U.S. patent application number 12/525270 was filed with the patent office on 2010-06-03 for redirected, genetically-engineered t regulatory cells and their use in suppression of autoimmune and inflammatory disease.
This patent application is currently assigned to YEDA RESEARCH AND DEVELOPMENT CO. LTD.. Invention is credited to Eran Elinav, Zelig Eshhar.
Application Number | 20100135974 12/525270 |
Document ID | / |
Family ID | 39674803 |
Filed Date | 2010-06-03 |
United States Patent
Application |
20100135974 |
Kind Code |
A1 |
Eshhar; Zelig ; et
al. |
June 3, 2010 |
REDIRECTED, GENETICALLY-ENGINEERED T REGULATORY CELLS AND THEIR USE
IN SUPPRESSION OF AUTOIMMUNE AND INFLAMMATORY DISEASE
Abstract
A redirected Treg cell is endowed with specificity toward a
selected target antigen or ligand. The cell contains a chimeric
receptor polypeptide that is expressed in a single, continuous
chain, with an extracellular recognition region displayed on the
surface of the cell, a transmembrane region and an intracellular
signaling region. The extracellular recognition region is specific
for the selected target antigen or ligand. The intracellular
signaling region includes a combination of T-cell signaling
polypeptide moieties, which combination, upon binding of the
extracellular recognition region to the selected target antigen or
ligand, triggers activation of the redirected Treg cells to cause
suppression of T-cell mediated immunity. Such redirected Treg cells
may be used to suppress undesired activity of T effector cells
thereby mediating an immune or inflammatory response. They are
particularly useful in treating T effector cell-mediated diseases,
such as inflammatory bowel disease, transplant rejection and GVH
disease.
Inventors: |
Eshhar; Zelig; (Rehovot,
IL) ; Elinav; Eran; (Jerusalem, IL) |
Correspondence
Address: |
BROWDY AND NEIMARK, P.L.L.C.;624 NINTH STREET, NW
SUITE 300
WASHINGTON
DC
20001-5303
US
|
Assignee: |
YEDA RESEARCH AND DEVELOPMENT CO.
LTD.
Rehovot
IL
|
Family ID: |
39674803 |
Appl. No.: |
12/525270 |
Filed: |
January 31, 2008 |
PCT Filed: |
January 31, 2008 |
PCT NO: |
PCT/US08/52724 |
371 Date: |
July 30, 2009 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
60898408 |
Jan 31, 2007 |
|
|
|
60951052 |
Jul 20, 2007 |
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Current U.S.
Class: |
424/93.71 ;
435/325; 435/375; 530/387.3; 536/23.5 |
Current CPC
Class: |
C12N 5/0636 20130101;
A61K 39/0011 20130101; A61P 37/00 20180101; A61P 29/00 20180101;
A61K 39/0008 20130101 |
Class at
Publication: |
424/93.71 ;
435/375; 536/23.5; 530/387.3; 435/325 |
International
Class: |
A61K 35/12 20060101
A61K035/12; C12N 5/10 20060101 C12N005/10; C07H 21/04 20060101
C07H021/04; C07K 16/00 20060101 C07K016/00; A61P 29/00 20060101
A61P029/00; A61P 37/00 20060101 A61P037/00 |
Claims
1. A redirected regulatory T lymphocyte (Treg cell) endowed with
specificity toward a selected target antigen or ligand, which cell
comprises a chimeric nucleic acid that encodes a chimeric receptor
polypeptide that comprises, expressed in a single, continuous
chain, an extracellular recognition region, a transmembrane region
and an intracellular signaling region, and is expressed in the
redirected Treg cell so as to display the extracellular region on
the cell surface, wherein (a) said extracellular recognition region
of said chimeric receptor is specific for the selected target
antigen or ligand; and (b) said intracellular signaling region
comprises a combination of T-cell signaling polypeptide moieties,
which combination of moieties, upon binding of the extracellular
recognition region to the selected target antigen or ligand,
triggers activation of the redirected Treg cells to cause
suppression of T-cell mediated immunity.
2. A redirected Treg cell in accordance with claim 1, wherein said
extracellular recognition region comprises an antibody-derived scFv
domain that is specific for a selected antigen.
3. A redirected Treg cell in accordance with claim 1, wherein said
extracellular recognition region comprises a member of a
ligand-receptor pair, which is specific for the other member of
that pair.
4. A redirected Treg cell in accordance with claim 1, wherein said
extracellular recognition region does not comprise an MHC protein
extracellular domain.
5. A redirected Treg cell in accordance with claim 1, wherein said
extracellular recognition region is linked to said transmembrane
region through a flexible spacer.
6. A redirected Treg cell in accordance with claim 5, wherein said
flexible spacer is a hinge from a molecule of the immunoglobulin
superfamily.
7. A redirected Treg cell in accordance with claim 1, wherein said
intracellular region comprises a signaling moiety from (i) a
polypeptide chain of an antigen-specific receptor of a T-cell
and/or (ii) a polypeptide chain of a receptor of a T-cell that has
a region comprising an immunoreceptor tyrosine-based activation
motif (ITAM).
8. A redirected Treg cell in accordance with claim 7, wherein said
intracellular region comprises a signaling moiety from (ii).
9. A redirected Treg cell in accordance with claim 7, wherein said
intracellular region comprises a signaling moiety from a
polypeptide chain selected from the group consisting of a chain of
the TCR/CD3 complex, and the .gamma. chain of an Ig Fc receptor
(FcR.gamma.).
10. A redirected Treg cell in accordance with claim 9, wherein said
polypeptide chain of an antigen-specific receptor of a T-cell is
the CD3-.zeta. chain or the FcR.gamma. chain.
11. A redirected Treg cell in accordance with claim 1, wherein said
intracellular region includes a signaling moiety of a T cell
costimulatory-receptor protein.
12. A redirected Treg cell in accordance with claim 11, wherein
said costimulatory-receptor protein is at least one protein
selected from the group consisting of CD28, OX40, CD40L, 4-1BB and
PD-1.
13. A redirected Treg cell in accordance with claim 11, wherein
said costimulatory-receptor protein is CD28.
14. A redirected Treg cell in accordance with claim 11, wherein
said costimulatory-receptor protein is 4-1BB.
15. A redirected Treg cell in accordance with claim 1, wherein said
intracellular region comprises at least two different ones of said
costimulatory-receptor protein signaling moieties.
16. A redirected Treg cell in accordance with claim 15, wherein
said intracellular region comprises an intracellular signaling
moiety from CD28 and from 4-1BB.
17. A redirected Treg cell in accordance with claim 1, wherein said
intracellular region comprises a signaling moiety from a T cell
cytokine receptor.
18. A redirected Treg cell in accordance with claim 17, wherein
said cytokine receptor is the IL-2 receptor.
19. A redirected Treg cell in accordance with claim 17, wherein
said cytokine receptor is the TGF-.beta. receptor.
20. A redirected Treg cell in accordance with claim 1, wherein said
intracellular signaling region includes a signal-transducing enzyme
that (a) is an enzyme in the signal transduction pathway of an
antigen-specific receptor of a T-cell or (b) is an enzyme with
corresponding specificity and activity as the enzyme of (a),
derived from a non-T-cell lymphocyte.
21. A redirected Treg cell in accordance with claim 20, wherein
said signal-transducing enzyme is a member of the Syk-kinase
family.
22. A redirected Treg cell in accordance with claim 1, wherein said
chimeric nucleic acid further includes a nucleotide sequence that
will cause the redirected Treg to express Foxp3.
23. A redirected Treg cell in accordance with claim 1, wherein said
selected target antigen or ligand is one that is present or
expressed at a site or target tissue of an autoimmune or
inflammatory response mediated by effector T-cells.
24. A redirected Treg cell in accordance with claim 23, wherein
said autoimmune or inflammatory response comprises an autoimmune
response or disease, an allograft or xenograft rejection, or
graft-vs. host (GVH) disease.
25. A redirected Treg cell in accordance with claim 1, wherein said
target antigen or ligand is an autoantigen or an antigen that is
cross-reactive with an autoantigen.
26. A redirected Treg cell in accordance with claim 25, wherein the
autoantigen is a pathogenic antigen in the pathophysiology of said
autoimmune disease.
27. The redirected Treg cell of claim 1, wherein said autoimmune or
inflammatory response and said target antigen or ligand are
selected from the group consisting of: (a) inflammatory bowel
disease (IBD), wherein said antigen or ligand is one that is
expressed in diseased colon or ileum; (b) rheumatoid arthritis,
wherein said antigen or ligand is an epitope of collagen or an
antigen present in joints; (c) Type I diabetes mellitus or
autoimmune insulitis, wherein said antigen or ligand is a
pancreatic .beta. cell antigen; (d) multiple sclerosis, wherein
said antigen or ligand is a myelin basic protein antigen, MOG-1,
MOG-2 or another neuronal antigen; (e) autoimmune thyroiditis,
wherein said antigen or ligand is a thyroid antigen; (f) autoimmune
gastritis, wherein said antigen or ligand is a gastric antigen; (g)
autoimmune uveitis or uveoretinitis, wherein said antigen or ligand
is S-antigen or another uveal or retinal antigen; (h) autoimmune
orchitis, wherein said antigen or ligand is a testicular antigen;
(i)) autoimmune oophoritis, wherein said antigen or ligand is an
ovarian antigen; (j) psoriasis; wherein said antigen or ligand is a
keratinocyte antigen or another dermal or epidermal antigen; (k)
vitiligo, where said antigen or ligand is a melanocyte antigen; (l)
autoimmune prostatitis, wherein said antigen or ligand is a
prostate antigen; (m) any undesired immune response, wherein said
antigen or ligand is an activation antigen expressed on T effector
cells present at the site of the undesired immune response; (n)
tissue rejection, wherein said antigen or ligand is the MHC
molecule having the haplotype of the transplanted tissue, or a
portion of that MHC molecule; and (o) an inflammatory condition,
wherein said antigen or ligand is one that is expressed on
nonlymphoid cells of the hemopoietic lineage that participate in
inflammation.
28. A redirected Treg cell in accordance with claim 27, wherein
said autoimmune disease is IBD and said antigen or ligand is
carcinoembryonic antigen (CEA), or an antigen of intestinal
bacterial flora.
29. A redirected Treg cell in accordance with claim 27, wherein the
activation antigen of (m) expressed on T effector cells is CD69 or
CD107a.
30. A redirected Treg cell in accordance with claim 27, wherein
said target antigen or ligand of (o) is one that is expressed on a
dendritic cell, macrophage/monocyte, granulocyte or eosinophil
present at said inflammation site.
31. An immunoregulatory pharmaceutical composition for suppressing
a T effector cell-mediated immune/inflammatory response or treating
a T effector cell-mediated immune/inflammatory disease or
condition, comprising: (a) a Treg cell according to claim 1, and
(b) a pharmaceutically and immunologically acceptable carrier,
excipient or diluent.
32. A method of producing the redirected Treg cell of claim 1,
comprising: (a) obtaining from a subject and, optionally enriching
or isolating and propagating, a population of lymphocytes or
T-cells; (b) inducing a Treg cell phenotype in said cells by
suitably stimulating or activating the cells by exposure to
TGF-.beta. or another cytokine that induces Foxp3 expression
thereby inducing the Treg phenotype; (c) before or after step (b),
transducing the cells ex vivo with an expression vector encoding
said chimeric receptor to be expressed on said Treg cell; and (d)
optionally, growing or expanding the cells obtained as above in
vitro.
33. A method of producing the redirected Treg cell of claim 1,
comprising: (a) obtaining from a subject and, optionally enriching
or isolating and propagating, a population of lymphocytes or
T-cells; (b) transducing the cells ex vivo with a vector encoding
the chimeric receptor; (c) before, after, or concomitantly with
step (b), transducing the cells ex vivo with a recombinant nucleic
acid expression construct encoding Foxp3; and (d) optionally,
growing or expanding the cells obtained as above in vitro.
34. A method of suppressing undesired activity of T effector cells
in mediating an immune or inflammatory response, comprising
delivering to a site of T effector cells to be suppressed an amount
of Treg cells according to claim 1 effective to suppress said T
effector cell activity.
35. The method of claim 34 wherein said Treg cells are administered
systemically.
36. The method of claim 34 wherein the redirected Treg cells are
administered regionally or locally to a site of inflammation.
37. The method of claim 34 wherein said undesired activity of T
effector cells is an autoimmune inflammatory response or disorder,
rejection of a transplant or GVH disease.
38. A method for treating or ameliorating symptoms of a disease or
condition in a mammalian subject that is mediated by undesired
activity of T effector cells, said method comprising administering
to said subject in need thereof an effective amount of Treg cells
in accordance with claim 1, wherein said target antigen or ligand
is one that is present at the site of said undesired T effector
cell activity.
39. The method of claim 38 wherein the redirected Treg cells are
produced by obtaining a population of T-cells from the mammalian
subject to be treated, transfecting said cells with said chimeric
nucleic acid and causing the cells to express Foxp3 by (i)
stimulating the transfected cells to induce Foxp3 expression, or
(ii) introducing an exogenous Foxp3-encoding construct into the
transfected cells.
40. The method of claim 38 wherein the redirected Treg cells are
produced by obtaining a mixed population of T-cells from the
mammalian subject to be treated, enriching or purifying Treg cells
from the mixed population of T-cells on the basis of the Treg
cells' expression of CD4 and CD25 and/or Foxp3, and transfecting
said enriched or purified Treg cells with said chimeric nucleic
acid.
41. The method of claim 40 wherein the redirected Treg cells are
produced by: (a) obtaining (i) peripheral blood mononuclear cells,
(ii) peripheral blood lymphocytes, (iii) T-cells enriched or
purified from (i) or (ii), or (iv) a subset of T-cells enriched or
purified from (iii); (b) exposing the cells obtained in (a), ex
vivo, to an amount of TGF-.beta. or other Treg-inducing cytokine or
agent that is effective to induce expression of Foxp3 and convert
T-cells to a Treg phenotype, (c) optionally, culturing and
expanding said exposed cells of (a); and (d) before after or
between said steps (a) and (b), transfecting said cells with said
chimeric nucleic acid.
42. The method of claim 38 wherein said disease or condition
comprises an autoimmune response or disease, an allograft or
xenograft rejection, or graft-vs. host (GVH) disease.
43. The method of claim 38, wherein said disease or condition is
IBD and said target antigen or ligand is CEA, or an antigen of
intestinal bacterial flora.
44. A method of claim 38, wherein said target antigen or ligand is
LPS.
45. A method of claim 44, wherein said extracellular recognition
region comprises an LPS-binding domain of MD-2 or an LPS-binding
domain of CD-14 or both an LPS-binding domain of MD-2 and an LPS
binding domain of CD-14.
46. The method of claim 38, further including the step of, either
before, concomitantly with, or after administration of the
redirected Treg cells, introducing to a subject to the site or
target tissue of said immune or inflammatory response, an exogenous
antigen or ligand and said target antigen or ligand is said
exogenous antigen or ligand.
47. A chimeric DNA molecule comprising: (a) a first DNA segment
comprising a sequence encoding an extracellular recognition region
specific for a selected target antigen or ligand, which region does
not comprise an MHC protein extracellular domain, said selected
target antigen or ligand being one that is present or expressed at
a site or tissue of a pathogenic or undesired immune response
mediated by effector T-cells; (b) a second DNA segment comprising a
sequence encoding a transmembrane region; and (c) a third DNA
segment comprising a sequence encoding an intracellular signaling
region comprising a combination of T-cell signaling polypeptide
moieties, which combination of moieties trigger activation of the
Treg cells to cause suppression of T-cell mediated immunity when
the chimeric DNA is transfected into a Treg cell and said
extracellular recognition region binds to the selected target
antigen or ligand, wherein, upon transfection of the chimeric DNA
into a Treg cell, the Treg cell expresses said extracellular
recognition region on its surface, said transmembrane region and
said intracellular signaling region in one single, continuous
chain.
48. A chimeric receptor polypeptide encoded by the chimeric DNA
molecule of claim 47.
Description
BACKGROUND OF THE INVENTION
[0001] 1. Field of the Invention
[0002] The invention in the field of immunology and medicine
relates to genetic modification of T regulatory cells with chimeric
receptors with antibody-type specificity, and the use of such cells
to suppress the action of T effector cells and treat any of a
number of diseases and conditions in which such suppression is
beneficial, primarily autoimmune and inflammatory diseases such as
inflammatory bowel diseases (IBD), organ-specific autoimmune
diseases, allograft rejection and Graft-vs. Host disease.
[0003] 2. Description of the Background Art
[0004] Regulatory T-Cells (Tregs)
[0005] One line of research that led to discovery of Treg cells was
the observation that thymectomy of mice of certain susceptible
strains on postnatal day 3 results in a spectrum of organ-specific
autoimmune effects, which were preventable by "reconstitution" of
these animals early in life with normal adult lymphocytes (Asano M
et al., J Exp Med 1996; 184:387-96). The effectors and suppressors
of autoimmunity in this model of multi-organ autoimmunity were
CD4+T-cells. It was subsequently shown that the regulatory
CD4+Tregs that prevent disease coexpressed CD25. CD4+CD25+ Tregs
represent 5-10% of total peripheral CD4+ cells in mice and 3-6% of
total peripheral blood CD4+T-cells in humans (Jonuleit H et al., J
Exp Med 2001; 193:1285-94).
[0006] Over the past decade, CD4+CD25+ Tregs have been studied for
their function in autoimmune disease. CD4+CD25+ Tregs suppressed
disease induced by cloned autoantigen-specific T effector cells
(Sufi-Payer, E et al., J. Immunol., 1998; 160:1212-18). The
CD4+CD25+ Treg cells appeared to be members of a unique lineage of
regulatory T-cells. These authors noted that, although the target
antigen(s) and mechanism of action of the CD4+CD25+T-cells remained
to be determined, they likely played an important role in
modulating other autoimmune diseases that are mediated by
activation of self-reactive T-cells. Tregs prevent organ-specific
autoimmune diseases including autoimmune thyroiditis, autoimmune
gastritis, insulitis and arthritis.
[0007] More recently, it was discovered that Tregs express a
transcription factor known as Foxp3 intracellularly. The absence of
the transcription factor called Scurfin (also forkhead box P3) and
encoded by the gene Foxp3 was known to cause a rapidly fatal
lymphoproliferative disease, similar to that seen in mice lacking
cytolytic T lymphocyte-associated antigen 4 (CTLA-4). Khattri R et
al. (Nat Immunol. 2003; 4:337-42) showed that Foxp3 was highly
expressed by Treg cells and was associated with their activity and
phenotype. Foxp3-deficient mice lacked Treg cells whereas mice that
overexpressed Foxp3 possess more Treg cells. Tregs constitutively
express the transcription factor Foxp3 and the inhibitory
costimulatory molecule CTLA-4 (Chen W et al., J Exp Med 1998;
188:1849-57.
[0008] Foxp3 is believed to act through negative transcriptional
regulation of cytokine genes, including IL2, IL4 and IFN-.gamma.
(Kasprowicz D J et al., J Immunol. 2003; 171:1216-23, 2003), though
many aspects of Foxp3 activity and regulation of its expression
remain obscure.
[0009] Loser K et al., Gene Ther 2005; 12:1294-304, generated Tregs
in vitro by infecting naive CD4+CD25-T-cells with a retrovirus
encoding Foxp3. Foxp3-infected T-cells were similar to naturally
occurring Treg cells as evidenced by surface marker expression and
function. These authors investigated the effects of Foxp3-infected
T-cells on contact hypersensitivity responses mediated by T
effector cells by injecting into sensitized mice Foxp3- or control
virus-infected T-cells. Only injection of Foxp3-infected T-cells
significantly inhibited CHS compared to controls, indicating that
Foxp3-infected T-cells are suppressive in vivo. The authors then
used Foxp3-infected T-cells to treat autoimmune-prone CD40L
transgenic (Tg) mice, which develop a severe systemic autoimmune
disease including autoreactive T-cells and autoantibodies.
Injection of Foxp3-infected T-cells into these mice inhibited the
ongoing development of autoimmune dermatitis and activation of
cytotoxic CD8+T-cells. This treatment also reduced serum
concentrations of antinuclear antibodies, which was paralleled with
reduced renal immunoglobulin depositions and increased kidney
function. The authors concluded that newly in vitro-generated
regulatory T-cells can be used to treat inflammatory and ongoing
autoimmune disorders successfully.
[0010] Suri-Payer E and Fritzsching B (Springer Semin Immunopathol.
2006; 28:3-16) recently summarized evidence for a role for Treg in
suppression of innate and adaptive immune responses in experimental
models of autoimmunity including arthritis, colitis, diabetes,
autoimmune encephalomyelitis, lupus, gastritis, oophoritis,
prostatitis, and thyroiditis. A common observation from such
studies is that Tregs are activated in an antigen-specific manner,
but exert their suppressive function in an antigen-independent
manner, mainly by producing and secreting suppressive cytokines
such as IL-10 and TGF-.beta.. Tregs can suppress "conventional"
T-cells in vitro by direct cell contact. It is appreciated,
however, that down-regulation of antigen-presenting cell (APC)
function, such as that of dendritic cells, and attenuation of
secretion of inhibitory cytokines such as IL-10 and TGF-.beta.
might be important for Treg function in vivo. The final outcome of
autoimmunity vs. tolerance depends on the balance between
stimulatory signals to T effector cells and inhibitory signals from
Treg. Whereas earlier studies analyzed the capacity of Tregs to
prevent onset of autoimmune disease, more recent reports indicate
successful treatment of ongoing disease.
[0011] In vivo, adoptive transfer of Tregs achieved the following
effects:
[0012] (1) suppressed development of autoimmunity;
[0013] (2) suppressed acute rejection of transplanted solid organs;
and
[0014] (3) suppressed anti-tumor immunity,
[0015] A review of Sakaguchi S et al., Immunol Rev. 2006; 212:8-27,
noted that naturally arising CD25+CD4+ Treg cells play key roles in
the maintenance of immunologic self-tolerance and negative control
of a variety of physiological and pathological immune responses.
Most of these cells are produced by the normal thymus as a
functionally mature T cell subpopulation. Natural Tregs
specifically express Foxp3, a transcription factor that plays a
critical role in their development and function. Complete depletion
of Foxp3-expressing natural Tregs (whether they are CD25+ or CD25-)
activated even weak or rare self-reactive T effector cell clones,
inducing severe and widespread autoimmune/inflammatory diseases.
Natural Tregs are highly dependent on exogenously provided
interleukin (IL)-2 for their survival in the periphery. In addition
to Foxp3 and IL-2/IL-2 receptor, a deficiency or functional
alteration of other molecules (expressed by T-cells or
non-T-cells), may affect the development or function of Tregs,
self-reactive T effector cells, or both, and consequently tip the
balance between the two populations in the periphery toward
autoimmunity. Thus, Tregs suppress the activity of T effector cells
that are a major cause of antigen-specific autoimmune inflammatory
disorders. Tregs induce anergy and promote suppression by a process
that involves both cell-cell contact, and probably more
importantly, by their secretion of TGF-.beta. and IL-10.
[0016] Sakaguchi et al., supra, stated that elucidation of the
molecular and cellular basis of Treg-mediated active maintenance of
self-tolerance will facilitate (1) our understanding of the
pathogenesis of autoimmune disease and (2) development of novel
methods of autoimmune disease prevention and treatment.
[0017] The present invention is directed to one such novel approach
to prevent or treat autoimmune disease and related
immune/inflammatory conditions by imposing Treg-mediated control
over T effector cells.
[0018] Engineering of Chimeric Receptors with Antibody Specificity
into T-Cells
[0019] Efforts to confer antibody-like specificity to T lymphocytes
arose as a response to certain basic discoveries and the failure to
convert them to therapeutic success. Human tumor-associated
antigens have been shown to exist as peptides associated with major
histocompatibility complex (MHC) proteins. The lack of success in
vaccinating tumor patients with a large variety of tumor-associated
antigen vaccines were thus frustrating (Rosenberg S A et al., Nat
Med 2004; 10:909-15). Passive vaccination with anti-tumor
antibodies (Abs), tumor infiltrating lymphocytes (TILs), or
lymphokine activated killer (LAK) cells showed very limited
effectiveness (primarily for vascular tumors). More recent
successes with the adoptive transfer of TILs into stage 1V melanoma
patients ((Dudley M E, et al., J Immunother 2001; 24:363-73; Dudley
M E, et al., Science 2002; 298:850-4) were still burdened by the
fact that the results were limited to a few cancers and to
individuals from whom it is possible to derive specific TILs.
[0020] To overcome such limitations in adoptive cellular
immunotherapy of cancer, and in order to develop an approach that
was not restricted to individual patients or limited to a specific
form of cancer, one of the present inventors and his colleagues
developed the "T-body" approach (Gross G et al., Proc Natl Acad Sci
USA, 1989; 86:10024-8; Gross G and Eshhar, Z, FASEB J 1992;
6:3370-8), See also the following patent publications by Eshhar and
colleagues, all of which are incorporated by reference in their
entirety: US Pat Pub 2002-0137697, U.S. Pat. No. 5,912,172, U.S.
Pat. No. 5,906,936, International Patent publications WO00/31239,
WO97/15669, WO95/14710 and WO93/19163. In these approaches, a
chimeric receptor (CR), was made, for example, by fusing the
variable portion of an antibody, such as an anti-tumor monoclonal
antibody (mAb), to a lymphocyte intracellular triggering domain, so
as to be expressed by the T-cell into which the gene has been
transfected as the extracellular domain of that T-cell triggering
molecule. Following the expression of such CR genes in immune
effector cells (T-cells and natural killer (NK) cells), the
resulting engineered cells (nicknamed "T-bodies") recognized their
tumor targets and efficiently killed them. Thus, the chimeric
immune receptor confers redirected antigenic specificity coupled to
direct, MHC-independent triggering of cellular activation in
response to binding of pre-defined target antigen.
[0021] Originally, the heterodimeric CR configuration comprised the
two T cell receptor (TCR) .alpha. and .beta. chains in which each
pair of TCR variable domains (V.sub..alpha. and V.sub..beta.) was
replaced with a pair of V.sub.H and V.sub.L domain derived from a
selected antibody. These two Ab-derived coding sequences were
co-transfected into T cell lines and were found to confer antibody
specificity (Gross et al., 1989, supra). Thus, T-cells could be
activated to effector function such as cell killing or cytotoxic
activity against an immunologically specific target in a manner
that was MHC independent (and thereby non-restricted) (Gross et
al., 1992, supra).
[0022] In "second generation" CR's, the single chain configuration
of the CR was further manipulated to obtain a configuration that
would be useful particularly for cancer or antiviral therapy. Here,
a single chain Fv (scFv) of an antibody was linked to transmembrane
and cytoplasmic domains of lymphocyte triggering moieties such as
the TCR/CD3 complex-associated .zeta. chain, or the Fc receptor
.gamma. chain (Eshhar Z et al., Proc Natl Acad Sci USA 1993;
90:720-4). This single chain configuration, which combined antibody
recognition and T cell signaling in a single continuous protein,
was a modular structure with functional domains that are simple to
manipulate, and could be readily expressed in human lymphocytes
using retrovirus-based vectors (Eshhar Z et al., J Imm Meth 2001;
248:67-76). Other such receptors designed by some of the present
inventors and colleagues, and discussed in more detail below, are
referred to as tripartite chimeric receptors (TpCR) that also
include a costimulatory domain(s) (e.g. CD28, 4-1BB).
[0023] With time, it has emerged that redirection of the
specificity of T effector cells using single chain CRs has become a
valid therapeutic option for cancer. Many investigators have
adapted this "T-body" approach to endow T-cells with various
specificities and functions (Gross & Eshhar, supra; Willemsen R
A et al., Hum Immunol 2003; 64:56-68; Baxevanis C N et al., Cancer
Immunol Immunother 2004; 53:893-903). For a recent review, see:
Eshhar, Z. "The T-Body Approach Redirecting T Cells with Antibody
Specificity," in Therapeutic Antibodies. Handbook of Experimental
Pharmacology 181, Chemajovsky & Nissim (eds.), Springer-Verlag,
2008, pp. 329-342.
[0024] In the present invention, the inventors have conceived
expanded uses of such CR's for the treatment of undesired
immune/inflammatory conditions such as autoimmune diseases (with a
particular initial emphasis on inflammatory bowel disease, IBD) and
graft rejection.
[0025] Chronic Inflammatory Disease and Animal Models
[0026] Inflammatory conditions, particularly chronic inflammatory
diseases, are of particular importance in clinical medicine. These
diseases, caused by actions of the immune system, involve
inappropriate or excessive activation of certain T-cells,
expression of regulatory cytokines and chemokines, loss of immune
tolerance, and the like. Examples of autoimmune and/or chronic
inflammatory diseases are multiple sclerosis, inflammatory bowel
diseases (IBD), joint diseases such as rheumatoid arthritis, and
systemic lupus erythematosus. Some of these diseases are rather
organ/tissue-specific as follows: intestine (Crohn's Disease), skin
(psoriasis), myelinated nerves (multiple sclerosis or MS),
pancreatic islet or .beta. cells (insulin dependent diabetes
mellitus (IDDM) or Type I Diabetes), salivary glands (Sjogren's
disease), skeletal muscle (myasthenia gravis), the thyroid
(Hashimoto's thyroiditis; Graves' Disease), the anterior chamber of
the eye (uveitis), joint tissue (rheumatoid arthritis), and various
cardiovascular diseases.
[0027] Inflammatory bowel disease (IBD) is a collective term used
to describe two intestinal disorders whose etiology is not
completely understood: Crohn's disease and ulcerative colitis. IBD
occurs worldwide and afflicts several million people (0.3% of
people in Western countries), and its incidence is on the rise
(Tsironi E, et al., Am J Gastroenterol. 2004; 99:1749-55). The
course and prognosis of IBD varies widely. Onset of IBD is
predominant in young adulthood and presents typically with
diarrhea, abdominal pain, and fever; anemia and weight loss are
also common signs. Between 10% and 15% of people with IBD require
surgery over a ten year period. Patients with IBD are also at
increased risk for the development of intestinal cancer. These
diseases are accompanied by a high frequency of psychological
symptoms, including anxiety and depression.
[0028] Although the pathogenesis of this common T cell-mediated
disorder remains uncertain, it is believed to result from loss of
tolerance in the intestinal immune system due to the presence of
constant antigenic stimulation provided by the very large numbers
of resident bacteria (Podolsky D K, N Engl J Med 2002; 347:417-29).
Unfortunately, new therapies for IBD are few, and both diagnosis
and treatment have been hampered by a lack of detailed knowledge of
the etiology. A combination of genetic factors, exogenous triggers
and endogenous microflora can contribute to the immune-mediated
damage of intestinal mucosa. Bacteria have been implicated in
initiation and progression of Crohn's Disease since intestinal
inflammation frequently responds to antibiotics. Common intestinal
colonists and novel pathogens have been implicated, either because
of direct detection or disease-associated anti-microbial immune
responses. In many genetically susceptible animal models of chronic
colitis, luminal microorganisms appear to be a necessary cofactor
for disease.
[0029] The initiating step in autoimmune disease pathology is often
obscure in humans where the diseases are largely sporadic, and
symptoms may appear years after the first pathogenic T cell is
activated. It has therefore been difficult to design effective
therapies to block induction of disease. In contrast, there are
common features in many of the later stages of these diseases.
Inflammation at the disease site/target organ is typically present,
caused by the release of inflammatory, also termed
"proinflammatory," cytokines (e.g. TNF-.alpha. and interferons) by
T-cells and by other cells that contribute to the activation steps
and effector pathways of immune/inflammatory processes. These cells
include (among others) macrophages, dendritic cells and their
precursors, B lymphocytes and plasma cells and NK cells (including
NKT-cells). These reactions often involve destruction of "target"
cells and tissue damage.
[0030] Studies using murine models of experimental chronic
inflammation are helping to define nature of the immunological
dysregulation that initiates inflammation and leads to destruction
of specific end organs as well as for testing therapies. See, for
example, Mombaerts et al. Cell, 1993; 75:274-82; Tarrant et al.,
2998; J Immunol, 161:122-7; Powrie et al., Immunity, 1994;
1:553-62; Hong et al., J Immunol, 1999; 162:7480-91; Horak, Clin
Immunol Immunopathol, 1995, 76(3 Pt 2):5172-173; Ehrhardt et al. J
Immunol, 1997; 158:566-73; Davidson et al., J Immunol, 1998;
161:3143-9; Kuhn et al. Cell, 1993. 75:263-74; Neurath et al., J
Exp Med, 1995. 182:1281-90. W. Strober, 2002; Annu. Rev. Immunol.
