mo.) and
5% heat-activated human AB serum (Pel-Freez, Brown Deer, Wis.). The
cells were incubated at 4.degree. C. on a rotator with saturating
amounts of monoclonal antibodies 60.1 (anti-CD11 a) (see Bernstein,
I. D. et al., "Leukocyte Typing II," Vol. 3, pgs. 1-25, ed.
Reinherz, E. L. et al., (1986); 1F5 (anti-CD20) (see Clark, E. A.
et al., PNAS(USA), 82:1766-1770 (1985)); FC-2 (anti-CD16) (see
June, C. H. et al., J. Clin. Invest., 77: 1224-1232 (1986)); and
anti-CD14 for 20 m. This mixture of antibodies coated all B cells,
monocytes, large granular lymphocytes and CD28.sup.- T cells with
mouse immunoglobulin. The cells were washed three times with PBS to
remove unbound antibody, and then incubated for 1 h at 4.degree. C.
with goat anti-mouse immunoglobulin-coated magnetic particles
(Dynal, Inc., Fort Lee, N.J.) at a ratio of 3 magnetic particles
per cell. Antibody-coated cells that were bound to magnetic
particles were then removed by magnetic separation as described by
Lea, T. et al., Scan. J. Immunol., 22:207-216 (1985). Typically,
approximately 700.times.10.sup.6 CD28.sup.+ T cells were recovered.
Cell purification was routinely monitored by flow cytometry and
histochemistry. Flow cytometry was performed as described by
Ledbetter, J. A. et al., "Lymphocyte Surface Antigens," pgs.
119-129 (ed. Heise, E., 1984). Briefly, CD28.sup.+ T cells were
stained with fluorescein isothiocyanate (FITC)-conjugated anti-CD2
mAb OKT11 (Coulter, Hialeah, Fla.) and with FITC-conjugated
anti-CD28 mAb 9.3 as described by Goding, J. W., "Monoclonal
Antibodies Principles and Practice," p. 230 (ed. Goding, J. W.,
1983). CD28.sup.+ T cells were over 99% positive with
FITC-conjugated monoclonal antibody OKT11 and over 98% positive
FITC-conjugated monoclonal antibody 9.3 when compared to a
non-binding, isotype-matched, FITC-labeled control antibody
(Coulter, Hialeah, Fla.). Residual monocytes were quantitated by
staining for non-specific esterase using a commercially available
kit obtained from Sigma Chemical Co., St. Louis, Mo., and were less
than 0.1% in all cell populations used in this study. Viability was
approximately 98% as measured by trypan blue exclusion as described
by Mishell, B. B. et al., "Selected Methods in Cell. Immunology,"
pgs.16-17 (1980).
SPECIFIC EXAMPLE III
Increased Cellular Production of Human T.sub.HCD28 Lymph kines by
CD28 Stimulation by Monoclonal Antibody 9.3
[0089] A. Increased Production of IL-2, TNF-.alpha., IFN-.gamma.
and GM-CSF.
[0090] CD28.sup.+ T cells were cultured at approximately
1.times.10.sup.5 cells/well in the presence of various combinations
of stimulators. The stimulators included phorbol myristate acetate
(PMA) (LC Services Corporation, Woburn, Mass.) at 3 ng/ml conc.;
anti-CD28 mAb 9.3 at 100 ng/ml; anti-CD3 mAb G19-4 at 200 ng/ml
which was immobilized by adsorbing to the surface of plastic tissue
culture plates as previously described by Geppert, et al., J.
Immunol., 138:1660-1666 (1987); also Ledbetter, et al., J.
Immunol., 135: 2331-2336 (1985); ionomycin (lono) (Calbiochem., San
Diego, Calif.) at 100 ng/ml. Culture supernatants were harvested at
24 h and serial dilutions assayed for the presence of T.sub.HCD28
lymphokines.
[0091] Specifically, IL-2 was assayed using a bioassay as
previously described by Gillis et al., Nature, 268:154-156 (1977).
One unit (U) was defined as the amount of IL-2 needed to induce
half maximal proliferation of 7.times.10.sup.3 CTLL-2 (a human
cytotoxic T cell line) cells at 24 h of culture. In separate
experiments, the relative levels of IL-2 for each of the culture
conditions above were independently confirmed using a commercially
available ELISA assay (Genzyme Corp., Boston, Mass.).
TNF-.alpha./LT levels were measured using a semi-automated L929
fibroblast lytic assay as previously described by Kunkel et al., J.
Biol. Chem., 263:5380-5384 (1988). Units of TNF-.alpha./LT were
defined using an internal standard for TNF-.alpha. (Genzyme Corp.,
Boston Mass.). The independent presence of both TNF-.alpha. and LT
was confirmed by the ability of a monoclonal antibody specific for
each cytokine to partially inhibit cell lysis mediated by the
supernatant from cells costimulated with immobilized anti-CD3 mAb
G19-4 and anti-CD28 mAb 9.3. IFN-.gamma. was measured by
radioimmunoassay using a commercially available kit (Centocor,
Malvern, Pa.). Units for IFN-.gamma. were determined from a
standard curve using .sup.125I-labeled human IFN-.gamma. provided
in the test kit. GM-CSF was detected by stimulation of
proliferation of the human GM-CSF-dependent cell line AML-193, as
described by Lange et al., Blood, 70:192-199 (1987), in the
presence of neutralizing monoclonal antibodies to TNF-.alpha. and
LT. The .sup.3H-thymidine uptake induced by 10 ng/ml of purified
GM-CSF (Genetics Institute, Cambridge, Mass.) was defined as 100 U.
Separate aliquots of cells were recovered 48 h after stimulation
and assayed for the percentage of cells in late stages of the cell
cycle (S+G.sub.2+M) by staining of cells with propidium iodide and
analysis by flow cytometry as previously described by Thompson et
al., Nature, 314:363-366 (1985).
[0092] As shown in Table 1, CD28 stimulation of CD3-stimulated T
cells resulted in marked increases in cellular production of IL-2,
TNF-.alpha., IFN-.gamma. and GM-CSF.
2TABLE 1 Increased Cellular Production of T.sub.HCD28 Lymphokines
by CD28 Stimulation IL-2 TNF-.alpha./LT IFN-.gamma. GM-CSF S +
G.sub.2 + M STIMULUS (U/ml) (U/ml) (U/ml) (U/ml) (%) Medium <2 0
0 0 4.6 PMA <2 0 0 NT 5.5 anti-CD28 <2 5 0 0 6.5 anti-CD28 +
435 300 24 150 48.9 PMA anti-CD3.sup.i 36 50 24 120 39.7
anti-CD3.sup.i + 1200 400 74 1050 44.7 anti-CD28 lonomycin <2 0
0 NT 6.6 lonomycin + 200 5 37 NT 43.6 PMA lonomycin + 1640 320 128
NT 43.5 PMA + anti- CD28 .sup.i= immobilized NT = not tested
[0093] B. Effects of Anti-CD28 Stimulation on T cell IL-4, IL-5 and
IL-3 Secretion.
[0094] Previous studies of the effects of anti-CD28 Mab stimulation
on T cell production of lymphokines of the T.sub.H2 type were
limited to the first few days of stimulation. In those studies IL-4
could not be detected after anti-CD28 stimulation (noted in
Thompson et al., PNAS 86:1333 (1989)). On reexamination of this
question, it was found that anti-CD28 can augment production of
IL-4 and of IL-5, and thus, augments production of T.sub.H2
lymphokines as well as the T.sub.H1 type lymphokines previously
described. As can be seen in Table 2, small amounts of both IL-4
and IL-5 can be detected after 24 h of stimulation with anti-CD3
plus anti-CD28. However, when cells are restimulated after 8 days
in culture, large amounts of both IL-4 and IL-5 are secreted. As
shown in Table 3, similar results were found when the CD28 subset
of T cells were stimulated with the combination of phorbol
myristate acetate (PMA) and anti-CD28 mAb. These results indicated
that small amounts of IL-4 and IL-5 can be detected after initial
stimulation of resting T cells with anti-CD28. However, with
continued stimulation, differentiation occurs, and large amounts of
IL-4 and, particularly, of IL-5 are produced, while lesser amounts
of IL-2 and .gamma.-IFN are also produced.
3TABLE 2 Effects of anti-CD3 and anti-CD28 Treatment f IL-4 and
IL-5 Production by T cells INITIAL RESTIMULATION IL-4 IL-5 IFN IL-2
IL-4 IL-5 IFN IL-2 STIMULUS pg/ml pg/ml pg/ml U/ml pg/ml pg/ml
pg/ml U/ml Medium <1 <1 <1 <0.1 <1 <1 <1
<0.1 anti-CD3 <1 <1 630 2.3 220 225 246 0.02 anti-CD3 +
207 137 887 7.0 498 2545 379 0.2 anti-CD28
[0095] The data in Table 2 above were obtained using the following
protocol: CD28.sup.+ T cells were isolated by negative selection
using monoclonal antibodies and magnetic immunobeads as described
in Specific Example II. The cells were cultured at
1.times.10.sup.6/ml in RPMI medium containing 10% FCS (Medium), or
in culture wells containing anti-CD3 monoclonal antibody G19-4
absorbed to the plastic, or plastic adsorbed anti-CD3 plus
anti-CD28 mAb 9.3 added in solution at 0.5 .mu.g/ml. Supernatants
from the cell culture were analyzed for lymphokine concentration
using commercially available ELISA kits and the values expressed as
pg/ml for IL-4, IL-5 and .gamma.-IFN, or as units per ml, for IL-2.
Supernatants were analyzed after 24 h of culture (Initial) or
alternatively, the cells were cultured for 8 days in the above
forms of stimulation, the cells were recovered, washed, and then
restimulated with their original treatment, and the supernatants
analyzed after a further 24 h of stimulation (Restimulation). The
values represent means of duplicate cultures.
4TABLE 3 Effects of PMA and anti-CD28 Treatment on IL-4 and IL-5
Production by T cells INITIAL RESTIMULATION IL-4 IL-5 IFN IL-2 IL-4
IL-5 IFN IL-2 STIMULUS pg/ml pg/ml pg/ml U/ml pg/ml pg/ml pg/ml
U/ml Medium <1 <1 <1 <0.1 <1 <1 <1 <0.1 PMA
<1 <1 152 .07 <1 <1 <1 0.6 anti-CD28 + <1 187 558
8.1 285 392 335 10.3 PMA
[0096] The data in Table 3 above were obtained using the following
protocol: CD28.sup.+ T cells were isolated by negative selection
using monoclonal antibodies and magnetic immunobeads as described
in Specific Example II. The cells were cultured at
1.times.10.sup.6/ml in RPMI medium containing 10% FSC (Medium), or
in medium plus PMA 3 ng/ml, or in PMA plus anti-CD28 Mab 9.3 at 0.5
.mu.g/ml. Supernatants from the cell culture were analyzed for
lymphokine concentration using commercially available ELISA kits
and the values expressed as pg/ml for IL-4, IL-5 and .gamma.-IFN.
Supernatants were analyzed after 24 h of culture (Initial) or,
alternatively, the cells were cultured for 8 days in the above
forms of stimulation, and then restimulated, and the supernatants
analyzed after a further 24 h period of stimulation
(Restimulation). The values represent means of duplicate
cultures.
[0097] Induction of IL-3 expression in T cells after anti-CD28
treatment. IL-3 is a multilineage hematopoietic growth factor that
is primarily produced by T cells, and is generally considered to be
produced by T.sub.H1 cells. The experimental protocol and the
findings described herein are described in detail in Guba, S. C. et
al., J Clin. Invest. 84(6):1701-1706 (1989), incorporated herein by
reference.
[0098] PBL were isolated as described previously. The CD28.sup.+
subset of T cells was isolated by negative selection as described
by June, C. H. et al., Mol. Cell. Biol. 7:4472-4481 (1987). In some
experiments, the CD28.sup.+ subset of T cells was isolated by
incubating PBL with mAB 9.3, and then removing the CD28.sup.+ cells
with goat anti-mouse coated magnetic beads (Advanced Magnetics
Institute, Cambridge, Mass.). Northern (RNA) blot analysis was done
as described by June, C. H. et al., Mol. Cell. Biol. 7:4472-4481
(1987). The IL-3 probe was a 1.0 kb Xho I cDNA fragment.
[0099] To determine if stimulation of the TCR/CD3 pathway of T cell
activation induced IL-3 gene expression, CD28.sup.+ T cells were
stimulated with maximal amounts of plastic immobilized anti-CD3 mAb
in the presence or absence of 9.3 mAb 1 .mu.g/ml for 1 to 36 h. As
shown in Table 4, anti-CD28 resulted in a 3 to 5-fold augmentation
of IL-3 mRNA expression over that induced by anti-CD3 alone. CD28
did not change the kinetics of IL-3 gene expression, which was at
peak levels at 6 h after anti-CD3 or after anti-CD3+anti-CD28
treatment. Further experiments showed that IL-3 gene expression was
restricted to the CD28.sup.+ subset of T cells, as determined by
Northern analysis (Table 4). The stability of IL-3 mRNA was also
determined. T cells were treated for 3 h with anti-CD3 or anti-CD3
plus anti-CD28 mAb to induce IL-3 mRNA expression. At 3 h,
actinomycin D was added to the culture to inhibit further RNA
synthesis. Total cellular RNA was isolated, and the remaining IL-3
mRNA determined by Northern analysis. The half-life of IL-3 mRNA
from anti-CD3 plus anti-CD28 treated cells was at least 8-fold
longer than the IL-3 mRNA from anti-CD3 treated cells (Table 4).
Thus, as would be expected from the previously described results of
anti-CD28 on other lymphokines, it can be concluded that the effect
of anti-CD28 on IL-3 gene induction can, in large part, be
explained by the ability of anti-CD28 to stabilize the IL-3
messenger RNA.
5TABLE 4 IL-3 GENE EXPRESSION (arbitrary densitometry CONDITION
units) EXPERIMENT #1 CD28.sup.+ T cells, 6 h, anti-CD3 1 CD28.sup.+
T cells, 6 h, anti-CD3 + anti-CD28 3-5 EXPERIMENT #2 CD28.sup.+ T
cells, 8 h, PMA 3 ng/ml + lonomycin >10 0.4 .mu.g/ml CD28.sup.-
T cells, 8 h, PMA 3 ng/ml + lonomycin <1 0.4 .mu.g/ml EXPERIMENT
#3 CD28.sup.+ T cells, anti CD3, then 1 actinomycin D 90 m
CD28.sup.+ T cells, anti-CD3 + anti-CD28, 8 then actinomycin D 90
m
SPECIFIC EXAMPLE IV
Comparison of CD28 Stimulation to Stimulation of Other T Cell
Surface Molecules
[0100] CD28.sup.+ T cells were cultured at approximately
1.times.10.sup.5 cells/well in RPMI media containing 5%
heat-inactivated fetal calf serum (FCS), PHA 10 pg/ml, PMA 3 ng/ml,
ionomycin at 100 ng/ml, anti-CD28 mAb 9.3 at 100 at ng/ml, or mAb
9.4 specific for CD45 at 1 .mu.g/ml or mAb 9.6 specific for CD2 at
1 .mu.g/ml, or immobilized mAb G19-4 specific for CD3 at 200
ng/well.
