U.S. patent application number 17/196451 was filed with the patent office on 2021-11-11 for compositions and methods for modulating an immune response.
This patent application is currently assigned to The Brigham and Women's Hospital, Inc.. The applicant listed for this patent is The Brigham and Women's Hospital, Inc.. Invention is credited to Kristi Baker, Richard S. Blumberg, Timo Rath.
Application Number | 20210347887 17/196451 |
Document ID | / |
Family ID | 1000005735782 |
Filed Date | 2021-11-11 |
United States Patent
Application |
20210347887 |
Kind Code |
A1 |
Blumberg; Richard S. ; et
al. |
November 11, 2021 |
COMPOSITIONS AND METHODS FOR MODULATING AN IMMUNE RESPONSE
Abstract
Described herein are compositions for increasing IL-12
production comprising IgG or a fragment thereof or a variant
thereof and uses of said compositions for treating cancer and
infectious diseases. Also described herein are compositions for
decreasing IL-12 production comprising an agent that inhibits
signaling mediated by interaction between FcRn and IgG and uses of
said compositions for treating autoimmune diseases. Further
described herein are methods for assessing efficacy of treatment by
monitoring levels of various cytokines in the subject.
Inventors: |
Blumberg; Richard S.;
(Waltham, MA) ; Baker; Kristi; (Brookline, MA)
; Rath; Timo; (Cambridge, MA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
The Brigham and Women's Hospital, Inc. |
Boston |
MA |
US |
|
|
Assignee: |
The Brigham and Women's Hospital,
Inc.
Boston
MA
|
Family ID: |
1000005735782 |
Appl. No.: |
17/196451 |
Filed: |
March 9, 2021 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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16598011 |
Oct 10, 2019 |
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17196451 |
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16280878 |
Feb 20, 2019 |
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16598011 |
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16023682 |
Jun 29, 2018 |
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16280878 |
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15039524 |
May 26, 2016 |
10035858 |
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PCT/US2014/067332 |
Nov 25, 2014 |
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16023682 |
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61909229 |
Nov 26, 2013 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61K 2039/507 20130101;
C07K 2317/31 20130101; C07K 2317/52 20130101; C07K 16/283 20130101;
C07K 2317/72 20130101; G01N 33/6854 20130101; C12N 15/1138
20130101; A61K 39/0011 20130101; C07K 16/244 20130101; G01N 2333/57
20130101; C07K 2317/21 20130101; G01N 33/6866 20130101; C07K 16/00
20130101; G01N 33/6869 20130101; G01N 2800/52 20130101; C12N
2310/14 20130101; G01N 2333/5434 20130101; G01N 2333/525 20130101;
G01N 33/6863 20130101; G01N 33/574 20130101; A61K 2039/505
20130101; C07K 2317/76 20130101; G01N 33/564 20130101 |
International
Class: |
C07K 16/28 20060101
C07K016/28; G01N 33/574 20060101 G01N033/574; G01N 33/68 20060101
G01N033/68; C07K 16/24 20060101 C07K016/24; A61K 39/00 20060101
A61K039/00; C07K 16/00 20060101 C07K016/00; C12N 15/113 20060101
C12N015/113; G01N 33/564 20060101 G01N033/564 |
Goverment Interests
GOVERNMENT RIGHTS
[0002] The invention was made with government support under Grant
No. DK53056 awarded by the National Institutes of Health. The
government has certain rights to the invention.
Claims
1. A method for determining the efficacy of treatment in a subject
in need thereof comprising: (a) administering to the subject a
treatment comprising a composition comprising an anti-FcRn antibody
that inhibits signaling mediated by interaction between FcRn and
IgG; (b) providing a sample from the subject, wherein the sample is
blood, plasma or tissue; (c) assaying the level of TNF-.alpha. in
the sample; and (d) determining that the treatment is efficacious
if the level of TNF-.alpha. in the sample from the subject is lower
relative to the level in a reference sample or determining that the
treatment is not efficacious if the level of TNF-.alpha. in the
sample from the subject is higher relative to the level in a
reference sample, wherein the subject has an autoimmune
disease.
2. The method of claim 1, wherein the antibody is selected from the
group consisting of a monoclonal antibody or a fragment thereof, a
polyclonal antibody or a fragment thereof, chimeric antibody,
humanized antibody and single chain antibody.
Description
CROSS-REFERENCE TO RELATED APPLICATION
[0001] This is application is a continuation under 35 U.S.C. .sctn.
120 of co-pending U.S. application Ser. No. 16/598,011 filed Oct.
10, 2019, which is a continuation of U.S. application Ser. No.
16/280,878 filed Feb. 20, 2019, which is a continuation of U.S.
application Ser. No. 16/023,682 filed Jun. 29, 2018, which is a
continuation of U.S. application Ser. No. 15/039,524 filed May 26,
2016, now U.S. Pat. No. 10,035,858 issued on Jul. 31, 2018, which
is a 35 U.S.C. .sctn. 371 National Phase Entry Application of
International Application No. PCT/US2014/067332 filed Nov. 25,
2014, which designated the U.S., and claims benefit under 35 U.S.C.
.sctn. 119(e) of U.S. Provisional Application No. 61/909,229 filed
Nov. 26, 2013, the contents of each of which are incorporated
herein by reference in their entireties.
FIELD OF INVENTION
[0003] The present invention relates to molecular immunology and
cell biology. Specifically, described herein are compositions for
increasing production of IL-12 by regulating the interactions
between IgG and FcRn and methods of using the composition for
treating cancer and infectious diseases in a subject. Also
described herein are compositions for decreasing production of
IL-12 by regulating the interactions between IgG and FcRn and
methods of using the composition for treating autoimmune diseases
in a subject. Also provided herein are methods for assessing
efficacy of treatment in a subject by monitoring the levels of
various cytokines.
BACKGROUND OF THE INVENTION
[0004] All publications cited herein are incorporated by reference
in their entirety to the same extent as if each individual
publication or patent application was specifically and individually
indicated to be incorporated by reference. The following
description includes information that may be useful in
understanding the present invention. It is not an admission that
any of the information provided herein is prior art or relevant to
the presently claimed invention, or that any publication
specifically or implicitly referenced is prior art.
[0005] Cancers arising at mucosal barrier sites, particularly the
lung, large intestine (LI), stomach and cervix, account for a
considerable fraction of human malignancies (Siegel et al., 2012).
One contributing factor to the colon's susceptibility to malignant
transformation is its immunosuppressive environment (MacDonald et
al., 2011) which is necessary for tolerance towards microbial and
dietary antigens but also results in dampened anti-cancer immune
responses (Revaz and Nardelli-Haefliger, 2005; Saleh and
Trinchieri, 2011). Identifying physiologic factors capable of
countering this inherent downside of local tolerance is critical
for understanding and manipulating carcinogenesis at this, and
possibly other, mucosal sites.
[0006] The production and handling of IgG are critical components
of mucosal immunity, particularly in the LI where IgG accounts for
a large fraction of homeostatic mucosal immunoglobulin secretion
(Kozlowski et al., 1997). The presence of IgG in the intestinal
lumen is associated with the actions of the bidirectional IgG
transport receptor, FcRn (neonatal Fc receptor for IgG), which is
expressed lifelong in most murine and human endothelial, epithelial
and hematopoietic cells (Claypool et al., 2004; Zhu et al., 2001).
FcRn is uniquely capable of delivering IgG into the lumen and also
retrieving lumenal IgG and IgG containing immune complexes (IgG IC)
which are delivered into the local immune system of the lamina
propria (LP) (Claypool et al., 2004; Yoshida et al., 2004). FcRn
within antigen presenting cells such as dendritic cells (DC) also
plays a critical role in the processing of antigens delivered as
IgG IC and actively promotes major histocompatibility complex (MHC)
class I and class II restricted T cell responses (Baker et al.,
2011; Qiao et al., 2008) which can alternatively promote
anti-bacterial IgG-driven colitis (Kobayashi et al., 2009) and
protect from mucosal pathogens (Qiao et al., 2008; Yoshida et al.,
2006).
[0007] It is well accepted that cytotoxic CD8+ T cell-mediated
responses are critical for efficient anti-tumor immunity (Pages et
al., 2005) and FcRn has recently been shown to enable highly
efficient cross-presentation of IgG-complexed antigens by
CD8-CD11b+ DC (Baker et al., 2011). Given the abundance of both IgG
and CD8-CD11b+ monocyte-derived DC in mucosal tissues, especially
in the context of malignancy (Kozlowski et al., 1997; Ma et al.,
2011; MacSween and Eastwood, 1980), the role of FcRn in homeostatic
CD8+ T cell responses and as an effector of anti-cancer immune
surveillance was examined. Described herein are findings showing
that FcRn ligation with IgG containing immune complexes (IgG IC) is
directly involved in the production of IL-12, a key regulator of an
immune response. Production of IL-12 may thus be targeted with
agents that increase IL-12 production via altered FcRn/IgG
interactions so as to treat cancer and/or infectious diseases or
with agents that decrease IL-12 production via altered FcRn/IgG
interactions so as to treat autoimmune diseases, therefore meeting
a need for therapeutic agents to treat cancer, infectious diseases
and autoimmune diseases.
SUMMARY OF THE INVENTION
[0008] The following embodiments and aspects thereof are described
and illustrated in conjunction with systems, compositions and
methods which are meant to be exemplary and illustrative, not
limiting in scope.
[0009] Provided herein are compositions and methods for increasing
IL-12 production in a subject in need thereof. Also provided herein
are compositions and methods for decreasing IL-12 production in a
subject in need thereof.
[0010] In some aspects, described herein is a composition for
increasing IL-12 production, the composition comprising
immunoglobulin G (IgG) or a variant thereof or a fragment
thereof.
[0011] In an embodiment, the composition comprising immunoglobulin
G (IgG) or a variant thereof or a fragment thereof increases
signaling mediated by interaction between IgG and FcRn.
[0012] In an embodiment, the composition comprising immunoglobulin
G (IgG) or a variant thereof or a fragment thereof increases an
immune response against an antigen.
[0013] In an embodiment, the IgG may be any isotype of IgG
including IgG1, IgG2, IgG3 and/or IgG4.
[0014] In an embodiment, the variant IgG comprises a methionine to
leucine substitution at position 428 and an asparagine to serine
substitution at position 434.
[0015] In an embodiment, the composition comprising immunoglobulin
G (IgG) or a variant thereof or a fragment thereof further
comprises an antigen conjugated to IgG or a variant thereof or a
fragment thereof so as to create a multimeric structure which can
cross-link FcRn.
[0016] In an embodiment, the composition comprising immunoglobulin
G (IgG) or a variant thereof or a fragment thereof further
comprises an antigen complexed to IgG or a variant thereof or a
fragment thereof so as to create a monomeric or multimeric
structure which can cross-link FcRn.
[0017] In various embodiments, the antigen is a tumor antigen, an
endogenous antigen, a cell-associated antigen, an apoptotic body, a
microbial antigen, a viral antigen, a parasitic antigen or a
combination thereof.
[0018] In various embodiments, the antigen is a protein or a
proteomimetic thereof, a peptide or a peptidomimetic thereof, a
lipid or a combination thereof.
[0019] In some embodiments, the IgG or a variant thereof or a
fragment thereof is mammalian.
[0020] In some embodiments, the IgG or a variant thereof or a
fragment thereof is human.
[0021] In some aspects, described herein are compositions for
decreasing IL-12 production comprising an agent that inhibits
signaling mediated by interaction between FcRn and IgG.
[0022] In some embodiments, the agent that inhibits signaling
mediated by interaction between FcRn and IgG is any one or more of
a peptide, protein, small molecule, nucleic acid, aptamer,
oligonucleotide, antibody or a combination thereof.
[0023] In some embodiments, the nucleic acid agent that inhibits
signaling mediated by interaction between FcRn and IgG is a siRNA
specific to FcRn.
[0024] In some embodiments, the antibody agent that inhibits
signaling mediated by interaction between FcRn and IgG is selected
from the group consisting of a monoclonal antibody or a fragment
thereof, a polyclonal antibody or a fragment thereof, chimeric
antibody, humanized antibody and single chain antibody.
[0025] In some embodiments, the agent that inhibits signaling
mediated by interaction between FcRn and IgG is a bispecific agent
comprising binding sites for IgG and FcRn.
[0026] In some embodiments, the agent that inhibits signaling
mediated by interaction between FcRn and IgG is a recombinant Fc
portion of IgG or a biologically active portion thereof or a
proteomimetic thereof.
[0027] In some embodiments, the agent that inhibits signaling
mediated by interaction between FcRn and IgG is a recombinant Fc
portion of IgG or a biologically active portion thereof or a
proteomimetic thereof, wherein the Fc portion of IgG or a
biologically active portion thereof is mammalian.
[0028] In some embodiments, the agent that inhibits signaling
mediated by interaction between FcRn and IgG is a recombinant Fc
portion of IgG or a biologically active portion thereof or a
proteomimetic thereof, wherein the Fc portion of IgG or a
biologically active portion thereof is human.
[0029] Also described are methods for modulating the interaction
between FcRn and IgG. The method comprises comprising contacting a
cell with an agent that binds FcRn and/or IgG and modulates binding
of FcRn to IgG.
[0030] In some embodiments, the agent use in the method for
modulating the interaction between FcRn and IgG, increases
signaling mediated by interaction of FcRn and IgG.
[0031] In some embodiments, the agent for use in the method
modulating the interaction between FcRn and IgG, decreases
signaling mediated by interaction of FcRn and IgG.
[0032] In some embodiments, the agent for use in the method
modulating the interaction between FcRn and IgG comprises binding
sites specific for IgG and FcRn.
[0033] In some embodiments, the agent for use in the method
modulating the interaction between FcRn and IgG comprises binding
sites specific for IgG and FcRn.
[0034] In some embodiments, the agent for use in the method
modulating the interaction between FcRn and IgG comprises binding
sites specific for Fc portion of IgG.
[0035] In some embodiments, the agent for use in the method
modulating the interaction between FcRn and IgG comprises a
bispecific polypeptide agent comprising binding sites specific for
IgG and FcRn.
[0036] In some embodiments, the bispecific polypeptide agent for
use in the method modulating the interaction between FcRn and IgG
comprises an antibody or antigen binding portion thereof that
specifically binds FcRn and an antibody or antigen binding portion
thereof that specifically binds IgG.
[0037] Also provided herein are methods for treating, inhibiting,
preventing metastasis of or preventing relapse of cancer in a
subject in need thereof comprising. The methods comprise providing
a composition comprising immunoglobulin G (IgG) or a variant
thereof or a fragment thereof and administering an effective amount
of the composition to the subject so as to treat, inhibit, prevent
metastasis or prevent relapse of cancer in the subject.
[0038] In some embodiments, the composition for use in the methods
for treating, inhibiting, preventing metastasis of or preventing
relapse of cancer in a subject in need thereof increases signaling
mediated by interaction of IgG and FcRn.
[0039] In some embodiments, the composition for use in the methods
for treating, inhibiting, preventing metastasis of or preventing
relapse of cancer in a subject in need thereof increases an immune
response against the antigen.
[0040] In some embodiments, the composition for use in the methods
for treating, inhibiting, preventing metastasis of or preventing
relapse of cancer in a subject in need thereof comprises a variant
IgG having a methionine to leucine substitution at position 428 and
an asparagine to serine substitution at position 434.
[0041] In some embodiments, the composition comprising
immunoglobulin G (IgG) or a variant thereof or a fragment thereof
for treating, inhibiting, preventing metastasis of or preventing
relapse of cancer in a subject in need thereof further comprises an
antigen. In some embodiments, the antigen is conjugated to the IgG
or a variant thereof or a fragment thereof. In some embodiments,
the antigen is complexed with the IgG or a variant thereof or a
fragment thereof.
[0042] Also provided herein are methods for treating, inhibiting or
reducing the severity of infectious diseases in a subject in need
thereof. The methods comprise providing a composition comprising
immunoglobulin G (IgG) or a variant thereof or a fragment thereof
and administering an effective amount of the composition to the
subject so as to treat, inhibit or reduce the severity of
infectious diseases in the subject.
[0043] In some embodiments, the composition for use in the methods
for treating, inhibiting or reducing the severity of infectious
diseases in a subject in need thereof increases signaling mediated
by interaction of IgG and FcRn.
[0044] In some embodiments, the composition for use in the methods
for treating, inhibiting or reducing the severity of infectious
diseases in a subject in need thereof increases an immune response
against the antigen.
[0045] In some embodiments, the composition for use in the methods
for treating, inhibiting or reducing the severity of infectious
diseases in a subject in need thereof comprises a variant IgG
having a methionine to leucine substitution at position 428 and an
asparagine to serine substitution at position 434.
[0046] In some embodiments, the composition comprising
immunoglobulin G (IgG) or a variant thereof or a fragment thereof
for treating, inhibiting or reducing the severity of infectious
diseases in a subject in need thereof further comprises an antigen.
In some embodiments, the antigen is conjugated to the IgG or a
variant thereof or a fragment thereof. In some embodiments, the
antigen is complexed with the IgG or a variant thereof or a
fragment thereof.
[0047] In various embodiments of the methods, the antigen is a
tumor antigen, an endogenous antigen, a cell-associated antigen, an
apoptotic body, a microbial antigen, a viral antigen, a parasitic
antigen or a combination thereof.
[0048] Also provided are methods for treating, inhibiting or
reducing the severity of autoimmune diseases in a subject in need
thereof The methods comprise providing a composition comprising an
agent that inhibits signaling mediated by interaction between FcRn
and IgG and administering an effective amount of the composition to
the subject so as to treat, inhibit or reduce the severity of
autoimmune diseases in the subject.
[0049] In some embodiments, the agent for use in the methods for
treating, inhibiting or reducing the severity of autoimmune
diseases in a subject in need thereof reduces or inhibits
production of IL-12.
[0050] In some embodiments, the agent for use in the methods for
treating, inhibiting or reducing the severity of autoimmune
diseases in a subject in need thereof is any one or more of a
peptide, protein, small molecule, nucleic acid, aptamer,
oligonucleotide, antibody or a combination thereof.
[0051] In some embodiments, the nucleic acid agent for use in the
methods for treating, inhibiting or reducing the severity of
autoimmune diseases in a subject in need thereof is siRNA specific
to FcRn.
[0052] In some embodiments, the antibody agent for use in the
methods for treating, inhibiting or reducing the severity of
autoimmune diseases in a subject in need thereof is selected from
the group consisting of a monoclonal antibody or a fragment
thereof, a polyclonal antibody or a fragment thereof, chimeric
antibody, humanized antibody and single chain antibody.
[0053] In some embodiments, the agent for use in the methods for
treating, inhibiting or reducing the severity of autoimmune
diseases in a subject in need thereof is a bispecific agent
comprising binding sites for IgG and FcRn.
[0054] In some embodiments, the agent for use in the methods for
treating, inhibiting or reducing the severity of autoimmune
diseases in a subject in need thereof the agent is a recombinant Fc
portion of IgG or a biologically active portion thereof or a
proteo-mimetic thereof.
[0055] Further provided herein is method for determining the
efficacy of treatment in a subject in need thereof. The method
includes providing a sample from a subject, wherein the subject has
been administered an effective amount of a composition comprising
immunoglobulin G (IgG) or a variant thereof or a fragment thereof
and assaying the levels of any one or more of IL-12, IL-2,
TNF-.alpha., IFN-.gamma., GM-CSF, IL-3, granzyme B, Tbet or a
combination thereof in the sample. In one embodiment, the treatment
is efficacious if the levels of any one or more of IL-12, IL-2,
TNF-.alpha., IFN-.gamma., GM-CSF, IL-3, granzyme B, Tbet or a
combination thereof in the sample from the subject is higher
relative to the levels in a reference sample. In another embodiment
the treatment is not efficacious if the levels of any one or more
of IL-12, IL-2, TNF-.alpha., IFN-.gamma., GM-CSF, IL-3, granzyme B,
Tbet or a combination thereof in the sample from the subject is
lower relative to the levels in a reference sample. In an
embodiment, the subject has cancer or an infectious disease. In
some embodiments, the sample is blood, plasma or tissue.
[0056] Also provided herein is a method for determining the
efficacy of treatment in a subject in need thereof The method
includes providing a sample from a subject, wherein the subject has
been administered a composition comprising an agent that inhibits
signaling mediated by interaction between FcRn and IgG and assaying
the levels of any one or more of IL-12, IL-2, TNF-.alpha.,
IFN-.gamma., GM-CSF, IL-3, granzyme B, Tbet or a combination
thereof in the sample. In an embodiment, the treatment is
efficacious if the levels of any one or more of IL-12, IL-2,
TNF-.alpha., IFN-.gamma., GM-CSF, IL-3, granzyme B, Tbet or a
combination thereof in the sample from the subject is lower
relative to the levels in a reference sample. In another
embodiment, the treatment is not efficacious if the levels of any
one or more of IL-12, IL-2, TNF-.alpha., IFN-.gamma., GM-CSF, IL-3,
granzyme B, Tbet or a combination thereof in the sample from the
subject is higher relative to the levels in a reference sample. In
an embodiment, the subject has an autoimmune disease. In some
embodiments, the sample is blood, plasma or tissue.
BRIEF DESCRIPTION OF FIGURES
[0057] This patent or application file contains at least one
drawing executed in color. Copies of this patent or patent
application publication with color drawings will be provided by the
Office upon request and payment of the necessary fee.
[0058] Exemplary embodiments are illustrated in the referenced
figures. It is intended that the embodiments and figures disclosed
herein are to be considered illustrative rather than
restrictive.
[0059] FIGS. 1A-1G depict, in accordance with various embodiments
of the present invention that FcRn protects against the development
of colorectal cancer through a mechanism independent of intestinal
microbiota. (FIG. 1A) Large intestine (LI) tumor incidence at 5
months of age and representative tumor histology in Apc.sup.Min/+
and Apc.sup.Min/+Fcgrt.sup.-/- mice. Scale bar=100 .mu.m. (FIG. 1B)
Tumor incidence in WT and Fcgrt.sup.-/- littermates treated with 8
doses of azoxymethane (AOM). (FIG. 1C) Tumor incidence in
AOM/DSS-treated WT and Fcgrt.sup.-/- littermates. (FIG. 1D) Tumor
incidence and maximum tumor diameter in WT and Fcgrt.sup.-/-
littermates in each of four independent experiments with n.gtoreq.3
mice per group per experiment. (FIG. 1E) Percent survival of WT and
Fcgrt.sup.-/- littermates treated with AOM/DSS. Significance was
assessed by Logrank test. (FIG. 1F) Richness indices of microbiota
associated with the distal LI of untreated 8-week old WT and
Fcgrt.sup.-/- littermates, as revealed by T-RFLP analysis. n=3-5
mice per group. (FIG. 1G) Abundance of specific microbial species
in the distal LI of untreated 7-week old WT and Fcgrt.sup.-/-
littermates as assessed by qPCR. n=9 mice per group. Representative
results of two (1A, 1B, 1E) or four (1D) independent experiments
each with n=4-10 mice per group. All data represent mean.+-.s.e.m.
* p.ltoreq.0.05, ** p.ltoreq.0.01, *** p.ltoreq.0.005.
[0060] FIGS. 2A-2D depict, in accordance with various embodiments
of the present invention that FcRn drives the activation and
retention of tumor-reactive cytotoxic CD8.sup.+ T cells which
confer tumor protection. (FIG. 2A) Frequency of CD8.sup.+ T cells
in the lamina propria lymphocyte (LPL) fraction of tumor and
adjacent LI tissue in WT and Fcgrt.sup.-/- littermates (upper
panels) following AOM/DSS treatment. Cytotoxic potential of cells
within the CD3.sup.+CD8.sup.+ gate was assessed by intracellular
staining for granzyme B (middle panels) or surface staining of
LAMP1 (lower panels). (FIG. 2B) Mean CD8.sup.+ T cell frequency and
cytotoxic potential in WT and Fcgrt.sup.-/- mice, as assessed by
flow cytometry, in each of three independent experiments. (FIG. 2C)
Cytokine secretion of sorted effector CD8.sup.+
CD44.sup.+CD62L.sup.- cells from the LP of tumor and adjacent
tissue of AOM/DSS treated WT and Fcgrt.sup.-/- mice following 24 h
restimulation with anti-CD3 and anti-CD28. (FIG. 2D) Tumor
incidence and tumor load (sum of the diameters of all tumors) in
recipient mice adoptively transferred with CD8.sup.+ T cells from
WT or Fcgrt.sup.-/- AOM/DSS-treated donors. Significance was
assessed by Mann-Whitney test. Representative results of three
independent experiments with n.gtoreq.4 mice per group per
experiment. All data represent mean.+-.s.e.m. NS=not significant.
ND=not detected. * p.ltoreq.0.05, ** p.ltoreq.0.01, ***
p.ltoreq.0.005.
[0061] FIGS. 3A-3G depict, in accordance with various embodiments
of the present invention that CD8.sup.-CD11b.sup.+ DC utilize FcRn
to efficiently prime protective anti-tumor CD8.sup.+ T cell
responses. (FIG. 3A) Tumor antigen-specific IgG in the serum or MLN
and LI homogenates of AOM/DSS treated WT or Fcgrt.sup.-/- mice.
ELISA plates coated with lysates from tumor epithelium were probed
with dilutions of serum or tissue homogenates from tumor bearing
mice. (FIG. 3B) Transcript profiles of sorted CD8.sup.-CD11b.sup.+
and CD8.sup.+CD11b.sup.- DC subsets isolated from the indicated
tissue compartment of AOM/DSS-treated WT and Fcgrt.sup.-/-
littermates. (FIG. 3C) Tumor incidence and survival in
Fcgrt.sup.-/- recipients adoptively transferred with DC from the
MLN and LP of AOM/DSS-treated WT or Fcgrt.sup.-/- donors. Endpoint
survival was assessed using a Chi-Squared test. (FIG. 3D) CD8.sup.+
T cell frequency in the LI LP following transfer of WT DC to
AOM/DSS-treated Fcgrt.sup.-/- recipients. (FIG. 3E) Tumor incidence
and LI LP CD8.sup.+ T cell frequency in
Itgax.sup.creFcgrt.sup.Fl/Fl mice and their littermate
Fcgrt.sup.Fl/Fl controls upon treatment with AOM/DSS. (FIGS. 3F-3G)
Tumor incidence (FIG. 3F) and survival (FIG. 3G) of CD8.sup.+ T
cell-depleted Fcgrt.sup.-/- mice adoptively transferred with WT DC.
CD8.sup.+ T cells were depleted by chronic i.p. administration of
anti-CD8 antibody (or isotype control). Representative results of
three (FIGS. 3B-3E) or two (FIG. 3A, 3F) independent experiments
with n=3-6 mice per group per experiment. All data represent
mean.+-.s.e.m. NS=not significant. * p.ltoreq.0.05, **
p.ltoreq.0.01, *** p.ltoreq.0.005.
[0062] FIGS. 4A-4F depict, in accordance with various embodiments
of the present invention that FcRn drives the induction of
endogenous tumor-reactive CD8.sup.+ T cells and can be
therapeutically targeted. (FIG. 4A) Incidence of pulmonary
metastatic nodules formed by i.v. administered OVA-expressing B16
melanoma cells (OVA-B16) in WT or Fcgrt.sup.-/- mice or
Fcgrt.sup.-/- mice pre-immunized with WT or Fcgrt.sup.-/- DC. (FIG.