20:495-54 reviews mucosal models of inflammation, and is
incorporated by reference in its entirety. One hallmark of the
better of these models is that the histopathology and
pathophysiology resembles that of the parallel human conditions,
further enhancing the models' utility in testing novel treatment
strategies. In the case of IBD this development has not been
uniform. Most emphasis has been placed on modulation of immune
mechanisms (Blumberg R S et al., Curr Opin Immunol. 1999;
11:648-56; Strober et al., supra) and recently of the enteric flora
(Sartor R B, Curr Opin Gastroenterol. 2001; 4:324-330).
[0031] Bhan A K et al., 1999 Immunol Rev 169:195-207 reviewed
studies of colitis in transgenic (Tg) and knockout (KO) animal
models for mucosal inflammation in IBD. Genetics and the
environment, particularly the normal enteric flora, were factors in
the development of mucosal inflammation, as stated above. Normal
mucosal homeostasis was disrupted by cytokine imbalance, abrogation
of oral tolerance, breach of epithelial barriers, and loss of
immunoregulatory cells. Some but not all immunodeficiencies, in the
appropriate setting, led to colitis. CD4+T effector cells have been
identified as the pathogenic lymphocytes in colitis, and can
mediate inflammation by either the Th1 or the Th2 pathway. The Th1
pathway dominates in most colitis models (and human Crohn's
Disease). In contrast, in the colitis observed in mice, the T cell
receptor (TCR) a chain knockout mice (TCR.alpha. KO mice) shared
many features of ulcerative colitis including the dominance of Th2
pathway in colon inflammation. Such models are important for the
development of therapeutic strategies to treat IBD. In a later
review, the same group (Mizoguchi A et al., 2003, Inflamm Bowel
Dis. 9:246-259) noted that exaggerated immune responses to normal
enteric microflora are involved in the initiation and perpetuation
of chronic intestinal inflammation. A major pathway involves
development of "acquired" immune responses by the interactions of
CD4+TCR.alpha..beta.+T-cells with antigen-presenting cells (APC),
particularly dendritic cells. CD4+CD25+ Treg cells attenuated
activated T cell responses.
[0032] The progression from the acute to the chronic phase of IBD
has not been well characterized in animal models and cannot be
easily evaluated in patients. Spencer D M et al. Gastroenterol.
2002; 122:94-105 reported changes in the mucosal immune response
over time in experimental colitis. Severity of colitis, body mass,
stool consistency and blood content, serum amyloid A, and tissue
histology were examined in mice deficient in interleukin-10 (IL-10)
over 35 weeks. The corresponding production of IL-12, IL-18,
IFN.gamma., TNF.alpha., IL-4, and IL-13 by lamina propria
mononuclear cells in the inflamed intestine was measured.
Administration of a neutralizing anti-IL-12 monoclonal antibody
(mAb) at distinct times during disease progression permitted
evaluation of the therapeutic potential of this agent. Lamina
propria mononuclear cells from mice with early disease synthesized
progressively more IL-12 and IFN.gamma., whereas production of both
cytokines declined dramatically and returned to pre-disease levels
in the late phase. Consistent with this pattern, the neutralizing
anti-IL-12 reversed early, but not late, disease. In contrast, IL-4
and IL-13 production increased progressively from pre- to early to
late disease. It was concluded that colitis developing in
IL-10-deficient mice evolves into two distinct phases. IL-12 plays
a pivotal role in early colitis, whereas other immune mechanisms,
presumably mediated by IL-4 and IL-13, predominate in late disease
to sustain chronic inflammation.
[0033] IL-10 and Chronic Inflammatory Disease
[0034] It has been known for some years that IL-10 affects the
growth and differentiation of many hemopoietic cell types in vitro
and is a particularly potent suppressor of macrophage and T cell
functions. These observation were based in part from use of
IL-10-deficient (knockout, KO) mutant mice by gene targeting (Kuhn
R et al., Cell 1993; 75:263-74). In these mice, lymphocyte
development and antibody responses are normal, but most animals are
growth retarded, anemic and suffer from chronic enterocolitis.
Alterations in the intestine include extensive mucosal hyperplasia,
inflammation, and aberrant epithelial expression of major
histocompatibility complex (MHC) class II molecules. In contrast,
IL-10 KO mutants kept under specific pathogen-free conditions,
develop only localized inflammation (limited to the proximal
colon). It was concluded that (1) bowel inflammation in these
mutants originated from uncontrolled immune responses stimulated by
enteric antigens and (2) IL-10 is an essential (negative) regulator
in the intestinal tract.
[0035] In a study validating this IL-10 KO mouse model of colitis,
T. Scheinin, T et al. (Clin Exp Immunol 2003; 133:38-43) emphasized
that a valuable animal model must respond to existing therapy in a
way that resembles the response of human disease. Since refractory
Crohn's Disease responded well to anti-TNF.alpha. antibody therapy,
the investigators examined responses of IL-10 KO mice to
anti-TNF.alpha. therapy, using a new scoring system similar to the
Crohn's Disease "Activity Index" in humans. Stool samples were
tested for cytokines and the findings compared with histology.
Results showed that anti-TNF antibody therapy starting at 4 weeks
markedly ameliorated the disease (as judged by the clinical score
or by gut histology). A marked diminution of inflammatory cytokines
in stool samples was noted, adding a further accurate measure of
clinical improvement. The authors concluded that this model is
useful for evaluating other therapeutic modalities of relevance to
Crohn's Disease.
[0036] Treg Cells and Inflammatory Bowel Disease
[0037] One of the commonly used animal models of IBD involves
adoptive transfer of CD45RB.sup.hiCD4+T-cells into SCID mice,
leading to the development of massive colon mononuclear cell
infiltrates, epithelial cell hyperplasia and ulceration (Thornton A
M, et al., J Immunol 2000; 164(1):183-90). Cotransfer of large
numbers of CD4+CD25+ Tregs prevented the development of colitis or
cured established colitis, an affect that required signaling
through CTLA-4 (Read S, et al., J Exp Med 2000; 192:295-302; 2000;
Morrissey P J, et al., J Exp Med 1993; 178:237-44). Even after the
development of immune-mediated colitis, adoptive transfer of
10.sup.6 CD4+CD25+ cells caused significant improvement of
intestinal inflammation (Fantini M C et al., Gut 2006; 55:671-80;
Mottet C, et al., J Immunol 2003; 170(8):3939-43; Uraushihara K, et
al., J Immunol 2003; 171:708-16).
[0038] In humans suffering from IBD, peripheral regulatory
CD4+CD25+ cells retain suppressive activity. However, in contrast
to other intestinal inflammatory disorders, the number of these
regulatory cells decreases in peripheral blood during active
inflammation and only slightly increases in intestinal lesions
(Maul J, et al., Gastroenterology 2005; 128(7):1868-78). This
aberration suggests that Treg homing defects, as well as
dysregulated in situ activation contribute to the pathogenesis of
IBD
[0039] There is therefore a recognized need in the art to find
modalities to suppress autoimmune/inflammatory reactions and
diseases, including but not limited to IBD, as well as to suppress
rejection of organ and tissue grafts and prevent Graft vs. Host
(GVH) disease. The present invention provides a novel approach,
that of redirecting Treg cells, as a means to recruit Tregs to
sites of inflammation, and activate them to suppress such
immune/inflammatory reactions and protect against, alleviate and
even cure such disease as IBD.
SUMMARY OF THE INVENTION
[0040] The present invention is based on the inventors' conception
that CR-mediated redirection and activation of Treg cells at sites
of inflammation results in suppression of inflammatory conditions,
commonly part of organ-specific autoimmune disease and exemplified
herein as inflammation in the colon in experimental IBD. The
inventors have further conceived of using these cells to overcome
rejection of mismatched cells and tissues by T effector cells that
arise in transplant recipients or to inhibit the pathogenic action
of transplanted immunocompetent cells in the case of GVH
disease.
[0041] The invention relies on the inventors' innovative T-body
approach that has thus far proven useful for immunotherapy of
cancer (and is currently in phase I/II clinical trials). The
invention provides a new approach to the exploitation of Treg cells
for amelioration of pathologic and undesired immune responses,
particularly immunotherapy of autoimmune and inflammatory
conditions, including various organ-predominant autoimmune
diseases, and other pathologic or undesirable immune responses such
as graft rejection and graft vs. host disease.
[0042] According to this invention, Treg cells are endowed with CRs
that are specific for a selected target antigen or ligand. Such
modification causes activation of redirected Tregs at sites of
inflammation to suppress the proinflammatory effector-type immune
responses. Based on the present inventors' (and their colleagues')
results with redirecting antitumor effector lymphocytes, it is
expected that Tregs, endowed with predefined specificity, will
migrate/home to and accumulate in, a targeted site, such as the
inflamed colon, where they will suppress disease-mediating T
effector cells. To avoid the necessity of migration or homing to
the targeted site, the Tregs may, where possible, be administered
directly at or to such site, where they will become activated and
suppress disease-mediating T effector cells.
[0043] Such redirected Treg cells, also referred to as "T-bodies,"
are Treg cells that have been genetically engineered to express a
CR, preferably a tripartite chimeric receptor (TpCR) that is made
of a single chain extracellular recognition unit, a transmembrane
region, and an intracellular signaling region.
[0044] The extracellular recognition region is specific for a
selected target antigen or ligand and may preferably be a single
chain antibody variable (scFv) region or another ligand that is
capable of binding to the target antigen or ligand. The
extracellular recognition region preferably does not comprise an
MHC protein extracellular domain. The redirected Tregs of the
present invention are sometimes referred to herein as "T-bodies"
despite the fact that the extracellular recognition region is not
necessarily an antibody domain. Thus, this term is not intended to
be limited to Tregs with antibody-like specificity, but also
includes Tregs with ligand-receptor-like specificity or
otherwise.
[0045] A flexible spacer region may be present between the
extracellular recognition region and the transmembrane region. Such
flexible spacer is preferably an immunoglobulin (Ig)-like hinge,
such as any hinge region derived from the Ig superfamily.
[0046] The intracellular region includes a combination of T-cell
signaling polypeptide moieties, fused in tandem, which combination
of moieties, upon binding of the extracellular recognition region
to the selected target antigen or ligand, triggers activation of
the Treg cells to cause suppression of T-cell mediated immunity.
The T-cell signaling moieties preferably include one or more
cytoplasmic domains of a costimulatory molecule (e.g., CD28) and a
cytoplasmic T-cell stimulatory domain, e.g., of FcR.gamma. or a
CD3-.zeta. chain. The redirected Treg cells become specifically
activated, upon binding of the extracellular recognition region of
the CR to its target antigen or ligand, in a manner that is
preferably (1) not restricted by, or dependent upon, the binding of
the target antigen or ligand to an MHC, nor is it otherwise
dependent in any way on the MHC (HL-A) haplotype of the recipient
and (2) independent of engagement of costimulatory ligand(s) on a
target cell.
[0047] A preferred target disease of this invention is an IBD such
as ulcerative colitis, in which the present methods, as used
successfully in an animal model, will permit Treg cells to reach
bowel lesions in IBD patients and become efficiently activated at
the inflammation site. The present invention results in
site-specific Treg accumulation, ultimately resulting in
CR-mediated, antigen-specific activation that results in the
production of suppressive cytokines which in turn suppress effector
autoimmune T-cells in an antigen-nonspecific manner, leading to
alleviation of symptoms and thereby treating the disease.
[0048] The present therapeutic approach has several unique
advantages. In contrast to other immunotherapeutic models, it
involves T-bodies redirected with a CR, and preferably a TpCR, that
combines antibody/antigen or ligand/receptor recognition with
stimulatory and costimulatory motifs. Thus, T-bodies can be fully
activated in a way that is not restricted by the MHC and is
independent of a requirement for costimulation. The second
advantage of the present invention stems from the fact that,
although Treg activation is antigen-dependent, the suppressive
action of these cells is antigen-, TCR-, and MHC-independent. By
exploiting this property, one can construct a chimeric receptor
that is specific for one or more tissue-associated antigens rather
than requiring specificity for an unknown number of yet undefined
autoimmune disease-specific antigens. Expression of such chimeric
receptors in Tregs redirects these cells and their activation to
the appropriate target tissue (in a preferred embodiment, the
colon) so that they are activated in an antigen-specific manner,
where their potent suppressive effects take place without a need
for further recognition of disease-associated-antigens (the
"bystander effect"). By using specifically-activated Tregs, many
fewer cells are required to treat autoimmune inflammatory
conditions; such as IBD, or allograft-associated reactions in
patients than would have been possible prior to this invention when
much higher numbers of non-specific Tregs would have been
needed.
[0049] The present inventors have constructed strains of transgenic
(Tg) mice whose T-cells and natural killer (NK) cells express an
antigen-specific TpCR. For exemplification of the invention, the
inventors selected the trinitrophenyl (TNP) hapten as the specific
target of the TpCRs. TNP specific Tregs isolated from these Tg mice
suppressed TNP-specific effector T-cells in vitro and in vivo and
were able to suppress trinitrobenzenesulfonic acid (TNBS)-induced
colitis in mice. In this embodiment, the target antigen for Treg
and the pathogenic antigen--the hapten TNP--are the same. In
another example, TNP-specific Tregs suppressed oxazolone-induced
colitis in mice in which a low dose of TNP was introduced into the
colon together with the oxazolone challenge. However, the
TNP-specific Tregs had no effect on the oxazolone-induced colitis
in the absence of TNP introduction. This establishes the
"bystander" effect of the present invention, i.e., that the target
antigen need not be the pathogenic antigen, as long as the
redirected Tregs are activated in the vicinity of the pathogenic or
undesired immune response.
[0050] One distinct advantage of the present invention is that it
provides cells and methods that permit antigen-specific activation
and antigen-nonspecific action of Treg cells used to suppress
effector T cell responses (and treat consequent pathologies) in a
way that does not require identity between the ligand (e.g., the
antigen) recognized by the TpCR (e.g., by its target recognition
portion) and the ligand/antigen that plays a pathogenic role in the
disease process. Thus, the antigen that is pathogenic does not have
to be recognized by the T effector cells being suppressed, and,
indeed, may be unrelated to the disease or condition being treated.
Thus, the invention exploits the "bystander" effect. As long as the
Treg is in the correct vicinity where T effector cells are located
and mediating their undesired effects, the redirected Tregs of the
present invention can be triggered or activated at that location to
release of suppressive cytokines (e.g., IL-10 and TGF-.beta.), that
will result in suppression of any "bystander" effector T-cells, and
by this mechanism, quell an ongoing inflammatory/autoimmune
response.
[0051] The unique characteristics of the Tg system used in the
present examples enables evaluation of the suppressive effect of
antigen-specific Tregs in IBD both in vitro and in vivo. Different
means are used to induce Tregs, allowing those of skill in the art
to select the optimal method for generating efficient numbers of
antigen-specific redirected Tregs for a desired antigen or
disease/condition. According to this invention, redirected human
Tregs constitute an effective cell-based therapeutic modality for
IBD or ulcerative colitis and, more broadly, for any T effector
cell-mediated disease or condition.
[0052] To overcome the scarcity of antigen-specific Tregs, the
present invention includes methods to induce these cells using
cytokines (e.g. TGF-.beta.) or by expression of transgenes (e.g.
encoding the Foxp3 transcription factor) that will, together with
the TpCR, allow antigen-specific Treg expansion.
[0053] According to the present invention, human Treg cells,
derived from either the subject with the autoimmune/inflammatory
disease or condition to be treated, or from an HLA-matched healthy
donor (or a universal cell that is not recognized by the
recipient's immune system), are endowed with
antigen/ligand-specificity, by transduction with the
antigen/ligand-specific TpCR as disclosed herein. Alternatively,
the cells being endowed with antigen-specificity are the entire
T-cell population and the nucleic acid construct including the
sequence encoding the TpCR further includes a Foxp3 transgene that
is present as an independently transcribed cistron. In another such
alternative, a Foxp3 transgene is separately transfected into the
T-cell population, to turn the T-cells into Treg cells. Examples
provided below include studies using murine colonoscopy, in vivo
imaging and immunofluorescence, and provide the basis for a novel
cell-based therapeutic modality for IBD, and, by extension, for
other pathologic and undesired immune responses mediated by antigen
specific T effector cells.
[0054] Various embodiments of the invention are described more
specifically below. [0055] The present invention is directed to a
redirected regulatory T lymphocyte (Treg cell) endowed with
specificity toward a selected target antigen or ligand, which cell
comprises a chimeric nucleic acid that encodes a chimeric receptor
(CR) polypeptide that comprises, expressed in a single, continuous
chain, an extracellular recognition region, a transmembrane region
and an intracellular signaling region, and is expressed in the Treg
cell so as to display the extracellular region on the cell surface,
wherein [0056] (a) the extracellular recognition region of the
chimeric receptor is specific for the selected target antigen or
ligand, and does not comprise an MHC protein extracellular domain;
and [0057] (b) the intracellular region comprises a combination of
T-cell signaling polypeptide moieties which combination of
moieties, upon binding of the extracellular recognition region to
the selected target antigen or ligand, triggers activation of the
Treg cells to cause suppression of T-cell mediated immunity.
[0058] In preferred embodiments of the present invention, the
extracellular recognition region is an antibody-derived scFv domain
that is specific for a selected antigen. In another preferred
embodiment, the extracellular recognition region is a member of a
ligand-receptor pair, which is specific for the other member of
that pair.
[0059] Preferably, the extracellular recognition region is linked
to the transmembrane region through a flexible spacer, which, more
preferably, is a hinge from a molecule of the immunoglobulin
family.
[0060] The intracellular signaling region preferably includes a
signaling moiety from a chain of an antigen-specific T-cell
receptor, which more preferably is one having a polypeptide region
comprising an immunoreceptor tyrosine-based activation motif
(ITAM). Non-limiting examples of antigen-specific T-cell receptors
are chains of the TCR/CD3 complex, a TCR .alpha., .beta., .gamma.
or .delta. chain, and the .gamma. chain of an Ig Fc receptor
(FcR.gamma.). The chain of an antigen-specific T-cell receptor is
preferably the CD3/.zeta. chain or an FcR.gamma. subunit.
[0061] The intracellular signaling region further preferably
includes a signaling moiety of a costimulatory-receptor protein of
a T-cell. The costimulatory-receptor protein is preferably selected
from CD28, OX40, CD40L (gp39), 4-1BB and PD-1 (or preferably the
human form or homolog of these costimulatory molecules). Most
preferred among these is CD28 or 4-1BB. In another preferred
embodiment, the intracellular signaling region includes more than
one of the costimulatory-receptor protein signaling moieties. For
example, the combination of T-cell signaling polypeptide moieties
in the intracellular signaling region may include both CD28 and
4-1BB. In a particularly preferred embodiment, the extracellular
hinge and transmembrane regions of CD28 are used as the
extracellular hinge and transmembrane regions of the chimeric
receptor.
[0062] The intracellular signaling region may also include a
signaling moiety from a cytokine receptor of a T-cell, such as the
IL-2 receptor or the TGF-.beta. receptor. The latter may help to
induce the T-cell containing the chimeric receptor to adopt the
characteristics of a Treg cell.
[0063] The intracellular region may also include a
signal-transducing enzyme that (a) is an enzyme in the signal
transduction pathway of an antigen-specific T-cell receptor or (b)
is an enzyme with corresponding specificity and activity as the
enzyme of (a), derived from a non-T-cell lymphocyte. Such enzyme is
preferably a kinase, such as the Syk kinase.
[0064] The chimeric nucleic acid encoding the CR may also include a
nucleotide sequence that encodes Foxp3 arranged such that Foxp3 is
expressed by the Treg cell independently of the chimeric receptor.
In other words, the chimeric nucleic acid may be bicistronic such
that the Foxp3 transgene is present as an independently transcribed
cistron.
[0065] The target antigen or ligand is preferably one that is
present or expressed at a site or target tissue of an immune or
inflammatory response mediated by effector T-cells. The autoimmune
or inflammatory response may comprise an autoimmune response or
disease, an allograft or xenograft response or rejection, or graft
vs. host (GVH) disease. In an alternative preferred embodiment, the
target antigen or ligand may be an autoantigen or an antigen that
is cross-reactive with an autoantigen, i.e., is also bound by an
antibody that is specific to the autoantigen. The autoantigen may
be a pathogenic antigen in the pathophysiology of the autoimmune
disease.
[0066] The antigen is not necessarily an autoantigen, but can be,
for example, an antigen that is part of the bacterial flora, such
as LPS derived from the bacteria native to the colon.
[0067] The autoimmune disease or graft response and the
antigen/ligand or antigens/ligands against which the Treg is
specific is preferably selected from the following group: [0068]
(a) inflammatory bowel disease (IBD), wherein the antigen or ligand
is one that is expressed in diseased colon or ileum; [0069] (b)
rheumatoid arthritis, wherein the antigen or ligand is an epitope
of collagen or an antigen present in joints; [0070] (c) Type I
diabetes mellitus or autoimmune insulitis, wherein the antigen or
ligand is a pancreatic .beta. cell antigen; [0071] (d) multiple
sclerosis, wherein the antigen or ligand is, for example, a myelin
basic protein (MBP) antigen or MOG-1 or MOG2-2, or a neuronal
antigen. [0072] (e) autoimmune thyroiditis, wherein the antigen or
ligand is a thyroid antigen; [0073] (f) autoimmune gastritis,
wherein the antigen or ligand is a gastric antigen; [0074] (g)
autoimmune uveitis or uveoretinitis, wherein the antigen or ligand
is S-antigen or another uveal or retinal antigen [0075] (h)
autoimmune orchitis, wherein the antigen or ligand is a testicular
antigen; [0076] (i)) autoimmune oophoritis, wherein the antigen or
ligand is an ovarian antigen; [0077] (j) psoriasis, wherein the
antigen or ligand is a keratinocyte antigen or another antigen
present in dermis or epidermis; [0078] (k) vitiligo, where the
antigen or ligand is a melanocyte antigen such as melanin or
tyrosinase; [0079] (l) autoimmune prostatitis, wherein the antigen
or ligand is a prostate antigen; [0080] (m) any undesired immune
response, wherein the antigen or ligand is an activation antigen or
other antigen expressed on T effector cells present at the site of
the undesired response; [0081] (n) tissue rejection, wherein the
antigen or ligand is the MHC specific to the transplanted tissue;
and [0082] (o) an inflammatory condition, wherein the antigen or
ligand is one that is expressed on nonlymphoid cells of the
hemopoietic lineage that participate in inflammation.
[0083] Most preferred is a Treg cell that is able to act and
suppress IBD or ulcerative colitis, and may be specific for an
antigen associated with IBD such as carcinoembryonic antigen (CEA)
or an antigen of intestinal bacterial flora such as bacterial
lipopolysaccharide (LPS) or a component thereof, preferably a Lipid
A component.
[0084] The Treg may be specific for an activation antigen expressed
on T effector cells such as CD69 or CD107a. The Treg may be
specific for an antigen expressed on a dendritic cell,
macrophage/monocyte, granulocyte or eosinophil present at the
inflammation site.
[0085] In a preferred embodiment, the above Treg cell is specific
for an antigen that is introduced exogenously to a subject to the
site or target tissue of the immune or inflammatory response,
either before, concomitantly with, or after administration of the
Treg cell.
[0086] The above Treg cell preferably is one that expresses CD4 or
CD8, along with CD25 on its surface and expresses the Foxp3
transcription factor intracellularly. The Foxp3 transcription
factor may be expressed in the cell endogenously (i.e., from the
cells' own Foxp3 gene); this expression is enhanced by exposure of
cells to TGF-.beta. or another cytokine that induces Foxp3
expression and induces a Treg phenotype in T-cells. In another
embodiment of the above Treg cell, Foxp3 is expressed from a
nucleic acid that has been introduced into the cell exogenously
(i.e., transduced) as a recombinant nucleic acid expression
construct encoding Foxp3 and regulating its expression. The above
Treg may be obtained from a mammalian subject prior to introduction
of the chimeric nucleic acid and prior to stimulation that induces
Foxp3 expression or prior to transducing the exogenous
Foxp3-encoding construct. The chimeric nucleic acid encoding the
chimeric receptor and the nucleic acid construct encoding Foxp3 may
be co-transduced into the cell. In one embodiment, co-transduction
is achieved using a bicistronic vector that includes, in a single
vector, a sequence of (i) the chimeric nucleic acid encoding the
chimeric receptor and (ii) the nucleic acid construct encoding
Foxp3, under the control of a common (or separate) promoter and
regulatory sequences.
[0087] The above Treg cells may be enriched or purified from a
mixed population of lymphocytes or T-cells on the basis of the Treg
cells' expression of CD4 (or CD8) and CD25 and/or Foxp3. The cell
may be subjected to the following treatment: [0088] (a) exposure ex
vivo of: [0089] (i) peripheral blood mononuclear cells, [0090] (ii)
peripheral blood lymphocytes, [0091] (iii) T-cells enriched or
purified from (i) or (ii), or [0092] (iv) a subset of T-cells
enriched or purified from (iii); to an amount of TGF-.beta. or
other Treg-inducing cytokine or agent that is effective to convert
T-cells to a Treg phenotype and to induce expression of Foxp3; and
[0093] (b) optionally, culturing and expanding the exposed cells of
step (a). Preferred Treg cells comprise the above cell that has
been transduced with an expression vector encoding Foxp3.
[0094] Also provided herein is an immunoregulatory pharmaceutical
composition for suppressing a T effector cell-mediated
immune/inflammatory response or treating a T effector cell-mediated
immune/inflammatory disease or condition, comprising a redirected
Treg as described above and a pharmaceutically and immunologically
acceptable carrier, excipient or diluent.
[0095] This invention is also directed to a method for producing
the above redirected Treg that expresses a chimeric receptor as
described. This method preferably comprises: [0096] (a) obtaining
from a subject and, optionally, enriching or isolating and
propagating, a population of lymphocytes or T-cells; [0097] (b)
inducing the Treg phenotype in these lymphocytes by suitably
stimulating or activating the cells by exposure to TGF-.beta. or
another cytokine or agent that induces Foxp3 expression and a Treg
phenotype; [0098] (c) before or after step (b), transducing the
cells ex vivo with an expression vector encoding the chimeric
receptor to be expressed in the Treg; and [0099] (d) optionally,
growing or expanding in vitro the cells obtained as above.
[0100] In another embodiment, this method comprises: [0101] (a)
obtaining from a subject and, optionally, enriching or isolating
and propagating, a population of lymphocytes or T-cells; [0102] (b)
transducing the cells ex vivo with a vector encoding the chimeric
receptor; [0103] (c) before, after, or concomitantly with step (b),
transducing the cells ex vivo with a recombinant nucleic acid
expression construct encoding Foxp3; and [0104] (d) optionally,
growing or expanding in vitro the cells obtained as above.
[0105] This invention further is directed to a method of
suppressing undesired activity of T effector cells in mediating an
immune or inflammatory response, comprising delivering to a
population of T effector cells to be suppressed (or to a site where
such T effector cells are present) an amount/number of redirected
Tregs as above, effective to suppress activity of the T effector
cells.
[0106] Also intended is a method of suppressing undesired activity
of T effector cells as indicated above, which method comprises
delivering to a population of T effector cells to be suppressed (or
to a site where such T effector cells are present) an amount/number
of redirected Tregs produced according to the above methods that
are effective for suppressing the T effector cell activity.
[0107] This delivering is preferably in vivo. The redirected Treg
cells are delivered by injection or infusion to a subject in whom
the T effector cell activity is to be suppressed, preferably by a
route selected from intravenous, intramuscular, subcutaneous,
intraperitoneal, intra-articular, intrathecal, intraluminal,
intracerebroventricularly, rectal, and topical. In one embodiment,
the Treg cells are delivered regionally or locally to a site of
inflammation.
[0108] The above method is intended for use in situations wherein
the T effector cells mediate an autoimmune inflammatory response or
disorder, rejection of a transplant or GVH disease.
[0109] In one embodiment, the method for treating or ameliorating
symptoms of a disease or condition in a subject that is mediated by
undesired activity of T effector cells comprises administering to
the subject in need thereof an effective amount/number of Treg
cells as described above, or a pharmaceutical composition described
above, wherein the target recognition domain of the redirected Treg
cells is specific for an antigen/ligand present in the subject in
the vicinity of the T effector cells so that, upon recognizing and
binding the antigen, these redirected Treg cells are activated to
secrete suppressive cytokines that suppress the T effector cells in
an antigen-nonspecific manner. As noted above, the Treg cell
activation occurs in a manner that is not restricted by the MHC and
does not require costimulation by a ligand for the costimulatory
signaling protein.
[0110] Also included is a method for treating or ameliorating
symptoms of a disease or condition in a subject that is mediated by
undesired activity of T effector cells, the method comprising: (a)
producing redirected Treg cells using the production methods
described above; (b) administering to the subject in need thereof
an effective amount/number of these Treg cells, thereby treating or
ameliorating symptoms of the disease or condition.
[0111] Stated more generally, the invention is directed to a method
for suppressing a T effector cell-mediated immune/inflammatory
process in a subject in need thereof, comprising administering to
the subject an effective amount/number of redirected Treg cells
that express on their surface an antigen-specific chimeric receptor
that includes portions that activate Treg cells upon contact with
the antigen for which the receptor is specific, the antigen being
one that is present in the vicinity of the immune/inflammatory
activity. The disease or condition to be treated or ameliorated is
preferably: (a) IBD; (b) rheumatoid arthritis; (c) Type I diabetes
mellitus or autoimmune insulitis; (d) multiple sclerosis; (e)
thyroiditis; (f) gastritis; (g) uveitis or uveoretinitis; (h)
orchitis; (i)) oophoritis; (j) psoriasis; (k) prostatitis; (l)
encephalomyelitis; (m) vitiligo; (n) rejection of a mismatched
cell, tissue or organ graft; or (o) GVH disease.
[0112] The present method is used to inhibit the rejection of
transplanted cells, tissue, or an organ (alto- or xeno-) that is,
for example, mismatched for a major and/or one or more minor
histocompatibility antigens. In the case of GVH disease, the
recipient generally has received a transplant of allogeneic,
semi-allogeneic or non-MHC-mismatched bone marrow cells or enriched
or isolated hematopoietic stem cells that are responsible for
mediating pathogenic effects.
[0113] The present invention is further directed to the novel
chimeric DNA that can be used to produce the redirected T-cells
described above, as well as to the chimeric receptor protein
encoded thereby. Such chimeric DNA comprises:
[0114] a first DNA segment encoding an extracellular recognition
region specific for a selected target antigen or ligand, which does
not comprise an MHC protein extracellular domain, the selected
target antigen or ligand being one that is present or expressed at
a site or target tissue of a pathogenic or undesired immune
response mediated by effector T-cells;
[0115] a second DNA segment encoding a transmembrane region;
and
[0116] a third DNA segment encoding an intracellular signaling
region comprising a combination of T-cell signaling polypeptide
moieties, which combination of moieties, upon transfection of the
chimeric DNA into a regulatory T lymphocyte (Treg cell) and binding
of the extracellular recognition region to the selected target
antigen or ligand thereof, triggers activation of the Treg cells to
cause suppression of T-cell mediated immunity, which chimeric DNA,
upon transfection into a Treg cell, expresses the extracellular
recognition region, the transmembrane region and the intracellular
signaling region in one single, continuous chain on the surface of
the transfected cell such that the transfected Treg is triggered to
activate and cause suppression of T-cell mediated immunity when the
expressed extracellular recognition region binds to its selected
target antigen or ligand.
BRIEF DESCRIPTION OF THE DRAWINGS
[0117] FIG. 1: Schematic diagram of TNP-specific TPCR structure.
(A) Schematic presentation of the TNP-specific chimeric receptors.
The TNP-specific CR encompasses a scFv derived from the anti-TNP
mAb, Sp6. In the tripartite configuration, the scFv is joined in
tandem to a short portion of CD28 (lacking the ligand-binding site)
of the extracellular and including the transmembrane, and
cytoplasmic domains fused to the FcR.gamma. ITAM domain. (B)
Chimeric receptor transgene constructs. Constructs used to generate
the transgenic mice were placed under the control of the human CD2
promoter/enhancer that directs expression only in T and NK cells.
CYT indicates cytoplasmic domain; H, hinge domain; L,
immunoglobulin leader; LCR, locus control region; P, promoter; pL,
plasmid sequence; TM, transmembrane domain; VH and VL,
immunoglobulin heavy and light-chain variable domains,
respectively; ACD28, truncated CD28 containing part of the
extracellular and the transmembrane domain, and lacking the
cytoplasmic signaling moiety.
[0118] FIG. 2: Flow cytometry results of Foxp3 staining of
TNP-specific Tregs. Splenocytes isolated from WT and TNP-Tg mice
were stained for intracellular Foxp3 and for TNP-specific chimeric
receptor using antiidiotypic antibody to the Sp6 scFv.
Representative flow cytometry analyses are shown for an individual
mouse out of five tested mice. Percentages indicate double-stained
cells.