[0101] CD28.sup.+ T cells were cultured in quadruplicate samples in
flat-bottomed 96-well microtiter plates in RPMI media containing 5%
heat-inactivated fetal calf serum. Equal aliquots of cells were
cultured for 18 h and then pulsed for 6 h with 1 .mu.Ci/well of
.sup.3H-uridine, or for 72 h and then pulsed for 6 h with 1
.mu.Ci/well of .sup.3H-thymidine. The means and standard deviations
(in cpm) were determined by liquid scintillation counting after
cells were collected on glass fiber filters.
[0102] All cultures containing cells immobilized to plastic by
anti-CD3 monoclonal antibodies were visually inspected to ensure
complete cell harvesting. The failure of cells in these cultures to
proliferate in response to PHA is the result of rigorous depletion
of accessory cells, in vivo activated T cells, B cells, and
CD11.sup.+ (CD28.sup.-) T cells by negative immunoabsorption as
described in Specific Example II above. In each experiment, cells
were stained with fluorescein-conjugated anti-CD2 mAb OKT11 and
fluorescein-conjugated anti-CD28 mAb 9.3 and were shown to be over
99% and over 98% surface positive, respectively.
[0103] A representative experiment is illustrated in FIGS. 1 and 2.
As shown in FIGS. 1 and 2, anti-CD28 by itself had no significant
effect on uridine or thymidine incorporation, nor did it serve to
augment proliferation induced either by immobilized anti-CD3 mAb
G19-4 or chemically-induced T cell proliferation involving phorbol
myristate acetate (PMA) and ionomycin (lono). However, as shown in
FIG. 2, anti-CD28 did significantly increase the uridine
incorporation of both sets of cells. In contrast, other monoclonal
antibodies including anti-CD2 mAb OKT11 and anti-CD45 mAb 9.4 had
no significant effect on uridine incorporation of anti-CD3
stimulated cells. This was not due to lack of effect of these
antibodies on the cells, since anti-CD2 monoclonal antibodies
significantly augmented the proliferation of anti-CD3 stimulated
cells. In separate experiments, the binding of isotype-matched mAbs
to other T cell surface antigens (CD4, CD6, CD7 or CD8) failed to
mimic the effects observed with anti-CD28.
[0104] These data serve to confirm that the stimulation of
activated T cells by CD28 has a unique phenotype which appears to
directly enhance the rate of incorporation of a radioactive marker
into the steady state RNA of T cells without directly enhancing T
cell proliferation.
SPECIFIC EXAMPLE V
[0105] Increased Cellular Production of Human T.sub.HCD28
Lymphokines by CD28 Stimulation Ex Vivo
[0106] Based on evidence from the in vitro systems it appeared that
CD28 did not have a significant effect on cellular production of
lymphokines unless they had undergone prior antigen activation or
its equivalent. However, CD28 binding by the 9.3 mAb significantly
enhanced the ability of anti-TCR/CD3 activated T cells to sustain
production of human T.sub.H1-type lymphokines. To test this effect
in a physiologic setting, the activation of T lymphocytes in an ex
vivo whole blood model was studied.
[0107] 50-100 ml of venous blood was obtained by standard aseptic
procedures from normal volunteers after obtaining informed consent.
The blood was heparinized with 25 U/ml of preservative-free heparin
(Spectrum, Gardenia, Calif.) to prevent clotting. Individual 10 ml
aliquots were then placed on a rocking platform in a 15 ml
polypropylene tube to maintain flow and aeration of the sample.
[0108] To assay for the effectiveness of CD28 stimulation on the
induction of lymphokine gene expression, the production of
TNF-.alpha. molecule was chosen as a model because of the extremely
short half-life (approximately 15 minutes) of the protein in whole
blood. 10 ml of whole blood isolated as described above was
incubated with soluble anti-CD3 mAb G19-4 at a concentration of 1
.mu.g/ml or anti-CD28 mAb 9.3 at a concentration of 1 .mu.g/ml or a
combination of the two antibodies. The plasma was assayed for
TNF-.alpha. as described in Specific Example III at one and four h.
An example of one such experiment is shown in Table 5, which
illustrates the significant increase in sustained production of
TNF-.alpha. by maximal stimulation of CD3 and costimulation of
CD28.
6 TABLE 5 TNF-.alpha. (pg/ml) STIMULUS 0 h 1 h 4 h anti-CD3
4.5.sup.a 65.0 2.1 anti-CD28 4.5.sup.a 1.6 3.3 anti-CD3 + anti-CD28
4.5.sup.a 35.0 75.0 .sup.avalue determined prior to addition of
monoclonal antibody to aliquots of the venous sample
SPECIFIC EXAMPLE VI
Resistance of CD28-Induced T Cell Proliferation to Cyclosporine
[0109] The protocol used and results described herein are described
in detail in June, C. H. et al., Mol. Cell. Biol., 7:4472-4481
(1987), herein incorporated by reference.
[0110] T cells, enriched by nylon wool filtration as described by
Julius, et al., Euro. J. Immunol., 3:645-649 (1973), were cultured
at approximately 5.times.10.sup.4/well in the presence of
stimulators in the following combinations: anti-CD28 mAb 9.3 (100
ng/ml) and PMA 1 (ng/ml); or immobilized anti-CD3 mAb G19-4 (200
ng/well); or PMA (100 ng/ml). The above combinations also included
fourfold titrations (from 25 ng/ml to 1.6 .mu.g/ml) of cyclosporine
(CSP) (Sandoz, Hanover, N.J.) dissolved in ethanol-Tween 80 as
described by Wiesinger, et al., Immunobiol., 156:454-463
(1979).
[0111] .sup.3H-thymidine incorporation was measured on day 3 of
culture and the results representative of eight independent
experiments are depicted in FIG. 3. The arithmetic mean
(91,850.+-.1300 (mean.+-.SD)) CD28-induced T cell proliferation
exhibits nearly complete cyclosporine resistance when accompanied
by the administration of PMA. Table 6 below illustrates the effects
of cyclosporine on CD3-induced proliferation of CD28.sup.+ T cells
cultured at approximately 5.times.10.sup.4 cells/well in
flat-bottomed 96-well microtiter plates (CoStar, Cambridge, Mass.)
under the following conditions: immobilized Mab G19-4; or
immobilized mAb G19-4 and mAb 9.30 100 ng/ml; or immobilized mAb
G19-4 and PMA 1 ng/ml; or Mab 9.3 100 ng/ml and PMA 1 ng/ml.
Cyclosporine was prepared as above and included in the cultures at
0, 0.2, 0.4, 0.8, 1.2 .mu.g/ml.
[0112] .sup.3H-thymidine incorporation was determined on day 3 of
culture as above. The percent inhibition of proliferation was
calculated between CD28.sup.+ T cells cultured in medium only or in
cyclosporine at 1.2 .mu.g/ml. CD28.sup.+ T cells cultured in the
absence of cyclosporine were given cyclosporine diluent.
.sup.3H-thymidine incorporation of cells cultured in medium, or
PMA, or monoclonal antibody 9.3 only were less than 150 cpm. As
shown in Table 6, costimulation of CD3 and CD28 resulted in a
marked increase in the resistance of T cell proliferation to
cyclosporine and the stimulation of CD28 in the presence of PMA
resulted in a complete absence of cyclosporine suppression of T
cell proliferation. As shown in Table 7, stimulation of CD28
together with immobilized anti-CD3 also resulted in resistance to
suppression of T cell proliferation by the immunosuppressant
dexamethasone.
7TABLE 7 Effects of CD28 Stimulation on Dexamethasone Resistance on
T cell Proliferation .sup.3HTdR + Dexamethasone (Nm) STIMULUS 0 25
% INHIBIT CD3 mAb G194 14,700 770 97 CD3 mAb + IL-2 21,700 1,900 93
CD28 mAb + PMA 181,600 197,700 <0 PMA 5,000 1,400 72
[0113]
8TABLE 6 Effects of CD28 Stimulation on Cyclosporine Resistance on
T Cell Proliferation Mean [.sup.3H]thymidine incorporation (kcpm)
.+-. 1 SD at Cyclosporine Conc (ug/ml) STIMULUS 0 0.2 0.4 0.8 1.2 %
INHIBIT CD3 mAb G19-4 77 .+-. 26 61 .+-. 6.8 52 .+-. 4.4 10 .+-.
3.4 8.2 .+-. 1.2 90 CD3 + CD28 123 .+-. 18 86 .+-. 2.3 63 .+-. 4.4
44 .+-. 6.4 43 .+-. 5.2 65 mAb 9.3 CD3 + PMA 145 .+-. 12 132 .+-.
2.8 123 .+-. 6.4 55 .+-. 3.6 56 .+-. 6.4 62 CD28 mAb 111 .+-. 12 97
.+-. 5.6 107 .+-. 12 99 .+-. 14 112 .+-. 2.4 <0 9.3 + PMA
SPECIFIC EXAMPLE VII
Human T.sub.HCD28 Lymphokine Secretion in the Presence of
Cyclosporine
[0114] As described in Specific Example III, CD28.sup.+ T cells
were cultured in the presence of various stimulators. Culture
supernatants were harvested at 24 h and serial dilutions assayed
for IL-2, TNF-.alpha./LT, IFN-.gamma., and GM-CSF as previously
described. Separate aliquots of cells were recovered 48 h after
stimulation and assayed for the percentage of cells in late stages
of the cell cycle (S+G.sub.2+M).
[0115] When cyclosporine at 0.6 .mu.g/ml was included in the test
protocol, as shown in Table 8 (which also incorporates the data of
Specific Example III for comparison), CD28.sup.+ T cells were found
to secrete the T.sub.HCD28 lymphokines in the presence of
cyclosporine in cultures stimulated with mAb 9.3 and PMA; or
immobilized mAb G19-4 and mAb 9.3; or PMA and ionomycin and mAb
9.3. T.sub.HCD28 lymphokine production induced by immobilized mAb
G19-4; or by PMA with ionomycin was, however, completely suppressed
in the presence of cyclosporine.
9TABLE 8 GM- S + IL-2 TNF-.alpha./LT IFN-.gamma. CSF G.sub.2 + M
STIMULUS (U/ml) (U/ml) (U/ml) (U/ml) (%) Medium <2 0 0 0 4.6 PMA
<2 0 0 NT 5.5 anti-CD28 <2 5 0 0 6.5 anti-CD28 + PMA 435 300
24 150 48.9 anti-CD28 + PMA + 192 200 12 NT 49.3 CSP anti-CD3.sup.i
36 50 24 120 39.7 anti-CD3.sup.i + CSP <2 0 0 NT 14.5
anti-CD3.sup.i + anti- 1200 40 74 1050 44.7 CD28 anti-CD3.sup.i +
anti- 154 200 9 NT 48.6 CD28 + CSP lonomycin <2 0 0 NT 6.6
lonomycin + PMA 200 5 37 NT 43.6 lonomycin + PMA + <2 0 0 NT 8.1
CSP lonomycin + PMA + 1640 320 128 NT 43.5 anti-CD28 lonomycin +
PMA + 232 120 15 NT 47.6 anti-CD28 + CSP .sup.i= immobilized NT =
not tested
SPECIFIC EXAMPLE VIII
Human T.sub.HCD28 Lymphokine mRNA Expression in the Presence of
Cyclosporine
[0116] In order to further examine whether CD28 stimulation led to
cyclosporine-resistant T.sub.HCD28 lymphokine gene expression as
well as secretion, the ability of cyclosporine to suppress
induction of IL-2, TNF-.alpha., LT, INF-.gamma., and GM-CSF
following stimulation by various stimulators was tested.
Specifically, CD28.sup.+ T cells were cultured at
2.times.10.sup.6/ml in complete RPM1 medium (GIBCO, Grand Island,
N.Y.) with 5% FCS (MED). Individual aliquots of CD28.sup.+ T cells
were incubated for 6 h in the presence or absence of 1.0 .mu.g/ml
cyclosporine with PMA 3 ng/ml and anti-CD28 mAb 9.3 (1 mg/ml); or
with immobilized anti-CD3 mAb G19-4 (1 .mu.g/well); or with
immobilized mAb G19-4 (1 .mu.g/well) and mAb 9.3 (1 ng/ml).
CD28.sup.+ T cells were harvested, total cellular RNA isolated and
equalized for ribosomal RNA as previously described by Thompson, et
al., Nature, 314:363-366 (1985).
[0117] Northern blots were prepared and hybridized sequentially
with .sup.32P-labeled, nick-translated gene specific probes as
described by June, C. H. et al., Mol. Cell. Biol., 7:4472-4481
(1987). The IL-2 probe was a 1.0 kb Pst I cDNA fragment as
described by June, C. H. et al., Mol Cell. Biol., 7:4472-4481
(1987); the IFN-.gamma. probe was a 1.0 kb Pst I cDNA fragment as
described by Young, et al., J. Immunol., 136:4700-4703 (1986). The
GM-CSF probe was a 700 base pair EcoR I-Hind III cDNA fragment as
described by Wong, et al., Science, 228:810-815 (1985); the 4F2
probe was a 1.85 kb EcoR I cDNA fragment as described by Lindsten,
et al., Mol. Cell. Biol., 8:3820-3826 (1988); the IL4 probe was a
0.9 kb Xho I cDNA fragment as described by Yokota, et al., PNAS
(USA), 83:5894-5898 (1986); and the human leukocyte antigen (HLA)
probe was a 1.4 kb Pst I fragment from the HLA-B7 gene as described
by Lindsten, et al., Mol. Cell. Biol., 8:3820-3826 (1988).
TNF-.alpha. and LT specific probes were synthesized as
gene-specific 30 nucleotide oligomers as described by Steffen, et
al., J. Immunol., 140:2621-2624 (1988) and Wang, et al., Science,
228:149-154 (1985). Following hybridization, blots were washed and
exposed to autoradiography at -70.degree. C. Quantitation of band
densities was performed by densitometry as described in Lindsten,
et al., Mol. Cell. Biol., 8:3820-3826 (1988).