4B) Frequency of endogenously occurring OVA-specific CD8.sup.+ T
cells in WT and Fcgrt.sup.-/- metastasis-bearing mice. Left panel
demonstrates results from individual animals in a single
experiment. Right panel shows the results of three independent
experiments each with n=3-6 mice per group. (FIG. 4C) Frequency of
pulmonary metastases from mice treated as in (FIG. 4A) and given
either a CD8.sup.+ T cell-depleting antibody or isotype control.
(FIG. 4D) Frequency of pulmonary metastatic nodules and
OVA-specific CD8.sup.+ T cells in the lungs of Fcgrt.sup.Fl/Fl and
Itgax.sup.creFcgrt.sup.Fl/Fl littermates. (FIG. 4E) Incidence of
pulmonary metastatic nodules in WT or Fcgrt.sup.-/- mice or
Fcgrt.sup.-/- mice adoptively transferred with OVA-specific
CD8.sup.+ T cells primed ex vivo by DC loaded with OVA-containing
IgG IC, FcRn non-binding IHH-IgG IC or soluble OVA. (FIG. 4F)
Incidence of pulmonary nodules in OVA-B16-treated WT and
Fcgrt.sup.-/- mice pre-immunized with WT DC loaded ex vivo with
OVA-containing IC formed with IgG or enhanced FcRn-binding LS-IgG.
Representative results of three (FIG. 4A, 4B, 4D) or two (FIG. 4C,
4E, 4F) independent experiments with n=3-6 mice per group per
experiment. All data represent mean.+-.s.e.m. NS=not significant.
(*) p=0.09, * p.ltoreq.0.05, ** p.ltoreq.0.01, ***
p.ltoreq.0.005.
[0063] FIGS. 5A-5G depict depicts, in accordance with various
embodiments of the present invention that FcRn within DC enables
homeostatic CD8.sup.+ T cell activation and IL-12 production in the
LI. (FIG. 5A) IgG isotype content of the serum and LI or MLN
homogenates in untreated WT and Fcgrt.sup.-/- littermates. (FIG.
5B) CD8.sup.+ T cell frequency of the LI LPL fraction of untreated
WT and Fcgrt.sup.-/- littermates in a single experiment (left
panels) or across three independent experimental repeats (right
panel). (FIG. 5C) Frequency of CD8.sup.+ T cells in the LPL
fraction of Fcgrt.sup.Fl/Fl and Itgax.sup.creFcgrt.sup.Fl/Fl
littermates. (FIG. 5D) Cytokine secretion by CD8.sup.+ T cells
sorted from LI LP of untreated WT and Fcgrt.sup.-/- mice following
24 h restimulation with anti-CD3 and anti-CD28. (FIG. 5E)
Transcript profiles of CD8.sup.+ T cells sorted from LI LP of
untreated littermate control mice. (FIG. 5F) Cytokine secretion
from 24 h tissue explant cultures of the MLN and LI of untreated WT
and Fcgrt.sup.-/- mice. (FIG. 5G) Transcript profiles of sorted
CD8.sup.-CD11b.sup.+ DC from the MLN of untreated littermates.
Representative results of three independent experiments with n=3-5
mice per group per experiment. All data represent mean.+-.s.e.m.
NS=not significant. * p.ltoreq.0.05, ** p.ltoreq.0.01, ***
p.ltoreq.0.005.
[0064] FIGS. 6A-6F depict, in accordance with various embodiments
of the present invention that IgG IC ligation of FcRn in
CD8.sup.-CD11b.sup.+ DC induced IL-12 production via activation of
a signaling cascade. (FIG. 6A) Induction of IL-12p35 upon ex vivo
stimulation of WT CD8.sup.-CD11b.sup.+ DC from the spleen or MLN
with IgG IC or FcRn non-binding IHH-IgG IC for 6 h. (FIG. 6B) IL-12
secretion after 24 h IgG IC stimulation of CD8.sup.-CD11b.sup.+ and
CD8.sup.+CD11b.sup.- DC sorted from the MLN of AOM/DSS-treated WT
and Fcgrt.sup.-/- mice. (FIG. 6C) Phosphorylation of STAT-1 and
nuclear translocation of IRF-1 and NF-.kappa.B p65 upon IgG IC
stimulation of DC isolated from WT or Fcgrt.sup.-/- mice. (FIG. 6D)
IL-12 transcript production by WT or Stat-1.sup.-/-
CD8.sup.-CD11b.sup.+ DC following stimulation with IgG or IHH-IgG
IC for 6 h. (FIG. 6E) Binding of IRF-1 and NF-.kappa.B p65 to the
promoters of IL-12p35 and IL-12p40 upon stimulation of WT or
Fcgrt.sup.-/- DC with IgG IC or IHH-IgG IC for 4 h. (FIG. 6F) Tumor
incidence in mice adoptively transferred with WT DC and treated
with a neutralizing anti-IL-12 antibody or isotype control.
Representative results of three (FIGS. 6A-6E) or one (FIG. 6F)
independent experiments with n=3-7 mice per group per experiment.
All data represent mean.+-.s.e.m. * p.ltoreq.0.05, **
p.ltoreq.0.01, *** p.ltoreq.0.005.
[0065] FIGS. 7A-7F depict, in accordance with various embodiments
of the present invention that FcRn expressing DC predict survival
in human CRC and secrete IL-12 upon FcRn stimulation. (FIG. 7A)
Double immunohistochemical staining of FcRn.sup.+CD11c.sup.+ DC in
the stroma of CRC (upper panels) and CRC-adjacent normal LI (lower
panels). FcRn=brown, CD11c=red. Scale bar left panels=100 .mu.m.
Scale bar right panels=20 .mu.m. (FIG. 7B) Colocalization of
FcRn.sup.+ DC and CD8.sup.+ T cells in stroma of CRC (upper panels)
and CRC-adjacent normal LI (lower panels). Arrowheads indicate
areas of colocalization. (FIG. 7C) Kaplan Meier survival curves of
183 patients with high (.gtoreq.10 per core) and low (.ltoreq.10
per core) tumor infiltration by CD11c.sup.+FcRn.sup.+ cells. (FIG.
7D) Incidence of tumors in chimeric mice treated with AOM/DSS. WT
recipients were reconstituted with WT bone marrow. Fcgrt.sup.-/-
recipients were reconstituted with Fcgrt.sup.-/-, WT or
hFCGRT-hB2M-mFcgrt.sup.-/- bone marrow. Representative result of
two independent experiments with n=4-5 mice per group per
experiment. (FIG. 7E) hIL-12p35 and hIL-12p40 transcript expression
in hMoDC upon stimulation with FcRn-binding (IgG IC) or FcRn
non-binding (IHH-IgG IC) immune complexes. (FIG. 7F) Nuclear
translocation of IRF-1 and phosphorylation of STAT-1 in hMoDC upon
stimulation with IgG IC or IHH-IgG IC. Data in panels FIGS. 7A-7B
are representative of a total of 50 matched CRC and adjacent normal
LI pairs. Data in panels FIGS. 7E-7F are representative of six
donors processed in pairs in each of three independent experiments.
All data represent mean.+-.s.e.m. * p.ltoreq.0.05, **
p.ltoreq.0.01, *** p.ltoreq.0.005.
[0066] FIGS. 8A-8K depict, in accordance with various embodiments
of the present invention that loss of FcRn predisposes to the
development of more severe inflammation-and
non-inflammation-associated colorectal cancer (CRC) via a mechanism
that is independent of intestinal microbiota. (FIG. 8A)
Histological classification of adenomas present in the large
intestine (LI) of Apc.sup.Min/+ and Apc.sup.Min/+Fcgrt.sup.-/- mice
assessed at 5 months of age. (FIG. 8B) Frequency of adenomas in the
small intestine (SI) of Apc.sup.Mn/+ and Apc.sup.Mn/+ Fcgrt.sup.-/-
mice. n=4-6 mice per group in each of two independent experiments.
(FIG. 8C) Maximum tumor diameter measured in WT and Fcgrt.sup.-/-
mice treated with eight weekly doses of azoxymethane (AOM) in one
representative experiment with n.gtoreq.3 mice per group. (FIG. 8D)
Details of the azoxymethane/dextran sodium sulfate (AOM/DSS)
treatment used for the induction of inflammation-associated
colorectal cancer. Mice were treated with a single 10 mg/kg dose of
AOM via i.p. injection (Day-7). Seven days later (Day 0), mice were
given 1.5% DSS in their drinking water for a period of seven days.
DSS was withdrawn and mice were allowed to drink regular water for
14 days (Day 7-21). The cycle of one week on DSS (Day 21-28) and
two weeks on regular water (Day 28-42) was repeated once. Mice were
sacrificed on Day 42. (FIG. 8E) Representative histology of the
tumors present in both WT and Fcgrt.sup.-/- mice showing severe
dysplasia and invasion (arrows) through the lamina propria in a
lesion from an Fcgrt.sup.-/- mouse. Scale bar=100 .mu.m. (FIG. 8F)
Weight curves during the first 20 days of treatment of mice
undergoing AOM/DSS regimen. Mice were weighed every 1-2 days. Data
are representative of two independent experiments with n=5 mice per
group per experiment. (FIG. 8G) Richness indices of microbiota
found in the feces of untreated 8-week old WT and Fcgrt.sup.-/-
littermates, as revealed by T-RFLP analysis. n=3-5 mice per group.
(FIG. 8H) Richness indices of microbiota found associated with the
distal LI or feces of untreated 2-week old WT and Fcgrt.sup.-/-
littermates, as revealed by T-RFLP analysis. n=4-9 mice per group.
(FIG. 8I) Multidimensional scaling (MDS) plots demonstrating
microbial community composition in each of the indicated tissue
compartments of untreated 8-week old and 2-week old WT and
Fcgrt.sup.-/- mice as assessed by T-RFLP. ANOSIM analysis of Bray
Curtis similarity matrices revealed no significant differences
between WT and Fcgrt.sup.-/- mice for any age or tissue
compartment. (FIG. 8J) ANOSIM results for analysis of microbial
differences between tissue compartments within each age group,
regardless of genotype. (FIG. 8K) Abundance of specific microbial
species associated with the feces of untreated 7-week old WT and
Fcgrt.sup.-/- mice as assessed by qPCR. Values from each species
have been normalized to total bacteria (16S). Each dot represents
an individual animal. n=9 mice per group. All data represent
mean.+-.s.e.m. ND=none detected. * p.ltoreq.0.05.
[0067] FIGS. 9A-9G depict, in accordance with various embodiments
of the present invention that FcRn-mediated tumor immune
surveillance is mediated by selective activation and retention of
CD8.sup.+T cells in the intestinal LP. (FIG. 9A) Flow cytometric
analysis of the frequency of CD4.sup.+ I cells, NK cells (NK1.1) or
macrophages (F4/80) in the lamina propria lymphocyte (LPL) fraction
of tumor and adjacent LI tissue in WI and Fcgrt.sup.-/- mice
following AOM/DSS treatment. (FIG. 9B) Absolute number of CD8.sup.+
I cells isolated from the LP tissue of the adjacent, tumor or
untreated (baseline) LI in AOM/DSS-treated mice as assessed by flow
cytometric staining and acquisition of fixed volumes of sample.
(FIGS. 9C-9D) Flow cytometric analysis of the frequency of
CD8.sup.+ I cells in the lamina propria (LP) fraction of
tumor-adjacent LI tissue in untreated Apc.sup.Min/+ and
Apc.sup.Min/+Fcgrt.sup.-/- mice (FIG. 9C) and in AOM-treated WI and
Fcgrt.sup.-/- littermates (FIG. 9D). Representative results from
one of three experiments with n=3 mice per group per experiment.
(FIG. 9E) Flow cytometric analysis of the extent of CD8.sup.+ I
cell proliferation (Ki-67) and apoptosis (Annexin V) in the LP
fraction of tumor and adjacent LI tissue in WI and
Fcgrt.sup.-/-littermates following AOM/DSS treatment. Plots depict
cells within the CD3.sup.+CD8.sup.+ gate of cells. Representative
plots from three independent experiments with n=3 mice per group
per experiment. (FIG. 9F) Phenotype of CD8.sup.+ I cells in the
indicated tissue compartment of WI and Fcgrt.sup.-/- littermates
treated with AOM/DSS. Representative plots from three independent
experiments with n=3-4 mice per group per experiment. (FIG. 9G)
Survival rates of AOM/DSS-treated recipient mice adoptively
transferred with CD4.sup.+ I cells taken from the MLN and LI LP of
AOM/DSS-treated WI and Fcgrt.sup.-/- donors. Representative results
from one of two independent experiments with n=4 mice per group per
experiment. Significance of survival curves was assessed by Logrank
test. All data represent mean.+-.s.e.m. * p.ltoreq.0.05.
[0068] FIGS. 10A-10L depict, in accordance with various embodiments
of the present invention that FcRn-dependent dendritic cell
(DC)-mediated tumor protection is associated with the presence of
tumor-antigen reactive IgG in intestinal tissues and the generation
of a local Th1/Tc1 polarizing cytokine environment. (FIG. 10A)
Isotype distribution of tumor antigen-specific IgG in the serum or
LI homogenates of AOM/DSS treated WT or Fcgrt.sup.-/- mice. ELISA
plates coated with lysates from tumor epithelium were probed with
dilutions of serum or tissue homogenates from tumor bearing mice
and developed with isotype-specific secondary antibodies. (FIG.
10B) Immunoblots demonstrating tumor antigen-specific IgG in the
serum and LI homogenates of each of eight AOM/DSS treated mice.
IgG-depleted lysates prepared from tumor intestinal epithelial
cells (IEC) or non-tumor control IEC were resolved under reducing
conditions by SDS-PAGE and membranes of the transferred lysates
were probed with serum or LI homogenates from tumor bearing mice.
Representative blots from two independent experiments with n=4 mice
per group per experiment. 2.sup.nd Ab=anti-mouse IgG-HRP. (FIG.
10C) Fold increase above baseline values in serum
anti-phosphatidylserine (.alpha.-PS) and anti-cardiolipin
(.alpha.-CL) IgG content in WT and Fcgrt.sup.-/- littermates. (FIG.
10D) IgG isotype content of the serum, LI homogenates and MLN
homogenates in AOM/DSS-treated WT and Fcgrt.sup.-/- mice. (FIG.
10E) Flow cytometric analysis of the frequency of CD8.sup.+ versus
CD11b.sup.+ DC (top row, gated on CD11c.sup.+ cells) and
characterization of the CD11c.sup.+CD8.sup.-CD11b.sup.+ DC (bottom
four rows) in the mucosal tissues of AOM/DSS-treated WT and
Fcgrt.sup.-/- littermates. (FIGS. 10F-10H) Whole tissue cytokine
transcript profiles of the indicated tissue compartments from
AOM/DSS-treated WT and Fcgrt.sup.-/- littermates (FIG. 10F),
untreated Apc.sup.Min/+ and Apc.sup.Min/+Fcgrt.sup.-/- littermates
(FIG. 10G) and AOM-treated WT and Fcgrt.sup.-/-littermates (FIG.
10H). Representative results from 2-4 independent experiments with
n=3-6 mice per group per experiment. Data represent mean.+-.s.e.m.
(FIG. 10I) Purity (top panel) and subset distribution (bottom
panel) of the DC transferred in FIGS. 3C, 3F. (FIG. 10J) Frequency
of congenic (CD45.2) WT or Fcgrt.sup.-/- DC in the MLN and LI LP of
recipient (CD45.1) mice 3 days and 7 days after intraperitoneal
transfer. Recipient mice injected with PBS are shown as controls.
(FIG. 10K) Ex vivo antigen cross-presentation with DC isolated from
the MLN of the indicated AOM/DSS-treated DC recipient mice and
loaded with IC containing FcRn-binding (IgG) immune complex (IC),
non-FcRn binding (IHH-IgG) IC or soluble antigen (OVA) and
cocultured with OT-I CD8.sup.+ T cells. (FIG. 10L) Relative Fcgrt
transcript levels as assessed by qPCR in DC (CD11c), macrophages
(CD11b) and hepatocytes purified from WT, Fcgrt.sup.-/-,
Fcgrt.sup.Fl/Fl or Itgax.sup.creFcgrt.sup.Fl/Fl mice. Data are
representative of two independent experiments with n=3-5 mice per
group per experiment. All data represent mean.+-.s.e.m. * p 5 0.05,
** p 5 0.01, *** p 5 0.005.
[0069] FIGS. 11A-11F depict, in accordance with various embodiments
of the present invention that both CD8.sup.+ T cells and
tumor-specific IgG are required for FcRn-mediated protection from
pulmonary metastases. (FIG. 11A) CD8.sup.+ T cell frequency in the
lungs of untreated WT or Fcgrt.sup.-/- mice. (FIGS. 11B-11C)
Tumor-specific anti-OVA IgG in the lung homogenates (FIG. 11B) or
serum (FIG. 11C) of WT and Fcgrt.sup.-/- littermates bearing lung
metastases. Lungs were harvested 2 weeks after i.v. injection of
0.5.times.10.sup.6 OVA-expressing B16 melanoma cells (OVA-B16).
Anti-OVA IgG content was evaluated by ELISA and normalized to
protein content of the homogenates for (FIG. 11B). (FIG. 11D)
Frequency of OVA-specific IgG producing B cells in the lymph nodes
(LN) or spleens of OVA-B16 lung metastasis-bearing WT and
Fcgrt.sup.-/- mice. B cells were isolated and cultured on
OVA-coated ELISpot plates for 24 h. (FIG. 11E) Representative lobes
from immunized or non-immunized mice injected with OVA-B16 cells.
(FIG. 11F) Ex vivo antigen cross-presentation using DC stimulated
with immune complexes (IC) formed with NIP-OVA and either
FcRn-binding (IgG IC), non-FcRn binding (IHH-IgG IC) or enhanced
FcRn-binding (LS-IgG IC) immunoglobulin and cocultured with OT-I
CD8.sup.+ T cells. All data are representative of the results of
one of three independent experiments with n=3-6 mice per group per
experiment. Data represent mean.+-.s.e.m. NS=not significant. *
p.ltoreq.0.05.
[0070] FIGS. 12A-12E depict, in accordance with various embodiments
of the present invention that FcRn in DC drives homeostatic local
activation of CD8.sup.+ T cells and the Th1 polarization of
CD4.sup.+ T cells within the LI LP by facilitating the production
of Tc1 and Th1 polarizing cytokines. (FIG. 12A) Frequency (upper
panels) and effector status (lower panels) of adoptively
transferred congenic CD8.sup.+ I cells (CD45.1) in the LI LP and
MLN of untreated WI and Fcgrt.sup.-/- recipient mice (CD45.2).
1.times.10.sup.6 CD8.sup.+ I cells were transferred into recipient
mice via i.v. injection and their distribution and phenotype was
assessed 10 days later. (FIG. 12B) Frequency of adoptively
transferred congenic CD8.sup.+ I cells from WI and Fcgrt.sup.-/-
donor mice (CD45.2) in the LI LP and MLN of untreated recipient
mice (CD45.1) 7 days after transfer. (FIG. 12C) Whole tissue
transcript profiles of the LI from untreated WI and Fcgrt.sup.-/-
littermates (left panel) or Fcgrt.sup.Fl/Fl and
Itgax.sup.creFcgrt.sup.Fl/Fl littermates (right panel), as assessed
by qPCR. (FIG. 12D) Cytokine secretion by CD4.sup.+ I cells
isolated via magnetic sorting from the LI LP of untreated WI and
Fcgrt.sup.-/- mice and restimulated for 24 h with anti-CD3
(.alpha.CD3) and anti-CD28 (.alpha.CD28). (FIG. 12E) CD4.sup.+ I
cell frequency in the LI LP of untreated WI and Fcgrt.sup.-/- mice
(left panels) or Fcgrt.sup.Fl/Fl and Itgax.sup.creFcgrt.sup.Fl/Fl
mice (right panels). Representative results from three independent
experiments with n=3-5 mice per group per experiment. Data
represent mean.+-.s.e.m. * p.ltoreq.0.05, ** p.ltoreq.0.01,***
p.ltoreq.0.005.
[0071] FIGS. 13A-13C depict, in accordance with various embodiments
of the present invention that FcRn-dependent induction of IL-12 is
not dependent on MYD88 and is not required for FcRn-mediated
cross-priming. (FIG. 13A) Induction of IL-12p35 upon ex vivo
stimulation of Myd88.sup.-/- CD8.sup.- CD11b.sup.+ DC with
FcRn-binding IC (IgG IC) or FcRn non-binding IC (IHH-IgG IC) for 6
h. (FIG. 13B) Binding of IRF-1 to the IL-12p35 promoter upon
stimulation of Myd88.sup.-/-CD8.sup.-CD11b.sup.+ DC with IgG IC or
IHH-IgG IC for 4 h. (FIG. 13C) Ex vivo antigen cross-presentation
in the presence of an IL-12 neutralizing antibody (.alpha.IL-12) or
isotype control. DC were stimulated with IgG IC or IHH-IgG IC and
co-cultured with OT-I CD8.sup.+ T cells. Representative data shown
from one of three independent experiments. Data represent
mean.+-.s.e.m. * p.ltoreq.0.05, ** p.ltoreq.0.01.
[0072] FIGS. 14A-14H depict, in accordance with various embodiments
of the present invention that human DC strongly express FcRn and
localize to the stroma of both normal and CRC-associated LI. (FIG.
14A) Double immunohistochemical staining of FcRn.sup.+CD11c.sup.+
DC in the stroma of CRC-bearing (upper panels) and tumor-adjacent
normal (lower panels) LI of additional cases of human CRC. See also
FIG. 7A. Scale bar left panels=100 .mu.m. Scale bar right panels=20
.mu.m. (FIG. 14B) Colocalization of FcRn.sup.+ DC and CD8.sup.+ T
cells in the stroma of CRC-bearing (upper panels) and
tumor-adjacent normal (lower panels) LI of additional cases of
human CRC. Arrowheads indicate areas of colocalization. See also
FIG. 7B. Data in FIGS. 14A-14B are representative of 50 matched
normal LI and CRC assessed. (FIG. 14C) Correlation between the
number of FcRn.sup.+CD11c.sup.+ DC and CD8.sup.+ T cells in the
stroma of normal LI adjacent to CRC. Significance was assessed
using Spearman's rank correlation. (FIG. 14D) Multivariable
analysis of the impact of colonic LP CD11c.sup.+FcRn.sup.+ cells on
patient survival in 183 human CRC patients. An increasing number of
CD11+FcRn+ cells has a positive effect on patient survival
(univariate analysis p=0.0333) and this effect is maintained in
multivariable analysis with the indicated parameters. See also FIG.
7C. (FIG. 14E) CD8.sup.+ T cell frequency in the tumor LP of
chimeric mice treated with AOM/DSS. WT recipients were
reconstituted with bone marrow from WT donors. Fcgrt.sup.-/-
recipients were reconstituted with bone marrow from either
Fcgrt.sup.-/-, WT or hFCGRT-hB2M-mFcgrt.sup.-/- donors.
Representative results from one of two independent experiments with
n=4-5 mice per group per experiment. (FIG. 14F) Phenotype of human
monocyte derived DC (hMoDC) as determined by flow cytometric
analysis. Shaded curve represents isotype control. (FIG. 14G)
Expression of FcRn in hMoDC, as assessed by the same antibody
utilized for immunohistochemical staining of CRC cases. (FIG. 14H)
Densitometric analysis of Western blots of hMoDC stimulated with
IgG IC depicted in FIG. 7F. Data in panels FIGS. 14F-14H are
representative of six donors processed in pairs in each of three
independent experiments. All data represent mean.+-.s.e.m.
[0073] FIGS. 15A-15B depict, in accordance with various embodiments
of the present invention that antibody mediated blockade of FcRn
decreases Th1 cytokine transcript levels during IgG-mediated
colitis. (FIG. 15A) Whole colon cytokine transcript levels from
flagellin immunized hFCGRT/hB2M/mFcgrt-/- chimeric mice treated
with either DVN24 or an isotype control before and during the onset
of DSS colitis. (FIG. 15B) Cytokine transcript levels from CD8+ T
cells isolated from the lamina propria of hFCGRT/hB2M/mFcgrt-/- BM
chimeras treated with DVN24 or an isotype control or mFcgrt-/- BM
chimeras treated with PBS during DSS colitis. *P<0.05.
**P<0.01.
[0074] FIG. 16 depicts, in accordance with various embodiments of
the present invention, FcRn-mediated upregulation of IL-12p35 is
dependent upon ERK and calmodulin but not cytoskeletal
rearrangements. Primary mouse dendritic cells were stimulated with
IgG containing immune complexes against chicken ovalbumin that were
wild type and able to bind FcRn or mutant and unable to bind FcRn
(IHH-IC) due to three mutations in the Fc domain of IgG. RNA was
isolated after 5 or 11 hours from such cells treated with
inhibitors for cytoskeleton (cytochalasin D and Latrunculin),
calmodulin (W7) or ERK. The RNA was reverse transcribed and IL-12
p35 was quantified by qPCR.
DETAILED DESCRIPTION OF THE INVENTION
[0075] All references cited herein are incorporated by reference in
their entirety as though fully set forth. Unless defined otherwise,
technical and scientific terms used herein have the same meaning as
commonly understood by one of ordinary skill in the art to which
this invention belongs. Singleton et al., Dictionary of
Microbiology and Molecular Biology 3.sup.rd ed., J. Wiley &
Sons (New York, N.Y. 2001); March, Advanced Organic Chemistry
Reactions, Mechanisms and Structure 5.sup.th ed., J. Wiley &
Sons (New York, N.Y. 2001); and Sambrook and Russel, Molecular
Cloning: A Laboratory Manual 3rd ed., Cold Spring Harbor Laboratory
Press (Cold Spring Harbor, N.Y. 2001), provide one skilled in the
art with a general guide to many of the terms used in the present
application.
[0076] One skilled in the art will recognize many methods and
materials similar or equivalent to those described herein, which
could be used in the practice of the present invention. Indeed, the
present invention is in no way limited to the methods and materials
described. For purposes of the present invention, the following
terms are defined below.
[0077] As used herein, the term "antibody" refers to an intact
immunoglobulin or to a monoclonal or polyclonal antigen-binding
fragment with the Fc (crystallizable fragment) region or FcRn
binding fragment of the Fc region, referred to herein as the "Fc
fragment" or "Fc domain". Antigen-binding fragments may be produced
by recombinant DNA techniques or by enzymatic or chemical cleavage
of intact antibodies. Antigen-binding fragments include, inter
alia, Fab, Fab', F(ab')2, Fv, dAb, and complementarity determining
region (CDR) fragments, single-chain antibodies (scFv), single
domain antibodies, chimeric antibodies, diabodies, tetrabodies and
other multimerized scFv moieties and polypeptides that contain at
least a portion of an immunoglobulin that is sufficient to confer
specific antigen binding to the polypeptide. The Fc domain includes
portions of two heavy chains contributing to two or three classes
of the antibody. The Fc domain may be produced by recombinant DNA
techniques or by enzymatic (e.g. papain cleavage) or via chemical
cleavage of intact antibodies. The amino acid sequence of the Fc
domain can be modified to increase its affinity with FcRn to enable
better induction of secreted proteins such as IL-12,
interferon-gamma, IL-2, tumor necrosis factor, granulocyte
macrophage colony stimulating factor, IL-3 and granzyme B or
transcription factors such as t-bet or modified to decrease its
affinity for FcRn and the induction of these aforementioned
cytokines and transcription factors.
