U.S. patent application number 11/033984 was filed with the patent office on 2006-02-23 for immunomodulatory methods using oligosaccharides.
Invention is credited to Donald A. Harn, Palanivel Velupillai.
Application Number | 20060040893 11/033984 |
Document ID | / |
Family ID | 24391863 |
Filed Date | 2006-02-23 |
United States Patent
Application |
20060040893 |
Kind Code |
A1 |
Harn; Donald A. ; et
al. |
February 23, 2006 |
Immunomodulatory methods using oligosaccharides
Abstract
Methods for modulating immune responses are provided. The
methods involve contacting an immune cell with an agent that
modulates interaction of a compound comprising a Lewis antigen with
the immune cell such that production by the immune cell of at least
one cytokine that regulates development of a T helper type 1 or T
helper type 2 response is modulated. In one embodiment, the agent
is a stimulatory form of a compound comprising a Lewis antigen,
such as a Lewis.sup.y, Lewis.sup.x or Lewis.sup.a oligosaccharide,
or a derivative thereof. In another embodiment, the agent is an
inhibitory form of a compound comprising a Lewis antigen, such as a
Lewis.sup.y, Lewis.sup.x or Lewis.sup.a oligosaccharide, or a
derivative thereof. In various embodiments, the immune cell is a
human immune cell, a macrophage or a T cell. Pharmaceutical
compositions for modulating immune responses are also provided.
Inventors: |
Harn; Donald A.; (Pembroke,
MA) ; Velupillai; Palanivel; (Boston, MA) |
Correspondence
Address: |
LAHIVE & COCKFIELD, LLP.
28 STATE STREET
BOSTON
MA
02109
US
|
Family ID: |
24391863 |
Appl. No.: |
11/033984 |
Filed: |
January 11, 2005 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
08597518 |
Jan 31, 1996 |
6841543 |
|
|
11033984 |
Jan 11, 2005 |
|
|
|
Current U.S.
Class: |
514/54 |
Current CPC
Class: |
C07H 15/00 20130101;
A61P 37/00 20180101; A61P 37/08 20180101; A61K 31/739 20130101;
C07K 14/5428 20130101; Y10S 530/813 20130101; A61K 39/39 20130101;
A61K 31/702 20130101; A61K 38/38 20130101; A61K 2039/55511
20130101; A61K 2039/57 20130101 |
Class at
Publication: |
514/054 |
International
Class: |
A61K 31/739 20060101
A61K031/739 |
Goverment Interests
GOVERNMENT FUNDING
[0002] Work described herein was supported under grant AI 27448
awarded by the National Institutes of Health. The U.S. government
therefore may have certain rights in this invention.
Claims
1. An immunomodulatory method comprising contacting a human immune
cell with an agent that modulates interaction of a compound
comprising a Lewis antigen with the human immune cell such that
production by the human immune cell of at least one cytokine that
regulates development of a T helper type 1 (Th1 ) or T helper type
2 (Th2) response is modulated.
2. The method of claim 1, wherein production of IL-10 by the human
immune cell is stimulated.
3. (canceled)
4. The method of claim 1, wherein the agent comprises a Lewis.sup.y
oligosaccharide or a derivative thereof; a Lewis.sup.x
oligosaccharide or a derivative thereof; a Lewis.sup.a
oligosaccharide or a derivative thereof; or a Lewis.sup.b
oligosaccharide or a derivative thereof.
5-6. (canceled)
7. The method of claim 1, wherein the agent is administered to a
human subject such that production by human immune cells of the
human subject of at least one cytokine that regulates development
of a Th1 or Th2 response is stimulated.
8. The method of claim 1, wherein production of IL-10 by the human
immune cell is inhibited.
9-12. (canceled)
13. The method of claim 1, wherein the agent is administered to a
human subject such that production by human immune cells of the
human subject of at least one cytokine that regulates development
of a Th1 or Th2 response is inhibited.
14. The method of claim 1, wherein the human immune cell is a T
cell.
15. The method of claim 1, wherein the human immune cell is a
macrophage.
16. The method of claim 1, wherein the human immune cell is a B
cell.
17-22. (canceled)
23. The method of claim 15, wherein the agent is administered to a
subject such that production by macrophages of the subject of at
least one cytokine that regulates development of a Th1 or Th2
response is stimulated.
24-28. (canceled)
29. The method of claim 15, wherein the agent is administered to a
subject such that production by macrophages of the subject of at
least one cytokine that regulates development of a Th1 or Th2
response is inhibited.
30-35. (canceled)
36. The method of claim 14, wherein the agent is administered to a
subject such that production by T cells of the subject of at least
one cytokine that regulates development of a Th1 or Th2 response is
stimulated.
37-41. (canceled)
42. The method of claim 14, wherein the agent is administered to a
subject such that production by T cells of the subject of at least
one cytokine that regulates development of a Th1 or Th2 response is
inhibited.