[0119] FIG. 3: Graph showing the ratio of CD4+CD25+ cells to CD4+
cells in splenocytes of wildtype and Tg mice. The groups are:
wildtype mice, mice Tg for a chimeric receptor specific for an
"irrelevant" antigen, ErbB2 (also referred to as ErbB2-Tg mice),
TNP-Tg mice that have been transfected with a vector lacking the
transgenic costimulatory CD28 domain (also referred to as
TNP.DELTA.CD28-Tg mice), and TNP-Tg. TNP-Tg Tregs fully express the
TNP-specific TpCR
[0120] FIG. 4: Graph (left) showing Foxp3+/CD4 cell ratio in
wildtype, ErbB2-Tg, TNP.DELTA.CD28-Tg, and TNP-Tg mice. Flow
cytograms (right) showing splenic Foxp3 expression. Results compare
wildtype, ErbB2-Tg, TNP-Tg and TNP.DELTA.CD28-Tg mice.
[0121] FIG. 5: Flow cytograms showing Foxp3 staining in sorted
wildtype, ErbB2-Tg, TNP-Tg and TNP-.DELTA.CD28-Tg CD4+CD25+
effector T-cells.
[0122] FIG. 6: Graph (left) showing ratio of Foxp3+ to CD25+/Foxp3+
cells. Flow cytogram (right) showing co-staining of Foxp3 and CD25.
Results compare wildtype, ErbB2-Tg, TNP-Tg and TNP-.DELTA.CD28-Tg
splenocytes.
[0123] FIG. 7: Graph showing percentage of Foxp3+ splenocytes in
the total CD3+T-cell population following induction of TNBS
colitis. Splenic lymphocytes were isolated from WT and TNP-Tg mice
prior to or 48 hours following induction of TNP colitis, and
double-stained with anti-Foxp3 and anti-CD3 antibodies.
[0124] FIG. 8: Graph showing percentage of Foxp3+ lymphocytes
extracted from colonic lamina propria following induction of TNBS
colitis. Lymphocytes were isolated from WT and TNP-Tg mice prior to
and 48 hours following induction of TNP colitis, and double-stained
as in FIG. 7. The percentage of Foxp3+ lymphocytes in the CD3+
population is presented as the average Foxp3/CD3 ratio .+-.s.d. of
each five-mouse group. Data shown are averages of two independent
experiments performed. Differences in ratios between naive and
colitis-induced TNP-Tg mice were significant (P<0.05).
[0125] FIG. 9: Series of 6 graphs showing stimulation of
proliferation of redirected Tregs (left) and T effector cells
(right) by an antigen-nonspecific stimulus (anti CD3 and anti CD28
mAbs) and antigen-specific (TNP) stimulus.
[0126] FIG. 10: Graph showing polyclonal activation with
Concanavalin A (Con A) of co-cultures of Tregs and T effectors
cells (and control cultures of individual cell populations)
[0127] FIG. 11: Graphs showing Specific activation of TNP-Tg Tregs
and their suppression of effector T-cells requires TNP and
CD28-co-stimulation. In the left panel, specific (TNP) activation
of Tregs is shown. WT or TNP-Tg Tregs (5.times.10.sup.4) were
cocultured with WT or TNP-Tg Teff (5.times.10.sup.4) in the
presence of irradiated, T-cell depleted, TNPylated splenic APC
(1.5.times.10.sup.5). Teff proliferation was measured after 48
hours by .sup.3H-Thymidine incorporation. Right panel: TNP-loaded
APCs as stimulus.
[0128] FIG. 12: Graph showing dose-response of TNP-specific
stimulation of Treg cell+T effector cell cocultures. APCs were
TNP-modified stimulator P815 mastocytoma cells, which do not
express B7 (P815-TNP) or TNP-modified P815 cells into which the B7
gene was stably transfected (B7-TNP).
[0129] FIG. 13: Graph showing Specific activation of TNP-Tg Tregs
and their suppression of effector T-cells requires TNP and
CD28-co-stimulation. To establish whether costimulatory signaling
is required for TNP-Tg Treg activation, coculture experiments were
repeated using as APC irradiated P815 mastocytoma cells
(1.5.times.10.sup.5) that were either stably transfected (or not)
with B7 cDNA. Teff cell proliferation was measured after 48 hours
by .sup.3H-Thymidine incorporation. Each group was cultured in
triplicate and the experiment was repeated three times. The data
shown represent mean (+s.d.) of triplicate cultures of a
representative experiment. Differences in stimulation index between
Teff+WT Tregs and Teff+TNP-Tg Tregs were significant
(P<0.01).
[0130] FIG. 14: Photograph of colon of wildtype mice (left) and
TNP-Tg mice (right) four days after induction of high-dose TNBS
colitis by intrarectal instillation of TNBS at day 0.
[0131] FIG. 15: Mortality curve of wild-type (WT), TNP-Tg, ErbB2-Tg
and TNP-.DELTA.CD28-Tg mice following induction of TNBS colitis by
intrarectal instillation of TNBS at day 0.
[0132] FIG. 16: Photomicrograph of stained tissue (H&E,
40.times.) of the colons from FIG. 14.
[0133] FIG. 17: Mortality curve of WT, TNP-Tg, ErbB2-Tg and
TNP-.DELTA.CD28-Tg mice following induction of colitis with
oxazolone (OXA; a hapten/antigen that is distinct from TNP). These
results serve as a control for Treg specificity in the experiment
the results of which are shown in FIGS. 14-16. Colitis was induced
using the unrelated hapten, oxazolone, which was intrarectally
instilled in similar strains of mice (n=10).
[0134] FIG. 18: Flow cytograms of Foxp3 staining of wildtype,
ErbB2-Tg, TNP-.DELTA.CD28-Tg and TNP-Tg T effector cells after a
week of culture in the presence of the following "stimuli" (across
top): anti-CD3, TGF-.beta., anti-CD3+TGF-.beta., TNP, or
TNP+TGF-.beta..
[0135] FIG. 19: Graphs showing mortality or survival rate of
wildtype mice subjected to induction of moderate (left panel) and
severe (right panel) TNBS colitis following adoptive transfer of
the following Treg populations: WT, ErbB2-Tg, TNP-Tg and
TNP-.DELTA.CD28-Tg.
[0136] FIG. 20: Graph showing Wallach Colitis Severity Score of
wildtype mice subjected to induction of TNBS colitis after adoptive
transfer of cells from WT, ErbB2-Tg or TNP-Tg donors. TNBS colitis
was induced in WT mice (n=8) on day 0. After 16 hours, Tregs
(1.times.10.sup.5) from TNP-Tg, ErbB2-Tg or WT mice were adoptively
transferred to the recipient mice. Each experiment was repeated
three times. The data shown represent the average of a
representative experiment.
[0137] FIG. 21: Photograph of excised colons of wildtype mice in
which TNBS colitis was induced, following adoptive transfer of
wildtype, ErbB2-Tg and TNP-Tg mice in the experiment described in
FIG. 20.
[0138] FIG. 22: Photomicrograph of stained colon tissue sections
(H&E, 40.times.) from wildtype mice with TNBS colitis after
adoptive transfer of Tregs from the following donors: (A) wildtype
(B) ErbB2-Tg and (C) TNP-Tg. Panel D shows normal control
colon.
[0139] FIG. 23: Localization of Tregs in the colon. Flow cytograms
of fluorescent staining Tregs labeled with the intracellular dye
carboxyfluorescein diacetate succinimidyl ester (CFSE) in the
colonic lamina propria of naive mice or mice with TNBS-colitis.
Labeled Tregs were intraperitoneally injected 24 hours following
induction of TNBS colitis. Lymphocytes from lamina propria were
obtained 16 hrs after adoptive transfer of 10.sup.6 wildtype or
TNP-Tg Tregs to indicated recipients. CFSE-labeled Tregs were
9-fold more abundant in diseased colons. Data shown represent the
percentages of CFSE-positive cells in the corresponding gates of
one representative mouse of each four-mouse group. Each experiment
was repeated twice.
[0140] FIG. 24: Localization of Tregs in the colon. In-vivo imaging
of WT mice receiving DiR-labeled wildtype and TNP-Tg Tregs
(1.times.10.sup.6) 16 hours following induction of TNP colitis
(n=3). Mice were subjected to a whole body imaging (IVIS.RTM. 100
Series Imaging System) at 12 hour intervals. A single
representative mouse out of three in each group is shown at all
time points. Two independent experiments were performed, with
similar results. (
[0141] FIG. 25: Localization of Tregs in the colon. In-situ
fluorescent microendoscopic (Cell Vizio) evaluation of CFSE-labeled
Tregs accumulating at the colonic pre-luminal mucosal layer. The
experimental design is identical to the one described in FIG. 23.
The figure shows representative frames taken 48 hours following
adoptive transfer. Each group consisted of four mice, and each
experiment was repeated twice.
[0142] FIG. 26: Intrarectal administration of TNBS results in
TNP-Tg Treg-mediated protective effect from oxazolone colitis. (a)
Mortality rates of wildtype and TNP-Tg mice administered
oxazolone.+-.low doses of TNBS, 1 week following presensitization
with oxazolone only. (b) Murine colonoscopy images of
representative WT and TNP-Tg mice. (c) Macroscopic appearance of
representative colons from various mouse groups. (d) Microscopic
appearance of colons shown in c. (e) Adoptive transfer of Tregs
(Tr) to oxazolone (O) pre-sensitized mice induced one week later
with oxazolone (O) colitis in the presence of low dose of TNBS (T).
WT or TNP-Tg Tregs were administered to mice (n=8) 16 hours after
the induction of colitis.
[0143] FIG. 27 shows 8 schematic drawings of T cells which are
transduced with a retroviral vector that carried one of 8 CR
constructs that include the detectable fluorescent protein, eGFP.
Those depicted in the lower half of the Figure are bicistronic
constructs that encode a fusion of GFP and the transcription factor
Foxp3. Light and fluorescence microscopy were used to follow
expression of the GFP in the cytoplasm of nucleus of the transduced
cells. The constructs are labeled as follows (where "TPCR" refers
to "tripartite chimeric receptor" even though, some of these CR's
were "more" than tripartite). [0144] a. TNP-TPCR: extracellular
recognition region comprised an scFv of a TNP-specific mAb. [0145]
b. MD2-TPCR: extracellular recognition region comprised an LPS
binding fragment or motif of the human MD2 protein, an LPS
co-receptor (that interacts with TLR4 receptors). The fragment of
MD2 corresponds to residues 120-132 of SEQ ID NO:5. The sequences
of such chimeric nucleic acids used here are SEQ ID NO:8 and 9.
[0146] c. CD14-TPCR: extracellular recognition region comprised an
LPS binding fragment of the human CD14 protein, a known LPS
receptor. The fragment of CD14 corresponds to residues 100-119 of
SEQ ID NO:4. The sequences of such chimeric nucleic acids used here
are SEQ ID NO:6 and 7. [0147] d. MD2-CD14-TPCR: the extracellular
recognition region comprised both the MD2 fragment and the CD14
fragments described above. The sequences of such chimeric nucleic
acids used here are SEQ ID NO:10 and 11. Each of the constructs
encoded as stimulatory and costimulatory moieties, tandemly linked
sequences encoding CD28 and FcR.gamma.. Results are shown as
side-by-side light and fluorescence micrographs.
[0148] FIG. 28. An annotated nucleotide sequence (SEQ ID NO:1) and
amino acid sequence (SEQ ID NO:2) of a TNP-specific tripartite CR
as used herein. The annotations include the origin of the regions
(scFv, here the "Sp6" mAb), the "CD28" region, and the FcR.gamma.
regions (indicated as "GAMMA"), as well as restriction sites,
leader sequence, etc. The mature protein begins at amino acid
residue 23.
[0149] FIG. 29. An annotated nucleotide sequence (SEQ ID NO:3) of a
pBullet plasmid that includes a CR-encoding construct that
comprises a nucleotide sequence encoding the scFv of mAb HB 9081
(i.e., produced by a hybridoma given ATCC Accession No. HB9081)
fused to C28/FcR.gamma.. This mAb and, hence, the scFv, is specific
for LPS. Annotations show various restriction enzyme recognition
sites, the leader sequence, and plasmid sequences.
[0150] FIG. 30A-30B. FIG. 30A is an annotated amino acids sequence
of Human CD14 (SEQ ID NO:4). See GenBank Accession No. P08571. A
signal sequence and an LPS-binding motif (residues 100-119) are
noted. This protein serves as an LPS receptor on cells. FIG. 30B is
an annotated amino acids sequence of Human MD-2 protein (SEQ ID
NO:5). See GenBank Accession No. NP.sub.--056179. A signal sequence
and an LPS-binding motif (residues 120-132) are noted. This
LPS-binding protein interacts with TLR-4 as a co-receptor.
[0151] FIG. 31A-31B. FIG. 31A is an annotated nucleotide sequence
(SEQ ID NO:6) showing the nucleotide sequence of a Chimeric
Receptor comprising CD14 motif-CD28-FcR.gamma.. FIG. 31B is an
annotated nucleotide sequence (SEQ ID NO:7) showing the nucleotide
sequence of a chimeric, bicistronic receptor: CD14
motif-CD28-FcR.gamma.-IRES-GFP-Foxp3. Also shown is the amino acid
sequence (single letter code) of the CD14 motif (residues 110-119
of SEQ ID NO:4). Annotations show various restriction sites,
beginnings and ends of protein regions, IRES region, etc.
[0152] FIG. 32A-32B. FIG. 32A is an annotated nucleotide sequence
(SEQ ID NO:8) showing the nucleotide sequence of a Chimeric
Receptor comprising MD2 motif-CD28-FcR.gamma.. FIG. 32B is an
annotated nucleotide sequence (SEQ ID NO:9) showing the nucleotide
sequence of a chimeric, bicistronic receptor: MD2
motif-CD28-FcR.gamma.-IRES-GFP-Foxp3. Also shown is the amino acid
sequence of the MD2 motif (residues 120-132 of SEQ ID NO:4.
Annotations show various restriction sites, beginnings and ends of
protein regions, IRES region, etc.
[0153] FIGS. 33A and 33B. FIG. 33A is an annotated nucleotide
sequence (SEQ ID NO:10) showing the nucleotide sequence of a
Chimeric Receptor comprising MD2 motif-CD14 motif-CD28-FcR.gamma..
FIG. 33B is an annotated nucleotide sequence (SEQ ID NO:11) showing
the nucleotide sequence of a chimeric, bicistronic receptor: MD2
motif-CD14 motif-CD28-FcR.gamma.-IRES-GFP-Foxp3. Also shown is the
amino acid sequence of the MD2 motif (residues 120-132 of SEQ ID
NO:4) and the amino acid sequence of the CD14 motif (residues
100-119 of SEQ ID NO:3). Nucleotides 106-148 of SEQ ID NO:11
(double underlined) encode a flexible linker (14 amino acids, SEQ
ID NO:12, also double underlined). Annotations show various
restriction sites, beginnings and ends of protein regions, IRES
region, etc.
[0154] FIG. 34 is an annotated nucleotide sequence (SEQ ID NO:13)
showing the nucleotide sequence of a Chimeric Receptor comprising
MD2-CD28-FcR.gamma. (SEQ ID NO:13). Also shown is the amino acid
sequence of the full length MD2 protein (SEQ ID NO:4). The
LPS-binding region of this amino acid sequence is underscored.
Annotations show various restriction sites, beginnings and ends of
protein regions, etc.
[0155] FIG. 35 is an annotated nucleotide sequence (SEQ ID NO:14)
showing the nucleotide sequence of a chimeric, bicistronic
receptor: MD2-CD28-FcR.gamma.-IRES-GFP-Foxp3. Also shown is the
amino acid sequence of the full length MD2 protein (SEQ ID NO:4).
The LPS-binding region of this amino acid sequence is underscored.
Annotations show various restriction sites, beginnings and ends of
protein coding regions, IRES, vector sequence, etc.
DESCRIPTION OF THE PREFERRED EMBODIMENTS
Definitions
[0156] "Regulatory T lymphocyte" or "Treg cell" or "Treg," as used
in the present specification and claims are synonymous and are
intended to have its standard definition as used in the art. Treg
cells are a specialized subpopulation of T cells that act in a
"regulatory" way to suppress activation of the immune system and
thereby maintain immune system homeostasis and tolerance to
self-antigens. Tregs have sometimes been referred to suppressor
T-cells. Treg cells are characterized by expression of the forkhead
family transcription factor Foxp3 (forkhead box p3). They may also
express CD4 or CD8 surface proteins. They usually also express
CD25. As used in the present specification and claims, and unless
otherwise specified, Tregs include natural Tregs and induced or
adaptive Tregs and Tregs that have been created using recombinant
DNA technology. Naturally-occurring Treg cells (CD4+CD25+Foxp3+)
arise like all other T cells in the thymus. In contrast, adaptive
Treg cells (also known as Trl cells or Th3 cells) may originate
during a normal immune response. Antigen-specific activation of
human effector T-cells leads to inducible expression of Foxp3 in a
subgroup of the activated effector cells, and this subgroup can
develop a regulatory (Treg) phenotype. One way to induce Tregs is
by prolonged exposure of T effector cells to TGF-.beta.. T-cells
may also be converted to Treg cells by transfection or transduction
of the Foxp3 gene into a mixed population of T-cells. A T-cell that
is caused to express Foxp3 adopts the Treg phenotype and such
recombinant Tregs are also defined herein as "Tregs".
[0157] "Redirected Treg" is intended to be a comprehensive term for
Tregs carrying a chimeric receptor (CR) as described and claimed
herein which confers on the cells the ability to bind to and be
activated by a target antigen or ligand that is different from that
to which a Treg population may have been previously specific (as
controlled by its endogenous antigen-specific TCR). Redirected
Tregs are "MHC-independent" and "non-MHC restricted" in the process
of their activation and in their actions as they do not require
association of a peptide derived from their target antigen or
ligand with MHC in order to recognize it. However, for special
purposes, it may be possible to design a redirected Treg that
recognizes a specific epitope of an MHC molecule per se, e.g.,
functioning as a transplantation antigen. In such a case these
redirected Tregs are still non-MHC restricted.
[0158] The term "selected target antigen or ligand" means a
molecule to which the extracellular recognition region of the
redirected Treg is intended to bind so as to activate that Treg. If
the selected target is an antigen, then an antibody can be raised
against it and the binding regions of such an antibody used to
construct the extracellular recognition region of the redirected
Treg. If the target molecule is a member of a receptor/ligand pair
(defined below), then the other member of that pair can be used as
part of the extracellular recognition region of the redirected
Treg. Generally, in designing a redirected Treg for use according
to this invention, the intended target tissue or site where the
Treg is to be employed is first identified, and then, an antigen or
ligand that is present on or near this intended target tissue or
site is identified. An antibody or ligand/receptor that binds
thereto is then identified, or, if necessary, created or
constructed for use on the redirected Treg.
[0159] The term "ligand" as used herein, and particularly as part
of the term "target antigen or ligand" or the term "receptor/ligand
pair" refers to a molecule that is able to bind to and form a
complex with another biomolecule to serve a biological purpose.
Often the binding partner of a ligand is called a receptor so that
the two binding partners are termed a "receptor/ligand pair." For
the purpose of the present specification and claims, the term
"receptor," when used in the sense of a "receptor/ligand pair," has
a broader meaning than, for example, a typical definition of a
"receptor" as a protein in or on a cell that binds to a specific
ligand. It is rather intended to mean any binding partner for a
ligand. Either member of a binding pair can be considered the
"ligand" while the other member is considered the "receptor." Thus,
a classical receptor may qualify as a "ligand" when used herein in
the term "target antigen or ligand" as it is one member of a
receptor/ligand binding pair. For example, IL-2 can be a ligand
because it binds to and forms a complex with another biomolecule,
i.e., an IL-2 receptor (IL-2R), to serve a biological purpose.
However, IL-2R is also a "ligand" because it is a molecule that
binds to and forms a complex with another biomolecule, i.e., IL-2,
to serve a biological purpose. Thus, under the present usage, if
IL-2R is considered a ligand in the IL2R/IL-2 binding pair, IL-2
may be considered the receptor, and vice versa.
[0160] A "chimeric receptor," as used in the present specification
and claims, is a recombinant polypeptide that includes an
extracellular recognition region that is derived from one molecule
and at least one intracellular signaling moiety that is derived
from a different molecule. In that sense it is chimeric.
[0161] A "chimeric nucleic acid" is a recombinant polynucleotide
that includes a sequence that encodes a chimeric receptor.
[0162] The terms "recombinant" or "recombinantly" when applied to a
polynucleotide, polypeptide or cell means that the molecule or cell
is made using genetic engineering techniques and would not exist
but for the hand of man.
[0163] The term "T-cell signaling polypeptide moiety" means that
portion of a molecule endogenous to a T-cell that mediates
signaling. It may be a portion of a T-cell receptor molecule that
mediates signaling, or a downstream signal-transducing enzyme or a
portion thereof that mediates signaling, i.e., that has enzymatic
activity.
[0164] The term "antibody-derived scFv domain" means a single-chain
antibody in which the V.sub.L of a specific antibody is linked to
the V.sub.H thereof by a flexible linker or spacer.
[0165] The term "an MHC protein extracellular domain" refers to the
disclosure of Meal D J et al. (Proc Nall Acad Sci USA 2005;
102:11817-22), discussed below, and Jodi M D et al., Nat.
Biotechnol., 2002, 20:1215-1220. These publications describe Treg
cells redirected against T-cells in a murine system. They used the
class II MHC Is .alpha. and Is .beta. chains as extracellular
regions of two separate chimeric receptors for use in a redirected
Treg. The term MHC protein extracellular domain is defined so as to
encompass what was used in the Meal et al. and Jodi et al.
publications and any analogs or fractions thereof that would have
been obvious to a person of ordinary skill in the art to substitute
for such domains for the purpose disclosed by Meal et al. and by
Jodi et al., i.e., to cause binding of the redirected Treg to a T
lymphocyte specifically directed to a particular autoantigen.
[0166] The term "flexible spacer" means any flexible peptide moiety
that will facilitate the functionality of the extracellular
recognition region. When this region is not rigidly attached to the
transmembrane region, but is allowed some degree of flexibility
with respect to the cell membrane, the ability of the recognition
region to recognize and bind to its target antigen or ligand is
facilitated. Small neutral amino acids, such as glycine and serine,
confer such flexibility. Examples are Gly.sub.4Ser and
Gly.sub.4Ser.sub.3.
[0167] The "immunoglobulin superfamily" (Igs) means the large group
of cell surface and soluble proteins that are involved in the
recognition, binding, or adhesion processes of cells. Molecules are
categorized as members of this superfamily based on shared
structural features with immunoglobulins (also known as
antibodies); they all possess a domain known as an immunoglobulin
domain or fold. Members of the Igs include various cell surface
antigen receptors, co-receptors and co-stimulatory molecules of the
immune system, molecules involved in antigen presentation to
lymphocytes, cell adhesion molecules and certain cytokine
receptors. They are commonly, though not exclusively, associated
with roles in the immune system.
[0168] The term "hinge" when referring to a region of a molecule of
the Igs means the region between the C.sub.H1 and C.sub.H2 domains
consisting of a small number of amino acids. The hinge is flexible
and allows the binding region to move freely relative to the rest
of the molecule. At the hinge region are the disulfide bridges
which link the two dimers, creating the tetramer structural unit.
Examples of such immunoglobulin hinge sequences may be found in
U.S. Pat. No. 6,165,476, which is incorporated herein by
reference.
[0169] The term "antigen-specific receptor of a T-cell" refers to a
receptor that is found on a T-cell that is antigen-specific, i.e.,
naturally has an extracellular region that binds specifically to a
particular antigen in preference to another. Examples of such
antigen-specific receptors of a T-cell are the TCR .alpha., .beta.,
.gamma. or .delta. chains, the TCR.alpha..beta. dimer and TCR
dimer.
[0170] The term "TCR/CD3 complex" is sometimes called the "TCR
complex." CD3 is a protein complex composed of four chains in
mammals (CD3.gamma., CD3.delta. and two CD38 chains), that
associate with molecules known as the T cell receptor (TCR; see
above) and with the .zeta.-chain and .eta.-chain (as homo- or
heterodimers) to generate an activation signal in T lymphocytes.
The intracellular tails of these CD3 molecules contain a single
conserved motif known as an "immunoreceptor tyrosine-based
activation motif" or ITAM for short, which is essential for the
signaling capacity of the TCR. The CD3-.gamma., -.delta., and
-.epsilon. chains and the .zeta.- and .eta.-chains, also known as
CD3-.zeta. and CD3-.eta. chains, together with the TCR, form what
is known as the T cell receptor complex.
[0171] The term "T-cell costimulatory-receptor protein" means a
receptor of the T-cell that provides a costimulatory signal. During
the activation of T cells, costimulation is often crucial to the
generation of an effective immune response. T cells require two
signals to become fully activated. A first, antigen-specific,
signal is provided through the T cell receptor/CD3 complex. A
second signal, the costimulatory signal, is antigen-nonspecific and
is provided by costimulatory molecules expressed on the T cell
membrane. Examples of T-cell costimulatory-receptor proteins are
CD28, OX40, CD40L, 4-1BB and PD-1.
[0172] The present invention is based on the conception that
regulatory T-cells (Treg) that have been modified to possess
antibody-type antigen specificity, can be harnessed to suppress T
effector cells function in vivo. The action of these Treg cells is
mediated in an antigen-nonspecific manner, primarily by release of
suppressive cytokines in the vicinity where the Tregs are activated
or stimulated by an antigen recognized by their TpCR. Once
activated, Tregs can suppress bystander T cell responses. Thus,
transfer of these cells that have been engineered to express the
CRs (as described herein) to a subject in whom it is desired to
suppress a T effector cell response and its attendant or consequent
inflammation, and their delivery to, and activation at, the site of
such inflammatory activity, results in therapeutic effects.
[0173] Thus, preferred target diseases or conditions for this
invention are autoimmune diseases, more preferably, organ-specific,
T cell-mediated autoimmune diseases. Other examples of undesired
immune responsiveness to be targeted herein are graft rejection of
solid tissue and organ grafts as well as grafts of suspended cells
(e.g. bone marrow (BM) transplants or hemopoietic stem cell (HSV)
transplant). Another target disease is graft-vs-host disease (GVH)
that is a common consequence of a mismatched BM or HSV transplant.
An additional condition targeted by this invention is transplant
rejection (e.g., of a mismatched kidney) where the recipient's
immune effector cells reject the graft.
[0174] Because the Foxp3 transcription factor (a member of the
forkhead family) appears to be essential for Treg development and
function, and is a distinctive marker for these cells (along with
CD4 and CD25), the present invention provides methods for producing
Tregs, as well as providing the Tregs produced by those methods,
that are based on induction of Foxp3 in T-cells in a process of
driving cells along the pathway to Treg status.
[0175] In another embodiment, DNA encoding Foxp3 is transduced or
transfected into T-cells using any suitable expression vector as a
delivery vehicle in a process to drive these cells to become Tregs.
Further support for this conception is found in reports that
prevention of Foxp3 expression in vivo results in animals with a
propensity for development of autoimmune and lymphoproliferative
disorders (Sakaguchi S, et al., J Immunol 1995; 155:1151-64; Hori S
et al., Science 2003; 299:1057-61; Khattri R, et al., supra;
Fontenot J D et al., Nat Immunol. 2003; 4:330-6.).
[0176] The starting population can be total PBL, T-cells that have
been enriched or isolated from PBL, or CD4+T-cells that have been
enriched or isolated from such T-cells (either expressing CD25 or
not). These cells can be redirected by transducing the TpCR prior
to, concomitantly with, or after transducing DNA encoding Foxp3
DNA, preferably in the form of a Foxp3 expression vector. Walker M
R. et al., 2005, Proc Natl Acad Sci USA. 102:4103-8, have shown
that antigen-specific human CD4+CD25+ Treg cells can be generated
de novo from CD4+CD25- cells. The advantage of the present
invention over the approach described by Walker et al. is that the
antigen-specificity and Treg activation requirements are
independent of the MHC. This important improvement makes isolation
and activation of antigen-specific Tregs simpler and allows for
therapeutic methods (described below) in which the antigen can be
conveniently administered together with the transferred Treg cells
to a desired site, such as an inflammatory site, exemplified by the
colon in IBD.
Naturally-Occurring vs. Inducible Tregs
[0177] "Classical" naturally-occurring Tregs are thymus-derived,
express high levels of Foxp3 and suppress activation of effector
lymphocytes. Antigen-specific activation of human effector T-cells
leads to inducible expression of Foxp3 in a subgroup of the
activated effector cells, which subgroup can develop a regulatory
(Treg) phenotype. These induced regulatory T-cells can suppress
(independently of cell contact) freshly isolated effector cells
(Walker M R., et al., 2005, supra; Walker M R., et al., 2003, J
Clin Invest. 112:1437-43). In mice, both in vitro and in vivo
induction of Tregs is achieved by prolonged exposure of effector
cells to TGF-.beta. (Wan Y R, et al., 2005, Proc Natl Acad Sci USA.
102:5126-31; Mantini M C, et al., 2004, J Immunol. 72:5149-53;
Mantini et al., 2006, supra). This small, peripherally generated
population of inducible Tregs are believed to play a central role
in regulating and containing ongoing immune responses just as the
lack of Treg induction is associated with a propensity for
autoimmunity.
Genetic Manipulation of CD4+CD25+T Regulatory Cells
[0178] According to the present invention, approaches that
specifically redirect regulatory T-cells to suppress the activity
of pathological T-cells are beneficial in inflammatory conditions
by facilitating localization of Tregs to inflammatory sites and
their specific activation by inflammation-associated antigens. When
specifically activated in inflammatory lesions, such Tregs are
expected to attenuate inflammatory disease by suppressing
pathogenic effector T lymphocytes in an antigen-nonspecific,
MHC-unrestricted, manner.
[0179] The MHC-independent activation and action of Treg cells
according to the present invention is an important advantage. Such
action is contrasted with the report of Meal D J et al. (Proc Natl
Acad Sci USA 2005; 102:11817-22) of a study of experimental
allergic encephalomyelitis (EAE) which described CD4+25+ Treg cells
redirected against myelin basic protein (MBP) epitope
89-101-reactive T cells by a CR that included the MBP epitope
linked to MHC class II protein. By enforcing the interaction
between a Treg cell and the autoreactive T cells directed against
MBP epitope 89-101, the Treg activity is antigen-specifically
focused against the autoreactive T-cells. Such a model requires
some degree of MHC-dependency as a single CR can only have domains
of a single MHC and thus can only be used for patients with that
HLA characteristic.
[0180] In contrast, the Treg cells of the present invention act to
suppress pathogenic T effector cells in an MHC-independent manner,
making them more advantageous for treating autoimmune/inflammatory
conditions because they can target common target antigens shared
among many individuals. In the model of Meal et al., supra, Tregs
acted by MHC- and antigen-restricted engagement. As such these
Tregs, which express the ligand that is recognized by the TCR of
the autoreactive T-cells, are stimulated by such interaction and
suppress the effector cells. However, as a clinical approach this
suffers from the disadvantage that it would require full
donor-recipient MHC compatibility in the human population in which
MHC (HLA) diversity is substantial. Moreover, such an approach
would be limited to suppression of clones that are autoreactive
against a single, recognizable peptide epitope in the context of
defined MHC-II (HLA-DR). An additional significant advantage of the
present invention is that it overcomes the requirement that the
pathogenic antigen be known. Indeed, disease-associated antigens in
a large number of human autoimmune disorders, including human IBD,
are not yet known and may be multiple in number.
The "T-Body" Approach of the Present Invention
[0181] The "T-body" approach was designed by one of the present
inventors and his colleagues as a novel modality for specific
redirection and activation of effector T lymphocytes towards
pre-defined targets, mostly those associated with neoplastic
processes (e.g., Pin thus JHU et al., J Clin Invest 2004;
114:1774-81) and infectious diseases (Bitton N, et al., Curr Top
Microbiol Immunol 2001; 260:271-300). The T-body approach was
intended to overcome the relative inaccessibility of antibodies to
certain sites (such as solid tumors) and the general
ineffectiveness of tumor-infiltrating lymphocytes to combat solid
tumors by combining into one effector cell population the
properties of the humoral and cellular arms of the immune system
(Gross G et al., 1989; supra Eshhar Z, et al., Br J Cancer Suppl,
1990; 10:27-9).
[0182] The preferred T-body chimeric receptors comprise a ligand
binding portion, preferably (1) a single chain antibody variable
region (scFv) directed against a disease-associated antigen, linked
to (2) an optional extracellular spacer and a transmembrane region
and (3) one or more intracytoplasmic moieties of T cell
costimulatory and stimulator/signaling molecules. Such CRs as
initially developed enable non-MHC restricted, specific
antibody-type recognition, homing and penetration of neoplastic
tissues. Within the target tissues, antigen-specific activation of
chimeric-receptor bearing T effector cells enabled T cell-mediated
destruction of tumor cells either by direct cytotoxicity or by
induction of a local inflammatory response.