[0078] The term "antibody fragment," as used herein, refer to a
protein fragment that comprises only a portion of an intact
antibody, generally including an antigen binding site of the intact
antibody and thus retaining the ability to bind antigen. Examples
of antibody fragments encompassed by the present definition
include: (i) the Fab fragment, having VL, CL, VH and CH1 domains;
(ii) the Fab' fragment, which is a Fab fragment having one or more
cysteine residues at the C-terminus of the CH1 domain; (iii) the Fd
fragment having VH and CH1 domains; (iv) the Fd' fragment having VH
and CH1 domains and one or more cysteine residues at the C-terminus
of the CH1 domain; (v) the Fv fragment having the VL and VH domains
of a single arm of an antibody; (vi) the dAb fragment (Ward et al.,
Nature 341, 544-546 (1989)) which consists of a VH domain; (vii)
isolated CDR regions; (viii) F(ab')2 fragments, a bivalent fragment
including two Fab' fragments linked by a disulphide bridge at the
hinge region; (ix) single chain antibody molecules (e.g., single
chain Fv; scFv) (Bird et al., Science 242:423-426 (1988); and
Huston et al., PNAS (USA) 85:5879-5883 (1988)); (x) "diabodies"
with two antigen binding sites, comprising a heavy chain variable
domain (VH) connected to a light chain variable domain (VL) in the
same polypeptide chain (see, e.g., EP 404,097; WO 93/11161; and
Hollinger et al., Proc. Natl. Acad. Sci. USA, 90:6444-6448 (1993));
(xi) "linear antibodies" comprising a pair of tandem Fd segments
(VH-CH1-VH-CH1) which, together with complementary light chain
polypeptides, form a pair of antigen binding regions (Zapata et al.
Protein Eng. 8(10):1057-1062 (1995); and U.S. Pat. No.
5,641,870).
[0079] As described herein, an "antigen" is a molecule that is
bound by a binding site on a polypeptide agent, such as an antibody
or antibody fragment thereof. Typically, antigens are bound by
antibody ligands and are capable of raising an antibody response in
vivo. An antigen can be a polypeptide, protein, nucleic acid, lipid
or other molecule. In the case of conventional antibodies and
fragments thereof, the antibody binding site as defined by the
variable loops (L1, L2, L3 and H1, H2, H3) is capable of binding to
the antigen. The term "antigenic determinant" refers to an epitope
on the antigen recognized by an antigen-binding molecule, and more
particularly, by the antigen-binding site of said molecule.
[0080] An "Fv" fragment is an antibody fragment which contains a
complete antigen recognition and binding site. This region consists
of a dimer of one heavy and one light chain variable domain in
tight association, which can be covalent in nature, for example in
scFv. It is in this configuration that the three CDRs of each
variable domain interact to define an antigen binding site on the
surface of the VH-VL dimer. Collectively, the six CDRs or a subset
thereof confer antigen binding specificity to the antibody.
However, even a single variable domain (or half of an Fv comprising
only three CDRs specific for an antigen) has the ability to
recognize and bind antigen, although usually at a lower affinity
than the entire binding site.
[0081] A "cancer" or "tumor" as used herein refers to an
uncontrolled growth of cells which interferes with the normal
functioning of the bodily organs and systems. A subject that has a
cancer or a tumor is a subject having objectively measurable
neoplastic cells present in the subject's body. Included in this
definition are benign and malignant cancers, as well as dormant
tumors or micrometastases. Cancers which migrate from their
original location and seed vital organs can eventually lead to the
death of the subject through the functional deterioration of the
affected organs. Hematopoietic cancers, such as leukemia, are able
to out-compete the normal hemopoietic compartments in a subject,
thereby leading to hemopoietic failure (in the form of anemia,
thrombocytopenia and neutropenia) ultimately causing death.
[0082] As used herein, "complexed" refers to the non-covalent
interactions between any two molecules. Examples include but are
not limited to complexes formed between and an antigen and an
antibody (for example, IgG or a variant thereof or a fragment
thereof) wherein the antigen and the antibody interact via
non-covalent bonds. Examples of non-covalent interactions include
but are not limited to electrostatic interactions (for example,
ionic interactions, hydrogen bonds, halogen bonds), van der Waals
forces (dipole-dipole, dipole-induced, London dispersion forces),
pi-effects (pi-pi interactions, cation-pi, anion-pi, polar-pi)
and/or hydrophobic interactions. In various embodiments, the
complex between the IgG or a fragment thereof or a variant thereof
and the antigen forms multimeric structures that can
cross-link/bind with FcRn.
[0083] As used herein, "conjugated" refers to covalent interactions
between any two molecules. Examples include but are not limited to
fusion proteins comprising an antigen and an antibody (for example,
IgG or a variant thereof or a fragment thereof) or any other
antigen-antibody complex that may be covalently linked. In various
embodiments, the conjugation between the IgG or a fragment thereof
or a variant thereof forms and the antigen forms monomeric or
multimeric structures that can cross-link/bind with FcRn.
[0084] As used herein, an "epitope" can be formed both from
contiguous amino acids, or noncontiguous amino acids juxtaposed by
tertiary folding of a protein. Epitopes formed from contiguous
amino acids are typically retained on exposure to denaturing
solvents, whereas epitopes formed by tertiary folding are typically
lost on treatment with denaturing solvents. An epitope typically
includes at least 3, and more usually, at least 5, about 9, or
about 8-10 amino acids in a unique spatial conformation. An
"epitope" includes the unit of structure conventionally bound by an
immunoglobulin VH/VL pair. Epitopes define the minimum binding site
for an antibody, and thus represent the target of specificity of an
antibody. In the case of a single domain antibody, an epitope
represents the unit of structure bound by a variable domain in
isolation. The terms "antigenic determinant" and "epitope" can also
be used interchangeably herein. In various embodiments, an epitope
may be protein, peptide, nucleic acid, lipid, other molecules or
combinations thereof.
[0085] As used herein, an "immune response" being modulated refers
to a response by a cell of the immune system, such as a B cell, T
cell (CD4 or CD8), regulatory T cell, antigen-presenting cell,
dendritic cell, monocyte, macrophage, NKT cell, NK cell, basophil,
eosinophil, or neutrophil, to a stimulus. In some embodiments, the
response is specific for a particular antigen (an "antigen-specific
response"), and refers to a response by a CD4 T cell, CD8 T cell,
or B cell via their antigen-specific receptor. In some embodiments,
an immune response is a T cell response, such as a CD4+ response or
a CD8+ response. Such responses by these cells can include, for
example, cytotoxicity, proliferation, cytokine or chemokine
production, trafficking, or phagocytosis, and can be dependent on
the nature of the immune cell undergoing the response. In some
embodiments of the compositions and methods described herein, an
immune response being modulated is T-cell tolerance.
[0086] By "metastasis" is meant the spread of cancer from its
primary site to other places in the body. Cancer cells can break
away from a primary tumor, penetrate into lymphatic and blood
vessels, circulate through the bloodstream, and grow in a distant
focus (metastasize) in normal tissues elsewhere in the body.
Metastasis can be local or distant. Metastasis is a sequential
process, contingent on tumor cells breaking off from the primary
tumor, traveling through the bloodstream, and stopping at a distant
site. At the new site, the cells establish a blood supply and can
grow to form a life-threatening mass. Both stimulatory and
inhibitory molecular pathways within the tumor cell regulate this
behavior, and interactions between the tumor cell and host cells in
the distant site are also significant.
[0087] The term "monoclonal antibody" as used herein refers to an
antibody obtained from a population of substantially homogeneous
antibodies, i.e., the individual antibodies comprising the
population are identical except for possible naturally occurring
mutations that can be present in minor amounts. Monoclonal
antibodies are highly specific, being directed against a single
antigen. Furthermore, in contrast to polyclonal antibody
preparations that typically include different antibodies directed
against different determinants (epitopes), each monoclonal antibody
is directed against a single determinant on the antigen. The
modifier "monoclonal" is not to be construed as requiring
production of the antibody by any particular method. For example,
the monoclonal antibodies to be used in accordance with the
invention can be made by the hybridoma method first described by
Kohler et al., Nature 256:495 (1975), or can be made by recombinant
DNA methods (see, e.g., U.S. Pat. No. 4,816,567). The "monoclonal
antibodies" can also be isolated from phage antibody libraries
using the techniques described in Clackson et al., Nature
352:624-628 (1991) or Marks et al., J. Mol. Biol. 222:581-597
(1991), for example.
[0088] As used herein, "selectively binds" or "specifically binds"
refers to the ability of an antibody or antibody fragment thereof
described herein to bind to a target, such as a molecule present on
the cell-surface, with a KD 10-5 M (10000 nM) or less, e.g.,
10.sup.-6 M, 10.sup.-7 M, 10.sup.-8 M, 10.sup.-9 M, 10.sup.-10 M,
10.sup.-11 M, 10.sup.-12 M, or less. Specific binding can be
influenced by, for example, the affinity and avidity of the
polypeptide agent and the concentration of polypeptide agent. The
person of ordinary skill in the art can determine appropriate
conditions under which the polypeptide agents described herein
selectively bind the targets using any suitable methods, such as
titration of a polypeptide agent in a suitable cell binding
assay.
[0089] "Subject" or "individual" or "animal" or "patient" or
"mammal," is meant any subject, particularly a mammalian subject,
for whom diagnosis, prognosis, or therapy is desired. In some
embodiments, the subject has cancer. In some embodiments, the
subject had cancer at some point in the subject's lifetime. In
various embodiments, the subject's cancer is in remission, is
re-current or is non-recurrent. In some embodiments the subject has
an infectious disease. In some embodiments, the subject has an
autoimmune disease.
[0090] "Mammal" as used herein refers to any member of the class
Mammalia, including, without limitation, humans, domestic animals,
farm animals, zoo animals, sport animals, pet animals such as dogs,
cats, guinea pigs, rabbits, rats, mice, horses, cattle, cows;
primates such as apes, monkeys, orangutans, and chimpanzees; canids
such as dogs and wolves; felids such as cats, lions, and tigers;
equids such as horses, donkeys, and zebras; food animals such as
cows, pigs, and sheep; ungulates such as deer and giraffes; rodents
such as mice, rats, hamsters and guinea pigs; and so on. In certain
embodiments, the mammal is a human subject. The term does not
denote a particular age or sex. Thus, adult and newborn subjects,
as well as fetuses, whether male or female, are intended to be
included within the scope of this term
[0091] As used herein, the term "target" refers to a biological
molecule (e.g., peptide, polypeptide, protein, lipid, carbohydrate)
to which a polypeptide domain which has a binding site can
selectively bind. The target can be, for example, an intracellular
target (e.g., an intracellular protein target) or a cell surface
target (e.g., a membrane protein, a receptor protein). Preferably,
a target is a cell surface target, such as a cell surface
protein.
[0092] As used herein, the terms "treat," "treatment," "treating,"
or "amelioration" refer to therapeutic treatments, wherein the
object is to reverse, alleviate, ameliorate, inhibit, slow down or
stop the progression or severity of a condition associated with, a
disease or disorder. The term "treating" includes reducing or
alleviating at least one adverse effect or symptom of a condition,
disease or disorder, such as an autoimmune disease, a chronic
infection or a cancer. Treatment is generally "effective" if one or
more symptoms or clinical markers are reduced. Alternatively,
treatment is "effective" if the progression of a disease is reduced
or halted. That is, "treatment" includes not just the improvement
of symptoms or markers, but also a cessation of at least slowing of
progress or worsening of symptoms that would be expected in absence
of treatment. Beneficial or desired clinical results include, but
are not limited to, alleviation of one or more symptom(s),
diminishment of extent of disease, stabilized (i.e., not worsening)
state of disease, delay or slowing of disease progression,
amelioration or palliation of the disease state, and remission
(whether partial or total), whether detectable or undetectable. The
term "treatment" of a disease also includes providing relief from
the symptoms or side-effects of the disease (including palliative
treatment).
[0093] A number of tumor antigens have been identified that are
associated with specific cancers. As used herein, the terms "tumor
antigen" and "cancer antigen" are used interchangeably to refer to
antigens which are differentially expressed by neoplastic cells and
can thereby be exploited in order to target neoplastic cells.
Cancer antigens are antigens which can potentially stimulate
apparently tumor-specific immune responses. Some of these antigens
are encoded, although not necessarily expressed, by normal cells.
These antigens can be characterized as those which are normally
silent (i.e., not expressed) in normal cells, those that are
expressed only at certain stages of differentiation and those that
are temporally expressed such as embryonic and fetal antigens.
Other cancer antigens are encoded by mutant cellular genes, such as
oncogenes (e.g., activated ras oncogene), suppressor genes (e.g.,
mutant p53), fusion proteins resulting from internal deletions or
chromosomal translocations. Still other cancer antigens can be
encoded by viral genes such as those carried on RNA and DNA tumor
viruses. Many tumor antigens have been defined in terms of multiple
solid tumors: MAGE 1, 2, & 3, defined by immunity;
MART-1/Melan-A, gp100, carcinoembryonic antigen (CEA), HER-2,
mucins (i.e., MUC-1), prostate-specific antigen (PSA), and
prostatic acid phosphatase (PAP). In addition, viral proteins such
as hepatitis B (HBV), Epstein-Barr (EBV), and human papilloma (HPV)
have been shown to be important in the development of
hepatocellular carcinoma, lymphoma, and cervical cancer,
respectively. However, due to the immunosuppression of patients
diagnosed with cancer, the immune systems of these patients often
fail to respond to the tumor antigens.
[0094] "Endogenous antigens" as used herein refers to antigens that
have been generated within the body. They include xenogenic
(heterologus), autologus, idiotypic or allogenic antigens and
autoantigens.
[0095] FcRn is a neonatal Fc receptor. It is similar in structure
to major histocompatibility complex I (MHC I). Human FcRn is very
stringent regarding its specificity and binds human Fc, but not
mouse, rat, bovine, or sheep Fc. The FcRn can bind to two sites of
the IgG (Sanchez et al., 1999; Schuck et al., 1999; West A. P. and
Bjorkman, 2000). Although mouse IgGs do not bind efficiently to
human FcRn and therefore have a short half-life in humans (Frodin
et al., 1990), mouse IgG as an immune complex is capable of binding
human FcRn and inducing signaling. In contrast, mouse FcRn binds
IgG from every species analyzed (Ober et al., 2001).
[0096] Most serum proteins have a short serum half-life (about 1-2
days). However, two types of serum proteins, namely albumin and
antibodies of the IgG class, have greatly extended serum
half-lives. For example, most subclasses of IgG have a half-life of
about 10-20 days in humans. The Fc region of IgG is required for
this extension of half-life. Thus, truncated IgG polypeptides
carrying only the Fc region, and potentially also proteins carrying
a short FcRn binding peptide sequence (FcBP) (Sockolosky et al.
Proc Natl Acad Sci USA 2012, 109, 16095-100), also show such
extended serum half-life. Moreover, when the Fc region is fused
with a fusion partner (e.g., a biologically active protein), this
Fc fusion protein shows an extended serum half-life due to its
interaction with FcRn.
[0097] The mechanism by which FcRn extends the serum half-life of
IgG and IgG Fc fusion proteins is well established (Ghetie and
Ward, 2000, 2002; Roopenian and Akilesh, 2007). FcRn is localized
in the endosomal compartments of many cell types, including
vascular endothelium. Serum proteins are constantly being
endocytosed and directed to the early endosomal vesicles. FcRn is
harbored primarily in this acidified vesicle. In this acidified
environment, the Fc region binds FcRn, and the IgG/FcRn complex is
then recycled either apically or basolaterally back to the plasma
membrane, whereupon exposure to the neutral pH 7.2 extracellular
environment results in its release into the circulation. In
contrast, other endocytosed proteins that do not bind FcRn are not
rescued, and thus continue through the endosomal route to catabolic
elimination, resulting in their short half-life. The biochemical
mechanism by which the Fc region of IgG binds FcRn in an acidic
environment is well understood. The CH2-CH3-hinge region of the Fc
region contains solvent exposed histidine residues, which when
protonated, engage residues on FcRn with sufficient affinity to
permit IgG to exploit the FcRn recycling pathway to escape
catabolic elimination.
[0098] As described herein, the inventors discovered that
cross-linking FcRn on dendritic cells with antigen/antibody immune
complexes, which function as ligands for FcRn, directly leads to
the production of IL-12 by these cells, as well as
interferon-gamma, tumor necrosis factor, granzyme B and t-bet by
CD8+ T cells. FcRn functions as a signaling molecule by organizing
the necessary proteins, including elements of the cytoskeleton and
mitogen activated protein kinases (MAPK) to directly promote the
transcription of IL-12 through factors such as IRF-1 and
NF.kappa.B. As demonstrated herein, FcRn-mediated upregulation of
IL-12p35 is dependent upon ERK and calmodulin but not cytoskeletal
rearrangements. The production of FcRn-dependent IL-12 is essential
for the generation of CD8+ effector T cells and their effector
function through factors such interferon-gamma, tumor necrosis
factor, granzyme B and t-bet. Such effector T cells mediate
anti-infectious immunity and mediate tumor immune-surveillance and
eradication. If IL-12 is neutralized, the FcRn-mediated effects on
CD8+ effector T cell functions that are associated with tumor
eradication are lost.
[0099] In a homologous manner, blockade of FcRn function is
associated with decreased FcRn-mediated signal transduction and
thereby decreased production of IL-12 and related factors which is
associated with protection from autoimmune disorders as shown in
animal models of inflammatory bowel disease. While not wishing to
be bound by any particular mechanism or theory, the aspects and
embodiments described herein are based on the finding that binding
of FcRn to antigen/antibody immune complexes regulates IL-12
production by dendritic cells, which can be manipulated for
therapeutic purposes. Since IL-12 is a master regulator of immune
responses associated with tumor and anti-infectious immunity on the
one hand, and inappropriate inflammation on the other, it is
desirable to increase IL-12 for anti-tumor immunity and decrease
IL-12 for anti-inflammatory purposes. Accordingly, described herein
are compositions and methods for increasing/enhancing/up-regulating
IL-12 production for treating cancer and infectious diseases and
compositions and methods for decreasing IL-12 production for
treating autoimmune disorders.
Compositions and Methods for Treating Cancer and/or Infectious
Diseases
[0100] In some aspects, the compositions and methods described
herein up-regulate/increase/enhance production of IL-12 by
increasing/enhancing interaction between IgG and FcRn (for example,
in response to an antigen bound to the IgG, or due to mutations in
IgG that increase the binding between IgG and FcRn). The
interactions between IgG and FcRn yield signals that result in
production of IL-12 by dendritic cells. The compositions provided
herein comprise IgG or a variant thereof or a fragment thereof so
as to increase IL-12 production. As described herein, the
compositions can further comprise antigens, including but not
limited to, tumor antigens, bacterial antigens, viral antigens,
parasitic antigens or combinations thereof, as to increase IL-12
production. As used herein, "IgG" can refer to any isotype of IgG
including IgG1, IgG2, IgG3 and/or IgG4.
[0101] In some embodiments, the variants of IgG in the compositions
and methods described herein are functional, non-naturally
occurring variants of IgG that enhance binding between IgG and
FcRn, so as to increase IL-12 production. "Functional variants of
IgG," as used herein, useful with the compositions and methods
described herein include molecules comprising mutations, such as
insertions, deletions and truncations in full-length IgG, or the
constant region of an IgG molecule, provided such molecules retain
the ability to bind to FcRn and increase IL-12 production. One
example of such a variant includes, but is not limited to, an IgG
comprising a methionine to leucine substitution at position 428 and
an asparagine to serine substitution at position 434 according to
the Kabat numbering scheme. Another example of a functional IgG
variant useful with the compositions and methods described herein
is an engineered variant of human IgG1 omprising mutations of Met
252, Ser254, Thr256, His433, and Asn434 to Tyr252, Thr254, Glu256,
Lys433, and Phe434, as described in "Engineering the Fc region of
immunoglobulin G to modulate in vivo antibody levels," Nature
Biotechnology, 2005 (23):10, pp 1283-88, the contents of which are
herein incorporated by reference in their entireties. Other
functional IgG variants useful with the compositions and methods
described herein are described in U.S. Pat. Nos. 8,188,231,
8,802,823, US2011025068, US20060235208, WO2012106578 A1, the
contents of each of which are herein incorporated by reference in
their entireties.
[0102] For example, functional IgG variants can comprise amino acid
modifications at Fc positions 230, 240, 244, 245, 247, 262, 263,
266, 273, 275, 299, 302, 313, 323, 325, 328, and 332, wherein the
numbering of the residues in the Fc region is that of the EU index
as in Kabat. In some embodiments, functional IgG variants can
comprise at least one amino acid substitution in the Fc region at a
position selected from the group consisting of 221, 222, 223, 224,
225, 227, 228, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239,
240, 241, 243, 244, 245, 246, 247, 249, 255, 258, 260, 262, 263,
264, 265, 266, 267, 268, 269, 270, 271, 272, 273, 274, 275, 276,
278, 280, 281, 282, 283, 284, 285, 286, 288, 290, 291, 292, 293,
294, 295, 296, 297, 298, 299, 300, 301, 302, 303, 304, 305, 313,
317, 318, 320, 322, 323, 324, 325, 326, 327, 328, 329, 330, 331,
332, 333, 334, 335, 336, and 337, wherein the numbering of the
residues in the Fc region is that of the EU index as in Kabat.
[0103] In some embodiments, said IgG variants comprise at least one
substitution selected from the group consisting of D221K, D221Y,
K222E, K222Y, T223E, T223K, H224E, H224Y, T225E, T225K, T225W,
P227E, P227G, P227K, P227Y, P228E, P228G, P228K, P228Y, P230A,
P230E, P230G, P230Y, A231E, A231G, A231K, A231P, A231Y, P232E,
P232G, P232K, P232Y, E233A, E233D, E233F, E233G, E233H, E233I,
E233K, E233L, E233M, E233N, E233Q, E233R, E233S, E233T, E233V,
E233W, E233Y, L234A, L234D, L234E, L234F, L234G, L234H, L234I,
L234K, L234M, L234N, L234P, L234Q, L234R, L234S, L234T, L234V,
L234W, L234Y, L235A, L235D, L235E, L235F, L235G, L235H, L235I,
L235K, L235M, L235N, L235P, L235Q, L235R, L235S, L235T, L235V,
L235W, L235Y, G236A, G236D, G236E, G236F, G236H, G236I, G236K,
G236L, G236M, G236N, G236P, G236Q, G236R, G236S, G236T, G236V,
G236W, G236Y, G237D, G237E, G237F, G237H, G237I, G237K, G237L,
G237M, G237N, G237P, G237Q, G237R, G237S, G237T, G237V, G237W,
G237Y, P238D, P238E, P238F, P238G, P238H, P238I, P238K, P238L,
P238M, P238N, P238Q, P238R, P238S, P238T, P238V, P238W, P238Y,
S239D, S239E, S239F, S239G, S239H, S239I, S239K, S239L, S239M,
S239N, S239P, S239Q, S239R, S239T, S239V, S239W, S239Y, V240A,
V240I, V240M, V240T, F241D, F241E, F241L, F241R, F241S, F241W,
F241Y, F243E, F243H, F243L, F243Q, F243R, F243W, F243Y, P244H,
P245A, K246D, K246E, K246H, K246Y, P247G, P247V, D249H, D249Q,
D249Y, R255E, R255Y, E258H, E258S, E258Y, T260D, T260E, T260H,
T260Y, V262A, V262E, V262F, V262I, V262T, V263A, V263I, V263M,
V263T, V264A, V264D, V264E, V264F, V264G, V264H, V264I, V264K,
V264L, V264M, V264N, V264P, V264A, V264R, V264S, V264T, V264W,
V264Y, D265F, D265G, D265H, D265I, D265K, D265L, D265M, D265N,
D265P, D265Q, D265R, D265S, D265T, D265V, D265W, D265Y, V266A,
V266I, V266M, V266T, S267D, S267E, S267F, S267H, S267I, S267K,
S267L, S267M, S267N, S267P, S267Q, S267R, S267T, S267V, S267W,
S267Y, H268D, H268E, H268F, H268G, H268I, H268K, H268L, H268M,
H268P, H268Q, H268R, H268T, H268V, H268W, E269F, E269G, E269H,
E269I, E269K, E269L, E269M, E269N, E269P, E269R, E269S, E269T,
E269V, E269W, E269Y, D270F, D270G, D270H, D270I, D270L, D270M,
D270P, D270Q, D270R, D270S, D270T, D270W, D270Y, P271A, P271D,
P271E, P271F, P271G, P271H, P271I, P271K, P271L, P271M, P271N,
P271Q, P271R, P271S, P271T, P271V, P271W, P271Y, E272D, E272F,
E272G, E272H, E272I, E272K, E272L, E272M, E272P, E272R, E272S,
E272T, E272V, E272W, E272Y, V273I, K274D, K274E, K274F, K274G,
K274H, K274I, K274L, K274M, K274N, K274P, K274R, K274T, K274V,
K274W, K274Y, F275L, F275W, N276D, N276E, N276F, N276G, N276H,
N276I, N276L, N276M, N276P, N276R, N276S, N276T, N276V, N276W,
N276Y, Y278D, Y278E, Y278G, Y278H, Y278I, Y278K, Y278L, Y278M,
Y278N, Y278P, Y278Q, Y278R, Y278S, Y278T, Y278V, Y278W, D280G,
D280K, D280L, D280P, D280W, G281D, G281E, G281K, G281N, G281P,
G281Q, G281Y, V282E, V282G, V282K, V282P, V282Y, E283G, E283H,
E283K, E283L, E283P, E283R, E283Y, V284D, V284E, V284L, V284N,
V284Q, V284T, V284Y, H285D, H285E, H285K, H285Q, H285W, H285Y,
N286E, N286G, N286P, N286Y, K288D, K288E, K288Y, K290D, K290H,
K290L, K290N, K290W, P291D, P291E, P291G, P291H, P291I, P291Q,
P291T, R292D, R292E, R292T, R292Y, E293F, E293G, E293H, E293I,
E293L, E293M, E293N, E293P, E293R, E293S, E293T, E293V, E293W,
E293Y, E294F, E294G, E294H, E294I, E294K, E294L, E294M, E294P,
E294R, E294S, E294T, E294V, E294W, E294Y, Q295D, Q295E, Q295F,
Q295G, Q295H, Q295I, Q295M, Q295N, Q295P, Q295R, Q295S, Q295T,
Q295V, Q295W, Q295Y, Y296A, Y296D, Y296E, Y296G, Y296H, Y296I,
Y296K, Y296L, Y296M, Y296N, Y296Q, Y296R, Y296S, Y296T, Y296V,
N297D, N297E, N297F, N297G, N297H, N297I, N297K, N297L, N297M,
N297P, N297Q, N297R, N297S, N297T, N297V, N297W, N297Y, S298D,
S298E, S298F, S298H, S298I, S298K, S298M, S298N, S298Q, S298R,
S298T, S298W, S298Y, T299A, T299D, T299E, T299F, T299G, T299H,
T299I, T299K, T299L, T299M, T299N, T299P, T299Q, T299R, T299S,
T299V, T299W, T299Y, Y300A, Y300D, Y300E, Y300G, Y300H, Y300K,
Y300M, Y300N, Y300P, Y300Q, Y300R, Y300S, Y300T, Y300V, Y300W,
R301D, R301E, R301H, R301Y, V302I, V303D, V303E, V303Y, S304D,
S304H, S304L, S304N, S304T, V305E, V305T, V305Y, W313F, K317E,
K317Q, E318H, E318L, E318Q, E318R, E318Y, K320D, K320F, K320G,
K320H, K320I, K320L, K320N, K320P, K320S, K320T, K320V, K320W,
K320Y, K322D, K322F, K322G, K322H, K322I, K322P, K322S, K322T,
K322V, K322W, K322Y, V323I, S324D, S324F, S324G, S324H, S324I,
S324L, S324M, S324P, S324R, S324T, S324V, S324W, S324Y, N325A,
N325D, N325E, N325F, N325G, N325H, N325I, N325K, N325L, N325M,
N325P, N325Q, N325R, N325S, N325T, N325V, N325W, N325Y, K326I,
K326L, K326P, K326T, A327D, A327E, A327F, A327H, A327I, A327K,
A327L, A327M, A327N, A327P, A327R, A327S, A327T, A327V, A327W,
A327Y, L328A, L328D, L328E, L328F, L328G, L328H, L328I, L328K,
L328M, L328N, L328P, L328Q, L328R, L328S, L328T, L328V, L328W,
L328Y, P329D, P329E, P329F, P329G, P329H, P329I, P329K, P329L,
P329M, P329N, P329Q, P329R, P329S, P329T, P329V, P329W, P329Y,
A330E, A330F, A330G, A330H, A330I, A330L, A330M, A330N, A330P,
A330R, A330S, A330T, A330V, A330W, A330Y, P331D, P331F, P331H,
P331I, P331L, P331M, P331Q, P331R, P331T, P331V, P331W, P331Y,
I332A, I332D, I332E, I332F, I332H, I332K, I332L, I332M, I332N,
I332P, I332Q, I332R, I332S, I332T, I332V, I332W, I332Y, E333F,
E333H, E333I, E333L, E333M, E333P, E333T, E333Y, K334F, K334I,
K334L, K334P, K334T, T335D, T335F, T335G, T335H, T335I, T335L,
T335M, T335N, T335P, T335R, T335S, T335V, T335W, T335Y, 1336E,
1336K, I336Y, S337E, S337H, and S337N, wherein the numbering of the
residues in the Fc region is that of the EU index as in Kabat.
