U.S. patent application number 15/371843 was filed with the patent office on 2017-06-08 for immunoregulation by anti-ilts antibodies and ilts-binding antibody fragments.
This patent application is currently assigned to Merck Sharp & Dohme Corp.. The applicant listed for this patent is Merck Sharp & Dohme Corp.. Invention is credited to Irina Apostolou, Paul Ponath, Joe Ponte, Michael Rosenzweig, Lou Vaickus.
Application Number | 20170158765 15/371843 |
Document ID | / |
Family ID | 44307213 |
Filed Date | 2017-06-08 |
United States Patent
Application |
20170158765 |
Kind Code |
A1 |
Apostolou; Irina ; et
al. |
June 8, 2017 |
IMMUNOREGULATION BY ANTI-ILTS ANTIBODIES AND ILTS-BINDING ANTIBODY
FRAGMENTS
Abstract
Disclosed herein are methods of using anti-ILT5 antibodies and
ILT5-binding fragments thereof to induce an immunostimulatory
effect in a T cell when such a T cell is contacted with an antigen
presenting cell (APC) that has been previously contacted with the
anti-ILT5 antibody or ILT5-binding fragment. Also disclosed herein
are methods of using anti-ILT5 antibodies and ILT5-binding
fragments thereof to inhibit a response in a T cell (e.g., a
proliferative response) when such a T cell is concomitantly
contacted, or has previously been contacted, with an APC, which APC
is simultaneously contacted with the anti-ILT5 antibody or
ILT5-binding fragment. Also disclosed herein are methods of using
anti-ILT5 antibodies and ILT5-binding fragments thereof for the
treatment of various diseases and for use as immunostimulatory
adjuvants.
Inventors: |
Apostolou; Irina; (Norwell,
MA) ; Ponath; Paul; (San Francisco, CA) ;
Ponte; Joe; (Weymouth, MA) ; Rosenzweig; Michael;
(Boston, MA) ; Vaickus; Lou; (Boston, MA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Merck Sharp & Dohme Corp. |
Rahway |
NJ |
US |
|
|
Assignee: |
Merck Sharp & Dohme
Corp.
Rahway
NJ
|
Family ID: |
44307213 |
Appl. No.: |
15/371843 |
Filed: |
December 7, 2016 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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14462596 |
Aug 19, 2014 |
9534051 |
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15371843 |
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13574391 |
Oct 16, 2012 |
8846397 |
|
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PCT/US11/21943 |
Jan 20, 2011 |
|
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14462596 |
|
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61296788 |
Jan 20, 2010 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C07K 2317/55 20130101;
C07K 2317/622 20130101; C07K 2317/70 20130101; A61K 2039/505
20130101; C07K 2317/35 20130101; Y02A 50/30 20180101; Y02A 50/466
20180101; A61P 35/00 20180101; A61P 37/00 20180101; C07K 2317/74
20130101; A61K 35/15 20130101; C07K 16/2803 20130101; A61P 37/04
20180101; A61P 43/00 20180101; A61K 39/0011 20130101; C07K 2319/30
20130101; A61P 37/02 20180101; C07K 2317/24 20130101; A61P 31/12
20180101; C07K 2317/54 20130101 |
International
Class: |
C07K 16/28 20060101
C07K016/28; A61K 39/00 20060101 A61K039/00; A61K 35/15 20060101
A61K035/15 |
Claims
1. A method of inducing a response in a T cell, the method
comprising contacting the T cell with an APC that has been
contacted with or is in contact with a monovalent anti-ILT5
antibody or an ILT5-binding fragment of the antibody.
2. The method of claim 1, wherein the response is a proliferative
response.
3. The method of claim 1, wherein the level of the response is
proportional to the amount of antibody or fragment with which the
APC is contacted.
4. The method of claim 1, wherein the response does not require
recognition of a MHC molecule by a T cell receptor.
5. A method of inducing a naive T cell to express NKG2D on its
surface, comprising contacting the T cell with an APC that has been
contacted with an anti-ILT5 antibody or an ILT5-binding fragment of
the antibody.
6. A method of inducing a T cell to upregulate expression of a T
cell receptor:CD3 complex, comprising contacting the T cell with an
APC that has been contacted with an anti-ILT5 antibody or an
ILT5-binding fragment of the antibody.
7. A method of inducing a T cell to secrete Fas ligand, comprising
contacting the T cell with an APC that has been contacted with an
anti-ILT5 antibody or an ILT5-binding fragment of the antibody.
8. A method of endowing a T cell with cytotoxic potential,
comprising contacting the T cell with an APC that has been
contacted with an anti-ILT5 antibody or an ILT5-binding fragment of
the antibody.
9. The method of claim 8, further comprising contacting the T cell
with an antigen from a tumor cell or from a cell that is infected
with a bacterium, a virus, a fungus, a protozoan, or a parasite;
wherein the T cell becomes cytotoxic when it binds or recognizes
the antigen on a cell.
10. The method of claim 1, wherein the contacting is done in
vitro.
11. The method of claim 1, wherein the contacting is done in
vivo.
12. The method of claim 1, wherein the T cell is a CD4+ T cell.
13. The method of claim 1, wherein the T cell is a CD8+ T cell.
14. A method of inducing or enhancing an immune response in a
subject, the method comprising administering to the subject an
anti-ILT5 antibody or an ILT5-binding fragment of the antibody.
15. The method of claim 14, wherein the response does not require
recognition of a MHC molecule by a T cell receptor.
16. The method of claim 14, wherein the method induces a response
in a T cell in the subject.
17. The method claim 14, wherein a T cell in the subject is endowed
with cytotoxic potential upon contact with an APC that has been
contacted with or is in contact with the administered anti-ILT5
antibody or the ILT5-binding fragment of the antibody.
18. The method of claim 17, wherein the T cell or its progeny,
having gained cytotoxic potential, becomes cytotoxic when it binds
or recognizes an antigen.
19. The method of claim 18, wherein the antigen is selected from
one or both of an exogenous antigen and an endogenous antigen.
20. The method of claim 19, wherein the exogenous antigen is
selected from the group consisting of: a tumor antigen, a viral
antigen, a bacterial antigen, a fungal antigen, a protozoan
antigen, and a parasite antigen.
21. The method of claim 20, wherein the exogenous antigen is
administered to the subject.
22. The method of claim 21, wherein the anti-ILT5 antibody or the
ILT5-binding fragment of the antibody is administered at least once
before, together with, or very close in time to the administration
of the exogenous antigen.
23. The method of claim 21, wherein the anti-ILT5 antibody or the
ILT5-binding fragment of the antibody is administered at least once
after, together with, or very close in time to the administration
of the exogenous antigen.
24. The method of claim 21, wherein the anti-ILT5 antibody or the
ILT5-binding fragment of the antibody is administered separately
from the administration of the exogenous antigen.
25. The method of claim 18, wherein the antigen is a cellular
antigen.
26. The method of claim 25, wherein the subject has a tumor.
27. The method of claim 26, wherein the cellular antigen comprises
an endogenous antigen.
28. The method of claim 27, further comprising administering to the
subject a therapy, wherein the therapy inhibits or prevents the
function of cells, causes destruction of cells, or both.
29. The method of claim 28, wherein the therapy induces or enhances
release of the endogenous antigen.
30. The method of claim 28, wherein the therapy is administered at
least once after, together with, or very close in time to
administration of the anti-ILT5 antibody or the ILT5-binding
fragment.
31. The method of claim 29, wherein the therapy is administered at
least once before, together with, or very close in time to
administration of the anti-ILT5 antibody or the ILT5-binding
fragment.
32. The method of claim 28, wherein the anti-ILT5 antibody or the
ILT5-binding fragment of the antibody is administered separately
from the administration of the therapy.
33. The method of claim 27, wherein the method results in one or
more of: inhibition of tumor growth, reduction in tumor size,
reduction in the number of tumors, a decrease in tumor burden,
prolonging survival of the subject.
34. The method of claim 25, wherein the subject has a viral
infection.
35. The method of claim 18, wherein the T cell becomes cytotoxic
when it binds or recognizes the antigen on a cell.
36. The method of claim 17, wherein the T cell is a CD4+ T
cell.
37. The method of claim 17, wherein the T cell is a CD8+ T
cell.
38. The method of claim 17, wherein the method induces a response
in the T cell.
39. A method of inhibiting a response in a T cell, the method
comprising contacting the T cell with an antigen at the same time
as or very close in time to contacting the T cell with an APC that
has been contacted with a crosslinked anti-ILT5 antibody or a
crosslinked ILT5-binding fragment of the antibody.
40. The method of claim 39, wherein a proliferative response is
inhibited.
41. The method of claim 39, wherein the level inhibition of the
response is proportional to the amount of antibody or fragment with
which the APC is contacted.
42. The method of claim 39, wherein the inhibition occurs when the
antibody or fragment crosslinks or hypercrosslinks ILT5.
43. The method of claim 39, wherein the contacting is done in
vitro.
44. The method of claim 39, wherein the contacting is done in
vivo.
45. The method of claim 39, wherein the T cell is a CD4+ T
cell.
46. The method of claim 39, wherein the T cell is a CD8+ T
cell.
47. A method of inducing tolerance in a subject, comprising
administering to the subject a crosslinked anti-ILT5 antibody or a
crosslinked ILT5-binding fragment of the antibody such that the
antibody or fragment binds an APC, wherein a T cell in the subject
that has previously bound or recognized an antigen is tolerized
upon contact with the APC.
48. The method of claim 47, wherein the subject has a disease.
49. The method of claim 48, wherein the disease is an
immune-related disease.
50. The method of claim 1, wherein the anti-ILT5 antibody or
ILT5-binding fragment binds human ILT5.
51. The method of claim 1, wherein the anti-ILT5 antibody or
ILT5-binding fragment is chimeric.
52. The method of claim 1, wherein the anti-ILT5 antibody or
ILT5-binding fragment is humanized.
53. The method of claim 1, wherein the ILT5-binding fragment
comprises a Fab fragment, a F(ab').sub.2 fragment, or a scFv
fragment.
Description
[0001] This application is a division of U.S. patent application
Ser. No. 14/462,596, filed Aug. 19, 2014, which is a division of
Ser. No. 13/574,391, filed Oct. 16, 2012, no U.S. Pat. No.
8,846,397, which is the national stage of International Application
No. PCT/US11/21943, filed Jan. 20, 2011, which claims the benefit
of U.S. Provisional Application No. 61/296,788, filed Jan. 20,
2010; each of which is herein incorporated by reference in its
entirety.
BACKGROUND
[0002] The immune system has evolved multiple mechanisms to
preserve immune homeostasis and protective immunity while sparing
"self" from autoimmune destruction. The initiation of immunity is a
tightly controlled process that relies in part on interplay between
inhibitory and activating receptors. Immunoglobulin-like
transcripts (ILTs), which encompass both types of receptors, are
encoded by rapidly evolving genes found in human and non-human
primates. Immunoglobulin-like transcript 5 ("ILT5") is a cell
surface molecule that is a member of the immunoglobulin superfamily
and is highly expressed on antigen-presenting cells (APCs), such as
immature dendritic cells and monocytes.
SUMMARY
[0003] Disclosed herein are methods of using anti-ILT5 antibodies
and ILT5-binding fragments thereof to induce an immunostimulatory
effect in a T cell when such a T cell is contacted with an antigen
presenting cell (APC) that has been previously contacted with the
anti-ILT5 antibody or ILT5-binding fragment. Also disclosed herein
are methods of using anti-ILT5 antibodies and fragments thereof to
inhibit a response in a T cell (e.g., a proliferative response)
when such a T cell is concomitantly contacted, or has previously
been contacted, with an APC, which APC is simultaneously contacted
with the anti-ILT5 antibody or ILT5-binding fragment. Also
disclosed herein are methods of using anti-ILT5 antibodies and
ILT5-binding fragments thereof for the treatment of various
diseases and for use as immunostimulatory adjuvants.
[0004] In certain embodiments, provided herein are methods of
inducing a response in a T cell, comprising contacting the T cell
with an APC that has been contacted with or is in contact with a
monovalent anti-ILT5 antibody or an ILT5-binding fragment of the
antibody. In certain embodiments, such a response is a
proliferative response. In certain embodiments, such a response is
proportional to the amount of antibody or fragment with which the
APC is contacted. In certain embodiments, a response does not
require recognition of a MHC molecule by a T cell receptor.
[0005] In certain embodiments, provided herein are methods of
inducing a naive T cell to express NKG2D on its surface, comprising
contacting the T cell with an APC that has been contacted with an
anti-ILT5 antibody or an ILT5-binding fragment of the antibody. In
certain embodiments, provided herein are methods of inducing a T
cell to upregulate expression of a T cell receptor:CD3 complex,
comprising contacting the T cell with an APC that has been
contacted with an anti-ILT5 antibody or an ILT5-binding fragment of
the antibody. In certain embodiments, provided herein are methods
of inducing a T cell to secrete Fas ligand, comprising contacting
the T cell with an APC that has been contacted with an anti-ILT5
antibody or an ILT5-binding fragment of the antibody.
[0006] In certain embodiments, provided herein are methods of
endowing a T cell with cytotoxic potential, comprising contacting
the T cell with an APC that has been contacted with an anti-ILT5
antibody or an ILT5-binding fragment of the antibody. Such methods
can further comprise contacting the T cell with an antigen from a
tumor cell or from a cell that is infected with a bacterium, a
virus, a fungus, a protozoan, or a parasite, wherein the T cell
becomes cytotoxic when it binds or recognizes the antigen on a
cell.
[0007] In certain embodiments, any of the above methods can be
performed by contacting in vitro. In certain embodiments, any of
the above methods can be performed by contacting in vivo. In
certain embodiments, a T cell in any of the above methods is a
CD4.sup.+ T cell. In certain embodiments, a T cell in any of the
above methods is a CD8.sup.+ T cell.
[0008] In certain embodiments, provided herein are methods of
inducing or enhancing an immune response in a subject, comprising
administering to the subject an anti-ILT5 antibody or an
ILT5-binding fragment of the antibody. In certain embodiments, such
an immune response does not require recognition of a MHC molecule
by a T cell receptor. In certain embodiments, the method induces a
response in a T cell in the subject.
[0009] In certain embodiments, a T cell in the subject is endowed
with cytotoxic potential upon contact with an APC that has been
contacted with or is in contact with the administered anti-ILT5
antibody or the ILT5-binding fragment of the antibody. In certain
embodiments, the T cell or its progeny, having gained cytotoxic
potential, becomes cytotoxic when it binds or recognizes an
antigen. In certain embodiments, the antigen is selected from one
or both of an exogenous antigen and an endogenous antigen. In
certain embodiments, the exogenous antigen is selected from the
group consisting of: a tumor antigen, a viral antigen, a bacterial
antigen, a fungal antigen, a protozoan antigen, and a parasite
antigen. In certain embodiments, the exogenous antigen is
administered to the subject. In certain embodiments, he anti-ILT5
antibody or the ILT5-binding fragment of the antibody is
administered at least once before, together with, or very close in
time to the administration of the exogenous antigen. In certain
embodiments, the anti-ILT5 antibody or the ILT5-binding fragment of
the antibody is administered at least once after, together with,
very close in time to the administration of the exogenous antigen.
In certain embodiments, the anti-ILT5 antibody or the ILT5-binding
fragment of the antibody is administered separately from the
administration of the exogenous antigen.
[0010] In certain embodiments, the antigen is a cellular antigen.
In certain embodiments, the subject has a tumor. In certain
embodiments, the cellular antigen comprises an endogenous antigen
In certain embodiments, methods of inducing or enhancing an immune
response in a subject further comprise administering to the subject
a therapy, wherein the therapy inhibits or prevents the function of
cells, causes destruction of cells, or both. In certain
embodiments, the therapy induces or enhances release of the
cellular antigen. In certain embodiments, the therapy is
administered at least once after, together with, or very close in
time to administration of the anti-ILT5 antibody or the
ILT5-binding fragment. In certain embodiments, the therapy is
administered at least once before, together with, or very close in
time to administration of the anti-ILT5 antibody or the
ILT5-binding fragment. In certain embodiments, the anti-ILT5
antibody or the ILT5-binding fragment of the antibody is
administered separately from the administration of the therapy. In
certain embodiments, the method results in one or more of:
inhibition of tumor growth, reduction in tumor size, reduction in
the number of tumors, a decrease in tumor burden, prolonging
survival of the subject.
[0011] In certain embodiments, the subject has a viral
infection.
[0012] In certain embodiments, a T cell in the above methods
becomes cytotoxic when it binds or recognizes the antigen on a
cell. In certain embodiments, the T cell is a CD4.sup.+ T cell. In
certain embodiments, the T cell is a CD8.sup.+ T cell. In certain
embodiments, the method induces a response in the T cell.
[0013] In certain embodiments, provided herein are methods of
inhibiting a response in a T cell, comprising contacting the T cell
with an antigen at the same time as or very close in time to
contacting the T cell with an APC that has been contacted with a
crosslinked anti-ILT5 antibody or a crosslinked ILT5-binding
fragment of the antibody. In certain embodiments, a proliferative
response is inhibited. In certain embodiments, the level inhibition
of the response is proportional to the amount of antibody or
fragment with which the APC is contacted. In certain embodiments,
the inhibition occurs when the antibody or fragment crosslinks or
hypercrosslinks ILT5. In certain embodiments, the contacting is
done in vitro. In certain embodiments, the contacting is done in
vivo. In certain embodiments, the T cell is a CD4.sup.+ T cell. In
certain embodiments, the T cell is a CD8.sup.+ T cell.
[0014] In certain embodiments, provided herein are methods of
inducing tolerance in a subject, comprising administering to the
subject a crosslinked anti-ILT5 antibody or a crosslinked
ILT5-binding fragment of the antibody such that the antibody or
fragment binds an APC, wherein a T cell in the subject that has
previously bound or recognized an antigen is tolerized upon contact
with the APC. In certain embodiments, the subject has a disease,
e.g., an immune-related disease.
[0015] In certain embodiments, methods provided herein employ an
anti-ILT5 antibody or ILT5-binding fragment that binds human ILT5.
In certain embodiments, the anti-ILT5 antibody or ILT5-binding
fragment is chimeric. In certain embodiments, the anti-ILT5
antibody or ILT5-binding fragment is humanized. In certain
embodiments, the ILT5-binding fragment comprises a Fab fragment, a
F(ab')2 fragment, or a scFv fragment.
[0016] Unless otherwise defined, all technical and scientific terms
used herein have the same meaning as commonly understood by one
skilled in the art to which this invention belongs. Methods and
materials are described herein for use in the present invention;
other, suitable methods and materials known in the art can also be
used. The materials, methods, and examples are illustrative only
and not intended to be limiting. All publications, patent
applications, patents, sequences, database entries, and other
references mentioned herein are incorporated by reference in their
entirety. In case of conflict, the present specification, including
definitions, will control.
[0017] Other features and advantages of the invention will be
apparent from the following detailed description and figures, and
from the claims.
DESCRIPTION OF THE DRAWINGS
[0018] FIG. 1 (A-F) shows the expression of ILT5 by various
hematopoietic subsets in the form of two-dimensional flow cytometry
(FCM) representations called quantile contour plots (or probability
plots). The latter plots show the levels of two fluorescent
parameters (i.e., fluorescent antibodies) on various cell
subpopulations, quantitate the proportions of cells displaying
these parameters, and indicate the frequency of cells present at
each point in the plot. Here, peripheral blood mononuclear cells
(PBMCs) from a healthy human donor were stained with the TRX585
antibody as well as antibodies specific for the indicated cell
subset. In FIG. 1A, plots depict ILT5 (TRX585) expression by gated
CD56.sup.-CD3.sup.+CD4.sup.+ or CD56.sup.-CD3.sup.+CD8.sup.+ T
cells. In FIG. 1B, PBMCs were stained as in (A) and subsequently
stained for intracellular Foxp3. Plots show ILT5 expression by
gated CD56.sup.-CD3.sup.+CD4.sup.+Foxp3.sup.+ or
CD56.sup.-CD3.sup.+CD4.sup.+Foxp3.sup.- T cells. FIG. 1C shows ILT5
expression by CD4.sup.+, CD8.sup.+ or CD4.sup.-CD8.sup.- NKT
(CD56.sup.+CD3.sup.+) cells. FIG. 1D shows ILT5 expression by
myeloid (CD11c.sup.+HLA-DR.sup.+) and plasmacytoid
(CD123.sup.+BDCA-2.sup.+) DC cells. FIG. 1E shows expression of
ILT5 and CD11b by CD33.sup.hiCD34.sup.-, CD33.sup.hiCD34.sup.lo and
CD33.sup.intCD34.sup.- monocyte subsets as well as by
CD33.sup.loCD34.sup.+ myeloid progenitors. FIG. 1F shows ILT5
expression by CD33.sup.hiCD34.sup.loCD11b.sup.+CD14.sup.+ myeloid
derived suppressor cells (MDSCs). Percentages of gated cells and
mean fluorescence intensity of the ILT5-staining of ILT5.sup.+
cells are depicted inside and above plots, respectively.
Specificity of the ILT5 staining was ascertained by staining all of
the above cell subsets with a mIgG1 isotype control antibody. Data
are representative of 4 independent experiments.
[0019] FIG. 2 is a series of bar graphs showing TRX585
antibody-mediated hyperresponsiveness of allogeneic responder
lymphocytes in primary MLRs (mixed lymphocyte reactions).
2.times.10.sup.5 PBMCs (responder population)/well were cultured
with 2.times.10.sup.5 mitomycin-treated PBMCs (stimulator
population) from an unrelated blood donor in the presence or
absence of the indicated amounts of mIgG1 or TRX585 antibodies.
Proliferative activity in the MLR was measured at day 3.5 by the
incorporation of 1 .mu.Ci [.sup.3H]TdR (tritiated thymidine) per
well to the DNA of replicating cells during the last 12-18 hours of
the culture. The X axes indicate the concentration of antibody. The
Y axes indicate [.sup.3H]TdR uptake. Data shown are representative
of several experiments utilizing different responder/stimulator
pairs and are reported as the mean cpm.+-.standard error of
triplicate wells.
[0020] FIG. 3 is a pair of bar graphs showing that pretreatment of
peripheral blood monocytes or DCs with soluble TRX585 antibody
prior to their use in MLR assays resulted in antibody-mediated
enhancement of cell proliferation. DCs or monocytes that were
purified from PBMCs were incubated in the presence or absence of 50
.mu.g/ml mIgG1 isotype control or TRX585 antibody. Twenty four
hours later, precultured DCs and monocytes were washed to remove
unbound antibodies, seeded at 1000 and 120,000 cells per well,
respectively, and cocultured with 1.times.10.sup.5 T cells freshly
isolated from PBMCs. [.sup.3H] thymidine incorporation was measured
as described above. Data shown are representative of several
experiments utilizing different responder/stimulator pairs and are
reported as the mean cpm of triplicate wells. The X axes indicate
which antibody was used. The Y axes indicate [.sup.3H]TdR
uptake.
[0021] FIG. 4 is a pair of bar graphs showing that monovalent but
not divalent TRX585 antibody is endowed with stimulatory potential.
2.times.10.sup.5 PBMCs/well were cultured with 2.times.10.sup.5
mitomycin-treated allogeneic PBMCs in the presence or absence of 10
.mu.g/ml of goat anti-mouse F(ab')2 fragments and various
concentrations of soluble TRX585 or mIgG1, as indicated on the
figure. [.sup.3H]TdR-incorporation (1 .mu.Ci/well) in the last
12-18 hours of the cultured was measured at day 3.5. Shown are the
mean cpm of triplicate wells.
[0022] FIG. 5 is a pair of bar graphs showing that immobilization
of TRX585 antibody on plastic abolishes its immunostimulatory
potential. 2.times.10.sup.5 PBMCs/well were cultured with
2.times.10.sup.5 mitomycin-treated allogeneic PBMCs with either
soluble or tissue culture plate-bound TRX585, which was coated onto
the tissue culture plate well bottoms at the indicated
concentrations. [.sup.3H]TdR-incorporation (1 .mu.Ci/well) in the
last 12-18 hours of the culture was measured at day 3.5. The X axes
indicate the concentration of antibody. The Y axes indicate
[.sup.3H]TdR uptake. Shown is the mean cpm of triplicate wells.
[0023] FIG. 6 (A-B) is a series of two dimensional displays called
color density plots (or pseudo-color plots) and bar graphs showing
that TRX585 induces the proliferation of the majority of T cells
and that this proliferation requires the presence of non-T cells.