[0183] While the scFv domain is the preferred recognition unit of
the present invention, in other embodiments, it may be substituted
by another structure that serves as a targeting ligand (or ligand
binding partner) that will facilitate bringing the Treg cells
expressing the CR to a selected site or a selected antigen. Capon
and colleagues have disclosed a number of CRs of this sort, such as
one where a ligand binding partner polypeptide is fused at its
C-terminus to the N-terminus of an immunoglobulin constant region.
See, for example, Roberts M R. et al., Blood 1994; 84:2878-89;
Ashkenazi A et al., Int Rev Immunol. 1993; 10:219-27; Chamow S M et
al., Int J Cancer Suppl. 1992; 7:69-72. See also U.S. Pat. Nos.
6,710,169; 6,407,221; 6,406,697; 6,319,494; 6,117,655; 6,103,521;
6,077,947; 5,741,899; 5,714,147; 5,514,582; 5,455,165; 5,428,130;
5,359,046; 5,336,603; 5,225,538; and 5,116,964. All of these
documents are incorporated by reference in their entirety.
[0184] The CR polypeptide of the present invention is characterized
broadly as comprising (1) an extracellular portion or domain
capable of binding to a ligand (such as a target antigen) in a
non-MHC restricted manner, (2) an optional extracellular spacer and
a transmembrane domain and (3) a cytoplasmic region (one or more
domains) capable of activating an intracellular signaling
pathway.
[0185] Examples of preferred T cell CRs comprise a first binding
domain, a preferred example of which is an extracellular scFv
fragment derived from a monoclonal antibody (mAb) specific for a
selected antigen. The foregoing domain is fused to a spacer domain
(preferably a hinge domain of the Ig family that provides spacing
and flexibility), a transmembrane domain, a costimulatory region,
for example parts of a CD28 molecule, and a further intracellular
signaling moiety for T-cells. Examples of the latter include a
TCR/CD3 complex-associated .zeta. chain or .eta. chain, or an
ITAM-containing cytoplasmic region such as the .gamma. chain of an
Ig Fc receptor (FcR.gamma.). An ITAM is an "immunoreceptor
tyrosine-based activation motif; for reviews, see Humphrey M B et
al., Immunol Rev. 2005 December; 208:50-65; Pitcher L A et al.,
Trends Immunol. 2003; 24:554-60; Isakov N, Receptors Channels.
1998; 5:243-53; Daeron M, Annu Rev Immunol. 1997; 15:203-34; Isakov
N, J Leukoc Biol. 1997, 61:6-16; Cambier J C, J Immunol. 1995;
155:3281-5; Flaswinkel H et al., Semin Immunol. 1995; 7:21-7, all
of which are incorporated by reference in their entirety. It is
also possible to use the intracellular portions of TCR .alpha.,
.beta., .gamma. or .delta. receptor molecules in the CR for this
purpose. The signaling moiety of a cytokine receptor may also be
present in the chimeric receptor chain for use in the present
invention. For example, adding the signaling portion of the IL-2
receptor will cause the Treg cell to further act as if it had been
subjected to external IL-2 upon binding of the extracellular
targeting domain to the selected target antigen or ligand.
Furthermore, adding the signaling moiety of the TGF.beta. receptor
will induce a T effector cell to become a Treg cell and thus this
may also be a useful addition to the chimeric receptor chain of the
present invention. Such CRs expressed on T-cells are known to be
functional and, upon exposure to antigen, promote cytokine
production (and, when expressed on appropriate effector cell type
in the prior art, promoted lysis of antigen-bearing target cells
(Stancovski I, et al., J Immunol 1993; 151:6577-82)).
[0186] An early configuration of a scFv-based CR comprised an
extra-cellular recognition domain and an intracellular signaling
moiety. Full activation of such T-bodies through the CR required
either pre-stimulation of the T-body or activation of a
costimulatory pathway by exposure to CD08/CD86 (B7)-bearing antigen
presenting cells (APCs).
[0187] The creation of a tripartite chimeric receptor (TpCR) by one
of the present inventors and by others (Pule M A, et al., Mol.
Ther. 2005; 12:933-41)), in which the signaling domain of the
costimulatory CD28 molecule was added to the cytoplasmic domain of
the CR, enabled antigen-mediated activation of both the stimulatory
and costimulatory signaling pathways independent of B7-CD28
interactions (Eshhar et al., 2001, supra). This approach
facilitates full activation of scFv-expressing lymphocytes,
resulting in improved effects (in the case of T effector cells,
improved anti-tumor; Pin thus JHU et al., supra).
[0188] Another useful intracellular signaling domain for the
present invention is all or part of the cytoplasmic domain of a
phosphotyrosine kinase (e.g., a molecule of the Syk family) which
is fused to the CR. See, for example, Eshhar Z & Fitzer-Attas C
J, Adv Drug Deliv Rev. 1998; 31:171-82; Fitzer-Attas C J et al., J
Immunol. 1998; 160:145-54.; and Eshhar Z et al., Springer Semin
Immunopathol. 1996; 18(2):199-209. Use of such a signaling moiety
bypasses membrane-proximal signaling events that are often
defective in T-cells of subjects with acute or chronic inflammation
or cancer.
Costimulatory Domains/Regions and Signals in the Tripartite
Chimeric Receptor
[0189] Retroviral-mediated expression of CRs in T-cells in general
requires T cell activation which activation is commonly achieved by
combined use of anti-CD3 and anti-CD28 antibodies. Such
pre-activation was sufficient to prime the T-cells to respond to a
signal mediated through the CR upon interaction with the antigen
for which the CR is specific--both in vitro and in vivo (e.g.,
Schwartz R H; Annu Rev Immunol 2003; 21:305-34). A costimulatory
signal is advantageous for optimal and sustained T cell function
and antigen-driven re-activation, even by targets that often lack
ligands for costimulatory molecules.
[0190] Antigen stimulation alone of CRs that lack a structure or
mechanism for costimulatory signaling is generally inadequate to
activate resting or naive lymphocytes (Brocker T et al., J Exp Med
1995; 181:1653-9). Thus, in the absence of costimulatory signaling
by CD28, resting T lymphocytes typically undergo anergy or
apoptosis (Boussiotis V A et al., Immunol Rev 1996; 153:5-26). For
further discussion of CD28 and its interactions with B7, see also,
L. Chen (ed.) The B7-CD28 Family Molecules, Landes Bioscience,
2003, which is incorporated by reference in its entirety.
[0191] To overcome these limitations in the CR's used in the
present invention, the first (recognition) domain, preferably an
scFv domain, is linked through an Ig hinge spacer and transmembrane
segments to the intracellular segment of a costimulatory signaling
molecule, preferably CD28, and then to an intracellular activation
region, such as from the CD3.zeta. chain or the FcR.gamma. chain.
Co-expression of two CRs, each with the same scFv, the first linked
to CD3.zeta. and the second to CD28, was found to provide the
requisite stimulatory and costimulatory signals for T cell
activation (Beecham E J et al., J Immunother 2000; 23:631-42).
[0192] Thus, in one preferred embodiment herein, an extracellular
recognition site, preferably an antibody-based recognition site
such as an scFv, is linked to a CD28 intracellular domain "in
series" and further linked to the intracellular signaling region of
the TCR complex .zeta. chain. Such a construct was 20-fold more
potent in stimulating IL-2 production upon exposure to solid phase
antigen (compared with transfectants expressing CR's lacking the
CD28 domain (Finney H M et al., J Immunol 1998; 161:2791-7)).
Intracellularly, this domain in the CR binds the p85 subunit of
phosphatidylinositol 3'-kinase.
[0193] One of the present inventors designed a novel tripartite CR
composed of a scFv recognition moiety fused to the non-ligand
binding part of the extracellular domain (ECD) of CD28, the entire
transmembrane and intracellular domains of CD28, and the
intracellular stimulatory domain of FcR.gamma.
("scFv-CD28-.gamma.") (Eshhar et al., 2001, supra). Human PBL
transduced with a nucleic acid construct encoding this CR were
specifically stimulated to produce IL-2. Activation was dependent
on CD28 costimulatory activity.
[0194] The present inventors' laboratory has generated several
lines of Tg mice expressing CRs under control of T cell-specific
regulatory sequences. T lymphocytes from unprimed, naive mice that
are Tg for the scFv-CD28-.gamma. TpCR manifested potent responses
(proliferation, IL-2 secretion, and rescue from apoptosis) upon
stimulation solely by the cognate antigen in immobilized form
(Friedmann-Morvinski D et al., supra).
[0195] According to the present invention, molecules other than, or
in addition to, CD28 are exploited to provide costimulatory signals
when included in the present CR configuration. Preferred examples
of these are the members of the "inducible co-stimulator" (ICOS)
family, including OX40 (CD134), CD40 ligand (CD40L, CD154), PD-1
("programmed death receptor-1), and 4-1BB (CD137). Each of these
ligand/receptor pairs possess distinct functions that differ
according to the nature of the stimulus and the "antigenic history"
of the T-cells on which they are expressed. For example, CD28
signaling is accompanied by induction of ICOS, which, in turn,
co-stimulates CD4+T cell activation. The engagement of OX40
(studied in the context of tumor-specific adoptive immunotherapy)
improved survival and anti-metastatic actions of T effector cells
by CD4+T helper cell-dependent mechanism (Weinberg A D, Trends
Immunol 2002; 23:102-9). Activation of OX40 promotes expression of
anti-apoptotic proteins Bcl-XL and Bcl-2 and, accordingly, enhances
the survival and hence the number of antigen-specific CD4+ T-cells,
resulting in strong antigen-specific CD4+T cell memory. Engagement
of 4-1BB (CD137) costimulatory receptor with its ligand, 4-1BBL,
increased TCR-induced proliferation, survival, and cytokine
production in both CD4+ and CD8+T-cells (Cheuk A T et al., Cancer
Gene Ther 2004; 11:215-26). Cell survival was associated with
increased expression of the anti-apoptotic genes bcl-XL and bfl-1.
In general, the interacting ligand/receptor pair 4-1BB/4-1BBL acts
to amplify existing costimulatory signals, particularly those
emanating from CD28 (Guinn B A et al., J Immunol 1999;
162:5003-10). Human CD4+T-cells express PD-1 and its ligands, PD-L1
and PD-L2, upon activation. Antibodies to the receptor can be
agonists or antagonists of the apoptotic pathway. PD-1 engagement
can promote ICOS- or CD28-mediated costimulation. (e.g., Bennett F
et al., J Immunol. 2003; 170:711-8.
[0196] The activity of costimulatory domains of CD28, ICOS, OX40
(CD134), and 4-1BB (CD137) in CRs is also known in human CD4+ and
CD8+T-cells (Finney H M et al., J Immunol 2004; 172:104-13). In
that study, the tripartite genes were electroporated into cells to
avoid pre-activation of the cells. When CR-bearing T-cells were
stimulated by their specific antigen (CD33), cytokine release and
cytotoxic activity were dramatically enhanced compared to cells in
which the CRs lacked costimulatory signaling structures. Inclusion
of the 4-1BB signaling domain as the costimulatory moiety in a TpCR
on human T-cells with specificity against the CD19 antigen
(anti-CD19-1BB-.zeta.) led to potent cytotoxicity against
CD19-bearing acute lymphoblastic leukemia target cells in vitro
(Imai C, et al., 2004; 18:676-84).
[0197] While the present invention includes the use of an
intracellular domain or part of any of these costimulatory
sequences in the CR, it is not certain that signaling evoked by
these molecules has practical advantages over use of the CD28
costimulatory sequences alone. So, even though the performance of
CD28 appears thus far to be quite satisfactory both in vitro and in
vivo, the present invention includes within its scope the use of
additional or alternative costimulatory systems to CD28 for
generating Treg cells that perform optimally in suppressing T
effector cells and treating autoimmune/inflammatory and other
conditions as described herein. 4-1BB has been used successfully as
an alternative to CD28 in T-bodies. See Zhang et al., J. Immunol.,
2007; 179:4910-4918.
Transfer of Redirected Tregs to Recipient Subjects
[0198] Use of transferred T-cells in vivo in adoptive therapy
requires that transferred cells survive, overcome the host's
homeostatic control mechanisms that may serve to hinder the
acceptance of these cells, and migrate to (home to, or traffic to)
and accumulate or localize at, the desired target site(s).
[0199] The immune system utilizes internal stimuli to regulate the
total size of lymphocyte pools. The total number of peripheral T
lymphocytes remains fairly constant, despite production of new
cells, turnover of existing cells, and clonal expansion of
antigen-specific cells during an immune response (Jameson S C. Nat
Rev Immunol 2002; 2:547-56.). These "internal stimuli," include
cytokines and self-peptide-MHC ligands for the TCR. At least two
general mechanisms are believed to be responsible for homeostatic
effects of bystander T-cells in limiting proliferation: (1)
inhibition by physical T cell-T cell interactions; and/or (2)
competition for limited "resources" (e.g., IL-7 and access to APCs
with suitable self-MHC ligands). The most prominent cytokines in
this process are those that signal through receptors containing a
common .gamma. chain, termed collectively ".gamma.C cytokines."
These include IL-2, IL-4, IL-7, IL-9, IL-15, and IL-21. Homeostatic
control of naive T cell expansion (examined in vitro) is supported
by IL-4, IL-7, IL15 and IL21 through the CD28 transmembrane region,
whereas only IL-7 appears to be required in vivo (Jameson,
supra).
[0200] Lymphodepletion or "lymphoablation" is preferably performed
to condition a recipient of the transduced Tregs of the present
invention. Any method known in the art may be used, for example,
irradiation, treatment with certain antimetabolites such as
fludarabine, etc. Such treatments have been used in conjunction
with adoptive T cell therapy in other contexts. Lymphodepletion in
vivo performed as a precursor to adoptive cell transfer is known to
boost antitumor immunotherapeutic activity in mice and in humans
(as studied particularly with autologous, tumor-reactive T effector
cells; Klebanoff C A et al., Trends Immunol 2005; 26:111-7). In
clinical trials, objective response rates of 50% were seen in
patients with solid metastatic tumors who had first been subjected
to lymphodepletion. The mechanisms that are believed to underlie
such effects include: the elimination of cellular cytokine `sinks`
for homeostatic .gamma.C-cytokines (such as IL-7, IL-15 and
possibly IL-21 (which serve to activate and expand effector
T-cells)), induction of tumor cell apoptosis and necrosis in
conjunction with APC activation, and, most important for the
present invention, the impairment of CD4+CD25+ Treg cells that
suppress T effector cells.
[0201] As noted, treatment with homeostatic cytokines may be used
to maintain the Treg populations in the recipient.
[0202] The present inventors' group found that activation of
T-cells in general and T-bodies in particular (such as that
required during the ex vivo manipulations to express the CR with
certain vectors) down regulated expression of the chemokine
receptor CXCR4, thereby impairing T cell homing in response to the
chemokine SDF-1, for example. SDF-1 is a chemoattractant for
T-cells that express the CXCR4 (Bleul C C et al., J Exp Med 1996;
184:1101-9; Beider K et al., Blood 2003; 102:1951-8). Using
ErbB2-specific human T-bodies; these investigators showed that this
homing is an essential step for the T effector cells to act in
vivo, measured as inhibition of advanced prostate cancer
progression and even cure (Pin thus et al., supra). Based on the
foregoing knowledge, according to the present invention, redirected
Treg cells must either home/migrate to the desired target sites or
be administered to such sites.
Persistence of Responses of TpCR-Bearing T-Cells
[0203] A key factor for success of adoptively transferred T cell
therapy (which thus far has been examined most thoroughly with T
effector cells in cancer) is maintenance of the transduced T-cells'
(effector) function. In one embodiment of the present invention, it
is desired to maintain the function of Tregs that have been
administered to perform a suppressive function. In another
embodiment, it may be preferred that the Tregs act in shorter
"bursts" to curtail a more acute (rather than a chronic) T effector
response.
[0204] Because lymphocytes found in tumor patients include
CD4+CD25+ Treg cells that suppress T effector cells (Wang H Y et
al., Immunity 2004; 20:107-18; Curiel T J, et al., Nat Med 2004;
10:942-9), such "endogenous" suppressive activity must be overcome
to optimize the action of redirected T effector cells. In the
present invention, the objective is the converse: redirected Treg
cells are administered to a subject in need thereof to quell or
otherwise inhibit immune/inflammatory responses that characterize
autoimmune conditions, transplant rejection, etc.
Examples of Clinical Trials Using Redirected T-Cells
[0205] While clinical trials using Tregs in accordance with the
present invention have not yet been carried out, a number trials
using redirected, CR-bearing T effector cells are described below.
Advantage may be taken of various lessons learned in those trials
in practicing the present invention.
[0206] In a Phase I trial in HIV infected subjects, autologous
lymphocytes bearing a CD4-.zeta. CR were administered (Mitsuyasu R
T, et al., 2000, Blood 96:785-93). Out of 24 patients, 11 also
received concurrent IL-2 infusions for 5 days. The treatment was
well tolerated. In some patients, a transient decrease of the viral
load was observed in plasma and rectal mucosa (the tissue reservoir
for HIV). All subjects tested negative for replication-competent
retrovirus (the delivery vector) for up to 1 year after
infusion.
[0207] Cell Genesys, Inc. conducted phase I clinical trials in
colorectal cancer patients using an anti-TAG72-.zeta. CR made from
the humanized CC49 mAb (Warren R et al., In: 7th International
Conference on Gene Therapy of Cancer; 1998).
[0208] The group of Junghans tested 24 doses of CR-bearing
lymphocytes the antigen-specificity of which was directed to CEA in
colorectal patients. Up to 10.sup.11 cells/patient were given. The
treatment was adequately tolerated (Junghans R et al., Proc Am
Assoc Can Res, 2000, 41:543).
[0209] Hwu and co-workers at the National Cancer Institute
conducted a phase I clinical trial in ovarian cancer patients using
T-bodies expressing a CR directed against the MoV18-murine
anti-folate-binding protein. Large doses of the modified cells were
infused into patients together with controlled administration of
IL-2. No adverse side effects were reported. Neutralizing
antibodies specific to murine MoV18 mAb determinants were found in
the sera of several patients (Kershaw M H et al., Clin Canc Res,
2006; 12:6106-15.
[0210] A Phase I clinical trial in renal cell cancer (RCC) employed
autologous G250-specific genetically modified T lymphocytes (Lamers
C H J et al., Daniel den Hoed Cancer News, 2004, 2:8-10). Infusions
of these cells were clinically well-tolerated. After 4-5 infusions,
patients began to develop liver enzyme abnormalities, a finding
explained by the reactivity of the infused T-cells with G250L
expressed on bile duct epithelium, albeit at low levels. Treatment
was thus limited to only low doses of CR-expressing T-bodies. The
results showed in any case that the redirected T-cells did exert
CR-dictated functions in vivo.
[0211] Two other Phase I clinical trials have been initiated though
their results have not yet been reported to the best of the
inventors' knowledge. One Phase I trial treated neuroblastoma
patients with PBLs and Epstein Barr virus-specific CTLs, both
expressing GD-2 specific chimeric T cell receptors (Brenner M K.
World wide web URL clinicaltrials.gov/ct/show/NCT00085930, 2005).
The other trial employs genetically modified CD20-specific CD8+CTLs
for relapsed follicular lymphoma (Wang J, et al., Mol Ther 2004;
9:577-86
[0212] The present inventors recognize that certain events may
interfere with the efficacy of the therapy using Treg cells
expressing CR's in vivo in humans, for example: [0213] (1)
formation of neutralizing anti-idiotypic antibodies directed to an
idiotope of the scFv part of the CR that could reduced the
life-span or effectiveness of the Tregs; [0214] (2) the low
proportion of engineered cells that eventually reached the targeted
sites and; [0215] (3) the potential damage to healthy tissue that
expresses the targeted antigen.
[0216] The use of Tregs according to the present invention has a
much lower risk of (3). As described herein, direct administration
of Tregs to sites of inflammation should overcome the limitation of
(1)-(3). Adjustment of dose regimens (number of cells, frequency of
administration) using routine clinical considerations are expected
to limit the impact of the above limiting factors.
[0217] According to the present invention, an effective amount of
redirected Treg cells are administered to a subject. Preferred
carriers for the Treg cells are phosphate buffer, preferably
0.01-0.1M, more preferably 0.05M, or 0.8% saline. Acceptable
diluents or carriers for various routes of administration are
well-known.
[0218] While individual needs vary, determination of optimal ranges
of effective amounts of a given cell type for a particular disease
or condition is within the skill of the art. The dosage
administered will be dependent upon the age, health, and weight of
the recipient, kind of concurrent treatment, if any, frequency of
treatment, and the nature of the effect desired. Determination of
the effective amounts can readily be made empirically by those of
ordinary skill in the art without undue experimentation.
[0219] Typical dosages are between about 10.sup.6 and about
10.sup.11 Treg cells per injection or infusion, more preferably,
about 10.sup.7 to about 10.sup.10 cells. If an antigen is to be
administered with the cells (or separately, but to a site where it
is intended to activate the cells), a dose of about 0.01 to 100
mg/kg/body preferably, 0.1 to 50 mg/kg/body wt is preferred.
[0220] An effective amount of Treg cells is that needed to induce a
measurable change, generally a decrease, in the severity of any
measurable symptom of the disease, preferably more than one
symptom, and most preferably, would result in cessation of symptoms
and cure of the disease or condition. For example, without limiting
the invention, the above decrease may be at least about 10%, more
preferably at least about 20%, more preferably at least about 30%,
even more preferably, at least about 40%, and more preferably, at
least about 50%, 60%, 70%, 80%, 90%, 95%, or 99%. It is within the
skill of the clinical arts to determine when such therapeutic goals
have been achieved, and to adjust the dose or frequency of
administration accordingly, or to cease further treatment.
[0221] The Treg cells of the invention may be given once, or on
multiple occasions, via a single or multiple routes. The cells may
be administered daily, or preferably on alternate days, preferably
weekly or biweekly. Administration can range over an interval of
several days to weeks, or even months or years. The frequency and
duration of administration can be determined empirically, or based
on the clinical history and experience of the subject.
[0222] The cellular compositions of the present invention can be
administered by any of a number of means and routes known in the
art. Administration is preferably parenteral. Preferred routes
include, intravenous, intramuscular, subcutaneous, intraperitoneal,
intra-articular, intracerebroventricular, intraluminal (preferably
into the lumen of the ileum or colon), rectal or the topical route.
Also included is the "intrathecal" route, which is intended to
encompass injection, infusion or instillation directly into a
cavity or space (thecum) surrounding an organ or body region in
which an undesired immune/inflammatory response is occurring. Such
spaces include the pleural space, peritoneum, subarachnoid space or
dural space, or pericardial space. The generic term for
administration into a sheath encasing an organ is termed
"intrathecal (see, for example, definition in Dorland's Medical
Dictionary 29.sup.th Edition, WB Saunders (2000) and Stedman's
Medical Dictionary, 27.sup.th Edition, Lippincott, Williams &
Wilkins (2000)) as meaning "within a sheath." As used herein, this
term is intended to be broader than a more commonly used definition
which is limited to intracranial spaces.
[0223] The compositions, methods, and products of this invention
are applicable to human and veterinary uses. The preferred subject
is a human.
Transgenic Mice Expressing TNP-Specific Chimeric Receptors
[0224] Several Tg strains of mice that express the TNP-specific
TpCR, that were recently produced by the present inventors and
their colleagues (Friedmann-Morvinski D, 2005) are described
herein. These mice are the source of TNP-specific T effector and T
regulatory cells and are used as experimental animals in which the
induction of colitis is evaluated using the `classical` reactive
hapten, TNBS. As a control for these CR-bearing cells, cells from
erbB-2-specific TpCR Tg mice that were produced in the present
inventors' laboratory are used as they express a CR specific for an
irrelevant antigen.
[0225] All mature T-cells and NK cells in these Tg mice express the
scFv-CD28-FcR.gamma. construct. Naive Tg T-cells can be fully
activated by plastic-immobilized TNP without the need for
pre-sensitization. (Friedmann-Morvinski D, et al., supra). Results
in the Examples herein show that splenic CD4+CD25+ Tregs isolated
from such mice specifically suppress proliferation and cytokine
secretion by TNP-specific effector T-cells. Moreover, these Tregs
are responsible for the delayed development and attenuation of
TNBS-induced colitis in these animals. Of importance is the fact
that the level of Tregs in the periphery of the TNP-specific
TpCR-expressing strains is higher than in wild-type (WT) mice and
that the Tregs do not require pre-activation to exhibit their
suppressive activity in vivo. This is believed to result from the
cross-reactivity of the SP6 mAb, from which the scFv of the TpCR
was derived.
Delivery of DNA Encoding the CR into T-Cells
[0226] Genetic modification of human peripheral T-cells is achieved
in one embodiment using retroviral vectors (Eshhar Z, et al., 2001,
supra). As a non-limiting example, the pBullet vector is used, into
which the CR-encoding cDNA (Weijtens M E, et al., 1998) is
introduced. A bicistronic expression construct is used in which the
TpCR and eGFP cDNA are expressed under control of the LTR. This
serves to generate a packaging cell based on PG13 that is being
used to pseudotype the retroviral vector with the gibbon ape
leukemia virus (GALV). Flow cytometric sorting is done on the basis
of eGFP expression, and packaging cells producing high-titer
virions are selected to achieve high transduction efficacy. To
transduce human lymphocytes from healthy donors, lymphocytes are
activated in culture with plate-bound anti-CD3 and anti-CD28 mAbs
(or using commercial microbeads coated with these antibodies; e.g.
from Invitrogen, Miltenyi Biotec, Inc.,) and are transferred to
plates coated with Retronectin.TM. (fibronectin fragment CH-296)
plates together with fresh supernatants taken from the packaging
cells. At the end of the process that takes 5-8 days, cells are
propagated in the presence of IL-2 and then harvested and used.
Following this ex vivo procedure, 45-70% of the cells are positive
for CR (and GFP) expression.
[0227] Useful additional reagents are anti-idiotypic antibodies
against idiotopes of the scFv of the TpCR. These enable direct
labeling and visualization of TpCR on cell membranes. Such
antibodies against the SP6 scFv (exemplified below) have been made
and used by the present inventors.
[0228] An annotated nucleotide sequence (SEQ ID NO:1) and amino
acid sequence (SEQ ID NO:2) of the TNP-specific TpCR used herein is
shown in FIG. 27. The mature protein begins at amino acid residue
23 of SEQ ID NO:2.
[0229] A preferred sequence that excludes the scFv above, and that
can be linked to any other appropriate ligand binding region,
preferably a different scFv specific for another antigen, is that
defined by the above sequences beginning at the CD28 region. Thus,
a preferred nucleotide coding sequence is nucleotides 2203-2523 of
SEQ ID NO:1 and amino acids 260-367 of SEQ ID NO:2. Additional
nucleotides comprising a 5' restriction site, and amino acids
"inadvertently" encoded thereby, may also be included in a
preferred sequence. Additional coding sequence added at the 3' end
of 2203-2523 of SEQ ID NO:1, or additional amino acids encoded
thereby and added to at the C-terminus of 260-367 of SEQ ID NO:2,
may be present, provided that they permit the encoded sequence, as
expressed on the redirected Tregs, to function as a TpCR in ways
described herein. Those skilled in the art of cloning and
recombinant DNA technology will understand how to modify theses
sequences to achieve the desired objective without undue
experimentation.
[0230] Expression vectors comprising the foregoing sequences are
also used in the present invention, in the production of
redirected, TpCR-expressing Tregs.
Generation and Expression of TpCR and Foxp3-GFP Fusion Gene and its
Expression
[0231] Redirected T-cells are "converted" to Tregs by causing them
to express both Foxp3 transcription factor and the antigen-specific
TpCR. Such manipulation permits production of large numbers of
Tregs for evaluation and therapeutic use. Successful
co-transduction or co-expression is tested by including a Foxp3-GFP
fusion gene in the same construct as a TpCR to express both in the
same cells. This approach is particularly useful when the starting
cell populations are human PBL in which Tregs constitute only about
3-5% of CD4+T-cells. This avoids the complications of another
approach, also within the scope of the invention, in which large
scale Treg propagation is required for effective transduction with
retroviral vectors. Moreover it will simplify the isolation of the
Tregs and assessment of their fate in vivo.
[0232] In one non-limiting example, messenger RNA (mRNA) for Foxp3
is cloned from purified Tregs using PCR. Foxp3 cDNA is cloned into
an eGFP Clontech plasmid to create a Foxp3-GFP fusion protein. The
fusion protein is cloned into the pBullet vector containing TpCR
inserted after an IRES to create a bicistronic expression vector.
Both a Foxp3-GFP single gene retroviral vector and a bicistronic
TpCR--IRES-Foxp3-GFP double gene retroviral vector are transduced
into isolated CD4+CD25- human peripheral blood T-cells following
their activation with anti-CD3 and anti-CD28 antibodies. The
resulting cells are tested for expression of the three genes by
FACS using (1) antiidiotypic antibodies specific for the scFv
idiotype, or anti-hinge region antibodies and (2) intracellular GFP
and Foxp3 by staining fixed cells with primary antibodies specific
for Foxp3 (Alexis Biochemicals, Lausanne, Switzerland).
[0233] In another embodiment, sequential expression protocols are
used (first TpCR and then Foxp3-GFP genes) or co-expression
protocols. Once the genes are expressed, relatively large number of
Tregs can be obtained and separated using cell sorter (FACSaria
fluorescence-activated cell sorting (Becton Dickinson, Mountain
View, Calif.), sorting for GFP and TpCR co-expressing cells.
[0234] The Foxp3 construct may be in the form of a bicistronic
vector that includes DNA encoding a reporter molecule such as a
fluorescent protein. Suitable reporter molecules are well-known in
the art and include fluorescent, chemiluminescent or chromogenic
proteins, for example Green fluorescent protein (GFP) or enhanced
yellow fluorescent protein (EYFP) or a fluorescent homologue
thereof, firefly luciferase protein (encoded by the Luc gene) the
enzymes chloramphenicol acetyl-transferase (CAT), or bacterial
LacZ, (.beta.-galactosidase) or the thymidine kinase gene (encoded
by the HSV1 TK gene. GFP and EYFP are detected by fluorimetry or
fluorescence histochemistry; enzymes are detected by use of a
chromogenic substrate that is converted into a colored product
which can be used in histochemical colorimetric detection of
enzymatic activity. Luciferase is measured by activation of
luciferin which emits light at a known wavelength. Reporter
molecules may be detected in vivo by non-invasive detection
techniques such as fluorescence optical imaging (FOI),
bioluminescence optical imaging (BOI), cooled charged coupled
device (CCD) camera optical imaging (CCOI) and positron emission
tomography (PET).
[0235] Infection of human CD4+CD25- T lymphocytes with retroviral
vectors carrying the Foxp3 gene was shown to convert these cells
into ones with a Treg phenotype (Walker et al., 2005, supra; Wan et
al., 2005, supra).
[0236] Any method for introducing DNA into a cell and expressing it
may be used in the present invention, including, but not limited to
vectors such as retroviral or lentiviral vectors, electroporation,
lipofection, and the like.
[0237] The functionality of redirected Tregs can be determined
using co-culture tests as described in the Examples. If APCs are to
be used in such tests, a preferred source is irradiated monocytes.
The antigen is loaded into irradiated human APCs which will present
it to T effectors and Tregs. In the case of antigens such as CEA,
human colon carcinoma cells stably transfected with the CEA epitope
may be used. In such coculture tests, one may detect specific
activation of TpCR-bearing Tregs through the TpCR. Treg activation
is assessed by examining these cells' action on T effector cell (1)
proliferation and (2) cytokine secretion profile, focusing on IL2,
IL4, IL10, IFN-.gamma. and TGF-.beta. (using commercial ELISA kits,
e.g., Ready-Set Go ELISA kit, Ebioscience CA). It is preferred to
assay TGF-.beta. and/or IL-10 as an indication of the cells' Treg
phenotype.
[0238] In a preferred embodiment, the present invention redirects
Tregs to sites of colonic inflammation, by introducing into such
cells CRs with antibody-type specificity. In sites of inflammation
the redirected Tregs are activated to suppress IBD-associated
immune response. Tregs endowed with predefined specificity migrate
and home to inflamed sites in the colon where they undergo
activation and, as a result, suppress T effector cells that mediate
the disease processes.
[0239] The present redirected Tregs represent a novel form of the
`T-bodies` discussed above and are employed as a novel therapeutic
modality in IBD. These T-bodies are T-cells that have been
genetically engineered to express TpCR in which an antibody
variable region is the recognition unit linked to T-cell
costimulatory and stimulatory domains that enable specific
activation of these T-cells but in a manner that is MHC independent
and not MHC-restricted. Based on previous studies using tumor
models described above, these redirected Tregs are tested in murine
models of IBD models.