[0118] As illustrated by the Northern blot of FIG. 4, stimulation
by mAb 9.3 with PMA and by mAb 9.3 with mAb G19-4 led to human
T.sub.HCD28 lymphokine gene expression that exhibited resistance to
cyclosporine. In contrast, stimulation by TCR/CD3 mAb G19-4 alone
was completely suppressed in the presence of cyclosporine.
SPECIFIC EXAMPLE IX
In Vivo Activation of T Cells by CD28 Stimulation
[0119] A. Monoclonal Antibody 9.3 F(ab').sub.2.
[0120] F(ab').sub.2 fragments of mAb 9.3 were prepared as described
by Ledbetter, J. A. et al., J. Immunol., 135:2331-2336 (1985).
Purified and endotoxin-free F(ab').sub.2 fragments were injected
intravenously at 1 mg/kg of body weight over a 30 minute period
into a healthy macaque (M. nemestrina) monkey. On days 2 and 7
after injection, 5 ml of blood was drawn and tested.
[0121] Peripheral blood lymphocytes from the monkey's blood were
isolated by density gradient centrifugation as described in
Specific Example II. Proliferation of peripheral blood mononuclear
cells in response to PMA (1 ng/ml) was tested in the treated monkey
and a control animal (no F(ab').sub.2 fragment treatment) in
triplicate as described in Specific Example IV. Proliferation was
measured by the uptake of .sup.3H-thymidine during the last 6 h of
a three-day experiment and the results shown in FIG. 5. Means of
triplicate culture are shown, and standard errors of the mean were
less than 20% at each point. As shown in FIG. 5, in vivo
stimulation of CD28 by the F(ab').sub.2 mAb 9.3 fragment increased
T cell proliferation for at least 7 days.
[0122] B. Monoclonal Antibody 9.3
[0123] Two doses of mAb 9.3, 10 mg and 0.1 mg, were administered
intravenously to primates Macaca mulatta. The antibody wa infused
intravenously immediately after baseline (time zero) blod valves
were obtained. Three animals were evaluated as described below at
each dose.
[0124] Cell population changes. At the higher dose, immediate
effects were monitored over the first 120 m. In FIGS. 6A and 6B, a
representative result is depicted showing the change in the
lymphocyte counts over time. The ALC and distribution of CD28.sup.+
cells are depicted in FIG. 6A, while FIG. 6B illustrates the
absolute numbers of CD4.sup.+ and CD8.sup.+ cells. The absolute
lymphocyte count (ALC) decreased over the first 60 m and then
increased above baseline at 24 h (see FIG. 6A). In this case, the
number of circulating CD28 positive lymphocytes remained
essentially the same, as determined by adding goat anti-mouse
phycoerythrin (GAM-PE) only or mAb 9.3 plus GAM-PE. In this same
animal, the CD4 and CD8 positive populations were followed and the
increase at 24 h was the result of an increase in
CD8.sup.+CD28.sup.- cells (see FIG. 6B). Animals re-evaluated after
8 days had between 35 to 60% of the CD28.sup.+ cells coated with
antibody. There was no significant change in the circulating
lymphocyte counts in primates treated with 0.1 mg mAb 9.3.
[0125] Cytokine released after in vitro stimulation. PBLs isolated
at specific time points from primates previously immunized with
tetanus toxoid and treated with 10 mg mAb 9.3 were cultured in
vitro to determine the effect of antigenic stimulation on cytokine
production. FIG. 7A represents the in vitro production of TNF while
FIG. 7B represents the in vitro production of IL-6. PBLs stimulated
with Concanavalin-a (Con-a) are depicted by .DELTA.. PBLs
stimulated with tetanus toxoid (TT) are depicted by .circle-solid..
Unstimulated PBLs are depicted by .smallcircle.. As shown in FIGS.
7A and 7B, cultures of baseline cells did not respond to either
Con-a or TT stimulation. However, as shown in FIG. 7A, PBLs
isolated from animals 6 h after infusion of antibody showed an
increase in TNF production. As depicted in FIG. 7B, after 24 h,
unstimulated cultures produced TNF but not IL-6. TT stimulation of
PBLs produced a similar quantity of both TNF and IL-6 as mitogen
stimulated cultures. This response was consistent through 72 h.
[0126] Monoclonal antibody 9.3 was administered to the primates
either as a single day bolus of 10 mg (n=3) or as multiple daily
injections of 10 mg/d for 5 consecutive days (n=3) and the animals
were followed simultaneously. The changes in the peripheral blood
cell populations were not dramatic. ALC as previously observed
decreased with the first injection but recovered to above baseline
if no further injections were administered. However, for animals
treated with multiple injections, ALC remained .about.25% below
normal during the period of mAb administration. ALC in
multiple-treated animals did not recover to above normal levels
over the 21-day study period. Absolute neutrophil count decreased
by .about.30% in the 5 day treated group.
[0127] Cytokine levels in serum. Serum was analyzed for IL-6, TNF,
and IL-1 .beta.. The detection of TNF from the serum preparations
was not successful and therefore no results are available at this
time. FIGS. 8A and 8B demonstrate the serum concentration of IL-6
after infusion of mAb 9.3. As shown in FIGS. 8A and 8B, increased
IL-6 levels 24 h after mAb infusion were detected. In animals
injected one time, the IL-6 levels increased to a peak on day 4,
but a decrease was observed when remeasured on day 8 (see FIG. 8A).
In comparison, 5 day treated animals (multiple doses) demonstrated
continual increase(s) in IL-6 through day 8 (see FIG. 8B).
[0128] FIGS. 9A and 9B demonstrate the serum concentration of IL-1
after infusion of mAb 9.3. As shown in FIGS. 9A and 9B,
measurements of IL-1 .beta. in the serum, did not detect any IL-1
until after day 8 in single injected animals (see FIG. 9A) or
multiple injected animals (see FIG. 9A). The multiple injected
animals, however, had increasing levels of IL-l.beta. at day 21
post-infusion, while single injected animals had decreasing levels
at this time.
[0129] Cytokine release after in vitro stimulation. IL-6 production
was not detected in the PBLs of animals on day 3 after in vitro
stimulation with TT. (FIG. 10). This finding contrasted with the
previous in vitro results. However, an increased production of IL-6
was detected on day 7 and the more significant increase was
observed from PBLs isolated from the 5 day treated primates. This
increase in production was further observed in culture of PBLs
isolated on day 14.
[0130] IL-6 Production and Proliferation of PBLs. FIGS. 10A and 10B
illustrate IL-6 production of in vitro stimulated PBLs isolated
from monkeys. Days 1, 3 and 14 are depicted in FIGS. 10A and 10B
with .DELTA. representing the control and .smallcircle.
representing the stimulated PBL response. FIG. 10A illustrates the
response of a single injected animal and FIG. 10B illustrates the
response of a multiple injected animal. (Note that the quantity of
cells harvested from PBL limited the number of assays performed,
resulting in no day zero points and no day zero data.) PBLs were
isolated from the different treated groups and evaluated for their
proliferative response to Con-A, TT or no stimulus. Historically
the TT response was .about.5,000 cpm and the baseline Con-A
response was .about.35,000 cpm. The PBL proliferative response to
Con-A was reduced by about 80% and gradually recovered over time
(not shown). No proliferative response was observed when the PBL
were stimulated with TT. This contrasts with the lymphokine
production observed in in vitro cultures.
SPECIFIC EXAMPLE X
Immunoregulation with CTLA-4lg
[0131] A. In Vitro.
[0132] The effects of CTLA-4lg on the primary immune response to
alloantigen was initially examined in a one-way mixed lymphocyte
culture (MLC) between Lewis rats (RT1.sup.l, responder) and
Brown-Norway rats (RT1.sup.n, stimulator). Lymphocytes were
isolated from paratracheal and cervical lymph nodes. Cultures were
performed in quadruplicate in 96-well round bottomed plates as
described in Turka, L. A. et al., Transplant., 47:388-390 (1989).
Cultures were harvested after 4 days and 1 mCi/well of
.sup.3H-thymidine was added for the last 6 h of culture. In this
assay, Brown-Norway stimulator cells were irradiated at 30 Gy to
prevent their proliferation, and then added to cultures of Lewis
responder lymphocytes. A proliferative response will normally occur
in approximately 1-5% of cells as a result of activation through
their cell-surface TCR in response to allogeneic MHC as discussed
in Marrack, P. et al., Immunol. Today, 9:308-315 (1988). Graded
concentrations of CTLA-4lg or an isotype-matched control monoclonal
antibody L6 described in Fell, H. P. et al., J. Bio. Chem. (in
press), was added to the cultures.
[0133] FIG. 11 represents the effect of CTLA-4lg on a one-way mixed
lymphocyte culture. Spontaneous proliferation is the incorporation
of thymidine by Lewis cells in the absence of Brown-Norway
stimulators, and is depicted by closed triangles in FIG. 11. As
shown in FIG. 11, CTLA-4lg was able to block proliferation in a
dose dependent fashion with virtually complete inhibition observed
at a concentration of 1 mg/ml. (Results are expressed as counts per
minute of .sup.3H-thymidine incorporation.+-.standard deviation).
Consistent with these results, alloreactive T cell responses can
also be inhibited by non-stimulatory Fab fragments of an anti-CD28
monoclonal antibody as shown in Azuma, M. et al., J. Exp. Med.,
175:353-360 (1992). Together, these data suggest that in order to
mount a proliferative response in vitro, alloreactive T cells must
be stimulated not only through MHC engagement of the TCR but also
require costimulation by B7 engagement of the CD28 receptor.
[0134] B. In Vivo: Cardiac Allografts.
[0135] CTLA-4lg was next used in a rat model of organ
transplantation to ascertain its ability to block alloantigen
responses in vivo. Recipient animals received a heterotopic cardiac
allograft which was anastomosed to vessels in the neck as described
in Bolling, S. F. et al., Transplant., 53:283-286 (1992). Grafts
were monitored for mechanical function by palpation and for
electrophysiologic function by electrocardiogram. Graft rejection
was said to occur on the last day of palpable contractile function.
As an initial test, animals were treated with daily injections of
CTLA-4lg or an isotype-matched negative control monoclonal antibody
L6 for 7 days. CTLA-4lg was administered at doses of 0.015 mg/day
(5 animals), 0.05 mg/day (5 animals), and 0.5 mg/day (8 animals).
L6 was given at 0.5 mg/day. Untreated Lewis rats rejected the
heterotopic Brown-Norway allografts in 6.8.+-.0.3 days (n=10). The
allografts in CTLA-4lg-treated animals remained functional
following completion of drug administration, whereas untreated
animals, or animals treated with the L6 control antibody, uniformly
rejected their grafts by day 8 (p<0.0001) as shown in Table 9.
(p values were calculated by Chi-square analysis).
10 TABLE 9 GRAFT SURVIVAL DAY 8 SIGNIFICANCE Untreated 0/10 p <
0.0001 CTLA-4lg 18/18 p < 0.0001 Control Protein 0/5
[0136] CTLA-4lg-treated rats manifested no observable acute or
chronic side effects from administration of the protein. No gross
anatomic abnormalities were observed in CTLA-4lg-treated animals at
autopsy.
[0137] An untreated animal and a CTLA-4lg-treated animal were
sacrificed for histological examination. Cardiac allografts were
removed from an untreated animal (shown in FIG. 12A) and a
CTLA-4lg-treated animal (0.5 mg/day) (shown in FIG. 12B) four days
after transplantation. Allografts were fixed in formalin, and
tissue sections were stained with hematoxylin-eosin. (Original
photography at 200.times. magnification.) The donor heart removed
from the untreated animal showed histological findings of severe
acute cellular rejection, including a prominent interstitial
mononuclear cell infiltrate with edema formation, myocyte
destruction, and infiltration of arteriolar walls. In contrast, the
transplanted heart from the CTLA-4lg-treated animal revealed only a
mild lymphoid infiltrate. Frank myocyte necrosis and evidence of
arteriolar involvement were absent. The native heart from each
animal showed no histological abnormalities.
[0138] To determine whether CTLA-4lg therapy established a state of
graft tolerance that persisted following drug treatment, animals
treated for 7 days with daily injections of CTLA-4lg were observed
without additional therapy until cessation of graft function.
Animals received either no treatment, CTLA-4lg (0.5 mg/day.times.7
days), or an isotype-matched control monoclonal antibody, L6 (0.5
mg/day.times.7 days). In all cases treatment was initiated at the
time of transplantation. FIG. 13 is a graph showing allograft
survival in the treated and control rats. Graft survival was 18-40
days in animals treated with 0.05 mg/day of CTLA-4lg. Graft
survival was assessed daily. This failure to induce permanent
engraftment did not appear to be due to inadequate dosing of
CTLA-4lg, as animals treated with a ten-fold higher dose, 0.5
mg/day, showed a similar graft survival curve as depicted in FIG.
13, with one animal maintaining long-term graft function (>50
days). In FIG. 13, graft survival is displayed as the last day of
graft function. Animals treated with a dose of 0.015 mg/day.times.7
days (n=5) had a mean survival of 12.6.+-.2.1 days (n=5).
Furthermore, serum CTLA-4lg trough levels in this group as measured
in a quantitative ELISA assay were in excess of 10 .mu.g/ml, a
concentration which is maximally suppressive in vitro (see FIG.
11). Histological examination of the allografts from
CTLA-4lg-treated animals whose grafts ceased functioning after
18-43 days displayed typical signs of acute cellular rejection, of
the same degree of severity as seen in control animals that had
rejected their hearts after 7 days. The animal with continued graft
function was sacrificed on day 57, and the allograft from this
animal failed to reveal any histological abnormalities.
[0139] At the time of sacrifice, lymphocytes from the day 57
"tolerant" animal, and from a CTLA-4lg-treated animal that rejected
the heart at day 33, were tested for their functional responses.
These responses were compared with those of lymphocytes from a
control (non-transplanted) Lewis rat, and results were normalized
as a percentage of the control response. In comparison to control
animals, lymphocytes from both the "tolerant" and rejecting animal
had equivalent proliferative responses to Con-a, (tolerant, 62.5%;
rejecting, 51.1%; p=0.63, two-tailed T test) and to cells from a
third party ACI rat (RT1.sup.avl) (tolerant, 160%; rejecting, 213%;
p=0.58). However a significant disparity was seen in the response
to Brown-Norway cells (tolerant, 34.2%; rejecting, 238%;
p<0.005), suggesting that T cells from animals with functioning
grafts were specifically hyporesponsive to donor MHC antigens. The
thymus and spleen from the day 57 "tolerant" animal were similar in
size and cell number to the non-transplanted control rat, and flow
cytometric analyses of thymus, lymph nodes and spleen revealed
similar percentages of both CD4.sup.+ and CD8.sup.+ T cells in each
animal. Splenocytes adoptively transferred from the day 57
"tolerant" animal into a native Lewis recipient failed to affect
the rate at which that animal rejected a Brown-Norway cardiac
allograft. Thus, tolerance did not appear to be maintained by
suppressor cells in this animal.