Color density plots provide the same information as quantile
contour plots. In FIG. 6A, 1.times.10.sup.7 PBMCs per ml were
stained with 1 .mu.M CFSE (carboxyfluorescein succinimidyl ester)
in PBS 0.1% BSA for 20 min at 37.degree. C., followed by 2 washes
in cold PBS. 2.times.10.sup.5 CFSE-labeled PBMCs (responder
population)/well were cultured with 2.times.10.sup.5
mitomycin-treated, allogeneic or autologous PBMCs (stimulator
population) in the presence or absence of mIgG1 or TRX585
antibodies (50 .mu.g/ml). Three and a half days later, cells were
stained with CD4 and CD8 antibodies as well as the viability dye,
7-amino-actinomycin D (7AAD). Dilution of the CFSE dye in viable
CD4 and CD8 T cells, which is indicative of cell proliferation, was
examined by flow cytometry. In the experiment shown in FIG. 6B,
1.times.10.sup.5 T cells purified from PBMCs were either cultured
alone (upper graph) or mixed with 2.times.10.sup.5
mitomycin-treated PBMCs (bottom graph), in the presence or absence
of the indicated amount of mIgG1 isotype control or TRX585
antibody. T cell proliferation was measured at day 3.5 by means of
[.sup.3H]TdR-incorporation as reported above. In the experiment
shown in FIG. 6B, the X axes indicate the concentration of
antibody, while the Y axes indicate [.sup.3H]TdR uptake. Shown are
the mean.+-.standard error cpm of triplicate wells.
[0024] FIG. 7 is a series of FCM pseudo-color plots showing that
TRX585 antibody-induced T cell proliferation is TCR-independent.
Responder cells (CD4.sup.+ and CD8.sup.+ T cells) as well as
stimulator cells (CD4.sup.- and CD8.sup.- cells) were isolated,
using a fluorescence-activated cell sorter (FACSAria), from PBMCs
that were stained with CD4 and CD8 antibodies as well as 7AAD
(7-aminoactinomycin D). Stimulator cells were inactivated by
mitomycin treatment and subsequently incubated with saturating
amounts of W6/32 (anti-pan-HLA class I) or Tu139 (anti-pan-HLA
class II) or no blocking antibody for 30 minutes at 37.degree. C.,
followed by washes. Responder cells were CFSE-labeled as previously
described. 2.times.10.sup.5 CFSE-labeled responder cells were mixed
with 2.times.10.sup.5 mitomycin-treated stimulator cells in the
presence or absence of TRX585 antibody (50 .mu.g/ml). Three and an
half days later, CD4 and CD8 T cell proliferation was examined by
flow cytometry. Plots show the proliferation of CD4.sup.+ T cells
(left two columns of plots) and CD8.sup.+ T cells (right two
columns of plots) in the absence of blocking antibody (top row of
plots), or in the presence of pan anti-MHC class I antibody (middle
row of plots), and pan anti-MHC class II antibody (bottom row of
plots). Percentage of proliferating cells is depicted inside the
plots.
[0025] FIG. 8 (A-B) is a series of FCM pseudo-color plots showing
the phenotype of TRX585 antibody-activated T cells. FIG. 8A is a
representation of a two-parameter flow cytometry analysis of
CFSE-labeled PBMCs that were cultured for 3.5 days with or without
TRX585 antibody (50 .mu.g/ml) and subsequently stained with CD4,
CD8 and CD25 antibodies. FIG. 8B shows NKG2D expression by PBMCs
cultured as previously described and stained with CD4, CD8 and
NKG2D antibodies. The plot on the right hand side depicts NKG2D
expression by freshly isolated PBMCs. Numbers in plots indicate the
proportion of cells within a given quadrant. Dead cells were
systematically excluded from the flow cytometry analysis by means
of 7AAD staining.
[0026] FIG. 9 is a series of FCM pseudo-color plots showing
sustained expression of NKG2D by anti-ILT5-exposed T cells despite
persistent NKG2D engagement. 2.times.10.sup.5 CFSE-labeled PBMCs
(responder population)/well were cultured with 2.times.10.sup.5
mitomycin-treated, allogeneic PBMCs (stimulator population) in the
presence or absence of mIgG1 or TRX585 antibodies (50 .mu.g/ml).
When indicated, blocking anti-NKG2D antibodies (clone 1D11 or 5C6)
were added to the cultures. In the latter blocking experiments,
responder cells were incubated with 20 .mu.g/ml of anti-NKG2D
antibodies for 30 min at 37.degree. C. prior to being mixed with
stimulator cells. Anti-NKG2D antibodies were present at 10 .mu.g/ml
in the final cultures. After three and a half days, cells were
stained with NKG2D, CD4 and CD8 antibodies as well as 7AAD, and
analyzed by flow cytometry.
[0027] FIG. 10 is a bar graph showing Fas ligand (FasL) secretion
by T cells contacted with APCs that have previously been contacted
with TRX585 antibodies. 2.times.10.sup.5 PBMCs (responder
population)/well were cultured with 2.times.10.sup.5
mitomycin-treated, allogeneic PBMCs (stimulator population) in the
presence or absence of TRX585 antibody (50 .mu.g/ml). Fas ligand
was quantified in 24 hour culture supernatants using commercial
human Fas ligand-specific ELISA.
[0028] FIG. 11 (A-C) is a series of line and bar graphs showing
that T cells from TRX585 antibody-containing PBMC cultures are
cytotoxic to a variety of human tumor cell lines. In the experiment
shown in FIG. 11A, 4.times.10.sup.5 CFSE-labeled PBMCs were
cultured with mIgG1 or TRX585 antibody (50 .mu.g/ml). Three and a
half days later, precultured T cells (effector cells) were purified
using a FACSAria.TM., based on CD4 and CD8 expression. On the same
day, 50,000 CFSE-labeled U937 and KG1 tumor cells (target cells)
per well were mixed with effector cells at effector:target (E:T)
ratios of 4:1, 1:1, 0.25:1 and 0:1 and cultured overnight. Cells
were subsequently stained with CD3, 7AAD and Annexin V, and
occurrence of apoptosis among target cells (CD3.sup.-CFSE.sup.+
cells) was examined by flow cytometry. The percentage of
cytotoxicity was determined by summing the percentage of late
apoptotic (7AAD.sup.+Annexin V.sup.+) and early apoptotic
(7AAD.sup.-Annexin V.sup.+) cells. The left and right graphs show
the percentages of dead U937 and KG1 cells, respectively, when
mixed with the indicated proportion of either mIgG1 isotype control
(black diamond) or TRX585 (blue squares) antibody-precultured
effector T cells. The X axes indicate effector:target ratios. The Y
axes indicate percent cytotoxicity. In the experiment shown in FIG.
11B, effector T cells were obtained and assessed for cytotoxic
function against ARPE 19, U937, and HCT116 tumor cell lines
(E:T=4:1) as described for FIG. 11A. The X axes indicate which
tumor cell line was used. The Y axes indicate percent cytotoxicity.
In the experiment shown in FIG. 11C, CD4 and CD8 T cells were
cell-sorted from 3.5 day-PBMC cultures, set up as described above,
and assessed independently for cytotoxic function against ARPE 19,
U937, HCT116 and K562 tumor cell lines (E:T=4:1). The X axes and
the key to the right of the graphs indicate which tumor cell line
was used. The Y axes indicate percent cytotoxicity.
[0029] FIG. 12(A-B): FIG. 12A is a line graph showing that the
cytotoxicity of TRX585-preactivated T cells is MHC class
I-dependent. FIG. 12B is a bar graph showing that the cytotoxicity
of TRX585-preactivated T cells is Fas ligand-dependent. Effector T
cells were purified from mIgG1- or TRX585-containing PBMC cultures
as well as cultures that did not contain antibodies, and assessed
for cytotoxicity against U937 tumor cells according to the
described experimental procedure described. For MHC class I
blocking experiments, target cells were incubated with saturating
amounts of anti-pan human MHC class I antibody (20 .mu.g/ml; clone
W6/32) for 30 min and washed to remove unbound W6/32 antibody
before exposure to T cells. To neutralize Fas ligand, 10 .mu.g/ml
of blocking anti-human Fas ligand antibody (clone NOK-2) was added
to the cocultures of effector and target cells. Data are
representative of several experiments using a variety of tumor cell
lines and could be recapitulated when using either CD4 or CD8
effector T cells.
[0030] FIG. 13 is a series of FCM pseudo-color plots showing that,
while CD4.sup.+ and CD8.sup.+ T cells from PBMC cultures containing
only TRX585 antibodies divided actively, concomitant treatment of
PBMCs with anti-CD3 and TRX585 antibodies resulted in the
inhibition of T cell proliferation. Here, 4.times.10.sup.5
CFSE-labeled PBMCs were cultured with the indicated concentration
of either mIgG1 isotype control or TRX585 antibody, in the presence
or absence of soluble anti-CD3 antibody (10 .mu.g/ml). Three and an
half days later, dilution of the CFSE dye in CD4 and CD8 T cells
was examined by flow cytometry. Numbers in plots indicate the
proportion of dividing cells. Dead cells were systematically
excluded from the flow cytometry analysis by means of 7AAD
staining.
[0031] FIG. 14 (A-B) is a series of FCM pseudo-color plots (FIG.
14A) and line graphs (FIG. 14B) showing that pre-exposure of
CD4.sup.+ and CD8.sup.+ T cells to TRX585 antibodies increases
their responsiveness to subsequent TCR stimulation as well as
surface CD3 complexes. In the experiment shown in FIG. 14A,
4.times.10.sup.5 CFSE-labeled PBMCs were cultured in the presence
or absence of mIgG1 isotype control or TRX585 antibody (50
.mu.g/ml). After 3.5 days, the precultured T cells as well as T
cells from freshly isolated autologous PBMCs were cell-sorted using
a FACSAria, CFSE-labeled and exposed to anti-CD3 antibody-mediated
stimulation for another 2 days. The latter TCR stimulation used
solid-phase anti-CD3 antibody at concentrations of 0.03-10
.mu.g/ml. CD4+ and CD8+ T cell proliferation was examined by flow
cytometry as described previously. Numbers in plots indicate the
proportion of dividing cells. Dead cells were systematically
excluded from flow cytometry analyses by means of 7AAD staining.
FIG. 14B shows the amount of CD3 complexes at the surface of the
same T cells, expressed as the mean fluorescence intensity of the
cells when stained with a fluorescent anti-CD3 antibody.
DESCRIPTION OF CERTAIN EMBODIMENTS
[0032] Various aspects of the disclosure are described below.
DEFINITIONS
[0033] "Antibody" as the term is used herein refers to a protein
that generally comprises heavy chain polypeptides and light chain
polypeptides. Antigen recognition and binding occurs within the
variable regions of the heavy and light chains. Single domain
antibodies having one heavy chain and one light chain and heavy
chain antibodies devoid of light chains are also known. A given
antibody comprises one of five types of heavy chains, called alpha,
delta, epsilon, gamma and mu, the categorization of which is based
on the amino acid sequence of the heavy chain constant region.
These different types of heavy chains give rise to five classes of
antibodies, IgA (including IgA1 and IgA2), IgD, IgE, IgG (IgG1,
IgG2, IgG3 and IgG4) and IgM, respectively. A given antibody also
comprises one of two types of light chains, called kappa or lambda,
the categorization of which is based on the amino acid sequence of
the light chain constant domains. IgG, IgD, and IgE antibodies
generally contain two identical heavy chains and two identical
light chains and two antigen combining domains, each composed of a
heavy chain variable region (V.sub.H) and a light chain variable
region (V.sub.L). Generally IgA antibodies are composed of two
monomers, each monomer composed of two heavy chains and two light
chains (as for IgG, IgD, and IgE antibodies); in this way the IgA
molecule has four antigen binding domains, each again composed of a
V.sub.H and a V.sub.L. Certain IgA antibodies are monomeric in that
they are composed of two heavy chains and two light chains.
Secreted IgM antibodies are generally composed of five monomers,
each monomer composed of two heavy chains and two light chains (as
for IgG and IgE antibodies); in this way the IgM molecule has ten
antigen binding domains, each again composed of a V.sub.H and a
V.sub.L. A cell surface form of IgM also exists and this has two
heavy chain/two light chain structure similar to IgG, IgD, and IgE
antibodies.
[0034] "Chimeric antibody" as the term is used herein refers to an
antibody that has been engineered to comprise at least one human
constant region. For example, one or all the variable regions of
the light chain(s) and/or one or all the variable regions the heavy
chain(s) of a mouse antibody (e.g., a mouse monoclonal antibody)
may each be joined to a human constant region, such as, without
limitation an IgG1 human constant region. Chimeric antibodies are
typically less immunogenic to humans, relative to non-chimeric
antibodies, and thus offer therapeutic benefits in certain
situations. Those skilled in the art will be aware of chimeric
antibodies, and will also be aware of suitable techniques for their
generation. See, for example, Cabilly et al., U.S. Pat. No.
4,816,567; Shoemaker et al., U.S. Pat. No. 4,978,775; Beavers et
al., U.S. Pat. No. 4,975,369; and Boss et al., U.S. Pat. No.
4,816,397, each of which is incorporated herein by reference in its
entirety.
[0035] "Complementarity determining region" or "CDR" as the terms
are used herein refer to short polypeptide sequences within the
variable region of both heavy and light chain polypeptides that are
primarily responsible for mediating specific antigen recognition.
CDRs have been described by Kabat, et al., J. Biol. Chem. 252,
6609-6616 1977; by Chothia, et al., J. Mol. Biol. 196:901-917,
1987; and by MacCallum, et al., J. Mol. Biol. 262:732-745, 1996,
each of which is incorporated herein by reference in its entirety.
There are three CDRs (termed CDR1, CDR2, and CDR3) within each
V.sub.L and each V.sub.H.
[0036] "Fragment" or "antibody fragment" as the terms are used
herein in reference to an antibody refer to a polypeptide derived
from an antibody polypeptide molecule (e.g., an antibody heavy or
light chain polypeptide) that does not comprise a full length
antibody polypeptide, but which still comprises at least a portion
of a full length antibody polypeptide. Antibody fragments often
comprise polypeptides that comprise a cleaved portion of a full
length antibody polypeptide, although the term is not limited to
such cleaved fragments. Since a fragment, as the term is used
herein in reference to an antibody, encompasses fragments that
comprise single polypeptide chains derived from antibody
polypeptides (e.g. a heavy or light chain antibody polypeptides),
it will be understood that an antibody fragment may not, on its
own, bind an antigen. For example, an antibody fragment may
comprise that portion of a heavy chain antibody polypeptide that
would be contained in a Fab fragment; such an antibody fragment
typically will not bind an antigen unless it associates with
another antibody fragment derived from a light chain antibody
polypeptide (e.g., that portion of a light chain antibody
polypeptide that would be contained in a Fab fragment), such that
the antigen-binding site is reconstituted. Antibody fragments can
include, for example, polypeptides that would be contained in Fab
fragments, F(ab').sub.2 fragments, scFv (single chain Fv)
fragments, diabodies, linear antibodies, multispecific antibody
fragments such as bispecific, trispecific, and multispecific
antibodies (e.g., diabodies, triabodies, tetrabodies), minibodies,
chelating recombinant antibodies, tribodies or bibodies,
intrabodies, nanobodies, small modular immunopharmaceuticals
(SMIP), binding-domain immunoglobulin fusion proteins, camelized
antibodies, and V containing antibodies. It will be appreciated
that "antibody fragments" or "antibody polypeptide fragments"
include "antigen-binding antibody fragments" and "antigen-binding
antibody polypeptide fragments." "Antigen-binding antibody
fragments" and "antigen-binding antibody polypeptide fragments"
include, for example, "ILT5-binding antibody fragments" and
"ILT5-binding antibody polypeptide fragments" and "ILT5-binding
fragments."
[0037] "Framework region" as the term is used herein refers to
amino acid sequences within the variable region of both heavy and
light chain polypeptides that are not CDR sequences, and are
primarily responsible for maintaining correct positioning of the
CDR sequences to permit antigen binding. Although the framework
regions themselves typically do not directly participate in antigen
binding, as is known in the art, certain residues within the
framework regions of certain antibodies can directly participate in
antigen binding or can affect the ability of one or more amino
acids in CDRs to interact with antigen. Framework regions are
sometimes referred to as "FR."
[0038] "Humanized antibody" as the term is used herein refers to an
antibody that has been engineered to comprise one or more human
framework regions in the variable region together with non-human
(e.g., mouse, rat, or hamster) complementarity-determining regions
(CDRs) of the heavy and/or light chain. In certain embodiments, a
humanized antibody comprises sequences that are entirely human
except for the CDR regions. Humanized antibodies are typically less
immunogenic to humans, relative to non-humanized antibodies, and
thus offer therapeutic benefits in certain situations. Those
skilled in the art will be aware of humanized antibodies, and will
also be aware of suitable techniques for their generation. See for
example, Hwang, W. Y. K., et al., Methods 36:35, 2005; Queen et
al., Proc. Natl. Acad. Sci. USA, 86:10029-10033, 1989; Jones et
al., Nature, 321:522-25, 1986; Riechmann et al., Nature,
332:323-27, 1988; Verhoeyen et al., Science, 239:1534-36, 1988;
Orlandi et al., Proc. Natl. Acad. Sci. USA, 86:3833-37, 1989; U.S.
Pat. Nos. 5,225,539; 5,530,101; 5,585,089; 5,693,761; 5,693,762;
6,180,370; and Selick et al., WO 90/07861, each of which is
incorporated herein by reference in its entirety.
[0039] "Treg" or "regulatory T cell" as the terms are used herein
refer to a population of T cells that function to suppress
activation of the immune system and thereby maintain immune system
homeostasis and tolerance to self-antigens. While a majority of
regulatory T cells develops in the thymus, peripheral non-Treg
cells can be instructed to commit into the regulatory T cell
lineage as well. Regulatory T cells are enriched among cells
exhibiting a CD4.sup.+CD25.sup.hi or a CD8.sup.+CD28.sup.-
phenotype, and many express the forkhead family transcription
factor (FOXP3). Regulatory T cells are involved in modulating
immune responses in mammalian subjects after their immune systems
have successfully responded to foreign antigens, and are also
involved in regulating immune responses that may potentially attack
the subjects' own tissues, thereby resulting in autoimmune
diseases.
ILT5
[0040] Immunoglobulin-like transcripts (ILTs) are encoded by
rapidly evolving genes found in human and non-human primates.
Immunoglobulin-like transcript 5 ("ILT5") is a cell surface
molecule that is a member of the immunoglobulin superfamily that
includes ILT1, ILT2, ILT3, IL4, ILT5, ILT6, ILT7 and ILT8 (Colonna
et al., J. of Exp. Med., Volume 186, Number 11, 1809-1818, 1997,
incorporate herein by reference in its entirety). It has been
established that the extracellular domain of ILT3, an ILT receptor
with inhibitory function, has the capacity to induce T cell
hyporesponsiveness (see e.g., US Patent Application Publication No.
2008/0038260, incorporated herein by reference in its entirety).
This observation suggests that upon interaction with ILT3, ILT3
ligand expressed by T cells transduces an inhibitory signal.
[0041] ILT5 is also an inhibitory ILT receptor and is highly
expressed on antigen-presenting cells (APCs), such as immature DCs
and monocytes in humans. Human ILT5 gene has been cloned and
characterized (see e.g., Colonna et al., J. of Exp. Med., Volume
186, Number 11, 1809-1818, 1997; Pfistershammer et al., Blood,
September 10; 114(11):2323-32. Epub 2009 Jul. 17; U.S. Pat. No.
6,448,035, each of which is incorporated herein by reference in its
entirety).
Effects of Anti-ILT5 Antibodies and ILT5-Binding Fragments Thereof
on Immune Cells
[0042] Anti-ILT5 antibodies and ILT5-binding fragments thereof for
use in the presently disclosed methods induce T cell proliferation
when the anti-ILT5 antibodies or ILT5-binding fragments are in
monovalent form, but not when the anti-ILT5 antibodies or
ILT5-binding fragments are in polyvalent (crosslinked) form. As
used in reference to anti-ILT5 antibodies and ILT5-binding
fragments described herein, "monovalent" refers to a single
molecule of the anti-ILT5 antibody or ILT5-binding fragment that is
not crosslinked. For example a soluble anti-ILT5 antibody
comprising two antigen-binding sites is "monovalent" as the term is
used herein. Without wishing to be bound by theory, we hypothesize
that upon interaction of ILT5 ligand-expressing T cells with
ILT5-expressing steady state APCs, engagement of the ILT5 ligand by
ILT5 induces inhibitory signals that increase the activation
threshold of T cells. Alternatively, ILT5:ILT5 ligand engagement
may preclude the interaction of ILT5 ligand with an undefined
receptor of lower affinity, which would be immunostimulatory. By
occupying, as well as inducing some internalization of surface ILT5
molecules, monovalent anti-ILT5 antibodies and ILT5-binding
fragments for use in the presently disclosed methods prevent the
ILT5 ligand-ILT5 interaction, thereby either removing inhibitory
signals or allowing activating signals to take place, either of
which would lower the activation threshold of T cells. The fact
that monovalent anti-ILT5 antibodies (rather than antibodies
immobilized on the plastic bottom of tissue culture wells) can be
stimulatory (see Example 3) strongly indicates that ILT5 molecules,
which comprise functional inhibitory motifs in their cytoplasmic
domain, do not function as inhibitory receptors if not
hypercrosslinked. When ILT5 receptors are co-engaged by crosslinked
anti-ILT5 antibodies or ILT5-binding fragments (e.g., those bound
to Fc receptors on APC or to the bottom of plastic tissue culture
wells), they become fully internalized, which precludes ILT5:ILT5
ligand interactions. Furthermore, crosslinked anti-ILT5 antibodies
do not enhance T cell activation. This suggests that crosslinked
anti-ILT5 antibodies can induce an inhibitory cascade in
ILT5-expressing cells (e.g., APCs), which is strong enough to
reprogram APCs such that these cells now display a function that
counteracts the T cell-specific activation signals (i.e. a
tolerogenic function) that occur in the absence of ILT5:ILT5 ligand
interactions. Thus, we hypothesize that as a result of the
integration of activating and inhibitory signals (due to blockade
of ILT5:ILT5 ligand interactions and ILT5 signaling into the APC,
respectively), unchanged (opposing signals of similar strength) or
diminished immunity can be achieved.
[0043] As further described in more detail in the Examples section
below, engagement of ILT5 on APCs with monovalent anti-ILT5
antibodies in autologous as well as allogeneic settings results in
an upregulation of NKG2D in naive T cells exposed to such APCs.
NKG2D is an immune receptor with an important role in tumor and
viral immunity. Moreover, T cells exposed to APCs that had
previously been contacted with the anti-ILT5 antibody maintain
elevated levels of NKG2D under conditions that normally trigger its
internalization. Such conditions typically include contact of NKG2D
with its ligand or with an agonist antibody or fragment that binds
NKG2D. NKG2D internalization aids tumors and intracellular
pathogens (e.g., viruses) in evading recognition by the immune
system. Therefore, extending the time of NKG2D expression by
appropriate cells of the immune system (e.g., CD4+ and CD8+ T
cells) can be advantageous in treating cancer and/or infections
with intracellular pathogens. In addition, T cells that proliferate
as a result of having interacted with APCs that have been contacted
with anti-ILT5 antibodies or ILT5-binding fragments thereof
significantly upregulate surface TCR:CD3 complexes and present with
markedly increased responsiveness to subsequent TCR stimulation.
Upon interaction with APCs that have been or are contacted with the
anti-ILT5 antibody, both CD4.sup.+ and CD8.sup.+ T cells secrete
high levels of Fas-ligand and subsequently exert potent, MHC class
I--as well as Fas-L-dependent, anti-tumor cytotoxic effects.
[0044] In certain embodiments, an anti-ILT5 antibody or an
ILT5-binding antibody fragment for use in the presently disclosed
methods induces a response in a T cell, e.g., a CD4.sup.+ or
CD8.sup.+ T cell, either in vivo or in vitro, when contacted with
an APC, which APC has been or is contacted with the anti-ILT5
antibody or ILT5-binding fragment. In certain embodiments, such a
response is a proliferative response and/or a cytokine/chemokine
producing response. In certain embodiments, a T cell response (e.g.
a proliferative response) is proportional to the amount of
anti-ILT5 antibody or ILT5-binding fragment contacted with the APC.