[0240] An important aspect of this invention is the inventors'
conception that, in the context of treating IBD, the
colon-associated antigen(s) to which the T-bodies are redirected
and targeted are not necessarily the pathogenic autoantigens
recognized by the autoaggressive T effector cells. Thus, this
invention can exploit the phenomenon of "bystander"
reactivity--where the presence of the relevant antigens at the
sites of the inflammatory reactions serve to attract and "hold" or
localize the redirected Tregs, permitting them to be activated and
to exert their suppressive effects in a paracrine manner--acting on
target effector cells in the vicinity irrespective of differences
in the T effector cells' and Treg cells' antigen specificity.
CEA and LPS-Colonic Antigens as Targets for Redirected Human Tregs
in IBD
[0241] Advantage was taken of a hapten-specific IBD model that is
based on specificity to the hapten TNP to study the suppressive
effects of Tregs. In human disease, other antigens that are
expressed in intestinal or colonic tissue either normally or in the
relevant disease state are preferred targets. The include
carcinoembryonic antigen, CEA, and bacterial floral antigens such
as lipopolysaccharide, LPS.
[0242] Human IBD is idiopathic to the extent that pathogenic
antigen(s) remain unknown. Lack of knowledge of the antigen would
appear to be an obstacle to implementing the T-bodies clinically.
Nevertheless, according to the present invention, there is no
requirement that a pathogenic antigen must also be the target
antigen for Treg redirection and activation. Treg activation is
indeed antigen-specific and thus depends on TCRs, or in the present
Tregs, on antibody-based specificity, associated with costimulation
together with the activation/mediated by the intracellular
signaling moieties of the present constructs. However, once the
Tregs are activated, their suppressive action is
antigen-independent, and is carried out by secretion of suppressive
cytokines (e.g., TGF-.beta. and IL-10) even after the activating
antigen has been eliminated. Thus, inducing colonic Treg activation
by any local colon-associated antigen will promote potent Treg
activation and proliferation, while the action of these cells in
inhibiting local inflammatory processes proceeds independently of
antigen. CEA is significantly over-expressed in diseased colon
tissue in patients with active ulcerative colitis compared to
normal individuals and to patients with quiescent IBD (Smithson J E
et al., J Pathol. 1996; 180:146-51; Pavelic Z P et al., Anticancer
Res. 1991; 11:1671-5). This enhanced tissue expression of CEA was
independent of dysplastic changes and is a result of the mucosal
reaction to the inflammatory process itself. Thus, CEA is a
preferred candidate for Treg TpCR targeting in active ulcerative
colitis.
[0243] A second candidate antigen (or "non-antigen" ligand) to
which Tregs may be redirected is endotoxin or LPS, derived from the
outer membrane of Gram-negative bacteria resident in the colon. In
one embodiment, the antibody-like part (scFv) the CR's
extracellular recognition region may be derived from an anti-LPS
antibody, such as the mAb produced by the hybridoma with ATCC
Accession No. HB9081. The nucleotide sequence of an scFv made from
this mAb is shown as an annotation in FIG. 29 as part of the full
sequence of a plasmid (pBullet) comprising this scFv--SEQ ID NO:3.
Thus, a Treg expressing a TpCR that displays this scFv
extracellularly will, at a site where LPS is present such as
inflamed colon tissue (whether the gut lumen, the lamina propria or
even regional lymph nodes and other gut-associate lymphatic tissue)
bind the LPS and be activated to cause suppression of any T
effectors cells in the vicinity in an antigen-nonspecific and
MHC-independent manner.
[0244] Several types of non-antibody LPS receptors are known in the
art. CD14 (SEQ ID NO:4) is a class of LPS receptor that is a
GPI-anchored 356 aa glycoprotein. It contains a 19aa signal
peptide, an extracellular domain which contain 11 leucine-rich
repeat (LRR) domains, 4 N-glycosylation sites and an unknown number
of O-glycosylation sites. At least 2 soluble forms of CD14 have
been described, one retains GPI and is released from the cell
surface which results in an approximately 48 kDa molecule and the
other is released prior to the addition of the GPI anchor resulting
in a higher molecular weight (>48 kDa).
[0245] While LPS interacts with CD14, CD14 is not capable of
initiating a transmembrane activation signal because it is a
glycosylphosphatidylinositol (GPI)-anchored protein. Thus, LPS must
interact with a transmembrane receptor(s) that is responsible for
signal transduction. LPS is recognized by the toll-like receptor
TLR4 and MD-2 (SEQ ID NO:5; human), a molecule associated with the
extracellular domain of TLR4. CD14 greatly enhances the formation
of LPS-TLR4-MD-2 complexes, apparently by LPS loading onto
TLR4-MD-2 but not in the interaction itself between LPS and
TLR4-MD-2. (Akashi S, et al., J. Exp. Med. 198:1035-42 (2003)).
[0246] Interaction of LPS with MD-2 in a TLR4-MD-2 complex triggers
an intracellular signal transduction cascade that leads to the
production and release of proinflammatory cytokines, particularly
TNF-.alpha. (Dauphinee S M et al., 2006, Lab. Invest. 86, 9-22).
Patients with IBD show increased colon and serum levels of
endotoxin, LBP, CD14, and MD-2 (Pastor Rojo O, et al., 2006,
Inflamm Bowel Dis., December 19 (epub); Amati L et al., Curr Pharm
Des. 2003; 9:1937-45; Cario E et al., J Immunol. 2006;
176:4258-66). This change correlates with disease activity, and
proinflammatory cytokine levels return to normal after
treatment.
[0247] A motif of human MD-2, for example, from amino acids 119-132
(14 residues) of SEQ ID NO: ______ can substitute for MD-2 in
MD-2-TLR4 complex binding to the lipid A moiety of LPS, which
(Mancek M et al., Biochem Biophys Res Comm 2002; 292: 880-5;
Kobayashi M et al., J Immunol. 2006; 176:6211-8).
[0248] Thus, in one preferred TpCR of the present invention, the
extracellular recognition region comprises, in place of an
antibody-like structure (e.g., an scFv), a receptor that binds to a
ligand that is not acting as an "antigen." A preferred ligand in
the present invention is LPS. Thus, the extracellular recognition
region may comprise any of the following receptor structures:
[0249] (a) CD14 (SEQ ID NO:4), [0250] (b) an LPS-binding motif of
CD14, such as residues 100-119 of SEQ ID NO:4, [0251] (c) full
length MD-2 (SEQ ID NO:5), [0252] (d) an LPS-binding motif of MD-2
(residues 120-132 of SEQ ID NO:5), [0253] (e) a combination of a
CD14 and MD-2 or [0254] (f) a combination of a CD14-motif and an
MD-2 motif (as is encoded by the relevant segment of the chimeric
nucleic acid of SEQ ID NO:10.
[0255] Any of these constructs, when displayed on a Treg surface,
will allow the redirected Treg to bind to, and be activated by LPS
molecules, for example, at colon inflammatory sites, and thereby
exert their suppressive activities in that vicinity. Again, this is
an example of receptor-ligand binding/recognition that is not
"antibody-like" but nevertheless permits the TpCR to act in
accordance with this invention and activate Tregs in an
antigen-nonspecific (and MHC-independent) manner.
[0256] The present invention includes an embodiment in which
redirected Tregs bearing a TpCR are designed to be specific for an
antigen, referred to herein as "AgX," that may have no inherent
relationship with the tissue being targeted or the disease being
treated. In this embodiment, the Tregs specific for AgX are
activated specifically in a selected site by administering them
together with AgX to that site. The site is one where T effector
cells are situated and active, where the ongoing inflammation is to
be suppressed. The AgX-specific antibody-like receptor of the Tregs
will recognize AgX without a need for antigen presentation, MHC,
etc., and the linked signaling moieties on the TpCR will serve to
activate the Tregs to release inhibitory cytokines at that site.
This process will lead to nonspecific suppression of the ongoing T
effector cell and inflammatory activity.
[0257] The methods and compositions described herein are useful for
any of a number of autoimmune diseases which involve undesired
effector T-cells activity as an underlying cause or as a
consequence of the pathophysiology. Such diseases include, but are
not limited to, IBD, rheumatoid arthritis, Type I diabetes,
multiple sclerosis, autoimmune thyroiditis, autoimmune
uveoretinitis, autoimmune orchitis, autoimmune insulitis,
autoimmune oophoritis, psoriasis, autoimmune polymyositis and the
like. See, for example, Theofilopoulos, A., In: Stites, D P et al.,
eds., Basic and Clinical Immunology, Lange Medical Publications,
Los Altos, Calif., 1988)).
[0258] Having now generally described the invention, the same will
be more readily understood through reference to the following
examples which are provided by way of illustration, and are not
intended to be limiting of the present invention, unless
specified.
Example I
Materials and Methods
[0259] The following materials and methods are used in various of
the Examples that follow, as well as in carrying out certain
embodiments of the invention.
Cell Fractionation and Isolation
[0260] CD4+CD25+ Tregs were purified from splenic lymphocytes or
peripheral blood mononuclear cell populations using several
methods. One method utilized magnetic bead separation (MACS).
Spleens are mashed gently into HBSS/5% FCS to prepare single cell
suspensions.
[0261] CD4+T-cells were purified by negative selection by
incubation with biotin-conjugated CD4 MACS beads (Miltenyi Biotec,
Inc., Auburn, Calif.). Further purification of CD4+CD25+ cells was
conducted by incubation with phycoerythrin (PE)-conjugated
anti-CD25 antibodies or anti-CD45RB.sup.high, followed by
incubation with anti-PE microbeads (Miltenyi Biotec, Inc., Auburn,
Calif.). Magnetic separation was conducted using magnetic columns
according to manufacturer's instructions. For highly-purified
(>99%) Treg and effector T lymphocyte subpopulation, high-speed
cell sorting is be applied, using BD FACSaria (.RTM.) cell-sorting
system (BD Bioscience)
[0262] Lamina propria lymphocytes from colon were isolated as
previously described (Han X et al., Gastroenterology. 2005;
129:185-203). Briefly, colonic mucosa was dissected, followed by
incubation with Ca.sup.2+--Mg.sup.2+-free Hanks' balanced salt
(HBSS) solution containing 1 mM dithiothreitol (Sigma-Aldrich, St.
Louis, Mo.) for 30 min to remove mucus, and then serially incubated
twice times in medium containing 0.75 mM EDTA (Sigma-Aldrich) for
60 min at each incubation. The supernatants from these incubations
containing epithelium and intraepithelial lymphocyte population are
discarded, and the residual fragments pooled and treated with 2
mg/mL collagenase A (Worthington Biomedical, Freehold, N.J.) and
0.01% DNase (Worthington) in humidified air at 37.degree. C. for 2
hours. The cells are then be pelleted twice through a 40% isotonic
Percoll solution, after which they are purified further by
Ficoll-Hypaque density gradient centrifugation (40%/75%).
In Vitro Induction of Tregs
[0263] Naturally-occurring Tregs are thymus derived, express high
levels of Foxp3 forkhead transcription factor and suppress
activation of effector lymphocytes. It has been discovered that
antigen-specific activation of human effector T-cells may induce
expression of Foxp3 in a subgroup of the activated effector cells,
which in turn develop a regulatory phenotype. These induced
regulatory T-cells were shown to be capable of
cell-contact-dependent suppression of freshly isolated effector
cells (Walker et al., 2003, supra). In mice, prolonged exposure of
effector cells to TGF-.beta. induces Tregs both in vitro and in
vivo (Fantini et al., J Immunol. 2004 and 2006, supra). This small,
peripherally generated population of inducible Tregs may be central
in regulation and containment of ongoing immune response, while the
inability to induce such Tregs may be responsible for a propensity
to develop autoimmunity.
[0264] To test whether such induction occurred after stimulation of
T effector cells through the TpCR, wildtype, TNP-Tg, Erbb2-Tg and
TNP-CD28A-Tg T effector cells were isolated by FACS sorting and
cultured for 7 days in the presence of either (1) anti CD3 Ab, (2)
murine TGF-.beta., (3) mAb to TNP, (4) anti CD3 Ab+TGF-.beta., or
(5) anti TNP Ab+TGF-.beta.. Induction of Foxp3 in cells
"developing" from these effector T-cells was assessed after seven
days of culture using intracellular Foxp3 staining
[0265] Antigen-specific activation of human effector T-cells leads
to inducible expression of Foxp3 in a subgroup of activated
effector cells, which in turn develop regulatory phenotype. These
induced regulatory T-cells are capable of cell-contact-dependent
suppression of freshly isolated effector cells. In mice, both in
vitro and in vivo induction of Tregs can be achieved with prolonged
exposure of effector cells to TGF-.beta. (Fantini et al., 2004,
2006, supra). The present inventors adopted this technology to
induce murine redirected Tregs from redirected effector T-cells
(see FIG. 3).
[0266] Animals
[0267] Several mouse strains were used in the studies described
below and are used in various other embodiments of the invention.
These include transgenic mouse lines that specifically expresses
anti-TNP or anti-Erb B2 TpCRs (bearing CD28-FcR signaling chains)
under the control of a CD2 promoter, as well as a transgenic mouse
line expressing human CEA (Saha A et al., Immunology 2006,
118:483-496)
[0268] All transgenic mice were back-crossed to Balb/c. Balb/c
wild-type mice serve routinely as controls and recipients of
adoptively transferred cells.
[0269] One cell-transfer colitis model is used in immune deficient
Rag.sup.-/- and SCID mice.
[0270] All invasive procedures were and are conducted under
Ketamine and Xylazine general aesthesia (127.5 and 4.5 mg/kg,
respectively). Subcutaneous (S.C.) injections are conducted under
local anesthesia with 10% Xylocalne spray.
Colitis Induction and Assessment:
[0271] To induce TNP hapten-mediated colitis mice were sensitized
with 15 .mu.l of the haptenating agent
2,4,6-trinitrobenzenesulfonic acid (TNBS, Sigma-Aldrich) at a
concentration of 2.5% v/v in 50% ethanol by skin painting on day 1.
On day 8, 150 .mu.l of 1% TNBS in 50% ethanol was administered
intrarectally via a 3.5 F catheter under general anesthesia.
OXA-induced colitis was induced by sensitizing mice with oxazolone
(4-ethoxymethylene-2-phenyl-2-oxazolin-5-one; Sigma-Aldrich) at a
concentration of 3% v/v in 100% ethanol by skin painting on day 1,
followed by intrarectal administration of 150 .mu.l at a
concentration of 1% v/v in 50% ethanol on day 8.
[0272] In one preferred cell transfer colitis models,
CD45RB.sup.high (naive) T-cells are transferred to immune deficient
mice from syngeneic background (Powrie F et al., J Exp Med. 1994;
179:589-600. This model of mucosal inflammation allows separating T
effector and Treg cell function within an inflammatory site.
[0273] In all models, colitis is assessed following induction using
the following parameters: degree of colon ulcerations, intestinal
and peritoneal adhesions, wall thickness, and degree of mucosal
edema. Each parameter is graded on a scale from 0 (completely
normal) to 4 (most severe) by two experienced, blinded observers.
For histological evaluation of inflammation, distal colon tissue
(last 10 cm) is removed and fixed in 10% formaldehyde. Five
paraffin sections from each mouse are stained with
hematoxylin-eosin using standard techniques. The degree of
inflammation is graded semiquantitatively on microscopic cross
sections of the colon from 0 to 4 as follows: Grade 0: Normal with
no signs of inflammation; Grade 1: very low level of leukocyte
infiltration; Grade 2: Low level of leukocyte infiltration; and
Grade 3: High level of infiltration with high vascular density, and
bowel wall thickening; Grade 4: Transmural infiltrates with loss of
goblet cells, high vascular density, wall thickening, and
disruption of normal bowel architecture.
Murine Colonoscopy
[0274] For continuous monitoring of colitis pathology, a
newly-developed, high resolution mouse video endoscopic system has
been used Becker C et al., Gut. 2005; 54:950-4. The experimental
endoscopy system (from Karl Storz, Tuttlingen, Germany) consists of
a miniature endoscope (1.9 mm outer diameter), a xenon light
source, a triple chip camera, and an air pump. Parameters for
grading of colitis include bowel wall thickening, granularity,
fecal consistency, fibrin deposition and vascular pattern. Whole
colon methylene blue chromoendoscopy staining is used, when
appropriate, to visualize crypt pattern. A 3fr. Flexible biopsy
forceps is used for biopsy-taking. Biopsies are either placed in
formalin for paraffin embedding, sectioning and subsequent
immunohistochemistry, frozen in liquid nitrogen for cryosections,
or obtained and used for RNA isolation. A typical yield of a biopsy
specimen is approximately 2 .mu.g RNA
In Vivo Imaging:
[0275] To follow migration (also referred to as homing or
trafficking) of redirected Tregs in mice, a whole body CCD camera
(IVIS.RTM. 100 Series Imaging System, Xenogen, Alameda Calif.). was
used. Redirected Tregs were labeled with the near-infrared (NIR)
lipophilic carbocyanine dye 1,1'-dioctadecyl-3,3,3',
3'-tetramethylindotricarbocyanine iodide (DiR, Invitrogen, USA).
This dye has absorption and fluorescence maxima at 750 and 782 nm,
respectively, enables the safe direct labeling of membranes of
human lymphoid cells with very low light absorption and
autofluorescence levels in living tissues (Miller M J et al., Proc
Natl Acad Sci USA, 2003; 100:2604-9; Kalchenko V et al., submitted
for publication, 2007). Additional in vivo visualization of Tregs
labeled with carboxy fluorescein diacetate succinimide ester (CFSE)
at colonic mucosa was performed by intrarectal insertion of a 300
and 650 .mu.m diameter confocal microendoscope (Cell Vizio, MKT,
Paris, France). This unique modality, previously untested in
colitis models, allows repeated in vivo assessment of homing of
CFSE-labeled redirected Tregs to the most inner layers of colon
tissue following induction of inflammation.
Determination of Colon Cytokine Levels
[0276] Colon mRNA expression of selected cytokines is determined to
allow assessment of redirected Treg effects on local intestinal
immune response. in particular, levels of pro-inflammatory
(TNF.alpha. and IFN.gamma.) and anti-inflammatory cytokines
(TGF.beta. and IL10), as well as levels of the TH.sub.1
transcription factor Tbet and the TH.sub.2 transcription factor
GATA-3. Colon cytokine levels are assessed by measuring mRNA
expression and protein levels.
[0277] Samples for mRNA isolation are removed from colons of mice
using in vivo colonoscopy or during sacrifice. Total RNA is
isolated and processed and cDNA produced by RT-PCR. In all
experiments, mice are divided into the following groups: naive
mice, colitis-induced mice, and colitis-induced mice adoptively
transferred with Tregs (naturally occurring, induced, or
redirected, see detailed adoptive transfer experiments herein). The
following sets of oligonucleotides and amplification conditions are
used:
TABLE-US-00001 SEQ ID Amplification SEQUENCE NO: conditions
TNF-.alpha. sense 5'-AGTCCGGGCAGGTCTACTTT-3' 15 60.degree./30
cycles antisense 5'-GAGGCAACCTGACCACTCTC-3' 16 IFN-.gamma. sense
5'-TCTGGAGGAACTGGCAAAA-3' 17 63.degree./35 cycles antisense
5'-TGAGCTCATTGAATGCTTGG-3' 18 TGF-.beta. sense
5'-TACAGGGCTTTCGATTCAGC-3' 19 63.degree./35 cycles antisense
5'-CGCACACAGCAGTTCTTCTC-3' 20 IL-10 sense
5'-TCCTTGGGAAGCAATTGAAG-3' 21 63.degree./35 cycles antisense
5'-AACTGGCCACAGTTTTCAGG-3' 22 T-bet sense
5'-CTAAGCAAGGACGGCGAATGT-3' 23 60.degree./35 cycles antisense
5'-GGCTGGGAACAGGATACTGG-3' 24 GATA-3' sense
5'-GCCTGCGGACTCTACCATAA-3' 25 54.8.degree./30 cycles antisense
5'-CAGGGATGACATGTGTCTGG-3' 26 GAPDH sense
5'-GTGTTCCTACCCCCAATGTG-3' 27 60.degree./25 cycles Antisense
5'-CTTGCTCAGTGTCCTTGCTG-3' 28
The relative mRNA expression compared to the housekeeping GAPDH is
assessed using NIH image software and averaged from mice in each
group.
[0278] IL-10 and IFN-.gamma. protein expression levels in colon
tissue are quantified by a cytofluorimetry-based ELISA system. In
brief, whole proteins are isolated from colon specimens in the
absence of detergent. Proteins (100 .mu.g) are immediately used for
cytokine determination according to manufacturer's
instructions.
Foxp3 Immunohistochemistry of Colon Samples:
[0279] Foxp3 immunofluorescence is performed to estimate in situ
the targeting of Treg to diseased colon, using TSA Cy3 and a
fluorescence microscope (Olympus). In brief, cryosections are fixed
in cold acetone for 10 minutes, followed by sequential incubation
with methanol, avidin/biotin (Vector Laboratories, CA), and protein
blocking reagent to eliminate nonspecific background staining.
Slides are then incubated overnight with primary antibodies
specific for Foxp3 (e.g., from Alexis Biochemicals, Lausanne,
Switzerland). Subsequently, slides are incubated for 30 minutes at
room temperature with biotinylated secondary antibodies, and
treated with streptavidin-horseradish peroxidase and stained with
Tyramide (Cy3 or FITC). Before examination, nuclei are
counterstained with Hoechst 3342 (Molecular Probes, Ohio).
Example I
Phenotypic Characterization of TNP-Specific Tregs
[0280] The inventors have produced transgenic (Tg) mice expressing
a TNP-specific tripartite chimeric receptor (TpCR) that serve as a
source of redirected Treg cells specific for the trinitrophenyl
(TNP) hapten. This hapten has served as a "classical" antigen for
years in studying both antibodies and T cell-mediated immunity. A
chemically reactive form of this hapten, TNBS, is a contact
sensitizing agent that induces and evokes delayed-type
hypersensitivity (DTH) responses as well as inducing colitis in
animals, as described herein.
[0281] Generation of TNP-specific Tregs was achieved by the
creation of Tg mice that express TNP-specific TpCR that comprises
an scFv from the TNP-specific mAb Sp6 mAb linked to a truncated
CD28 molecule which was inserted between the scFv and the
cytoplasmic part of the FcR.gamma. chain (abbreviated as y herein
(see FIG. 1). This construct includes the hinge region,
transmembrane region, and cytoplasmic region of CD28 but lacks the
B7 (ligand) binding site.
[0282] For the truncated form of CD28 (TpCR/CD28, FIG. 1) that does
not include the CD28 intracellular signaling domain, the inventors
cloned the vector at the same site. As a control, a Tg mouse
expressing TpCR specific for another, irrelevant antigen (Erb-B2)
was used.
[0283] For expression of TpCR in T-cells of Tg mice, a construct
comprising an anti-TNP (Sp6-derived scFv-CD28-.gamma. was cloned
into a human CD2 promoter/enhancer minigene-based vector. Tg mice
were generated at the Weizmann Institute's Department for
Veterinary Resources by pronuclear microinjection of
(BALB/c.times.C57BL/6)F.sub.1 fertilized eggs derived from
hyperovulated donor females. Founder mice were screened by PCR of
DNA from tail samples. Several founder strains were obtained that
express high level of the TpCR on their cell surfaces. These were
backcrossed for more than nine generations to either BALB/c or
C57BL/6 mice to obtain MHC-homogeneous mice.
[0284] The studies below describe the characterization of various
Treg subpopulations in the different strains of TNP-CR transgenic
mice, and the expression of TpCR on these Tregs.
Example II
Isolation of Trees in which TNP-Specific TpCR are Highly
Expressed
[0285] Tregs were isolated using double magnetic bead separation
(Miltenyi Biotech) or by fluorescent cell sorting in which
fluorescently labeled CD4+CD25+ cells were sorted using the
FACSARIA cell sorting system.
[0286] Treg expression of TNP-specific TpCR was assessed by
containing cells for Foxp3 (considered the "gold standard" marker
of Tregs) and PE-labeled mAb specific for TNP antibody (generated
in the inventors' laboratory). Controls included groups stained
with the appropriate isotype controls. As is shown in FIG. 2, Tregs
from TNP-Tg mice, but not from wild-type mice, expressed high
levels of TNP-specific TpCR.
Example III
TNP-Tg Mice Posses Increased Numbers of Foxp3+Tree Population
[0287] Peripheral lymphocytes from the spleen as well as
gut-associated lymphocytes from the lamina propria of the colon
were stained. As shown in FIG. 3, a CD4+CD25+ cell population
(represented as the ratio of CD4+CD25+ cells among CD4+T-cells) was
elevated modestly in TNP-Tg mice in comparison to control mice
(wildtype, ErbB2-Tg and TNP-CD28 null-Tg mice). In contrast, higher
numbers of Foxp3+ cells were observed in TNP-Tg animals compared to
the control animals in comparison to all other mouse types (FIG.
4).
[0288] To resolve what may have appeared to be an inconsistency
between the highly elevated Foxp3+Treg population in TNP-Tg mice
and the modestly elevated CD4+CD25+ Treg population in these mice,
effector CD4+CD25- cells were isolated by cell sorting to a level
of 99% purity. Isolated cells were stained for Foxp3 (FIG. 5). As
expected, no positive Foxp3 staining was noted in T effector cells
from wildtype, ErbB2-Tg and TNP-CD28null-Tg mice. In contrast,
TNP-Tg T effector cells featured a significant population of Foxp3+
cells. This observation was further validated in whole spleen cell
populations that were co-staining for Foxp3 and CD25 (FIG. 6). The
presence of a significantly greater Foxp3+CD25- Treg population in
TNP-Tg mice is supported by other recent results by the inventors'
laboratory showing that the Sp6 mAb from which the scFv of the
TNP-specific TpCR was derived recognizes cross-reactive endogenous
thymic antigens. This results in either deletion or early release
from the thymus to the periphery before several other immature T
cell subsets, including immature CD25- Tregs.
Example IV
Induction of TNBS Colitis in TNP-Tg Mice Significant Elevated the
Numbers of Foxp3+ Expressing Cells in Peripheral and Colon-Derived
Lymphocyte Populations
[0289] Induction of TNBS colitis results in further elevation in
splenic (FIG. 7) and colon (FIG. 8) Foxp3+Tregs in TNP-Tg (FIGS. 7
& 8, respectively). These results demonstrated that
TNP-specific Treg expansion occurred following induction of colitis
in Tg mice, reflecting Treg proliferation following
antigen-specific activation by TNP.
Example V
In Vitro Functional Characterization of Redirected Tregs
[0290] A key prerequisite for the utility of Tregs expressing
TNP-specific TpCR in the treatment of autoimmunity is verification
of their regulatory activity, namely an ability to suppress T
effector cell proliferation in a dose-dependent manner. Also
examined was whether such Treg activation occurs as a result of
TpCR signaling, and whether it was indeed independent of CD28-B7
interaction. A series of coculture experiments examined Tregs from
the different Tg strains, as is outlined below.
Example VI
Tregs Bearing the TNP-Specific Chimeric Receptor Specifically
Suppressed the Activity of T Effector Cells
[0291] To characterize whether TNP-Tg Tregs retained their anergic
properties, CD4+CD25+ Treg cells and CD4+CD25- T effector cells
from different Tg mouse founders (anti-TNP, anti-Erb-b2 control and
wildtype (WT) mice) were purified from bulk splenocytes. 10.sup.5
cells were incubated in vitro for 24 h, 48 h or 72 hrs (FIG. 9) and
activated non-specifically with anti CD3 and anti-CD28 Abs, or
specifically with Fowl gamma globulin-modified TNP (FyG-TNP). T
cell proliferation was measured using either the uptake of a dye
(tetrazolium salt XTT) or radiolabeled Thymidine. IL2 secretion was
measured using XTT staining of the IL-2-dependent CTLL-2 cell
line.
[0292] All effector cell populations showed significantly increased
proliferation and IL2 secretion following non-specific stimulation
with anti-CD3+ anti-CD28 Abs. Specific stimulation by FyG-TNP
resulted in proliferation and IL2 secretion by T effector cells
bearing TNP-chimeric receptor, but not by such T-cells from WT or
anti-Erb-b2 Tg mice. In contrast, Tregs from wildtype mice,
TNP-chimeric receptor Tg mice and Erb-b2 Tg mice retained their
anergic properties: they did not undergo measurable proliferation
or IL2 secretion when subjected to the non-specific stimulus or
specific Ag.
[0293] To characterize whether polyclonal activation could trigger
the suppressive action of TNP-Tg Tregs, these Tregs were cocultured
in 96-well microplates (0.2 ml) with irradiated antigen presenting
cells (APCs) and T effector cells (CD4+CD25-) at 1:1 ratios. Cells
in these culture were activated either by (1) immobilized antigen
"mimic" (anti-CD3+ anti-CD28) or (2) soluble Concanavalin A (ConA).
T cell proliferation was measured as Thymidine uptake and IL2
secretion was measured as growth of cells of the IL-2-dependent
CTLL-2 cell line (XTT staining).
[0294] FIG. 10 shows a ConA experiment. Non-specific (polyclonal)
stimulation of Tregs induced these cells to exhibit potent
inhibition of T effector cell proliferation and IL2 secretion,
irrespective of the origin of the Tregs or the presence of the
chimeric receptor. Thus, genetic manipulation of Tregs of the type
described here preserves their suppressive properties.
Example VII
Antigen-Specific Stimulation of Redirected Tregs Cells with TNP
Results in Suppression of T Effector Cell Proliferation
[0295] To study the antigen-specific Treg stimulation through the
TpCR, coculture experiments were done in which TNP-loaded APCs
provided the Ag presentation (FIG. 11). Comparisons of TNP-specific
Treg stimulation was performed, comparing wildtype vs. TNP-Tg Tregs
(FIG. 11, left panel) or ErbB2-Tg and TNP-Tg Tregs (FIG. 11, right
panel). In the absence of TNP stimulation, T effector cell
proliferation did not occur (left-most bars in both graphs). In
contrast, incubation with TNP-modified APC's resulted in:
(1) marked proliferation of TNP-Tg but not of wildtype or ErbB2-Tg
effector T-cells in the absence of Tregs; and (2) activation of
TNP-Tg, but not of WT or Erb-b2-Tg Tregs, manifest as suppression
of effector cell proliferation by TNP-specific Tregs only.
[0296] These results proved the antigen-specific manner of
activation and function of TNP specific TpCR Tregs cells in
response to the antigen, TNP.
[0297] Co-culture of varying ratios of TNP-specific Tregs and
TNP-specific effector T-cells (FIG. 12) demonstrated successful
antigen-specific inhibition by Tregs at a ratio of 1 Treg to 8 T
effector cells.
[0298] Studies supporting the existence of the bystander effects
were carried out. Colitis was induced in mice as above using OXA as
described in Example I. Adoptive transfer of TNP-specific Tregs
alone did not protect these animals from colitis. However, in the
presence of trace amounts of TNP applied to the colon, animals were
protected from this OXA-induced colitis.
Example VIII
Suppressive Activity of TpCR-Redirected Tregs is Independent of
Costimulatory Receptors
[0299] To assess the role of costimulatory signaling in the above
TpCR-Tg model, coculture experiments as above were performed using
as APC's (a) TNP-modified P815 cells, a cell line that does not
express B7, or (b) TNP-loaded genetically modified P815 cells
stably expressing the B7 gene (FIG. 13). Stimulation of TNP-Tg
effector T-cells with TNP-P815 cells induced proliferation, which
was markedly suppressed by TNP-Tg Tregs. Expression of B7 on these
APC's did not promote any further Treg-mediated suppression. It was
concluded that maximal Treg suppression occurred independently of
B7. Some suppression was also noted with wildtype Tregs. This was
explained by the pre-activation of these cells prior to their
harvesting. Based on these results, it could be concluded that
inclusion of the intra-cytoplasmic signaling domain of CD28 in the
TpCR of redirected Treg cells results in full activation of their
suppressive activity when stimulated by Ag irrespective of the
presence of B7-CD28 costimulation.
Example IX
Functional Characterization of TNP-Specific Treg Activity In Vivo
in Murine Colitis
[0300] TNBS is a potent inducer of T-cell responses such as
DTH/contact sensitization. This reactive hapten also induces
autoimmune colitis when applied to the colon of pre-sensitized
mice. To determine whether TpCR-bearing Tregs could suppress
autoimmunity, the acute TNBS-mediated colitis model was employed.
Intra-rectal administration of TNBS leads to its binding to colon
proteins, rendering these modified proteins immunogenic so that
they elicited a T cell mediated immune response. The suppressive
effect of endogenous or exogenously transferred Tregs on autoimmune
inflammatory disease was tested in this model. A different hapten,
oxazolone (OXA) with similar sensitizing properties and which
induces experimental colitis was used as a specificity control in
vivo.