[0140] The fact that 4 of 5 allograft recipients treated with high
dose CTLA-4lg (0.5 mg/day.times.7 days) rejected their grafts
within the study period indicated that blockade of the
costimulatory molecule B7 did not consistently induce permanent
graft tolerance. One possible explanation was that CTLA-4lg induced
a temporary state of non-responsiveness, and that upon recovery,
recipient T cells could effect graft rejection. Alternatively,
CTLA-4lg treatment may have resulted in a state of permanent
non-responsiveness in circulating T cells by allowing target
antigen recognition without B7-dependent costimulation. Newly
matured T cells emerging from the thymus after cessation of
CTLA-4lg treatment could not be tolerized by this mechanism, and
could mediate graft rejection as a result of B7-costimulated T cell
alloreactivity. To differentiate between these two possibilities,
rats were thymectomized 3 days prior to cardiac transplantation,
and treated with daily injection of CTLA-4lg (0.5 mg/day.times.7
days) following transplantation. These animals rejected their
grafts between days 28 and 33, indicating that allograft recipients
were not dependent upon the influx of new T cells to initiate an
alloimmune response. Thus, it appears that T cells present during
the time of CTLA-4lg treatment can eventually induce graft
rejection. This may represent T cell recovery from a temporary
state of non-responsiveness, or may reflect the kinetics of T cell
trafficking during the CTLA-4lg treatment period.
[0141] C. Synergistic Effects with Cyclosporine.
[0142] Based on the ability to show that a soluble CD28 receptor
homologue, CTLA-4lg, is capable of suppressing cell-mediated
responses in vitro and in vivo, experimentation was performed to
determine whether or not this immunosuppressant has additive or
synergistic effect with cyclosporine. A mixed lymphocyte reaction
(MLR) with Brown-Norway rat lymph node cells as stimulators and
Lewis strain rat lymph node cells as responders was measured by
measuring tritiated thymidine incorporation 72 h after
cocultivation. The ability of CTLA-4lg at a concentration of 0.1
.mu.g/ml and cyclosporine at 30 ng/ml alone or in combination was
measured. FIG. 14 shows .sup.3H-thymidine incorporation under
various conditions. As can be seen in FIG. 14, although either
immunosuppressant led to only a partial reduction in the mixed
lymphocyte proliferative response (MLR+CTLA-4lg and MLR+CSP), the
combination of the two (MLR+CSP+CTLA-4lg) completely blocked the
mixed lymphocyte reaction between these MHC-incompatible strains.
This effect is greater than what would be expected from two
immunosuppressive reagents which have additive effects, suggesting
that CTLA-4lg and cyclosporine block T cell activation by
independent mechanisms and have a synergistic effect on T cell
activation in response to alloantigens.
SPECIFIC EXAMPLE XI
Control of Lymphokine Production by Second Messengers
[0143] A variety of second messengers in the regulation of
lymphokine production were examined. In particular, a role for the
two primary cell secondary messenger systems, the activation of
protein kinase C and elevation in intracellular calcium, were
characterized as being central regulators of the transcription of
lymphokine genes. In addition, specific tyrosine phosphorylation
events were identified that may correlate with the generation of
alterations in translation and/or MRNA stability. Further
investigations into serine and threonine kinases indicate that they
may also have a role in the signal transduction events involved in
lymphokine production. In contrast, experiments into the regulation
by cGMP showed that this agent has relatively non-specific effects
on lymphokine production.
[0144] Tyrosine phosphorylation events related to CD28 were further
studied as described below.
Protocol
[0145] Monoclonal antibodies. Anti-CD2 mAb G19-4 (IgG1), anti-CD28
mAb 9.3 (IgG2a), anti-CD5 mAb 10.2 (IgG2a), and anti-CD45 mAb 9.4
(IgG2a) were produced, purified and in some cases, biotinylated as
described in Ledbetter, J. A. et al., J. Immunol., 135:2331 (1985)
and Ledbetter, J. A. et al., J. Immunol., 137:3299 (1986). Anti-B7
mAb 133-(IgM) and dilutions of ascites as described in Freedman, A.
S. et al., J. Immunol., 139:3260 (1987), were used. Anti-CD3 mAb
OKT3 (IgG2a) was absorbed to goat anti-mouse IgG covalently linked
to microspheres (KPL, Gaithersburg, Md.), by incubation of a
1/10.sup.5 dilution of pooled ascites with 10.sup.7 beads/ml in
HBSS at room temperature, followed by extensive washing.
[0146] Cells. The CD28.sup.+ subset of T cells was isolated from
peripheral blood T lymphocytes by negative selection using
immunoabsorption with goat anti-mouse lg-coated magnetic particles
as previously described in June, C. H. et al., Mol. Cell. Biol.,
7:4472 (1987). This resulted in a population of resting T cells
that was >99% CD3.sup.+ and that did not contain
CD2.sup.+/CD3.sup.- cells such as NK cells. The Jurkat T leukemia
cell line E6-1 was a gift from Dr. A. Weiss and maintained in
complete media, i.e. RPMI 1640 containing 2 mM L-glutamine, 50
.mu.g/ml gentamycin, and 10% FCS (HyClone Laboratories, Logan,
Utha). In some instances, T cells or Jurkat cells were cultured in
complete media, or in complete media with 5 ng/ml PMA (Sigma
Chemical Co., St. Louis, Mo.) or OKT3 beads (.+-.15 beads/cell)
before experiments. The Jurkat J32 cell line (CD2.sup.+, CD3.sup.-,
CD28.sup.+) has been described in Makni, H. et al., J. Immunol.,
146:2522 (1991). J32 variants (CD2.sup.+, CD3.sup.-, CD28.sup.+)
were derived by .gamma. irradiation-induced mutagenesis and
immunoselection (see Makni, supra (1991)); one such cloned mutant,
J32-72.4 is stable in culture. The surface receptor expression of
these cells was quantitated by indirect immunofluorescence and
analyzed by flow cytometry. The mean log fluorescence intensity for
each sample was determined and was converted into linear relative
fluorescence units (.DELTA.FL) by the formula
.DELTA.FL=10.sup.[(E-C)/D]; where E is the mean log fluorescence
intensity of the experimental antibody sample, C is the mean log
fluorescence intensity of the control antibody sample, D is 50
channels/decade. For the TCR/CD3 and CD28 receptors, .DELTA.FL of
the J32 cells was 27.0 and 57.0, and for the J32-74.2 cells 1.1 and
40.7. Northern blot analysis of J32-72.4 revealed no detectable
TCR-.beta. mRNA, while the expression of the TCR-.alpha.,
CD3-.gamma., .delta., and .epsilon. and TCR .zeta. mRNA was similar
to that of the parental J32 cells (unpublished data).
[0147] B7 transfection of CHO cells. CHO cells were transfected
with B7 cDNA as previously described in Gimmi, C. D. et al., PNAS
(USA), 88:6575 (1991). These cells have previously been shown to
stimulate lymphocyte proliferation and lymphokine secretion in a
manner that mimics CD28 mAb-induced T cell activation. See Linsley,
P. S. et al., J. Exp. Med., 173:721 (1991) and Gimmi, supra (1
991). Transfected CHO cells showing no B7 expression were recloned
and are referred to as CHO-B7.sup.-. CHO cells were detached from
tissue culture plates by incubation in PBS with 0.5 mM EDTA for 30
m and fixed in 0.4% paraformaldehyde as described in Gimmi, supra
(1991). Fixed CHO-B7.sup.- cells were used as control cells.
[0148] Immunoblot analysis of protein tyrosine phosphorylation.
Details of the immunoblot assay with anti-phosphotyrosine
antibodies has been described in Hsi, E. D. et al., J. Biol. Chem.,
264:10836 (1989) and June, C. H. et al., J. Immunol., 144:1591
(1990). Cells were suspended at 5-10.times.10.sup.7 cells/ml in
reaction media, i.e., HBSS containing 0.8% FCS and 20 Mm Hepes at
37.degree. C. at time -3 m and stimulated at time 0 m. mAbs were
used at 10 .mu.g/ml final concentration. For crosslinking,
biotinylated mAbs were incubated with cells for 5-8 m at room
temperature, the cells prewarmed at time 3 min and stimulated with
avidin (Sigma Chemical Co.) at a final concentration of 40 .mu.g/ml
at time 0. Stimulation was terminated by the addition of ice-cold
10.times. lysis buffer, yielding a final concentration of 0.5%
Triton X-100. See June, J. Immunol., supra (1990). After lysis at
4.degree. C., nuclei were pelleted and postnuclear supernatants
were subject to SDS-PAGE on a 7.5% gel, transferred to
polyvinylidene difluoride microporous membrane (Millipore, Bedford,
Mass.) and the membranes probed with affinity-purified
anti-phosphotyrosine antibodies, labeled with .sup.125I
staphylococcal protein A (ICN, Irvine, Calif.) and exposed to x-ray
film.
Results
[0149] Herbimycin A prevents CD28-stimulated IL2 production.
Previous studies have shown that three distinct biochemical
signals, provided by phorbol esters, calcium ionophore, and
ligation of the CD28 receptor with mAb, are required to cause
optimal IL-2 secretion (see June, C. H. et al., J. Immunol.,
143:153 (1989)). Cells cultured in the presence of PMA, ionomycin,
or CD28 mAb alone produced no detectable IL-2 and, as previously
reported in June, J. Immunol., (1989) supra, and Fraser, J. D. et
al., Science (Wash., D.C.) 251:313 (1991), stimulation of the CD28
receptor strongly up-regulated IL-2 production of T cells
stimulated with immobilized anti-CD2 mAb, PMA, or PMA plus
ionomycin. To address the potential role of tyrosine kinases in
CD28-triggered signaling, the effect of herbimycin A, an inhibitor
of the src family protein tyrosine kinases (see Uehara, Y. et al.,
Biochem. Biophys. Res. Commun., 163:803 (1989)), on the
CD28-triggered enhancement of IL-2 production was investigated. T
cells were cultured overnight in the absence (depicted as open bars
in FIG. 15) or presence (depicted as filled bars in FIG. 15) of
herbimycin A (1 .mu.M). The cells were then cultured for a further
24 h period in the presence of medium-immobilized anti-CD3 mAb
(G19-4), PMA (3 ng/ml) (P), or PMA plus ionomycin (150 ng/ml) (P+l)
in the presence or absence of soluble anti-CD28 mAb 9.3 (1 ug/ml).
Cell-free supernatant was collected, dialized to remove herbimycin
A and serial dilutions were analyzed for IL-2 content by bioassay
as described in June, J. Immunol., supra (1989). FIG. 15 shows the
effect of herbimycin A on CD28-stimulated IL-2 production. The CD28
mAb mediated enhancement of IL-2 production in response to
stimulation with immobilized anti-CD3, or PMA was nearly completely
inhibited in the presence of herbimycin A. In contrast, cells
cultured in PMA, ionomycin or 9.3 mAb only produced <10 U/mI of
IL-2.
[0150] Disruption of the proximal signaling pathway triggered
through CD3 could potentially explain the effect of herbimycin on
cells stimulated with anti-CD3 and anti-CD28. Consistent with this,
CD3-triggered IL-2 production was previously shown to be
exquisitely sensitive to herbimycin A. See June, C. H. et al., PNAS
(USA), 87:7722 (1990). However, IL-2 production induced with the
combination of PMA plus ionomycin or PMA plus CD28 stimulation
permits, in principle, the ability to isolate the CD28 signal for
testing the effect of herbimycin A. PMA plus anti-CD28-stimulated
IL-2 production was sensitive to the effects of herbimycin A while,
as previously noted, PMA plus ionomycin-stimulated IL-2 secretion
was resistant to the effects of herbimycin A. The combination of
PMA plus ionomycin plus anti-CD28-stimulation resulted in more IL-2
secretion than optimal amounts of PMA plus ionomycin, consistent
with the previous reports of June, J. Immunol., supra (1989); and
Fraser, J. D. et al., Science (Wash. D.C.) 251:313 (1991). However,
in the presence of herbimycin A, PMA plus ionomycin plus
CD28-stimulated cells produced approximately equivalent amounts of
IL-2 as cells stimulated in the absence of herbimycin with PMA plus
ionomycin. Together, the above results suggest that the function of
both the TCR and CD28 receptors are sensitive to herbimycin, and
further suggest the independent effects of these three reagents on
IL-2 gene expression. See June, J. Immunol., supra (1989); and
Fraser, supra (1991).
[0151] CD28 receptor crosslinking with mAb induces protein tyrosin
phosphorylation in PMA-treated Jurkat cells. Given the above
functional results, the potential involvement of protein tyrosine
phosphorylation in CD28-mediated signal transduction was
investigated by immunoblot analysis of postnuclear supernatants of
whole cell lysates of the T cell leukemia line Jurkat E6-1. Jurkat
E-6 cells were cultured for 2 days in the presence or absence of
PMA (5 ng/ml). After washing, 10.sup.7 cells in 120 .mu.l were
stimulated with reaction media (control), anti-CD3 Mab (G19-4),
anti-CD28 mAb (9.3), or crosslinked anti-CD28 mAb (9.3) (final
concentration, 10 .mu.g/ml). For crosslinking, biotinylated mAb was
added at time 10 m, followed by avidin (40 .mu.g/ml) at time zero.
After 2 m, the reaction was terminated with ice-cold lysis buffer
and postnuclear supernatants were resolved by SDS-PAGE
electrophoresis, transferred to immobilon, and immunoblotted with
antiphosphotyrosine, followed by .sup.125I-protein A and
autoradiography.
[0152] In a previous report by Ledbetter, J. A. et al., Blood,
75:1531 (1990), increased tyrosine phosphorylation could not be
detected in resting T cells after crosslinking the CD28 receptor.
Consistent with that report, no changes in tyrosine phosphorylation
were detected in unstimulated Jurkat cells after the binding of
bivalent or crosslinked CD28 mAb. Previous studies have shown that
CD28 stimulation alone does not result in lymphokine production in
Jurkat cells or induce proliferation of primary T cells. See Weiss,
A. et al., J. Immunol., 137:819 (1986); Martin, P. J. et al., J.
Immunol., 136:3282 (1 986); and Hara, T. et al., J. Exp. Med.,
161:1513 (1985). Engagement of CD28 by CD28 mAbs or by B7, the
natural CD28 ligand, delivers a costimulatory signal provided T
cells are stimulated with PMA or with TCR/CD3 mAbs. See June, C. H.
et al., Immunol. Today, 11:211 (1990); Koulova, L. et al., J. Exp.