In certain embodiments, a T cell response (e.g. a proliferative
response) is induced in a T cell that has not been exposed to an
antigen (e.g., a naive T cell). In certain embodiments, the T cell
response (e.g. a proliferative response) is induced in a memory T
cell that has previously been exposed to an antigen. A T cell
response induced by an anti-ILT5 antibody or an ILT5-binding
antibody fragment for use in the presently disclosed methods, e.g.
a proliferative response, occurs in the absence of TCR recognition
of a MHC molecule. Thus, a T cell response mediated by such an
anti-ILT5 antibody or ILT5-binding fragment occurs in a
TCR-independent manner (e.g., the response does not require
recognition of a MHC molecule by a TCR).
[0045] In certain embodiments, a T cell, e.g., a CD4.sup.+ or
CD8.sup.+ T cell, either in vivo or in vitro, upregulates
expression of NKG2D on its surface when the T cell is contacted
with an APC, which APC has been previously contacted with an
anti-ILT5 antibody or an ILT5-binding antibody fragment. As used in
the present context, the term "upregulate" means that the T cell
expresses a protein (e.g., NKG2D on the cell surface) at a higher
level than a control T cell contacted with an APC that has not been
contacted with the anti-ILT5 antibody or an ILT5-binding antibody
fragment. "Upregulates" refers to the condition of expressing more
of a given protein (e.g., NKG2D on the cell surface) when the
control T cell expresses some level of that protein. "Upregulates"
also refers to the condition of expressing any amount, e.g. any
detectable amount, of a given protein (e.g., NKG2D on the cell
surface) when the control T cell does not expresses that protein at
all. In certain embodiments, a T cell maintains expression of NKG2D
on its surface under one or more conditions in which the NKG2D is
typically internalized from the cell surface. Non-limiting examples
of such conditions include engagement of the NKG2D with an NKG2D
ligand expressed on the surface of a cell, engagement of the NKG2D
with a secreted NKG2D ligand, and engagement of the NKG2D with an
antibody or fragment that binds NKG2D.
[0046] In certain embodiments, a T cell, e.g., a CD4.sup.+ or
CD8.sup.+ T cell, either in vivo or in vitro, upregulates
expression of a TCR and CD3 molecules (e.g., a TCR:CD3 complex) on
its surface when the T cell is contacted with an APC, which APC has
been previously contacted with an anti-ILT5 antibody or an
ILT5-binding antibody fragment. In certain embodiments, a T cell
secretes Fas ligand (FasL) at a higher level than a T cell
contacted with an APC that has not been contacted with the
anti-ILT5 antibody or the ILT5-binding fragment.
[0047] In certain embodiments, a T cell or its progeny, e.g., a
CD4.sup.+ or CD8.sup.+ T cell, either in vivo or in vitro,
contacted with an APC, which APC has been previously contacted with
an anti-ILT5 antibody or an ILT5-binding antibody fragment,
exhibits cytotoxic potential. "Cytotoxic potential" as the term is
used herein, refers to the state of being able to acquire cytotoxic
activity when exposed to an antigen. Thus, in certain embodiments,
a T cell or its progeny that exhibits cytotoxic potential (e.g., as
a result if having been contacted with an APC that was previously
contacted with an anti-ILT5 antibody or an ILT5-binding antibody
fragment) is induced to become cytotoxic when exposed to an
antigen. A T cell exhibiting cytotoxic potential (e.g., a T cell or
its progeny contacted with an APC, which APC has been previously
contacted with an anti-ILT5 antibody or an ILT5-binding antibody
fragment) may be induced to cytotoxicity upon exposure to any of a
variety of antigens. For example, such a T cell may be exposed to a
"cellular antigen". As used herein, the term "cellular antigen"
refers to an antigen that can be expressed on or in a cell of a
subject. The"cellular antigen" can be an exogenous antigen, an
endogenous antigen or both. As used herein, the term "exogenous
antigen" refers to an antigen that is administered to a subject.
The term "exogenous antigen" includes foreign, non-endogenous
antigens (see definition of "endogenous antigen" below), as well as
antigens that are identical to antigens present in vivo in the body
of a subject. As used herein, the term "endogenous antigen" refers
to an antigen that is not administered to a subject, e.g., is
present in the body of the subject. In certain embodiments, a
"cellular antigen" is released upon administration of a therapy
(e.g., radiation or a chemotherapeutic agent, as described more
fully below). In certain embodiments, a "cellular antigen"
comprises an antigen present on or in a tumor cell or an
antigen-bearing APC. Alternatively, a "cellular antigen" can be
cell-free, so long as it is capable of being expressed on or in a
cell of a subject. In certain embodiments, the contact with antigen
will generally have occurred at least one hour (e.g., at least two
hours, three hours, five hours, ten hours, 15 hours, 24 hours, two
days, fours days, one week, two weeks, or longer) after the contact
with the anti-ILT5 antibody or ILT5-binding fragment-contacted APC.
Contact with any of a variety of antigens will result in the
transition from having cytotoxic potential to cytotoxicity. In
certain embodiments, a T cell that exhibits cytotoxic potential may
become cytotoxic when it binds to or recognizes a cell that is
cancerous, or a cell that is infected with a bacterium, a virus, a
fungus (including, e.g., a yeast), a protozoan, or a parasite. For
example, a T cell that exhibits cytotoxic potential may become
cytotoxic when it binds to a cell that is infected with a
bacterium, a virus, a fungus, a protozoan, or a parasite. Exemplary
antigens of interest include those derived from infectious agents
and tumor antigens, wherein an immune response directed against the
antigen serves to prevent or treat disease caused by the agent.
Such antigens include, but are not limited to, proteins,
glycoproteins, lipoproteins, glycolipids, and the like. Antigens of
interest also include those which provide therapeutic benefit to a
subject who is at risk of acquiring, or who is diagnosed as having,
a tumor. In certain embodiments, such antigens are administered to
a subject who is at risk of acquiring, or who is diagnosed as
having, a tumor. Appropriate antigens include tumor vaccines,
proteins, markers and the like that are associated with disease. In
certain embodiments, a tumor vaccine introduces a costimulatory
protein with the aim of breaking the tolerogenic tumor
environment.
[0048] Non-limiting examples of tumor antigens include, for
example, tumor-associated glycoprotein TAG-72, HER-2, high M.sub.r
melanoma antigens that bind to the antibody 9.2.27, Lewis-Y-related
carbohydrate (found on epithelial carcinomas), the IL-2 receptor
p55 subunit (expressed on leukemia and lymphoma cells), the
erbB2/p185 carcinoma-related proto-oncogene (overexpressed in
breast cancer), gangliosides (e.g., GM2, GD2, and GD3), epithelial
tumor mucin (i.e., MUC-1), carcinoembryonic antigen, ovarian
carcinoma antigen MOv-18, squamous carcinoma antigen 17-1A, and
malignant melanoma MAGE antigens (e.g., MAGE-1 and MAGE-3), and the
like. Those skilled in the art will be aware of other suitable
tumor antigens that render cytotoxic T cells or their progeny
having cytotoxic potential as described herein.
[0049] Non-limiting examples of viral antigens include, but are not
limited to, the nucleoprotein (NP) of influenza virus and the Gag
proteins of HIV. Other antigens include, but are not limited to,
HIV Env protein or its component parts, gp120 and gp41, HIV Nef
protein, and the HIV Pol proteins, reverse transcriptase and
protease. In addition, other viral antigens such as Ebola virus
(EBOV) antigens, such as, for example, EBOV NP or glycoprotein
(GP), either full-length or GP deleted in the mucin region of the
molecule (Yang Z-Y, et al. (2000) Nat Med 6:886-9, 2000), small pox
antigens, hepatitis A, B or C virus, human rhinovirus such as type
2 or type 14, Herpes simplex virus, poliovirus type 2 or 3,
foot-and-mouth disease virus (FMDV), rabies virus, rotavirus,
influenza virus, coxsackie virus, human papilloma virus (HPV), for
example the type 16 papilloma virus, the E7 protein thereof, and
fragments containing the E7 protein or its epitopes; and simian
immunodeficiency virus (SIV) may be used. An antigen of interest
need not be limited to antigens of viral origin. Parasitic
antigens, such as, for example, malarial antigens are included, as
are fungal antigens, bacterial antigens and tumor antigens.
Non-limiting examples of bacterial antigens include: Bordetella
pertussis (e.g., P69 protein and filamentous haemagglutinin (FHA)
antigens), Vibrio cholerae, Bacillus anthracis, E. coli antigens
such as E. coli heat Labile toxin B subunit (LT-B), E. coli K88
antigens, and enterotoxigenic E. coli antigens, the Y.
enterocolitica heat shock protein 60 (Mertz et al., J. Immunol.
164(3):1529-1537, 2000)M. tuberculosis heat-shock proteins hsp60
and hsp70, Chlamydia trachomatis outer membrane protein (Ortiz et
al., Infect. Immun. 68(3):1719-1723, 2000), B. burgdorferi outer
surface protein (Chen et al., Arthritis Rheum. 42(9):1813-1823,
1999); L. major GP63 (White et al., Vaccine 17(17):2150-2161, 1999
(and published erratum in Vaccine 17(20-21):2755)), N. meningitidis
meningococcal serotype 15 PorB protein (Delvig et al., Clin.
Immunol. Immunopathol. 85(2); 134-142, 1997), P. gingivalis 381
fimbrial protein (Ogawa, J. Med. Microbiol. 41(5):349-358, 1994),
E. coli outer membrane protein F (Williams et al., Infect. Immun.
68(5):2535-2545, 2000). Other examples of microbial antigens
include Schistosoma mansoni P28 glutathione S-transferase antigens
(P28 antigens) and antigens of flukes, mycoplasma, roundworms,
tapeworms, Chlamydia trachomatis, and malaria parasites, e.g.,
parasites of the genus plasmodium or babesia, for example
Plasmodium falciparum, and peptides encoding immunogenic epitopes
from the aforementioned antigens. Each of the references disclosed
above is incorporated herein by reference in its entirety.
[0050] Any of a variety of anti-ILT5 antibodies or ILT5-binding
antibody fragments that mediate an indirect immunostimulatory
effect and/or proliferative response on naive CD4.sup.+ or naive
CD8.sup.+ T cells when such T cells are contacted with an APC that
has previously been contacted with the anti-ILT5 antibody or
ILT5-binding fragment (e.g., a monovalent form of the anti-ILT5
antibody or ILT5-binding fragment) can be used in the presently
disclosed methods. Moreover, any of a variety of anti-ILT5
antibodies or ILT5-binding antibody fragments that results in
upregulation of NKG2D or TCR:CD3 complexes in T cells (e.g., naive
T cells) exposed to such APCs can be used in the presently
disclosed methods. Moreover, any of a variety of anti-ILT5
antibodies or ILT5-binding antibody fragments that endow a T cell
or its progeny with cytotoxic potential when the T cell is
contacted with an APC that has previously been contacted with the
anti-ILT5 antibody or ILT5-binding fragment (e.g., a monovalent
form of the anti-ILT5 antibody or ILT5-binding fragment) can be
used in the presently disclosed methods. Those of ordinary skill in
the art will be able to choose suitable anti-ILT5 antibodies or
ILT5-binding fragments thereof for use in the presently disclosed
methods. For example, anti-IL5 antibodies and ILT5-binding
fragments that can be used in the presently disclosed methods
include, but are not limited to, the anti-IL5 antibodies and
ILT5-binding fragments described below, e.g., those comprising one
or more of SEQ ID NOs: 1-32.
Inhibition of T Cell Responses
[0051] In contrast to the immunoenhancing effects described above,
APCs that have been previously contacted with crosslinked anti-ILT5
antibodies may be tolerogenic since T cells interacting with such
APCs do not mount the T cell response that is observed when
monovalent antibody is used. Thus, in certain embodiments, an
anti-ILT5 antibody or ILT5-binding fragment thereof for use in the
presently disclosed methods might inhibit a response in a T cell,
e.g., a CD4.sup.+ or CD8.sup.+ T cell, either in vivo or in vitro,
when contacted with an APC, which APC has been contacted with a
crosslinked form of the ant-ILT5 antibody or ILT5-binding fragment,
the T cell being contacted with antigen at the same time as or very
close in time to the contact of the T cell with the APC previously
contacted with the anti-ILT5 antibody or ILT5-binding fragment. As
used in reference to inhibition of T cell responses, "very close in
time" means within a timeframe where the pharmacodynamic effects of
the anti-ILT5 antibodies on the APCs are still exerted. Without
wishing to be bound by any particular theory, it is hypothesized
that factors such as, but not limited to, the half-life of
anti-ILT5 antibodies or ILT5-binding fragments thereof will be
important in determining what such timeframe will be. In certain
embodiments, such a response is a proliferative response. In
certain embodiments, an inhibition of a T cell response (e.g. a
proliferative response) is proportional to the amount of
crosslinked anti-ILT5 antibody or ILT5-binding fragment contacted
with the APC. In certain embodiments, a T cell response (e.g. a
proliferative response) is inhibited when the anti-ILT5 antibody or
ILT5-binding fragment crosslinks or hypercrosslinks ILT5. As
described herein, such hypercrosslinking occurs when the ant-ILT5
antibody or ILT5-binding fragment is in polyvalent form (e.g.,
bound to a solid support or to Fc receptors (in vivo)), but not
when the anti-ILT5 antibody or ILT5-binding fragment is monovalent
form.
[0052] In certain embodiments, an anti-ILT5 antibody or
ILT5-binding antibody fragment for use in the presently disclosed
methods is used to induce tolerance in a subject (e.g., a human).
For example, an anti-ILT5 antibody or ILT5-binding antibody
fragment can be administered to a subject at the same time as or
very close in time to an antigen of interest.
Treatment of Diseases and Infections
[0053] Treatment Via Induction or Enhancement of a T Cell
Response
[0054] Monovalent anti-ILT5 antibodies and ILT5-binding antibody
fragments such as those described in the section entitled "Effects
of Anti-ILT5 Antibodies and ILT5-Binding Fragments Thereof on
Immune Cells" indirectly activate both CD4.sup.+ and CD8.sup.+ T
cells in a TCR-independent manner (e.g., activation does not
require recognition of a MHC molecule by a TCR), when such T cells
are contacted with an APC that has been or still is contacted with
the anti-ILT5 antibody or ILT5-binding fragment. Such T cells or
their progeny exhibit cytotoxic potential, which cytotoxic
potential can be exploited in the treatment of certain conditions
by exposing the T cells or their progeny to an antigen, thus
rendering the cells cytotoxic. Thus, such anti-ILT5 antibodies and
ILT5-binding fragments thereof may be used to treat any of a
variety of conditions in a subject (e.g., a human), including but
not limited to cancers and infections.
[0055] In certain embodiments, anti-ILT5 antibodies and
ILT5-binding fragments thereof for use in the presently disclosed
methods may be used to treat any of a variety of cancers in a
subject. Cancers are characterized by uncontrolled, abnormal growth
of cells, and include all types of hyperproliferative growth,
hyperplastic growth, oncogenic processes, metastatic tissues or
malignantly transformed cells, tissues, or organs, irrespective of
histopathologic type or stage of invasiveness. Cancers that can be
treated include, but are not limited to, pancreatic cancer,
melanomas, breast cancer, lung cancer, bronchus cancer, colorectal
cancer, prostate cancer, pancreas cancer, stomach cancer, ovarian
cancer, urinary bladder cancer, peripheral nervous system cancer,
esophageal cancer, cervical cancer, uterine or endometrial cancer,
cancer of the oral cavity or pharynx, liver cancer, kidney cancer,
testicular cancer, biliary tract cancer, small bowel or appendix
cancer, salivary gland cancer, thyroid gland cancer, adrenal gland
cancer, osteosarcoma, chondrosarcoma, and cancers of hematological
tissues.
[0056] In certain embodiments, an anti-ILT5 antibody or an
ILT5-binding fragment thereof is used in combination with another
therapy to treat cancer. For example, an anti-ILT5 antibody or
ILT5-binding fragment thereof may be used in combination with
radiation therapy. Additionally or alternatively, an anti-ILT5
antibody or ILT5-binding fragment may be used in combination with a
chemotherapeutic agent. In certain embodiments, the therapy is
administered, at least once, at the same time as or very close in
time to administration of the anti-ILT5 antibody or the
ILT5-binding fragment. As used in reference to a therapy
administered in combination with an anti-ILT5 antibody or
ILT5-binding fragment, "very close in time" means within a
timeframe within which T cells display pharmacodynamic effects due
to the anti-ILT5 antibodies or ILT5-binding fragments.
[0057] The timing of therapy administration relative to that of the
anti-ILT5 antibody or ILT5-binding fragment administration will
take into account several parameters: On one hand, factors that are
known to influence the in vivo kinetics of antigen-specific T cell
activation and responses, which are initiated in secondary lymphoid
tissues (e.g. lymph nodes) through contacts between T cells and
antigen-bearing DCs, will be considered. For instance, the
abundance, immunogenicity and availability of an antigen are
obviously important and dictate whether T cell responses will be
initiated, as well as their overall strength and duration. The
affinity of a single TCR for a peptide:WIC complex is another
parameter. In this regard and without wishing to be bound by any
particular theory, we hypothesize that by increasing TCR:CD3
complexes on the surface of T cells, anti-ILT5 antibodies or
ILT5-binding fragments thereof increase the avidity of T cells for
antigen-bearing DCs and thereby not only permit the recruitment of
T cells with low affinity TCRs into a response in which these T
cells would otherwise not have participated but also accelerate the
overall kinetics of T cell activation. The kinetics of T cell
activation also depend on whether DCs acquire the antigen of
interest within or outside the lymph nodes (e.g., in the blood
stream or a peripheral non-lymphoid tissue). In the latter case,
the time required for antigen-bearing DCs to traffic to the lymph
nodes increases the timing within which T cell activation occurs
relative to the time of antigen exposure. On the other hand, in
vitro data show that concomitant TCR stimulation and exposure of
CD4.sup.+ and CD8.sup.+ T cells to anti-ILT5 antibody abrogates
anti-ILT5 antibody-induced T cell responsiveness (see FIG. 13).
Thus and without wishing to be bound by any particular theory, it
is hypothesized that it is desirable to allow the anti-ILT5
antibodies or ILT5-binding fragments to exert their pharmacodynamic
effects on T cells prior to the initiation of antigen-specific T
cell activation, which activation is induced through antigen
exposure by means of therapy administration. In addition, it is
desirable to administer the anti-ILT5 antibody or ILT5-binding
fragment closer in time to the antigen of interest when the
anti-ILT5 antibody or ILT5-binding fragment has a relatively short
half-life. Determining the half-life of administered antibodies or
antigen-binding fragments is routine in the art, and can be
accomplished by a variety of methods including, but not limited to,
measuring the amount of antibody or fragment in serum levels (e.g.,
by ELISA) at various times post administration and fitting such
measurements to a half-life curve. Other factors that can influence
the length of time between administrations include, without
limitation, a patient's physiological reaction or lack thereof to
the anti-ILT5 antibody or ILT5-binding fragment, the nature of the
therapy and practical considerations such as minimizing the number
of clinic visits. Those skilled in the art are aware of the above
factors and will be able to determine which timing of therapy
administration is appropriate. In certain embodiments a therapy
(e.g. radiation or a chemotherapeutic agent) is administered
subsequent to administration of the anti-ILT5 antibody or
ILT5-binding fragment. For example, a therapy can be administered
several hours or days after the anti-ILT5 antibody or ILT5-binding
fragment. In other embodiments, a therapy (e.g. radiation or a
chemotherapeutic agent) is administered prior to or concomitantly
with administration of the anti-ILT5 antibody or ILT5-binding
fragment. For example, a therapy can be administered at the same
time as, or several hours or days prior to the anti-ILT5 antibody
or ILT5-binding fragment.
[0058] In certain embodiments, the therapy consists of a bone
marrow/hematopoietic cell transplant (BMT/HCT) in a subject with a
hematopoietic tumor. In such embodiments, anti-ILT5 antibodies or
ILT5-binding fragments thereof may be administered in a subject
showing signs of tumor recurrence, thus after to the therapy
(BMT/HCT). In the latter case and without wishing to be bound by
theory, it is hypothesized that anti-ILT5 antibodies or
ILT5-binding fragments thereof will induce and/or enhance T cell
responses including those targeting tumor cells. In such
embodiments, anti-ILT5 antibodies or ILT5-binding fragments thereof
are administered anytime after tumor relapse is diagnosed.
[0059] In certain embodiments, administration of a therapy (e.g.,
radiation or a chemotherapeutic agent) inhibits or prevents the
function of cells and/or causes destruction of cells, e.g., acts to
lyse or otherwise disrupt (e.g., by inducing apoptosis) cells that
comprise an antigen of interest, thereby providing a source of
antigen that will activate the cytotoxic potential of a T cell or
its progeny that has interacted with an APC contacted with an
anti-ILT5 antibody or an ILT5-binding fragment thereof. In certain
embodiments, a therapy releases a cellular antigen from a cell
(e.g., a tumor cell or a cell infected with a virus), thereby
providing a source of antigen that will activate the cytotoxic
potential of a T cell that has interacted with an APC contacted
with an anti-ILT5 antibody or an ILT5-binding fragment thereof. In
certain embodiments, a therapy (e.g. a tumor vaccine) introduces a
substantial amount of a tumor antigen, wherein the antigen does not
induce sufficient immunity to eradicate tumor cells when expressed
endogenously in the tumor environment. In certain embodiments, the
tumor vaccine can also introduce costimulatory molecules. When the
therapy is administered prior to the antibody or ILT5-binding
fragment, it will be understood that the kinetics of the
antigen-mediated T cell response may be slower than the kinetics of
anti-ILT5 antibody-mediated T cell responses, as described below,
and thus cytotoxicity of the T cell towards such an antigen is
induced or enhanced. In certain embodiments, anti-ILT5 antibodies
and ILT5-binding antibody fragments thereof may be used to treat a
subject suffering from a tumor. In certain embodiments, a tumor is
poorly immunogenic or even tolerogenic. In such embodiments, an
antigen of interest that may potentially trigger cytotoxicity of a
T cell as described herein may also be poorly immunogenic or even
tolerogenic, e.g., as a result of being in a tumor environment. In
certain embodiments, administration of a therapy (e.g., a
chemotherapeutic agent or radiation therapy) as described above
releases one or more antigens from the tumor in a manner such that
the antigen is no longer poorly immunogenic or tolerogenic. As a
result, APCs that have previously been bound by and anti-ILT5
antibodies or an ILT5-binding antibody fragment may bind T cells,
which T cells can be induced to become cytotoxic upon binding or
recognizing an antigen released by the therapy. In certain
embodiments, a tumor cell expresses ILT5 receptors, which bind to
ILT5 ligands on a T cell that recognizes an antigen on the tumor
cell. In such embodiments, ILT5:ILT5 ligand interactions prohibit
the activation of the T cell that is bound to the tumor cell. Thus
in certain embodiments, administration of anti-ILT5 antibodies or
ILT5-binding fragments thereof, with or without therapy, prevents
ILT5:ILT5 ligand interactions between the T cell and the tumor
cell, which permits the activation of the T cell that is bound to
the tumor cell.
[0060] In certain embodiments, a therapy is administered until a
desired endpoint is reached. For example, a therapy may be
administered until a desired level of inhibition of tumor growth,
reduction in tumor size, reduction in the number of tumors,
decrease in tumor burden, and/or prolonging of survival time is
reached. Those skilled in the art will be aware of these and other
desired endpoints, and will be able to determine when such an
endpoint is reached using standard methods.
[0061] A variety of radiation therapies are known in the art,
including for example, external beam radiotherapy (EBRT or XBRT) or
teletherapy which is applied from outside the body, brachytherapy
or sealed source radiotherapy in which sealed radioactive sources
are placed in the area under treatment, and systemic radioisotope
therapy or unsealed source radiotherapy which is administered by
infusion or oral ingestion. A variety of external beam
radiotherapies are known, including but not limited to,
conventional 2D external beam radiotherapy (2DXRT), stereotactic
radiation, 3-dimensional conformal radiotherapy (3DCRT), and
intensity-modulated radiation therapy (IMRT). Brachytherapy can
employ temporary or permanent placement of radioactive sources.
Those skilled in the art will be aware of these and other radiation
therapies and will be able to appropriately administer them.
[0062] A variety of chemotherapeutic agents are known in the art.