Example X
Transgenic Mice Whose Entire Treg Population Expresses the Chimeric
Anti-TNP Receptor are Resistant to TNBS-Induced Colitis
[0301] TNP hapten-mediated colitis was induced in Tg and WT mice by
first sensitizing the animals with 150 .mu.l of the
2,4,6-trinitrobenzenesulfonic acid (TNBS, Sigma-Aldrich) at a
concentration of 2.5% in 50% ethanol painted on the skin on day 1.
On day 8, the antigen was administered rectally (150 .mu.l of 1%
TNBS in 50% ethanol; high dose colitis). WT mice developed severe
colitis within 2-5 days of rectal TNBS administration (FIG. 14,
left panel). In contrast, 90% of the TNP-Tg mice had normal looking
colons (FIG. 14, right). Colitis severity scores were as
follows:
TABLE-US-00002 Colitis score Animals (Arbitrary Units) Mortality
Wildtype 12 .+-. 3.1 90 .+-. 20% TNP-.DELTA.CD28-Tg 11.1 .+-. 4
ErbB2-Tg 12.7 .+-. 3.2 TNP-Tg 2 .+-. 2 (p < 0.05) 20 .+-. 20% (p
< 0.01)
[0302] To produce mortality curves the above experiments were
repeated with lower doses of TNBS (75 .mu.l of 1% TNBS in 50%
ethanol). Similar differences in colitis severity and in mortality
were noted (FIG. 15). Microscopically, colons of wildtype,
TNP-.DELTA.CD28-Tg and ErbB2-Tg mice showed severe inflammation,
necrosis, hemorrhage and in some cases perforation, while those of
TNP-Tg mice appeared normal or near normal (FIG. 16).
[0303] Evidence of antigen specificity of the protection from
hapten-mediated colitis came from studies of OXA-induced colitis.
As shown in FIG. 17, no differences in mortality were noted between
wildtype, TNP-Tg, TNP-.DELTA.CD28-Tg and ErbB2-Tg mice. The same
was true for macroscopic and microscopic colitis scores. From these
in vivo experiments, it was concluded that the presence of a Treg
cell population that uniformly expresses the anti-TNP chimeric
receptor results in high grade protection against TNBS-induced
inflammation, manifest as reduced colon inflammation and
significantly improved survival. It is noteworthy that inclusion of
CD28 costimulatory signaling in the CR significantly enhances
TNP-Tg Treg suppressive function.
Example XI
Prolonged Stimulation with TNP Combined with TGF-.beta. Promotes
Conversion of Leads to TNP-Specific Effector T-Cells to
TNP-Specific Trees
[0304] Naturally-occurring Tregs are thymus derived, express high
levels of Foxp3 and suppress activation of effector lymphocytes.
Antigen-specific activation of human effector T-cells may induce
expression of Foxp3 in a subgroup of the activated effector cells,
which in turn develop a regulatory phenotype. These induced
regulatory T-cells were shown to be capable of
cell-contact-dependent suppression of freshly isolated effector
cells (Walker et al., 2003, supra). In mice, it has been
demonstrated that prolonged exposure of effector cells to
TGF-.beta. induces Tregs both in vitro and in vivo (Fantini et al.,
2004, 2006, supra). This small, peripherally generated population
of inducible Tregs may be central in regulation and containment of
ongoing immune response, while the inability to induce such Tregs
may be responsible for a propensity to develop autoimmunity.
[0305] To test whether such induction occurred after stimulation of
T effector cells through the TpCR, wildtype, TNP-Tg, Erbb2-Tg and
TNP-CD28null-Tg T effector cells were isolated by FACS sorting and
cultured for 7 days in the presence of either (1) anti CD3 Ab, (2)
murine TGF.beta., (3_mAb to TNP, (4) anti-CD3 Ab+TGF-.beta., or (5)
anti-TNP Ab+TGF-.beta.. Induction of Foxp3 in cells "developing"
from these effector T-cells was assessed after seven days of
culture using intracellular Foxp3 staining (FIG. 18).
[0306] At time 0 to the time of T effector cell sorting, no Foxp3
staining was noted. A week of stimulation with anti-CD3+TGF-.beta.,
but not with TNP+TGF-.beta., resulted in a 2-fold increase in
Foxp3+ cells in wildtype, ErbB2-tg and TNP-CD28null-Tg T effector
cells. In contrast, a dramatic 30-fold increase in Foxp3+ cells was
observed in TNP-Tg effector cells following exposure to
TNP+TGF-.beta.. Interestingly, no Foxp3 induction was noted in
TNP-Tg T effector cells following incubation with anti CD3 Ab or
with TGF-.beta., probably due to significant attenuation of CD3
expression in TNP-Tg T-cells (Morvinsky-Friedman et al, in
press).
[0307] The foregoing results demonstrated that antigen-specific
stimulation through the TpCR in the presence of TGF-.beta., led to
induction of Ag-specific Tregs from T effector cells, further
contributing to Treg expansion. This change was dependent both on
the antigen-specificity of the Ab recognition unit of the TpCR and
the intra-cytoplasmic CD28 signaling moiety.
[0308] According to the present invention, induction of Tregs in
this manner permits the generation of large populations of
TpCR-bearing Tregs that can be used in cell-based therapy of
autoimmunity.
Example XII
Adoptive Transfer of TNP-Tg Tregs to WT Mice with TNBS Colitis
Ameliorates Symptoms and Improved Survival
[0309] Studies were conducted to establish that TNP-Tg Tregs are
responsible for the resistance of TNP-Tg mice to TNBS colitis and
to evaluate their therapeutic capacity in autoimmunity. Wildtype,
TNP-Tg and Erb-b2-Tg Tregs were isolated and administered in
varying numbers to wildtype mice a day after induction of TNBS
colitis. As was previously described, adoptive transfer of large
numbers of Tregs of any origin (>2.times.10.sup.5) caused
nonspecific attenuation of TNBS colitis. This is believed to result
from the presence of a sufficiently large population of
pre-activated Tregs that can exert their suppressive activity in
the absence of antigen stimulation or specificity. In contrast,
adoptive transfer of smaller numbers (5.times.10.sup.4) of TNP-Tg
Tregs but not of wildtype or Erbb2-Tg Tregs, prolonged survival
(FIG. 19), improved Wallach colitis severity scores (FIG. 20), and
significantly improved macroscopic (FIG. 21) and microscopic (FIG.
22) appearance of colon tissue. FIG. 21 shows marked bowel
shortening, a manifestation of colon inflammation (in WT and
ErbB2-Tg, but not in TNP-Tg mice). FIG. 22 shows the severe
transmural inflammation, necrosis, mucosal bleeding and loss of
normal architecture in colons of WT mice with TNBS colitis that had
received control (WT and ErbB2-Tg), but not in TNP-Tg colons.
Example XIII
Migration/Trafficking of Redirected `Tregs to Sites of
Inflammation: Adoptively-Transferred TNP-TG Tregs Localize to
Colons in TNBS Induced Colitis
[0310] Studies were done to garner additional support for the role
of TNP-Tg Tregs in attenuating TNBS colitis by showing that these
cells indeed localize to inflamed colon tissue. WT and TNP-Tg Tregs
were isolated and stained with the fluorescent intracellular dye,
carboxyfluorescein diacetate succinimidyl ester (CFSE). Following
staining, 10.sup.6 Tregs were administered intraperitoneally (ip)
to control WT mice or to WT mice in which TNBS colitis had been
induced 12 hours earlier. Sixteen hours after this treatment, mice
were sacrificed and lamina propria lymphocytes isolated from their
colons. The protocol used was described above to isolate lamina
propria lymphocytes. Thereafter, the cells were examined for the
presence of CFSE-positive cells by FACS analysis. As shown in FIG.
23, very small numbers of adoptively-transferred WT or TNP-Tg Tregs
reached the colons of normal mice. Induction of colitis led to a
small increase (0.5% to 0.8%) in the number of WT CFSE-stained
Tregs in colon tissue. In contrast, adoptive transfer of
CFSE-labeled TNP-Tg Tregs to mice with colitis led to a significant
increase in colon Treg population, ranging from 0.4% to 3.6%. This
demonstrates that TNP-Tg Tregs localize in a TNBS-exposed target
organ, where they exert their suppressive function.
Example XIV
Accumulation of Adoptively Transferred TNP-Tg Tregs within the
Mucosal Layer of Colons of Mice with TNBS Colitis
[0311] An important aspect of understanding the role of Tregs for
adoptive therapy of autoimmune inflammation of the Treg in diseased
organs, where they are expected to exert their suppressive effects.
To demonstrate TNP-Tg-Treg localization, WT and TNP-Tg Tregs were
labeled with CFSE and transferred to WT mice 24 hours following
induction of TNBS colitis. While very small numbers of CFSE-labeled
WT Tregs were observed in cell extracted from colonic lamina
propria of naive or TNBS colitis-induced mice, a nine-fold increase
in TNP-Tg Tregs was noted (FIG. 23).
[0312] To study the kinetics of TNP-Tg Treg localization of in the
living animal, the IVIS.RTM. 100 Series Imaging System was employed
(Xenogen, Alameda Calif.). Wildtype and TNP-Tg Tregs,
1.5.times.10.sup.6, labeled with the near-infrared lipophilic
carbocyanine dye
1,1'-dioctadecyl-3,3,3',3'-tetramethylindotricarbocyanine iodide
(DiR, Invitrogen USA), were administered ip to WT mice with or
without TNBS colitis, who were monitored daily with the IVIS whole
body CCCD camera (FIG. 24). A strong anterior-abdominal fluorescent
signal, reflecting the bulk of injected Tregs, was noted in naive
mice 24-48 hours following ip injection of Tregs and disappeared
thereafter due to Treg redistribution. In mice with TNBS colitis, a
faint abdominal signal was noted for 72 hours, probably reflecting
inflammation-related auto-fluorescence. In mice with TNBS colitis
that received labeled WT Tregs, a week-moderate abdominal
fluorescent signal could be recognized up to 96 hours following
Treg transfer. In contrast, TNP-Tg Treg-administered to WT mice
with TNBS colitis featured a distinct abdominal fluorescent signal
for up to a week following cell transfer, substantially stronger
than that of wildtype Tregs at all time points. These results
reflect persistent TNP-Tg Treg localization within colons during
the colitis.
[0313] To determine whether TNP-Tg Tregs reach the inner colonic
mucosal layer, the location where most of TNBS-induced mucosal
damage takes place, the Cell Vizio confocal microendo-scopy system
was employed (Cell Vizio, MKT, Paris, France). An
intrarectally-inserted 650 .mu.m diameter confocal microendoscope
enabled visualization of CFSE-labeled cells in up to a 150 .mu.m
bowel wall thickness (FIG. 25). Numerous adoptively transferred
CFSE-labeled TNP-Tg, but no WT Tregs could be visualized in the
inner mucosal layer of WT mice with TNBS colitis as early as 12
hours following systemic Treg transfer. This result indicates that
TNP-Tg Tregs localize in response to colonic TNBS, within hours of
their administration, and that they reach the deepest colonic
mucosal layers, where they exert their suppressive functions.
Example XV
Administration of TNP-Tg Tregs Specific to a Bystander Antigen
(TNBS) Cures Colitis Mediated by a Pathogenic (Oxazolone)
Antigen
[0314] In contrast to hapten-mediated colitis, in which the
eliciting antigen is predefined, the disease-causing antigen in
inflammatory bowel disease (IBD) is unknown. To enable application
of the `T-body` approach in IBD, naive TPCR-bearing Tregs were
tested to determine whether they can be activated by a
predetermined `bystander` colon- or colitis-associated antigen, to
perform their antigen-nonspecific suppressive action. To this end,
WT and TNP-Tg mice were pre-sensitized to oxazolone only. A mixture
of oxazolone and low doses of TNBS were introduced intrarectally.
As is shown in FIG. 26a, concomitant challenge of WT mice with TNBS
and oxazolone, was associated with a 100% one-week mortality rate,
as compared to only a 15% one-week mortality of TNP-Tg mice
(P<0.01). Similarly, significant mucosal inflammation was
evident in both WT and TNP-Tg mice with oxazolone colitis (not
shown), and was most severe in wild-type mice given TNBS+oxazolone
(FIG. 26b, box I) resulting in severe bleeding, fibrin deposition
and sloughing off of colonic mucosa.
[0315] In utter contrast, TNP-Tg mice administered TNBS+oxazolone
featured normal-appearing colonic mucosa with scattered areas of
mild colitis (FIG. 26b, box II). Macroscopically and
microscopically, colons of concomitantly TNBS-treated and
oxazolone-treated WT mice featured severe colitis, as opposed to
the near-normal colons in TNP-Tg mice (FIGS. 26c and 26d,
respectively.)
[0316] Notably, this "bystander" protective effect also occurred
when TNP-Tg Tregs were adoptively transferred to
oxazolone-presensitized wild-type mice which were intrarectally
boosted with a mixture of oxazolone and low doses of TNBS (FIG.
26e, P<0.01)). In contrast, adoptively transferred WT Tregs did
not have this curative effect, and the very low TNBS doses in the
absence of pre-sensitization were insufficient by themselves to
induce TNBS colitis. These results demonstrate that Treg activation
by a bystander antigen (TNBS) cause an improvement in colitis that
has been induced by a different non-cross-reactive antigen
(oxazolone).
Example XVI
Delivery of Foxp3 to Cell Nucleus by Vectors Comprising Chimeric
Receptors
[0317] An experiment was conducted to verify that the Foxp3 can be
expressed following transduction of A273 cells with retroviral
vector constructs designed to transduce Treg cells. A fused gene
was generated that included eGFP sequences encoding green
fluorescent protein (referred to as eGFP or GFP). This was
engineered as a bicistronic construct with the GFP sequence alone
or linked with a Foxp3-encoding sequence (after an IRES) into
vectors that comprised a chimeric receptor construct with the
following extracellular recognition regions: See description of
FIG. 27 for discussion of the chimeric receptor constructs used.
273 cells. transduced with vectors comprising the same chimeric
receptors but with a bicistronic eGFP gene only (without Foxp3)
served as controls for Foxp2 expression.
[0318] The results are shown in FIG. 27. The upper half of the
Figure shows the GFP-only controls, whereas the lower half of the
Figure shows GFP-Foxp3 constructs. The two-paneled rectangles in
the Figure show light microscopic (left half) and fluorescence
microscopic (right half) images of the same material (to visualize
and localize the GFP).
[0319] All the control group expressed the eGFP in their cytoplasm
only. In contrast, in cells that were transduced with the
eGfP-Foxp3-fusion constructs, the nuclei were fluorescent
(appearing as bright nuclear images) due to the transport to and
expression of the Foxp3 transcription factor in the nuclei.
[0320] In another experiment not shown here, expression of chimeric
receptor made of the full length MD2 protein (SEQ ID NO:5) or the
CD14 protein (SEQ ID NO:4) was confirmed by the ability of
transduced cells, which expressed the extracellular region of the
CR on their surface, to bind the ligand of MD2 and CD14, bacterial
LPS, which was provided in biotinylated form and revealed by
secondary binding of fluorescent avidin.
Example XVII
Vectors Comprising Chimeric Receptors with LPS-Binding
Extracellular Regions
[0321] Nucleic acid constructs and vectors that encode
extracellular regions that comprise an anti-LPS antibody domain
(e.g., SEQ ID NO:3 or the scFv-coding portion thereof) have been
made and others can be made. Such vectors express extracellular
polypeptide domains that are shown to bind LPS, for example in an
assay using biotinylated LPS and detectably labeled (e.g.,
fluorescently labeled) avidin. See also Example XVI above.
[0322] Nucleic acid constructs and vectors that encode
extracellular regions that comprise a LPS-binding nonantibody
polypeptide have been made (e.g., SEQ ID NO:6-11, 13 and 14). Such
constructs include bicistronic ones that also comprise Foxp3. Other
such constructs can be made using the method described above along
with methods well-known in the art. Such constructs (such as SEQ ID
NO: 13 and 14) include those encoding full length CD14 (SEQ ID
NO:4) or MD2 (SEQ ID NO:5) protein, and constructs encoding
LPS-binding motifs therefrom (such as SEQ ID NO:6-9) and
combinations (such as SEQ ID NO:10 and 11). The constructs that are
made include those with CD28-FcR.gamma. intracellular
stimulatory/costimulatory regions and those that utilize others of
the type disclosed herein.
[0323] Treg cells are redirected as described herein using the
above constructs, including those that have been made and tested
and those that can be made.
[0324] Such Treg cells are administered into subjects suffering
from IBD, such as ulcerative colitis. Treg cells are administered
in numbers in accordance with the above examples, or in numbers
that are readily determined to be effective by those skilled in the
art using only routine experimentation, and via routes of
administration as exemplified above and disclosed throughout this
document. These redirected Treg cells that express an LPS binding
antibody region or another LPS-binding moiety on their surface
(CD14, MD2, fragments thereof, or combinations of these) as part of
their CR's are able to reduce the symptoms, intensity, severity and
duration of the IBD in the subject to a significant degree compared
to untreated control subjects or control subjects administered with
control Tregs. (Such control Treg cells are ones not transduced to
express the present CR's, or those redirected to be specific to
antigens or ligands not present at the site of the IBD.)
Introduction of LPS-related molecules or epitopes that bind to
these same extracellular receptors on the redirected Treg cells to
the sites of administration (and/or expected action) of the
redirected Treg cells further facilitates their therapeutic
activity.
[0325] The references cited above are all incorporated by reference
herein, whether specifically incorporated or not.
[0326] Having now fully described this invention, it will be
appreciated by those skilled in the art that the same can be
performed within a wide range of equivalent parameters,
concentrations, and conditions without departing from the spirit
and scope of the invention and without undue experimentation.
Sequence CWU 1
1
3311176DNAArtificial Sequencesynthetic 1tctagactgc c atg gat ttt
cag gtg cag att ttc agc ttc ctg cta atc 50 Met Asp Phe Gln Val Gln
Ile Phe Ser Phe Leu Leu Ile 1 5 10agt gcc tca gtc ata atg tct aga
gga gat att gtg atg acc cag tct 98Ser Ala Ser Val Ile Met Ser Arg
Gly Asp Ile Val Met Thr Gln Ser 15 20 25cca aaa ttc atg tcc aca tca
gta gga ggc agg gtc agc atc acc tgc 146Pro Lys Phe Met Ser Thr Ser
Val Gly Gly Arg Val Ser Ile Thr Cys30 35 40 45aag gcc agt cag aat
gtg ggt act gct gta gcc tgg tat caa cag aaa 194Lys Ala Ser Gln Asn
Val Gly Thr Ala Val Ala Trp Tyr Gln Gln Lys 50 55 60cca gga caa tct
cct aaa cta ctg att tac tcg gca tcc aat cgg tac 242Pro Gly Gln Ser
Pro Lys Leu Leu Ile Tyr Ser Ala Ser Asn Arg Tyr 65 70 75act gga gtc
cct gat cgc ttc aca ggc agt gga tct ggg aca gat ttc 290Thr Gly Val
Pro Asp Arg Phe Thr Gly Ser Gly Ser Gly Thr Asp Phe 80 85 90act ctc
acc atc agc aat atg cag tct gaa gac ctg gca gat tat ttc 338Thr Leu
Thr Ile Ser Asn Met Gln Ser Glu Asp Leu Ala Asp Tyr Phe 95 100
105tgc cag caa tat agc agc tat cct ctc acg ttc ggt gct ggc acc aag
386Cys Gln Gln Tyr Ser Ser Tyr Pro Leu Thr Phe Gly Ala Gly Thr
Lys110 115 120 125ctg gaa atw aaa ggg tcg act tcc ggt agc ggc aaa
tcc tct gaa ggc 434Leu Glu Xaa Lys Gly Ser Thr Ser Gly Ser Gly Lys
Ser Ser Glu Gly 130 135 140aaa ggt cag gtc cag ctg cag cag tct gga
cct gag ctg gtg aag cct 482Lys Gly Gln Val Gln Leu Gln Gln Ser Gly
Pro Glu Leu Val Lys Pro 145 150 155ggg gct tca gtg agg ata tcc tgc
aag gct tct ggc tac acc ttc aca 530Gly Ala Ser Val Arg Ile Ser Cys
Lys Ala Ser Gly Tyr Thr Phe Thr 160 165 170agc tac tat ata cac tgg
gtg aag cag agg cct gga cag gga ctt gag 578Ser Tyr Tyr Ile His Trp
Val Lys Gln Arg Pro Gly Gln Gly Leu Glu 175 180 185tgg att gga tgg
att tat cct gga aat gtt aat act aag tac aat gag 626Trp Ile Gly Trp
Ile Tyr Pro Gly Asn Val Asn Thr Lys Tyr Asn Glu190 195 200 205aag
ttc aag ggc aag gcc aca ctg act gca gac aaa tcc tcc agc aca 674Lys
Phe Lys Gly Lys Ala Thr Leu Thr Ala Asp Lys Ser Ser Ser Thr 210 215
220gcc tac atg cag ctc agc agc ctg acc tct gag gac tct gcg gtc tat
722Ala Tyr Met Gln Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr
225 230 235ttc tgt gca aga aac tac ggt agt agc tac ggg ctt gct tac
tgg ggc 770Phe Cys Ala Arg Asn Tyr Gly Ser Ser Tyr Gly Leu Ala Tyr
Trp Gly 240 245 250caa gga act acg gtc acc gtg aaa ggg aaa cac ctt
tgt cca agt ccc 818Gln Gly Thr Thr Val Thr Val Lys Gly Lys His Leu
Cys Pro Ser Pro 255 260 265cta ttt ccc gga cct tct aag ccc ttt tgg
gtg ctg gtg gtg gtt ggt 866Leu Phe Pro Gly Pro Ser Lys Pro Phe Trp
Val Leu Val Val Val Gly270 275 280 285gga gtc ctg gct tgc tat agc
ttg cta gta aca gtg gcc ttt att att 914Gly Val Leu Ala Cys Tyr Ser
Leu Leu Val Thr Val Ala Phe Ile Ile 290 295 300ttc tgg gtg agg agt
aag agg agc agg ctc ctg cac agt gac tac atg 962Phe Trp Val Arg Ser
Lys Arg Ser Arg Leu Leu His Ser Asp Tyr Met 305 310 315aac atg act
ccc cgc cgc ccc ggg ccc acc cgc aag cat tac cag ccc 1010Asn Met Thr
Pro Arg Arg Pro Gly Pro Thr Arg Lys His Tyr Gln Pro 320 325 330tat
gcc cca cca cgc gac ttc gca gcc tat aga tct caa gtg cga aag 1058Tyr
Ala Pro Pro Arg Asp Phe Ala Ala Tyr Arg Ser Gln Val Arg Lys 335 340
345gca gct ata acc agc tat gag aaa tca gat ggt gtt tac acg ggc ctg
1106Ala Ala Ile Thr Ser Tyr Glu Lys Ser Asp Gly Val Tyr Thr Gly
Leu350 355 360 365agc acc agg aac cag gag act tac gag act ctg aag
cat gag aaa cca 1154Ser Thr Arg Asn Gln Glu Thr Tyr Glu Thr Leu Lys
His Glu Lys Pro 370 375 380cca cag tag ctttagactc gag 1176Pro
Gln2383PRTArtificial SequenceSynthetic Construct 2Met Asp Phe Gln
Val Gln Ile Phe Ser Phe Leu Leu Ile Ser Ala Ser1 5 10 15Val Ile Met
Ser Arg Gly Asp Ile Val Met Thr Gln Ser Pro Lys Phe 20 25 30Met Ser
Thr Ser Val Gly Gly Arg Val Ser Ile Thr Cys Lys Ala Ser 35 40 45Gln
Asn Val Gly Thr Ala Val Ala Trp Tyr Gln Gln Lys Pro Gly Gln 50 55
60Ser Pro Lys Leu Leu Ile Tyr Ser Ala Ser Asn Arg Tyr Thr Gly Val65
70 75 80Pro Asp Arg Phe Thr Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu
Thr 85 90 95Ile Ser Asn Met Gln Ser Glu Asp Leu Ala Asp Tyr Phe Cys
Gln Gln 100 105 110Tyr Ser Ser Tyr Pro Leu Thr Phe Gly Ala Gly Thr
Lys Leu Glu Ile 115 120 125Lys Gly Ser Thr Ser Gly Ser Gly Lys Ser
Ser Glu Gly Lys Gly Gln 130 135 140Val Gln Leu Gln Gln Ser Gly Pro
Glu Leu Val Lys Pro Gly Ala Ser145 150 155 160Val Arg Ile Ser Cys
Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr Tyr 165 170 175Ile His Trp
Val Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile Gly 180 185 190Trp
Ile Tyr Pro Gly Asn Val Asn Thr Lys Tyr Asn Glu Lys Phe Lys 195 200
205Gly Lys Ala Thr Leu Thr Ala Asp Lys Ser Ser Ser Thr Ala Tyr Met
210 215 220Gln Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Phe
Cys Ala225 230 235 240Arg Asn Tyr Gly Ser Ser Tyr Gly Leu Ala Tyr
Trp Gly Gln Gly Thr 245 250 255Thr Val Thr Val Lys Gly Lys His Leu
Cys Pro Ser Pro Leu Phe Pro 260 265 270Gly Pro Ser Lys Pro Phe Trp
Val Leu Val Val Val Gly Gly Val Leu 275 280 285Ala Cys Tyr Ser Leu
Leu Val Thr Val Ala Phe Ile Ile Phe Trp Val 290 295 300Arg Ser Lys
Arg Ser Arg Leu Leu His Ser Asp Tyr Met Asn Met Thr305 310 315