Med., 173:759 (1991); Linsley, P. S. et al., J. Exp. Med., 173:721
(1991); and Gimmi, C. D. et al., PNAS (USA) 88:6575 (1991). It thus
appeared that CD28-induced protein tyrosine phosphorylation might
only occur in the context of a costimulatory signal.
[0153] To test this hypothesis, Jurkat cells were cultured in PMA
and then stimulated with anti-CD28 mAb as previously described. In
the PMA-stimulated cells, crosslinking of CD28 for 2 m induced
phosphotyrosine on substrates migrating with approximate molecular
masses of 47, 62, 75, 82, 100, 110, and 145 kD. Bivalent CD28 mAb
induced tyrosine phosphorylation, but to a lesser magnitude. In
agreement with June, C. H. et al., J. Immunol., 144:1591 (1990),
CD3 triggering of Jurkat cells induced tyrosine phosphorylation of
phosphoprotein (pp) 56, pp65, pp75, pp 100, pp110, and pp145 in
resting Jurkat cells and in PMA-treated Jurkat cells. Of particular
interest were pp75 and pp100, which were consistently
phosphorylated by CD28 stimulation under all conditions tested.
[0154] CD28 receptor crosslinking with Mab induces protein tyrosine
phosphorylation in normal T cells. Similar experiments with highly
purified peripheral blood T cells from normal human donors were
performed in order to determine if CD28 could increase tyrosine
phosphorylation in nontransformed cells. Peripheral blood
CD28.sup.+ T cells were cultured in PMA (5 ng/ml) for 6 h. After
washing, 10.sup.7 cells were stimulated for 2 m with media
(control), anti-CD3 mAb (G19.4), anti-CD28 mAb (9.3), crosslinked
anti-CD28 mAb (9.3), or crosslinked anti-CD5 mAb (1 0.2). Cells
were lysed and protein tyrosine phosphorylation was determined as
previously described. Crosslinking of CD28 on PMA-treated cells
induced the appearance of tyrosine phosphorylated substrates that
migrated at 45, 75, and 100 kD. Again, pp75 and pp100 were most
prominent and consistently reproduced.
[0155] The effects of CD28 stimulation observed after 24-48 h of
PMA stimulation were more pronounced than those seen after 6 h.
Ligation of CD28 by mAb on resting T cells caused the appearance of
weakly detected tyrosine phosphorylation. The induction of
increased responsiveness to anti-CD28 mAb stimulation by PMA is
slow in that 4-6 h of PMA treatment are required to consistently
observe CD28-induced tyrosine phosphorylation. Experiments with
cycloheximide indicate that new protein synthesis is required for
cells to become responsive to CD28. The specificity of the
CD28-induced tyrosine phosphorylation was investigated by
crosslinking CD5 with an isotype-matched mAb. Increased tyrosine
phosphorylation on the 75 kD substrate was occasionally induced by
CD5 crosslinking. In contrast, CD5 never induced tyrosine
phosphorylation on pp100. Similarly, crosslinking of the MHC class
I receptor also did not induce tyrosine phosphorylation of this
substrate.
[0156] CD28 receptor crosslinking induces protein tyrosine
phosphorylation in CD3-treated normal T cells. The above
experiments suggested that the CD28 receptor is relatively inactive
in quiescent cells, and becomes responsive consequent to protein
kinase C activation. To determine whether TCR stimulation could
also prime cells for the CD28 signal, T cells were cultured
overnight in medium or in the presence of anti-CD3-coated beads.
The cells were recovered, and 8.times.10.sup.6 cells were
stimulated with crosslinked anti-CD28 mAb for 0-5 m, the cells
lysed, and protein tyrosine phosphorylation determined as
previously described. Crosslinked CD28 mAb induced low level
tyrosine phosphorylation on multiple substrates in resting T cells
that peaked 2-5 m after CD28 stimulation. In contrast, CD28 mAb
induced marked tyrosine phosphorylation in CD3-primed cells that
was maximal within 1 m. Thus, costimulation of T cells with
anti-CD3 augmented CD28-induced tyrosine phosphorylation as
manifested by an increased magnitude of response and an accelerated
kinetics of response. This induction of responsiveness to CD28 did
not require DNA synthesis, as separate studies have shown that the
T cell blasts used for these studies were in the late G.sub.1 phase
of the cell cycle.
[0157] CD28 receptor-B7/BB1 receptor interaction induces specific
tyrosin phosphorylation in T cells. The above results indicate that
CD28 mAb can increase tyrosine phosphorylation in a variety of
substrates on preactivated T cells. Previous studies have indicated
that CD28 appears to deliver two biochemically distinct signals,
depending on the degree of crosslinking. See Ledbetter, J. A. et
al., Blood, 75:1531 (1990). The unique functional properties of
CD28 mAb observed after stimulation of T cells do not require
highly crosslinked CD28 mAb and are obtained using intact or
F(ab').sub.2 CD28 mAb. As discussed in Hara, T. et al., J. Exp.
Med., 161:1513 (1985), studies have shown that CHO cells expressing
the CD28 ligand mimic the functional effects of CD28 mAb. See
Linsley, supra (1991), and Gimmi, supra (1991). These cells
presumably represent a more physiologic means to study CD28
receptor-mediated signal transduction. CHO-B7.sup.+ cells were
incubated with PMA-treated T cells at a CHO/T cell ratio of 1:10
for 5-30 m. B7-transfected CHO cells not expressing B7 on the cell
surface (CHO-B7.sup.- cells) were used as controls. Before the
stimulation, CHO cells were fixed with paraformaldehyde to decrease
phosphotyrosine background. Previous studies have indicated that
this treatment leaves intact B7-CD28 interaction and the ensuing
functional effects. See Gimmi, supra (1991). For the time zero
point, lysis buffer was added to the T cells first, immediately
followed by addition of CHO cells to the mixture. CHO-B7.sup.+
cells induced specific tyrosine phosphorylation that was detected
primarily on a substrate that migrated at 100 kD. The
CHO-B7-induced tyrosine phosphorylation was detectable within 5 m
of stimulation and remained elevated at plateau levels for at least
30 m. CHO-B7-induced tyrosine phosphorylation was evident at a
variety of CHO-T cell ratios, and has been consistently observed
for only the 100 kD substrate. CHO-B7.sup.- cells did not induce
tyrosine phosphorylation of pp100. The B7-induced tyrosine
phosphorylation was dependent upon CD28-B7 interaction as
preincubation of CHO cells with anti-B7 mAb prevented CHO-B7
induced pp100 tyrosine phosphorylation. B7-CHO cells induced a
slight increase in pp100 tyrosine phosphorylation in some
experiments; however, this was not consistently observed.
[0158] In other experiments, alloantigen-induced T cell blasts were
tested for CD28-induced tyrosine phosphorylation. T cells were
culture for 8 days with allogeneic irradiated cells and then
stimulated with CD28 mAb. Tyrosine phosphorylation that was most
pronounced on the 74 and 100 kD substrates was observed. Thus, CD28
stimulation of T cells preactivated with alloantigen, CD3 mAb, or
PMA can induce tyrosine phosphorylation on a limited number of
substrates that is early in onset and brief in duration.
[0159] CD28-induced tyrosine phosphorylation prevented by CD45 and
by herbimycin. Given that protein tyrosine kinase inhibitor
herbimycin A could efficiently inhibit CD28-induced IL-2 secretion,
this inhibitor was tested for effects on CD28-induced tyrosine
phosphorylation. T cells were treated overnight with PMA (5 ng/ml)
in the presence of the indicated concentration of herbimycin A or
in control medium. The cells were collected, washed, and
8.times.10.sup.6 cells were stimulated with media or with
crosslinked anti-CD28 mAb for 2 m. Detergent-soluble proteins were
processed as previously described. Tyrosine phosphorylation induced
by anti-CD28 mAb was nearly completely prevented in
herbimycin-treated cells under conditions that specifically inhibit
CD28-induced IL-2 production.
[0160] The brief temporal course of CD28 mAb-induced tyrosine
phosphorylation suggested regulation by a phosphatase. To address
the effects of phosphatases on CD28-mediated signal transduction, T
cells were cultured overnight with PMA (5 ng/ml). 107 cells were
incubated for 10 m with media (control), biotinylated anti-CD45 mAb
(9.4), anti-CD28 mAb (9.3), or both. Monoclonal antibodies were
crosslinked with avidin at time 0. The reaction was terminated
after 2 m. Immunoblot analysis with antiphosphotyrosine antibodies
of detergent-soluble proteins was performed as previously
described. CD28 crosslinking induced tyrosine phosphorylation on
pp75 and pp100 that was completely prevented by CD45. Consistent
with previous results described in Samelson, L. E. et al., J.
Immunol., 145:2448 (1990), crosslinking of CD45 alone caused
increased tyrosine phosphorylation of a 120-135 kD substrate; this
effect is also seen in CD28 plus CD45-treated cells. Thus, the
above studies indicate that CD28-induced tyrosine phosphorylation
is sensitive to an inhibitor of src family protein tyrosine
kinases, and furthermore, that the CD45 protein tyrosine
phosphatase can prevent CD28-induced protein tyrosine
phosphorylation.
[0161] CTLA-4 expression predicts expression of IL-2 following CD28
pathway activation. Purified resting T cells were stimulated with
immobilized anti-CD2 Ab, anti-CD3+mAb 9.3, PMA+ionomycin, PMA+mAb
9.3 and PMA+ionomycin+mAb 9.3 in the presence or absence of the
protein-tyrosine kinase inhibitor herbimycin for 8 h. Duplicate
Northern blots were hybridized to CTLA-4, CD28, IL-2 or HLA
specific probes. Expression of CD28, CTLA-4 and IL-2 was then
analyzed by Northern blot. IL-2 expression correlated well with
CTLA-4 expression following CD28 pathway activation. CTLA-4 and
IL-2 expression were also suppressed to a similar degree with
herbimycin while CD28 expression remained unchanged. This suggests
that the suppressive effects of protein-tyrosine kinase inhibitors
on CD28 pathway activation may be mediated through suppression of
CTLA-4 expression.
SPECIFIC EXAMPLE XII
Induction of MHC Independent T Cell Proliferation by CD28 and
Staphylococcus Enterotoxins
Protocol
[0162] Isolation of T cells. Peripheral blood was drawn from normal
human volunteers. The mononuclear cell fraction was obtained by
density gradient centrifugation through a Ficoll-Hypaque
(Pharmacia) cushion. This fraction was used in experiments
utilizing peripheral blood mononuclear cells (PBMCs). Purified
resting T cells were obtained by incubating the mononuclear cells
with an antibody cocktail directed against B cells, monocytes and
activated T cells. The antibody coated cells were then removed by
incubation with goat anti-mouse immunoglobulin-coated magnetic
beads (Advanced Magnetics Inc.) as previously described in June,
supra (1987). This method has routinely yielded a population
>99% CD2.sup.+ by flow cytometry.
[0163] Proliferation assays. Proliferation was measured by
culturing 5.times.10.sup.5 purified T cells or PBMC's in each well
of a 96 well microtiter plate. The final culture volume was 200
.mu.l of RPMI 1640 (Gibco) supplemented with 10% FCS, penicillin
(100 U/ml), streptomycin (100 .mu.g/ml) and 2 Mm L-glutamine.
Staphylococcal Enterotoxin A (SEA), Staphylococcal Enterotoxin B
(SEB) (Toxin Technologies) and cyclosporine A (Sandoz) were added
in the indicated doses at the initiation of the culture. Anti-CD28
monoclonal antibody (mAb 9.3, gift from J. Ledbetter) and
anti-HLA-DR monoclonal antibody (mAb L243, gift from J. Ledbetter)
were added at the start of the culture period. Tritiated thymidine
(.sup.3H-TdR, ICN) was included at a concentration of 1 .mu.Ci per
well for the final 8 h of the culture. The cells were harvested
onto glass microfiber filter strips (Whatman) after 72 h using a
PHD cell harvester (Cambridge Technologies) and counted on a liquid
scintillation counter (LKB). All values are expressed as the mean
cpm.+-.standard deviation of triplicate or quadruplicate
cultures.
[0164] Flow Cytometry. One ml cultures of T cells were incubated
with media alone, SEA (100 ng/ml) or SEB (1 .mu.g/ml), SEA or SEB
plus anti-CD28 antibody (1 .mu.g/ml) or PMA (3 ng/ml) plus
anti-CD28 antibody (1 .mu.g/ml) at 37.degree. C. for 72 h. Aliquots
of each sample were stained with acridine orange (Polysciences) for
cell cycle analysis as described in Darzynkiewicz, Z., Meth. Cell.
Biol., 33:285 (1990), FITC conjugated anti-IL2 receptor antibody
(Coulter), FITC conjugated anti-HLA-DR antibody (Becton-Dickinson)
or an isotype-matched irrelevant antibody (Becton-Dickinson). Each
sample was analyzed on a FACScan flow cytometer
(Becton-Dickinson).
Results
[0165] CD28 provides costimulatory activity for
superantigen-activae depurified T cells. Highly purified T cells
were cultured with graded concentrations of either SEA (0.1 ng/ml
to 1.0 .mu.g/ml) or SEB (.01 .mu.g/ml to 100 .mu.g/ml). Replicate
cultures were prepared in which a stimulatory antibody to CD28 was
added. The cultures were pulsed with .sup.3H-TdR for the final 8 h
of a 72 h culture and incorporated thymidine determined by liquid
scintillation counting as described above. Each condition was
performed in quadruplicate. Treatment with SEB alone failed to
induce thymidine incorporation above control cultures. However, the
addition of anti-CD28 antibody resulted in significant
proliferation to graded doses of SEB. Treatment with anti-CD28
antibody alone had no effect. The lack of accessory cells was
verified by an absence of proliferation to PHA. Identical results
were obtained using SEA.