In certain embodiments, a chemotherapeutic agent used in
combination with an anti-ILT5 antibody or ILT5-binding antibody
fragment is an antimetabolite. Non-limiting examples of
anti-metabolites include Aminopterin, Methotrexate, Pemetrexed,
Raltitrexed, Cladribine, Clofarabine, Fludarabine, Mercaptopurine,
Pentostatin, Thioguanine, Capecitabine, Cytarabine, Fluorouracil,
Floxuridine, and Gemcitabine. In certain embodiments, an
antimetabolite is a nucleoside analogue such as, without
limitation, gemcitabine or fluorouracil. In certain embodiments, a
chemotherapeutic agent used in combination with an anti-ILT5
antibody or an ILT5-binding fragment thereof is an agent that
affects microtubule formation. Non-limiting examples of agents that
affects microtubule formation include paclitaxel, docetaxel,
vincristine, vinblastine, vindesine, vinorelbin, taxotere,
etoposide, and teniposide. In certain embodiments, the
chemotherapeutic agent used in combination with an anti-ILT5
antibody or an ILT5-binding fragment thereof is an alkylating agent
such as, e.g., cyclophosphamide. In certain embodiments, a
chemotherapeutic agent used in combination with an anti-ILT5
antibody or an ILT5-binding fragment thereof is a cytotoxic
antibiotic, e.g., a topoisomerase II inhibitor such as doxorubicin.
In certain embodiments, a chemotherapeutic agent comprises a toxin
such as a small-molecule toxin or an enzymatically active toxin of
bacterial, fungal, plant, or animal origin, or fragments thereof.
In certain embodiments. a chemotherapeutic agent used in
combination with an anti-ILT5 antibody or ILT5-binding fragment is
an anti-angiogenic agent such as e.g. avastin. In certain
embodiments. a chemotherapeutic agent used in combination with an
anti-ILT5 antibody or ILT5-binding fragment thereof is a biologic
agent such as e.g. herceptin.
[0063] In certain embodiments, anti-ILT5 antibodies and
ILT5-binding fragments thereof for use in the presently disclosed
methods may be used to treat or prevent (e.g. in combination with
vaccination) any of a variety of infections in a subject. Exemplary
infections include any of a variety of bacterial (e.g., an
intracellular bacterium), viral, fungal, protozoan, or parasitic
(e.g., an intracellular parasitic) infections. Viral infections
that can be treated include, but are not limited to, infections
caused by HIV (e.g., HIV-1 and HIV-2), human herpes viruses,
cytomegalovirus (esp. human), rotavirus, epstein-barr virus,
varicella zoster virus, hepatitis viruses, such as hepatitis B
virus, hepatitis A virus, hepatitis C virus and hepatitis E virus,
paramyxoviruses: respiratory syncytial virus, parainfluenza virus,
measles virus, mumps virus, human papilloma viruses (for example
HPV6, 11, 16, 18 and the like), flaviviruses (e.g. yellow fever
virus, dengue virus, tick-borne encephalitis virus, Japanese
encephalitis virus), and influenza virus.
[0064] Bacterial infections include, but are not limited to,
infections caused by Neisseria spp, including N. gonorrhea and N.
meningitidis, Streptococcus spp, including S. pneumoniae, S.
pyogenes, S. agalactiae, S. mutans; Haemophilus spp, including H.
influenzae type B, non typeable H. influenzae, H. ducreyi;
Moraxella spp, including M. catarrhalis, also known as Branhamella
catarrhalis; Bordetella spp, including B. pertussis, B.
parapertussis and B. bronchiseptica; Mycobacterium spp., including
M. tuberculosis, M. bovis, M. leprae, M. avium, M.
paratuberculosis, M. smegmatis; Legionella spp, including L.
pneumophila; Escherichia spp, including enterotoxic E. coli,
enterohemorragic E. coli, enteropathogenic E. coli; Vibrio spp,
including V. cholera, Shigella spp, including S. sonnei, S.
dysenteriae, S. flexnerii; Yersinia spp, including Y.
enterocolitica, Y. pestis, Y. pseudotuberculosis, Campylobacter
spp, including C. jejuni and C. coli; Salmonella spp, including S.
typhi, S. paratyphi, S. choleraesuis, S. enteritidis; Listeria
spp., including L. monocytogenes; Helicobacter spp, including H.
pylori; Pseudomonas spp, including P. aeruginosa, Staphylococcus
spp., including S. aureus, S. epidermidis; Enterococcus spp.,
including E. faecalis, E. faecium; Clostridium spp., including C.
tetani, C. botulinum, C. difficile; Bacillus spp., including B.
anthracis; Corynebacterium spp., including C. diphtherias; Borrelia
spp., including B. burgdorferi, B. garinii, B. afzelii, B.
andersonii, B. hermsii; Ehrlichia spp., including E. equi and the
agent of the Human Granulocytic Ehrlichiosis; Rickettsia spp,
including R. rickettsii; Chlamydia spp., including C. trachomatis,
C. neumoniae, C. psittaci; Leptsira spp., including L. interrogans;
Treponema spp., including T. pallidum, T. denticola, and T.
hyodysenteriae.
[0065] In certain embodiments, an anti-ILT5 antibody or an
ILT5-binding fragment thereof for use in the presently disclosed
methods can be used to induce and/or enhance an immune response in
a subject. In such embodiments, the anti-ILT5 antibody or
ILT5-binding fragment acts as an adjuvant by inducing or enhancing
an immune response (e.g., a cytotoxic cell immune response) against
an antigen of interest. For example, a subject may first be
administered an anti-ILT5 antibody or an ILT5-binding fragment
thereof without concomitant administration of an antigen of
interest. Without wishing to be bound by theory, it is hypothesized
that such administration endows certain naive T cells in the
subject with cytotoxic potential. Subsequently, a subject may be
administered an antigen of interest. Again without wishing to be
bound by theory, it is hypothesized that certain T cells exhibiting
cytotoxic potential are rendered cytotoxic to cells expressing that
antigen of interest upon exposure to the antigen. Thus, in certain
embodiments, cytotoxicity of at least one T cell in the subject
towards a cell that comprises the antigen is induced or enhanced.
In certain embodiments, one or more additional antigens are
provided by direct administration of the antigen to the subject. In
certain embodiments, an antigen provided by direct administration
is the same antigen as is released from a lysed or disrupted cell
upon administration of a therapy.
[0066] As discussed previously, a suitable length of time between
administration of the anti-ILT5 antibody or ILT5-binding fragment
and the antigen of interest will depend on various factors, each of
which can be determined by those skilled in the art. In certain
embodiments, the anti-ILT5 antibody or ILT5-binding fragment can be
administered 1-14 days (e.g., 3 days) before administration of the
antigen of interest. In certain embodiments, an antigen is
administered prior to or concomitantly with administration of the
anti-ILT5 antibody or ILT5-binding fragment. For example, a therapy
can be administered as the same time as, or several hours or days
prior to the anti-ILT5 antibody or ILT5-binding fragment.
[0067] In certain embodiments, a subject may be administered an
anti-ILT5 antibody or an ILT5-binding antibody fragment at or about
the same time as an antigen of interest to induce or enhance an
immune response in a subject. As described in more detail herein, a
proliferative T cell response is induced in vitro in allogeneic
mixed lymphocyte cultures that simultaneously contain both an
anti-ILT5 antibody and a foreign antigen present on a foreign
lymphocyte. In contrast, anti-ILT5 antibody-induced proliferation
of a T cell simultaneously exposed an anti-CD3 antibody-mediated
stimulation and an APC that has been affected by an anti-ILT5
antibody is inhibited. Without wishing to be bound by theory, it is
possible to reconcile these results by the differential kinetics of
the antigen-mediated and anti-ILT5 antibody-mediated T cell
responses. T cell proliferation in allogeneic mixed lymphocyte
reactions typically occurs at day 5-9 due to the fact that it takes
some time for the T cells to establish stable contacts with APCs,
which is an essential component of the T cell activation process.
In contrast, anti-ILT5 mediated T cell proliferation is detected as
soon as 24 hours, and peaks at day 3-3.5, similar to stimulation of
T cell proliferation observed with anti-CD3 antibodies. Thus, the
kinetics of the antigen-mediated T cell responses are slower than
the kinetics of anti-ILT5 antibody-mediated T cell responses.
Another possibility, again without wishing to be bound by theory,
is that one or more ILT5 ligands and TCR complexes in the mixed
lymphocyte culture compete for a molecule that is involved in the
observed T cell proliferation.
[0068] In certain embodiments, the subject is also administered an
adjuvant (e.g., administered with the antigen of interest) to
bolster the subject's immune response against the antigen. Suitable
adjuvants include, without limitation, CpG, alum, oil-in-water
emulsions (e.g., MF59.TM. (a sub-micron oil-in-water emulsion of a
squalene, polyoxyethylene sorbitan monooleate, and sorbitan
trioleate), Montanide (Seppic), Adjuvant 65, Lipovant), immune
stimulating complexes or ISCOMs (honeycomb-like structures composed
of typically Quillaja saponins, cholesterol, and phospholipids),
QS-21 (a natural product of the bark of the Quillaja saponaria tree
species), and inulin-based adjuvants.
[0069] In certain embodiments, an anti-ILT5 antibody or
ILT5-binding antibody fragment is used to induce or enhance an
immunostimulatory response in a T cell in vitro, which T cell is
then administered to a subject, either alone or in combination with
one or more therapeutic agents. For example, PBMCs from a given
subject can be cultured with an anti-ILT5 antibody or an
ILT5-binding fragment thereof. In these cultures, T cells (e.g., a
CD4.sup.+ or CD8.sup.+ T cell) will interact with APCs that have
been contacted with an anti-ILT5 antibody or ILT5-binding fragment
thereof and they or their progeny will acquire a cytotoxic
potential. The T cell can then be contacted with an antigen of
interest, rendering the T cell cytotoxic to cells having the
antigen of interest on their surface. This contacting with antigen
can be in vitro prior to the administration of the cells to the
subject or it can be in vivo after the administration of the cells
to the subject. The cytotoxicity of T cells generated by such
methods can be evaluated by those skilled in the art according to
routine methods. T cells that exhibit suitable cytotoxicity can be
be infused in the subject they came from to treat a disease or
condition associated with the antigen of interest. Alternatively, a
T cell having cytotoxic potential as a result of having been
contacted with an APC previously contacted with an anti-ILT5
antibody or ILT5-binding fragment thereof described herein in an
autologous setting, or its progeny having such cytotoxic potential,
may be infused in the subject it came from, wherein the T cell
become cytotoxic upon exposure to an antigen in vivo. In certain
embodiments, only CD4.sup.+ T cells are made cytotoxic and infused
in a subject. In certain embodiments, only CD8.sup.+ T cells are
made cytotoxic and infused in a subject. In certain embodiments, a
population of T cells comprising both CD4.sup.+ and CD8.sup.+ T
cells is made cytotoxic and infused in a subject.
[0070] In certain embodiments, a cell is obtained from a subject
and a nucleic acid molecule encoding an anti-ILT5 antibody or
ILT5-binding antibody fragment is introduced into the cell. For
example, the nucleic acid molecule may be operatively linked to a
promoter or other sequence that mediates expression of the
anti-ILT5 antibody or the ILT5-binding fragment in that cell. In
certain embodiments, the nucleic acid molecule may also comprise
one or more sequences encoding a polypeptide moiety that mediates
secretion, which sequences are operatively linked (e.g., in frame)
to the sequences encoding the ant-ILT5 antibody or the ILT5-binding
fragment, such that the anti-ILT5 antibody or ILT5-binding fragment
is secreted from the cell. Such cells obtained from a subject and
containing the introduced nucleic acid molecule, or the progeny of
such cells, can then be introduced back into the subject, such that
the cell secretes the anti-ILT5 antibody or ILT5-binding antibody
fragment in vivo. Naturally, where the progeny of the cells
obtained from the subject are used, they also should contain and
express the nucleic acid molecule. The cells to be administered
back to the subject can optionally be treated so as to prevent or
inhibit their proliferation after administration. They can be
treated with, for example, an appropriate dose of ionizing
radiation (e.g., x- or gamma-irradiation) or a drug such as
mitomycin-C.
[0071] As will be appreciated by those skilled in the art upon
reading the present disclosure, an anti-ILT5 antibody or
ILT5-binding antibody fragment produced by cells as described above
(e.g., produced by ex vivo methods) will be useful in inducing T
cell proliferation, and in inducing T cells to exhibit cytotoxic
potential and/or cytotoxicity.
[0072] Treatment Via Inhibition of a T Cell Response
[0073] In certain embodiments, an anti-ILT5 antibody or an
ILT5-binding antibody fragment for use in the presently disclosed
methods is used to induce tolerance as described above in order to
treat an immune-related disease. "Immune-related disease" as the
term is used herein refers to a disease that is associated with at
least one abnormal immune phenomenon. For example, one class of
immune-related diseases comprises autoimmune diseases. An
autoimmune disease typically results when the subject's immune
system is activated against one or more components (cells, tissues,
or cell/tissue-free molecules) of the subject and attacks that
subject's own organs, tissues or cells, instead of attacking, for
example, foreign bacteria, viruses and other infectious agents or
cancer cells. Every mammalian subject exhibits autoimmunity to some
extent, but such autoimmunity normally does not result in a disease
state since the immune system regulates and suppresses normal
autoimmunity. Autoimmune diseases develop when there is a
disruption in the immune system's regulation. Autoimmune diseases
can also result when there is a molecular alteration in a subject's
cell that is recognized by the immune system, such that the immune
system recognizes the altered cell as "foreign."
[0074] Another example of an immune-related disease is a disease
associated with the effects of organ, tissue, or cell
transplantation. Transplanted cells rarely exhibit that same
antigens on their surfaces as the recipient subject's endogenous
cells. Thus, a transplant subject's immune system often attacks and
rejects transplanted solid tissues, which can lead to organ failure
or other serious systemic complications. Certain immunosuppressive
drugs are typically used to mediate or prevent these immune
attacks, but such drugs often cause undesirable side effects,
including for example, the risk of developing opportunistic
infections as a result of decreased immune responses. Exemplary
immune-related diseases include, but are not limited to, adrenergic
drug resistance, alopecia areata, ankylosing spondylitis,
antiphospholipid syndrome, autoimmune Addison's disease, autoimmune
diseases of the adrenal gland, allergic encephalomyelitis,
autoimmune hemolytic anemia, autoimmune hepatitis, autoimmune
inflammatory eye disease, autoimmune neonatal thrombocytopenia,
autoimmune neutropenia, autoimmune oophoritis and orchitis,
autoimmune thrombocytopenia, autoimmune thyroiditis, Behcet's
disease, bullous pemphigoid, cardiomyopathy, cardiotomy syndrome,
celiac sprue-dermatitis, chronic active hepatitis, chronic fatigue
immune dysfunction syndrome (CFIDS), chronic inflammatory
demyelinating polyneuropathy, Churg-Strauss syndrome, cicatrical
pemphigoid, CREST syndrome, cold agglutinin disease, Crohn's
disease, dense deposit disease, diseases associated with effects
from organ transplantation, discoid lupus, essential mixed
cryoglobulinemia, fibromyalgia-fibromyositis, glomerulonephritis
(e.g., IgA nephrophathy), gluten-sensitive enteropathy,
Goodpasture's syndrome, GVHD, Graves' disease (including e.g.,
Graves thyroiditis and Graves ophthalmopathy), Guillain-Barre,
hyperthyroidism (i.e., Hashimoto's thyroiditis), idiopathic
pulmonary fibrosis, idiopathic Addison's disease, idiopathic
thrombocytopenia purpura (ITP), IgA neuropathy, Insulin Resistance
Syndrome, juvenile arthritis, lichen planus, lupus erythematosus,
Meniere's disease, Metabolic Syndrome, mixed connective tissue
disease, multiple sclerosis, Myasthenia Gravis, myocarditis,
diabetes (e.g., Type I diabetes or Type II diabetes), neuritis,
other endocrine gland failure, pemphigus vulgaris, pernicious
anemia, polyarteritis nodosa, polychrondritis,
Polyendocrinopathies, polyglandular syndromes, polymyalgia
rheumatica, polymyositis and dermatomyositis, post-MI, primary
agammaglobulinemia, primary biliary cirrhosis, psoriasis, psoriatic
arthritis, Raynauld's phenomenon, relapsing polychondritis,
Reiter's syndrome, rheumatic heart disease, rheumatoid arthritis,
sarcoidosis, scleroderma, Sjogren's syndrome, stiff-man syndrome,
systemic lupus erythematosus, takayasu arteritis, temporal
arteritis/giant cell arteritis, ulcerative colitis, urticaria,
uveitis, Uveitis Ophthalmia, vasculitides such as dermatitis
herpetiformis vasculitis, vitiligo, and Wegener's
granulomatosis.
[0075] In certain embodiments, a crosslinked anti-ILT5 antibody or
an ILT5-binding antibody fragment is administered in combination
with another therapeutic agent that induced tolerance. A
non-limiting example of such a tolerance-inducing therapeutic agent
is an antibody or antigen-binding fragment thereof that binds CD3,
e.g., otelixizumab (Keymeulen B, et al. N Engl J Med.;
352:2598-2608, 2005 incorporated herein by reference in its
entirety. Other anti-CD3 antibodies include, without limitation,
hOKT3 (humanized (IgG1 or IgG4) anti-human CD3), HUM291 (humanized
(IgG2) anti-human CD3; visilizumab; NUVIONTM), UCHT1 (mouse (IgG1)
anti-human CD3), Leu4 (mouse (IgG1) anti-human CD3), 500A2 (hamster
(IgG) anti-mouse CD3), CLB-T3/3 (mouse (IgG2a) anti-human CD3),
BMA030 (mouse (IgG2a) anti-human CD3), YTH 12.5 (rat (IgG2b)
anti-human CD3), and NI-0401 (fully human anti-human CD3). Those
skilled in the art will be aware of other anti-CD3 antibodies and
fragments that can be used in combination with anti-ILT5 antibodies
and ILT5-binding fragments thereof disclosed herein.
[0076] In certain embodiments, an anti-ILT5 antibody or an
ILT5-binding antibody fragment that is administered to a subject is
crosslinked or otherwise aggregated. As indicated above, without
wishing to be bound by any particular theory, it is hypothesized
that co-engagement of ILT5 receptors by crosslinked anti-ILT5
antibodies and ILT5-binding fragments described herein, initiates
an inhibitory cascade in ILT5-expressing APCs, which renders them
tolerogenic.
[0077] Treatment Generally
[0078] In certain embodiments, an anti-ILT5 antibody or
ILT5-binding antibody fragment is administered to a subject
directly. Routes of administration are described in more detail in
the section entitled "Pharmaceutical Compositions." A
therapeutically active amount of an anti-ILT5 antibody or
ILT5-binding fragment can be administered in an amount effective,
at dosages and for periods of time necessary to achieve the desired
result. For example, a therapeutically active amount of anti-ILT5
antibody or ILT5-binding fragment thereof may vary according to
factors such as the disease state, age, sex, and weight of the
subject, and the ability of the anti-ILT5 antibody or ILT5-binding
fragment to elicit a desired response in the subject. Dosage
regimens can be adjusted to provide the optimum therapeutic
response. For example, several divided doses can be administered
daily or the dose can be proportionally reduced as indicated by the
exigencies of the therapeutic situation. Those skilled in the art
will be aware of dosages and dosing regimens suitable for
administration of an anti-ILT5 antibody or ILT5-binding fragment to
a subject. See e.g., Physicians' Desk Reference, 63rd edition,
Thomson Reuters, Nov. 30, 2008, incorporated herein by reference in
its entirety.
[0079] Those skilled in the art will be aware of other diseases and
conditions that can be treated using any of a variety of anti-ILT5
antibodies and ILT5-binding fragments.
Pharmaceutical Formulations
[0080] Anti-ILT5 antibodies or ILT5-binding antibody fragments
described herein may be formulated for delivery by any available
route including, but not limited to parenteral (e.g., intravenous),
intradermal, subcutaneous, oral, nasal, bronchial, ophthalmic,
transdermal (topical), transmucosal, rectal, and vaginal routes.
Anti-ILT5 antibodies or ILT5-binding fragments thereof may include
a delivery agent (e.g., a cationic polymer, peptide molecular
transporter, surfactant, etc., as described above) in combination
with a pharmaceutically acceptable carrier. As used herein the term
"pharmaceutically acceptable carrier" includes solvents, dispersion
media, coatings, antibacterial and antifugal agents, isotonic and
absorption delaying agents, and the like, compatible with
pharmaceutical administration. Supplementary active compounds can
also be incorporated into pharmaceutical formulations comprising an
anti-ILT5 antibody or an ILT5-binding fragment thereof as described
herein.
[0081] A pharmaceutical composition is formulated to be compatible
with its intended route of administration. Solutions or suspensions
used for parenteral, intradermal, or subcutaneous application can
include the following components: a sterile diluent such as water
for injection, saline solution, fixed oils, polyethylene glycols,
glycerine, propylene glycol or other synthetic solvents;
antibacterial agents such as benzyl alcohol or methyl parabens;
antioxidants such as ascorbic acid or sodium bisulfite; chelating
agents such as ethylenediaminetetraacetic acid; buffers such as
acetates, citrates or phosphates and agents for the adjustment of
tonicity such as sodium chloride or dextrose. pH can be adjusted
with acids or bases, such as hydrochloric acid or sodium hydroxide.
The parenteral preparation can be enclosed in ampoules, disposable
syringes or multiple dose vials made of glass or plastic.
[0082] Pharmaceutical compositions suitable for injectable use
typically include sterile aqueous solutions (where water soluble)
or dispersions and sterile powders for the extemporaneous
preparation of sterile injectable solutions or dispersion. For
intravenous administration, suitable carriers include physiological
saline, bacteriostatic water, Cremophor EL' (BASF, Parsippany,
N.J.) or phosphate buffered saline (PBS). In all cases, the
composition should be sterile and should be fluid to the extent
that easy syringability exists. Pharmaceutical formulations are
ideally stable under the conditions of manufacture and storage and
should be preserved against the contaminating action of
microorganisms such as bacteria and fungi. In general, the relevant
carrier can be a solvent or dispersion medium containing, for
example, water, ethanol, polyol (for example, glycerol, propylene
glycol, and liquid polyetheylene glycol, and the like), and
suitable mixtures thereof. The proper fluidity can be maintained,
for example, by the use of a coating such as lecithin, by the
maintenance of the required particle size in the case of dispersion
and by the use of surfactants. Prevention of the action of
microorganisms can be achieved by various antibacterial and
antifungal agents, for example, parabens, chlorobutanol, phenol,
ascorbic acid, thimerosal, and the like. In many cases, it will be
advantageous to include isotonic agents, for example, sugars,
polyalcohols such as manitol, sorbitol, or sodium chloride in the
composition. Prolonged absorption of the injectable compositions
can be brought about by including in the composition an agent which
delays absorption, for example, aluminum monostearate and
gelatin.
[0083] Sterile injectable solutions can be prepared by
incorporating the anti-ILT5 antibody or ILT5-binding fragment in
the required amount in an appropriate solvent with one or a
combination of ingredients enumerated above, as required, followed
by filtered sterilization. Generally, dispersions are prepared by
incorporating the purified anti-ILT5 antibody or ILT5-binding
fragment into a sterile vehicle which contains a basic dispersion
medium and the required other ingredients from those enumerated
above. In the case of sterile powders for the preparation of
sterile injectable solutions, exemplary methods of preparation are
vacuum drying and freeze-drying which yields a powder of the active
ingredient plus any additional desired ingredient from a previously
sterile-filtered solution thereof.
[0084] Oral compositions generally include an inert diluent or an
edible carrier. For the purpose of oral therapeutic administration,
an anti-ILT5 antibody or an ILT5-binding antibody fragment can be
incorporated with excipients and used in the form of tablets,
troches, or capsules, e.g., gelatin capsules. Oral compositions can
also be prepared using a fluid carrier for use as a mouthwash.
Pharmaceutically compatible binding agents, and/or adjuvant
materials can be included as part of the composition. The tablets,
pills, capsules, troches and the like can contain any of the
following ingredients, or compounds of a similar nature: a binder
such as microcrystalline cellulose, gum tragacanth or gelatin; an
excipient such as starch or lactose, a disintegrating agent such as
alginic acid, Primogel, or corn starch; a lubricant such as
magnesium stearate or Sterotes; a glidant such as colloidal silicon
dioxide; a sweetening agent such as sucrose or saccharin; or a
flavoring agent such as peppermint, methyl salicylate, or orange
flavoring. Formulations for oral delivery may advantageously
incorporate agents to improve stability within the gastrointestinal
tract and/or to enhance absorption.
[0085] For administration by inhalation, an anti-ILT5 antibody or
an ILT5-binding antibody fragment and a delivery agent are
preferably delivered in the form of an aerosol spray from a
pressured container or dispenser which contains a suitable
propellant, e.g., a gas such as carbon dioxide, or a nebulizer. The
present disclosure particularly contemplates delivery of the
compositions using a nasal spray, inhaler, or other direct delivery
to the upper and/or lower airway. Intranasal administration of DNA
vaccines directed against influenza viruses has been shown to
induce CD8 T cell responses, indicating that at least some cells in
the respiratory tract can take up DNA when delivered by this route,
and the delivery agents of the invention will enhance cellular
uptake. According to certain embodiments, an anti-ILT5 antibody or
ILT5-binding fragment thereof and a delivery agent are formulated
as large porous particles for aerosol administration.