[0104] In some embodiments, said IgG variants comprise any one of
the following combinations of substitutions: S239D/A330L/I332E,
S239D/A330Y/I332E/L234I, S239D/A330Y/I332EN266I,
S239D/D265F/N297D/I332E, S239D/D265H/N297D/I332E,
S239D/D265I/N297D/I332E, S239D/D265L/N297D/I332E,
S239D/D265T/N297D/I332E, S239D/D265Y/N297D/I332E,
S239D/E272I/A330L/I332E, S239D/E272I/I332E,
S239D/E272K/A330L/I332E, S239D/E272K/I332E,
S239D/E272S/A330L/I332E, S239D/E272S/I332E,
S239D/E272Y/A330L/I332E, S239D/E272Y/I332E,
S239D/F241S/F243H/V262T/N264T/N297D/A330Y/I332E, S239D/H268D,
S239D/H268E, S239D/I332D, S239D/I332E, S239D/I332E/A327D,
S239D/I332E/A330I, S239D/I332E/A330Y, S239D/I332E/E272H,
S239D/I332E/E272R, S239D/I332E/E283H, S239D/I332E/E283L,
S239D/I332E/G236A, S239D/I332E/G236S, S239D/I332E/H268D,
S239D/I332E/H268E, S239D/I332E/K246H, S239D/I332E/R255Y,
S239D/I332E/S267E, S239D/I332E/V264I, S239D/I332E/V264I/A330L,
S239D/I332E/V264I/S298A, S239D/I332E/V284D, S239D/I332E/V284E,
S239D/I332E/V284E, S239D/I332N, S239D/I332D,
S239D/K274E/A330L/I332E, S239D/K274E/I332E,
S239D/K326E/A330L/I332E, S239D/K326E/A330Y/I332E,
S239D/K326E/I332E, S239D/K326T/A330Y/I332E, S239D/K326T/I332E,
S239D/N297D/A330Y/I332E, S239D/N297D/I332E,
S239D/N297D/K326E/I332E, S239D/S267E/A330L/I332E,
S239D/S267E/I332E, S239D/S298A/K326E/I332E,
S239D/S298A/K326T/I332E, S239D/V240I/A330Y/I332E,
S239D/V264T/A330Y/I332E, S239D/Y278T/A330L/I332E,
S239D/Y278T/I332E, S239E, S239E/D265G, S239E/D265N, S239E/D265Q,
S239E/I332D, S239E/I332E, S239E/I332N, S239E/I332Q,
S239E/N297D/I332E, S239E/V264I/A330Y/I332E, S239E/V264I/I332E,
S239E/V264I/S298A/A330Y/I332E, S239F, S239G, S239H, S239I, S239K,
S239L, S239M, S239N, S239N/I332D, S239N/I332E, S239N/1332E/A330L,
S239N/I332E/A330Y, S239N/I332N, S239N/1332Q, S239P, S239Q,
S239Q/I332D, S239Q/I332E, S239Q/I332N, S239Q/I332Q,
S239Q/V264I/I332E, S239R, S239T, S239V, S239W, S239Y, V240A, V240I,
V240I/V266I, V240M, V240T, F241D, F241E,
F241E/F243Q/V262T/V264E/I332E, F241E/F243Q/V262T/V264E,
F241E/F243R/V262E/B264R/I332E, F241E/F243R/V262E/V264R,
F241E/F243Y/V262T/V264R/I332E, F241E/F243Y/V262T/V264R, F241L,
F241L/F243L/V262I/V264I, F241L/V262I,
F241R/F243Q/V262T/V264R/I332E, F241R/F243Q/V262T/V264R, F241W,
F241W/F243W, F241W/F243W/V262A/V264A, F241Y,
F241Y/F243Y/V262T/V264T/N297D/I332E, F241Y/F243Y/V262T/V264T,
F243E, F243L, F243L/V262I/V264W, F243L/V264I, F243W, P244H,
P244H/P245A/P247V, P245A, K246D, K246E, K246H, K246Y, P247G, P247V,
D249H, D249Q, D249Y, R255E, R255Y, E258H, E258S, E258Y, T260D,
T260E, T260H, T260Y, V262E, V262F, V263A, V263I, V263M, V263T,
V264A, V264D, V264E, V264E/N297D/I332E, V264F, V264G, V264H, V264I,
V264I/A330L/I332E, V264I/A330Y/I332E, V264I/1332E, V264K, V264L,
V264M, V264N, V264P, V264Q, V264R, V264S, V264T, V264W, V264Y,
D265F, D265F/N297E/I332E, D265G, D265H, D265I, D265K, D265L, D265M,
D265N, D265P, D265Q, D265R, D265S, D265T, D265V, D265W, D265Y,
D265Y/N297D/I332E, D265Y/N297D/T299L/I332E, V266A, V266I, V266M,
V266T, S267D, S267E, S267E, S267E/A327D, S267E/P331D, S267E/S324I,
S267E/V282G, S267F, S267H, S267I, S267K, S267L, S267L/A327S, S267M,
S267N, S267P, S267Q, S267Q/A327S, S267R, S267T, S267V, S267W,
S267Y, H268D, H268E, H268F, H268G, H268I, H268K, H268L, H268M,
H268P, H268Q, H268R, H268T, H268V, H268W, E269F, E269G, E269H,
E269I, E269K, E269L, E269M, E269N, E269P, E269R, E269S, E269T,
E269V, E269W, E269Y, D270F, D270G, D270H, D270I, D270L, D270M,
D270P, D270Q, D270R, D270S, D270T, D270W, D270Y, P271A, P271D,
P271E, P271F, P271G, P271H, P271I, P271K, P271L, P271M, P271N,
P271Q, P271R, P271S, P271T, P271V, P271W, P271Y, E272D, E272F,
E272G, E272H, E272I, E272K, E272L, E272M, E272P, E272R, E272S,
E272T, E272V, E272W, E272Y, V273I, K274D, K274E, K274F, K274G,
K274H, K274I, K274L, K274M, K274N, K274P, K274R, K274T, K274V,
K274W, K274Y, F275L, F275W, N276D, N276E, N276F, N276G, N276H,
N276I, N276L, N276M, N276P, N276R, N276S, N276T, N276V, N276W,
N276Y, Y278D, Y278E, Y278G, Y278H, Y278I, Y278K, Y278L, Y278M,
Y278N, Y278P, Y278Q, Y278R, Y278S, Y278T, Y278V, Y278W, Y278W,
Y278W/E283R/V302I, Y278W/V302I, D280G, D280K, D280L, D280P, D280W,
G281D, G281D/V282G, G281E, G281K, G281N, G281P, G281Q, G281Y,
V282E, V282G, V282G/P331D, V282K, V282P, V282Y, E283G, E283H,
E283K, E283L, E283P, E283R, E283R/V302I/Y278W/E283R, E283Y, V284D,
V284E, V284L, V284N, V284Q, V284T, V284Y, H285D, H285E, H285K,
H285Q, H285W, H285Y, N286E, N286G, N286P, N286Y, K288D, K288E,
K288Y, K290D, K290H, K290L, K290N, K290W, P291D, P291E, P291G,
P291H, P291I, P291Q, P291T, R292D, R292E, R292T, R292Y, E293F,
E293G, E293H, E293I, E293L, E293M, E293N, E293P, E293R, E293S,
E293T, E293V, E293W, E293Y, E294F, E294G, E294H, E294I, E294K,
E294L, E294M, E294P, E294R, E294S, E294T, E294V, E294W, E294Y,
Q295D, Q295E, Q295F, Q295G, Q295H, Q295I, Q295M, Q295N, Q295P,
Q295R, Q295S, Q295T, Q295V, Q295W, Q295Y, Y296A, Y296D, Y296E,
Y296G, Y296I, Y296K, Y296L, Y296M, Y296N, Y296Q, Y296R, Y296S,
Y296T, Y296V, N297D, N297D/I332E, N297D/I332E/A330Y,
N297D/I332E/S239D/A330L, N297D/I332E/S239D/D265V,
N297D/I332E/S298A/A330Y, N297D/I332E/T299E, N297D/I332E/T299F,
N297D/I332E/T299H, N297D/I332E/T299I, N297D/I332E/T299L,
N297D/I332E/T299V, N297D/I332E/Y296D, N297D/I332E/Y296E,
N297D/I332E/Y296H, N297D/I332E/Y296N, N297D/I332E/Y296Q,
N297D/I332E/Y296T, N297E/I332E, N297F, N297G, N297H, N297I, N297K,
N297L, N297M, N297P, N297Q, N297R, N297S, N297S/I332E, N297T,
N297V, N297W, N297Y, S298A/I332E, S298A/K326E, S298A/K326E/K334L,
S298A/K334L, S298D, S298E, S298F, S298H, S298I, S298K, S298M,
S298N, S298Q, S298R, S298T, S298W, S298Y, T299A, T299D, T299E,
T299F, T299G, T299H, T299I, T299K, T299L, T299M, T299N, T299P,
T299Q, T299R, T299S, T299V, T299W, T299Y, Y300A, Y300D, Y300E,
Y300G, Y300H, Y300K, Y300M, Y300N, Y300P, Y300Q, Y300R, Y300S,
Y300T, Y300V, Y300W, R301D, R301E, R301H, R301Y, V302I, V303D,
V303E, V303Y, S304D, S304H, S304L, S304N, S304T, V305E, V305T,
V305Y, W313F, K317E, K317Q, E318H, E318L, E318Q, E318R, E318Y,
K320D, K320F, K320G, K320H, K320I, K320L, K320N, K320P, K320S,
K320T, K320V, K320W, K320Y, K322D, K322F, K322G, K322H, K322I,
K322P, K322S, K322T, K322V, K322W, K322Y, V323I, S324D, S324F,
S324G, S324H, S324I, S324I/A327D, S324L, S324M, S324P, S324R,
S324T, S324V, S324W, S324Y, N325A, N325D, N325E, N325F, N325G,
N325H, N325I, N325K, N325L, N325M, N325P, N325Q, N325R, N325S,
N325T, N325V, N325W, N325Y, K326I, K326L, K326P, K326T, A327D,
A327E, A327F, A327H, A327I, A327K, A327L, A327M, A327N, A327P,
A327R, A327S, A327T, A327V, A327W, A327Y, L328A, L328D,
L328D/I332E, L328E, L328E/1332E, L328F, L328G, L328H, L328H/I332E,
L328I, L328I/1332E, L328I/I332E, L328K, L328M, L328M/I332E, L328N,
L328N/I332E, L328P, L328A, L328Q/I332E, L328Q/I332E, L328R, L328S,
L328T, L328T/I332E, L328V, L328V/I332E, L328W, L328Y, P329D, P329E,
P329F, P329G, P329H, P329I, P329K, P329L, P329M, P329N, P329Q,
P329R, P329S, P329T, P329V, P329W, P329Y, A330E, A330F, A330G,
A330H, A330I, A330L, A330L/I332E, A330M, A330N, A330P, A330R,
A330S, A330T, A330V, A330W, A330Y, A330Y/I332E, P331D, P331F,
P331H, P331I, P331L, P331M, P331Q, P331R, P331T, P331V, P331W,
P331Y, I332A, I332D, I332E, I332E/G281D, I332E/H268D, I332E/H268E,
I332E/S239D/S298A, I332E/S239N/S298A, I332E/V264I/S298A,
I332E/V284E, I332F, I332H, I332K, I332L, I332M, I332N, I332P,
I332Q, I332R, I332S, I332T, I332V, I332W, I332Y, E333F, E333H,
E333I, E333L, E333M, E333P, E333T, E333Y, K334F, K334I, K334P,
K334T, T335D, T335F, T335G, T335H, T335I, T335L, T335M, T335N,
T335P, T335R, T335S, T335V, T335W, T335Y, I336E, I336K, I336Y,
S337E, S337H, and S337N, wherein the numbering of the residues in
the Fc region is that of the EU index as in Kabat.
[0105] The IgG or Fc variants described herein are defined
according to the amino acid modifications that compose them. Thus,
for example, I332E is an Fc variant with the substitution I332E
relative to the parent Fc polypeptide. Likewise, S239D/A330L/I332E
(also referred to as 239D/330L/332E) defines an Fc variant with the
substitutions S239D, A330L, and I332E (239D, 330L, and 332E)
relative to the parent Fc polypeptide. It is noted that the order
in which substitutions are provided is arbitrary, that is to say
that, for example, S239D/A330L/I332E is the same Fc variant as
S239D/I332E/A330L, and so on. For all positions discussed in the
present invention, numbering is according to the EU index or EU
numbering scheme (Kabat et al., 1991, Sequences of Proteins of
Immunological Interest, 5th Ed., United States Public Health
Service, National Institutes of Health, Bethesda, incorporated by
reference). The EU index or EU index as in Kabat refers to the
numbering of the EU antibody (Edelman et al., 1969, Proc Natl Acad
Sci USA 63:78-85, incorporated by reference).
[0106] Any other IgG mutants/variants (such as insertions,
deletions, substitutions, truncations or frameshift mutations) that
increase the interactions between the IgG and FcRn and increase
IL-12 production can be used in the compositions and methods
described herein. In some embodiments, the IgG or a variant thereof
or a fragment thereof is mammalian. In some embodiments, the IgG or
a variant thereof or a fragment thereof is human. In some
embodiments, the IgG or a variant thereof or a fragment thereof is
recombinant.
[0107] In some embodiments, fragments of IgG that can be used in
the compositions described herein include wild type Fc fragments
IgG or mutant Fc fragments of IgG, provided such molecules retain
the ability to bind to FcRn and increase IL-12 production.
[0108] In some embodiments, the composition comprising IgG or a
variant thereof or a fragment thereof comprises the variable region
of an FcRn specific antibody or a multimeric form (such as a
triabody) of the variable region. In some embodiments, the
composition comprising IgG or a variant thereof or a fragment
thereof comprises multimerized scFv structures such as
tetrabodies.
[0109] In some embodiments, the compositions comprising IgG or a
variant thereof or a fragment thereof further comprise an antigen
that can be conjugated to or complexed with the IgG or a variant
thereof or a fragment thereof The interaction between FcRn and the
IgG complexed with an antigen or the IgG conjugated to an antigen
results in increased production of IL-12 in response to the
antigen. In various embodiments, the antigen is a tumor antigen, a
microbial antigen, a viral antigen, a parasitic antigen or a
combination thereof. In some embodiments, the antigen is a protein
or a proteomimetic thereof, a peptide or a peptidomimentic thereof,
a lipid or a combination thereof. In some embodiments, a
composition in which the IgG or a variant thereof or a fragment
thereof is complexed to an antigen, the antigen is non-covalently
bound to the IgG or a fragment thereof or a variant thereof. In
some embodiments, a composition in which the IgG or a variant
thereof or a fragment thereof is conjugated to an antigen, the
antigen is covalently bound to the IgG or a fragment thereof or a
variant thereof.
[0110] As used herein, in various embodiments, IL-12 production is
"increased" if IL-12 levels are increased by a statistically
significant amount, for example by at least 10%, at least 20%, at
least 30%, at least 40%, at least 50%, at least 60%, at least 70%,
at least 80%, at least 90%, at least 95%, at least 97%, at least
98%, or more, up to and including at least 100% or more, at least
2-fold, at least 3-fold, at least 4-fold, at least 5-fold, at least
6-fold, at least 7-fold, at least 8-fold, at least 9-fold, at least
10-fold, at least 50-fold, at least 100-fold, or more, in the
presence of an agent or stimulus, relative to the absence of such
an agent or stimulus. The agent or stimulus can be the binding of
FcRn to the IgG or a fragment thereof or a variant thereof so as to
increase IL-12 production. In some embodiments, IgG or a fragment
thereof or a variant thereof can be complexed or conjugated to an
antigen, as described herein.
[0111] The compositions described herein are used to treat,
inhibit, prevent relapse of and/or prevent metastasis of cancer in
a subject in need thereof The methods for treating, inhibiting,
preventing relapse of and/or preventing metastasis of cancer in the
subject comprise providing a composition comprising IgG or a
variant thereof or a fragment and administering an effective amount
of the composition to the subject. In some embodiments, the IgG
variant comprises a methionine to leucine substitution at position
428 and an asparagine to serine substitution at position 434
according to the Kabat numbering scheme. The compositions can
further comprise an antigen conjugated or complexed with the IgG or
a variant thereof or a fragment. In some embodiments, the IgG or a
variant thereof or a fragment thereof is mammalian. In some
embodiments, the IgG or a variant thereof or a fragment thereof is
human. In some embodiments, the IgG or a variant thereof or a
fragment thereof is recombinant. In some embodiments, the methods
for treating, inhibiting, preventing relapse of and/or preventing
metastasis of cancer in the subject further comprise administering
to the subject a chemotherapeutic aigggent and/or radiation
therapy, concurrently or sequentially with the composition.
[0112] Examples of cancer include but are not limited to,
carcinoma, lymphoma, blastoma, sarcoma, and leukemia. More
particular examples of such cancers include, but are not limited
to, basal cell carcinoma, biliary tract cancer; bladder cancer;
bone cancer; brain and CNS cancer; breast cancer; cancer of the
peritoneum; cervical cancer; choriocarcinoma; colon and rectum
cancer; connective tissue cancer; cancer of the digestive system;
endometrial cancer; esophageal cancer; eye cancer; cancer of the
head and neck; gastric cancer (including gastrointestinal cancer);
glioblastoma; hepatic carcinoma; hepatoma; intra-epithelial
neoplasm; kidney or renal cancer; larynx cancer; leukemia; liver
cancer; lung cancer (e.g., small-cell lung cancer, non-small cell
lung cancer, adenocarcinoma of the lung, and squamous carcinoma of
the lung); lymphoma including Hodgkin's and non-Hodgkin's lymphoma;
melanoma; myeloma; neuroblastoma; oral cavity cancer (e.g., lip,
tongue, mouth, and pharynx); ovarian cancer; pancreatic cancer;
prostate cancer; retinoblastoma; rhabdomyosarcoma; rectal cancer;
cancer of the respiratory system; salivary gland carcinoma;
sarcoma; skin cancer; squamous cell cancer; stomach cancer;
testicular cancer; thyroid cancer; uterine or endometrial cancer;
cancer of the urinary system; vulval cancer; as well as other
carcinomas and sarcomas; as well as B-cell lymphoma (including low
grade/follicular non-Hodgkin's lymphoma (NHL); small lymphocytic
(SL) NHL; intermediate grade/follicular NHL; intermediate grade
diffuse NHL; high grade immunoblastic NHL; high grade lymphoblastic
NHL; high grade small non-cleaved cell NHL; bulky disease NHL;
mantle cell lymphoma; AIDS-related lymphoma; and Waldenstrom's
Macroglobulinemia); chronic lymphocytic leukemia (CLL); acute
lymphoblastic leukemia (ALL); Hairy cell leukemia; chronic
myeloblastic leukemia; and post-transplant lymphoproliferative
disorder (PTLD), as well as abnormal vascular proliferation
associated with phakomatoses, edema (such as that associated with
brain tumors), and Meigs' syndrome.
[0113] In some embodiments described herein, the methods further
comprise administering a tumor or cancer antigen to a subject being
administered the composition for increasing IgG and FcRn
interactions described herein. In exemplary embodiments,
tumor-specific antigens that can be conjugated or complexed with
IgG or a variant thereof or a fragment thereof include, but are not
limited to, any one or more of 4-1BB, 5T4, adenocarcinoma antigen,
alpha-fetoprotein, BAFF, B-lymphoma cell, C242 antigen, CA-125,
carbonic anhydrase 9 (CA-IX), C-MET, CCR4, CD152, CD19, CD20,
CD200, CD22, CD221, CD23 (IgE receptor), CD28, CD30 (TNFRSF8),
CD33, CD4, CD40, CD44 v6, CD51, CD52, CD56, CD74, CD80, CEA,
CNT0888, CTLA-4, DRS, EGFR, EpCAM, CD3, FAP, fibronectin extra
domain-B, folate receptor 1, GD2, GD3 ganglioside, glycoprotein 75,
GPNMB, HER2/neu, HGF, human scatter factor receptor kinase, IGF-1
receptor, IGF-I, IgG1, L1-CAM, IL-13, IL-6, insulin-like growth
factor I receptor, integrin .alpha.5.beta.1, integrin
.alpha.v.beta.3, MORAb-009, MS4A1, MUC1, mucin CanAg,
N-glycolylneuraminic acid, NPC-1C, PDGF-R .alpha., PDL192,
phosphatidylserine, prostatic carcinoma cells, RANKL, RON, ROR1,
SCH 900105, SDC1, SLAMF7, TAG-72, tenascin C, TGF beta 2,
TGF-.beta., TRAIL-R1, TRAIL-R2, tumor antigen CTAA16.88, VEGF-A,
VEGFR-1, VEGFR2 or vimentin. Other antigens specific for cancer
will be apparent to those of skill in the art and can be used in
connection with alternate embodiments of the invention.
[0114] In some embodiments of the methods described herein, the
methods further comprise administering a chemotherapeutic agent to
the subject being administered a composition for increasing IgG and
FcRn interactions. Non-limiting examples of chemotherapeutic agents
can include alkylating agents such as thiotepa and CYTOXAN.RTM.
cyclosphosphamide; alkyl sulfonates such as busulfan, improsulfan
and piposulfan; aziridines such as benzodopa, carboquone,
meturedopa, and uredopa; ethylenimines and methylamelamines
including altretamine, triethylenemelamine,
trietylenephosphoramide, triethiylenethiophosphoramide and
trimethylolomelamine; acetogenins (especially bullatacin and
bullatacinone); a camptothecin (including the synthetic analogue
topotecan); bryostatin; callystatin; CC-1065 (including its
adozelesin, carzelesin and bizelesin synthetic analogues);
cryptophycins (particularly cryptophycin 1 and cryptophycin 8);
dolastatin; duocarmycin (including the synthetic analogues, KW-2189
and CB1-TM1); eleutherobin; pancratistatin; a sarcodictyin;
spongistatin; nitrogen mustards such as chlorambucil,
chlornaphazine, cholophosphamide, estramustine, ifosfamide,
mechlorethamine, mechlorethamine oxide hydrochloride, melphalan,
novembichin, phenesterine, prednimustine, trofosfamide, uracil
mustard; nitrosureas such as carmustine, chlorozotocin,
fotemustine, lomustine, nimustine, and ranimnustine; antibiotics
such as the enediyne antibiotics (e.g., calicheamicin, especially
calicheamicin gamma1I and calicheamicin omegaI1 (see, e.g., Agnew,
Chem. Intl. Ed. Engl., 33: 183-186 (1994)); dynemicin, including
dynemicin A; bisphosphonates, such as clodronate; an esperamicin;
as well as neocarzinostatin chromophore and related chromoprotein
enediyne antiobiotic chromophores), aclacinomysins, actinomycin,
authramycin, azaserine, bleomycins, cactinomycin, carabicin,
caminomycin, carzinophilin, chromomycinis, dactinomycin,
daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine,
ADRIAMYCIN.RTM. doxorubicin (including morpholino-doxorubicin,
cyanomorpholino-doxorubicin, 2-pyrrolino-doxorubicin and
deoxydoxorubicin), epirubicin, esorubicin, idarubicin,
marcellomycin, mitomycins such as mitomycin C, mycophenolic acid,
nogalamycin, olivomycins, peplomycin, potfiromycin, puromycin,
quelamycin, rodorubicin, streptonigrin, streptozocin, tubercidin,
ubenimex, zinostatin, zorubicin; anti-metabolites such as
methotrexate and 5-fluorouracil (5-FU); folic acid analogues such
as denopterin, methotrexate, pteropterin, trimetrexate; purine
analogs such as fludarabine, 6-mercaptopurine, thiamiprine,
thioguanine; pyrimidine analogs such as ancitabine, azacitidine,
6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine,
enocitabine, floxuridine; androgens such as calusterone,
dromostanolone propionate, epitiostanol, mepitiostane,
testolactone; anti-adrenals such as aminoglutethimide, mitotane,
trilostane; folic acid replenisher such as frolinic acid;
aceglatone; aldophosphamide glycoside; aminolevulinic acid;
eniluracil; amsacrine; bestrabucil; bisantrene; edatraxate;
defofamine; demecolcine; diaziquone; elformithine; elliptinium
acetate; an epothilone; etoglucid; gallium nitrate; hydroxyurea;
lentinan; lonidainine; maytansinoids such as maytansine and
ansamitocins; mitoguazone; mitoxantrone; mopidanmol; nitraerine;
pentostatin; phenamet; pirarubicin; losoxantrone; podophyllinic
acid; 2-ethylhydrazide; procarbazine; PSK.RTM. polysaccharide
complex (JHS Natural Products, Eugene, Oreg.); razoxane; rhizoxin;
sizofuran; spirogermanium; tenuazonic acid; triaziquone;
2,2',2''-trichlorotriethylamine; trichothecenes (especially T-2
toxin, verracurin A, roridin A and anguidine); urethan; vindesine;
dacarbazine; mannomustine; mitobronitol; mitolactol; pipobroman;
gacytosine; arabinoside ("Ara-C"); cyclophosphamide; thiotepa;
taxoids, e.g., TAXOL.RTM. paclitaxel (Bristol-Myers Squibb
Oncology, Princeton, N.J.), ABRAXANE.RTM. Cremophor-free,
albumin-engineered nanoparticle formulation of paclitaxel (American
Pharmaceutical Partners, Schaumberg, Ill.), and TAXOTERE.RTM.
doxetaxel (Rhone-Poulenc Rorer, Antony, France); chloranbucil;
GEMZAR.RTM. gemcitabine; 6-thioguanine; mercaptopurine;
methotrexate; platinum analogs such as cisplatin, oxaliplatin and
carboplatin; vinblastine; platinum; etoposide (VP-16); ifosfamide;
mitoxantrone; vincristine; NAVELBINE; vinorelbine; novantrone;
teniposide; edatrexate; daunomycin; aminopterin; xeloda;
ibandronate; irinotecan (Camptosar, CPT-11) (including the
treatment regimen of irinotecan with 5-FU and leucovorin);
topoisomerase inhibitor RFS 2000; difluoromethylornithine (DMFO);
retinoids such as retinoic acid; capecitabine; combretastatin;
leucovorin (LV); oxaliplatin, including the oxaliplatin treatment
regimen (FOLFOX); lapatinib (Tykerb); inhibitors of PKC-alpha, Raf,
H-Ras, EGFR (e.g., erlotinib (Tarceva.RTM.)) and VEGF-A that reduce
cell proliferation and pharmaceutically acceptable salts, acids or
derivatives of any of the above. In addition, the methods of
treatment can further include the use of radiation therapy.