320Pro Arg Arg Pro Gly Pro Thr Arg Lys His Tyr Gln Pro Tyr Ala Pro
325 330 335Pro Arg Asp Phe Ala Ala Tyr Arg Ser Gln Val Arg Lys Ala
Ala Ile 340 345 350Thr Ser Tyr Glu Lys Ser Asp Gly Val Tyr Thr Gly
Leu Ser Thr Arg 355 360 365Asn Gln Glu Thr Tyr Glu Thr Leu Lys His
Glu Lys Pro Pro Gln 370 375 38037881DNAArtificial Sequencesynthetic
3gattgactga gtcgcccggg tacccgtgta tccaataaac cctcttgcag ttgcatccga
60cttgtggtct cgctgttcct tgggagggtc tcctctgagt gattgactac ccgtcagcgg
120gggtctttca tttgggggct cgtccgggat cgggagaccc ctgcccaggg
accaccgacc 180caccaccggg aggtaagctg gccagcaact tatctgtgtc
tgtccgattg tctagtgtct 240atgactgatt ttatgcgcct gcgtcggtac
tagttagcta actagctctg tatctggcgg 300acccgtggtg gaactgacga
gttcggaaca cccggccgca accctgggag acgtcccagg 360gacttcgggg
gccgtttttg tggcccgacc tgagtcctaa aatcccgatc gtttaggact
420ctttggtgca ccccccttag aggagggata tgtggttctg gtaggagacg
agaacctaaa 480acagttcccg cctccgtctg aatttttgct ttcggtttgg
gaccgaagcc gcgccgcgcg 540tcttgtctgc tgcagcatcg ttctgtgttg
tctctgtctg actgtgtttc tgtatttgtc 600tgaaaatatg ggcccgggct
agcctgttac cactccctta agtttgacct taggtcactg 660gaaagatgtc
gagcggatcg ctcacaacca gtcggtagat gtcaagaaga gacgttgggt
720taccttctgc tctgcagaat ggccaacctt taacgtcgga tggccgcgag
acggcacctt 780taaccgagac ctcatcaccc aggttaagat caaggtcttt
tcacctggcc cgcatggaca 840cccagaccag gtcccctaca tcgtgacctg
ggaagccttg gcttttgacc cccctccctg 900ggtcaagccc tttgtacacc
ctaagcctcc gcctcctctt cctccatccg ccccgtctct 960cccccttgaa
cctcctcgtt cgaccccgcc tcgatcctcc ctttatccag ccctcactcc
1020ttctctaggc gcccccatat ggccatatga gatcttatat ggggcacccc
cgccccttgt 1080aaacttccct gaccctgaca tgacaagagt tactaacagc
ccctctctcc aagctcactt 1140acaggctctc tacttagtcc agcacgaagt
ctggagacct ctggcggcag cctaccaaga 1200acaactggac cgaccggtgg
tacctcaccc ttaccgagtc ggcgacacag tgtgggtccg 1260ccgacaccag
actaagaacc tagaacctcg ctggaaagga ccttacacag tcctgctgac
1320cacccccacc gccctcaaag tagacggcat cgcagcttgg atacacgccg
cccacgtgaa 1380ggctgccgac cccgggggtg gaccatcctc tagactgcc atg gat
ttt cag gtg 1434 Met Asp Phe Gln Val 1 5cag att ttc agc ttc ctg cta
atc agt gcc tca gtc ata atg tct aga 1482Gln Ile Phe Ser Phe Leu Leu
Ile Ser Ala Ser Val Ile Met Ser Arg 10 15 20gga gat att gtg ctc aca
cag tct cca tcc tcc ctg gct gtg tca gca 1530Gly Asp Ile Val Leu Thr
Gln Ser Pro Ser Ser Leu Ala Val Ser Ala 25 30 35gga gag aag gtc act
atg agc tgc aaa tcc agt cag agt ctg ctc aac 1578Gly Glu Lys Val Thr
Met Ser Cys Lys Ser Ser Gln Ser Leu Leu Asn 40 45 50agt aga acc cga
aag aac tac ttg gct tgg tac cag cag aaa cca ggg 1626Ser Arg Thr Arg
Lys Asn Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly 55 60 65cag tct cct
aaa ctg ctg atc tac tgg gca tcc act agg gaa tct ggg 1674Gln Ser Pro
Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg Glu Ser Gly70 75 80 85gtc
cct gat cgc ttc aca ggc agt gga tct ggg aca gat ttc act ctc 1722Val
Pro Asp Arg Phe Thr Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu 90 95
100acc atc agc agt gtg cag gct gaa gac ctg gca gtt tat tac tgc aag
1770Thr Ile Ser Ser Val Gln Ala Glu Asp Leu Ala Val Tyr Tyr Cys Lys
105 110 115caa tct tat aat ctg tac acg ttc gga ggg ggg acc aag ctg
gaa ata 1818Gln Ser Tyr Asn Leu Tyr Thr Phe Gly Gly Gly Thr Lys Leu
Glu Ile 120 125 130aaa ggg tcg act tcc ggt agc ggc aaa tcc tct gaa
ggc aaa ggt gag 1866Lys Gly Ser Thr Ser Gly Ser Gly Lys Ser Ser Glu
Gly Lys Gly Glu 135 140 145gtc cag ctg cag cag tct gga ggt ggc ctg
gtg cag cct gga gga tcc 1914Val Gln Leu Gln Gln Ser Gly Gly Gly Leu
Val Gln Pro Gly Gly Ser150 155 160 165ctg aaa ctc tcc tgt gca gcc
tca gga ttc gat ttt agt aga tac tgg 1962Leu Lys Leu Ser Cys Ala Ala
Ser Gly Phe Asp Phe Ser Arg Tyr Trp 170 175 180atg agt tgg gtc cgg
cag gct cca ggg aaa ggg cta gaa tgg att gga 2010Met Ser Trp Val Arg
Gln Ala Pro Gly Lys Gly Leu Glu Trp Ile Gly 185 190 195gaa att aat
cca gat agc agt acg ata aac tat acg cca tct cta aag 2058Glu Ile Asn
Pro Asp Ser Ser Thr Ile Asn Tyr Thr Pro Ser Leu Lys 200 205 210gat
aaa ttc atc atc tcc aga gac aac gcc aaa aat acg ctg tac ctg 2106Asp
Lys Phe Ile Ile Ser Arg Asp Asn Ala Lys Asn Thr Leu Tyr Leu 215 220
225caa atg agc aaa gtg aga tct gag gac aca gcc ctt tat tac tgt gca
2154Gln Met Ser Lys Val Arg Ser Glu Asp Thr Ala Leu Tyr Tyr Cys
Ala230 235 240 245aga cgt tat ggt aac tac tgg tac ttc gat gtc tgg
ggc gca ggg acc 2202Arg Arg Tyr Gly Asn Tyr Trp Tyr Phe Asp Val Trp
Gly Ala Gly Thr 250 255 260acg gtc acc gtg aaa ggg aaa cac ctt tgt
cca agt ccc cta ttt ccc 2250Thr Val Thr Val Lys Gly Lys His Leu Cys
Pro Ser Pro Leu Phe Pro 265 270 275gga cct tct aag ccc ttt tgg gtg
ctg gtg gtg gtt ggt gga gtc ctg 2298Gly Pro Ser Lys Pro Phe Trp Val
Leu Val Val Val Gly Gly Val Leu 280 285 290gct tgc tat agc ttg cta
gta aca gtg gcc ttt att att ttc tgg gtg 2346Ala Cys Tyr Ser Leu Leu
Val Thr Val Ala Phe Ile Ile Phe Trp Val 295 300 305agg agt aag agg
agc agg ctc ctg cac agt gac tac atg aac atg act 2394Arg Ser Lys Arg
Ser Arg Leu Leu His Ser Asp Tyr Met Asn Met Thr310 315 320 325ccc
cgc cgc ccc ggg ccc acc cgc aag cat tac cag ccc tat gcc cca 2442Pro
Arg Arg Pro Gly Pro Thr Arg Lys His Tyr Gln Pro Tyr Ala Pro 330 335
340cca cgc gac ttc gca gcc tat aga tct caa gtg cga aag gca gct ata
2490Pro Arg Asp Phe Ala Ala Tyr Arg Ser Gln Val Arg Lys Ala Ala Ile
345 350 355acc agc tat gag aaa tca gat ggt gtt tac acg ggc ctg agc
acc agg 2538Thr Ser Tyr Glu Lys Ser Asp Gly Val Tyr Thr Gly Leu Ser
Thr Arg 360 365 370aac cag gag act tac gag act ctg aag cat gag aaa
cca cca cag tag 2586Asn Gln Glu Thr Tyr Glu Thr Leu Lys His Glu Lys
Pro Pro Gln 375 380 385ctttagactc gagcgggatc aattccgccc cccccctaac
gttactggcc gaagccgctt 2646ggaataaggc cggtgtgcgt ttgtctatat
gttattttcc accatattgc cgtcttttgg 2706caatgtgagg gcccggaaac
ctggccctgt cttcttgacg agcattccta ggggtctttc 2766ccctctcgcc
aaaggaatgc aaggtctgtt gaatgtcgtg aaggaagcag ttcctctgga
2826agcttcttga agacaaacaa cgtctgtagc gaccctttgc aggcagcgga
accccccacc 2886tggcgacagg tgcctctgcg gccaaaagcc acgtgtataa
gatacacctg caaaggcggc 2946acaaccccag tgccacgttg tgagttggat
agttgtggaa agagtcaaat ggctctcctc 3006aagcgtattc aacaaggggc
tgaaggatgc ccagaaggta ccccattgta tgggatctga 3066tctggggcct
cggtgcacat gctttacatg tgtttagtcg aggttaaaaa aacgtctagg
3126ccccccgaac cacggggacg tggttttcct ttgaaaaaca cgataatagc
atgctgagca 3186agggcgagga gctgttcacc ggggtggtgc ccatcctggt
cgagctggac ggcgacgtaa 3246acggccacaa gttcagcgtg tccggcgagg
gcgagggcga tgccacctac ggcaagctga 3306ccctgaagtt catctgcacc
accggcaagc tgcccgtgcc ctggcccacc ctcgtgacca 3366ccttcgccta
cggcctgcag tgcttcgccc gctaccccga ccacatgaag cagcacgact
3426tcttcaagtc cgccatgccc gaaggctacg tccaggagcg caccatcttc
ttcaaggacg 3486acggcaacta caagacccgc gccgaggtga agttcgaggg
cgacaccctg gtgaaccgca 3546tcgagctgaa gggcatcgac ttcaaggagg
acggcaacat cctggggcac aagctggagt 3606acaactacaa cagccacaac
gtctatatca tggccgacaa gcagaagaac ggcatcaagg 3666tgaacttcaa
gatccgccac aacatcgagg acggcagcgt gcagctcgcc gaccactacc
3726agcagaacac ccccatcggc gacggccccg tgctgctgcc cgacaaccac
tacctgagca 3786cccagtccgc cctgagcaaa gaccccaacg agaagcgcga
tcacatggtc ctgctggagt 3846tcgtgaccgc cgccgggatc actctcggca
tggacgagct gtacaagtga gtgtaaactc 3906gaggatccgc gccgctcgcg
actcgagaga tccggattag tccaatttgt taaagacagg 3966atatcagtgg
tccaggctct agttttgact caacaatatc accagctgaa gcctatagag
4026tacgagccat agataaaata aaagatttta tttagtctcc agaaaaaggg
gggaatgaaa 4086gaccccacct gtaggtttgg caagctagct taagtaacgc
cattttgcag gcatggaaaa 4146atacataact gagaatagag aagttcagat
caaggtcagg aacagatgga acagctgaat 4206atgggccaaa caggatatct
gtggtaagca gttcctgccc cggctcaggg ccaagaacag 4266atggaacagc
tgaatatggg ccaaacagga tatctgtggt aagcagttcc tgccccggct
4326cagggccaag aacagatggt ccccagatgc ggtccagccc tcagcagttt
ctagagaacc 4386atcagatgtt tccagggtgc cccaaggacc tgaaatgacc
ctgtgcctta tttgaactaa 4446ccaatcagtt cgcttctcgc ttctgttcgc
gcgcttctgc tccccgagct caataaaaga 4506gcccacaacc cctcactcgg
ggcgccagtc ctccgattga ctgagtcgcc cgggtacccg 4566tgtatccaat
aaaccctctt gcagttgcat ccgacttgtg gtctcgctgt tccttgggag
4626ggtctcctct gagtgattga ctacccgtca gcgggggtct ttcacatgca
gcatgtatca 4686aaattaattt ggtttttttt cttaagtatt tacattaaat
ggccatagtc tgctcgatcg 4746aggagctttt tgcaaaagcc taggcctcca
aaaaagcctc ttcactactt ctggaatagc 4806tcagaggccg aggcggcctc
ggcctctgca taaataaaaa aaattagtca gccatgcatg 4866gtaatagcga
tgactaatac gtagatgtac tgccaagtag gaaagtccca taaggtcatg
4926tactgggcat aatgccaggc gggccattta ccgtcattga cgtcaatagg
gggcgtactt 4986ggcatatgat acacttgatg tactgccaag tgggcagttt
accgtaaata ctccacccat 5046tgacgtcaat ggaaagtccc tattggcgtt
actatgggaa catacgtcat tattgacgtc 5106aatgggcggg ggtcgttggg
cggtcagcca ggcgggccat ttaccgtaag ttatgtaacg 5166gactctagcc
catcgatggg aattccggtc tccctatagt gagtcgtatt aatttcgata
5226agccagacca ttccttgcgg cggcggtgct caacggcctc aacctactac
tgggctgctt 5286cctaatgcag gagtcgcata agggagagcg tcgaatggtg
cactctcagt acaatctgct 5346ctgatgccgc atagttaagc cagccccgac
acccgccaac acccgctgac gcgccctgac 5406gggcttgtct gctcccggca
tccgcttaca gacaagctgt gaccgtctcc gggagctgca 5466tgtgtcagag
gttttcaccg tcatcaccga aacgcgcgag acgaaagggc ctcgtgatac
5526gcctattttt ataggttaat gtcatgataa taatggtttc ttagacgtca
ggtggcactt 5586ttcggggaaa tgtgcgcgga acccctattt gtttattttt
ctaaatacat tcaaatatgt 5646atccgctcat gagacaataa ccctgataaa
tgcttcaata atattgaaaa aggaagagta 5706tgagtattca acatttccgt
gtcgccctta ttcccttttt tgcggcattt tgccttcctg 5766tttttgctca
cccagaaacg ctggtgaaag taaaagatgc tgaagatcag ttgggtgcac
5826gagtgggtta catcgaactg gatctcaaca gcggtaagat ccttgagagt
tttcgccccg 5886aagaacgttt tccaatgatg agcactttta aagttctgct
atgtggcgcg gtattatccc 5946gtattgacgc cgggcaagag caactcggtc
gccgcataca ctattctcag aatgacttgg 6006ttgagtactc accagtcaca
gaaaagcatc ttacggatgg catgacagta agagaattat 6066gcagtgctgc
cataaccatg agtgataaca ctgcggccaa cttacttctg acaacgatcg
6126gaggaccgaa ggagctaacc gcttttttgc acaacatggg ggatcatgta
actcgccttg 6186atcgttggga accggagctg aatgaagcca taccaaacga
cgagcgtgac accacgatgc 6246ctgtagcaat ggcaacaacg ttgcgcaaac
tattaactgg cgaactactt actctagctt 6306cccggcaaca attaatagac
tggatggagg cggataaagt tgcaggacca cttctgcgct 6366cggcccttcc
ggctggctgg tttattgctg ataaatctgg agccggtgag cgtgggtctc
6426gcggtatcat tgcagcactg gggccagatg gtaagccctc ccgtatcgta
gttatctaca 6486cgacggggag tcaggcaact atggatgaac gaaatagaca
gatcgctgag ataggtgcct 6546cactgattaa gcattggtaa ctgtcagacc
aagtttactc atatatactt tagattgatt 6606taaaacttca tttttaattt
aaaaggatct aggtgaagat cctttttgat aatctcatga 6666ccaaaatccc
ttaacgtgag ttttcgttcc actgagcgtc agaccccgga gaaaagatca
6726aaggatcttc ttgagatcct ttttttctgc gcgtaatctg ctgcttgcaa
acaaaaaaac 6786caccgctacc agcggtggtt tgtttgccgg atcaagagct
accaactctt tttccgaagg 6846taactggctt cagcagagcg cagataccaa
atactgttct tctagtgtag ccgtagttag 6906gccaccactt caagaactct
gtagcaccgc ctacatacct cgctctgcta atcctgttac 6966cagtggctgc
tgccagtggc gataagtcgt gtcttaccgg gttggactca agacgatagt
7026taccggataa ggcgcagcgg tcgggctgaa cggggggttc gtgcacacag
cccagcttgg 7086agcgaacgac ctacaccgaa ctgagatacc tacagcgtga
gctatgagaa agcgccacgc 7146ttcccgaagg gagaaaggcg gacaggtatc
cggtaagcgg cagggtcgga acaggagagc 7206gcacgaggga gcttccaggg
ggaaacgcct ggtatcttta tagtcctgtc gggtttcgcc 7266acctctgact
tgagcgtcga tttttgtgat gctcgtcagg ggggcggagc ctatggaaaa
7326acgccagcaa cgcggccttt ttacggttcc tggccttttg ctggcctttt
gctcacatgt 7386tctttcctgc gttatcccct gattctgtgg ataaccgtat
taccgccttt gagtgagctg 7446ataccgctcg ccgcagccga acgaccgagc
gcagcgagtc agtgagcgag gaagcggaag 7506agcgcccaat acgcaaaccg
cctctccccg cgcgttggcc gattcattaa tgcagcaatt 7566agtcagcaac
catagtcccg cccctaactc cgcccatccc gcccctaact ccgcccagtt
7626ccgcccattc tccgccccat gcatggtgat gcggttttgg cagtacatca
atgggcgtgg 7686atagcggttt gactcacggg gatttccaag tctccacccc
attgacgtca atgggagttt 7746gttttggcac caaaatcaac gggactttcc
aaaatgtcgt aacaactccg ccccattgac 7806gcaaatgggc ggtaggcgtg
tacggtggga ggtctatata agcagagctc gtttagtgaa 7866ccgcgccagt cctcc
78814375PRTHomo sapiensCD14 protein 4Met Glu Arg Ala Ser Cys Leu
Leu Leu Leu Leu Leu Pro Leu Val His1 5 10 15Val Ser Ala Thr Thr Pro
Glu Pro Cys Glu Leu Asp Asp Glu Asp Phe 20 25 30Arg Cys Val Cys Asn
Phe Ser Glu Pro Gln Pro Asp Trp Ser Glu Ala 35 40 45Phe Gln Cys Val
Ser Ala Val Glu Val Glu Ile His Ala Gly Gly Leu 50 55 60Asn Leu Glu
Pro Phe Leu Lys Arg Val Asp Ala Asp Ala Asp Pro Arg65 70 75 80Gln
Tyr Ala Asp Thr Val Lys Ala Leu Arg Val Arg Arg Leu Thr Val 85 90
95Gly Ala Ala Gln Val Pro Ala Gln Leu Leu Val Gly Ala Leu Arg Val
100 105 110Leu Ala Tyr Ser Arg Leu Lys Glu Leu Thr Leu Glu Asp Leu
Lys Ile 115 120 125Thr Gly Thr Met Pro Pro Leu Pro Leu Glu Ala Thr
Gly Leu Ala Leu 130 135 140Ser Ser Leu Arg Leu Arg Asn Val Ser Trp
Ala Thr Gly Arg Ser Trp145 150 155 160Leu Ala Glu Leu Gln Gln Trp
Leu Lys Pro Gly Leu Lys Val Leu Ser 165 170 175Ile Ala Gln Ala His
Ser Pro Ala Phe Ser Cys Glu Gln Val Arg Ala 180 185 190Phe Pro Ala
Leu Thr Ser Leu Asp Leu Ser Asp Asn Pro Gly Leu Gly 195 200 205Glu
Arg Gly Leu Met Ala Ala Leu Cys Pro His Lys Phe Pro Ala Ile 210 215
220Gln Asn Leu Ala Leu Arg Asn Thr Gly Met Glu Thr Pro Thr Gly
Val225 230 235 240Cys Ala Ala Leu Ala Ala Ala Gly Val Gln Pro His
Ser Leu Asp Leu 245 250 255Ser His Asn Ser Leu Arg Ala Thr Val Asn
Pro Ser Ala Pro Arg Cys 260 265 270Met Trp Ser Ser Ala Leu Asn Ser
Leu Asn Leu Ser Phe Ala Gly Leu 275 280 285Glu Gln Val Pro Lys Gly
Leu Pro Ala Lys Leu Arg Val Leu Asp Leu 290 295 300Ser Cys Asn Arg
Leu Asn Arg Ala Pro Gln Pro Asp Glu Leu Pro Glu305 310 315 320Val
Asp Asn Leu Thr Leu Asp Gly Asn Pro Phe Leu Val Pro Gly Thr 325 330
335Ala Leu Pro His Glu Gly Ser Met Asn Ser Gly Val Val Pro Ala Cys
340 345 350Ala Arg Ser Thr Leu Ser Val Gly Val Ser Gly Thr Leu Val
Leu Leu 355 360 365Gln Gly Ala Arg Gly Phe Ala 370 3755159PRTHomo
sapiensMD-2 protein 5Met Leu Pro Phe Leu Phe Phe Ser Thr Leu Phe
Ser Ser Ile Phe Thr1 5 10 15Glu Ala Gln Lys Gln Tyr Trp Val Cys Asn
Ser Ser Asp Ala Ser Ile 20 25 30Ser Tyr Thr Tyr Cys Asp Lys Met Gln
Tyr Pro Ile Ser Ile Asn Val 35 40 45Asn Pro Cys Ile Glu Leu Lys Gly
Ser Lys Gly Leu Leu His Ile Phe 50 55 60Tyr Ile Pro Arg Arg Asp Leu
Lys Gln Leu Tyr Phe Asn Leu Tyr Ile65 70 75 80Thr Val Asn Thr Met
Asn Leu Pro Lys Arg Lys Glu Val Ile Cys Arg 85 90 95Gly Ser Asp Asp
Asp Tyr Ser Phe Cys Arg Ala Leu Lys Gly Glu Thr 100 105 110Val Asn
Thr Thr Ile Ser Phe Ser Phe Lys Gly Ile Lys Phe Ser Lys 115 120
125Gly Lys Tyr Lys Val Val Glu Ala Ile Ser Gly Ser Pro Glu Glu Met
130 135 140Leu Phe Cys Leu Glu Phe Val Ile Leu His Gln Pro Asn Ser
Asn145 150 1556504DNAArtificial Sequencesynthetic 6atg gat ttt cag
gtg cag att ttc agc ttc ctg cta atc agt gcc tca 48Met Asp Phe Gln
Val Gln Ile Phe Ser Phe Leu Leu Ile Ser Ala Ser1 5 10 15gtc ata atg
tct aga cag gtt cct gct cag cta ctg gta ggc gcc ctg 96Val Ile Met
Ser Arg Gln Val Pro Ala Gln Leu Leu Val Gly Ala Leu 20 25 30cgt gtg
cta gcg tac tcc cgc ctc aag gtc acc gtg aaa ggg aaa cac 144Arg Val
Leu Ala Tyr Ser Arg Leu Lys Val Thr Val Lys Gly Lys His 35 40 45ctt
tgt cca agt ccc cta ttt ccc gga cct tct aag ccc ttt tgg gtg 192Leu
Cys Pro Ser Pro Leu Phe Pro Gly Pro Ser Lys Pro Phe Trp Val 50 55
60ctg gtg gtg gtt ggt gga gtc ctg gct tgc tat agc ttg cta gta aca
240Leu Val Val Val Gly Gly Val Leu Ala Cys Tyr Ser Leu Leu Val
Thr65 70 75 80gtg gcc ttt att att ttc tgg gtg agg agt aag agg agc
agg ctc ctg 288Val Ala Phe Ile Ile Phe Trp Val Arg Ser Lys Arg Ser
Arg Leu Leu 85 90 95cac agt gac tac atg aac atg act ccc cgc cgc ccc
ggg ccc acc cgc 336His Ser Asp Tyr Met Asn Met Thr Pro Arg Arg Pro
Gly Pro Thr Arg 100 105 110aag cat tac cag ccc tat gcc cca cca cgc
gac ttc gca gcc tat aga 384Lys His Tyr Gln Pro Tyr Ala Pro Pro Arg
Asp Phe Ala Ala Tyr Arg 115 120 125tct caa gtg cga aag gca gct ata
acc agc tat gag aaa tca gat ggt 432Ser Gln Val Arg Lys Ala Ala Ile
Thr Ser Tyr Glu Lys Ser Asp Gly 130 135 140gtt tac acg ggc ctg agc
acc agg aac cag gag act tac gag act ctg 480Val Tyr Thr Gly Leu Ser
Thr Arg Asn Gln Glu Thr Tyr Glu Thr Leu145 150 155 160aag cat gag
aaa cca cca cag tag 504Lys His Glu Lys Pro Pro Gln
16573984DNAArtificial Sequencesynthetic 7atggattttc aggtgcagat
tttcagcttc ctgctaatca gtgcctcagt cataatgtct 60agacaggttc ctgctcagct
actggtaggc gccctgcgtg tgctagcgta ctcccgcctc 120aaggtcaccg
tgaaagggaa acacctttgt ccaagtcccc tatttcccgg accttctaag
180cccttttggg tgctggtggt ggttggtgga gtcctggctt gctatagctt
gctagtaaca 240gtggccttta ttattttctg ggtgaggagt aagaggagca
ggctcctgca cagtgactac 300atgaacatga ctccccgccg ccccgggccc
acccgcaagc attaccagcc ctatgcccca 360ccacgcgact tcgcagccta
tagatctcaa gtgcgaaagg cagctataac cagctatgag 420aaatcagatg
gtgtttacac gggcctgagc accaggaacc aggagactta cgagactctg
480aagcatgaga aaccaccaca gtagctttag actcgagcgg gatcaattcc
gccccccccc 540taacgttact ggccgaagcc gcttggaata aggccggtgt
gcgtttgtct atatgttatt 600ttccaccata ttgccgtctt ttggcaatgt
gagggcccgg aaacctggcc ctgtcttctt 660gacgagcatt cctaggggtc
tttcccctct cgccaaagga atgcaaggtc tgttgaatgt 720cgtgaaggaa
gcagttcctc tggaagcttc ttgaagacaa acaacgtctg tagcgaccct
780ttgcaggcag cggaaccccc cacctggcga caggtgcctc tgcggccaaa
agccacgtgt 840ataagataca cctgcaaagg cggcacaacc ccagtgccac
gttgtgagtt ggatagttgt 900ggaaagagtc aaatggctct cctcaagcgt
attcaacaag gggctgaagg atgcccagaa 960ggtaccccat tgtatgggat
ctgatctggg gcctcggtgc acatgcttta catgtgttta 1020gtcgaggtta
aaaaaacgtc taggcccccc gaaccacggg gacgtggttt tcctttgaaa
1080aacacgataa tagcatgcag cgctaccggt cgccaccatg gtgagcaagg
gcgaggagct 1140gttcaccggg gtggtgccca tcctggtcga gctggacggc
gacgtaaacg gccacaagtt 1200cagcgtgtcc ggcgagggcg agggcgatgc
cacctacggc aagctgaccc tgaagttcat 1260ctgcaccacc ggcaagctgc
ccgtgccctg gcccaccctc gtgaccaccc tgacctacgg 1320cgtgcagtgc
ttcagccgct accccgacca catgaagcag cacgacttct tcaagtccgc
1380catgcccgaa ggctacgtcc aggagcgcac catcttcttc aaggacgacg
gcaactacaa 1440gacccgcgcc gaggtgaagt tcgagggcga caccctggtg
aaccgcatcg agctgaaggg 1500catcgacttc aaggaggacg gcaacatcct
ggggcacaag ctggagtaca actacaacag 1560ccacaacgtc tatatcatgg
ccgacaagca gaagaacggc atcaaggtga acttcaagat 1620ccgccacaac
atcgaggacg gcagcgtgca gctcgccgac cactaccagc agaacacccc
1680catcggcgac ggccccgtgc tgctgcccga caaccactac ctgagcaccc
agtccgccct 1740gagcaaagac cccaacgaga agcgcgatca catggtcctg
ctggagttcg tgaccgccgc 1800cgggatcact ctcggcatgg acgagctgta
caagtccggc cggactcaga tctcgagctc 1860aagcttcgaa ttcatgccca
accccaggcc tggcaagccc tcggcccctt ccttggccct 1920tggcccatcc
ccaggagcct cgcccagctg gagggctgca cccaaagcct cagacctgct
1980gggggcccgg ggcccagggg gaaccttcca gggccgagat cttcgaggcg
gggcccatgc 2040ctcctcttct tccttgaacc ccatgccacc atcgcagctg
cagctgccca cactgcccct 2100agtcatggtg gcaccctccg gggcacggct
gggccccttg ccccacttac aggcactcct 2160ccaggacagg ccacatttca
tgcaccagct ctcaacggtg gatgcccacg cccggacccc 2220tgtgctgcag
gtgcaccccc tggagagccc agccatgatc agcctcacac cacccaccac
2280cgccactggg gtcttctccc tcaaggcccg gcctggcctc ccacctggga
tcaacgtggc 2340cagcctggaa tgggtgtcca gggagccggc actgctctgc
accttcccaa atcccagtgc 2400acccaggaag gacagcaccc tttcggctgt
gccccagagc tcctacccac tgctggcaaa 2460tggtgtctgc aagtggcccg
gatgtgagaa ggtcttcgaa gagccagagg acttcctcaa 2520gcactgccag
gcggaccatc ttctggatga gaagggcagg gcacaatgtc tcctccagag
2580agagatggta cagtctctgg agcagcagct ggtgctggag aaggagaagc
tgagtgccat 2640gcaggcccac ctggctggga aaatggcact gaccaaggct
tcatctgtgg catcatccga 2700caagggctcc tgctgcatcg tagctgctgg
cagccaaggc cctgtcgtcc cagcctggtc 2760tggcccccgg gaggcccctg
acagcctgtt tgctgtccgg aggcacctgt ggggtagcca 2820tggaaacagc
acattcccag agttcctcca caacatggac tacttcaagt tccacaacat
2880gcgaccccct ttcacctacg ccacgctcat ccgctgggcc atcctggagg
ctccagagaa 2940gcagcggaca ctcaatgaga tctaccactg gttcacacgc
atgtttgcct tcttcagaaa 3000ccatcctgcc acctggaaga acgccatccg
ccacaacctg agtctgcaca agtgctttgt 3060gcgggtggag agcgagaagg
gggctgtgtg gaccgtggat gagctggagt tccgcaagaa 3120acggagccag
aggcccagca ggtgttccaa ccctacacct ggcccctgag gatccgcgcc
3180gctcgcgctc gagagatccg gattagtcca atttgttaaa gacaggatat
cagtggtcca 3240ggctctagtt ttgactcaac aatatcacca gctgaagcct
atagagtacg agccatagat 3300aaaataaaag attttattta gtctccagaa
aaagggggga atgaaagacc ccacctgtag 3360gtttggcaag ctagcttaag
taacgccatt ttgcaggcat ggaaaaatac ataactgaga 3420atagagaagt
tcagatcaag gtcaggaaca gatggaacag ctgaatatgg gccaaacagg
3480atatctgtgg taagcagttc ctgccccggc tcagggccaa gaacagatgg
aacagctgaa 3540tatgggccaa acaggatatc tgtggtaagc agttcctgcc
ccggctcagg gccaagaaca 3600gatggtcccc agatgcggtc cagccctcag
cagtttctag agaaccatca gatgtttcca 3660gggtgcccca aggacctgaa
atgaccctgt gccttatttg aactaaccaa tcagttcgct 3720tctcgcttct
gttcgcgcgc ttctgctccc cgagctcaat aaaagagccc acaacccctc
3780actcggggcg ccagtcctcc gattgactga gtcgcccggg tacccgtgta
tccaataaac 3840cctcttgcag ttgcatccga cttgtggtct cgctgttcct
tgggagggtc tcctctgagt 3900gattgactac ccgtcagcgg gggtctttca
catgcagcat gtatcaaaat taatttggtt 3960ttttttctta agtatttaca ttaa
39848489DNAArtificial Sequencesynthetic 8atg gat ttt cag gtg cag
att ttc agc ttc ctg cta atc agt gcc tca 48Met Asp Phe Gln Val Gln
Ile Phe Ser Phe Leu Leu Ile Ser Ala Ser1 5 10 15gtc ata atg tct aga
ttc tcc ttc aag gga ata aaa ttt tct aag gga 96Val Ile Met Ser Arg
Phe Ser Phe Lys Gly Ile Lys Phe Ser Lys Gly 20 25 30aaa tac aaa ggt
cac ctc gtg aaa ggg aaa cac ctt tgt cca agt ccc 144Lys Tyr Lys Gly
His Leu Val Lys Gly Lys His Leu Cys Pro Ser Pro 35 40 45cta ttt ccc
gga cct tct aag ccc ttt tgg gtg ctg gtg gtg gtt ggt 192Leu Phe Pro
Gly Pro Ser Lys Pro Phe Trp Val Leu Val Val Val Gly 50 55 60gga gtc
ctg gct tgc tat agc ttg cta gta aca gtg gcc ttt att att 240Gly Val
Leu Ala Cys Tyr Ser Leu Leu Val Thr Val Ala Phe Ile Ile65 70 75
80ttc tgg gtg agg agt aag agg agc agg ctc ctg cac agt gac tac atg
288Phe Trp Val Arg Ser Lys Arg Ser Arg Leu Leu His Ser Asp Tyr Met
85 90 95aac atg act ccc cgc cgc ccc ggg ccc acc cgc aag cat tac cag
ccc 336Asn Met Thr Pro Arg Arg Pro Gly Pro Thr Arg Lys His Tyr Gln
Pro 100 105 110tat gcc cca cca cgc gac ttc gca gcc tat aga tct caa
gtg cga aag 384Tyr Ala Pro Pro Arg Asp Phe Ala Ala Tyr Arg Ser Gln
Val Arg Lys 115 120 125gca gct ata acc agc tat gag aaa tca gat ggt
gtt tac acg ggc ctg 432Ala Ala Ile Thr Ser Tyr Glu Lys Ser Asp Gly
Val Tyr Thr Gly Leu 130 135 140agc acc agg aac cag gag act tac gag
act ctg aag cat gag aaa cca 480Ser Thr Arg Asn Gln Glu Thr Tyr Glu
Thr Leu Lys His Glu Lys Pro145 150 155 160cca cag tag 489Pro
Gln93160DNAArtificial Sequencesynthetic 9atggattttc aggtgcagat
tttcagcttc ctgctaatca gtgcctcagt cataatgtct 60agattctcct tcaagggaat
aaaattttct aagggaaaat acaaaggtca cctcgtgaaa 120gggaaacacc
tttgtccaag tcccctattt cccggacctt ctaagccctt ttgggtgctg
180gtggtggttg gtggagtcct ggcttgctat agcttgctag taacagtggc
ctttattatt 240ttctgggtga ggagtaagag gagcaggctc ctgcacagtg
actacatgaa catgactccc 300cgccgccccg ggcccacccg caagcattac
cagccctatg ccccaccacg cgacttcgca 360gcctatagat ctcaagtgcg
aaaggcagct ataaccagct atgagaaatc agatggtgtt 420tacacgggcc
tgagcaccag gaaccaggag acttacgaga ctctgaagca tgagaaacca
480ccacagtagc tttagactcg agcgggatca attccgcccc ccccctaacg
ttactggccg 540aagccgcttg gaataaggcc ggtgtgcgtt tgtctatatg
ttattttcca ccatattgcc 600gtcttttggc aatgtgaggg cccggaaacc
tggccctgtc ttcttgacga gcattcctag 660gggtctttcc cctctcgcca
aaggaatgca aggtctgttg aatgtcgtga aggaagcagt 720tcctctggaa
gcttcttgaa gacaaacaac gtctgtagcg accctttgca ggcagcggaa
780ccccccacct ggcgacaggt gcctctgcgg ccaaaagcca cgtgtataag
atacacctgc 840aaaggcggca caaccccagt gccacgttgt gagttggata
gttgtggaaa gagtcaaatg 900gctctcctca agcgtattca acaaggggct