[0086] Systemic administration can also be by transmucosal or
transdermal means. For transmucosal or transdermal administration,
penetrants appropriate to the barrier to be permeated are used in
the formulation. Such penetrants are generally known in the art,
and include, for example, for transmucosal administration,
detergents, bile salts, and fusidic acid derivatives. Transmucosal
administration can be accomplished through the use of nasal sprays
or suppositories. For transdermal administration, the purified
polypeptide or protein and delivery agents are formulated into
ointments, salves, gels, or creams as generally known in the
art.
[0087] In certain embodiments, compositions are prepared with
carriers that will protect an anti-ILT5 antibody or an ILT5-binding
antibody fragment against rapid elimination from the body, such as
a controlled release formulation, including implants and
microencapsulated delivery systems. Biodegradable, biocompatible
polymers can be used, such as ethylene vinyl acetate,
polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and
polylactic acid. Methods for preparation of such formulations will
be apparent to those skilled in the art. The materials can also be
obtained commercially from Alza Corporation and Nova
Pharmaceuticals, Inc. Liposomal suspensions (including liposomes
targeted to infected cells with monoclonal antibodies to viral
antigens) can also be used as pharmaceutically acceptable carriers.
These can be prepared according to methods known to those skilled
in the art, for example, as described in U.S. Pat. No. 4,522,811,
incorporated herein by reference in its entirety.
[0088] It is advantageous to formulate oral or parenteral
compositions in dosage unit form for ease of administration and
uniformity of dosage. "Dosage unit form" as used herein refers to
physically discrete units suited as unitary dosages for the subject
to be treated; each unit containing a predetermined quantity of
active anti-ILT5 antibody or ILT5-binding fragment thereof
calculated to produce the desired therapeutic effect in association
with the required pharmaceutical carrier.
[0089] An anti-ILT5 antibody or an ILT5-binding antibody fragment
can be administered at various intervals and over different periods
of time as required, e.g., one time per week for between about 1 to
10 weeks, between 2 to 8 weeks, between about 3 to 7 weeks, about
4, 5, or 6 weeks, etc. Those skilled in the art will appreciate
that certain factors can influence the dosage and timing required
to effectively treat a subject, including but not limited to the
severity of the disease or disorder, previous treatments, the
general health and/or age of the subject, and other diseases
present. Generally, treatment of a subject with an anti-ILT5
antibody or an ILT5-binding antibody fragment as described herein
can include a single treatment or, in many cases, can include a
series of treatments. It is furthermore understood that appropriate
doses may depend upon the potency of the anti-ILT5 antibody or
ILT5-binding fragment and may optionally be tailored to the
particular recipient, for example, through administration of
increasing doses until a preselected desired response is achieved.
It is understood that the specific dose level for any particular
animal subject may depend upon a variety of factors including the
activity of the specific polypeptide or protein employed, the age,
body weight, general health, gender, and diet of the subject, the
time of administration, the route of administration, the rate of
excretion, any drug combination, and the degree of expression or
activity to be modulated.
[0090] Pharmaceutical formulations as described herein can be
included in a container, pack, or dispenser together with
instructions for administration.
Detection and Diagnostic Assays
[0091] Given their ability to bind to ILT5, anti-ILT5 antibodies
and ILT5-binding antibody fragments can be used to detect ILT5
(e.g., in a biological sample, such as serum or plasma), using any
of a variety of immunoassays including, but not limited to, enzyme
linked immunosorbent assays (ELISAs), radioimmunoassays (RIAs),
cell sorting assays (e.g. fluorescent activation cell sorting, or
FACS), FCM or tissue immunohistochemistry assays. In certain
embodiments, methods for detecting ILT5 (e.g., human ILT5) in a
biological sample are provided, certain of such methods comprising
contacting a biological sample (e.g. a cell or tissue such as
blood) with an anti-ILT5 antibody or ILT5-binding fragment thereof,
and detecting either the anti-ILT5 antibody or ILT5-binding
fragment bound to ILT5 or unbound antibody or fragment, to thereby
detect ILT5 in the biological sample. The anti-ILT5 antibody or
ILT5-binding fragment thereof may be directly or indirectly labeled
with a detectable label to facilitate detection of the bound or
unbound anti-ILT5 antibody or ILT5-binding fragment. Suitable
detectable labels include various enzymes, prosthetic labels,
fluorescent labels, luminescent labels and radioactive labels.
Non-limiting examples of suitable enzymes include horseradish
peroxidase, alkaline phosphatase, beta-galactosidase, and
acetylcholinesterase. Non-limiting examples of suitable prosthetic
labels include streptavidin/biotin and avidin/biotin. Non-limiting
examples of suitable fluorescent labels include umbelliferone,
fluorescein, fluorescein isothiocyanate, rhodamine,
dichlorotriazinylamine fluorescein, dansyl chloride or
phycoerythrin. A non-limiting example of a luminescent label
includes luminal. Non-limiting examples of suitable radioactive
labels include .sup.125I, .sup.131I, .sup.35S and .sup.3H.
[0092] In certain embodiments, ILT5 can be assayed in a biological
sample by a competition immunoassay utilizing ILT5 standards
labeled with a detectable substance and an unlabeled anti-ILT5
antibody or ILT5-binding fragment thereof. In such an assay, the
biological sample, the labeled ILT5 standards and the anti-ILT5
antibody or ILT5-binding fragment are combined and the amount of
labeled ILT5 standard bound to the anti-ILT5 unlabeled antibody or
ILT5-binding fragment is determined. The amount of ILT5 in the
biological sample is inversely proportional to the amount of
labeled ILT5 standard bound to the anti-ILT5 antibody or
ILT5-binding.
[0093] Other detection assays that utilize antibodies or fragments
will be known to those skilled in the art. Any of the antibodies or
fragments described herein may be used in accordance with such
assays.
Cells
[0094] As described in the Examples below, culturing of human PBMCs
and anti-ILT5 antibody resulted in the production in the cultures
of distinct CD4+ and CD8+ T cells populations. Based on these
findings, this disclosure provides the isolated or cultured cells
described below in this section.
[0095] In certain embodiments, a cell of the present disclosure is
a human CD4.sup.+ T cell that expresses CD25 and NKG2D (a
CD4.sup.+CD25.sup.+NKG2D.sup.+ T cell). In certain embodiments, a
cell of the present disclosure is a human CD8.sup.+ T cell that
expresses CD25, and also expresses NKG2D at a level higher than the
level of NKG2D observed on steady state natural killer cells
(CD8.sup.+CD25.sup.+ NKG2D.sup.hi T cell). In certain embodiments,
such human T cells proliferate in a manner that does not require
recognition of a MHC molecule by the T cell, e.g., in a
TCR-independent manner (e.g., proliferation does not require
recognition of a MHC molecule by a TCR). In certain embodiments,
such human T cells secrete Fas ligand (FasL) at a higher level than
a naive T cell. In certain embodiments, such T cells express a
TCR:CD3 complex at a higher level than would be observed on a naive
T cell. In certain embodiments, such human T cells express MHC
Class II (DR) at a higher level than would be observed on a naive T
cell.
[0096] In certain embodiments, a CD4.sup.+CD25.sup.+NKG2D.sup.+ or
CD8.sup.+CD25.sup.+ NKG2D.sup.hi T cell maintains NKG2D on its cell
surface under a condition in which the NKG2D is typically
internalized from the cell surface. Non-limiting examples of such
conditions include engagement of the NKG2D with an NKG2D ligand
expressed on the surface of a cell, engagement of the NKG2D with a
secreted NKG2D ligand, and engagement of the NKG2D with an antibody
or fragment that binds NKG2D.
[0097] In certain embodiments, a CD4.sup.+CD25.sup.+NKG2D.sup.+ or
CD8.sup.+CD25.sup.+ NKG2D.sup.hi T cell is produced by a method
comprising contacting a naive T cell with an antigen presenting
cell (APC) that has previously been contacted with an anti-ILT5
antibody or an ILT5-binding antibody fragment. In certain
embodiments, a CD4.sup.+CD25.sup.+NKG2D.sup.+ or
CD8.sup.+CD25.sup.+ NKG2D.sup.hi T cell is produced by a method
comprising contacting a memory T cell with an antigen presenting
cell (APC) that has previously been contacted with an anti-ILT5
antibody or an ILT5-binding fragment of the antibody in the absence
of TCR stimulation.
[0098] In certain embodiments, such a
CD4.sup.+CD25.sup.+NKG2D.sup.+ or CD8.sup.+CD25.sup.+ NKG2D.sup.hi
T cell, or its progeny, is endowed with cytotoxic potential, as
described herein. In certain embodiments, a
CD4.sup.+CD25.sup.+NKG2D.sup.+ or CD8.sup.+CD25.sup.+ NKG2D.sup.hi
T cell is induced to become cytotoxic when it binds to or
recognizes an antigen. Binding or recognition with any of a variety
of antigens will result in the transition from having cytotoxic
potential to cytotoxicity. For example, a
CD4.sup.+CD25.sup.+NKG2D.sup.+ or CD8.sup.+CD25.sup.+ NKG2D.sup.hi
T cell that exhibits cytotoxic potential may become cytotoxic when
it binds to or recognizes a purified or isolated antigen.
Similarly, a CD4.sup.+CD25.sup.+NKG2D.sup.+ or CD8.sup.+CD25.sup.+
NKG2D.sup.hi T cell that exhibits cytotoxic potential may become
cytotoxic when it binds to or recognizes an unpurified or
non-isolated antigen such as, for example, an antigen derived from
a cellular lysate, or an antigen present in a blood or serum
sample. In certain embodiments, a CD4.sup.+CD25.sup.+NKG2D.sup.+ or
CD8.sup.+CD25.sup.+ NKG2D.sup.hi T cell having cytotoxic potential
is induced to become cytotoxic when the cell binds to or recognizes
a cancerous cell, or a cell that is infected with a bacterium, a
virus, a fungus, a protozoan, or a parasite. In certain
embodiments, a CD4.sup.+CD25.sup.+NKG2D.sup.+ or
CD8.sup.+CD25.sup.+ NKG2D.sup.hi T cell having cytotoxic potential
is induced to become cytotoxic when the cell binds to or recognizes
an antigen present on a cancerous cell, or a cell that is infected
with a bacterium, a virus, a fungus, a protozoan, or a
parasite.
[0099] In certain embodiments, in cell cultures comprising a
CD4.sup.+CD25.sup.+NKG2D.sup.+ or CD8.sup.+CD25.sup.+ NKG2D.sup.hi
T cell the cytokines TNF-alpha (tumor necrosis factor alpha) and
IL5, and/or the chemokines, Rantes, IP-10 (interferon-inducible
protein 10), or MIP-1 (macrophage inflammatory protein 1) are
produced. In certain embodiments, a CD4.sup.+CD25.sup.+NKG2D.sup.+
or CD8.sup.+CD25.sup.+ NKG2D.sup.hi T cell present in such a cell
culture produces one or more of Rantes, IP-10, TNF-alpha, IL5, or
MIP-1 itself. In certain embodiments, a CD4+CD25+NKG2D+ or
CD8+CD25+ NKG2D.sup.hi T cell in a cell culture induces another
type of cell in the culture to produce one or more of Rantes,
IP-10, TNF-alpha, IL5, or MIP-1. For example, a CD4+CD25+NKG2D+ or
CD8+CD25+ NKG2D.sup.hi T cell can induce one or more of a monocyte,
a macrophage, a T cell other than CD4+CD25+NKG2D+ or CD8+CD25+
NKG2D.sup.hi T cell, a B cell, a mast cell, an endothelial cell,
and/or a fibroblast to produce one or more of Rantes, IP-10,
TNF-alpha, IL5, or MIP-1. Thus, methods of using CD4+CD25+NKG2D+ or
CD8+CD25+ NKG2D.sup.hi cells for inducing the production of these
soluble mediators (cytokine and chemokines) by these cell types
(monocytes, macrophages, T cells other than CD4+CD25+NKG2D+ or
CD8+CD25+ NKG2D.sup.hi T cells, or B cells) are provided.
[0100] CD4.sup.+CD25.sup.+NKG2D.sup.+ or CD8.sup.+CD25.sup.+
NKG2D.sup.hi T cells as described in this section may be used in
any of a variety of applications, including any of the applications
described in the section entitled "Treatment of Diseases and
Infections" above.
Anti-ILT5 Antibodies and ILT5-Binding Fragments Thereof
[0101] Disclosed herein are a variety of anti-ILT5 antibodies and
ILT5-binding antibody fragments thereof. In certain embodiments, an
anti-ILT5 antibody or an ILT5-binding antibody fragment thereof can
be used for one or more applications described herein (e.g.,
inducing an immunostimulatory response in T cells, thereby causing
them to proliferate or display a cytotoxic function). In certain
embodiments, such T cells produce cytokines and/or induce other
cells to produce cytokines and/or chemokines. In certain
embodiments, such T cells may be used in the treatment of various
diseases or infections. In certain embodiments, the antibody is
monoclonal. In certain embodiments, an anti-ILT5 antibody or an
ILT5-binding antibody fragment is chimeric in that it contains
human heavy and/or light chain constant regions. See, for example,
Cabilly et al., U.S. Pat. No. 4,816,567; Shoemaker et al., U.S.
Pat. No. 4,978,775; Beavers et al., U.S. Pat. No. 4,975,369; and
Boss et al., U.S. Pat. No. 4,816,397, each of which is incorporated
herein by reference in its entirety. In certain embodiments, an
anti-ILT5 antibody or ILT5-binding fragment thereof is humanized in
that it contains one or more human framework regions in the
variable region together with non-human (e.g., mouse, rat, or
hamster) complementarity-determining regions (CDRs) of the heavy
and/or light chain. Humanized antibodies can be produced using
recombinant DNA techniques well known to those skilled in the art.
See for example, Hwang, W. Y. K., et al., Methods 36:35, 2005;
Queen et al., Proc. Natl. Acad. Sci. USA, 86:10029-10033, 1989;
Jones et al., Nature, 321:522-25, 1986; Riechmann et al., Nature,
332:323-27, 1988; Verhoeyen et al., Science, 239:1534-36, 1988;
Orlandi et al., Proc. Natl. Acad. Sci. USA, 86:3833-37, 1989; U.S.
Pat. Nos. 5,225,539; 5,530,101; 5,585,089; 5,693,761; 5,693,762;
6,180,370; and Selick et al., WO 90/07861, each of which is
incorporated herein by reference in its entirety.
[0102] In certain embodiments, a fragment (e.g., an antigen-binding
fragment) is derived from a whole antibody molecule, such as a
monoclonal or a polyclonal antibody. The antibody can be, e.g.,
cleaved on the carboxy terminal side of its hinge region (e.g.,
with pepsin) to generate a F(ab').sub.2 fragment, or on the amino
terminal side of its hinge region (e.g., with papain) to generate
Fab fragments. In certain embodiments, an anti-ILT5 antibody or an
ILT5-binding antibody fragment binds human ILT5.
[0103] In certain embodiments, an anti-ILT5 antibody fragment is a
Fab fragment, a F(ab').sub.2 fragment, a scFv fragment, a diabody,
a linear antibody, a multispecific antibody fragment such as a
bispecific, a trispecific, or a multispecific antibody (e.g., a
diabody, a triabody, a tetrabody), a minibody, a chelating
recombinant antibody, a tribody or bibody, an intrabody, a
nanobody, a small modular immunopharmaceutical (SMIP), a
binding-domain immunoglobulin fusion protein, a camelid antibody,
or a V containing antibody. Those skilled in the art will be aware
of how to engineer or construct such antibodies or fragments
without undue experimentation.
[0104] In certain embodiments, an anti-ILT5 antibody or an
ILT5-binding antibody fragment thereof comprises a light chain
variable region comprising the amino acid sequence of SEQ ID NO: 1
[DIQMTQSPASLSVSVGETVTITCRASENIYSNLAWYQQKQGKSPQVLVYAATNLADGV
PSRFSGSGSGTQFSLKINSLQSEDFGNYFCQHFWRIPWTFGGGTKLEIK]. In certain
embodiments, an anti-ILT5 antibody or an ILT5-binding antibody
fragment comprises a heavy chain variable region comprising the
amino acid sequence of SEQ ID NO: 2
[DVQLQESGPGLVKPSQSLFLTCSVTGYSISSSYYWNWIRQFPGNKLEWMGYISFDGSNN
YNPSLKNRISITRDTSKNQFFLKLNSVTTEDTATYYCAREKENYYGSSFYYFDYWGLGT
SLTVSS]. In certain embodiments, an anti-ILT5 antibody or an
ILT5-binding antibody fragment comprises a heavy chain variable
region comprising the amino acid sequence of SEQ ID NO: 3
[EVQLQESGPGLVKPSQSLFLTCSVTGYSISSSYYWNWIRQFPGNKLEWMGYISFDGSNN
YNPSLKNRISITRDTSKNQFFLKLNSVTTEDTATYYCAREKENYYGSSFYYFDYWGLGT
SLTVSS].
[0105] In certain embodiments, an anti-ILT5 antibody or
ILT5-binding fragment thereof comprises a light chain variable
region comprising the amino acid sequence of SEQ ID NO: 1 and a
heavy chain variable region comprising the amino acid sequence of
SEQ ID NO: 2. In certain embodiments, an anti-ILT5 antibody or
ILT5-binding fragment thereof comprises a light chain variable
region comprising the amino acid sequence of SEQ ID NO: 1 and a
heavy chain variable region comprising the amino acid sequence of
SEQ ID NO: 3.
[0106] In certain embodiments, an anti-ILT5 antibody or an
ILT5-binding antibody fragment, e.g., a humanized or chimeric
antibody or fragment, comprises one or more of the following CDRs:
SEQ ID NO: 4 [SSYYWN] (VH CDR1), SEQ ID NO: 5 [YISFDGSNNYNPSLKN]
(VH CDR2), SEQ ID NO: 6 [EKENYYGSSFYYFDY] (VH CDR3), SEQ ID NO: 7
[RASENIYSNLA] (VL CDR1), SEQ ID NO: 8 [AATNLAD] (VL CDR2), and SEQ
ID NO: 9 [QHFWRIPWT] (VL CDR3).
[0107] In certain embodiments, an anti-ILT5 antibody or an
ILT5-binding antibody fragment, e.g., a humanized or chimeric
antibody or fragment, comprises a heavy chain variable region (VH)
comprising: a VH CDR1 comprising the amino acid sequence of SEQ ID
NO: 4, a VH CDR2 comprising the amino acid sequence of SEQ ID NO:
5, and a VH CDR3 comprising the amino acid sequence of SEQ ID NO:
6. In certain embodiments, an anti-ILT5 antibody or ILT5-binding
fragment thereof, e.g., a humanized or chimeric antibody or
fragment, comprises a light chain variable region (VL) comprising:
a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 7, a VL
CDR2 comprising the amino acid sequence of SEQ ID NO: 8, and a VL
CDR3 comprising the amino acid sequence of SEQ ID NO: 9. In certain
embodiments, an anti-ILT5 antibody or an ILT5-binding antibody
fragment, e.g., a humanized or chimeric antibody or fragment,
comprises a heavy chain variable region comprising: a VH CDR1
comprising the amino acid sequence of SEQ ID NO: 4, a VH CDR2
comprising the amino acid sequence of SEQ ID NO: 5, a VH CDR3
comprising the amino acid sequence of SEQ ID NO: 6, a VL CDR1
comprising the amino acid sequence of SEQ ID NO: 7, a VL CDR2
comprising the amino acid sequence of SEQ ID NO: 8, and a VL CDR3
comprising the amino acid sequence of SEQ ID NO: 9.
[0108] In certain embodiments, an anti-ILT5 antibody or an
ILT5-binding antibody fragment comprises a heavy chain variable
region comprising: a VH CDR1 consisting of the amino acid sequence
of SEQ ID NO: 4, a VH CDR2 consisting of the amino acid sequence of
SEQ ID NO: 5, and a VH CDR3 consisting of the amino acid sequence
of SEQ ID NO: 6. In certain embodiments, an anti-ILT5 antibody or
an ILT5-binding antibody fragment comprises a light chain variable
region comprising: a VL CDR1 consisting of the amino acid sequence
of SEQ ID NO: 7, a VL CDR2 consisting of the amino acid sequence of
SEQ ID NO: 8, and a VL CDR3 consisting of the amino acid sequence
of SEQ ID NO: 9.
[0109] In certain embodiments, an anti-ILT5 antibody or an
ILT5-binding antibody fragment comprises a heavy chain variable
region comprising: a VH CDR1 comprising the amino acid sequence of
SEQ ID NO: 4, a VH CDR2 comprising the amino acid sequence of SEQ
ID NO: 5, a VH CDR3 comprising the amino acid sequence of SEQ ID
NO: 6, a VL CDR1 consisting of the amino acid sequence of SEQ ID
NO: 7, a VL CDR2 consisting of the amino acid sequence of SEQ ID
NO: 8, and a VL CDR3 consisting of the amino acid sequence of SEQ
ID NO: 9. In certain embodiments, an anti-ILT5 antibody or an
ILT5-binding antibody fragment comprises a heavy chain variable
region comprising: a VH CDR1 consisting of the amino acid sequence
of SEQ ID NO: 4, a VH CDR2 consisting of the amino acid sequence of
SEQ ID NO: 5, a VH CDR3 consisting of the amino acid sequence of
SEQ ID NO: 6, a VL CDR1 comprising the amino acid sequence of SEQ
ID NO: 7, a VL CDR2 comprising the amino acid sequence of SEQ ID
NO: 8, and a VL CDR3 comprising the amino acid sequence of SEQ ID
NO: 9. In certain embodiments, an anti-ILT5 antibody or an
ILT5-binding antibody fragment thereof comprises a heavy chain
variable region comprising: a VH CDR1 consisting of the amino acid
sequence of SEQ ID NO: 4, a VH CDR2 consisting of the amino acid
sequence of SEQ ID NO: 5, a VH CDR3 consisting of the amino acid
sequence of SEQ ID NO: 6, a VL CDR1 consisting of the amino acid
sequence of SEQ ID NO: 7, a VL CDR2 consisting of the amino acid
sequence of SEQ ID NO: 8, and a VL CDR3 consisting of the amino
acid sequence of SEQ ID NO: 9.
[0110] In certain embodiments, an anti-ILT5 antibody or an
ILT5-binding antibody fragment thereof is humanized in that it
comprises one or more human framework regions, e.g. a human heavy
chain framework region and/or a human light chain framework region.
In certain embodiments, an anti-ILT5 antibody or an ILT5-binding
antibody fragment comprises one or more human framework regions
from a heavy chain variable region comprising an amino acid
sequence selected from the group consisting of the amino acid
sequence of
TABLE-US-00001 SEQ ID NO: 10
[QVQLQESGPGLVKPPGTLSLTCAVSGGSISSSYYWNWVRQPPGKGLEWI
GYISFDGSNNYNPSLKNRVTISVDKSKNQFSLKLSSVTAADTAVYCCARE
KENYYGSSFYYFDYWGQGTLVTVSS], SEQ ID NO: 11
[QVQLQESGPGLVKPSGTLSLTCAVSGGSISSSYYWNWVRQPPGKGLEWI
GYISFDGSNNYNPSLKNRVTISVDKSKNQFSLKLSSVTAADTAVYCCARE
KENYYGSSFYYFDYWGQGTLVTVSS], SEQ ID NO: 12
[QVQLQESGPGLVKPPGTLSLTCAVSGGSISSSYYWNWVRQPPGKGLEWI
GYISFDGSNNYNPSLKNRVTISVDKSKNQFSLKLSSVTAADTAVYYCARE
KENYYGSSFYYFDYWGQGTLVTVSS], SEQ ID NO: 13
[QVQLQESGPGLVKPSDTLSLTCAVSGYSISSSYYWNWIRQPPGKGLEWI
GYISFDGSNNYNPSLKNRVTMSVDTSKNQFSLKLSSVTAVDTAVYYCARE
KENYYGSSFYYFDYWGQGTLVTVSS], SEQ ID NO: 14
[QLQLQESGPGLVKPSETLSLTCTVSGGSISSSYYWNWIRQPPGKGLEWI
GYISFDGSNNYNPSLKNRVTISVDTSKNQFSLKLSSVTAADTAVYYCARE
KENYYGSSFYYFDYWGQGTLVTVSS], SEQ ID NO: 15
[QVQLQESGPGLVKPSETLSLTCTVSGGSISSSYYWNWIRQPPGKGLEWI
GYISFDGSNNYNPSLKNRVTISVDTSKNQFSLKLSSVTAADTAVYYCARE
KENYYGSSFYYFDYWGQGTLVTVSS], SEQ ID NO: 16
[QVQLQESGPGLVKPSETLSLTCTVSGGSVSSSYYWNWIRQPPGKGLEWI
GYISFDGSNNYNPSLKNRVTISVDTSKNQFSLKLSSVTAADTAVYYCARE
KENYYGSSFYYFDYWGQGTLVTVSS], and SEQ ID NO: 17
[QVQLQESGPGLVKPSETLSLTCAVSGYSISSSYYWNWIRQPPGKGLEWI
GYISFDGSNNYNPSLKNRVTISVDTSKNQFSLKLSSVTAADTAVYYCARE
KENYYGSSFYYFDYWGQGTLVTVSS]
(CDR sequences are underlined in SEQ ID NOs: 10-17, while framework
regions lack underlining. The four framework regions in each
sequence are numbered 1-4 (FW1, FW2, FW3, and FW4) starting from
the N-terminal ends of the sequences.). In certain embodiments, an
anti-ILT5 antibody or an ILT5-binding antibody fragment comprises
one or more human framework regions from a heavy chain variable
region comprising an amino acid sequence selected from the group
consisting of the amino acid sequence of SEQ ID NO: 10.