[0115] The compositions described herein are also used to treat,
inhibit and/or reduce the severity of infectious diseases in a
subject in need thereof The methods for treating, inhibiting and/or
reducing the severity of infectious diseases in the subject include
providing a composition comprising IgG or a variant thereof or a
fragment and administering an effective amount of the composition
to the subject. The compositions can further comprise an antigen
conjugated or complexed with the IgG or a variant thereof or a
fragment, as described herein. In some embodiments, the IgG or a
variant thereof or a fragment thereof is mammalian. In some
embodiments, the IgG or a variant thereof or a fragment thereof is
human. In some embodiments, the IgG or a variant thereof or a
fragment thereof is recombinant.
[0116] In various embodiments, bacterial antigens can be any
antigen present in infectious bacteria and that induce an immune
response in a subject. Examples of infectious bacteria include:
Helicobacterpyloris, Borelia burgdorferi, Legionella pneumophilia,
Mycobacteria sps (such as M. tuberculosis, M. avium, M.
intracellulare, M. kansaii, M. gordonae), Staphylococcus aureus,
Neisseria gonorrhoeae, Neisseria meningitidis, Listeria
monocytogenes, Streptococcus pyogenes (Group A Streptococcus),
Streptococcus agalactiae (Group B Streptococcus), Streptococcus
(viridans group), Streptococcus faecalis, Streptococcus bovis,
Streptococcus (anaerobic sps.), Streptococcus pneumoniae,
pathogenic Campylobacter sp., Enterococcus sp., Haemophilus
influenzae, Bacillus anthracia, corynebacterium diphtheriae,
corynebacterium sp., Erysipelothrix rhusiopathiae, Clostridium
perfringens, Clostridium tetani, Enterobacter aerogenes, Klebsiella
pneumoniae, Pasturella multocida, Bacteroides sp., Fusobacterium
nucleatum, Streptobacillus moniliformis, Treponema pallidium,
Treponema pertenue, Leptospira, and Actinomyces Israelli. The
compositions and methods described herein are contemplated for use
in treating infections with these bacterial agents. Other
infectious organisms (such as protists) include: Plasmodium
falciparum and Toxoplasma gondii. The compositions and methods
described herein are contemplated for use in treating infections
with these agents.
[0117] In various embodiments, viral antigens can be any antigens
present in infectious viruses and that induce an immune response in
a subject. Examples of infectious viruses include: Retroviridae
(for example, HIV); Picornaviridae (for example, polio viruses,
hepatitis A virus; enteroviruses, human coxsackie viruses,
rhinoviruses, echoviruses); Calciviridae (such as strains that
cause gastroenteritis); Togaviridae (for example, equine
encephalitis viruses, rubella viruses); Flaviridae (for example,
dengue viruses, encephalitis viruses, yellow fever viruses);
Coronaviridae (for example, coronaviruses); Rhabdoviridae (for
example, vesicular stomatitis viruses, rabies viruses); Filoviridae
(for example, ebola viruses); Paramyxoviridae (for example,
parainfluenza viruses, mumps virus, measles virus, respiratory
syncytial virus); Orthomyxoviridae (for example, influenza
viruses); Bungaviridae (for example, Hantaan viruses, bunga
viruses, phleboviruses and Nairo viruses); Arena viridae
(hemorrhagic fever viruses); Reoviridae (e.g., reoviruses,
orbiviurses and rotaviruses); Birnaviridae; Hepadnaviridae
(Hepatitis B virus); Parvoviridae (parvoviruses); Papovaviridae
(papilloma viruses, polyoma viruses); Adenoviridae (most
adenoviruses); Herpesviridae (herpes simplex virus (HSV) 1 and
HSV-2, varicella zoster virus, cytomegalovirus (CMV), herpes
viruses); Poxviridae (variola viruses, vaccinia viruses, pox
viruses); and Iridoviridae (such as African swine fever virus); and
unclassified viruses (for example, the etiological agents of
Spongiform encephalopathies, the agent of delta hepatitis (thought
to be a defective satellite of hepatitis B virus), the agents of
non-A, non-B hepatitis (class 1=internally transmitted; class
2=parenterally transmitted (i.e., Hepatitis C); Norwalk and related
viruses, and astroviruses). The compositions and methods described
herein are contemplated for use in treating infections with these
viral agents.
[0118] Examples of fungal infections that can be treated with the
compositions and methods described herein include but are not
limited to: aspergillosis; thrush (caused by Candida albicans);
cryptococcosis (caused by Cryptococcus); and histoplasmosis. Thus,
examples of infectious fungi include, but are not limited to,
Cryptococcus neoformans, Histoplasma capsulatum, Coccidioides
immitis, Blastomyces dermatitidis, Chlamydia trachomatis, Candida
albicans. The compositions and methods described herein are
contemplated for use in treating infections with these fungal
agents.
[0119] In some embodiments of the aspects described herein, the
methods further comprise administering an effective amount of a
viral, bacterial, fungal, or parasitic antigen in conjunction with
the compositions comprising IgG or a variant thereof or a fragment
thereof. Non-limiting examples of suitable viral antigens include:
influenza HA, NA, M, NP and NS antigens; HIV p24, pol, gp41 and
gp120; Metapneumovirus (hMNV) F and G proteins; Hepatitis C virus
(HCV) E1, E2 and core proteins; Dengue virus (DEN1-4) E1, E2 and
core proteins; Human Papilloma Virus L1 protein; Epstein Barr Virus
gp220/350 and EBNA-3A peptide; Cytomegalovirus (CMV) gB
glycoprotein, gH glycoprotein, pp65, IE1 (exon 4) and pp 150;
Varicella Zoster virus (VZV) 1E62 peptide and glycoprotein E
epitopes; Herpes Simplex Virus Glycoprotein D epitopes, among many
others. The antigenic polypeptides can correspond to polypeptides
of naturally occurring animal or human viral isolates, or can be
engineered to incorporate one or more amino acid substitutions as
compared to a natural (pathogenic or non-pathogenic) isolate.
Compositions and Methods for Treating Autoimmune Diseases
[0120] In some aspects, described herein are compositions for
decreasing/down-regulating production of IL-12 by altering or
inhibiting interactions between IgG and FcRn. The compositions
comprise agents that inhibit (reduce or block) signaling mediated
by interaction between FcRn and IgG. Such agents include, but are
not limited to, antibodies ("antibodies" includes antigen-binding
portions of antibodies such as epitope- or antigen-binding
peptides, paratopes, functional CDRs; recombinant antibodies;
chimeric antibodies; tribodies; midibodies; or antigen-binding
derivatives, analogs, variants, portions, or fragments thereof),
protein-binding agents, small molecules, recombinant protein,
peptides, aptamers, avimers and protein-binding derivatives,
portions or fragments thereof. In some embodiments, the composition
comprises agents that inhibit FcRn-mediated downstream signaling
such as signaling mediated by interactions between, for example,
FcRn and WAVE2, or FcRn and calmodulin.
[0121] Antisense oligonucleotides represent another class of agents
that are useful in the compositions and methods described herein,
particularly as IgG and/or FcRn antagonists. This class of agents
and methods for preparing and using them are all well-known in the
art, as are ribozyme and miRNA molecules. See, e.g., PCT
US2007/024067 for a thorough discussion. Alternatively, an agent
that inhibits interactions between IgG and FcRn can, in some
embodiments of the compositions and methods described herein,
include recombinant Fc or conjugates, or protein or antibody, small
interfering RNA specific for or targeted to FcRn mRNA, and
antisense RNA that hybridizes with the mRNA of FcRn, for
example.
[0122] Other agents for use in the compositions and methods
described herein that inhibit IgG interaction with FcRn include,
for example, antibodies against FcRn, specifically reactive or
specifically binding to FcRn. In some embodiments, the antibody is
a blocking antibody and can be any of a monoclonal antibody or a
fragment thereof, a polyclonal antibody or a fragment thereof, a
chimeric antibody, humanized antibody or a single chain antibody.
In some embodiments, the agent is a bispecific agent comprising
binding sites for IgG and FcRn. In some embodiments, the agent is a
recombinant Fc portion of IgG or a biologically active portion
thereof or a proteo-mimetic thereof. The Fc portion or a
biologically active portion thereof can be mammalian. The Fc
portion or a biologically active portion thereof can be human.
[0123] As used herein, a "blocking" antibody or an antibody
"antagonist" is one which inhibits or reduces the biological
activity of the antigen(s) it binds. For example, a IgG/FcRn
bispecific blocking or antagonist antibody binds IgG and FcRn and
inhibits the ability of IgG and FcRn to induce IL-12 production by
dendritic cells. In certain embodiments, the blocking antibodies or
antagonist antibodies or portions thereof described herein
completely inhibit the interaction between IgG and FcRn. In certain
embodiments, the blocking antibodies or antagonist antibodies or
portions thereof described herein reduce/decrease the interaction
between IgG and FcRn. In an embodiment, the antibody is a
monoclonal antibody that specifically binds FcRn. In an embodiment,
the monoclonal antibody that specifically binds FcRn is DVN24. In
an embodiment, DVN24 is humanized.
[0124] Simple binding assays can be used to screen for or detect
agents that bind to FcRn or IgG, or disrupt the interaction between
a FcRn and IgG. Further, agents that inhibit the FcRn/IgG
interaction for use in the compositions and methods described
herein, including recombinant FcRn or IgG peptido-mimetics, can be
identified by, for example, transfecting cells with expression
vectors expressing FcRn or IgG or portions thereof; contacting the
cells with an agent; lysing the cells; and characterizing the
FcRn/IgG interaction in comparison with cells not contacted with
agent. Cells can be characterized using, for example,
co-immunoprecipitation.
[0125] Another variation of assays to determine binding of a FcRn
protein to a IgG protein is through the use of affinity biosensor
methods. Such methods can be based on the piezoelectric effect,
electrochemistry, or optical methods, such as ellipsometry, optical
wave guidance, and surface plasmon resonance (SPR). For example,
efficacy of an siRNA on the expression of FcRn or IgG can be
monitored using methods known in the art such as quantitative
RT-PCR with specific oligonucleotide primers for each gene
respectively, or ELISA for FcRn and/or IgG from a sample of
peripheral blood. Alternatively, the population of blood cells can
be determined by FACS analysis using the markers characteristic of
particular populations and subpopulations known in the art or
disclosed herein.
[0126] In some aspects, provided herein are methods for suppressing
an immune response in vivo (for example by decreasing IL-12
production), comprising administering to the subject a
therapeutically effective amount of an inhibitor of interaction
between FcRn and IgG, or an inhibitor of the generation of its
downstream signaling, as described herein. Reducing or inhibiting
the interaction between IgG and FcRn is useful for specifically
suppressing an immune response in vivo, which can be useful for the
treatment of conditions related to immune function including
autoimmune disease and transplantation (e.g., bone marrow or
organs). The inhibitors that decrease IgG/FcRn interactions can be
used alone as a primary therapy or in combination with other
therapeutics as a combination therapy to enhance the therapeutic
benefits of other medical treatments.
[0127] In various embodiments, as used hrerein, interaction between
IgG and FcRn is "decreased" if one or more signaling activities or
downstream read-outs of FcRn activity, such as IL-12 production, is
reduced by a statistically significant amount, for example by at
least 10%, at least 20%, at least 30%, at least 40%, at least 50%,
at least 60%, at least 70%, at least 80%, at least 90%, at least
95%, at least 97%, at least 98%, or more, up to and including at
least 100% or more, at least 2-fold, at least 3-fold, at least
4-fold, at least 5-fold, at least 6-fold, at least 7-fold, at least
8-fold, at least 9-fold, at least 10-fold, at least 50-fold, at
least 100-fold, or more, in the presence of an inhibitor, relative
to the absence of such an inhibitor. In various embodiments, the
inhibitor alters FcRn/IgG mediated signaling so as to decrease
IL-12 production.
[0128] In some embodiments of the methods described herein, the
subject being administered an inhibitor for decreasing IgG/FcRn
interactions is diagnosed with, has, or suffers from an autoimmune
disease. Accordingly, provided herein, in some aspects, are methods
of treating a subject having or diagnosed with an autoimmune
disorder comprising administering an effective amount of an agent
for decreasing FcRn/IgG interactions. Also provided herein are
methods for inhibiting or reducing the severity of an autoimmune
disorder in a subject administering an effective amount of an agent
for decreasing FcRn/IgG interactions.
[0129] "Autoimmune disease" refers to a class of diseases in which
a subject's own antibodies react with host tissue or in which
immune effector T cells are autoreactive to endogenous
self-peptides and cause destruction of tissue. Thus an immune
response is mounted against a subject's own antigens, referred to
as self-antigens. A "self-antigen" as used herein refers to an
antigen of a normal host tissue. Normal host tissue does not
include neoplastic cells.
[0130] Accordingly, in some embodiments, the autoimmune diseases to
be treated or prevented using the methods described herein,
include, but are not limited to: rheumatoid arthritis, Crohn's
disease, ulcerative colitis, multiple sclerosis, primary sclerosing
cholangitis, systemic lupus erythematosus (SLE), autoimmune
encephalomyelitis, myasthenia gravis (MG), Hashimoto's thyroiditis,
Goodpasture's syndrome, pemphigus (e.g., pemphigus vulgaris),
Grave's disease, autoimmune hemolytic anemia, autoimmune
thrombocytopenic purpura, scleroderma with anti-collagen
antibodies, mixed connective tissue disease, polymyositis,
pernicious anemia, idiopathic Addison's disease, autoimmune-
associated infertility, Kawasaki's disease, glomerulonephritis
(e.g., crescentic glomerulonephritis, proliferative
glomerulonephritis), bullous pemphigoid, Sjogren's syndrome,
insulin resistance, and autoimmune diabetes mellitus (type 1
diabetes mellitus; insulin-dependent diabetes mellitus). Autoimmune
disease has been recognized also to encompass atherosclerosis and
Alzheimer's disease. In one embodiment of the aspects described
herein, the autoimmune disease is selected from the group
consisting of multiple sclerosis, type-I diabetes, Hashinoto's
thyroiditis, Crohn's disease, rheumatoid arthritis, systemic lupus
erythematosus, gastritis, autoimmune hepatitis, hemolytic anemia,
autoimmune hemophilia, autoimmune lymphoproliferative syndrome
(ALPS), autoimmune uveoretinitis, glomerulonephritis,
Guillain-Barre syndrome, psoriasis and myasthenia gravis.
[0131] The term "effective amount" as used herein refers to the
amount of an agent for modulating (increasing or decreasing) the
interactions between FcRn and IgG or a fragment thereof or a
variant thereof needed to alleviate at least one or more symptom of
the disease or disorder, and relates to a sufficient amount of
pharmacological composition to provide the desired effect, for
example increasing IL-12 production to so as to treat cancer or
infectious diseases, or decreasing IL-12 production to treat
autoimmune diseases. The term "therapeutically effective amount"
therefore refers to an amount of an agent for modulating the
interactions between FcRn and IgG or a fragment thereof or a
variant thereof using the methods as disclosed herein, that is
sufficient to effect a particular effect when administered to a
typical subject. An effective amount as used herein would also
include an amount sufficient to delay the development of a symptom
of the disease, alter the course of a symptom of disease (for
example but not limited to, slow the progression of a symptom of
the disease), or reverse a symptom of disease. Thus, it is not
possible to specify the exact "effective amount". However, for any
given case, an appropriate "effective amount" can be determined by
one of ordinary skill in the art using only routine
experimentation.
[0132] Effective amounts, toxicity, and therapeutic efficacy can be
determined by standard pharmaceutical procedures in cell cultures
or experimental animals, e.g., for determining the LD50 (the dose
lethal to 50% of the population) and the ED50 (the dose
therapeutically effective in 50% of the population). The dosage can
vary depending upon the dosage form employed and the route of
administration utilized. The dose ratio between toxic and
therapeutic effects is the therapeutic index and can be expressed
as the ratio LD50/ED50. Compositions and methods that exhibit large
therapeutic indices are preferred. A therapeutically effective dose
can be estimated initially from cell culture assays. Also, a dose
can be formulated in animal models to achieve a circulating plasma
concentration range that includes the IC50 (i.e., the concentration
of the agent for modulating interactions between FcRn and IgG or a
fragment thereof or a variant thereof, which achieves a
half-maximal inhibition of symptoms) as determined in cell culture,
or in an appropriate animal model. Levels in plasma can be
measured, for example, by high performance liquid chromatography.
The effects of any particular dosage can be monitored by a suitable
bioassay. The dosage can be determined by a physician and adjusted,
as necessary, to suit observed effects of the treatment.
[0133] The agents useful according to the compositions and methods
described herein, including antibodies and other polypeptides, are
isolated agents, meaning that the agents are substantially pure and
are essentially free of other substances with which they may be
found in nature or in vivo systems to an extent practical and
appropriate for their intended use. In particular, the agents are
sufficiently pure and are sufficiently free from other biological
constituents of their host cells so as to be useful in, for
example, producing pharmaceutical preparations. Because an isolated
agent may be admixed with a pharmaceutically acceptable carrier in
a pharmaceutical preparation, the agents may comprise only a small
percentage by weight of the preparation.
[0134] The agents described herein for modulating the interactions
between FcRn and IgG or a fragment thereof or a variant thereof can
be administered to a subject in need thereof by any appropriate
route which results in an effective treatment in the subject. As
used herein, the terms "administering," and "introducing" are used
interchangeably and refer to the placement of a polypeptide agent
into a subject by a method or route which results in at least
partial localization of such agents at a desired site, such as a
site of inflammation, such that a desired effect(s) is
produced.
[0135] In some embodiments, the agents described herein for
modulating the interactions between FcRn and IgG or a fragment
thereof or a variant thereof are administered to a subject by any
mode of administration that delivers the agent systemically or to a
desired surface or target, and can include, but is not limited to,
injection, infusion, instillation, and inhalation administration.
To the extent that polypeptide agents can be protected from
inactivation in the gut, oral administration forms are also
contemplated. "Injection" includes, without limitation,
intravenous, intramuscular, intraarterial, intrathecal,
intraventricular, intracapsular, intraorbital, intracardiac,
intradermal, intraperitoneal, transtracheal, subcutaneous,
subcuticular, intraarticular, sub capsular, subarachnoid,
intraspinal, intracerebro spinal, and intrasternal injection and
infusion. In preferred embodiments, the agents for modulating
interactions between FcRn and IgG or a fragment thereof or a
variant thereof for use in the methods described herein are
administered by intravenous infusion or injection.
[0136] The phrases "parenteral administration" and "administered
parenterally" as used herein, refer to modes of administration
other than enteral and topical administration, usually by
injection. The phrases "systemic administration," "administered
systemically", "peripheral administration" and "administered
peripherally" as used herein refer to the administration of an
agent for modulating interactions between FcRn and IgG or a
fragment thereof or a variant thereof other than directly into a
target site, tissue, or organ, such as a tumor site, such that it
enters the subject's circulatory system and, thus, is subject to
metabolism and other like processes.
[0137] For the clinical use of the methods described herein,
administration of an agent for modulating interactions between FcRn
and IgG or a fragment thereof or a variant thereof can include
formulation into pharmaceutical compositions or pharmaceutical
formulations for parenteral administration, e.g., intravenous;
mucosal, e.g., intranasal; ocular, or other mode of administration.
In some embodiments, an agent for modulating interactions between
FcRn and IgG or a fragment thereof or a variant thereof described
herein can be administered along with any pharmaceutically
acceptable carrier compound, material, or composition which results
in an effective treatment in the subject. Thus, a pharmaceutical
formulation for use in the methods described herein can contain an
agent for modulating interactions between FcRn and IgG or a
fragment thereof or a variant thereof as described herein in
combination with one or more pharmaceutically acceptable
ingredients.
[0138] The phrase "pharmaceutically acceptable" refers to those
compounds, materials, compositions, and/or dosage forms which are,
within the scope of sound medical judgment, suitable for use in
contact with the tissues of human beings and animals without
excessive toxicity, irritation, allergic response, or other problem
or complication, commensurate with a reasonable benefit/risk ratio.
The phrase "pharmaceutically acceptable carrier" as used herein
means a pharmaceutically acceptable material, composition or
vehicle, such as a liquid or solid filler, diluent, excipient,
solvent, media, encapsulating material, manufacturing aid (e.g.,
lubricant, talc magnesium, calcium or zinc stearate, or steric
acid), or solvent encapsulating material, involved in maintaining
the stability, solubility, or activity of, an agent for modulating
interactions between FcRn and IgG or a fragment thereof or a
variant thereof. Each carrier must be "acceptable" in the sense of
being compatible with the other ingredients of the formulation and
not injurious to the patient. Some examples of materials which can
serve as pharmaceutically-acceptable carriers include: (1) sugars,
such as lactose, glucose and sucrose; (2) starches, such as corn
starch and potato starch; (3) cellulose, and its derivatives, such
as sodium carboxymethyl cellulose, methylcellulose, ethyl
cellulose, microcrystalline cellulose and cellulose acetate; (4)
powdered tragacanth; (5) malt; (6) gelatin; (7) excipients, such as
cocoa butter and suppository waxes; (8) oils, such as peanut oil,
cottonseed oil, safflower oil, sesame oil, olive oil, corn oil and
soybean oil; (9) glycols, such as propylene glycol; (10) polyols,
such as glycerin, sorbitol, mannitol and polyethylene glycol (PEG);
(11) esters, such as ethyl oleate and ethyl laurate; (12) agar;
(13) buffering agents, such as magnesium hydroxide and aluminum
hydroxide; (14) alginic acid; (15) pyrogen-free water; (16)
isotonic saline; (17) Ringer's solution; (19) pH buffered
solutions; (20) polyesters, polycarbonates and/or polyanhydrides;
(21) bulking agents, such as polypeptides and amino acids (22)
serum components, such as serum albumin, HDL and LDL; (23) C2-C12
alchols, such as ethanol; and (24) other non-toxic compatible
substances employed in pharmaceutical formulations. Release agents,
coating agents, preservatives, and antioxidants can also be present
in the formulation. The terms such as "excipient", "carrier",
"pharmaceutically acceptable carrier" or the like are used
interchangeably herein.
[0139] The agents for modulating (increasing or decreasing)
interactions between FcRn and IgG or a fragment thereof or a
variant thereof described herein can be specially formulated for
administration of the compound to a subject in solid, liquid or gel
form, including those adapted for the following: (1) parenteral
administration, for example, by subcutaneous, intramuscular,
intravenous or epidural injection as, for example, a sterile
solution or suspension, or sustained-release formulation; (2)
topical application, for example, as a cream, ointment, or a
controlled-release patch or spray applied to the skin; (3)
intravaginally or intrarectally, for example, as a pessary, cream
or foam; (4) ocularly; (5) transdermally; (6) transmucosally; or
(79) nasally. Additionally, a bispecific or multispecific
polypeptide agent can be implanted into a patient or injected using
a drug delivery system. See, for example, Urquhart, et al., Ann.
Rev. Pharmacol. Toxicol. 24: 199-236 (1984); Lewis, ed. "Controlled
Release of Pesticides and Pharmaceuticals" (Plenum Press, New York,
1981); U.S. Pat. Nos. 3,773,919; and 35 3,270,960.
[0140] Further embodiments of the formulations and modes of
administration of an agent for modulating interactions between FcRn
and IgG or a fragment thereof or a variant thereof that can be used
in the methods described herein are illustrated below.
[0141] Parenteral Dosage Forms. Parenteral dosage forms of an agent
for modulating (increasing or decreasing) interactions between FcRn
and IgG or a fragment thereof or a variant thereof can also be
administered to a subject by various routes, including, but not
limited to, subcutaneous, intravenous (including bolus injection),
intramuscular, and intraarterial. Since administration of
parenteral dosage forms typically bypasses the patient's natural
defenses against contaminants, parenteral dosage forms are
preferably sterile or capable of being sterilized prior to
administration to a patient. Examples of parenteral dosage forms
include, but are not limited to, solutions ready for injection, dry
products ready to be dissolved or suspended in a pharmaceutically
acceptable vehicle for injection, suspensions ready for injection,
controlled-release parenteral dosage forms, and emulsions.
[0142] Suitable vehicles that can be used to provide parenteral
dosage forms of the disclosure are well known to those skilled in
the art. Examples include, without limitation: sterile water; water
for injection USP; saline solution; glucose solution; aqueous
vehicles such as but not limited to, sodium chloride injection,
Ringer's injection, dextrose Injection, dextrose and sodium
chloride injection, and lactated Ringer's injection; water-miscible
vehicles such as, but not limited to, ethyl alcohol, polyethylene
glycol, and propylene glycol; and non-aqueous vehicles such as, but
not limited to, corn oil, cottonseed oil, peanut oil, sesame oil,
ethyl oleate, isopropyl myristate, and benzyl benzoate.
[0143] Aerosol formulations. An agent for modulating (increasing or
decreasing) interactions between FcRn and IgG or a fragment thereof
or a variant thereof can be packaged in a pressurized aerosol
container together with suitable propellants, for example,
hydrocarbon propellants like propane, butane, or isobutane with
conventional adjuvants. An agent for modulating interactions
between FcRn and IgG or a fragment thereof or a variant thereof
interactions can also be administered in a non-pressurized form
such as in a nebulizer or atomizer. An agent for modulating
interactions between FcRn and IgG or a fragment thereof or a
variant thereof can also be administered directly to the airways in
the form of a dry powder, for example, by use of an inhaler.
[0144] Suitable powder compositions include, by way of
illustration, powdered preparations of an agent for modulating
interactions between FcRn and IgG or a fragment thereof or a
variant thereof thoroughly intermixed with lactose, or other inert
powders acceptable for intrabronchial administration. The powder
compositions can be administered via an aerosol dispenser or
encased in a breakable capsule which can be inserted by the subject
into a device that punctures the capsule and blows the powder out
in a steady stream suitable for inhalation. The compositions can
include propellants, surfactants, and co-solvents and can be filled
into conventional aerosol containers that are closed by a suitable
metering valve.