[0166] Stimulation with SEA or SEB leads to cell cycle entry. Since
CD28 stimulation alone does not induce T cell cycle entry, the
observation that CD28 provided costimulatory activity for T cells
treated with either SEA or SEB suggested that these enterotoxins
could induce cell cycle entry in purified T cells. In order to
examine this, purified T cell cultures were stimulated with SEB (1
.mu.g/ml) alone or with SEB (1 .mu.g/ml) plus anti-CD28 monoclonal
antibody (1 .mu.g/ml) for 48 h and stained with acridine orange for
cell cycle analysis. Unstimulated cells were run simultaneously in
order to determine the G.sub.0/G.sub.1 interface. Those with an
increased RNA content but unchanged DNA content were considered
G.sub.1 phase cells. Cells with increases in both RNA and DNA
content were considered in S, G.sub.2 or M phases. Concomitantly,
aliquots were stained with FITC-conjugated anti-IL-2 receptor
antibody. Treatment with enterotoxin alone for 48 h resulted in
progression of greater than 10% of the T cells from G.sub.0 to
G.sub.1 as determined by an increase in RNA staining with no
increase in DNA content. Similarly, enterotoxin alone induced IL-2
receptor expression in 15% of the cells at 72 h. In contrast, when
the anti-CD28 monoclonal antibody was present, a significant
proportion of the cells that had left the G.sub.0 stage of the cell
cycle were found to have increased their DNA content and thus are
in either the S, G.sub.2 or M phases of the cell cycle. These data
indicate that stimulation with SEB alone is sufficient to activate
the T cell but delivers an inadequate signal for complete
progression through the cell cycle. Provision of a second signal by
simultaneous stimulation of the CD28 pathway allowed the cell to
progress to S-phase and proliferate.
[0167] Proliferation of T cells stimulated by SEA and anti-CD28 is
resistant to cycl sporine A. CD28 has been shown to utilize a
signal transduction pathway that is resistant to the effects of
cyclosporine A (CsA) when the initial signal is provided by PMA,
and partially resistant to CsA when cells are initially activated
through the T cell receptor. See June, supra (1987). In order to
further examine the pathways involved in T cell activation by
enterotoxin, cyclosporine A (1 .mu.g/ml) was included in cultures
activated by SEA and SEA plus anti-CD28 antibody (.sup.3H-TdR
incorporation determined as described above). As for SEB, SEA alone
did not induce thymidine incorporation whereas addition of antibody
against CD28 resulted in significant proliferation. Even in the
presence of cyclosporine A (1 .mu.g/ml), there was a dose dependent
increase in proliferation when cultures were activated by a
combination of SEA and anti-CD28 antibody. Control cultures
activated with PMA plus anti-CD28 antibody were resistant to
cyclosporine A and activation by PMA plus ionomycin was sensitive
to cyclosporine A.
[0168] Activation by SEB and anti-CD28 is independent of class II
MHC. Previous work has demonstrated that the staphylococcal
enterotoxins are capable of simultaneously binding the TCR and
class II MHC molecules on the surface of antigen presenting cells
(APCs). See Herrmann, T. et al., Eur. J. Immunol., 19:2171 (1989);
and Chintagumpala, M. M. et al., J. Immunol., 147:3876 (1991). The
observation that proliferation was not observed unless APCs were
present in the culture was interpreted to mean that T cell
activation by superantigen is dependent upon class II MHC
expression as discussed in Fleischer, B. et al., J. Exp. Med.,
167:1697 (1988); Carlsson, R. et al., J. Immunol., 140:2484 (1988);
and Herman, A. et al., J. Exp. Med., 172:709 (1990). Our
observation that highly purified T cells could be induced to
proliferate by simultaneous stimulation with enterotoxin and
anti-CD28 antibody suggested that class II MHC may not be
absolutely required for superantigen activation of T cells.
Alternatively, activated T cells can express class II MHC and thus
might provide class II-dependent superantigen presentation to other
T cells in trans.
[0169] To examine this possibility, a blocking antibody against
HLA-DR, monoclonal antibody L243, was included in cultures of T
cells and PBMCs activated by enterotoxin or enterotoxin plus
anti-CD28 antibody as shown in FIG. 16. Each point is expressed as
the mean.+-.the standard deviation of triplicate or quadruplicate
cultures. No significant proliferation was observed with SEB alone.
As shown previously, inclusion of anti-CD28 antibody allows SEB to
induce T cell proliferation in a dose-dependent manner. There was
no decrease in proliferation when anti-class II antibody was
included in the cultures at doses of 1.0 or 10 .mu.g/ml (data for
10 .mu.g/ml not shown). In contrast, as shown in FIG. 17, the
proliferation of PBMCs isolated from the same donor and stimulated
with enterotoxin was significantly inhibited by anti-class II
antibody. In addition, HLA-DR expression at 24 and 72 h was
examined by purified T cells activated with SEA (0.1 or 1.0 ng/ml)
with and without CD28 costimulation. There was no expression of
HLA-DR in either condition as determined by flow cytometry. This
indicated that the T cell proliferation induced by
enterotoxin+anti-CD28 is not dependent on presentation by an MHC
class II molecule.
SPECIFIC EXAMPLE XIII
Prevention of Programmed Cell Death
[0170] A series of experiments were done to test whether anti-CD28
mAb might prevent cell death in mature T cells. Jurkat leukemia
cells are commonly used as an example of mature T cells that mimic
physiologic effects found in peripheral blood T cells. For example,
Jurkat cells can be induced to secrete IL-2 with anti-CD3 mAb and
anti-CD28 mAb, and Jurkat cells can be infected and killed by
HIV-1. The Jurkat line JHMI-2.2 was obtained from A. Weiss (UCSF);
the muscarinic M.sub.1 receptor subtype has been transfected and is
stably expressed in these cells. JHMI-2.2 cells,
0.3.times.10.sup.6/well, were added to culture wells in complete
medium, or to wells that contained plastic-adsorbed anti-CD3 mAb
G19-4, in the presence or absence of 9.3 mAb 10 .mu.g/ml, or 9.3
mAb alone. Cell death was scored after 1 to 3 days of culture and
graded as 0 (none), 1+ (20 to 70% of cells dead), and 2+ (70 to
100% of cells dead), and was determined by visual inspection of the
wells, and confirmed by trypan blue permeability. As shown in Table
10, cells in medium continued to grow and remain viable while cells
in anti-CD3-treated wells died. In contrast, the cells in wells
containing anti-CD3 plus anti-CD28 continued to proliferate. The
ability of anti-CD28 to rescue cells from anti-CD3-induced cell
death was specific, because carbacol (30 .mu.M) (a specific agonist
of M.sub.1 receptors that is believed to activate signal
transduction in cells via a mechanism distinct from the T cell
receptor/CD3 complex), also induced cell death in Jurkat cells.
Anti-CD28 did not prevent carbacol-induced cell death.
11 TABLE 10 CONDITION CELL DEATH (GRADE 0-2) EXPERIMENT #1 Medium 0
anti-CD3 2+ anti-CD3 + anti-CD28 0 EXPERIMENT #2 Medium 0 Carbacol
1+ Carbacol + anti-CD28 2+
SPECIFIC EXAMPLE XIV
Bone Marrow Studies
[0171] Proliferation of T cells after activation of T cells with
soluble or immobilized anti-CD3 (OKT3). A series of titration
studies were performed using soluble or immobilized OKT3 to
activate and induce T cell proliferation. Immobilized OKT3 (2
.mu.g/ml precoated plates for 1 h at 37.degree. C.) and soluble
OKT3 (10 ng/ml) consistently induced T cell proliferative responses
from E-rosette (E.sup.+) purified T cells or PBL. PBL or purified T
cells were activated by incubation for 1 h to 7 days on immobilized
OKT3 or by adding 10 ng/ml of soluble OKT3 at the beginning of
culture. In a series of experiments, proliferation after activation
of T cells with immobilized OKT3 was comparable to proliferative
responses by PBL after activation with soluble OKT3.
[0172] Cytotoxicity mediated by anti-CD3 and anti-CD28 triggered
PBL The cytotoxicity of anti-CD3 activated PBL after 7 days of
culture in the presence of low doses of IL-2 or anti-CD28 was
tested. In this set of experiments, the ability of CD28 to induce
increases in lymphokine production to substitute for previously
reported immune augmented effects of in vitro T cell treatment with
IL-2 has been examined. One lytic unit is equivalent to 20% lysis
of 5.times.10.sup.3 target cells per 1.times.10.sup.6 effector
cells as discussed in Press, H. F. et al., J. Clin. Immunol.
1:51-83 (1981). Various targets, including Daudi and K562, were
tested. Cytotoxicity results of a representative experiment are
shown in Table 11.
12 TABLE 11 CELL GROWTH Target - Daudi Target - K562 STIMULUS (LU)
(LU) anti-CD3 plus IL-2 14.9 10.6 anti-CD3 plus anti-CD28 12.1
6.1
[0173] OKT3-induced cytotoxicity in PBL comparable to T cells. In 5
different subjects, PBL were compared with T cells (E.sup.+) in
their ability to kill Daudi, K562, and BSB cells 8 days after being
activated with OKT3. These experiments were performed in X-Vivo 10
supplemented with 5% human serum (HS). The mean cytotoxicity in 5
normal subjects using PBL directed at Daudi, K562, and BSB were
15.0, 7.4, and 9.2 LU, respectively. In T cells from the same 5
subjects, the mean cytotoxicity directed at Daudi, K562, and BSB
were 14.3, 7.7, and 9.3 LU, respectively.
[0174] Cytotoxicity as a function of in vitro time in cell culture.
In order to test for optimal cytotoxicity, T cells were cultured
with anti-CD3 and IL-2 for 31 days and tested at weekly intervals
for cytotoxicity against Daudi and K562. Logarithmic cell growth
was maintained during this time, with a 300-fold expansion in cell
number. Cytotoxicity was a strong function of culture duration
however, with <0.1, 24, 3.5, 0.5, 1.0 LU at 0, 8, 15, 22, and 29
days of culture. Similar results were found when Daudi was the
target, with <0.01, 23, 5, 2, and 5 LU at 0, 8, 15, 22, and 29
days of culture.
[0175] Cytotoxicity induced by soluble or immobilized OKT3. In 7
experiments, cytotoxicity mediated by T cells after activation was
compared with soluble or immobilized OKT3. Both methods induced
cytotoxicity directed at Daudi and K562. Soluble OKT3 activated T
cells mediated a mean cytotoxicity of 27 LU (SD-18) directed at
Daudi and a mean cytotoxicity of 21 LU (SD -16) directed at K562.
Immobilized OKT3 activated T cells mediated a mean cytotoxicity of
22 LU (SD-16) directed at Daudi and a mean cytotoxicity of 12 LU
(SD-8) directed at K562. These experiments were performed with
E.sup.+ cells in RPMI 1640 supplemented with 10% fetal bovine serum
(FBS). Cytotoxicity was assessed 7 to 8 days after triggering with
soluble (10 ng/ml) or immobilized (2 .mu.g/ml) OKT3.
[0176] Eff cts f IL-2 concentrations. The dose of IL-2 was titrated
after establishing the optimal time of OKT3 activation. In several
experiments, the doses of IL-2 were gradually reduced from 6000
IU/ml to 60 IU/ml. Proliferation, as measured by tritiated
thymidine incorporation after 3 days of culture, remained constant
as IL-2 was titrated in this range. In contrast, cytotoxicity
varied with the dose of IL-2, and was maximal at lower doses of
IL-2 (lytic units with Daudi targets were 28, 9, 8.5, 9 and 4.8 LU
after culture in 30, 150, 300, 600 and 6000 IU/ml of rIL-2). The
data show that both proliferative and cytotoxic responses of the
anti-CD3 triggered T cells can be obtained and maintained in low
doses of IL-2.
[0177] Effects of serum and medium on proliferation and
cytotoxicity. The ability of HS, FBS, and serum free media were
compared for their ability to support growth and maintain
cytotoxicity. The data show that proliferation and cytotoxicity
directed at Daudi, K562, and BSB rapidly decreased below a serum
concentration of 2%. There were no significant differences between
X-Vivo 10 and RPMI 1640.
[0178] Bone marrow mononuclear cells (BMMNC) as a source of CTC
after anti-CD3 and anti-CD28 treatment. To test whether BMMNC might
serve as a source of T cells for therapy in patients with
malignancies, BMMNC were cultured in X-Vivo 10 medium after OKT3
and IL-2 or anti-CD28 stimulation. Although the BMMNC population
initially contained only 25% CD3.sup.+ cells, proliferative and
cytotoxic responses were excellent after 2 weeks of culture. T
cells expanded more than 40-fold after CD3 and IL-2 stimulation and
cytotoxicity was between 5 and 12 LU at 8 to 15 days of culture
when tested against Daudi and K562. These data show that BMMNC
obtained from autologous bone marrow harvest from a patient before
bone marrow transplantation provide a suitable source of cytotoxic
T cells. Table 12 shows that both normal and patient bone marrows
provide satisfactory sources of cytotoxicity after CD3 and IL-2
treatment. In 4 experiments using normal bone marrow or autologous
bone marrow, there was a median of 89-fold expansion of cells
(range 18- to 173-fold) after 9 to 19 days of culture. Mean
cytotoxicity directed at Daudi and K562 mediated by BMMNC
stimulated with OKT3 was 5.5 LU (range 2-11) and 4.3 LU (range
2-8), respectively. All cultures were tested 14 days after
activation with OKT3 and expanded in the presence of 50 IU/ml of
IL-2. Either soluble OKT3 10 ng/ml(s) or immobilized OKT3 (coated
with 2 .mu.g/ml)(l) were added as indicated.
13TABLE 12 Daudi K562 Source OKT3 (S or I) Fold Increase LU LU PBL
1 I 47 6 3 2 I 4 11 11 3 S 12 9 6 4 S 176 5 4 BMMNC 1 I 89 2 2 2 I
18 11 8 3 S 22 6 5 4 S 173 3 2
[0179] Anti-CD3 plus anti-CD28 treatment of BMMNC as a source of
effector T cells. Proliferative and cytotoxic responses from 3
patients were tested. The patients had received extensive
chemotherapy and yet their PBL or BMMNC maintained strong
proliferative and cytotoxic responses after anti-CD3 plus anti-CD28
treatment. PBL or BMMNC (1.5.times.10.sup.5) were cultured in RPMI
plus 5% human serum in the presence of immobilized OKT3 mAb or 50
IU/ml rIL-2 or 9.3 mAb 0.5 .mu.g/ml. Proliferation was assessed on
day 3 of culture and cytotoxicity on day 7. Table 13 summarizes the
results for BMMNC.