[0111] In certain embodiments, an anti-ILT5 antibody or an
ILT5-binding antibody fragment comprises one or more human
framework regions from a light chain variable region comprising an
amino acid sequence selected from the group consisting of the amino
acids sequence of
TABLE-US-00002 SEQ ID NO: 18
[AIRMTQSPSSFSASTGDRVTITCRASENIYSNLAWYQQKPGKAPKLLIY
AATNLADGVPSRFSGSGSGTDFTLTISCLQSEDFATYYFATYYCQHFWRI PWTFGQGTKVEIK],
SEQ ID NO: 19 [DIQLTQSPSFLSASVGDRVTITCRASENIYSNLAWYQQKPGKAPKLLIY
AATNLADGVPSRFSGSGSGTEFTLTISSLQPEDFATYYCQHFWRIPWTFG QGTKVEIK], SEQ
ID NO: 20 [DIQMTQSPSSVSASVGDRVTITCRASENIYSNLAWYQQKPGKAPKLLIY
AATNLADGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQHFWRIPWTFG QGTKVEIK], SEQ
ID NO: 21 [DIQMTQSPSSVSASVGDRVTITCRASENIYSNLAWYQQKPGKAPKLLIY
AATNLADGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQHFWRIPWTFG QGTKVEIK], SEQ
ID NO: 22 [AIQLTQSPSSLSASVGDRVTITCRASENIYSNLAWYQQKPGKAPKLLIY
AATNLADGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQHFWRIPWTFG QGTKVEIK], SEQ
ID NO: 23 [AIQLTQSPSSLSASVGDRVTITCRASENIYSNLAWYQQKPGKAPKLLIY
AATNLADGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQHFWRIPWTFG QGTKVEIK], and
SEQ ID NO: 24 [DIQMTQSPSSLSASVGDRVTITCRASENIYSNLAWYQQKPEKAPKSLIY
AATNLADGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQHFWRIPWTFG QGTKVEIK]
(CDR sequences are underlined in SEQ ID NOs: 18-24, while framework
regions lack underlining. The four framework regions in each
sequence are numbered 1-4 (FW1, FW2, FW3, and FW4) starting from
the N-terminal ends of the sequences.). In certain embodiments, an
anti-ILT5 antibody or an ILT5-binding antibody fragment thereof
comprises one or more human framework regions from a light chain
variable region comprising an amino acid sequence selected from the
group consisting of the amino acid sequence of SEQ ID NO: 18.
[0112] In certain embodiments, an anti-ILT5 antibody or an
ILT5-binding antibody fragment comprises one or more human
framework regions from a heavy chain variable region comprising an
amino acid sequence selected from the group consisting of the amino
acid sequence of SEQ ID NO: 10-17, and a light chain variable
region comprising an amino acid sequence selected from the group
consisting of the amino acid sequence of SEQ ID NOs: 18-24.
[0113] In certain embodiments, a CDR homology based method is used
for humanization (see, e.g., Hwang, W. Y. K., et al., Methods
36:35, 2005). This method generally involves substitution of
non-human CDRs into a human framework based on similarly structured
non-human and human CDRs, rather than similarly structured
non-human and human frameworks. The similarity of the non-human and
human CDRs is generally determined by identifying human genes of
the same chain type (light or heavy) that have the same combination
of canonical CDR structures as the mouse binding molecules and thus
retain three-dimensional conformation of CDR peptide backbones.
Secondly, for each of the candidate variable region gene segments
with matching canonical structures, residue to residue homology
between the non-human and candidate human CDRs is evaluated.
Finally, to generate a humanized binding molecule, CDR residues of
the chosen human candidate CDR not already identical to the
non-human CDR are converted to the non-human sequence. In certain
embodiments, no mutations of the human framework are introduced
into the humanized binding molecule.
[0114] In certain embodiments, the substitution of non-human CDRs
into a human framework is based on the retention of the correct
spatial orientation of the non-human framework by identifying human
frameworks which will retain the same conformation as the non-human
frameworks from which the CDRs were derived. In certain
embodiments, this is achieved by obtaining the human variable
regions from human antibodies whose framework sequences exhibit a
high degree of sequence identity with the non-human framework
regions from which the CDRs were derived. See Kettleborough et al.,
Protein Engineering 4:773, 1991; Kolbinger et al., Protein
Engineering 6:971, 1993; and Carter et al., WO 92/22653, each of
which is incorporated herein by reference in its entirety.
[0115] In certain embodiments, one or more human framework residues
can be changed or substituted to residues at the corresponding
positions in the original non-human (e.g. murine) antibody so as to
preserve the binding affinity of the humanized antibody to the
antigen. Such a change is sometimes called "backmutation". Certain
amino acids from the human framework residues are selected for
backmutation based on their possible influence on CDR conformation
and/or binding to antigen. For example, residues immediately
surrounding one or more CDRs can be backmutated to ensure proper
spatial positioning of the CDRs. The placement of non-human (e.g.
murine) CDR regions within human framework regions can result in
conformational restraints, which, unless corrected by substitution
of certain amino acid residues, lead to loss of binding affinity.
Thus, in certain embodiments, backmutations can be made in residues
that affect proper conformation of the anti-ILT5 antibody or
ILT5-binding fragment to ensure adequate affinity to ILT5.
[0116] In certain embodiments, the selection of amino acid residues
for backmutation can be determined, in part, by computer modeling,
using art recognized techniques. In general, molecular models are
produced starting from solved structures for immunoglobulin chains
or domains thereof. The chains to be modeled are compared for amino
acid sequence similarity with chains or domains of solved
three-dimensional structures, and the chains or domains showing the
greatest sequence similarity is/are selected as starting points for
construction of the molecular model. Chains or domains sharing at
least 50% sequence identity are selected for modeling, and
preferably those sharing at least 60%, 70%, 80%, 90% sequence
identity or more are selected for modeling. The solved starting
structures are modified to allow for differences between the actual
amino acids in the immunoglobulin chains or domains being modeled,
and those in the starting structure. The modified structures are
then assembled into a composite immunoglobulin. Finally, the model
is refined by energy minimization and by verifying that all atoms
are within appropriate distances from one another and that bond
lengths and angles are within chemically acceptable limits.
[0117] The selection of amino acid residues for substitution can
also be determined, in part, by examination of the characteristics
of the amino acids at particular locations, or empirical
observation of the effects of substitution or mutagenesis of
particular amino acids. For example, when an amino acid differs
between a non-human (e.g. murine) framework residue and a selected
human framework residue, the human framework amino acid may be
substituted by the equivalent framework amino acid from the
non-human binding molecule when it is reasonably expected that the
amino acid: (1) noncovalently binds antigen directly, (2) is
adjacent to a CDR region, (3) otherwise interacts with a CDR region
(e.g., is within about 3-6 angstroms of a CDR region as determined
by computer modeling), or (4) participates in the VL-VH
interface.
[0118] In certain embodiments, an anti-ILT5 antibody or an
ILT5-binding antibody fragment comprises a human heavy chain
constant region. For example, an anti-ILT5 antibody or an
ILT5-binding antibody fragment may comprise an IgG (K) heavy chain
constant region such as a IgG1 (K1) heavy chain constant region, an
IgG2 (K2) heavy chain constant region, an IgG3 (K3) heavy chain
constant region, or an IgG4 (K4) heavy chain constant region.
Moreover, they can comprise an IgA (I) heavy chain constant region,
an IgE (M) heavy chain constant region, an IgM (T) heavy chain
constant region, or an IgD (A) heavy chain constant region. In
certain embodiments, an anti-ILT5 antibody or an ILT5-binding
antibody fragment comprises a heavy chain constant region
comprising the amino acid sequence of SEQ ID NO: 25
[ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQS
SGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLG
GPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQ
YNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPS
RDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVD
KSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK]. In certain embodiments, an
anti-ILT5 antibody or an ILT5-binding antibody fragment comprises a
heavy chain constant region comprising the amino acid sequence of
SEQ ID NO: 26
[ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQS
SGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLG
GPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQ
YASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPS
RDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVD
KSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK]. SEQ ID NO: 26 differs from SEQ
ID NO: 25 in that an asparagine has been altered to an alanine (the
relevant amino acid is underlined in each sequence), which
alteration results in elimination of N-linked in vivo glycosylation
of anti-ILT5 antibodies and ILT5-binding fragments thereof
comprising SEQ ID NO: 26. Absence of N-linked glycosylation at the
relevant residue results in drastically decreased binding of the Fc
region of the relevant anti-ILT5 antibody or ILT5-binding fragment
to a Fc receptor.
[0119] Any of a variety of other modifications may be made that
result in reduced binding of an anti-ILT5 antibody or an
ILT5-binding fragment to a Fc receptor. For example, a humanized
OTK3-derived antibody in which two amino acid residues at positions
234 and 235 of the Fc domain have been modified to alanine residues
(referred to as hOKT3-gamma-1 (ala-ala)) is disclosed in United
States Patent Publication numbers 2007/0077246 and 2008/0095766,
the disclosures of which are incorporated herein by reference in
their entirety. The hOKT3-gamma-1 (ala-ala) antibody is described
as exhibiting reduced binding to Fc (gamma) receptors, even though
its Fc domain comprises residues that are N-linked glycosylated. In
certain embodiments, an ant-ILT5 antibody or an ILT5-binding
fragment thereof that exhibits reduced binding to at least one Fc
(gamma) receptor is modified in that it lacks some or all of an Fc
domain. For example, Fab fragments and F(ab').sub.2 fragments lack
some or all of an Fc domain. In certain embodiments, an antibody or
antigen-binding fragment thereof is modified in some other way such
that it exhibits reduced binding to at least one Fc (gamma)
receptor. For example, the anti-ILT5 antibody or ILT5-binding
fragment may be modified by covalent linkage of a chemical moiety
that prevents the anti-ILT5 antibody or ILT5-binding fragment from
binding, or decreases its ability to bind, to least one Fc (gamma)
receptor. As another example, the anti-ILT5 antibody or
ILT5-binding fragment may be modified by non-covalent linkage of a
chemical moiety that prevents the anti-ILT5 antibody or
ILT5-binding fragment from binding, or decreases its ability to
bind, to least one Fc (gamma) receptor. Any of a variety of
moieties may be covalently or non-covalently linked to the
anti-ILT5 antibody or ILT5-binding fragment thereof to prevent or
decrease binding to at least one Fc (gamma) receptor. Those skilled
in the art will be aware of suitable moieties that can be linked to
an antibody or fragment, and will be able to employ such moieties
in accordance with the teachings herein.
[0120] In certain embodiments, any of a variety of modifications
may be made to an anti-ILT5 antibody or an ILT5-binding antibody
fragment, which modification results in alteration of the a
physical or in vivo property of the anti-ILT5 antibody or
ILT5-binding fragment. For example, any of a variety of
modifications may be made that affect the stability of the
anti-ILT5 antibody or ILT5-binding fragment (e.g., in vivo).
Additionally and/or alternatively, any of a variety of
modifications may be made that affect the halflife of an anti-ILT5
antibody or ILT5-binding fragment thereof in vivo. As is known in
the art, FcRn protects IgG-type antibodies from degradation,
resulting in longer half-life of this class of antibody in the
serum (see Roopenian and Akilesh, Nature Reviews Immunology 7,
715-725, 2007, incorporated herein by reference in its entirety).
Thus, in certain embodiments, an IgG-type antiI-ILT5 antibody or
fragment thereof is modified by altering amino acid residues in its
Fc region such that it bind differently to FcRn. Alterations that
result in improved binding to FcRn will result in the anti-ILT5
antibody or ILT5-binding fragment having a longer halflife in vivo.
Alterations that result in decreased binding to FcRn will result in
the anti-ILT5 antibody or ILT5-binding fragment having a shorter
halflife in vivo. Those skilled in the art will be aware of
suitable alterations that can be made, such as pegylation and/or
amino acid substitutions, and will be able to make such
corresponding alterations in anti-ILT5 antibodies and ILT5-binding
fragments thereof disclosed herein without undue
experimentation.
[0121] In certain embodiments, an anti-ILT5 antibody or an
ILT5-binding antibody fragment comprises a human light chain
constant region. For example, an anti-ILT5 antibody or an
ILT5-binding antibody fragment may comprise a human kappa or human
lambda light chain constant region. In certain embodiments, an
anti-ILT5 antibody or an ILT5-binding antibody fragment comprises a
light chain constant region comprising the amino acid sequence of
SEQ ID NO: 27
[RTVAAPSVFIFPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQ
DSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC].
[0122] In certain embodiments, an anti-ILT5 antibody or an
ILT5-binding antibody fragment comprises a light chain comprising
or consisting of the amino acid sequence of SEQ ID NO: 28
[DIQMTQSPASLSVSVGETVTITCRASENIYSNLAWYQQKQGKSPQVLVYAATNLADGV
PSRFSGSGSGTQFSLKINSLQSEDFGNYFCQHFWRIPWTFGAGTKLEIKRTVAAPSVFIFPP
SDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSST
LTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC].
[0123] In certain embodiments, an anti-ILT5 antibody or an
ILT5-binding antibody fragment comprises a heavy chain comprising
or consisting of the amino acid sequence of SEQ ID NO: 29
[DVQLQESGPGLVKPSQSLFLTCSVTGYSISSSYYWNWIRQFPGNKLEWMGYISFDGSNN
YNPSLKNRISITRDTSKNQFFLKLNSVTTEDTATYYCAREKENYYGSSFYYFDYWGAGT
LVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFP
AVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPA
PELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTK
PREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQV
YTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYS
KLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK] or SEQ ID NO: 30:
[EVQLQESGPGLVKPSQSLFLTCSVTGYSISSSYYWNWIRQFPGNKLEWMGYISFDGSNN
YNPSLKNRISITRDTSKNQFFLKLNSVTTEDTATYYCAREKENYYGSSFYYFDYWGAGT
LVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFP
AVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPA
PELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTK
PREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQV
YTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYS
KLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK]. In certain embodiments,
an anti-ILT5 antibody or an ILT5-binding antibody fragment
comprises a heavy chain comprising or consisting of the amino acid
sequence of SEQ ID NO: 31
[DVQLQESGPGLVKPSQSLFLTCSVTGYSISSSYYWNWIRQFPGNKLEWMGYISFDGSNN
YNPSLKNRISITRDTSKNQFFLKLNSVTTEDTATYYCAREKENYYGSSFYYFDYWGAGT
LVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFP
AVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPA
PELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTK
PREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQV
YTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYS
KLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK] or SEQ ID NO: 32
[EVQLQESGPGLVKPSQSLFLTCSVTGYSISSSYYWNWIRQFPGNKLEWMGYISFDGSNN
YNPSLKNRISITRDTSKNQFFLKLNSVTTEDTATYYCAREKENYYGSSFYYFDYWGAGT
LVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFP
AVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPA
PELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTK
PREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQV
YTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYS
KLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK]. SEQ ID NOs: 29 and 30
differs from SEQ ID NOs: 31 and 32 in that an asparagine has been
altered to an alanine (the relevant amino acid is underlined in
each sequence), which alteration results in decreased in vivo
glycosylation of anti-ILT5 antibodies and ILT5-binding fragments
thereof comprising SEQ ID NOs: 31 and 32.
[0124] In certain embodiments, an anti-ILT5 antibody or an
ILT5-binding antibody fragment comprises a light chain comprising
or consisting of the amino acid sequence of SEQ ID NO: 28, and a
heavy chain comprising or consisting of the amino acid sequence of
SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, or SEQ ID NO: 32.
[0125] In certain embodiments, an anti-ILT5 antibody or an
ILT5-binding antibody fragment comprises an amino acid sequence
that is at least 75% identical to one or more of SEQ ID NOs: 1-32,
e.g., at least 80% identical, at least 85% identical, at least 90%
identical, at least 95% identical, at least 96% identical, at least
97% identical, at least 98% identical, or at least 99% identical to
one or more of SEQ ID NOs: 1-32. In certain embodiments, an
anti-ILT5 antibody or ILT5-binding fragment thereof comprises an
amino acid sequence comprising at least 5 contiguous amino acid
residues of one or more of SEQ ID NOs: 1-32, e.g., at least 6, at
least 7, at least 8, at least 9, at least 10, at least 11, at least
12, at least 13, at least 14, at least 15, at least 20, at least
25, at least 30, at least 40, at least 50, or more contiguous amino
acid residues.
[0126] In certain embodiments, an anti-ILT5 antibody or an
ILT5-binding antibody fragment comprises a polypeptide having one
or more amino acid substitutions, deletions or insertions as
compared to a polypeptide having an amino acid sequence of one or
more of SEQ ID NOs: 1-32. For example, an anti-ILT5 antibody or an
ILT5-binding antibody fragment may have 1, 2, 3, 4, 5, 6, 7, 8, 9,
10, or more amino acid substitutions, deletions or insertions.
Substitutions, deletions or insertions may be introduced by
standard techniques, such as site-directed mutagenesis or
PCR-mediated mutagenesis of a nucleic acid molecule encoding a
polypeptide of an anti-ILT5 antibody or an ILT5-binding antibody
fragment. In certain embodiments, conservative amino acid
substitutions are made at one or more positions. A "conservative
amino acid substitution" is one in which the amino acid residue is
replaced with an amino acid residue having a similar side chain.
Families of amino acid residues having similar side chains have
been defined in the art, including basic side chains (e.g., lysine,
arginine, histidine), acidic side chains (e.g., aspartic acid,
glutamic acid), uncharged polar side chains (e.g., glycine,
asparagine, glutamine, serine, threonine, tyrosine, cysteine),
nonpolar side chains (e.g., alanine, valine, leucine, isoleucine,
proline, phenylalanine, methionine, tryptophan), beta-branched side
chains (e.g., threonine, valine, isoleucine) and aromatic side
chains (e.g., tyrosine, phenylalanine, tryptophan; histidine).
Thus, an amino acid residue in a polypeptide of an anti-ILT5
antibody or an ILT5-binding antibody fragment may be replaced with
another amino acid residue from the same side chain family. In
certain embodiments, a string of amino acids can be replaced with a
structurally similar string that differs in order and/or
composition of side chain family members. Those skilled in the art
will be able to evaluate whether an anti-ILT5 antibody or an
ILT5-binding antibody fragment comprising a polypeptide having one
or more amino acid substitutions, deletions or insertions as
compared to a polypeptide having an amino acid sequence of one or
more of SEQ ID NOs: 1-32 binds ILT5 by utilizing routine,
art-recognized methods including, but not limited to, ELISAs,
Western blots, phage display, etc.
[0127] Anti-ILT5 antibodies and ILT5-binding fragments thereof can
be produced by any of a variety of methods known to those skilled
in the art. In certain embodiments, anti-ILT5 antibodies and
ILT5-binding antibody fragments can be produced recombinantly. For
example, nucleic acid sequences encoding one or more of SEQ ID NOs:
1-32, or portions thereof, may be introduced into a bacterial cell
(e.g., E. coli, B. subtilis) or a eukaryotic cell (e.g., a yeast
such as S. cerevisiae, or a mammalian cell such as a CHO cell line,
various Cos cell lines, a HeLa cell, various myeloma cell lines, or
a transformed B-cell or hybridoma), or into an in vitro translation
system, and the translated polypeptide may be isolated. One of
ordinary skill in the art will recognize that antibody light chain
proteins and heavy chain proteins are produced in the cell with a
leader sequence that is removed upon production of a mature
anti-ILT5 antibody or ILT5-binding fragment thereof.
[0128] Anti-ILT5 antibodies and ILT5-binding antibody fragments can
be prepared by recombinant expression of immunoglobulin light and
heavy chain genes in a host cell. For example, a host cell is
transfected with one or more recombinant expression vectors
carrying DNA fragments encoding the light and heavy chains of the
anti-ILT5 antibody or ILT5-binding fragment such that the light and
heavy chains are expressed in the host cell and, preferably,
secreted into the medium in which the host cells are cultured, from
which medium the anti-ILT5 antibody or ILT5-binding fragment can be
recovered. Standard recombinant DNA methodologies are used to
obtain antibody heavy and light chain genes, incorporate these
genes into recombinant expression vectors, and introduce the
vectors into host cells (e.g., methodologies such as those
described in Sambrook, Fritsch and Maniatis (eds), Molecular
Cloning; A Laboratory Manual, Second Edition, Cold Spring Harbor,
N.Y., 1989; Ausubel, F. M. et al. (eds.) Current Protocols in
Molecular Biology, Greene Publishing Associates, 1989; and in U.S.
Pat. No. 4,816,397, each of which is incorporated herein by
reference in its entirety.
[0129] As is understood in the art, an expression vector comprises
sequences that mediate replication and often comprises one or more
selectable markers. An expression vector is transfected into a host
cell by standard techniques. Non-limiting examples include
electroporation, calcium-phosphate precipitation, DEAE-dextran
transfection and the like.
[0130] To express an anti-ILT5 antibody or an ILT5-binding antibody
fragment, DNA (e.g., cDNA) molecules encoding partial or
full-length light and heavy chains (e.g., human or humanized heavy
and light chains) may be inserted into expression vectors such that
the genes are operatively linked to transcriptional and
translational control sequences. In this context, the term
"operatively linked" means that a nucleic acid sequence encoding
the anti-ILT5 antibody or ILT5-binding fragment is inserted into a
vector such that transcriptional and translational control
sequences within the vector serve their intended function of
regulating the transcription and translation of the nucleic acid
sequence. In certain embodiments, the expression vector and
expression control sequences are chosen to be compatible with the
expression host cell used. Nucleic acid sequences encoding the
light and heavy chains may be inserted into separate vectors or
both genes may be inserted into the same expression vector. The
nucleic acid sequences may be inserted into the expression vector
by standard methods (e.g., ligation of complementary restriction
sites on the binding molecule gene fragment and vector, or blunt
end ligation if no restriction sites are present). Prior to
insertion of light and/or heavy chain-encoding sequences, the
expression vector may already comprise a nucleic acid sequence
encoding a constant region. For example, one approach to converting
VH and VL sequences to full-length antibody-encoding sequences is
to insert them into expression vectors already encoding heavy chain
constant and light chain constant regions, respectively, such that
the VH segment is operatively linked to the CH segment(s) within
the vector and the VL segment is operatively linked to the CL
segment within the vector. Additionally or alternatively, the
recombinant expression vector can encode a signal peptide fused in
frame to the heavy and/or light chain that facilitates secretion of
the binding molecule chain from a host cell. The signal peptide can
be an immunoglobulin signal peptide or a heterologous signal
peptide (i.e., a signal peptide from a non-immunoglobulin
protein).
[0131] Those skilled in the art will be able to determine whether
an antibody or fragment comprising a given polypeptide sequence
binds to ILT5 without undue experimentation using standard
methodologies such as, without limitation, Western blots, ELISA
assays, and the like.
[0132] Certain embodiments of methods and compositions provided
herein are further illustrated by the following examples. The
examples are provided for illustrative purposes only. They are not
to be construed as limiting the scope or content of the invention
in any way.