[0145] Aerosols for the delivery to the respiratory tract are known
in the art. See for example, Adjei, A. and Garren, J. Pharm. Res.,
1: 565-569 (1990); Zanen, P. and Lamm, J.-W. J. Int. J. Pharm.,
114: 111-115 (1995); Gonda, I. "Aerosols for delivery of
therapeutic an diagnostic agents to the respiratory tract," in
Critical Reviews in Therapeutic Drug Carrier Systems, 6:273-313
(1990); Anderson et al., Am. Rev. Respir. Dis., 140: 1317-1324
(1989)) and have potential for the systemic delivery of peptides
and proteins as well (Patton and Platz, Advanced Drug Delivery
Reviews, 8:179-196 (1992)); Timsina et. al., Int. J. Pharm., 101:
1-13 (1995); and Tansey, I. P., Spray Technol. Market, 4:26-29
(1994); French, D. L., Edwards, D. A. and Niven, R. W., Aerosol
Sci., 27: 769-783 (1996); Visser, J., Powder Technology 58: 1-10
(1989)); Rudt, S. and R. H. Muller, J. Controlled Release, 22:
263-272 (1992); Tabata, Y, and Y. Ikada, Biomed. Mater. Res., 22:
837-858 (1988); Wall, D. A., Drug Delivery, 2: 10 1-20 1995);
Patton, J. and Platz, R., Adv. Drug Del. Rev., 8: 179-196 (1992);
Bryon, P., Adv. Drug. Del. Rev., 5: 107-132 (1990); Patton, J. S.,
et al., Controlled Release, 28: 15 79-85 (1994); Damms, B. and
Bains, W., Nature Biotechnology (1996); Niven, R. W., et al.,
Pharm. Res., 12(9); 1343-1349 (1995); and Kobayashi, S., et al.,
Pharm. Res., 13(1): 80-83 (1996), contents of all of which are
herein incorporated by reference in their entirety.
[0146] The formulations of the agents for modulating (increasing or
decreasing) interactions between FcRn and IgG or a fragment thereof
or a variant thereof described herein further encompass anhydrous
pharmaceutical compositions and dosage forms comprising the
disclosed compounds as active ingredients, since water can
facilitate the degradation of some compounds. For example, the
addition of water (e.g., 5%) is widely accepted in the
pharmaceutical arts as a means of simulating long-term storage in
order to determine characteristics such as shelf life or the
stability of formulations over time. See, e.g., Jens T. Carstensen,
Drug Stability: Principles & Practice, 379-80 (2nd ed., Marcel
Dekker, NY, N.Y.: 1995). Anhydrous pharmaceutical compositions and
dosage forms of the disclosure can be prepared using anhydrous or
low moisture containing ingredients and low moisture or low
humidity conditions. Pharmaceutical compositions and dosage forms
that comprise lactose and at least one active ingredient that
comprise a primary or secondary amine are preferably anhydrous if
substantial contact with moisture and/or humidity during
manufacturing, packaging, and/or storage is expected. Anhydrous
compositions are preferably packaged using materials known to
prevent exposure to water such that they can be included in
suitable formulary kits. Examples of suitable packaging include,
but are not limited to, hermetically sealed foils, plastics, unit
dose containers (e.g., vials) with or without desiccants, blister
packs, and strip packs.
[0147] Controlled and Delayed Release Dosage Forms. In some
embodiments of the methods described herein, an agent for
modulating (increasing or decreasing) interactions between FcRn and
IgG or a fragment thereof or a variant thereof can be administered
to a subject by controlled- or delayed-release means. Ideally, the
use of an optimally designed controlled-release preparation in
medical treatment is characterized by a minimum of drug substance
being employed to cure or control the condition in a minimum amount
of time. Advantages of controlled-release formulations include: 1)
extended activity of the drug; 2) reduced dosage frequency; 3)
increased patient compliance; 4) usage of less total drug; 5)
reduction in local or systemic side effects; 6) minimization of
drug accumulation; 7) reduction in blood level fluctuations; 8)
improvement in efficacy of treatment; 9) reduction of potentiation
or loss of drug activity; and 10) improvement in speed of control
of diseases or conditions. (Kim, Cherng-ju, Controlled Release
Dosage Form Design, 2 (Technomic Publishing, Lancaster, Pa.:
2000)). Controlled-release formulations can be used to control a
compound'sonset of action, duration of action, plasma levels within
the therapeutic window, and peak blood levels. In particular,
controlled- or extended-release dosage forms or formulations can be
used to ensure that the maximum effectiveness of a compound of
formula (I) is achieved while minimizing potential adverse effects
and safety concerns, which can occur both from under-dosing a drug
(i.e., going below the minimum therapeutic levels) as well as
exceeding the toxicity level for the drug.
[0148] A variety of known controlled- or extended-release dosage
forms, formulations, and devices can be adapted for use with the
agents for modulating interactions between FcRn and IgG or a
fragment thereof or a variant thereof described herein. Examples
include, but are not limited to, those described in U.S. Pat. Nos.
3,845,770; 3,916,899; 3,536,809; 3,598,123; 4,008,719; 5674,533;
5,059,595; 5,591 ,767; 5,120,548; 5,073,543; 5,639,476; 5,354,556;
5,733,566; and 6,365,185 B1, each of which is incorporated herein
by reference in their entireties. These dosage forms can be used to
provide slow or controlled-release of one or more active
ingredients using, for example, hydroxypropylmethyl cellulose,
other polymer matrices, gels, permeable membranes, osmotic systems
(such as OROS.RTM. (Alza Corporation, Mountain View, Calif. USA)),
multilayer coatings, microparticles, liposomes, or microspheres or
a combination thereof to provide the desired release profile in
varying proportions. Additionally, ion exchange materials can be
used to prepare immobilized, adsorbed salt forms of the disclosed
compounds and thus effect controlled delivery of the drug. Examples
of specific anion exchangers include, but are not limited to,
Duolite.RTM. A568 and Duolite.RTM. AP143 (Rohm&Haas, Spring
House, Pa. USA).
[0149] In some embodiments, an agent for modulating (increasing or
decreasing) interactions between FcRn and IgG or a fragment thereof
or a variant thereof for use in the methods described herein is
administered to a subject by sustained release or in pulses. Pulse
therapy is not a form of discontinuous administration of the same
amount of a composition over time, but comprises administration of
the same dose of the composition at a reduced frequency or
administration of reduced doses. Sustained release or pulse
administrations are particularly preferred when the disorder occurs
continuously in the subject, for example where the subject has
continuous or chronic symptoms of a viral infection. Each pulse
dose can be reduced and the total amount of the agent for
modulating interactions between FcRn and IgG or a fragment thereof
or a variant thereof administered over the course of treatment to
the patient is minimized.
[0150] The interval between pulses, when necessary, can be
determined by one of ordinary skill in the art. Often, the interval
between pulses can be calculated by administering another dose of
the composition when the composition or the active component of the
composition is no longer detectable in the subject prior to
delivery of the next pulse. Intervals can also be calculated from
the in vivo half-life of the composition. Intervals can be
calculated as greater than the in vivo half-life, or 2, 3, 4, 5 and
even 10 times greater the composition half-life. Various methods
and apparatus for pulsing compositions by infusion or other forms
of delivery to the patient are disclosed in U.S. Pat. Nos.
4,747,825; 4,723,958; 4,948,592; 4,965,251 and 5,403,590.
Assays for Assessing Efficacy of Therapeutic Agents
[0151] Provided herein are methods for determining the efficacy of
a treatment in a subject in need thereof. The method includes
providing a sample from a subject, assaying the sample for levels
of any one or more of IL-12, TNF-.alpha., IFN-.gamma., GM-CSF,
IL-3, IL-2, granzyme B, Tbet or a combination thereof and
determining whether the treatment is efficacious.
[0152] In one embodiment, the subject is diagnosed with cancer or
an infectious disease and is receiving or has received a treatment
that includes a composition comprising immunoglobulin G (IgG) or a
variant thereof or a fragment thereof. In the subject receiving a
composition comprising immunoglobulin G (IgG) or a variant thereof
or a fragment thereof, the treatment is determined to be
efficacious if the levels of any one or more of IL-12, TNF-.alpha.,
IFN-.gamma., GM-CSF, IL-3, IL-2, granzyme B, Tbet or a combination
thereof in the sample from the subject is higher relative to the
levels in a reference sample. In the subject receiving a
composition comprising immunoglobulin G (IgG) or a variant thereof
or a fragment thereof, the treatment is determined to be not
efficacious if the levels of any one or more of IL-12, TNF-.alpha.,
IFN-.gamma., GM-CSF, IL-3, IL-2, granzyme B, Tbet or a combination
in the sample from the subject is lower relative to the levels in a
reference sample
[0153] In one embodiment, the subject is diagnosed with an
autoimmune disease and is receiving or has received a treatment
that includes a composition comprising an agent that inhibits
signaling mediated by interaction between FcRn and IgG. In the
subject receiving the composition comprising an agent that inhibits
signaling mediated by interaction between FcRn and IgG, the
treatment is determined to be efficacious if the levels of any one
or more of IL-12, TNF-.alpha., IFN-.gamma., GM-CSF, IL-3, IL-2,
granzyme B, Tbet or a combination in the sample from the subject is
lower relative to the levels in a reference sample. In the subject
receiving the composition comprising an agent that inhibits
signaling mediated by interaction between FcRn and IgG, the
treatment is determined to be not efficacious if the levels of any
one or more of IL-12, TNF-.alpha., IFN-.gamma., GM-CSF, IL-3, IL-2,
granzyme B, Tbet or a combination in the sample from the subject is
higher relative to the levels in a reference sample.
[0154] In various embodiments, the sample is blood, plasma or
tissue.
[0155] In various embodiments, the methods for assaying the levels
of IL-12, TNF-.alpha., IFN-.gamma., GM-CSF, IL-3, IL-2, granzyme B,
or Tbet in a sample will be apparent to a person of skill in the
art. For example, specific antibodies may be used to detect the
presence of one or more proteins of interest. Any suitable
immunoassay method may be utilized, including those which are
commercially available, to ascertain the presence of, and
optionally quantify the amount of, the protein of interest present
in the sample. In various embodiments, the antibody is any one or
more of a monoclonal antibody or fragment thereof, a polyclonal
antibody or a fragment thereof, chimeric antibodies, humanized
antibodies, human antibodies, and a single chain antibody.
Extensive discussion of the known immunoassay techniques is not
required here since these are known to those of skill in the art.
Typical suitable immunoassay techniques include Western blots,
sandwich enzyme-linked immunoassays (ELISA), radioimmunoassays
(RIA), competitive binding assays, homogeneous assays,
heterogeneous assays, etc. Various known immunoassay methods are
reviewed, e.g., in Methods in Enzymology, 70, pp. 30-70 and 166-198
(1980).
[0156] Further, "sandwich-type" assays may be used with the methods
described herein. Some examples of sandwich-type assays are
described in U.S. Pat. Nos. 4,168,146 and 4,366,241. Alternatively,
"competitive-type" assays may be used with the methods described
herein. In a competitive assay, the labeled probe is generally
conjugated with a molecule that is identical to, or an analog of,
the analyte. Thus, the labeled probe competes with the analyte of
interest for the available receptive material. Examples of
competitive immunoassay devices are described in U.S. Pat. Nos.
4,235,601, 4,442,204 and 5,208,535.
[0157] Techniques that may be used to assess the amount of nucleic
acid encoding any one or more of IL-12, TNF-.alpha., IFN-.gamma.,
GM-CSF, IL-3, IL-2, granzyme B, or Tbet from a sample obtained from
a subject include but are not limited to in situ hybridization
(e.g., Angerer (1987) Meth. Enzymol 152: 649). Preferred
hybridization-based assays include, but are not limited to,
traditional "direct probe" methods such as Southern blots or in
situ hybridization (e.g., FISH and FISH plus SKY), and "comparative
probe" methods such as comparative genomic hybridization (CGH),
e.g., cDNA-based or oligonucleotide-based CGH. The methods can be
used in a wide variety of formats including, but not limited to,
substrate (e.g. membrane or glass) bound methods or array-based
approaches. Probes that may be used for nucleic acid analysis are
typically labeled, e.g., with radioisotopes or fluorescent
reporters. Preferred probes are sufficiently long so as to
specifically hybridize with the target nucleic acid(s) under
stringent conditions. The preferred size range is from about 200
bases to about 1000 bases. Hybridization protocols suitable for use
with the methods of the invention are described, e.g., in Albertson
(1984) EMBO J. 3: 1227-1234; Pinkel (1988) Proc. Natl. Acad. Sci.
USA 85: 9138-9142; EPO Pub. No. 430,402; Methods in Molecular
Biology, Vol. 33: In situ Hybridization Protocols, Choo, ed.,
Humana Press, Totowa, N.J. (1994), Pinkel, et al. (1998) Nature
Genetics 20: 207-211, and/or Kallioniemi (1992) Proc. Natl Acad Sci
USA 89:5321-5325 (1992).
[0158] Methods of "quantitative" amplification are well known to
those of skill in the art. For example, quantitative PCR involves
simultaneously co-amplifying a known quantity of a control sequence
using the same primers. This provides an internal standard that may
be used to calibrate the PCR reaction. Detailed protocols for
quantitative PCR are provided in Innis, et al. (1990) PCR
Protocols, A Guide to Methods and Applications, Academic Press,
Inc. N.Y.). Measurement of DNA copy number at microsatellite loci
using quantitative PCR anlaysis is described in Ginzonger, et al.
(2000) Cancer Research 60:5405-5409. The known nucleic acid
sequence for the genes is sufficient to enable one of skill in the
art to routinely select primers to amplify any portion of the gene.
Fluorogenic quantitative PCR may also be used in the methods of the
invention. In fluorogenic quantitative PCR, quantitation is based
on amount of fluorescence signals, e.g., TaqMan and sybr green.
[0159] Other suitable amplification methods include, but are not
limited to, ligase chain reaction (LCR) (see Wu and Wallace (1989)
Genomics 4: 560, Landegren, et al. (1988) Science 241:1077, and
Barringer et al. (1990) Gene 89: 117), transcription amplification
(Kwoh, et al. (1989) Proc. Natl. Acad. Sci. USA 86: 1173),
self-sustained sequence replication (Guatelli, et al. (1990) Proc.
Nat. Acad. Sci. USA 87: 1874), dot PCR, and linker adapter PCR,
etc.
[0160] In certain embodiments, other techniques may be used to
determine expression of a polynucleotide gene product, including
microarray analysis (Han, M., et al., Nat Biotechnol, 19: 631-635,
2001; Bao, P., et al., Anal Chem, 74: 1792-1797, 2002; Schena et
al., Proc. Natl. Acad. Sci. USA 93:10614-19, 1996; and Heller et
al., Proc. Natl. Acad. Sci. USA 94:2150-55, 1997) and SAGE (serial
analysis of gene expression). Like MPSS, SAGE is digital and can
generate a large number of signature sequences. (see e.g.,
Velculescu, V. E., et al., Trends Genet, 16: 423-425., 2000; Tuteja
R. and Tuteja N. Bioessays. 2004 August; 26(8):916-22), although
orders of magnitude fewer than that are available from techniques
such as MPSS. Examples of nucleic acid microarrays may be found in,
for example, U.S. Pat. Nos. 6,391,623, 6,383,754, 6,383,749,
6,380,377, 6,379,897, 6,376,191, 6,372,431, 6,351,712 6,344,316,
6,316,193, 6,312,906, 6,309,828, 6,309,824, 6,306,643,
6,300,063.
[0161] In various embodiments of the methods described herein, the
reference value is based on the presence and/or amount of protein
of interest including any one or more of IL-12, TNF-.alpha.,
IFN-.gamma., GM-CSF, IL-3, IL-2, granzyme B, or Tbet in a sample
obtained from the subject (for example, a subject having cancer,
infectious diseases or autoimmune diseases).
[0162] In some embodiments, the reference value is the mean or
median presence of the protein of interest (or nucleic acid
encoding the protein of interest) in a population of subjects that
do not have a disease-state. For example, in subjects with cancer,
the reference value is the mean or median presence of any one or
more of IL-12, TNF-.alpha., IFN-.gamma., GM-CSF, IL-3, IL-2,
granzyme B, or Tbet in a population of subjects that do not have
the cancer. For example, in subjects with an infectious disease,
the reference value is the mean or median presence of any one or
more of IL-12, TNF-.alpha., IFN-.gamma., GM-CSF, IL-3, IL-2,
granzyme B, or Tbet in a population of subjects that do not have
the infectious disease. For example, in subjects with an autoimmune
disease, the reference value is the mean or median presence of any
one or more of IL-12, TNF-.alpha., IFN-.gamma., GM-CSF, IL-3, IL-2,
granzyme B, or Tbet in a population of subjects that do not have
the the autoimmune disease. In various embodiments, the levels of
any one or more of IL-12, TNF-.alpha., IFN-.gamma., GM-CSF, IL-3,
IL-2, granzyme B, or net is altered (increased if the subject has
cancer or an infectious disease and decreased if the subject has an
autoimmune disease) relative to the reference value by at least or
about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 100%. In
various embodiments the levels of any one or more of IL-12,
TNF-.alpha., IFN-.gamma., GM-CSF, IL-3, IL-2, granzyme B, or net is
altered (increased if the subject has cancer or an infectious
disease and decreased if the subject has an autoimmune disease)
relative to the reference value by at least or about 1-fold,
2-fold, 3-fold, 4-fold, 5-fold, 10-fold, 15-fold, 20-fold, 25-fold,
30-fold, 35-fold, 40-fold, 45-fold, 50-fold, 55-fold, 60-fold,
65-fold, 70-fold, 75-fold, 80-fold, 85-fold, 90-fold, 95-fold,
100-fold or a combination thereof.
[0163] While particular embodiments of various aspects disclosed
herein have been shown and described, it will be obvious to those
skilled in the art that, based upon the teachings herein, changes
and modifications may be made without departing from this invention
and its broader aspects and, therefore, the appended claims are to
encompass within their scope all such changes and modifications as
are within the true spirit and scope of this invention.
[0164] All patents and other publications identified are expressly
incorporated herein by reference for the purpose of describing and
disclosing, for example, the methodologies described in such
publications that might be used in connection with the present
invention. These publications are provided solely for their
disclosure prior to the filing date of the present application.
Nothing in this regard should be construed as an admission that the
inventors are not entitled to antedate such disclosure by virtue of
prior invention or for any other reason. All statements as to the
date or representation as to the contents of these documents is
based on the information available to the applicants and does not
constitute any admission as to the correctness of the dates or
contents of these documents.
[0165] Exemplary embodiments of the various aspects disclosed
herein can be described by one or more of the following numbered
paragraphs: [0166] A. A composition for increasing IL-12
production, the composition comprising immunoglobulin G (IgG) or a
variant thereof or a fragment thereof. [0167] B. The composition of
paragraph A, wherein the composition increases signaling mediated
by interaction between IgG and FcRn. [0168] C. The composition of
paragraph A, wherein the composition increases an immune response
against an antigen. [0169] D. The composition of paragraph A,
wherein the variant IgG comprises a methionine to leucine
substitution at position 428 and an asparagine to serine
substitution at position 434. [0170] E. The composition of
paragraph A, further comprising an antigen conjugated to IgG or a
variant thereof or a fragment thereof so as to create a monomeric
or a multimeric structure which can cross-link FcRn. [0171] F. The
composition of paragraph A, further comprising an antigen complexed
to IgG or a variant thereof or a fragment thereof so as to create a
multimeric structure which can cross-link FcRn. [0172] G. The
composition of paragraphs E or F, wherein the antigen is a tumor
antigen, an endogenous antigen, a cell-associated antigen, an
apoptotic body, a microbial antigen, a viral antigen, a parasitic
antigen or a combination thereof. [0173] H. The composition of
paragraph G, wherein the antigen is a protein or a proteomimetic
thereof, a peptide or a peptidomimetic thereof, a lipid or a
combination thereof. [0174] I. The composition of paragraph A,
wherein the IgG or a variant thereof or a fragment thereof is
mammalian. [0175] J. The composition of paragraph A, wherein the
IgG or a variant thereof or a fragment thereof is human. [0176] K.
A composition for decreasing IL-12 production, the composition
comprising an agent that inhibits signaling mediated by interaction
between FcRn and IgG. [0177] L. The composition of paragraph K,
wherein the agent is any one or more of a peptide, protein, small
molecule, nucleic acid, aptamer, oligonucleotide, antibody or a
combination thereof. [0178] M. The composition of paragraph L,
wherein the nucleic acid is siRNA specific to FcRn. [0179] N. The
composition of paragraph L, wherein the antibody is selected from
the group consisting of a monoclonal antibody or a fragment
thereof, a polyclonal antibody or a fragment thereof, chimeric
antibody, humanized antibody and single chain antibody. [0180] O.
The composition of paragraph K, wherein the agent is a bispecific
agent comprising binding sites for IgG and FcRn. [0181] P. The
composition of paragraph K, wherein the agent is a recombinant Fc
portion of IgG or a biologically active portion thereof or a
proteo-mimetic thereof. [0182] Q. The composition of paragraph P,
wherein the Fc portion of IgG or a biologically active portion
thereof is mammalian. [0183] R. The composition of paragraph P,
wherein the Fc portion of IgG or a biologically active portion
thereof is human. [0184] S. A method for modulating the interaction
between FcRn and IgG comprising contacting a cell with an agent
that binds FcRn and/or IgG and modulates binding of FcRn to IgG.
[0185] T. The method of paragraph S, wherein the agent increases
signaling mediated by interaction of FcRn and IgG. [0186] U. The
method of paragraph S, wherein the agent decreases signaling
mediated by interaction of FcRn and IgG. [0187] V. The method of
paragraph S, wherein the agent comprises binding sites specific for
IgG and FcRn. [0188] W. The method of paragraph S, wherein the
agent comprises binding sites specific for IgG or FcRn. [0189] X.
The method of paragraph S, wherein the agent comprises binding
sites specific for Fc portion of IgG. [0190] Y. The method of
paragraph S, wherein agent comprises a bispecific polypeptide agent
comprising binding sites specific for IgG and FcRn. [0191] Z. The
method of paragraph Y, wherein the bispecific polypeptide agent
comprises an antibody or antigen binding portion thereof that
specifically binds FcRn and an antibody or antigen binding portion
thereof that specifically binds IgG. [0192] AA. A method for
treating, inhibiting, preventing metastasis of or preventing
relapse of cancer in a subject in need thereof comprising: [0193]
(a) providing a composition comprising immunoglobulin G (IgG) or a
variant thereof or a fragment thereof; and [0194] (b) administering
an effective amount of the composition to the subject so as to
treat, inhibit, prevent metastasis or prevent relapse of cancer in
the subject. [0195] BB. A method for treating, inhibiting or
reducing the severity of infectious diseases in a subject in need
thereof comprising: [0196] (a) providing a composition comprising
immunoglobulin G (IgG) or a variant thereof or a fragment thereof;
and [0197] (b) administering an effective amount of the composition
to the subject so as to treat, inhibit or reduce the severity of
infectious diseases in the subject. [0198] CC. The method of
paragraphs AA or BB, wherein the composition increases signaling
mediated by interaction of IgG and FcRn. [0199] DD. The method of
paragraphs AA or BB, wherein the composition increases an immune
response against the antigen. [0200] EE. The method of paragraphs
AA or BB, wherein the variant IgG comprises a methionine to leucine
substitution at position 428 and an asparagine to serine
substitution at position 434. [0201] FF. The method of paragraphs
AA or BB, wherein the composition further comprises an antigen,
wherein the antigen is conjugated to the IgG or a variant thereof
or a fragment thereof, or wherein the antigen is complexed with the
IgG or a variant thereof or a fragment thereof. [0202] GG. The
method of paragraph FF, wherein the antigen is a tumor antigen, a
microbial antigen, a viral antigen, a parasitic antigen or a
combination thereof. [0203] HH. The method of paragraph FF, wherein
the antigen is a protein or a proteomimetic thereof, a peptide or a
peptidomimetic thereof, a lipid or a combination thereof. [0204]
II. A method for treating, inhibiting or reducing the severity of
autoimmune diseases in a subject in need thereof comprising: [0205]
(a) providing a composition comprising an agent that inhibits
signaling mediated by interaction between FcRn and IgG; and [0206]
b) administering an effective amount of the composition to the
subject so as to treat, inhibit or reduce the severity of
autoimmune diseases in the subject. [0207] JJ. The method of
paragraph II, wherein the agent reduces or inhibits production of
IL-12. [0208] KK. The method of paragraph II, wherein the agent is
any one or more of a peptide, protein, small molecule, nucleic
acid, aptamer, oligonucleotide, antibody or a combination thereof.
[0209] LL. The method of paragraph KK, wherein the nucleic acid is
siRNA specific to FcRn. [0210] MM. The method of paragraph KK,
wherein the antibody is selected from the group consisting of a
monoclonal antibody or a fragment thereof, a polyclonal antibody or
a fragment thereof, chimeric antibody, humanized antibody and
single chain antibody. [0211] NN. The method of paragraph II,
wherein the agent is a bispecific agent comprising binding sites
for IgG and FcRn. [0212] OO. The method of paragraph KK, wherein
the agent is a recombinant Fc portion of IgG or a biologically
active portion thereof or a proteo-mimetic thereof. [0213] PP. A
method for downregulating expression of IL-12 in a subject
comprising: [0214] (a) providing a composition comprising an FcRn
antibody; and [0215] (b) administering an effective amount of the
composition to the subject so as to downregulate expression of
IL-12 in the subject. [0216] QQ. A method for determining the
efficacy of treatment in a subject in need thereof comprising:
[0217] (a) providing a sample from a subject, wherein the subject
has been administered an effective amount of a composition
comprising immunoglobulin G (IgG) or a variant thereof or a
fragment thereof; [0218] (b) assaying the levels of any one or more
of IL-12, TNF-.alpha., IFN-.gamma., GM-CSF, IL-3, IL-2, granzyme B,
Tbet or a combination thereof in the sample; and [0219] (c)
determining that the treatment is efficacious if the levels of any
one or more of IL-12, TNF-.alpha., IFN-.gamma., GM-CSF, IL-3, IL-2,
granzyme B, Tbet or a combination thereof in the sample from the
subject is higher relative to the levels in a reference sample or
determining that the treatment is not efficacious if the levels of
any one or more of IL-12, TNF-.alpha., IFN-.gamma., GM-CSF, IL-3,
IL-2, granzyme B, Tbet or a combination thereof in the sample from
the subject is lower relative to the levels in a reference sample,
wherein the subject has cancer or an infectious disease. [0220]
RR.A method for determining the efficacy of treatment in a subject
in need thereof comprising: [0221] (a) providing a sample from a
subject, wherein the subject has been administered a composition
comprising an agent that inhibits signaling mediated by interaction
between FcRn and IgG; [0222] (b) assaying the levels of any one or
more of IL-12, TNF-.alpha., IFN-.gamma., GM-CSF, IL-3, IL-2,
granzyme B, Tbet or a combination thereof in the sample; and [0223]
(c) determining that the treatment is efficacious if the levels of
any one or more of IL-12, TNF-.alpha., IFN-.gamma., GM-CSF, IL-3,
IL-2, granzyme B, Tbet or a combination thereof in the sample from
the subject is lower relative to the levels in a reference sample
or determining that the treatment is not efficacious if the levels
of any one or more of IL-12, TNF-.alpha., IFN-.gamma., GM-CSF,
IL-3, IL-2, granzyme B, Tbet or a combination thereof in the sample
from the subject is higher relative to the levels in a reference
sample, wherein the subject has an autoimmune disease. [0224] SS.