14TABLE 13 Proliferation .sup.3H Cytotoxicity Cytotoxicity SAMPLE
incorp. (cpm) Daudi (LU) K562 (LU) Bone Marrow #171 AML, Relapsed
OKT3 23,479 -- -- OKT3 + anti-CD28 mAb 58,563 18.0 4.8 OKT3 + IL-2
48,745 15.1 4.6 PBL P#632 non-Hodgkins lymphoma, pre-transplant
OKT3 58,670 -- -- OKT3 + anti-CD28 mAb 77,046 14.8 12.7 OKT3 + IL-2
63,603 18.8 18.1 PBL P#635 Hodgkins lymphoma, pre-transplant OKT3
27,758 -- -- OKT3 + anti-CD28 mAb 46,133 -- -- OKT3 + IL-2 43,918
-- --
[0180] OKT3-activated T cells (CTC) do not inhibit hematopoietic
progenitor growth. In order to determine whether BMMNC mixed with
OKT3-activated T cells (CTC) in hematopoietic progenitor assays
would inhibit the development of CFU-GM, CTC obtained from PBL
after a week of growth was mixed with fresh BMMNC and plated the
mixtures into the CFU-GM assay. The autologous CTC were mixed with
BMMNC in various ratios, incubated for 1 h at 37.degree., and then
plated in a standard CFU-GM assay. The CTC had no deleterious
effect on colony formation, as the number of CFU-GM colonies was
within 75% of control over a wide variety CTC:BMMNC ratios (ratios
of 1:25 to 5). The number of CFU-GM colonies was not inhibited
greater than 90% (an accepted % inhibition of CFU-GM in purged
autologous marrow grafts) even at a ratio of 1 CTC to 1 BMMNC.
These data suggest that CTC will not inhibit or delay engraftment
of autologous bone marrow transplants.
[0181] Expansion and cytotoxic functions of PBL or BMMNC from
patients before BMT. In order to determine whether PBL or BMMNC
from patients heavily pretreated for AML or lymphoma could be
activated with anti-CD3 and grown in low dose IL-2, PBL or BMMNC
obtained prior to BMT on several patients was tested. The PBL and
BMMNC of the patients tested proliferated and exhibited cytoxicity
in a fashion comparable to that seen in normal PBL or BMMNC
obtained from normal allogeneic marrow donors. It was anticipated
that some patients that had been heavily treated with chemotherapy
or radiation would have low counts or have poor responses to
anti-CD3 activation. Thus, the number of starting cells was
increased in the protocol to compensate for cell loss or inability
to proliferate. The use of 9.3 as a costimulant to anti-CD3
activated T cells to enhance helper activity or enhance
cytotoxicity could result in improved in vitro expansion of
activated T. cells. Data presented in this example (Specific
Example XIV) show that mAb 9.3 can correct proliferative defects in
post-transplant lymphocytes further supporting the rationale for
using OKT3/9.3 costimulation approach to accelerate immune
reconstitution and enhance cytotoxicity directed at mallignant
cells in ABMT recipients. A recent study by Katsanis, E. et al.,
Blood 78:1286-1291 (1991) shows that T cells from BMT recipients
can be expanded by stimulation with OKT3 and IL-2.
[0182] Messenger RNA levels for IL-2 receptors (IL-2R), IL-2, and
IL-3 in PBL from short and long-term BMT recipients. Earlier
studies (see Lum, L. G. et al., Blood Suppl. (Abstract) (1991))
suggested that T cells from BMT recipients fail to secrete IL-2 or
express IL-2R. Such defects may be due to failure of mRNA synthesis
for lymphokines or lymphokine receptors. A determination was made
whether T cells from BMT patients failed to express detectable
levels of mRNA for IL-2, IL-2R and IL-3. PBL from 11 allogeneic (3
short-term, ST, and 8 long-term, LT) and 4 autologous recipients (2
ST and 2 LT) were tested for levels of IL-2R, IL-2, and IL-3 mRNAs
without stimulation (-) or after phytohemagglutinin (PHA) and
phorbol ester (TPA) stimulation (+). cDNA synthesized by reverse
transcriptase (RTase) from total RNA was amplified by PCR using
specific primers and the PCR products run on 1.5% agarose gels
containing ethidium bromide. Table 14 shows the fraction and
percent of recipients whose PBL had detectable levels of mRNA for
IL-2R, IL-2, and IL-3.
15TABLE 14 STIMULATION IL-2R (%) IL-2 (%) IL-3 (%) Allogeneic
Recipients - 2/11 (18) 1/9 (11) 8/11 (73) + 9/11 (82) 8/9 (89) 7/10
(70) Autologous Recipients - 2/4 (50) 3/4 (75) 4/4 (100) + 4/4
(100) 3/4 (75) 3/4 (75)
[0183] PBL from a high proportion of ST and LT autologous and
allogeneic BMT recipients expressed levels of mRNAs for IL-2R,
IL-2, and IL-3 after stimulation with PHA+TPA. In the ST recipients
tested, 2 of 2 ABMT recipients and 3 of 3 allogeneic recipients
tested had PBL that expressed mRNA levels of IL-2R and IL-3; 2 of 2
allogeneic recipients tested expressed mRNA for IL-2. In most
cases, defective mRNA synthesis for IL-2R, IL-2, and IL-3 may not
be responsible for defects in IL-2 secretion and IL-2R expression.
Posttranscriptional events may play a more important role in
defective lymphokine secretion by T cells from BMT recipients.
[0184] CTC help Ig synthesis and express mRNA for lymphokines and
perforin. As discussed in Ueda, M. et al., J. Cell. Biochem.
(Abstract) (submitted 1992), helper activity was assessed by adding
normal T cells or T cells activated with OKT3 to normal B cells
after PW stimulation as measured by an ELISA-Plaque (PFC) assay.
The number of PFC per million B cells cultured was 3200, 4100, 8800
when 25, 50 or 75.times.10.sup.3 normal T cells were added. When
the same numbers of CTC were added, the number of PFC were 220,
2100, and 2600. Thus, CTC exhibit substantial helper activity.
Furthermore, CTC did not suppress normal autologous or allogeneic T
and B cells in a suppressor assay for Ig synthesis. Helper activity
was radioresistant. Messenger RNA for IL-2, IL-3, IL-6 and perforin
was detected from 6 h to more than 3 days after OKT3 activation
using a Rtase-PCR method. In summary, CTC help B cells produced Ig
and did not suppress Ig synthesis by normal T and B cells. Thus,
adoptive transfer of CTC after BMT may not only mediate a GVL
effect but may accelerate immune reconstitution. patients or paired
by adding anti-CD28. In vitro data on anti-CD3 and
anti-CD3/anti-CD28 stimulated proliferative responses of T cells
from BMT recipients support the premise that using Mab 9.3 in
combination with OKT3 may have potent in vivo clinical effects as
reported. See Joshi, I. et al., Blood Suppl. (Abstract) (1991). T
cells from BMT recipients have defects in proliferation after
mitogen or anti-CD3 stimulation. Previous studies show that
costimulation of normal T cells with anti-CD3 (G19-4) and 9.3
enhance anti-CD3-induced proliferation by stabilizing lymphokine
mRNAs. Experimentation to assess the ability of anti-CD3 (G19-4 or
OKT3)+9.3 to correct defective anti-CD3-induced proliferative
responses in PBL from autologous and allogeneic BMT recipients
(53-605 days post BMT) was performed. PBL from recipients or
controls were stimulated for 3 days with G19-4, G19-4+9.3, OKT3, or
OKT3+9.3. 9.3 was added at a final concentration of 100 ng/ml.
Fifteen tests were performed on ABMT recipients and sixteen tests
were performed on allogeneic recipients. Table 15 shows the number
of recipients whose PBL increased (.Arrow-up bold.), or decreased
(.dwnarw.), or did not change () their proliferative responses
after the addition of 9.3 to anti-CD3 stimulated PBL. The
parenthesis indicate percent of recipients whose proliferative
responses increased after the addition of 9.3.
16 TABLE 15 TREATMENT BTM RECIPIENTS CHANGE G19-4 + 9.3 OKT3 + 9.3
Autologous BMT .Arrow-up bold. 9 (60%) 9 (82%) .dwnarw. 3 (20%) 2
(18%) 3 (20%) 0 (0%) Allogeneic BMT .Arrow-up bold. 11 (69%) 8
(62%) .dwnarw. 5 (31%) 5 (38%) 0 (0%) 0 (0%)
[0185] Costimulation of G19-4+9.3 or OKT3+9.3 significantly
increased proliferative responses induced by G19-4 or OKT3 alone
(p<0.05, paired rank-sum) in ABMT recipients. In summary,
defects in anti-CD3-induced T cell proliferation in BMT recipients
were repaired by costimulation with 9.3. These findings have
therapeutic implications for patients with immune defects manifest
with impaired T cell proliferation. Indeed similar results have
been obtained which indicate that the proliferative defect of T
cells from patients with HIV infection can be repaired with
anti-CD28 treatment. See Lane, H. C. et al., J. Engl. J. Med.,
313:85 (1985) regarding proliferate defects in HIV.
[0186] Costimulation with anti-CD3 OKT3 and anti-CD28 9.3 enhanced
dectectable mRNA levels for IL-2 in PBL from ABMT recipient. PBL
from a short-term ABMT recipient were studied for expression of
mRNA levels for IL-2 after stimulation with OKT3 and costimulation
with OKT3/9.3 using RTase-PCR. cDNA synthesized by RTase from total
RNA was amplified by PCR using specific primers for IL-2 and the
PCR products run on 1.5% agarose gels containing ethidium bromide.
Consistent with the findings in the previous paragraph, activated T
cells from the ABMT recipient did not have detectable levels of
mRNA for IL-2 after OKT3 stimulation alone, whereas the same T
cells costimulated with OKT3/9.3 had a distinct band for IL-2 of
458 bp detected on ethidium bromide stained agarose gel. This is an
example of how OKT3/9.3 costimulation can repair an apparent defect
in the expression of mRNA for IL-2 in T cells from ABMT
recipients.
[0187] Stimulation of negatively selected CD4.sup.+ cells with
anti-CD28 9.3 after activation with anti-CD3 induces IL-2
independent proliferative responses. CD4.sup.+ cells were purified
by a series of negative selection steps as previously described in
Thompson, C. B. et al., PNAS (USA) 86:1333-1337 (1989). PBL were
incubated with a cocktail of mAbs directed at non-CD28.sup.+ cells,
washed, and incubated with immunomagnetic bead coated with goat
anti-mouse antibody. The CD28.sup.+ enriched cells were further
purified by removing the CD8.sup.+ cells by treatment with anti-CD8
and binding the CD8.sup.+ cells to the immunomagnetic beads.
[0188] The remaining CD28.sup.+, CD4.sup.+ T cells from a normal
donor were cultured by adding cells to culture dishes containing
plastic adsorbed OKT3. After 48 h, the cells were removed and
placed in flasks containing either rIL-2 (200 IU/ml) or anti-CD28
mAb (100 ng/ml). The cells were fed with fresh medium as required
to maintain a cell density of 0.5.times.10.sup.6/ml, and
restimulated at approximately weekly intervals by culture on
plastic adsorbed OKT3 for 24 h. The cells could be maintained in
logarithmic growth, with a 4 to 5 log.sub.1o expansion in cells
number. As shown in FIG. 18, cells propagated with anti-CD3 and
anti-CD28 routinely expanded 10 to 30-fold more than cells grown in
optimal amounts of anti-CD3 and IL-2. When synthetic medium (X-Vivo
10) not containing FBS was used, anti-CD3 plus anti-CD28 treated
cells also expanded 10-fold better than anti-CD3 plus IL-2 treated
cells. The highly enriched CD4 cells did not proliferate in the
presence of optimal amounts of the lectin phytohemagglutinin (PHA).
Thus, the in vitro expansion of CD4 cells using anti-CD28 has an
advantage over previously described methods, in that it is
independent of the addition of exogenous growth factors, as no IL-2
or any other growth factors were added to these cells. In addition,
this system does not require the presence of accessory cells, which
is advantageous in clinical situations where accessory cells are
limiting or defective.
[0189] Phenotypes of anti-CD3 activated T cells. Populations of CTC
cells grown in IL-2 for 6 to 12 days contained predominantly
CD3.sup.+ cells (greater than 84%, median 88%). The proportion of
CD56.sup.+ cells (a marker for NK cells) was less than 1.3%.
Triggering of E.sup.+ cells with OKT3 is preferentially selecting
CD3.sup.+ cells. CD4.sup.+ cells were 18% or less and CD8.sup.+
cells were greater than 66%. Lytic activity did not correlate with
the proportions of CD56.sup.+ cells in the cultures.
[0190] Immunophenotype of T cells differs after anti-CD28 and
IL-2-mediated cellular growth. To examine the subsets of T cells
that are expanded, PBL were propagated for 16 days using either
anti-CD3 and IL-2 or anti-CD3 and anti-CD28. The percentage of CD4
and CD8 cells was 23.8 and 84.5 in the cells grown in IL-2, and
56.0 and 52.6 in the cells grown in CD28. These results suggests
that CD28 expansion favors the CD4.sup.+ cells, in contrast to the
well established observation that CD8.sup.+ cells predominate in
cells grown in IL-2 (for example, see above paragraph; see also
Cantrell, D. A. et al., J. Exp. Med. 158:1895 (1983)). To further
test this possibility, CD4 cells were enriched to 98% purity using
negative selection with monoclonal antibodies and magnetic
immunobeads as described elsewhere in this example. The cells were
cultured for one month using anti-CD3 and either IL-2 or anti-CD28
to propagate the cells. There was equal expansion of the cells for
the first 26 days of the culture, however, as can be seen in Table
16, the phenotype of cells diverged progressively with increasing
time in culture.
17TABLE 16 CULTURE METHOD DAYS CD3 (%) CD4 (%) CD8 (%) anti-CD3 +
IL-2 0 >99 >99 <1 6 >99 98 1 12 >99 85 10 20 >99
45 40 26 >99 12 78 anti-CD3 + IL-2 0 >99 >99 <1 6
>99 >99 <1 12 >99 >99 <1 20 >99 >99 <1
26 >99 >99 <1
[0191] Use of anti-CD28 for in vitro expansion of TIL The use of
IL-2 and inactivated tumor cells to expand tumor infiltrating
lymphocytes (TIL cells) for later adoptive immunotherapy with a
variety of neoplasms has demonstrated promise (e.g., see Rosenberg,
S. A. et al., NEJM 323:570-578 (1990)). TIL cells were isolated
from a nephrectomy specimen from a patient with renal cell
carcinoma. The cells were cultured with tumor cells, and either
IL-2 or anti-CD28 and IL-2 with mAb OKT3 added at weekly intervals,
beginning at day 14. Table 17 demonstrates that anti-CD28 is an
improved method for the propagation of these cells in some
patients, with a 20-fold greater yield of cells. Immunophenotype
analysis also reveals that CD4 T cells are expanded in "TIL"
cultures. Furthermore, these cells also exhibited potent cytotoxic
activity against DAUDI targets, with 82.8, 69.7, 78.8 and 101.5
percent specific lysis at effector to target ratios of 40:1, 20:1,
10:1 and 5:1.