gaaggatgcc cagaaggtac cccattgtat 960gggatctgat ctggggcctc
ggtgcacatg ctttacatgt gtttagtcga ggttaaaaaa 1020acgtctaggc
cccccgaacc acggggacgt ggttttcctt tgaaaaacac gataatagca
1080tgcagcgcta ccggtcgcca ccatggtgag caagggcgag gagctgttca
ccggggtggt 1140gcccatcctg gtcgagctgg acggcgacgt aaacggccac
aagttcagcg tgtccggcga 1200gggcgagggc gatgccacct acggcaagct
gaccctgaag ttcatctgca ccaccggcaa 1260gctgcccgtg ccctggccca
ccctcgtgac caccctgacc tacggcgtgc agtgcttcag 1320ccgctacccc
gaccacatga agcagcacga cttcttcaag tccgccatgc ccgaaggcta
1380cgtccaggag cgcaccatct tcttcaagga cgacggcaac tacaagaccc
gcgccgaggt 1440gaagttcgag ggcgacaccc tggtgaaccg catcgagctg
aagggcatcg acttcaagga 1500ggacggcaac atcctggggc acaagctgga
gtacaactac aacagccaca acgtctatat 1560catggccgac aagcagaaga
acggcatcaa ggtgaacttc aagatccgcc acaacatcga 1620ggacggcagc
gtgcagctcg ccgaccacta ccagcagaac acccccatcg gcgacggccc
1680cgtgctgctg cccgacaacc actacctgag cacccagtcc gccctgagca
aagaccccaa 1740cgagaagcgc gatcacatgg tcctgctgga gttcgtgacc
gccgccggga tcactctcgg 1800catggacgag ctgtacaagt ccggccggac
tcagatctcg agctcaagct tcgaattcat 1860gcccaacccc aggcctggca
agccctcggc cccttccttg gcccttggcc catccccagg 1920agcctcgccc
agctggaggg ctgcacccaa agcctcagac ctgctggggg cccggggccc
1980agggggaacc ttccagggcc gagatcttcg aggcggggcc catgcctcct
cttcttcctt 2040gaaccccatg ccaccatcgc agctgcagct gcccacactg
cccctagtca tggtggcacc 2100ctccggggca cggctgggcc ccttgcccca
cttacaggca ctcctccagg acaggccaca 2160tttcatgcac cagctctcaa
cggtggatgc ccacgcccgg acccctgtgc tgcaggtgca 2220ccccctggag
agcccagcca tgatcagcct cacaccaccc accaccgcca ctggggtctt
2280ctccctcaag gcccggcctg gcctcccacc tgggatcaac gtggccagcc
tggaatgggt 2340gtccagggag ccggcactgc tctgcacctt cccaaatccc
agtgcaccca ggaaggacag 2400caccctttcg gctgtgcccc agagctccta
cccactgctg gcaaatggtg tctgcaagtg 2460gcccggatgt gagaaggtct
tcgaagagcc agaggacttc ctcaagcact
gccaggcgga 2520ccatcttctg gatgagaagg gcagggcaca atgtctcctc
cagagagaga tggtacagtc 2580tctggagcag cagctggtgc tggagaagga
gaagctgagt gccatgcagg cccacctggc 2640tgggaaaatg gcactgacca
aggcttcatc tgtggcatca tccgacaagg gctcctgctg 2700catcgtagct
gctggcagcc aaggccctgt cgtcccagcc tggtctggcc cccgggaggc
2760ccctgacagc ctgtttgctg tccggaggca cctgtggggt agccatggaa
acagcacatt 2820cccagagttc ctccacaaca tggactactt caagttccac
aacatgcgac cccctttcac 2880ctacgccacg ctcatccgct gggccatcct
ggaggctcca gagaagcagc ggacactcaa 2940tgagatctac cactggttca
cacgcatgtt tgccttcttc agaaaccatc ctgccacctg 3000gaagaacgcc
atccgccaca acctgagtct gcacaagtgc tttgtgcggg tggagagcga
3060gaagggggct gtgtggaccg tggatgagct ggagttccgc aagaaacgga
gccagaggcc 3120cagcaggtgt tccaacccta cacctggccc ctgaggatcc
316010588DNAArtificial Sequencesynthetic 10atg gat ttt cag gtg cag
att ttc agc ttc ctg cta atc agt gcc tca 48Met Asp Phe Gln Val Gln
Ile Phe Ser Phe Leu Leu Ile Ser Ala Ser1 5 10 15gtc ata atg tct aga
ttc tcc ttc aag gga ata aaa ttt tct aag gga 96Val Ile Met Ser Arg
Phe Ser Phe Lys Gly Ile Lys Phe Ser Lys Gly 20 25 30aaa tac aaa ggg
tcg act tcc ggt agc ggc aaa tcc tct gaa ggc aaa 144Lys Tyr Lys Gly
Ser Thr Ser Gly Ser Gly Lys Ser Ser Glu Gly Lys 35 40 45ggt cag gtt
cct gct cag cta ctg gta ggc gcc ctg cgt gtg cta gcg 192Gly Gln Val
Pro Ala Gln Leu Leu Val Gly Ala Leu Arg Val Leu Ala 50 55 60tac tcc
cgc ctc aag gtc acc gtg aaa ggg aaa cac ctt tgt cca agt 240Tyr Ser
Arg Leu Lys Val Thr Val Lys Gly Lys His Leu Cys Pro Ser65 70 75
80ccc cta ttt ccc gga cct tct aag ccc ttt tgg gtg ctg gtg gtg gtt
288Pro Leu Phe Pro Gly Pro Ser Lys Pro Phe Trp Val Leu Val Val Val
85 90 95ggt gga gtc ctg gct tgc tat agc ttg cta gta aca gtg gcc ttt
att 336Gly Gly Val Leu Ala Cys Tyr Ser Leu Leu Val Thr Val Ala Phe
Ile 100 105 110att ttc tgg gtg agg agt aag agg agc agg ctc ctg cac
agt gac tac 384Ile Phe Trp Val Arg Ser Lys Arg Ser Arg Leu Leu His
Ser Asp Tyr 115 120 125atg aac atg act ccc cgc cgc ccc ggg ccc acc
cgc aag cat tac cag 432Met Asn Met Thr Pro Arg Arg Pro Gly Pro Thr
Arg Lys His Tyr Gln 130 135 140ccc tat gcc cca cca cgc gac ttc gca
gcc tat aga tct caa gtg cga 480Pro Tyr Ala Pro Pro Arg Asp Phe Ala
Ala Tyr Arg Ser Gln Val Arg145 150 155 160aag gca gct ata acc agc
tat gag aaa tca gat ggt gtt tac acg ggc 528Lys Ala Ala Ile Thr Ser
Tyr Glu Lys Ser Asp Gly Val Tyr Thr Gly 165 170 175ctg agc acc agg
aac cag gag act tac gag act ctg aag cat gag aaa 576Leu Ser Thr Arg
Asn Gln Glu Thr Tyr Glu Thr Leu Lys His Glu Lys 180 185 190cca cca
cag tag 588Pro Pro Gln 195113259DNAArtificial Sequencesynthetic
11atggattttc aggtgcagat tttcagcttc ctgctaatca gtgcctcagt cataatgtct
60agattctcct tcaagggaat aaaattttct aagggaaaat acaaagggtc gacttccggt
120agcggcaaat cctctgaagg caaaggtcag gttcctgctc agctactggt
aggcgccctg 180cgtgtgctag cgtactcccg cctcaaggtc accgtgaaag
ggaaacacct ttgtccaagt 240cccctatttc ccggaccttc taagcccttt
tgggtgctgg tggtggttgg tggagtcctg 300gcttgctata gcttgctagt
aacagtggcc tttattattt tctgggtgag gagtaagagg 360agcaggctcc
tgcacagtga ctacatgaac atgactcccc gccgccccgg gcccacccgc
420aagcattacc agccctatgc cccaccacgc gacttcgcag cctatagatc
tcaagtgcga 480aaggcagcta taaccagcta tgagaaatca gatggtgttt
acacgggcct gagcaccagg 540aaccaggaga cttacgagac tctgaagcat
gagaaaccac cacagtagct ttagactcga 600gcgggatcaa ttccgccccc
cccctaacgt tactggccga agccgcttgg aataaggccg 660gtgtgcgttt
gtctatatgt tattttccac catattgccg tcttttggca atgtgagggc
720ccggaaacct ggccctgtct tcttgacgag cattcctagg ggtctttccc
ctctcgccaa 780aggaatgcaa ggtctgttga atgtcgtgaa ggaagcagtt
cctctggaag cttcttgaag 840acaaacaacg tctgtagcga ccctttgcag
gcagcggaac cccccacctg gcgacaggtg 900cctctgcggc caaaagccac
gtgtataaga tacacctgca aaggcggcac aaccccagtg 960ccacgttgtg
agttggatag ttgtggaaag agtcaaatgg ctctcctcaa gcgtattcaa
1020caaggggctg aaggatgccc agaaggtacc ccattgtatg ggatctgatc
tggggcctcg 1080gtgcacatgc tttacatgtg tttagtcgag gttaaaaaaa
cgtctaggcc ccccgaacca 1140cggggacgtg gttttccttt gaaaaacacg
ataatagcat gcagcgctac cggtcgccac 1200catggtgagc aagggcgagg
agctgttcac cggggtggtg cccatcctgg tcgagctgga 1260cggcgacgta
aacggccaca agttcagcgt gtccggcgag ggcgagggcg atgccaccta
1320cggcaagctg accctgaagt tcatctgcac caccggcaag ctgcccgtgc
cctggcccac 1380cctcgtgacc accctgacct acggcgtgca gtgcttcagc
cgctaccccg accacatgaa 1440gcagcacgac ttcttcaagt ccgccatgcc
cgaaggctac gtccaggagc gcaccatctt 1500cttcaaggac gacggcaact
acaagacccg cgccgaggtg aagttcgagg gcgacaccct 1560ggtgaaccgc
atcgagctga agggcatcga cttcaaggag gacggcaaca tcctggggca
1620caagctggag tacaactaca acagccacaa cgtctatatc atggccgaca
agcagaagaa 1680cggcatcaag gtgaacttca agatccgcca caacatcgag
gacggcagcg tgcagctcgc 1740cgaccactac cagcagaaca cccccatcgg
cgacggcccc gtgctgctgc ccgacaacca 1800ctacctgagc acccagtccg
ccctgagcaa agaccccaac gagaagcgcg atcacatggt 1860cctgctggag
ttcgtgaccg ccgccgggat cactctcggc atggacgagc tgtacaagtc
1920cggccggact cagatctcga gctcaagctt cgaattcatg cccaacccca
ggcctggcaa 1980gccctcggcc ccttccttgg cccttggccc atccccagga
gcctcgccca gctggagggc 2040tgcacccaaa gcctcagacc tgctgggggc
ccggggccca gggggaacct tccagggccg 2100agatcttcga ggcggggccc
atgcctcctc ttcttccttg aaccccatgc caccatcgca 2160gctgcagctg
cccacactgc ccctagtcat ggtggcaccc tccggggcac ggctgggccc
2220cttgccccac ttacaggcac tcctccagga caggccacat ttcatgcacc
agctctcaac 2280ggtggatgcc cacgcccgga cccctgtgct gcaggtgcac
cccctggaga gcccagccat 2340gatcagcctc acaccaccca ccaccgccac
tggggtcttc tccctcaagg cccggcctgg 2400cctcccacct gggatcaacg
tggccagcct ggaatgggtg tccagggagc cggcactgct 2460ctgcaccttc
ccaaatccca gtgcacccag gaaggacagc accctttcgg ctgtgcccca
2520gagctcctac ccactgctgg caaatggtgt ctgcaagtgg cccggatgtg
agaaggtctt 2580cgaagagcca gaggacttcc tcaagcactg ccaggcggac
catcttctgg atgagaaggg 2640cagggcacaa tgtctcctcc agagagagat
ggtacagtct ctggagcagc agctggtgct 2700ggagaaggag aagctgagtg
ccatgcaggc ccacctggct gggaaaatgg cactgaccaa 2760ggcttcatct
gtggcatcat ccgacaaggg ctcctgctgc atcgtagctg ctggcagcca
2820aggccctgtc gtcccagcct ggtctggccc ccgggaggcc cctgacagcc
tgtttgctgt 2880ccggaggcac ctgtggggta gccatggaaa cagcacattc
ccagagttcc tccacaacat 2940ggactacttc aagttccaca acatgcgacc
ccctttcacc tacgccacgc tcatccgctg 3000ggccatcctg gaggctccag
agaagcagcg gacactcaat gagatctacc actggttcac 3060acgcatgttt
gccttcttca gaaaccatcc tgccacctgg aagaacgcca tccgccacaa
3120cctgagtctg cacaagtgct ttgtgcgggt ggagagcgag aagggggctg
tgtggaccgt 3180ggatgagctg gagttccgca agaaacggag ccagaggccc
agcaggtgtt ccaaccctac 3240acctggcccc tgaggatcc
32591214PRTArtificial Sequencesynthetic 12Gly Ser Thr Ser Gly Ser
Gly Lys Ser Ser Glu Gly Lys Gly1 5 1013927DNAArtificial
Sequencesynthetic 13atg gat ttt cag gtg cag att ttc agc ttc ctg cta
atc agt gcc tca 48Met Asp Phe Gln Val Gln Ile Phe Ser Phe Leu Leu
Ile Ser Ala Ser1 5 10 15gtc ata atg tct aga atg tta cca ttt ctg ttt
ttt tcc acc ctg ttt 96Val Ile Met Ser Arg Met Leu Pro Phe Leu Phe
Phe Ser Thr Leu Phe 20 25 30tct tcc ata ttt act gaa gct cag aag cag
tat tgg gtc tgc aac tca 144Ser Ser Ile Phe Thr Glu Ala Gln Lys Gln
Tyr Trp Val Cys Asn Ser 35 40 45tcc gat gca agt att tca tac acc tac
tgt gat aaa atg caa tac cca 192Ser Asp Ala Ser Ile Ser Tyr Thr Tyr
Cys Asp Lys Met Gln Tyr Pro 50 55 60att tca att aat gtt aac ccc tgt
ata gaa ttg aaa gga tcc aaa gga 240Ile Ser Ile Asn Val Asn Pro Cys
Ile Glu Leu Lys Gly Ser Lys Gly65 70 75 80tta ttg cac att ttc tac
att cca agg aga gat tta aag caa tta tat 288Leu Leu His Ile Phe Tyr
Ile Pro Arg Arg Asp Leu Lys Gln Leu Tyr 85 90 95ttc aat ctc tat ata
act gtc aac acc atg aat ctt cca aag cgc aaa 336Phe Asn Leu Tyr Ile
Thr Val Asn Thr Met Asn Leu Pro Lys Arg Lys 100 105 110gaa gtt att
tgc cga gga tct gat gac gat tac tct ttt tgc aga gct 384Glu Val Ile
Cys Arg Gly Ser Asp Asp Asp Tyr Ser Phe Cys Arg Ala 115 120 125ctg
aag gga gag act gtg aat aca aca ata tca ttc tcc ttc aag gga 432Leu
Lys Gly Glu Thr Val Asn Thr Thr Ile Ser Phe Ser Phe Lys Gly 130 135
140ata aaa ttt tct aag gga aaa tac aaa tgt gtt gtt gaa gct att tct
480Ile Lys Phe Ser Lys Gly Lys Tyr Lys Cys Val Val Glu Ala Ile
Ser145 150 155 160ggg agc cca gaa gaa atg ctc ttt tgc ttg gag ttt
gtc atc cta cac 528Gly Ser Pro Glu Glu Met Leu Phe Cys Leu Glu Phe
Val Ile Leu His 165 170 175caa cct aat tca aat ggt cac ctc gtg aaa
ggg aaa cac ctt tgt cca 576Gln Pro Asn Ser Asn Gly His Leu Val Lys
Gly Lys His Leu Cys Pro 180 185 190agt ccc cta ttt ccc gga cct tct
aag ccc ttt tgg gtg ctg gtg gtg 624Ser Pro Leu Phe Pro Gly Pro Ser
Lys Pro Phe Trp Val Leu Val Val 195 200 205gtt ggt gga gtc ctg gct
tgc tat agc ttg cta gta aca gtg gcc ttt 672Val Gly Gly Val Leu Ala
Cys Tyr Ser Leu Leu Val Thr Val Ala Phe 210 215 220att att ttc tgg
gtg agg agt aag agg agc agg ctc ctg cac agt gac 720Ile Ile Phe Trp
Val Arg Ser Lys Arg Ser Arg Leu Leu His Ser Asp225 230 235 240tac
atg aac atg act ccc cgc cgc ccc ggg ccc acc cgc aag cat tac 768Tyr
Met Asn Met Thr Pro Arg Arg Pro Gly Pro Thr Arg Lys His Tyr 245 250
255cag ccc tat gcc cca cca cgc gac ttc gca gcc tat aga tct caa gtg
816Gln Pro Tyr Ala Pro Pro Arg Asp Phe Ala Ala Tyr Arg Ser Gln Val
260 265 270cga aag gca gct ata acc agc tat gag aaa tca gat ggt gtt
tac acg 864Arg Lys Ala Ala Ile Thr Ser Tyr Glu Lys Ser Asp Gly Val
Tyr Thr 275 280 285ggc ctg agc acc agg aac cag gag act tac gag act
ctg aag cat gag 912Gly Leu Ser Thr Arg Asn Gln Glu Thr Tyr Glu Thr
Leu Lys His Glu 290 295 300aaa cca cca cag tag 927Lys Pro Pro
Gln305144408DNAArtificial Sequencesynthetic 14atggattttc aggtgcagat
tttcagcttc ctgctaatca gtgcctcagt cataatgtct 60agaatgttac catttctgtt
tttttccacc ctgttttctt ccatatttac tgaagctcag 120aagcagtatt
gggtctgcaa ctcatccgat gcaagtattt catacaccta ctgtgataaa
180atgcaatacc caatttcaat taatgttaac ccctgtatag aattgaaagg
atccaaagga 240ttattgcaca ttttctacat tccaaggaga gatttaaagc
aattatattt caatctctat 300ataactgtca acaccatgaa tcttccaaag
cgcaaagaag ttatttgccg aggatctgat 360gacgattact ctttttgcag
agctctgaag ggagagactg tgaatacaac aatatcattc 420tccttcaagg
gaataaaatt ttctaaggga aaatacaaat gtgttgttga agctatttct
480gggagcccag aagaaatgct cttttgcttg gagtttgtca tcctacacca
acctaattca 540aatggtcacc tcgtgaaagg gaaacacctt tgtccaagtc
ccctatttcc cggaccttct 600aagccctttt gggtgctggt ggtggttggt
ggagtcctgg cttgctatag cttgctagta 660acagtggcct ttattatttt
ctgggtgagg agtaagagga gcaggctcct gcacagtgac 720tacatgaaca
tgactccccg ccgccccggg cccacccgca agcattacca gccctatgcc
780ccaccacgcg acttcgcagc ctatagatct caagtgcgaa aggcagctat
aaccagctat 840gagaaatcag atggtgttta cacgggcctg agcaccagga
accaggagac ttacgagact 900ctgaagcatg agaaaccacc acagtagctt
tagactcgag cgggatcaat tccgcccccc 960ccctaacgtt actggccgaa
gccgcttgga ataaggccgg tgtgcgtttg tctatatgtt 1020attttccacc
atattgccgt cttttggcaa tgtgagggcc cggaaacctg gccctgtctt
1080cttgacgagc attcctaggg gtctttcccc tctcgccaaa ggaatgcaag
gtctgttgaa 1140tgtcgtgaag gaagcagttc ctctggaagc ttcttgaaga
caaacaacgt ctgtagcgac 1200cctttgcagg cagcggaacc ccccacctgg
cgacaggtgc ctctgcggcc aaaagccacg 1260tgtataagat acacctgcaa
aggcggcaca accccagtgc cacgttgtga gttggatagt 1320tgtggaaaga
gtcaaatggc tctcctcaag cgtattcaac aaggggctga aggatgccca
1380gaaggtaccc cattgtatgg gatctgatct ggggcctcgg tgcacatgct
ttacatgtgt 1440ttagtcgagg ttaaaaaaac gtctaggccc cccgaaccac
ggggacgtgg ttttcctttg 1500aaaaacacga taatagcatg cagcgctacc
ggtcgccacc atggtgagca agggcgagga 1560gctgttcacc ggggtggtgc
ccatcctggt cgagctggac ggcgacgtaa acggccacaa 1620gttcagcgtg
tccggcgagg gcgagggcga tgccacctac ggcaagctga ccctgaagtt
1680catctgcacc accggcaagc tgcccgtgcc ctggcccacc ctcgtgacca
ccctgaccta 1740cggcgtgcag tgcttcagcc gctaccccga ccacatgaag
cagcacgact tcttcaagtc 1800cgccatgccc gaaggctacg tccaggagcg
caccatcttc ttcaaggacg acggcaacta 1860caagacccgc gccgaggtga
agttcgaggg cgacaccctg gtgaaccgca tcgagctgaa 1920gggcatcgac
ttcaaggagg acggcaacat cctggggcac aagctggagt acaactacaa
1980cagccacaac gtctatatca tggccgacaa gcagaagaac ggcatcaagg
tgaacttcaa 2040gatccgccac aacatcgagg acggcagcgt gcagctcgcc
gaccactacc agcagaacac 2100ccccatcggc gacggccccg tgctgctgcc
cgacaaccac tacctgagca cccagtccgc 2160cctgagcaaa gaccccaacg
agaagcgcga tcacatggtc ctgctggagt tcgtgaccgc 2220cgccgggatc
actctcggca tggacgagct gtacaagtcc ggccggactc agatctcgag
2280ctcaagcttc gaattcatgc ccaaccccag gcctggcaag ccctcggccc
cttccttggc 2340ccttggccca tccccaggag cctcgcccag ctggagggct
gcacccaaag cctcagacct 2400gctgggggcc cggggcccag ggggaacctt
ccagggccga gatcttcgag gcggggccca 2460tgcctcctct tcttccttga
accccatgcc accatcgcag ctgcagctgc ccacactgcc 2520cctagtcatg
gtggcaccct ccggggcacg gctgggcccc ttgccccact tacaggcact
2580cctccaggac aggccacatt tcatgcacca gctctcaacg gtggatgccc
acgcccggac 2640ccctgtgctg caggtgcacc ccctggagag cccagccatg
atcagcctca caccacccac 2700caccgccact ggggtcttct ccctcaaggc
ccggcctggc ctcccacctg ggatcaacgt 2760ggccagcctg gaatgggtgt
ccagggagcc ggcactgctc tgcaccttcc caaatcccag 2820tgcacccagg
aaggacagca ccctttcggc tgtgccccag agctcctacc cactgctggc
2880aaatggtgtc tgcaagtggc ccggatgtga gaaggtcttc gaagagccag
aggacttcct 2940caagcactgc caggcggacc atcttctgga tgagaagggc
agggcacaat gtctcctcca 3000gagagagatg gtacagtctc tggagcagca
gctggtgctg gagaaggaga agctgagtgc 3060catgcaggcc cacctggctg
ggaaaatggc actgaccaag gcttcatctg tggcatcatc 3120cgacaagggc
tcctgctgca tcgtagctgc tggcagccaa ggccctgtcg tcccagcctg
3180gtctggcccc cgggaggccc ctgacagcct gtttgctgtc cggaggcacc
tgtggggtag 3240ccatggaaac agcacattcc cagagttcct ccacaacatg
gactacttca agttccacaa 3300catgcgaccc cctttcacct acgccacgct
catccgctgg gccatcctgg aggctccaga 3360gaagcagcgg acactcaatg
agatctacca ctggttcaca cgcatgtttg ccttcttcag 3420aaaccatcct
gccacctgga agaacgccat ccgccacaac ctgagtctgc acaagtgctt
3480tgtgcgggtg gagagcgaga agggggctgt gtggaccgtg gatgagctgg
agttccgcaa 3540gaaacggagc cagaggccca gcaggtgttc caaccctaca
cctggcccct gaggatccgc 3600gccgctcgcg actcgagaga tccggattag
tccaatttgt taaagacagg atatcagtgg 3660tccaggctct agttttgact
caacaatatc accagctgaa gcctatagag tacgagccat 3720agataaaata
aaagatttta tttagtctcc agaaaaaggg gggaatgaaa gaccccacct
3780gtaggtttgg caagctagct taagtaacgc cattttgcag gcatggaaaa
atacataact 3840gagaatagag aagttcagat caaggtcagg aacagatgga
acagctgaat atgggccaaa 3900caggatatct gtggtaagca gttcctgccc
cggctcaggg ccaagaacag atggaacagc 3960tgaatatggg ccaaacagga
tatctgtggt aagcagttcc tgccccggct cagggccaag 4020aacagatggt
ccccagatgc ggtccagccc tcagcagttt ctagagaacc atcagatgtt
4080tccagggtgc cccaaggacc tgaaatgacc ctgtgcctta tttgaactaa
ccaatcagtt 4140cgcttctcgc ttctgttcgc gcgcttctgc tccccgagct
caataaaaga gcccacaacc 4200cctcactcgg ggcgccagtc ctccgattga
ctgagtcgcc cgggtacccg tgtatccaat 4260aaaccctctt gcagttgcat
ccgacttgtg gtctcgctgt tccttgggag ggtctcctct 4320gagtgattga
ctacccgtca gcgggggtct ttcacatgca gcatgtatca aaattaattt
4380ggtttttttt cttaagtatt tacattaa 44081520DNAArtificial
Sequencesynthetic 15agtccgggca ggtctacttt 201620DNAArtificial
Sequencesynthetic 16gaggcaacct gaccactctc 201719DNAArtificial
Sequencesynthetic 17tctggaggaa ctggcaaaa 191820DNAArtificial
Sequencesynthetic 18tgagctcatt gaatgcttgg 201920DNAArtificial
Sequencesynthetic 19tacagggctt tcgattcagc 202020DNAArtificial
Sequencesynthetic 20cgcacacagc agttcttctc 202120DNAArtificial
Sequencesynthetic 21tccttgggaa gcaattgaag 202220DNAArtificial
Sequencesynthetic 22aactggccac agttttcagg 202321DNAArtificial
Sequencesynthetic 23ctaagcaagg acggcgaatg t 212420DNAArtificial
Sequencesynthetic 24ggctgggaac aggatactgg 202520DNAArtificial
Sequencesynthetic 25gcctgcggac tctaccataa 202620DNAArtificial
Sequencesynthetic 26cagggatgac atgtgtctgg 202720DNAArtificial
Sequencesynthetic 27gtgttcctac ccccaatgtg 202820DNAArtificial
Sequencesynthetic 28cttgctcagt gtccttgctg 2029388PRTArtificial
Sequencesynthetic 29Met Asp Phe Gln Val Gln Ile Phe Ser Phe Leu Leu
Ile
Ser Ala Ser1 5 10 15Val Ile Met Ser Arg Gly Asp Ile Val Leu Thr Gln
Ser Pro Ser Ser 20 25 30Leu Ala Val Ser Ala Gly Glu Lys Val Thr Met
Ser Cys Lys Ser Ser 35 40 45Gln Ser Leu Leu Asn Ser Arg Thr Arg Lys
Asn Tyr Leu Ala Trp Tyr 50 55 60Gln Gln Lys Pro Gly Gln Ser Pro Lys
Leu Leu Ile Tyr Trp Ala Ser65 70 75 80Thr Arg Glu Ser Gly Val Pro
Asp Arg Phe Thr Gly Ser Gly Ser Gly 85 90 95Thr Asp Phe Thr Leu Thr
Ile Ser Ser Val Gln Ala Glu Asp Leu Ala 100 105 110Val Tyr Tyr Cys
Lys Gln Ser Tyr Asn Leu Tyr Thr Phe Gly Gly Gly 115 120 125Thr Lys
Leu Glu Ile Lys Gly Ser Thr Ser Gly Ser Gly Lys Ser Ser 130 135
140Glu Gly Lys Gly Glu Val Gln Leu Gln Gln Ser Gly Gly Gly Leu
Val145 150 155 160Gln Pro Gly Gly Ser Leu Lys Leu Ser Cys Ala Ala
Ser Gly Phe Asp 165 170 175Phe Ser Arg Tyr Trp Met Ser Trp Val Arg
Gln Ala Pro Gly Lys Gly 180 185 190Leu Glu Trp Ile Gly Glu Ile Asn
Pro Asp Ser Ser Thr Ile Asn Tyr 195 200 205Thr Pro Ser Leu Lys Asp
Lys Phe Ile Ile Ser Arg Asp Asn Ala Lys 210 215 220Asn Thr Leu Tyr
Leu Gln Met Ser Lys Val Arg Ser Glu Asp Thr Ala225 230 235 240Leu
Tyr Tyr Cys Ala Arg Arg Tyr Gly Asn Tyr Trp Tyr Phe Asp Val 245 250
255Trp Gly Ala Gly Thr Thr Val Thr Val Lys Gly Lys His Leu Cys Pro
260 265 270Ser Pro Leu Phe Pro Gly Pro Ser Lys Pro Phe Trp Val Leu
Val Val 275 280 285Val Gly Gly Val Leu Ala Cys Tyr Ser Leu Leu Val
Thr Val Ala Phe 290 295 300Ile Ile Phe Trp Val Arg Ser Lys Arg Ser
Arg Leu Leu His Ser Asp305 310 315 320Tyr Met Asn Met Thr Pro Arg
Arg Pro Gly Pro Thr Arg Lys His Tyr 325 330 335Gln Pro Tyr Ala Pro
Pro Arg Asp Phe Ala Ala Tyr Arg Ser Gln Val 340 345 350Arg Lys Ala
Ala Ile Thr Ser Tyr Glu Lys Ser Asp Gly Val Tyr Thr 355 360 365Gly
Leu Ser Thr Arg Asn Gln Glu Thr Tyr Glu Thr Leu Lys His Glu 370 375
380Lys Pro Pro Gln38530167PRTArtificial Sequencesynthetic 30Met Asp
Phe Gln Val Gln Ile Phe Ser Phe Leu Leu Ile Ser Ala Ser1 5 10 15Val
Ile Met Ser Arg Gln Val Pro Ala Gln Leu Leu Val Gly Ala Leu 20 25
30Arg Val Leu Ala Tyr Ser Arg Leu Lys Val Thr Val Lys Gly Lys His
35 40 45Leu Cys Pro Ser Pro Leu Phe Pro Gly Pro Ser Lys Pro Phe Trp
Val 50 55 60Leu Val Val Val Gly Gly Val Leu Ala Cys Tyr Ser Leu Leu
Val Thr65 70 75 80Val Ala Phe Ile Ile Phe Trp Val Arg Ser Lys Arg
Ser Arg Leu Leu 85 90 95His Ser Asp Tyr Met Asn Met Thr Pro Arg Arg
Pro Gly Pro Thr Arg 100 105 110Lys His Tyr Gln Pro Tyr Ala Pro Pro
Arg Asp Phe Ala Ala Tyr Arg 115 120 125Ser Gln Val Arg Lys Ala Ala
Ile Thr Ser Tyr Glu Lys Ser Asp Gly 130 135 140Val Tyr Thr Gly Leu
Ser Thr Arg Asn Gln Glu Thr Tyr Glu Thr Leu145 150 155 160Lys His
Glu Lys Pro Pro Gln 16531162PRTArtificial Sequencesynthetic 31Met
Asp Phe Gln Val Gln Ile Phe Ser Phe Leu Leu Ile Ser Ala Ser1 5 10
15Val Ile Met Ser Arg Phe Ser Phe Lys Gly Ile Lys Phe Ser Lys Gly
20 25 30Lys Tyr Lys Gly His Leu Val Lys Gly Lys His Leu Cys Pro Ser
Pro 35 40 45Leu Phe Pro Gly Pro Ser Lys Pro Phe Trp Val Leu Val Val
Val Gly 50 55 60Gly Val Leu Ala Cys Tyr Ser Leu Leu Val Thr Val Ala
Phe Ile Ile65 70 75 80Phe Trp Val Arg Ser Lys Arg Ser Arg Leu Leu
His Ser Asp Tyr Met 85 90 95Asn Met Thr Pro Arg Arg Pro Gly Pro Thr
Arg Lys His Tyr Gln Pro 100 105 110Tyr Ala Pro Pro Arg Asp Phe Ala
Ala Tyr Arg Ser Gln Val Arg Lys 115 120 125Ala Ala Ile Thr Ser Tyr
Glu Lys Ser Asp Gly Val Tyr Thr Gly Leu 130 135 140Ser Thr Arg Asn
Gln Glu Thr Tyr Glu Thr Leu Lys His Glu Lys Pro145 150 155 160Pro
Gln32195PRTArtificial Sequencesynthetic 32Met Asp Phe Gln Val Gln
Ile Phe Ser Phe Leu Leu Ile Ser Ala Ser1 5 10 15Val Ile Met Ser Arg
Phe Ser Phe Lys Gly Ile Lys Phe Ser Lys Gly 20 25 30Lys Tyr Lys Gly
Ser Thr Ser Gly Ser Gly Lys Ser Ser Glu Gly Lys 35 40 45Gly Gln Val
Pro Ala Gln Leu Leu Val Gly Ala Leu Arg Val Leu Ala 50 55 60Tyr Ser
Arg Leu Lys Val Thr Val Lys Gly Lys His Leu Cys Pro Ser65 70 75
80Pro Leu Phe Pro Gly Pro Ser Lys Pro Phe Trp Val Leu Val Val Val
85 90 95Gly Gly Val Leu Ala Cys Tyr Ser Leu Leu Val Thr Val Ala Phe
Ile 100 105 110Ile Phe Trp Val Arg Ser Lys Arg Ser Arg Leu Leu His
Ser Asp Tyr 115 120 125Met Asn Met Thr Pro Arg Arg Pro Gly Pro Thr
Arg Lys His Tyr Gln 130 135 140Pro Tyr Ala Pro Pro Arg Asp Phe Ala
Ala Tyr Arg Ser Gln Val Arg145 150 155 160Lys Ala Ala Ile Thr Ser
Tyr Glu Lys Ser Asp Gly Val Tyr Thr Gly 165 170 175Leu Ser Thr Arg
Asn Gln Glu Thr Tyr Glu Thr Leu Lys His Glu Lys 180 185 190Pro Pro
Gln 19533308PRTArtificial Sequencesynthetic 33Met Asp Phe Gln Val
Gln Ile Phe Ser Phe Leu Leu Ile Ser Ala Ser1 5 10 15Val Ile Met Ser
Arg Met Leu Pro Phe Leu Phe Phe Ser Thr Leu Phe 20 25 30Ser Ser Ile
Phe Thr Glu Ala Gln Lys Gln Tyr Trp Val Cys Asn Ser 35 40 45Ser Asp
Ala Ser Ile Ser Tyr Thr Tyr Cys Asp Lys Met Gln Tyr Pro 50 55 60Ile
Ser Ile Asn Val Asn Pro Cys Ile Glu Leu Lys Gly Ser Lys Gly65 70 75
80Leu Leu His Ile Phe Tyr Ile Pro Arg Arg Asp Leu Lys Gln Leu Tyr
85 90 95Phe Asn Leu Tyr Ile Thr Val Asn Thr Met Asn Leu Pro Lys Arg
Lys 100 105 110Glu Val Ile Cys Arg Gly Ser Asp Asp Asp Tyr Ser Phe
Cys Arg Ala 115 120 125Leu Lys Gly Glu Thr Val Asn Thr Thr Ile Ser
Phe Ser Phe Lys Gly 130 135 140Ile Lys Phe Ser Lys Gly Lys Tyr Lys
Cys Val Val Glu Ala Ile Ser145 150 155 160Gly Ser Pro Glu Glu Met
Leu Phe Cys Leu Glu Phe Val Ile Leu His 165 170 175Gln Pro Asn Ser
Asn Gly His Leu Val Lys Gly Lys His Leu Cys Pro 180 185 190Ser Pro
Leu Phe Pro Gly Pro Ser Lys Pro Phe Trp Val Leu Val Val 195 200
205Val Gly Gly Val Leu Ala Cys Tyr Ser Leu Leu Val Thr Val Ala Phe
210 215 220Ile Ile Phe Trp Val Arg Ser Lys Arg Ser Arg Leu Leu His
Ser Asp225 230 235 240Tyr Met Asn Met Thr Pro Arg Arg Pro Gly Pro
Thr Arg Lys His Tyr 245 250 255Gln Pro Tyr Ala Pro Pro Arg Asp Phe
Ala Ala Tyr Arg Ser Gln Val 260 265 270Arg Lys Ala Ala Ile Thr Ser
Tyr Glu Lys Ser Asp Gly Val Tyr Thr 275 280 285Gly Leu Ser Thr Arg
Asn Gln Glu Thr Tyr Glu Thr Leu Lys His Glu 290 295 300Lys Pro Pro
Gln305
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