The method of paragraph QQ or RR, wherein the sample is blood,
plasma or tissue.
EXAMPLES
[0225] The following examples are provided to better illustrate the
claimed invention and are not to be interpreted as limiting the
scope of the invention. To the extent that specific materials are
mentioned, it is merely for purposes of illustration and is not
intended to limit the invention. One skilled in the art may develop
equivalent means or reactants without the exercise of inventive
capacity and without departing from the scope of the invention.
Example 1
Experimental Methods
[0226] Mice and tumor models. Fcgrt-/- mice (Roopenian et al.,
2003), deficient in FcRn, on a C57BL/6 background were originally
purchased from The Jackson Laboratory. FcgrtFl/Fl mice were a kind
gift of Dr. E. Sally Ward (University of Texas Southwestern Medical
Center) (Montoyo et al., 2009). hFCGRT-hB2M-mFcgrt-/- mice have
been described previously (Yoshida et al., 2004). All procedures
were approved by the Harvard Medical Area Standing Committee on
Animals. AOM, AOM/DSS, Apcmin/+ and lung metastasis tumor models
were performed using previously described protocols
(LeibundGut-Landmann et al., 2008; Meunier et al., 2009; Wirtz et
al., 2007).
Induction of Colorectal Cancer
[0227] Inflammation-associated CRC was induced by a single i.p.
dose of 10 mg/kg azoxymethane (AOM) (Sigma Aldrich) and the
subsequent administration of two 7-day courses of 1.5% dextran
sodium sulfate (DSS) (MP Biomedicals) in drinking water, as
outlined in FIG. 8D and described previously (Wirtz et al., 2007).
Tumor burden was assessed two weeks after withdrawal of the final
course of DSS. Tumor load was calculated as the sum of all tumor
diameters, as described previously (Grivennikov et al., 2012). Bone
marrow chimeras were generated by lethal irradiation of recipients
(2.times.6 Gy) followed by reconstitution with 1.times.10.sup.6
bone marrow cells from the appropriate donor. Treatment of the
chimeras with AOM/DSS was begun 8 weeks after reconstitution.
Non-inflammation-associated CRC was induced by eight weekly i.p.
injections of 10 mg/kg AOM. Tumor burden was assessed 12 weeks
after administration of the final dose of AOM. Tumor burden in
ApcMin/+ and ApcMin/+Fcgrt-/- mice was assessed at 5-6 months of
age in untreated mice. In all experiments, tumor evaluation 14 was
carried out blindly by counting macroscopically visible tumors
(>1 mm in diameter) and measuring the largest diameter of each
lesion with digital calipers.
Adoptive Transfer Experiments
[0228] CD8+ T cells or DC from the MLN and LI lamina propria were
isolated from donor mice at day 21 of the AOM/DSS treatment course
(see FIG. 8D). Isolation of cells was carried out by sequential
collagenase digestions, as previously described (Baker et al.,
2011). CD8+ T cells were subsequently purified using negative
magnetic selection (Miltenyi Biotec). DC were purified using
positive magnetic selection with CD11c-microbeads (Miltenyi Biotec)
and yielded predominantly CD8-CD11b+DC (FIG. 10I). 1.times.10.sup.6
CD8+ T cells or DC were adoptively transferred to recipient mice
via i.p. injection, on days 0 and 21 for CD8+ T cells and days -7
and 14 for DC. Appropriate localization of the transferred cells to
the MLN and LI lamina propria of the recipient mice was verified in
separate experiments using mice congenic for Ly5.1 expression (FIG.
10J). For depletion of CD8+ T cells, mice were initially treated
i.p. on consecutive days with three doses of 0.5 mg of an anti-CD8
antibody (clone 53-6.72, BWHBRI Antibody Core Facility) (or isotype
control) beginning 3 days prior to the initial DC transfer
(Kruisbeek, 1991). Thereafter, CD8+ T cell depletion was maintained
by administration of 0.5 mg anti-CD8 antibody (or isotype) every
three days until termination of the experiment. For IL-12
neutralization, mice were initially treated on consecutive days
with three i.p. doses of 0.5 mg of an anti-IL-12p40 antibody (clone
C17.8) (kindly provided by Dr. Giorgio Trinchieri, National Cancer
Institute) (or isotype control) beginning 3 days prior to the
initial DC transfer. Neutralization was maintained by
administration of 0.5 mg anti-IL-12p40 antibody (or isotype) twice
per week until termination of the experiment.
Lung Metastasis Experiments
[0229] Lung metastases were induced by the i.v. injection of
0.5.times.10.sup.6 OVA-transfected B16 melanoma cells (OVA-B16
cells, a generous gift of Dr. Kenneth Rock, University of
Massachusetts Medical School) in log phase growth (Falo et al.,
1995; 15 LeibundGut-Landmann et al., 2008). Evaluation of lung
nodules was carried out following lung inflation and fixation in
10% formalin. For DC immunization experiments, DC were isolated by
collagenase digestion from the lung and draining lymph nodes of
metastasis-bearing donor mice and 1.times.10.sup.6 DC were injected
s.c. into the hind footpad of recipient mice. Two weeks later,
recipients were given OVA-B16 cells, as above. For CD8+ T cell
protection experiments, OVA-specific OT-I CD8+ T cells were
stimulated with DC pulsed with IgG IC or IHH-IgG IC, as described
above, in the presence of 20 U/ml IL-2 for 5 days before
purification and i.v. injection of 1.times.10.sup.6 T cells into
recipient mice having received OVA-B16 cells 24 h earlier. For
immunization with ex vivo loaded DC, LS-IgG was generated and WT DC
were loaded for 3 h ex vivo with OVA-containing IgG IC or LS-IgG IC
and then washed extensively before s.c. footpad injection. For
quantification of CD8+ T cells specific for the tumor antigen OVA,
lungs were digested with collagenase II as previously described
(Olszak et al., 2012) in order to create a single cell suspension.
Cells were stained with anti-CD3, anti-CD8 and the SIINFEKL-H2-Kb
tetramer or a control LCMV-H2-Kb tetramer (Beckman-Coulter).
Frequency of cells positive for the SIINFEKL-H2-Kb tetramer within
the CD3+CD8+ gate was assessed by flow cytometry.
Microbiota Analysis
[0230] Analysis of the microbiota was conducted as using previously
published methods (Uronis et al., 2011). Briefly, for T-RFLP
analysis, samples were collected from the feces, proximal LI or
distal LI of adult (8-week old) and pre-weaning (2-week old)
littermates and snap frozen in liquid nitrogen. Samples were
processed for T-RFLP analysis as previously described (Uronis et
al., 2011). Analysis was conducted using Sequentix Gelquest to
assign size (length of fragment) and peak height (abundance) to
each TRF. Using PRIMER v6, TRF abundance was standardized by total
and transformed by square root. The standardized transformed
abundances were compiled into a Bray Curtis similarity matrix and
Analysis of Similarity (ANOSIM) was used to test for statistically
significant differences in overall community composition between
genotypes. Diversity was measured using the Shannon diversity (H),
Margalef richness (d), and Pielou evenness (J) indices and
differences were assessed by Student's t test. For qPCR, samples
were collected from the feces, proximal LI or distal LI of 7-week
old littermates and snap frozen in liquid nitrogen. Samples were
processed as described above. qPCR was performed using previously
published primer sets (Arthur et al., 2012; Miyamoto et al., 2002;
Periasamy and Kolenbrander, 2009; Rabizadeh et al., 2007; Shames et
al., 1995).
Human DC and Tissue Experiments
[0231] Human leukopacks were obtained from the Kraft Family Blood
Donor Center of the Dana-Farber Cancer Institute and Brigham and
Women's Hospital. hMoDC were derived as previously described
(Zeissig et al., 2010) for 5 days in 1000 U/ml hGM-CSF and 500 U/ml
hIL-4. During the final 24 h of culture, 100 U/ml IFN-.gamma. was
added. IgG and IHH-IgG stimulations were carried out as described
above. One set of human CRC tissue micro-arrays containing 50
samples of matched tumor and adjacent normal tissue from the same
donors were obtained from BioMax USA. A second TMA containing
multiple punches from each of 220 patients and for which survival
data was available has previously been described (Karamitopoulou et
al., 2011). Tissue was stained using the EnVision G2 Doublestain
System, Rabbit/Mouse (DAB+/Permanent Red) Kit from Dako following
heat-induced epitope retrieval in 10 mM citrate, 1 mM EDTA, 0.05%
Tween (pH 6.0). Primary antibodies were anti-hFCGRT (HPA012122,
Sigma Aldrich), anti-hCD11c (Novocastra) and anti-hCD8 (Dako) all
of which were used at 1/50. Experiments were performed under
Brigham and Women's Hospital Review Board approval.
Biochemical Methods
[0232] Flow cytometry, RNA analysis, IgG quantification, ChIP,
Western blotting, ELISpot and in vitro co-culture experiments were
conducted as described herein and as previously described (Baker et
al., 2011).
Flow Cytometry
[0233] Cells were isolated from the spleen, MLN or colon using
collagenase digestion, as previously described (Baker et al.,
2011). All antibodies used for flow cytometric staining were
purchased from BioLegend except the following: Ki-67 (BD
Pharmingen), granzyme B (eBioscience), FCGR4 (Sino Biologicals).
Intracellular staining for Granzyme B was carried out using the
Cytofix/Cytoperm kit (BD Pharmingen) following a 4 h restimulation
by PMA (30 ng/ml) and ionomycin (2 .mu.g/ml) (Sigma) and GolgiStop
(BD Pharmingen).
RNA Isolation and qPCR
[0234] RNA was isolated directly from flow cytometrically sorted
cell populations (CD8+ T cells or DC) using an RNeasy MicroKit
(Qiagen) or from snap-frozen tissue using an RNeasy MiniKit
(Qiagen). RNA was reverse-transcribed using SuperScriptIII (Life
Technologies) and quantified by qPCR using SYBR Green technology
(Roche).
In Vitro and Ex Vivo Cultures
[0235] CD8+ or CD4+ T cell activation was assessed following 24 h
of restimulation with plate-bound anti-CD3 and anti-CD28 using a
cytometric bead array (BD Pharmingen). Immune complexes were formed
using ovalbumin conjugated to the hapten NIP
(4-hydroxy-3-iodo-5-nitrophenylacetic acid) and NIP-specific
chimeric IgG (IgG), IHH-IgG or LS-IgG. IHH-IgG is a mutational
variant of the chimeric IgG protein which contains a NIP-specific
mouse Fab fragment and a human IgG1 Fc fragment and which has been
rendered incapable of FcRn binding due to the introduction of
mutations in three critical amino acids in the Fc region which are
required for FcRn ligation as previously described (Baker et al.,
2011). LS-IgG was generated by introduction of the M428L/N434S
mutations which are known to enhance FcRn binding (Zalevsky et al.,
2010) into a previously described chimeric antibody specific for
the hapten 4-hydroxy-3-iodo-5-nitrophenylacetic acid (NIP) and
containing a human IgG1 Fc (Ober et al., 2001) using the QuikChange
site-directed mutagenesis kit (Strategene). In vitro cross
presentation assays were carried out by pulsing 1.times.10.sup.5
isolated DC with preformed immune complexes (0.5 .mu.g/ml
NIP-conjugated OVA+100 .mu.g/ml anti-NIP IgG or anti-NIP IHH-IgG)
for 2-3 h followed by extensive washing and the addition of
2.times.10.sup.5 purified OT-I CD8+ T cells (Baker et al., 2011).
Cytokine secretion was measured after 24 h or 48 h by ELISA (BD
Pharmingen). For IL-12 neutralization experiments, the indicated
concentration of IL-12 neutralizing goat IgG (RnD Systems) or goat
isotype control IgG (RnD Systems) was added to DC following IgG IC
pulsing. DC were incubated in the presence of the neutralizing
antibody for 1 h before addition of the OT-I CD8+ T cells.
IgG Analysis
[0236] IgG isotypes in the serum of untreated or treated mice was
quantified using isotype specific ELISAs (Southern Biotech). For
quantification of tissue IgG, snap frozen tissue was briefly thawed
and then homogenized in PBS containing protease inhibitors (Roche).
Insoluble material was removed by centrifugation and protein
concentration in the clarified supernatant was assessed by BCA
assay (Thermo Scientific). Equivalent quantities of protein were
used in IgG isotype ELISAs and IgG concentration was normalized to
mg of total protein. Tumor specific IgG was evaluated using lysates
made from purified tumor epithelial cells, isolated by dispase and
collagenase digestion (Baker et al., 2011; Olszak et al., 2012).
Tumor lysates were depleted of IgG by overnight incubation with
Protein G Sepharose beads (GE Healthcare Life Sciences) at
4.degree. C. For Western blotting, 10 .mu.g lysate from tumor
epithelium or normal intestinal epithelium was resolved by SDS-PAGE
under reducing conditions and transferred to nitrocellulose
membranes. Membrane strips were then probed with 1/10 dilutions of
serum or intestinal homogenates from individual mice and developed
with anti-mouse IgG-HRP. Blots were developed with ECL Western
Blotting Reagent (GE Healthcare). For ELISA, plates were coated
with 1/10 dilution of tumor lysate in coating buffer before
application of serial dilutions of serum or tissue homogenates and
development with anti-mouse IgG-HRP. OVA-specific IgG secreting B
cells from OVA-B16 metastasis-bearing mice were quantified by
ELISpot (mAbTech) according to the manufacturer's instructions
following isolation of B cells with CD19-microbeads (Miltenyi
Biotech).
Nuclear Translocation and ChIP
[0237] Nuclear tanslocation of transcription factors was assessed
following stimulation of isolated DC with IgG IC (formed as above
with FcRn-binding IgG or non-FcRn binding IHH-IgG and NIP-OVA) for
the indicated times. Nuclei and cytoplasmic fractions were isolated
using the NE-PER Nuclear and Cytoplasmic Extraction Kit (Thermo
Scientific). Anti-NF-.kappa.B p65, anti-IRF-1 and anti-HDAC
antibodies were all purchased from Cell Signaling Technologies.
ChIP was performed following IC stimulation with the SimpleChIP
Plus Enzymatic Chromatin IP Kit (Magnetic Beads) from Cell
Signaling Technologies.
Statistical Analyses
[0238] All data are expressed as mean.+-.s.e.m. Unless otherwise
specified, data was analyzed using two-tailed unpaired Student's t
tests. Significance of results across independent experiments was
assessed by pairwise Student's t test. As indicated where relevant,
non-normally distributed data was assessed using Mann-Whitney test
and survival for mouse experiments was evaluated using Logrank test
or Chi-Squared test. The human survival analysis was performed with
the Kaplan-Meier method and the two curves were compared with the
log rank test. Subsequently, FcRn+CD11c+ status was entered into
uni- and multivariate Cox regression analysis. Hazard ratios (HR)
and 95% confidence intervals (CI) were used to determine the
prognostic effect of FcRn+CD11c+ cell numbers on survival time. All
analyses were carried out using GraphPad Prism software (GraphPad
Software, Inc.).
Example 2
FcRn Protects Against the Development of Colorectal Cancer
[0239] The majority of sporadic colorectal cancers (CRC) arise
following a defined series of mutational events often involving
inactivation of the adenomatous polyposis coli (APC) gene (Walther
et al., 2009). We thus began by investigating whether FcRn could
contribute to the development of CRC in ApcMin/+ mice which possess
an abnormal copy of Apc and spontaneously develop large numbers of
small intestinal adenomas (Saleh and Trinchieri, 2011). Typically,
ApcMin/+mice do not develop colonic lesions in the absence of
further insults, such as the additional loss of a tumor suppressor
gene (Aoki et al., 2003; Saleh and Trinchieri, 2011). However,
ApcMin/+ mice crossed with mice deficient in FcRn (Fcgrt-/-)
spontaneously developed significantly more LI tumors than their
ApcMin/+ littermates (FIG. 1A). Importantly, high grade dysplasia
and local invasion through the LP were detected only in lesions
from ApcMin/+Fcgrt-/- but not ApcMin/+ animals (FIGS. 1A and 8A).
No differences were detected in the frequency of tumors in the
small intestine (SI) (FIG. 8B), where tumor development in ApcMin/+
mice does not depend on a second genetic event (Aoki et al., 2003;
Saleh and Trinchieri, 2011). We next investigated the role of FcRn
in the development of CRC induced by the chronic administration of
a chemical carcinogen, azoxymethane (AOM), which, upon repeated
administration, drives the development of colorectal malignancies
(Meunier et al., 2009). We observed that Fcgrt-/- mice subjected to
a standard regimen of AOM administration developed significantly
more abundant and larger tumors (FIGS. 1B and 8C) than did WT
littermates. These data demonstrate the importance of FcRn in
determining susceptibility to the development of sporadic CRC.
[0240] Knowing that inflammatory bowel disease is associated with a
heightened risk of CRC and that inflammation plays an important
role in driving even sporadic neoplasias (Coghill et al., 2012;
Herrinton et al., 2012), we examined whether FcRn-mediated tumor
protection extended to inflammation-associated CRC. We found that
Fcgrt-/- mice treated with AOM and dextran sodium sulfate (AOM/DSS)
(FIG. 8D) (Wirtz et al., 2007) developed significantly larger and
more abundant colorectal adenocarcinomas than WT littermates (FIGS.
1C, 1D, 8E). Additionally, at higher concentrations of DSS,
Fcgrt-/- mice experienced significantly poorer survival rates
compared to their WT littermates (FIG. 1E), indicating that
FcRn-mediated anti-tumor immunity is potent enough to influence
disease outcome. The smaller initial weight loss in the Fcgrt-/-
mice compared to WT controls (FIG. 8F) was consistent with previous
findings that Fcgrt-/- mice are protected from IgG-induced colitis
(Kobayashi et al., 2009), suggesting that tumor development in the
context of FcRn-deficiency is not simply due to increased
inflammation.
[0241] Certain intestinal microbes may play a role in promoting the
development of CRC (Arthur and Jobin, 2011; Arthur et al., 2012).
We thus profiled the intestinal microbiota in our WT and Fcgrt-/-
littermate mice in order to determine whether FcRn was exerting
tumor protection through the regulation of gut microbial
composition. Terminal restriction fragment length polymorphism
(T-RFLP) analysis of the overall microbial community composition
and diversity from WT and Fcgrt-/- littermates revealed no
significant differences in either post-weaning, eight week old mice
or pre-weaning, two week old mice (FIGS. 1F and 8G, 8H) in any of
three separate intestine-associated tissue compartments (proximal
LI, distal LI and feces). However, regardless of genotype, the
microbiota were found to differ between these three tissue sites,
thereby confirming that our analysis had sufficient power to detect
differences in microbial composition (FIGS. 8I, 8J). In order to
exclude differences in specific organisms previously associated
with CRC development (Arthur and Jobin, 2011), we also assessed the
abundance of these microbes in a separate cohort of seven week old
mice using genus or species specific qPCR and found no significant
differences in either the distal LI (FIG. 1G) or feces (FIG. 8K) of
WT and Fcgrt-/- littermates. Together, these data demonstrate that
FcRn does not protect against colorectal tumor development by
regulating intestinal microbial diversity or decreasing the
presence of tumor-promoting microbes.
Example 3
FcRn Promotes the Retention and Activation of Tumor Protective CD8+
T Cells in the Large Intestine
[0242] In seeking to better understand the nature of FcRn-driven
anti-tumor immunity, we examined the immunological composition of
LP lymphocytes (LPL) in both dissected tumors and macroscopically
tumor-free adjacent tissue. While no differences were noted in the
numbers of CD4+ T cells, natural killer (NK) cells or macrophages
(FIG. 9A), significantly greater numbers of CD8+ T cells were
consistently found both within tumor tissue and adjacent tissue of
WT AOM/DSS-treated mice in comparison to their Fcgrt-/- littermates
(FIGS. 2A, 2B, upper panels, and FIG. 9B). This same deficiency in
CD8+ T cell infiltration into the tumor microenvironment of
FcRn-deficient mice was also seen in ApcMin/+Fcgrt-/- animals in
comparison to their ApcMin/+ littermates (FIG. 9C) as well as in
Fcgrt-/- mice treated with AOM alone (FIG. 9D). Furthermore, a
greater percentage of the CD8+ T cells from AOM/DSS treated WT
animals expressed intracellular granzyme B or surface lysosomal
associated membrane protein-1 (LAMP1) (FIGS. 2A, 2B, lower panels)
than did those from their Fcgrt-/- littermates. We confirmed this
using anti-CD3 and anti-CD28 restimulation of sorted effector
CD8+CD44+CD62L- T cells from the LI of AOM/DSS-treated mice (FIG.
2C). CD8+ T cells from Fcgrt-/- tumor-bearing mice secreted only
small amounts of interferon-.gamma. (IFN-.gamma.), tumor necrosis
factor (TNF) and interleukin-10 (IL-10), the latter of which has
recently been shown to be critical for efficient cytotoxic CD8+ T
cell-mediated anti-viral and anti-tumor immunity (Mumm et al.,
2011; Zhang and Bevan, 2011). While no differences were seen in the
rates of CD8+ T cell proliferation or apoptosis, as assessed by
Ki-67 and annexin V staining, respectively (FIG. 9E), both
upregulation of CD103, an integrin associated with T cell retention
(Le Floc'h et al., 2007), and increased expression of
activation-associated CD44 on CD62L+CD8+ T cells were observed in
CD8+ T cells infiltrating the LP of WT but not Fcgrt-/- littermates
(FIG. 9F). This was specific for the tumor-associated tissues as
these differences were not observed in the mesenteric lymph nodes
(MLN) (FIG. 9F) and is notable because the presence of high numbers
of CD8+CD44+CD62L+ cells bearing an effector memory (TEM) cell
phenotype has been associated with improved prognosis in human CRC
patients (Pages et al., 2005). These data are thus most consistent
with a role for FcRn in driving anti-tumor immunity by promoting
the retention and activation of cytotoxic T cells having homed to
the LI.
[0243] We next sought to confirm that FcRn-mediated activation of
CD8+ T cells was critical for its tumor protective function using
adoptive transfer of CD8+ T cells isolated from the MLN and LP of
AOM/DSS-treated WT or Fcgrt-/- mice into recipient Fcgrt-/-
AOM/DSS-treated animals. By transferring CD8+ T cells isolated from
both the MLN and total LI LP, we aimed to minimize the number of T
cells likely to have been exposed to a tolerizing tumor
microenvironment (Chen and Mellman, 2013). Fcgrt-/- recipient mice
that received CD8+ T cells from WT donors developed significantly
fewer tumors than non-T cell (PBS) treated control Fcgrt-/- mice
(FIG. 2D). While the transfer of CD8+ T cells from Fcgrt-/- donors
into Fcgrt-/- recipients did exert a non-significant decrease in
tumor frequency compared to Fcgrt-/- controls, it also led to a
significant increase in tumor size and thus of total tumor load, as
measured by the total surface of the colon which was neoplastic
(FIG. 2D) (Grivennikov et al., 2012). Similar experiments performed
with adoptively transferred CD4+ T cells revealed that CD4+ T cells
are not sufficient for FcRn-mediated tumor protection. Rather, our
data are consistent with activation of cytotoxic CD8+ T cells being
a primary mechanism by which FcRn-driven tumor immune surveillance
operates.
Example 4
FcRn-Dependent Cross Priming by Dendritic Cells Induces Effective
Anti-Tumor CD8+ T Cell Responses
[0244] In light of our recent demonstration that FcRn in CD8-CD11b+
DC, in which the acidic endosomal and phagosomal pH favor FcRn-IgG
binding, drives the cross-presentation of IgG IC-delivered antigens
and the resulting activation of CD8+ T cells (Baker et al., 2011),
which lack FcRn, we sought to determine whether FcRn-dependent
cross-priming by DC was required for its anti-tumor effects. Given
that this mechanism would necessitate the presence of
tumor-reactive IgG to form IC and that tumor-reactive IgG has
previously been documented in human CRC (Auer et al., 1988; Kijanka
et al., 2010), we first confirmed the presence of these effector
molecules in our model. Both ELISA (FIGS. 3A and 10A) and Western
blotting (FIG. 10B) assays using IgG-depleted tumor epithelium
lysates from AOM/DSS-treated mice verified that tumor-reactive IgG
was present in the serum as well as in MLN and LI tissue
homogenates of AOM/DSS-treated WT and Fcgrt-/- littermates but not
non-tumor bearing controls. We also noted increases of similar
magnitude in anti-phosphatidylserine and anti-cardiolipin IgG,
which could promote the formation of IC containing apoptotic bodies
or mitochondria released by dying tumor cells (Kepp et al., 2009),
in the serum of both WT and Fcgrt-/- littermates (FIG. 10C).
Moreover, both tumor-reactive IgG (FIGS. 3A and 10A) and total IgG
(FIG. 10D) were also present at similar levels in the MLN and
intestinal tissues of AOM/DSS-treated WT and Fcgrt-/- mice. Thus,
although FcRn is critically important in protecting circulating IgG
from catabolism and our Fcgrt-/- mice were predictably systemically
hypogammaglobulinemic (FIG. 10D) (Roopenian et al., 2003), this was
not the case in tissues for either total IgG (FIG. 10D) or
tumor-specific IgG (FIG. 3A) where local IgG production by resident
plasma cells is likely sufficient to normalize tissue IgG levels.
Thus, the extreme susceptibility of Fcgrt-/- mice to tumor
development cannot simply be attributed to a local deficiency in
the IgG ligand for FcRn.
[0245] Having confirmed the presence of IgG capable of binding
tumor antigens in both WT and Fcgrt-/- mice, we verified that there
were no differences in the distribution of DC subsets or DC
Fc.gamma.R expression in the LI LP of WT and Fcgrt-/- littermates
(FIG. 10E). This was particularly important given that
FcRn-dependent cross-presentation requires Fc.gamma.R for the
initial IgG IC internalization (Baker et al., 2011). Evaluation of
the functional characteristics of sorted CD8-CD11b+ and
CD8+CD11b-DC from the MLN, adjacent, and tumor tissues of
AOM/DSS-treated mice revealed that Fcgrt-/- mice were significantly
deficient in the production of cytokines (IFN-.gamma., IL-12) and
transcription factors (T-bet) known to drive effective cytotoxic T
cell-mediated immunity (FIG. 3B) (Garrett et al., 2009; Gerosa et
al., 1996; Trinchieri, 2003; Zhang and Bevan, 2011). These
differences were greatest in the CD8-CD11b+ DC subset which are
highly efficient at FcRn-dependent cross-presentation (Baker et
al., 2011). Furthermore, analysis of whole tissue transcripts taken
from the tumor and adjacent tissue of tumor-bearing mice (FIGS.
10F-10H) clearly demonstrated decreased transcripts at the tissue
level of these pro-cytotoxicity cytokines in FcRn-deficient
animals. These data thus indicate that FcRn within DC is required
for the establishment of a tissue level cytokine environment within
the LI that is conducive to effective CD8+ T cell activation.