EXAMPLES
Example 1: Preparation of Anti-ILT5 Antibodies
[0133] A human ILT5-mouse Ig fusion construct was generated using
standard molecular biology techniques. Soluble ILT5-Ig fusion
protein was purified from the cell culture supernatant of
transiently transfected 293 cells by Protein A/G-Sepharose
chromatography. Expression plasmid DNA (100 .mu.g) encoding the
ILT5-Ig fusion protein was coated onto gold beads (1 .mu.M)
according to instructions from the manufacturer (Bio-Rad, Hercules,
Calif.). Mice were immunized with hILT5-Ig expression
plasmid-coated gold beads every other day for 10 days using a
Helios.RTM. Gene Gun. Sera from immunized mice were tested for
reactivity by ELISA against purified hILT5-Ig protein. Mice with
demonstrated serum immunoreactivity were boosted with recombinant
hILT5-Ig fusion protein (20 .mu.g/200 .mu.l) three days prior to
fusion. Hybridoma supernatants were screened by ELISA for
immunoreactivity against purified hILT5-Ig and an irrelevant Ig
fusion protein. Hybridomas producing antibody reactive with
hILT5-Ig but not the irrelevant fusion protein were cloned by
limiting dilution and soft agar. The 8G6 mAb (IgG1, .kappa.,
hereafter referred to as "TRX585") was purified from hybridoma
culture supernatant by Protein G Sepharose column chromatography
and dialyzed against Dulbecco's Phosphate Buffered Saline overnight
at 2-8.degree. C. The purified TRX585 mAb was stored at -80.degree.
C. until use.
Example 2: Expression of ILT5 Receptor
[0134] Surface Expression of ILT5:
[0135] Peripheral blood mononuclear cells (PBMCs) from healthy
human blood donors were stained with a murine monoclonal antibody
(TRX585) comprising a heavy chain variable region comprising the
amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 3, and a light
chain variable region comprising the amino acid sequence of SEQ ID
NO: 1. Sequencing analysis of the heavy chain variable region was
inconclusive as to whether the first amino acid residue was D or E.
As the two amino acids are very similar and the first residue is
not in a CDR, it is highly likely that VH with D or E at the first
position would have very similar if not the same ILT5-binding
properties. PBMCs were also stained with antibodies specific for
defined hematopoietic cell lineages and analyzed for ILT5
expression by flow cytometry.
[0136] ILT5 Expression by CD4.sup.+ and CD8.sup.+ T Cells:
[0137] The surface expression of ILT5 was observed on about 1% of
CD56.sup.-CD4.sup.+CD3.sup.+ T cells but not on
CD56.sup.-CD8.sup.+CD3.sup.+ T cells (FIG. 1A). Experimental
details can be found in FIG. 1A and the description thereof.
[0138] ILT5 Expression by Tregs:
[0139] ILT5 expression by regulatory T cells (Tregs) was examined
by staining with the TRX585 antibody. Naive
Foxp3.sup.+CD4.sup.+CD3.sup.+ cells were found to display about 10
fold more ILT5 than their Foxp3- counterpart (see FIG. 1B).
Furthermore, 5% of CD4.sup.+ natural killer T (NKT) cells, another
immunomodulatory T cell subset that is restricted by the
non-classical MHC class I molecule, CD1d, also exhibited surface
expression of ILT5 (FIG. 1C). Experimental procedures can be found
in FIG. 1 (panels B and C) and the description thereof.
[0140] ILT5 Expression by APCs:
[0141] ILT5 expression was measured on APCs by staining with the
TRX585 antibody. Steady state myeloid dendritic cells (DCs)
(CD11c.sup.+HLA.sup.-DR.sup.+ cells) showed ILT5 expression whereas
plasmacytoid DCs did not express surface ILT5 (see FIG. 1D). In
contrast, the majority of monocytic subsets were found to express
ILT5, albeit at varying levels (FIG. 1E). Further experimental
details can be found in FIG. 1 (panels D, E) and the description
thereof.
[0142] ILT5 Expression by MDSCs:
[0143] Cancer patients often show an increase in myeloid-derived
suppressor cells (MDSCs), which can suppress T cell responses in
peripheral blood, as well as within the tumors. Increasing evidence
suggests that MDSCs contribute to the induction of tolerance in
cancer and some other pathologies. ILT5 expression on steady state
peripheral blood MDSCs, defined as
CD33.sup.hiCD34.sup.loCD11b.sup.+CD14.sup.+ cells, was detected by
staining with the TRX585 antibody. These cells were found to
express high levels of surface ILT5 (FIG. 1F). Experimental
procedures are detailed in FIG. 1F and the description thereof.
Example 3: Characteristics and Biological Activity of the TRX585
Anti-ILT5 Antibody
[0144] Immunoregulatory Properties of TRX585 Antibody:
[0145] Stimulation of peripheral blood mononuclear cells (PBMC)
with mitomycin C-treated allogeneic PBMCs is an established in
vitro model for T cell responsiveness. Because ILT molecules are
thought to be immunomodulatory, it was tested whether crosslinking
of ILT5 by means of the mouse anti-human ILT5 TRX585 antibody would
modulate a mixed lymphocyte response (MLR). To this end, the
proliferation of allogeneic PBMCs in primary MLRs performed in the
presence or absence of increasing doses of soluble TRX585 antibody
or a mouse IgG1 isotype control antibody (mIgG1) was compared.
There was no change in the intensity of the MLR responses for
varying concentrations of control mIgG1 (see FIG. 2). In contrast,
TRX585 antibody-mediated a dose-dependent enhancement of cell
proliferation (FIG. 2). Further detail can be found in FIG. 2 and
the description thereof.
[0146] These observations were established under conditions in
which TRX585 antibody was left in the culture for the entire
duration of the assay. To examine whether the TRX585
antibody-mediated increase of T cell proliferation was dependent on
the level of antibodies present in the culture, ILT-expressing
antigen-presenting cells (APCs) were pretreated with soluble (50
.mu.g/ml) TRX585 antibody for 24-48 hours, washed, and utilized as
stimulators in allogeneic MLRs. Pretreatment of monocytes or
peripheral blood DCs with soluble TRX585 antibody prior to its use
in MLR assays recapitulated the antibody-mediated enhancement of
cell proliferation that was observed in initial experiments (FIG.
3). Further detail can be found in FIG. 3 and the description
thereof.
[0147] Myeloid/monocytic cells express both activation and
inhibitory Fc receptors, the engagement of which by antibodies can
either enhance or downregulate immunity and, thus, either increase
or decrease the potency of antibodies with immunoregulatory
properties. To test whether the biological effect of TRX585
antibody could be enhanced by Fc crosslinking, primary MLR assays
were conducted as described above in the presence or absence of 10
.mu.g/ml F(ab').sub.2 goat anti-mouse IgG antibody. At
subsaturating concentrations (<10 .mu.g/ml), monovalent (i.e.,
in the absence of F(ab').sub.2 goat anti-mouse IgG antibody) but
not divalent TRX585 antibody (i.e., in the presence of F(ab').sub.2
goat anti-mouse IgG antibody) induced cell proliferation (see FIG.
4). However, addition of TRX585 antibody at concentrations higher
than that of F(ab').sub.2 fragments restored hyperresponsiveness,
albeit at levels much lower than that observed with identical
concentrations of monovalent TRX585 antibody (i.e., in the absence
of F(ab').sub.2 goat anti-mouse IgG antibody) (see FIG. 4).
Experimental procedures are detailed in FIG. 4 and the description
thereof.
[0148] To determine whether the above observations could be
extended to antibodies of higher valency, allogeneic PBMCs were
seeded either with soluble or solid phase TRX585 antibody.
Antibody-induced proliferation was observed with soluble but not
TRX585 antibody immobilized on plastic (see FIG. 5 and the
description thereof). In accordance with these observations,
pretreatment of APCs with solid phase antibody did not lead to an
enhancement of T cell responses (data not shown). Addition of
soluble TRX585 antibody to APCs resulted in occupancy and partial
internalization of surface ILT5, whereas addition of solid phase
TRX585 antibody to APCs induced complete internalization of ILT5
(data not shown).
[0149] Overall, these observations indicate that monovalent and
polyvalent TRX585 antibodies have a differential effect on APCs and
APC-mediated regulation of T cell responses. Furthermore, the above
findings suggest that in vivo hypercrosslinking of ILT5 antigens on
APCs by TRX585 antibody may decrease the effectiveness of the
latter reagents. Crosslinking can be reduced by modification of the
antibody to reduce or eliminate binding via the Fc receptors.
[0150] Closer examination of proliferating cells in MLR assays
revealed that TRX585 antibodies induced the proliferation of the
vast majority of CD4.sup.+ and CD8.sup.+ T cells in allogeneic as
well as autologous settings. This was determined by examining cell
content CFSE dye by flow cytometry since this fluorescent dye gets
diluted as cells divide (see FIG. 6A and the description thereof).
In another experiment, purified T cells were cultured with the
TRX585 or mIgG1, with or without allogeneic stimulator cells. The
lack of proliferation of the purified T cells, which was observed,
ruled out the possibility that TRX585 antibody was directly
mitogenic to T cells, and demonstrated that TRX585 antibody-induced
T cell proliferation required the presence of non-T cells (see FIG.
6B and the description thereof).
[0151] Because not all T cells can undergo simultaneous
proliferation as a consequence of self and/or non-self recognition,
the previous observations suggested that TRX585 antibody-induced T
cell proliferation was achieved in a TCR-independent manner (e.g.,
proliferation does not require recognition of a MHC molecule by a
TCR). Indeed, blocking TCR: MHC/peptide complex interactions by
means of pan anti-MHC antibodies did not abrogate TRX585
antibody-induced T cell proliferation (see FIG. 7). Further detail
can be found in FIG. 7 and the description thereof.
[0152] Generation of T Cells with Altered Phenotype:
[0153] Upon culturing PBMCs with the TRX585 antibody as described
above, proliferating CD4.sup.+ and CD8.sup.+ T cells acquired a
unique phenotype. In addition to upregulating CD25, T cells also
upregulated expression of NKG2D, a major innate activating immune
receptor that plays an important role in anti-tumor and anti-viral
immunity (see FIGS. 8A and 8B and the description thereof).
[0154] Ligands for NKG2D are rarely detected on healthy tissues and
cells, but are often expressed by tumor cells as well as
virus-infected cells. In humans and mice, local as well as systemic
(through shedding of NKG2D ligands) down-regulation of NKG2D as a
consequence of persistent expression of NKG2D ligands is one
mechanism by which tumors and viruses escape immune surveillance.
We thus examined NKG2D expression on T cells that were subjected to
both TRX585 antibodies and signals mimicking NKG2D persistent
engagement by NKG2D ligands. Remarkably, TRX585 antibody-exposed T
cells not only upregulated NKG2D at levels higher than that
observed on steady state natural killer (NK) cells (see FIG. 8B and
the description thereof for further detail), but presented with a
sustained expression of NKG2D under conditions that normally
trigger its internalization and subsequent degradation (e.g., via
NKG2D engagement by either soluble MICA antigen (a NKG2D ligand) or
anti-NKG2D mAbs, clones 1D11 and 5C6) (see FIG. 9 and the
description thereof for further detail).
[0155] The tight control of NKG2D-mediated effector functions by
microenvironmental factors, such as NKG2D ligands and cytokines,
should provide an additional safeguard mechanism to prevent the
development of unwanted immune responses.
[0156] Production of Cytokines and Chemokines:
[0157] Supernatants from MLR assay cultures conducted in the
presence of soluble TRX585 antibody contained increased amounts of
TNF-alpha and IL5 as compared to control cultures (data not shown).
Addition of TRX585 antibody did not result in overproduction of
other major cytokines such as IL2, IFN-gamma, IL17, IL6, and IL12.
In contrast, TRX585 antibody-containing cultures contained
increased amounts of Rantes, IP-10 and MIP-1 chemokines, which play
an active role in recruiting leukocytes into inflammatory sites and
can elicit powerful antitumor effect in vivo (data not shown). In
cultures containing TRX585 antibody, T cells also overproduced
soluble Fas ligand, a factor that participates in essential
effector functions of the immune system and is, for example, a
potent mediator of cytotoxicity. Further detail can be found in
FIG. 10 and the description thereof. The above observation prompted
investigation into whether TRX585 antibody-activated T cells were
endowed with cytotoxic activity. To this end, PBMCs were cultured
in the presence of TRX585 or mIgG1 antibodies for 3.5 days.
Proliferating T cells (effector cells) were subsequently
cell-sorted and mixed with a variety of tumor cells (target cells)
at different effector:target (E:T) ratios for 12-18 hours.
Examination of tumor cell viability after the incubation showed
that T cells from TRX585 antibody-containing but not
mIgG1-containing precultures exerted a potent anti-tumor cytotoxic
effect (see FIGS. 11A and B). Although the presence of human
cytotoxic T cells has been reported in a number of viral infections
and rheumatoid arthritis, acquisition of lytic activity by
CD4.sup.+ T cells is a rare event. Yet, preactivation of PBMCs with
TRX585 antibodies was found to confer a cytotoxic activity to both
CD4.sup.+ and CD8.sup.+ T cell subsets (see FIG. 11C and the
description thereof for experimental procedures). Of note, the
cytotoxic activity of CD4.sup.+ and CD8.sup.+ T cells appeared to
be specific to tumor cells but not healthy cells since the same T
cells did not kill autologous or allogeneic PBMCs using the same
killing assay (not shown).
[0158] TRX585 antibody-induced cytotoxic CD4.sup.+ and CD8.sup.+ T
cells did not express perforin or granzyme A (data not shown),
ruling out these molecules as possible mediators of the observed
cytotoxicity. In contrast, blocking MHC class I molecules or Fas
ligand by means of a pan anti-MHC class I antibody or a
neutralizing anti-Fas ligand antibody, respectively, markedly
diminished the anti-tumor cytotoxic effect of T cells from TRX585
antibody-containing cultures. Further detail can be found in FIG.
12 and the description thereof. In addition, both CD4.sup.+ and
CD8.sup.+ T cells from TRX585 antibody-containing cultures were
found to express high levels of granzyme B.
[0159] Overall, these data demonstrate that while T cells that have
been cultured with APC and TRX585 antibody are able to exert a
potent cytotoxic effect, such cells do not exhibit cytotoxic
function in the absence of an appropriate trigger.
[0160] To determine whether the sequence of administration of
TRX585 antibody impacted the modulation of immune responses, we
conducted a series of in vitro experiments in which either TRX585
antibody and TCR stimulation were given simultaneously, or TRX585
antibody was added prior to the delivery of TCR stimulus. FIG. 13
shows that, while CD4.sup.+ and CD8.sup.+ T cells from PBMC
cultures containing TRX585 antibody divided actively, concomitant
treatment of PBMCs with anti-CD3 and TRX585 antibodies resulted in
the inhibition of TRX585-induced T cell proliferation. In contrast,
when T cells that were induced to proliferate in TRX585
antibody-containing PBMC cultures were subsequently purified and
subjected to anti-CD3 stimulation, such T cells showed markedly
increased responsiveness to TCR stimulation and upregulated TCR:CD3
complexes on the cell surface (see FIGS. 14A and 14B and the
description thereof for additional detail).
[0161] Overall, these results indicate that TRX585 antibody may be
used to overcome tumor-specific tolerance, enhance immune responses
and/or induce tumor cell killing. Such effects may result from
acquisition of anti-tumor cytotoxic function by the T cells
resulting from administration of TRX585 antibodies followed by
another therapeutic agent or antigen.
Example 4: Use of Anti-ILT5 Antibodies and Fragments as
Immunostimulatory Adjuvants
[0162] An anti-ILT5 antibody or an ILT5-binding antibody fragment
is used as an immunostimulatory agent to enhance an immune response
to an antigen of interest. To stimulate an antibody or cellular
immune response to an antigen of interest in vivo (e.g., for
vaccination purposes), the antigen and an anti-ILT5 antibody or an
ILT5-binding antibody fragment are administered to a human subject
such that an enhanced immune response occurs in the subject. The
antigen of interest and the anti-ILT5 antibody or ILT5-binding
fragment are formulated appropriately, e.g., in separate
pharmaceutical compositions. In certain situations, it may be
desirable to administer the antibody at or about the same time as
the antigen. In certain situations, it may be desirable to
administer the antibody first, followed by the antigen, wherein a
priming dose of the antibody is administered prior to
administration of the antigen of interest to allow pharmacodynamic
effect on the T-cells. For example, the anti-ILT5 antibody or
ILT5-binding fragment can be administered 1-14 days (e.g., 3 days)
before administration of the antigen of interest. It is expected
that upon administration of the antigen of interest, a robust
immune response against the antigen will be induced.
Example 5: Use of Anti-ILT5 Antibodies and Fragments to Increase a
Specific Immune Response to Tumor Cells
[0163] An anti-ILT5 antibody or an ILT5-binding antibody fragment
is administered to a subject having tumor cells to overcome
tumor-specific tolerance in the subject and to upmodulate an immune
response to inhibit tumor growth, metastasis or to trigger tumor
eradication. The tumor may be, for example, of the hematopoietic
system, such as, a leukemia, lymphoma, or other malignancy of blood
cells, or of a solid tumor, such as, a melanoma, gastric, lung,
breast, and prostate cancers. In certain embodiments, such
anti-ILT5 antibodies or ILT5-binding fragments are used as part of
a combination therapy with another therapeutic treatment in a
subject as adjuvants used to enhance an immune response such as in
combination with chemotherapeutic agents. It is expected that upon
administration of the anti-ILT5 antibody or ILT5-binding fragment
thereof, tumor-specific tolerance will be reduced, resulting in
diminished tumor growth or metastasis, and tumors will be
eradicated or reduced in size or number. Following administration
of the anti-ILT5 antibody, one or more appropriate tumor antigens
(see above) or vaccines may also be administered.
Example 6: Use of Anti-ILT5 Antibodies and Fragments to Increase a
Specific Immune Response to Cells Infected with a Virus
[0164] An anti-ILT5 antibody or an ILT5-binding antibody fragment
is administered to a subject suffering from a viral infection to
upmodulate an immune response against cells infected with the
virus. In certain embodiments, such anti-ILT5 antibodies or
ILT5-binding fragments are used as part of a combination therapy
with another therapeutic treatment in a subject as adjuvants used
to enhance an immune response. It is expected that upon
administration of the anti-ILT5 antibody or ILT5-binding fragment
thereof, cells infected with the virus are eliminated or reduced in
number. Following administration of the anti-ILT5 antibody, one or
more appropriate viral antigens (see above) or vaccines may also be
administered.
Example 7: Use of Crosslinked or Aggregated Anti-ILT5 Antibodies
and Fragments to Induce Tolerance
[0165] A crosslinked or otherwise aggregated anti-ILT5 antibody or
an ILT5-binding antibody fragment is administered to a subject to
inhibit a cellular immune response to an antigen of interest in
vivo. Without wishing to be bound by theory, it is hypothesized
that co-engagement of ILT5 receptors by such crosslinked, but not
monovalent, anti-ILT5 antibodies and ILT5-binding fragments,
initiates an inhibitory cascade in ILT-expressing APCs, which
decreases their stimulatory potential or might render them
tolerogenic. In certain embodiments, a crosslinked or otherwise
aggregated anti-ILT5 antibody or ILT5-binding fragment may be
administered at the same time as administration of another
therapeutic agent or antigen to inhibit immune response to the
antigen. It will be appreciated that the effects of concomitant
removal of ILT ligand-transduced inhibitory signals in T cells and
decreased DC-immunostimulatory capacities counterbalance each other
and lead to diminished immunity. It is expected that upon
administration of the anti-ILT5 antibody or ILT5-binding fragment
thereof, tolerance will be induced.
[0166] Procedures such as those described in Example 6 would be
useful as treatments of, for example, autoimmune diseases and
immunological rejection of allogeneic and xenogeneic organ, tissue,
or cell transplants.