18TABLE 17 Use of anti-CD28 to Expand TIL cells Tumor cells + IL-2
Tumor cells + IL-2 Day of Culture (1000 U/ml) anti-CD3, then
anti-CD28 0 3.6 .times. 10.sup.7 3.6 .times. 10.sup.7 5 5.8 .times.
10.sup.7 1.3 .times. 10.sup.8 12 1.7 .times. 10.sup.8 1.4 .times.
10.sup.8 16 2.0 .times. 10.sup.8 2.6 .times. 10.sup.8 19 3.8
.times. 10.sup.8 4.8 .times. 10.sup.8 25 2.9 .times. 10.sup.8 9.0
.times. 10.sup.8 36 3.7 .times. 10.sup.8 1.8 .times. 10.sup.9 43
3.0 .times. 10.sup.8 3.2 .times. 10.sup.9 48 2.3 .times. 10.sup.8
5.1 .times. 10.sup.9
[0192] Anti-CD3 and anti-CD28 costimulation enhances expression of
mRNA for IL-2 and TNF-.alpha. in CD4+ cells. To examine whether CD4
cells propagated in vitro by anti-CD28 might be an effective source
of lymphokines, resting CD4 cells were stimulated by anti-CD3 mAb
for 48 h followed by the addition of 50 IU/ml of recombinant IL-2
and compared with CD4 T cells costimulated with anti-CD3 and
anti-CD28. Total RNA was harvested from each combination. A total
of 10 .mu.g of RNA was loaded into each lane. A class I probe (HLA
B7) was used to show uniform loading. The bolt was hybridized with
.sup.32P labeled probes specific for IL-2, TNF-.alpha. and HLA in
succession. On days 1 and 8, the cultures were restimulated with
anti-CD3 mAb in the presence of IL-2 or anti-CD28 mAb 9.3 (0.1
.mu.g/ml). The blots were stripped, and rehybridized with a probe
for a constant region of HLA class I mRNA, to demonstrate equal
loading of the lanes. As illustrated by the Northern Blot of FIG.
19, there was a clear enhancement of mRNA for IL-2 and TNF-.alpha.
after costimulation with anti-CD3 and anti-CD28 and the IL-2 and
TNF-.alpha. mRNA exceeded that of the IL-2 propagated cell by 10 to
50-fold. Similar results were obtained when the culture was
examined for one month of culture, after weekly restimulation with
anti-CD3 and IL-2 or anti-CD28.
[0193] Supernatants from long-term anti-CD3 and anti-CD28
stimulated cultures of CD4.sup.+ cells contain substantial amounts
of IL-2, GM-CSF, and TNF-.alpha.. After anti-CD-3 stimulation in
the presence of 200 IU/ml of IL-2 or the combination of anti-CD3
and anti-CD28 in the absence of IL-2, supernatants were tested for
IL-2 content using the CTLL-2 cell line, GM-CSF content by ELISA,
and TNF-.alpha. content by ELISA. The CD4.sup.+ cells were
restimulated with anti-CD3 and IL-2 or anti-CD3 and anti-CD28
respectively at approximately weekly intervals. Anti-CD3 and
anti-CD28 cultures of CD4.sup.+ cells produced roughly the same
amount of IL-2 found in supernatant obtained from anti-CD3
activated cells grown in exogenous IL-2. The amount of GM-CSF
produced by anti-CD3 and anti-CD28 stimulated CD4.sup.+ cells was
also substantial. Although there were variations in levels of
TNF-.alpha. depending on when the supernatants were tested,
costimulation with anti-CD3 and anti-CD28 was superior to
stimulation with anti-CD3 and IL-2 for inducing mRNA for
TNF-.alpha.. These data indicate that anti-CD28 costimulation with
anti-CD3 may not only replace some of the functions of IL-2 but may
enhance other synthetic functions of CD4.sup.+ cells.
SPECIFIC EXAMPLE XV
CD28 and CTLA-4 Expression Studies
[0194] CTLA-4 expression limited to CD28.sup.+ T cells. Utilizing
site-specific primers and DNA PCR of human genomic DNA, a 348 bp
fragment corresponding to exon II of human CTLA-4 was generated,
gel purified and used as a .sup.32P-labeled probe. Purified human T
cells were separated into CD28.sup.+ and CD28.sup.- fractions by
negative selection with magnetic bead immunoabsorbtion. CD28.sup.+
T cells were either tested in media or stimulated with
PMA+ionomycin or PMA+anti-CD28 mAb (mAb 9.3) for 12 h. CD28.sup.+
cells were stimulated with the last two conditions for 12 h. RNA
was extracted by guanidinium isothicyanate and purified over cesium
chloride gradients. Equal amounts of RNA (as determined by ethidium
bromide staining) were loaded and separated on a
formaldehyde-agarose gel and transferred to nitrocellulose to
demonstrate equal loading of RNA. This blot was subsequently probed
with the CTLA-4 probe generated above. CTLA-4 was expressed in
CD28.sup.+ cells following PMA or PMA+mAb 9.3 stimulation but not
expressed in resting or stimulated CD28.sup.- cells. The same blot
was hybridized to a HLA probe to confirm equal loading of RNA.
[0195] The expression of CTLA-4 induced under conditions causing
CD28 pathway activation. Purified resting CD28.sup.+ T cells were
stimulated with PMA alone, ionomycin alone, and PMA+ionomycin for 1
h, 6 h, 12 h and 24 h. RNA was extracted and analyzed by
hybridization to CD28 and CTLA-4 probes. Northern blot analysis
showed that CTLA-4 expression was induced by PMA or PMA+ionomycin,
conditions which are costimulatory with CD28 pathway activation.
CTLA-4 expression was not induced by ionomycin, which is not
costimulatory with CD28 pathway activation. In contrast, CD28
expression was constant with ionomycin or PMA and even appeared to
be suppressed with PMA and ionomycin stimulation. It should also be
noted that the induction of CTLA-4 expression occurred as soon as 1
h after stimulation, compared to 6-12 h with IL-2 expression
following CD28 pathway activation. Since expression of CTLA-4
precedes the biological events caused by CD28 pathway activation
(i.e. enhanced IL-2 expression), CTLA-4 expression likely plays a
role in the generation of later events.
[0196] Purified human T cells were either untreated or stimulated
for 1 h, 4 h or 23 h with PMA+PHA, anti-CD28 mAb crosslinked with a
second antibody (goat anti-mouse Ig), or anti-CD5 mAb crosslinked
in the same manner. Northern blot analysis showed that crosslinking
of CD28 receptors, which also can activate the CD28 pathway through
mechanisms distinct from PMA and ionomycin, also induced CTLA-4
expression.
[0197] CD28.sup.+ cell lines slightly or not responsive to CD28
pathway activation do not express CTLA-4. Two cell lines that are
CD28.sup.+ but responded poorly (T cell line Jurkat C J) or not at
all (myeloma cell line RPMI-8226) to CD28 costimulation as
discussed in Kozbor, D. et al., J. Immunol., 138:4128-4132 (1987)
and Ledbetter, J. A. et al., PNAS (USA), 84:1384-1388 (1987), were
stimulated with PMA+ionomycin+mAb 9.3 and subsequently analyzed by
Northern blot for CD28 and CTLA-4 expression. Northern blot
analysis of the T cell leukemia cell line Jurkat C J and of the
myeloma cell line RPMI 8226 showed that these cell lines did not
express CTLA-4 despite CD28 expression.
[0198] T cells were incubated with various combinations of mitogens
including phytohemagglutinin (PHA), phorbol myristate acetate
(PMA), and ionomycin (IONO), or anti-CD28 monoclonal antibodies
(.alpha.-CD28), and examined for the ability to induce CTLA-4 mRNA
expression. As shown in FIG. 20, the combination of the mitogens
PHA and PMA readily induce CTLA-4 expression in normal human T
cells, but all combinations tested failed to induce the expression
of the CD28-isoform CTLA-4 in the Jurkat T cell line. Both cell
types express significant levels of CD28 mRNA under all conditions
tested.
[0199] Resting T cells were costimulated with anti-CD28 monoclonal
antibodies to examine activation in normal human cells. As shown in
FIG. 21, normal human T cells stimulated by plate-adherent anti-CD3
(.alpha.-CD3) monoclonal antibodies at a concentration of 1
.mu.g/ml induced only low levels of CTLA-4 mRNA expression first
observable at 1 h after stimulation. In contrast, costimulation of
cells with anti-CD3 monoclonal antibodies (1 .mu.g/ml) soluble
anti-CD28 (.alpha.-CD28) monoclonal antibody (1 .mu.g/ml) led to a
dramatic increase in the induction of CTLA-4 mRNA expression.
SPECIFIC EXAMPLE XVI
Efficacy of Administration of CTLA-4lg as a Treatment for
Autoimmune Disease
[0200] Experimental Autoimmune Encephalomyelitis (EAE) is a rodent
and primate model for multiple sclerosis. Data has been generated
on the effect of administration of CTLA-4lg in both passive
(indirect) and active (direct) models of EAE. CTLA-4lg is a fusion
protein consisting of the extracellular domain of human CTLA-4
fused to the constant region of human IgG1 (referred to here as
huCTLA-4lg).
[0201] Adoptively Transferred (Passive) EAE. In the passive EAE
model, donor mice are immunized with 0.4 mg Myelin Basic Protein
(MBP) in Complete Freund's Adjuvant (CFA), divided over four
quadrants. The draining axillary and inguinal lymph nodes are
removed eleven days later. Lymph node cells (4.times.10.sup.6/ml)
are plated in 2 ml cultures in 24 well plates, in the presence of
25 .mu.g/ml MBP. After four days in culture, 30.times.10.sup.6 of
the treated cells are injected into the tail vein of each naive,
syngeneic recipient mouse.
[0202] The recipient mice develop a remitting, relapsing disease
and are evaluated utilizing the following criteria:
[0203] 0--normal, healthy
[0204] 1--limp tail, incontinence; occasionally the first sign of
the disease is a "tilt"
[0205] 2--hind limb weakness, clumsiness
[0206] 3--mild paraparesis
[0207] 4--severe paraparesis
[0208] 5--quadriplegia
[0209] 6--death
[0210] Using this passive model of EAE, the effect of huCTLA-4lg
treatment of the donor cells on disease severity was tested in
PLSJLF1/J mice. Treatment of lymph node cells in vitro with MBP was
performed either in the presence or the absence of 30 .mu.g/ml
huCTLA-4lg. The treated cells were then introduced into a syngeneic
recipient mouse. As shown in FIG. 22, mice receiving
huCTLA-4lg-treated cells (designated PPIB CTLA-4) showed a
significantly reduced severity of their first episode of disease as
compared to mice receiving untreated cells (designated PPIA
control). In addition, ensuing relapses in the mice receiving
huCTLA-4lg-treated cells were less severe than in mice receiving
cells not exposed to huCTLA-4lg. In fact, all five mice receiving
huCTLA-4lg-treated cells stopped relapsing, and no longer showed
signs of disease at 80-100 days post transfer.
[0211] Clinical disease severity was reduced even further by
treating both the donor mice and the cultured cells with huCTLA-4lg
(FIG. 23). In these experiments, donor mice of the SJL/J strain
were given either 100 .mu.g huCTLA-4lg or 100 .mu.g chimeric
control IgG1 intraperitoneally each day for eleven days. T cells
were then isolated from lymph nodes of these donors and cultured
with MBP in vitro in the presence of either 30 .mu.g/ml huCTLA-4lg
or chimeric control IgG1. The treated cells were then introduced
into a syngeneic recipient. Treatment of either the donor mice or
the in vitro cultures resulted in significantly reduced clinical
disease severity. Treatment of both the donor mice and the cultured
cells with huCTLA-4lg was the most effective protocol for reducing
clinical disease severity.
[0212] Direct administration of huCTLA-4lg to mice receiving
adoptively transferred cells was also examined. As shown in FIG.
24, when PLSJLFI/J recipient mice were given 100 .mu.g of either
huCTLA-4lg or human IgG in PBS intraperitoneally on days 1 to 9
post transfer, no difference in disease severity Was observed
between the two groups of mice. However, in experiments utilizing
SJL/J mice, reduced disease severity during relapse was noted in
mice treated with 100 .mu.g huCTLA-4lg intraperitoneally on days 1
to 5 post transfer (FIG. 25). Ongoing experiments are examining the
effect of administration of a single dose of huCTLA-4lg to SJL/J
recipient mice on day 2 post transfer. Table 18 shows results of
such an experiment, compared to the results obtained when recipient
mice are given either huCTLA-4lg or human IgG1 on days 1 to 5 post
transfer. While severity of the first episode of disease did not
appear to be significantly altered by treatment with huCTLA-4lg
either on day 2 or days 1-5, the duration of the first episode of
disease was shorter for mice given huCTLA-4lg treatment (Table 18).
In addition, huCTLA-4lg treatment on days 1-5 resulted in delayed
onset of the first episode of disease.
19TABLE 18 Effect of Administration of CTLA-4Ig to Mice with
Adoptively Transferred EAE CTLA-4Ig CTLA-4Ig IgG Control (day 2)
(days 1-5) (days 1-5) Mean day of onset 11.3 16.3 10.8 Maximum
score of first 3.2 2.8 2.8 episode Maximum score to date 3.5 3.2
3.0 (day 35) Average of first episode 5.2 6.7 9.8 (days)
[0213] Direct (Active) Model of EAE. Studies using a direct
(active) model of EAE have also been conducted. In these
experiments, huCTLA-4lg was directly administered to mice immunized
with MBP and treated with pertussis toxin (PT). PLSJLFI/J mice
immunized with MBP on day 0 and injected with PT intravenously on
days 0 and 2 were given either huCTLA-4lg or control IgG1 on days 0
to 7. Five of ten mice given huCTLA-4lg died on days 5 to 7, for
reasons related to the PT administered, and not the experimental
design. The results of the experiment for the remaining five mice
are as shown in FIG. 27. Administration of huCTLA-4lg markedly
reduced the mean clinical severity of disease in these animals, as
compared to the mice treated with IgG1. These findings indicate
that direct administration of soluble human huCTLA-4lg can provide
an effective therapeutic strategy in the treatment of autoimmune
disease.
[0214] It should be appreciated that a latitude of modification,
change or substitution is intended in the foregoing disclosure and,
accordingly, it is appropriate that the appended claims be
construed broadly and in a manner consistent with the spirit and
scope of the invention herein.
[0215] All publications and applications cited herein are
incorporated by reference.
* * * * *