[0246] In order to demonstrate that FcRn-sufficient DC were
directly involved in driving anti-tumor immunity, we first
conducted a series of adoptive transfer experiments. Fcgrt-/- mice
receiving CD8-CD11b+ DC (FIG. 10I) from AOM/DSS treated WT donors
developed significantly fewer tumors than control PBS-treated
Fcgrt-/- mice or Fcgrt-/- mice given Fcgrt-/- DC (FIG. 3C, left)
despite equivalent homing and persistence of donor DC from both
genotypes (FIG. 10J). Furthermore, administration of WT DC, but not
Fcgrt-/- DC, protected Fcgrt-/- recipients from AOM/DSS-induced
mortality (FIG. 3C, right). The transfer of even a small number of
FcRn-sufficient WT CD8-CD11b+ DC was able to normalize the
infiltration of CD8+ T cells into adjacent and tumor LI tissue of
Fcgrt-/- mice (FIG. 3D), thereby confirming that no primary defect
in CD8+ T cells is operating in Fcgrt-/- mice. Ex vivo assays on DC
isolated from the MLN of Fcgrt-/- recipients 7 days after the
transfer of WT DC further confirmed that FcRn-dependent
cross-priming capacity had been restored (FIG. 10K). We validated
these findings using mice bearing a floxed Fcgrt gene (FcgrtFl/Fl)
(Montoyo et al., 2009) which were bred with Itgaxcre animals in
order to specifically delete FcRn in DC (FIG. 10L). Treatment of
ItgaxcreFcgrtFl/Fl mice with AOM/DSS induced significantly more
colorectal tumors than were found in their FcgrtFl/Fl littermates
(FIG. 3E). ItgaxcreFcgrtFl/Fl mice were also deficient in LI LP
CD8+ T cell infiltration compared (FIG. 3E, bottom), thereby
further supporting our hypothesis that FcRn specifically within DC
could orchestrate CD8+ T cell activation within the intestine. We
validated this by performing simultaneous DC transfer and CD8+ T
cell depletion experiments which revealed that removal of CD8+ T
cells from Fcgrt-/- recipients of WT CD8-CD11b+ DC undergoing
AOM/DSS treatment abrogated the improvement in tumor incidence and
cancer survival conferred by the WT DC (FIGS. 3F,G). These data
thus confirm that an important mechanism of FcRn-mediated tumor
protection is DC-dependent activation of CD8+ T cells via
cross-priming of IgG IC-delivered tumor antigens and conditioning
of the cytokine environment.
Example 5
FcRn Within DC Enables Activation of Endogenous CD8+ T Cells
Towards Defined Cognate Tumor Antigens
[0247] To confirm the individual components of FcRn-mediated tumor
immune surveillance in an antigen specific system and demonstrate
the effectiveness of targeting a single defined tumor antigen to an
FcRn-enabled pathway, we used a pulmonary metastasis model using a
melanoma cell line (B16) expressing the OVA antigen (OVA-B16) (Falo
et al., 1995). Knowing that FcRn is highly expressed in the lung
(Spiekermann et al., 2002), we first verified that the lungs of WT
mice were enriched in CD8+ T cells in comparison to those of their
Fcgrt-/- littermates (FIG. 11A). These data extend the range of
FcRn-regulated mucosal CD8+ T cell responses to a second site which
is frequently affected by cancer (Siegel et al., 2012) and is known
to engage in both FcRn-dependent immune responses (Yoshida et al.,
2004) and immunological crosstalk with the intestine (Keely et al.,
2011). Subsequent to i.v. administration of OVA-B16, we observed a
rise in anti-OVA IgG in lung homogenates and serum from both WT and
Fcgrt-/- littermates (FIGS. 11B, 11C) and detected equivalent
numbers of anti-OVA IgG-secreting B cells in the LN and spleens of
WT and Fcgrt-/- mice (FIG. 11D), thereby confirming that there is
no defect in the local production of tumor antigen-specific IgG in
FcRn deficient animals. WT mice developed considerably fewer
pulmonary nodules than Fcgrt-/- mice and subcutaneous vaccination
at a distant site with WT DC, but not Fcgrt-/- DC conferred
protection from pulmonary metastatic seeding to Fcgrt-/-
recipients. (FIGS. 4A and 11E). Using SIINFEKL/H2kb tetramer
staining, we established that a greater proportion of endogenous
CD8+ T cells with OVA tumor antigen specificity arose in the lungs
of WT mice receiving OVA-B16 tumor cells than in their Fcgrt-/-
littermates (FIG. 4B). In order to confirm that DC-based,
FcRn-mediated tumor protection was dependent upon activation of
CD8+ T cells, we chronically administered a depleting anti-CD8
antibody to Fcgrt-/- recipients immunized with WT CD8-CD11b+ DC and
given OVA-B16. Whereas the transfer of WT DC significantly
decreased the incidence of metastatic pulmonary nodules in Fcgrt-/-
recipients, this protection was abrogated by depletion of CD8+ T
cells (FIG. 4C). We further confirmed that the main locus of
FcRn-mediated tumor immune surveillance was the DC by showing that
ItgaxcreFcgrtFl/Fl mice developed greater numbers of pulmonary
nodules than did their FcgrtFl/Fl littermates and were less
efficient in driving the expansion of tumor specific CD8+ T cells
(FIG. 4D). These findings identify both endogenously arising
tumor-reactive IgG and cognate endogenously derived CD8+ T cells as
important components of the mechanism by which DC exert
FcRn-dependent tumor immune surveillance.
[0248] We next sought to demonstrate that targeting FcRn-mediated
cross-presentation with a single IgG-complexed tumor antigen could
be a viable and attractive strategy for anti-tumor immunotherapy.
In order to do so, we made use of a non-FcRn binding IHH-IgG
containing three point mutations in the Fc domain which disable
FcRn, but not Fc.gamma. receptor, binding (Baker et al., 2011) and
an enhanced FcRn binding LS-IgG containing the `LS` mutation
(M428L/N434S), which increase FcRn binding while maintaining pH
dependency (Claypool et al., 2004; Zalevsky et al., 2010). When
OVA-reactive OT-I CD8+ T cells were stimulated ex vivo with WT DC
primed with OVA-containing IgG or IHH-IgG IC and adoptively
transferred to Fcgrt-/- recipient mice 24 h after i.v.
administration of OVA-B16 cells, only CD8+ T cells primed by IgG
IC-pulsed DC protected against the development of pulmonary
metastases (FIG. 4E). Furthermore, immunizing mice with DC loaded
with OVA-containing LS-IgG IC conferred significantly greater
protection from metastasis development than did immunization with
DC loaded with IgG IC (FIG. 4F). This is consistent with our
finding that LS-IgG IC was more potent than native IgG IC at
inducing cross-priming of low dose antigen in vitro (FIG. 11F).
Collectively, these data demonstrate that targeting the
immunostimulatory potential of FcRn using complexes formed from a
single defined tumor antigen and IgG or FcRn-binding-enhanced IgG
is a tractable and effective anti-tumor therapeutic approach.
Example 6
Dendritic Cell FcRn Enables Homeostatic CD8+ T Cell Activation and
Tc1 Cytokine Secretion in the LI
[0249] Our discovery of FcRn-mediated anti-tumor immunity arising
in the LI in the absence of preexisting inflammation suggested that
FcRn might be playing an active role in intestinal immune
surveillance before the onset of cancer development and thus led us
to investigate whether FcRn regulates CD8+ T cell activation in the
LI under homeostatic conditions. Similar to our observation in
AOM/DSS-treated mice, and despite the well-known differences in
circulating IgG concentrations (Roopenian et al., 2003), similar
quantities of IgG were present in the LI and MLN tissue of both WT
and Fcgrt-/- littermates under steady-state conditions (FIG. 5A).
These results confirmed that the susceptibility of Fcgrt-/- mice to
tumor development could not be attributed to homeostatic local IgG
deficiency. In spite of these comparable tissue IgG amounts,
however, the LI LP of WT mice contained greater quantities of CD8+
T cells, but not other lymphocyte subsets, relative to that
observed in Fcgrt-/- mice (FIG. 5B). A similar deficiency in CD8+ T
cell infiltration into the LI LP was present in untreated
ItgaxcreFcgrtFl/Fl mice compared to their littermate controls (FIG.
5C). Moreover, CD8+ T cells from the LI LP of WT mice secreted more
IFN-.gamma., IL-10 and TNF upon restimulation in comparison to T
cells obtained from Fcgrt-/- littermates (FIG. 5D) and expressed
more activation and cytotoxicity associated cytokines when assessed
immediately after isolation (FIG. 5E). Adoptive transfer of
congenic CD8+ T cells into WT and Fcgrt-/- recipients indicated
that not only did a greater number of transferred T cells
accumulate in the LI LP of WT mice 10 days after transfer but that
these also upregulated significantly more of the activation marker
CD44 (FIG. 12A), consistent with our findings of deficient
CD8+CD44+CD62L+ T cells in Fcgrt-/- tumor-bearing mice (FIG. 9F).
In contrast, CD8+ T cells from WT or Fcgrt-/- donors transferred to
congenic WT recipients homed equally well to the LI LP (FIG. 12B),
thereby confirming that the effect of FcRn is within the local
tissue microenvironment rather than being intrinsic to the T
cells.
[0250] Given that efficient CD8+ T cell activation requires an
appropriate cytokine environment, we next examined the local tissue
cytokine milieu of Fcgrt-/- mice under homeostatic conditions
(Gerosa et al., 1996; Zhang and Bevan, 2011). Tissue explant
cultures (FIG. 5F) and analysis of tissues RNA transcripts (FIG.
12C) indicated that in the absence of FcRn, the MLN and LI were
deficient in their ability to produce cytotoxicity-promoting IL-12
and TNF. By examining the cytokine profiles of sorted DC from the
MLN of untreated WT and Fcgrt-/- littermates, we observed a similar
dependence for FcRn on the expression of IFN-.gamma., IL-12p35,
T-bet and TNF, but not IL-23p19, in CD8-CD11b+ DC (FIG. 5G). This
suggests that FcRn within the CD8-CD11b+ subset of
tissue-associated DC is responsible for establishing a cytokine
milieu conducive to CD8+ T cell activation and thus promoting tumor
immune surveillance in the LI. Similarly, CD4+ T cells from the LI
of untreated Fcgrt-/- mice were deficient in secretion of Th1
cell-associated cytokines upon anti-CD3 and anti-CD28
re-stimulation despite being present in equal amounts in WT and
Fcgrt-/- mice (FIGS. 12D, 12E). These data suggest that IgG IC
ligation of FcRn in DC contributes to the establishment of
homeostatic Th1 and T cytotoxic-1 (Tc1) cell polarization as well
as CD8+ T cell function in the LI.
Example 7
Multimeric IgG IC Ligation of FcRn in DC Induces the Production of
IL-12
[0251] Knowing that IL-12 is a potent enhancer of CD8+ T
cell-mediated immunity (Gerosa et al., 1996; Trinchieri, 2003) and
having consistently observed greater quantities of IL-12 in DC,
particularly the CD8-CD11b+ subset, from the mucosal tissues of WT
compared to Fcgrt-/- littermates, we next investigated the effects
of ligation of FcRn by IgG IC or IgG IC incapable of binding FcRn
(IHH-IgG IC) on IL-12 secretion. Incubation of WT CD8-CD11b+ DC
from the MLN and spleen of untreated WT mice with FcRn binding IgG
IC, but not FcRn-non-binding IHH-IgG IC, led to the direct
induction of IL-12p35 transcripts (FIG. 6A). Similarly, IgG IC
stimulation of WT but not Fcgrt-/- CD8-CD11b+ DC isolated from the
MLN of AOM/DSS treated mice resulted in increased IL-12 secretion
(FIG. 6B). We observed that stimulation of Fcgrt-/- DC with IgG IC
led to considerably less phosphorylation of the Th1 cell-associated
transcription factor STAT-1 (Antonios et al., 2010) than was seen
in WT DC (FIG. 6C). We confirmed that STAT-1 activation was an
important component of FcRn-induced IL-12 production by treating
CD8-CD11b+ DC isolated from Stat1-/- mice with FcRn-binding IgG IC
and FcRn-non-binding IHH-IgG IC and observing that IgG IC failed to
induce IL-12p35 in the absence of STAT-1 (FIG. 6D). We further
showed that (Liu et al., 2003; Murphy et al., 1995) stimulation of
WT CD8-CD11b+ DC with IgG IC led to significantly greater nuclear
translocation and IL-12p35 promoter binding of both interferon
regulatory factor-1 (IRF-1) and NF-.kappa.B p65 than was observed
in Fcgrt-/- DC or upon stimulation with IHH-IgG IC (FIGS. 6C, 6E)
and confirmed that this was MYD88 independent (FIGS. 13A, 13B). IgG
IC ligation of FcRn in DC is thus able to directly induce the
production of the potent Th1 and Tc1 cell-associated cytokine
IL-12.
[0252] In order to demonstrate that FcRn-mediated induction of
IL-12 by DC contributes to the ability of this receptor to promote
anti-tumor immune surveillance, we next performed an IL-12
neutralization experiment in which Fcgrt-/- mice adoptively
transferred with WT DC were subjected to AOM/DSS treatment in the
presence of a neutralizing anti-IL-12 antibody or isotype control
(Wysocka et al., 1995). Whereas WT DC were able to decrease the
incidence of colorectal tumors in Fcgrt-/- recipients down to the
numbers seen for WT control mice, this protection was completely
abrogated when mice were treated with anti-IL-12 (FIG. 6F).
However, in vitro co-culture assays demonstrated that
FcRn-dependent CD8+ T cell priming was independent of IL-12
production (FIG. 13C) indicating that the cross-presenting and
cytokine inducing functions of FcRn are independent of each other.
Collectively, these data indicate that FcRn-driven DC-mediated
tumor protection results from a dual ability to promote
cross-presentation of IgG IC-delivered antigen to CD8+ T cells as
well as to induce secretion of IL-12 which, notably, can augment
the cytotoxic capacity of the primed T cells.
Example 8
FcRn Expressing DC Predict Survival in Human CRC and Secrete
FcRn-Dependent IL-12
[0253] To definitively establish that our observations in mice were
relevant to the development of human CRC, we evaluated the presence
of FcRn-expressing DC in 50 cases of human CRC and matched adjacent
normal tissue utilizing immunohistochemical staining for FcRn and
CD11c. As shown in FIGS. 7A and 14A, FcRn+CD11c+ cells were clearly
present in the stroma of both tumor LI (upper panels) and adjacent
normal LI (lower panels) of CRC patients. Furthermore, a direct
interaction of FcRn+ stromal cells with CD8+ T cells was observed
in both tumor LI (upper panels) and CRC-adjacent normal LI (lower
panels) (FIGS. 7B and 14B). Importantly, the frequency of
FcRn+CD11c+ DC correlated positively with the presence of CD8+ T
cells in the CRC-adjacent normal stroma (FIG. 14C). In order to
determine if the presence of FcRn+CD11c+ cells in the tumor
microenvironment had an impact on patient survival, we stained a
well characterized tissue microarray of 183 human CRC cases for
these cells and analyzed their impact on disease outcome.
Kaplan-Meier survival curves indicated that patients with
.gtoreq.10 FcRn+CD11c+ cells per punch had significantly longer
survival times over a 70 month follow up than did those with <10
FcRn+CD11c+ cells (FIG. 7C). Furthermore, increasing numbers of
FcRn+CD11c+ cells were found to have a positive effect on patient
survival in univariate proportional hazard analysis (p=0.0333), an
effect which was maintained in a multivariable analysis (p=0.0388)
when adjusting for the indicated clinical parameters (FIG. 14D).
Collectively, these studies demonstrate that FcRn-expressing DC
localize to both the CRC and CRC-associated adjacent
microenvironment, correlate with the infiltration of CD8+ T cells
into the tumor tissue and predict improved prognosis for CRC
patients.
[0254] In order to demonstrate a direct causative link between
human FcRn and anti-tumor immune surveillance, we generated
chimeric mice in which irradiated Fcgrt-/- recipients were
reconstituted with bone marrow from donors that were either WT,
Fcgrt-/- or expressed human FcRn and .beta.2-microglobulin
(.beta.2M) on an Fcgrt-/- background (hFCGRT-hB2M-mFcgrt-/-) and
thus possess only the human form of the receptor (Roopenian et al.,
2003). When the chimeras were subjected to AOM/DSS treatment,
Fcgrt-/- mice reconstituted with Fcgrt-/- bone marrow developed far
greater numbers of colorectal tumors than did Fcgrt-/- mice
reconstituted with either WT or hFCGRT-hB2M-mFcgrt-/- bone marrow
(FIG. 7D). Furthermore, CD8+ T cell infiltration in the LI LP of
tumor-bearing mice was restored to WT levels in Fcgrt-/- mice
possessing human-FcRn expressing hematopoietic cells (FIG. 14E).
Thus, human FcRn is equally as capable of orchestrating anti-tumor
immune surveillance as is its murine ortholog.
[0255] We lastly sought to confirm that the intracellular
mechanisms we had demonstrated in mouse DC were also operative in
their human equivalents using human monocyte-derived DC (hMoDC),
which phenotypically akin to the murine CD8-CD11b+ DC subset which
engage in efficient FcRn-dependent cross-priming (FIGS. 14F, 14G)
(Baker et al., 2011; Collin et al., 2011). As shown in FIG. 7E,
stimulation of hMoDC by IgG IC leads to greater production of both
IL-12p35 and IL-12p40 than stimulation with non-FcRn-binding
IHH-IgG IC. Furthermore, IgG IC induced greater phosphorylation of
STAT-1 and nuclear translocation of IRF-1 after both 1 h and 3 h
than did IHH-IgG IC (FIGS. 7F and 14H). Together, they support the
importance of human FcRn function in DC in enabling anti-tumor
immunity through its ability to regulate IL-12 production and CD8+
T cell activation.
Example 9
[0256] As shown in FIG. 15, blockade with the FcRn specific
monoclonal antibody DVN24 decreases the levels of Th1
cytokines.
[0257] The findings presented here identify a key physiological
role for FcRn-mediated cross-priming in driving homeostatic
activation of CD8+ T cells and in CD8+ T cell-mediated anti-tumor
immune surveillance in the LI and lung. We have clearly established
in multiple tumor models that genetic deletion of FcRn increases
susceptibility to carcinogenesis at these mucosal sites. Transfer
of CD8+ T cells primed under FcRn-sufficient conditions or of WT
FcRn-bearing DC was capable of rescuing FcRn-deficient animals from
both a high tumor burden and tumor-induced mortality. This depended
upon IgG IC-mediated ligation of FcRn within mucosal DC which
enabled the priming of tumor antigen-specific endogenous CD8+ T
cells via its dual induction of antigen cross-presentation and the
production of immune-enhancing cytokines such as IL-12. We have
also demonstrated the human relevance of our findings by
demonstrating that FcRn-expressing human DC respond to IgG IC
stimulation with the production of cytotoxicity-promoting IL-12,
that FcRn+ DC localize to the CRC microenvironment where their
ability to induce anti-tumor immunity contributes to improved
patient survival and that human FcRn in hematopoietic cells can
substitute for its mouse ortholog in protecting against the
development of colorectal carcinoma. Our findings are furthermore
consistent with a model in which IgG IC binding to FcRn within
mucosal DC directs not only the intracellular trafficking of IgG IC
but also the previously unrecognized organization of a signaling
cascade which enhances the secretion of cytotoxicity-promoting
cytokines. These features of FcRn biology uniquely enable potent
anti-tumor immune surveillance requiring only small amounts of
antigen and capable of overcoming the immunoregulatory environment
characteristic of intestinal and, potentially, other mucosal
tissues (MacDonald et al., 2011). Moreover, our demonstration that
FcRn-deficiency does not result in decreased levels of
tissue-associated IgG but rather diminished levels of CD8+ T cells
and decreased CD8+ T cell function under homeostatic conditions
further suggests that a major function of FcRn in tissues is in the
regulation of cell mediated immunity rather than solely the
protection of IgG from catabolism which is observed
systemically.
[0258] An important prerequisite for FcRn-mediated tumor protection
is the presence of IgG capable of recognizing a tumor antigen and
initiating a cascade of FcRn-dependent anti-tumor responses which
feed in to the recently described "Cancer-Immunity Cycle" (Brichory
et al., 2001; Chen and Mellman, 2013; Desmetz et al., 2011). The
presence of appreciable quantities of endogenous IgG autoantibodies
reactive or cross-reactive towards altered or abnormally expressed
tumor antigens is well documented and these are likely to serve as
the initiators for FcRn-mediated tumor protection. Specifically,
the accelerated release of tumor-associated antigens either alone
or as part of cellular debris or apoptotic bodies caused by
increased rates of tumor cell death (Kepp et al., 2009), will
promote the formation of immune complexes with endogenous
tumor-reactive or phospholipid-specific autoantibodies.
Subsequently, the concomitant induction of IL-12 production
resulting from FcRn ligation by IgG IC can be expected to amplify
local FcRn-dependent anti-tumor immune responses even further since
IL-12 itself is a potent inducer of humoral immunity (Metzger,
2010). A key physiological role for FcRn within DC therefore
appears to be the integration of humoral and cellular adaptive
immune responses capable of targeting mucosal malignancies and,
undoubtedly, a host of intracellular microbial infections.
[0259] Several intriguing aspects of FcRn biology emerge from our
work. The first of these is that FcRn-dependent immune regulation
is operative under homeostatic conditions and is critical for
establishing baseline colonic CD8+ T cell activation and function.
We predict that such homeostatic responses are largely directed at
microbial antigens given that IgG with antibacterial specificities
are abundantly present in the intestine (Macpherson et al., 1996)
and that the pathways described here may also play a critical role
in immune surveillance against acute and chronic microbial
infections (Yoshida et al., 2006) Secondly, our work highlights the
differential role played by FcRn in different body compartments.
Whereas FcRn is critical for maintaining IgG persistence within the
circulatory system (Roopenian et al., 2003), our observations
indicate that within tissues, FcRn is predominantly involved in the
regulation of local immune responses. In addition to the inadequate
immune activation seen in the intestines of Fcgrt-/- mice, this
idea is supported by our findings that Fcgrt-/- mice were only
minimally deficient in IgG quantities within tissues where equal
amounts of IgG producing cells were able to compensate for the lack
of FcRn-mediated IgG protection. Finally, we have demonstrated the
feasibility and effectiveness of targeting FcRn-mediated
anti-cancer immunosurveillance pathways using a single defined
tumor antigen in complex with native IgG or IgG engineered for
enhanced FcRn binding. In addition to enabling antigen specific
CD8+ T cell mediated immunity after tumor onset, such therapies
also have the potential to promote tumor immune surveillance in
healthy high risk individuals by enhancing the baseline cytotoxic
potential of the intestine. While a large body of knowledge exists
pertaining to the dynamics of FcRn-IgG interaction (Vaughn et al.,
1997) and the ability to engineer IgG with increased affinity for
FcRn is currently available (Mi et al., 2008; Zalevsky et al.,
2010), targeting of FcRn has yet to be exploited by current
DC-based vaccination strategies (Tacken et al., 2007) despite the
growing interest in DC antibody vaccines (Palucka and Banchereau,
2013).
[0260] The pleiotropic nature of FcRn and its wide-reaching
influence on normal physiology remain poorly understood. Whereas
the main function of FcRn systemically is the protection of
monomeric IgG from catabolism (Roopenian et al., 2003), a major
role in tissues, particularly mucosal tissues replete with IgG,
appears to be one of immunological activation upon ligation by
multimeric IgG IC. To this effect, FcRn participates in the
organization not only of an antigen presentation cascade but also
of a signaling cascade that is associated with innate effector
immune function. As shown here, a major consequence of this role
for FcRn is the efficient induction of anti-tumor immunity. These
studies show that FcRn functions in anti-tumor immunity through the
induction (via IL-12) and instruction (via cross-presentation) of
CD8+ T cells. Developing a greater understanding of the nuances of
FcRn-modulated immune activation, particularly at the tissue level
where FcRn in dendritic cells promotes the immunogenic catabolism
of IgG-complexed antigens, holds considerable promise for the
development of new therapies against mucosal diseases.
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[0336] The various methods and techniques described above provide a
number of ways to carry out the application. Of course, it is to be
understood that not necessarily all objectives or advantages
described can be achieved in accordance with any particular
embodiment described herein. Thus, for example, those skilled in
the art will recognize that the methods can be performed in a
manner that achieves or optimizes one advantage or group of
advantages as taught herein without necessarily achieving other
objectives or advantages as taught or suggested herein. A variety
of alternatives are mentioned herein. It is to be understood that
some preferred embodiments specifically include one, another, or
several features, while others specifically exclude one, another,
or several features, while still others mitigate a particular
feature by inclusion of one, another, or several advantageous
features.
[0337] Furthermore, the a person of ordinary skill in the art will
recognize the applicability of various features from different
embodiments. Similarly, the various elements, features and steps
discussed above, as well as other known equivalents for each such
element, feature or step, can be employed in various combinations
by one of ordinary skill in this art to perform methods in
accordance with the principles described herein. Among the various
elements, features, and steps some will be specifically included
and others specifically excluded in diverse embodiments.
[0338] Although the application has been disclosed in the context
of certain embodiments and examples, it will be understood by those
skilled in the art that the embodiments of the application extend
beyond the specifically disclosed embodiments to other alternative
embodiments and/or uses and modifications and equivalents
thereof.
[0339] In some embodiments, the terms "a" and "an" and "the" and
similar references used in the context of describing a particular
embodiment of the application (especially in the context of certain
of the following claims) can be construed to cover both the
singular and the plural. The recitation of ranges of values herein
is merely intended to serve as a shorthand method of referring
individually to each separate value falling within the range.
Unless otherwise indicated herein, each individual value is
incorporated into the specification as if it were individually
recited herein. All methods described herein can be performed in
any suitable order unless otherwise indicated herein or otherwise
clearly contradicted by context. The use of any and all examples,
or exemplary language (for example, "such as") provided with
respect to certain embodiments herein is intended merely to better
illuminate the application and does not pose a limitation on the
scope of the application otherwise claimed. No language in the
specification should be construed as indicating any non-claimed
element essential to the practice of the application.
[0340] Preferred embodiments of this application are described
herein, including the best mode known to the inventors for carrying
out the application. Variations on those preferred embodiments will
become apparent to those of ordinary skill in the art upon reading
the foregoing description. It is contemplated that skilled artisans
can employ such variations as appropriate, and the application can
be practiced otherwise than specifically described herein.
Accordingly, many embodiments of this application include all
modifications and equivalents of the subject matter recited in the
claims appended hereto as permitted by applicable law. Moreover,
any combination of the above-described elements in all possible
variations thereof is encompassed by the application unless
otherwise indicated herein or otherwise clearly contradicted by
context.
[0341] All patents, patent applications, publications of patent
applications, and other material, such as articles, books,
specifications, publications, documents, things, and/or the like,
referenced herein are hereby incorporated herein by this reference
in their entirety for all purposes, excepting any prosecution file
history associated with same, any of same that is inconsistent with
or in conflict with the present document, or any of same that may
have a limiting affect as to the broadest scope of the claims now
or later associated with the present document. By way of example,
should there be any inconsistency or conflict between the
description, definition, and/or the use of a term associated with
any of the incorporated material and that associated with the
present document, the description, definition, and/or the use of
the term in the present document shall prevail.
[0342] It is to be understood that the embodiments of the
application disclosed herein are illustrative of the principles of
the embodiments of the application. Other modifications that can be
employed can be within the scope of the application. Thus, by way
of example, but not of limitation, alternative configurations of
the embodiments of the application can be utilized in accordance
with the teachings herein. Accordingly, embodiments of the present
application are not limited to that precisely as shown and
described.
* * * * *