Sequence CWU 1
1
321107PRTMus musculus 1Asp Ile Gln Met Thr Gln Ser Pro Ala Ser Leu
Ser Val Ser Val Gly1 5 10 15 Glu Thr Val Thr Ile Thr Cys Arg Ala
Ser Glu Asn Ile Tyr Ser Asn 20 25 30 Leu Ala Trp Tyr Gln Gln Lys
Gln Gly Lys Ser Pro Gln Val Leu Val 35 40 45 Tyr Ala Ala Thr Asn
Leu Ala Asp Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser
Gly Thr Gln Phe Ser Leu Lys Ile Asn Ser Leu Gln Ser65 70 75 80 Glu
Asp Phe Gly Asn Tyr Phe Cys Gln His Phe Trp Arg Ile Pro Trp 85 90
95 Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys 100 105 2124PRTMus
musculus 2Asp Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro
Ser Gln1 5 10 15 Ser Leu Phe Leu Thr Cys Ser Val Thr Gly Tyr Ser
Ile Ser Ser Ser 20 25 30 Tyr Tyr Trp Asn Trp Ile Arg Gln Phe Pro
Gly Asn Lys Leu Glu Trp 35 40 45 Met Gly Tyr Ile Ser Phe Asp Gly
Ser Asn Asn Tyr Asn Pro Ser Leu 50 55 60 Lys Asn Arg Ile Ser Ile
Thr Arg Asp Thr Ser Lys Asn Gln Phe Phe65 70 75 80 Leu Lys Leu Asn
Ser Val Thr Thr Glu Asp Thr Ala Thr Tyr Tyr Cys 85 90 95 Ala Arg
Glu Lys Glu Asn Tyr Tyr Gly Ser Ser Phe Tyr Tyr Phe Asp 100 105 110
Tyr Trp Gly Leu Gly Thr Ser Leu Thr Val Ser Ser 115 120 3124PRTMus
musculus 3Glu Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro
Ser Gln1 5 10 15 Ser Leu Phe Leu Thr Cys Ser Val Thr Gly Tyr Ser
Ile Ser Ser Ser 20 25 30 Tyr Tyr Trp Asn Trp Ile Arg Gln Phe Pro
Gly Asn Lys Leu Glu Trp 35 40 45 Met Gly Tyr Ile Ser Phe Asp Gly
Ser Asn Asn Tyr Asn Pro Ser Leu 50 55 60 Lys Asn Arg Ile Ser Ile
Thr Arg Asp Thr Ser Lys Asn Gln Phe Phe65 70 75 80 Leu Lys Leu Asn
Ser Val Thr Thr Glu Asp Thr Ala Thr Tyr Tyr Cys 85 90 95 Ala Arg
Glu Lys Glu Asn Tyr Tyr Gly Ser Ser Phe Tyr Tyr Phe Asp 100 105 110
Tyr Trp Gly Leu Gly Thr Ser Leu Thr Val Ser Ser 115 120 46PRTMus
musculus 4Ser Ser Tyr Tyr Trp Asn1 5 516PRTMus musculus 5Tyr Ile
Ser Phe Asp Gly Ser Asn Asn Tyr Asn Pro Ser Leu Lys Asn1 5 10 15
615PRTMus musculus 6Glu Lys Glu Asn Tyr Tyr Gly Ser Ser Phe Tyr Tyr
Phe Asp Tyr1 5 10 15 711PRTMus musculus 7Arg Ala Ser Glu Asn Ile
Tyr Ser Asn Leu Ala1 5 10 87PRTMus musculus 8Ala Ala Thr Asn Leu
Ala Asp1 5 99PRTMus musculus 9Gln His Phe Trp Arg Ile Pro Trp Thr1
5 10124PRTHomo sapiens 10Gln Val Gln Leu Gln Glu Ser Gly Pro Gly
Leu Val Lys Pro Pro Gly1 5 10 15 Thr Leu Ser Leu Thr Cys Ala Val
Ser Gly Gly Ser Ile Ser Ser Ser 20 25 30 Tyr Tyr Trp Asn Trp Val
Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp 35 40 45 Ile Gly Tyr Ile
Ser Phe Asp Gly Ser Asn Asn Tyr Asn Pro Ser Leu 50 55 60 Lys Asn
Arg Val Thr Ile Ser Val Asp Lys Ser Lys Asn Gln Phe Ser65 70 75 80
Leu Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Cys Cys 85
90 95 Ala Arg Glu Lys Glu Asn Tyr Tyr Gly Ser Ser Phe Tyr Tyr Phe
Asp 100 105 110 Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser 115
120 11124PRTHomo sapiens 11Gln Val Gln Leu Gln Glu Ser Gly Pro Gly
Leu Val Lys Pro Ser Gly1 5 10 15 Thr Leu Ser Leu Thr Cys Ala Val
Ser Gly Gly Ser Ile Ser Ser Ser 20 25 30 Tyr Tyr Trp Asn Trp Val
Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp 35 40 45 Ile Gly Tyr Ile
Ser Phe Asp Gly Ser Asn Asn Tyr Asn Pro Ser Leu 50 55 60 Lys Asn
Arg Val Thr Ile Ser Val Asp Lys Ser Lys Asn Gln Phe Ser65 70 75 80
Leu Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Cys Cys 85
90 95 Ala Arg Glu Lys Glu Asn Tyr Tyr Gly Ser Ser Phe Tyr Tyr Phe
Asp 100 105 110 Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser 115
120 12124PRTHomo sapiens 12Gln Val Gln Leu Gln Glu Ser Gly Pro Gly
Leu Val Lys Pro Pro Gly1 5 10 15 Thr Leu Ser Leu Thr Cys Ala Val
Ser Gly Gly Ser Ile Ser Ser Ser 20 25 30 Tyr Tyr Trp Asn Trp Val
Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp 35 40 45 Ile Gly Tyr Ile
Ser Phe Asp Gly Ser Asn Asn Tyr Asn Pro Ser Leu 50 55 60 Lys Asn
Arg Val Thr Ile Ser Val Asp Lys Ser Lys Asn Gln Phe Ser65 70 75 80
Leu Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys 85
90 95 Ala Arg Glu Lys Glu Asn Tyr Tyr Gly Ser Ser Phe Tyr Tyr Phe
Asp 100 105 110 Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser 115
120 13124PRTHomo sapiens 13Gln Val Gln Leu Gln Glu Ser Gly Pro Gly
Leu Val Lys Pro Ser Asp1 5 10 15 Thr Leu Ser Leu Thr Cys Ala Val
Ser Gly Tyr Ser Ile Ser Ser Ser 20 25 30 Tyr Tyr Trp Asn Trp Ile
Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp 35 40 45 Ile Gly Tyr Ile
Ser Phe Asp Gly Ser Asn Asn Tyr Asn Pro Ser Leu 50 55 60 Lys Asn
Arg Val Thr Met Ser Val Asp Thr Ser Lys Asn Gln Phe Ser65 70 75 80
Leu Lys Leu Ser Ser Val Thr Ala Val Asp Thr Ala Val Tyr Tyr Cys 85
90 95 Ala Arg Glu Lys Glu Asn Tyr Tyr Gly Ser Ser Phe Tyr Tyr Phe
Asp 100 105 110 Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser 115
120 14124PRTHomo sapiens 14Gln Leu Gln Leu Gln Glu Ser Gly Pro Gly
Leu Val Lys Pro Ser Glu1 5 10 15 Thr Leu Ser Leu Thr Cys Thr Val
Ser Gly Gly Ser Ile Ser Ser Ser 20 25 30 Tyr Tyr Trp Asn Trp Ile
Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp 35 40 45 Ile Gly Tyr Ile
Ser Phe Asp Gly Ser Asn Asn Tyr Asn Pro Ser Leu 50 55 60 Lys Asn
Arg Val Thr Ile Ser Val Asp Thr Ser Lys Asn Gln Phe Ser65 70 75 80
Leu Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys 85
90 95 Ala Arg Glu Lys Glu Asn Tyr Tyr Gly Ser Ser Phe Tyr Tyr Phe
Asp 100 105 110 Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser 115
120 15124PRTHomo sapiens 15Gln Val Gln Leu Gln Glu Ser Gly Pro Gly
Leu Val Lys Pro Ser Glu1 5 10 15 Thr Leu Ser Leu Thr Cys Thr Val
Ser Gly Gly Ser Ile Ser Ser Ser 20 25 30 Tyr Tyr Trp Asn Trp Ile
Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp 35 40 45 Ile Gly Tyr Ile
Ser Phe Asp Gly Ser Asn Asn Tyr Asn Pro Ser Leu 50 55 60 Lys Asn
Arg Val Thr Ile Ser Val Asp Thr Ser Lys Asn Gln Phe Ser65 70 75 80
Leu Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys 85
90 95 Ala Arg Glu Lys Glu Asn Tyr Tyr Gly Ser Ser Phe Tyr Tyr Phe
Asp 100 105 110 Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser 115
120 16124PRTHomo sapiens 16Gln Val Gln Leu Gln Glu Ser Gly Pro Gly
Leu Val Lys Pro Ser Glu1 5 10 15 Thr Leu Ser Leu Thr Cys Thr Val
Ser Gly Gly Ser Val Ser Ser Ser 20 25 30 Tyr Tyr Trp Asn Trp Ile
Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp 35 40 45 Ile Gly Tyr Ile
Ser Phe Asp Gly Ser Asn Asn Tyr Asn Pro Ser Leu 50 55 60 Lys Asn
Arg Val Thr Ile Ser Val Asp Thr Ser Lys Asn Gln Phe Ser65 70 75 80
Leu Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys 85
90 95 Ala Arg Glu Lys Glu Asn Tyr Tyr Gly Ser Ser Phe Tyr Tyr Phe
Asp 100 105 110 Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser 115
120 17124PRTHomo sapiens 17Gln Val Gln Leu Gln Glu Ser Gly Pro Gly
Leu Val Lys Pro Ser Glu1 5 10 15 Thr Leu Ser Leu Thr Cys Ala Val
Ser Gly Tyr Ser Ile Ser Ser Ser 20 25 30 Tyr Tyr Trp Asn Trp Ile
Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp 35 40 45 Ile Gly Tyr Ile
Ser Phe Asp Gly Ser Asn Asn Tyr Asn Pro Ser Leu 50 55 60 Lys Asn
Arg Val Thr Ile Ser Val Asp Thr Ser Lys Asn Gln Phe Ser65 70 75 80
Leu Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys 85
90 95 Ala Arg Glu Lys Glu Asn Tyr Tyr Gly Ser Ser Phe Tyr Tyr Phe
Asp 100 105 110 Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser 115
120 18112PRTHomo sapiens 18Ala Ile Arg Met Thr Gln Ser Pro Ser Ser
Phe Ser Ala Ser Thr Gly1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg
Ala Ser Glu Asn Ile Tyr Ser Asn 20 25 30 Leu Ala Trp Tyr Gln Gln
Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Ala Ala Thr
Asn Leu Ala Asp Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly
Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Cys Leu Gln Ser65 70 75 80
Glu Asp Phe Ala Thr Tyr Tyr Phe Ala Thr Tyr Tyr Cys Gln His Phe 85
90 95 Trp Arg Ile Pro Trp Thr Phe Gly Gln Gly Thr Lys Val Glu Ile
Lys 100 105 110 19107PRTHomo sapiens 19Asp Ile Gln Leu Thr Gln Ser
Pro Ser Phe Leu Ser Ala Ser Val Gly1 5 10 15 Asp Arg Val Thr Ile
Thr Cys Arg Ala Ser Glu Asn Ile Tyr Ser Asn 20 25 30 Leu Ala Trp
Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr
Ala Ala Thr Asn Leu Ala Asp Gly Val Pro Ser Arg Phe Ser Gly 50 55
60 Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln
Pro65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln His Phe Trp Arg
Ile Pro Trp 85 90 95 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys
100 105 20107PRTHomo sapiens 20Asp Ile Gln Met Thr Gln Ser Pro Ser
Ser Val Ser Ala Ser Val Gly1 5 10 15 Asp Arg Val Thr Ile Thr Cys
Arg Ala Ser Glu Asn Ile Tyr Ser Asn 20 25 30 Leu Ala Trp Tyr Gln
Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Ala Ala
Thr Asn Leu Ala Asp Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser
Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro65 70 75
80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln His Phe Trp Arg Ile Pro Trp
85 90 95 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys 100 105
21107PRTHomo sapiens 21Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Val
Ser Ala Ser Val Gly1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala
Ser Glu Asn Ile Tyr Ser Asn 20 25 30 Leu Ala Trp Tyr Gln Gln Lys
Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Ala Ala Thr Asn
Leu Ala Asp Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser
Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro65 70 75 80 Glu
Asp Phe Ala Thr Tyr Tyr Cys Gln His Phe Trp Arg Ile Pro Trp 85 90
95 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys 100 105 22107PRTHomo
sapiens 22Ala Ile Gln Leu Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser
Val Gly1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Glu Asn
Ile Tyr Ser Asn 20 25 30 Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys
Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Ala Ala Thr Asn Leu Ala Asp
Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp
Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro65 70 75 80 Glu Asp Phe Ala
Thr Tyr Tyr Cys Gln His Phe Trp Arg Ile Pro Trp 85 90 95 Thr Phe
Gly Gln Gly Thr Lys Val Glu Ile Lys 100 105 23107PRTHomo sapiens
23Ala Ile Gln Leu Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly1
5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Glu Asn Ile Tyr Ser
Asn 20 25 30 Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys
Leu Leu Ile 35 40 45 Tyr Ala Ala Thr Asn Leu Ala Asp Gly Val Pro
Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu
Thr Ile Ser Ser Leu Gln Pro65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr
Cys Gln His Phe Trp Arg Ile Pro Trp 85 90 95 Thr Phe Gly Gln Gly
Thr Lys Val Glu Ile Lys 100 105 24107PRTHomo sapiens 24Asp Ile Gln
Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly1 5 10 15 Asp
Arg Val Thr Ile Thr Cys Arg Ala Ser Glu Asn Ile Tyr Ser Asn 20 25
30 Leu Ala Trp Tyr Gln Gln Lys Pro Glu Lys Ala Pro Lys Ser Leu Ile
35 40 45 Tyr Ala Ala Thr Asn Leu Ala Asp Gly Val Pro Ser Arg Phe
Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser
Ser Leu Gln Pro65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln His
Phe Trp Arg Ile Pro Trp 85 90 95 Thr Phe Gly Gln Gly Thr Lys Val
Glu Ile Lys 100 105 25330PRTHomo sapiens 25Ala Ser Thr Lys Gly Pro
Ser Val Phe Pro Leu Ala Pro Ser Ser Lys1 5 10 15 Ser Thr Ser Gly
Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr 20 25 30 Phe Pro
Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 35 40 45
Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser 50
55
60 Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln
Thr65 70 75 80 Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys
Val Asp Lys 85 90 95 Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His
Thr Cys Pro Pro Cys 100 105 110 Pro Ala Pro Glu Leu Leu Gly Gly Pro
Ser Val Phe Leu Phe Pro Pro 115 120 125 Lys Pro Lys Asp Thr Leu Met
Ile Ser Arg Thr Pro Glu Val Thr Cys 130 135 140 Val Val Val Asp Val
Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp145 150 155 160 Tyr Val
Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu 165 170 175
Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu 180
185 190 His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser
Asn 195 200 205 Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys
Ala Lys Gly 210 215 220 Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro
Pro Ser Arg Asp Glu225 230 235 240 Leu Thr Lys Asn Gln Val Ser Leu
Thr Cys Leu Val Lys Gly Phe Tyr 245 250 255 Pro Ser Asp Ile Ala Val
Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn 260 265 270 Asn Tyr Lys Thr
Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe 275 280 285 Leu Tyr
Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn 290 295 300
Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr305
310 315 320 Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 325 330
26330PRTHomo sapiens 26Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu
Ala Pro Ser Ser Lys1 5 10 15 Ser Thr Ser Gly Gly Thr Ala Ala Leu
Gly Cys Leu Val Lys Asp Tyr 20 25 30 Phe Pro Glu Pro Val Thr Val
Ser Trp Asn Ser Gly Ala Leu Thr Ser 35 40 45 Gly Val His Thr Phe
Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser 50 55 60 Leu Ser Ser
Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr65 70 75 80 Tyr
Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys 85 90
95 Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys
100 105 110 Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe
Pro Pro 115 120 125 Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro
Glu Val Thr Cys 130 135 140 Val Val Val Asp Val Ser His Glu Asp Pro
Glu Val Lys Phe Asn Trp145 150 155 160 Tyr Val Asp Gly Val Glu Val
His Asn Ala Lys Thr Lys Pro Arg Glu 165 170 175 Glu Gln Tyr Ala Ser
Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu 180 185 190 His Gln Asp
Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn 195 200 205 Lys
Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly 210 215
220 Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp
Glu225 230 235 240 Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val
Lys Gly Phe Tyr 245 250 255 Pro Ser Asp Ile Ala Val Glu Trp Glu Ser
Asn Gly Gln Pro Glu Asn 260 265 270 Asn Tyr Lys Thr Thr Pro Pro Val
Leu Asp Ser Asp Gly Ser Phe Phe 275 280 285 Leu Tyr Ser Lys Leu Thr
Val Asp Lys Ser Arg Trp Gln Gln Gly Asn 290 295 300 Val Phe Ser Cys
Ser Val Met His Glu Ala Leu His Asn His Tyr Thr305 310 315 320 Gln
Lys Ser Leu Ser Leu Ser Pro Gly Lys 325 330 27106PRTHomo sapiens
27Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Ser Asp Glu Gln1
5 10 15 Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe
Tyr 20 25 30 Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala
Leu Gln Ser 35 40 45 Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp
Ser Lys Asp Ser Thr 50 55 60 Tyr Ser Leu Ser Ser Thr Leu Thr Leu
Ser Lys Ala Asp Tyr Glu Lys65 70 75 80 His Lys Val Tyr Ala Cys Glu
Val Thr His Gln Gly Leu Ser Ser Pro 85 90 95 Val Thr Lys Ser Phe
Asn Arg Gly Glu Cys 100 105 28214PRTArtificial
Sequencerecombinantly produced 28Asp Ile Gln Met Thr Gln Ser Pro
Ala Ser Leu Ser Val Ser Val Gly1 5 10 15 Glu Thr Val Thr Ile Thr
Cys Arg Ala Ser Glu Asn Ile Tyr Ser Asn 20 25 30 Leu Ala Trp Tyr
Gln Gln Lys Gln Gly Lys Ser Pro Gln Val Leu Val 35 40 45 Tyr Ala
Ala Thr Asn Leu Ala Asp Gly Val Pro Ser Arg Phe Ser Gly 50 55 60
Ser Gly Ser Gly Thr Gln Phe Ser Leu Lys Ile Asn Ser Leu Gln Ser65
70 75 80 Glu Asp Phe Gly Asn Tyr Phe Cys Gln His Phe Trp Arg Ile
Pro Trp 85 90 95 Thr Phe Gly Ala Gly Thr Lys Leu Glu Ile Lys Arg
Thr Val Ala Ala 100 105 110 Pro Ser Val Phe Ile Phe Pro Pro Ser Asp
Glu Gln Leu Lys Ser Gly 115 120 125 Thr Ala Ser Val Val Cys Leu Leu
Asn Asn Phe Tyr Pro Arg Glu Ala 130 135 140 Lys Val Gln Trp Lys Val
Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln145 150 155 160 Glu Ser Val
Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser 165 170 175 Ser
Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr 180 185
190 Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser
195 200 205 Phe Asn Arg Gly Glu Cys 210 29454PRTArtificial
Sequencerecombinantly produced 29Asp Val Gln Leu Gln Glu Ser Gly
Pro Gly Leu Val Lys Pro Ser Gln1 5 10 15 Ser Leu Phe Leu Thr Cys
Ser Val Thr Gly Tyr Ser Ile Ser Ser Ser 20 25 30 Tyr Tyr Trp Asn
Trp Ile Arg Gln Phe Pro Gly Asn Lys Leu Glu Trp 35 40 45 Met Gly
Tyr Ile Ser Phe Asp Gly Ser Asn Asn Tyr Asn Pro Ser Leu 50 55 60
Lys Asn Arg Ile Ser Ile Thr Arg Asp Thr Ser Lys Asn Gln Phe Phe65
70 75 80 Leu Lys Leu Asn Ser Val Thr Thr Glu Asp Thr Ala Thr Tyr
Tyr Cys 85 90 95 Ala Arg Glu Lys Glu Asn Tyr Tyr Gly Ser Ser Phe
Tyr Tyr Phe Asp 100 105 110 Tyr Trp Gly Ala Gly Thr Leu Val Thr Val
Ser Ser Ala Ser Thr Lys 115 120 125 Gly Pro Ser Val Phe Pro Leu Ala
Pro Ser Ser Lys Ser Thr Ser Gly 130 135 140 Gly Thr Ala Ala Leu Gly
Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro145 150 155 160 Val Thr Val
Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr 165 170 175 Phe
Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val 180 185
190 Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn
195 200 205 Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val
Glu Pro 210 215 220 Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys
Pro Ala Pro Glu225 230 235 240 Leu Leu Gly Gly Pro Ser Val Phe Leu
Phe Pro Pro Lys Pro Lys Asp 245 250 255 Thr Leu Met Ile Ser Arg Thr
Pro Glu Val Thr Cys Val Val Val Asp 260 265 270 Val Ser His Glu Asp
Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly 275 280 285 Val Glu Val
His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn 290 295 300 Ser
Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp305 310
315 320 Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu
Pro 325 330 335 Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln
Pro Arg Glu 340 345 350 Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp
Glu Leu Thr Lys Asn 355 360 365 Gln Val Ser Leu Thr Cys Leu Val Lys
Gly Phe Tyr Pro Ser Asp Ile 370 375 380 Ala Val Glu Trp Glu Ser Asn
Gly Gln Pro Glu Asn Asn Tyr Lys Thr385 390 395 400 Thr Pro Pro Val
Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys 405 410 415 Leu Thr
Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys 420 425 430
Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu 435
440 445 Ser Leu Ser Pro Gly Lys 450 30454PRTArtificial
Sequencerecombinantly produced 30Glu Val Gln Leu Gln Glu Ser Gly
Pro Gly Leu Val Lys Pro Ser Gln1 5 10 15 Ser Leu Phe Leu Thr Cys
Ser Val Thr Gly Tyr Ser Ile Ser Ser Ser 20 25 30 Tyr Tyr Trp Asn
Trp Ile Arg Gln Phe Pro Gly Asn Lys Leu Glu Trp 35 40 45 Met Gly
Tyr Ile Ser Phe Asp Gly Ser Asn Asn Tyr Asn Pro Ser Leu 50 55 60
Lys Asn Arg Ile Ser Ile Thr Arg Asp Thr Ser Lys Asn Gln Phe Phe65
70 75 80 Leu Lys Leu Asn Ser Val Thr Thr Glu Asp Thr Ala Thr Tyr
Tyr Cys 85 90 95 Ala Arg Glu Lys Glu Asn Tyr Tyr Gly Ser Ser Phe
Tyr Tyr Phe Asp 100 105 110 Tyr Trp Gly Ala Gly Thr Leu Val Thr Val
Ser Ser Ala Ser Thr Lys 115 120 125 Gly Pro Ser Val Phe Pro Leu Ala
Pro Ser Ser Lys Ser Thr Ser Gly 130 135 140 Gly Thr Ala Ala Leu Gly
Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro145 150 155 160 Val Thr Val
Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr 165 170 175 Phe
Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val 180 185
190 Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn
195 200 205 Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val
Glu Pro 210 215 220 Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys
Pro Ala Pro Glu225 230 235 240 Leu Leu Gly Gly Pro Ser Val Phe Leu
Phe Pro Pro Lys Pro Lys Asp 245 250 255 Thr Leu Met Ile Ser Arg Thr
Pro Glu Val Thr Cys Val Val Val Asp 260 265 270 Val Ser His Glu Asp
Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly 275 280 285 Val Glu Val
His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn 290 295 300 Ser
Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp305 310
315 320 Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu
Pro 325 330 335 Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln
Pro Arg Glu 340 345 350 Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp
Glu Leu Thr Lys Asn 355 360 365 Gln Val Ser Leu Thr Cys Leu Val Lys
Gly Phe Tyr Pro Ser Asp Ile 370 375 380 Ala Val Glu Trp Glu Ser Asn
Gly Gln Pro Glu Asn Asn Tyr Lys Thr385 390 395 400 Thr Pro Pro Val
Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys 405 410 415 Leu Thr
Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys 420 425 430
Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu 435
440 445 Ser Leu Ser Pro Gly Lys 450 31454PRTArtificial
Sequencerecombinantly produced 31Asp Val Gln Leu Gln Glu Ser Gly
Pro Gly Leu Val Lys Pro Ser Gln1 5 10 15 Ser Leu Phe Leu Thr Cys
Ser Val Thr Gly Tyr Ser Ile Ser Ser Ser 20 25 30 Tyr Tyr Trp Asn
Trp Ile Arg Gln Phe Pro Gly Asn Lys Leu Glu Trp 35 40 45 Met Gly
Tyr Ile Ser Phe Asp Gly Ser Asn Asn Tyr Asn Pro Ser Leu 50 55 60
Lys Asn Arg Ile Ser Ile Thr Arg Asp Thr Ser Lys Asn Gln Phe Phe65
70 75 80 Leu Lys Leu Asn Ser Val Thr Thr Glu Asp Thr Ala Thr Tyr
Tyr Cys 85 90 95 Ala Arg Glu Lys Glu Asn Tyr Tyr Gly Ser Ser Phe
Tyr Tyr Phe Asp 100 105 110 Tyr Trp Gly Ala Gly Thr Leu Val Thr Val
Ser Ser Ala Ser Thr Lys 115 120 125 Gly Pro Ser Val Phe Pro Leu Ala
Pro Ser Ser Lys Ser Thr Ser Gly 130 135 140 Gly Thr Ala Ala Leu Gly
Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro145 150 155 160 Val Thr Val
Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr 165 170 175 Phe
Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val 180 185
190 Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn
195 200 205 Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val
Glu Pro 210 215 220 Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys
Pro Ala Pro Glu225 230 235 240 Leu Leu Gly Gly Pro Ser Val Phe Leu
Phe Pro Pro Lys Pro Lys Asp 245 250 255 Thr Leu Met Ile Ser Arg Thr
Pro Glu Val Thr Cys Val Val Val Asp 260 265 270 Val Ser His Glu Asp
Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly 275 280 285 Val Glu Val
His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Ala 290 295 300 Ser
Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp305 310
315 320 Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu
Pro 325 330 335 Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln
Pro Arg Glu 340 345 350 Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp
Glu Leu Thr Lys Asn 355 360 365 Gln Val Ser Leu Thr Cys Leu Val Lys
Gly Phe Tyr Pro Ser Asp Ile 370 375 380 Ala Val Glu Trp Glu Ser Asn
Gly Gln Pro Glu Asn Asn Tyr Lys Thr385 390 395 400 Thr Pro Pro Val
Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys 405 410 415 Leu Thr
Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys 420 425 430
Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu 435
440 445 Ser Leu Ser Pro Gly Lys 450
32454PRTArtificial Sequencerecombinantly produced 32Glu Val Gln Leu
Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Gln1 5 10 15 Ser Leu
Phe Leu Thr Cys Ser Val Thr Gly Tyr Ser Ile Ser Ser Ser 20 25 30
Tyr Tyr Trp Asn Trp Ile Arg Gln Phe Pro Gly Asn Lys Leu Glu Trp 35
40 45 Met Gly Tyr Ile Ser Phe Asp Gly Ser Asn Asn Tyr Asn Pro Ser
Leu 50 55 60 Lys Asn Arg Ile Ser Ile Thr Arg Asp Thr Ser Lys Asn
Gln Phe Phe65 70 75 80 Leu Lys Leu Asn Ser Val Thr Thr Glu Asp Thr
Ala Thr Tyr Tyr Cys 85 90 95 Ala Arg Glu Lys Glu Asn Tyr Tyr Gly
Ser Ser Phe Tyr Tyr Phe Asp 100 105 110 Tyr Trp Gly Ala Gly Thr Leu
Val Thr Val Ser Ser Ala Ser Thr Lys 115 120 125 Gly Pro Ser Val Phe
Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly 130 135 140 Gly Thr Ala
Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro145 150 155 160
Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr 165
170 175 Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser
Val 180 185 190 Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr
Ile Cys Asn 195 200 205 Val Asn His Lys Pro Ser Asn Thr Lys Val Asp
Lys Lys Val Glu Pro 210 215 220 Lys Ser Cys Asp Lys Thr His Thr Cys
Pro Pro Cys Pro Ala Pro Glu225 230 235 240 Leu Leu Gly Gly Pro Ser
Val Phe Leu Phe Pro Pro Lys Pro Lys Asp 245 250 255 Thr Leu Met Ile
Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp 260 265 270 Val Ser
His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly 275 280 285
Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Ala 290
295 300 Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp
Trp305 310 315 320 Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn
Lys Ala Leu Pro 325 330 335 Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala
Lys Gly Gln Pro Arg Glu 340 345 350 Pro Gln Val Tyr Thr Leu Pro Pro
Ser Arg Asp Glu Leu Thr Lys Asn 355 360 365 Gln Val Ser Leu Thr Cys
Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile 370 375 380 Ala Val Glu Trp
Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr385 390 395 400 Thr
Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys 405 410
415 Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys
420 425 430 Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys
Ser Leu 435 440 445 Ser Leu Ser Pro Gly Lys 450
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