43. A pharmaceutical composition comprising an agent that modulates
interaction of a compound comprising a Lewis antigen with human
immune cells of a human subject in an amount sufficient to modulate
production by the human immune cells of at least one cytokine that
regulates development of a T helper type 1 (Th1) or a T helper type
2 (Th2) response when the agent is administered to the human
subject, and a pharmaceutically acceptable carrier.
44. The pharmaceutical composition of claim 43, wherein the agent
is a stimulatory form of a compound comprising a Lewis antigen.
45. The pharmaceutical composition of claim 44, wherein the Lewis
antigen comprises a Lewis.sup.y oligosaccharide or a derivative
thereof; a Lewis.sup.x oligosaccharide or a derivative thereof; a
Lewis.sup.a oligosaccharide or a derivative thereof or a
Lewis.sup.b oligosaccharide or a derivative thereof.
46-47. (canceled)
48. The pharmaceutical composition of claim 43, wherein the agent
is an inhibitory form of a compound comprising a Lewis antigen.
49. The pharmaceutical composition of claim 48, wherein the Lewis
antigen comprises a Lewis.sup.y oligosaccharide or a derivative
thereof; a Lewis.sup.x oligosaccharide or a derivative thereof; a
Lewis.sup.a oligosaccharide or a derivative thereof; or a
Lewis.sup.b oligosaccharide or a derivative thereof.
50-54. (canceled)
55. A pharmaceutical composition comprising a conjugate of a Lewis
antigen, or derivative thereof, and a carrier molecule, such that
the Lewis antigen, or derivative thereof, comprises 10-25% of the
conjugate by weight, in an amount sufficient to stimulate
production of at least one cytokine that regulates development of a
T helper type 1 (Th1) or a T helper type 2 (Th2) response when the
agent is administered to the subject, and a pharmaceutically
acceptable carrier.
Description
RELATED APPLICATION
[0001] This application is a continuation of U.S. application Ser.
No. 08/597518 filed Jan. 31, 1996 (now U.S. Pat. No. 6841543,
issued Jan., 11, 2005), the entire contents of which are hereby
incorporated by reference.
BACKGROUND OF THE INVENTION
[0003] The T lymphocyte compartment of the immune system can be
divided into a variety of cell subsets. For example, CD4+ T cells
represent the T helper cell subset, whereas CD8+ T cells represent
the cytotoxic T cell subset. Additionally, CD4+ T helper cells
mature into distinct subpopulations that produce different panels
of cytokines: the T helper type 1 (Th1) subset produces
interleukin-2 (IL-2), interferon-.gamma. (IFN-.gamma.) and tumor
necrosis factor-.uparw. (TNF-.beta.), whereas the T helper type 2
(Th2) subset produces interleukin-4 (IL-4), interleukin-5 (IL-5),
interleukin-6 (IL-6) and interleukin-10 (IL-10). The Th1 and Th2
subsets also have differing functional activities. Th1 cells are
involved in inducing delayed type hypersensitivity responses,
whereas Th2 cells are involved in providing efficient "help" to B
lymphocytes and stimulate production of IgG1 and IgE antibodies.
For a review of Th1 and Th2 subsets, see Seder, R. A. and Paul, W.
E. (1994) Ann. Rev. Immunol. 12:635-673.
[0004] Cytokines are thought to play a dominant role in controlling
the differentiation of .tau. helper precursors (Thp) to either the
Th1 or Th2 lineage. Th1-associated cytokines, such as IFN-.gamma.,
can enhance the development of Th1 cells and inhibit the
development of Th2 cells, whereas Th2-associated cytokines, such as
IL-4 and IL-10, can enhance the development of Th2 cells and
inhibit the development of Th1 cells. Thus, cytokines can
reciprocally regulate the development and/or progression of either
a Th1 or a Th2 response.
[0005] The course of certain disease states is influenced by
whether a predominant Th1 response or Th2 response is mounted. For
example, in experimental leishmania infections in mice, animals
that are resistant to infection mount predominantly a Th1 response,
whereas animals that are susceptible to progressive infection mount
predominantly a Th2 response (Heinzel, F. P., et al. (1989) J. Exp.
Med 169:59-72; Locksley, R. M. and Scott, P. (1992)
Immunoparasitology Today 1:A58-A61). In murine schistosomiasis, a
Th1 to Th2 switch is observed coincident with the release of eggs
into the tissues by female parasites and is associated with a
worsening of the disease condition (Pearce, E. J., et al. (1991) J.
Exp. Med. 173:159-166; Grzych, J-M., et al. (1991)J. Immunol.
141:1322-1327; Kullberg, M. C., et al. (1992) J. Immunol.
148:3264-3270). Many human diseases, including chronic infections
(such as with human immunodeficiency virus (HIV) or tuberculosis)
and certain metastatic carcinomas, also are characterized by a Th1
to Th2 switch, with elevated expression of IL-10 (see e.g.,
Shearer, G. M. and Clerici, M. (1992) Prog. Chem. Immunol.
54:21-43; Clerici, M and Shearer, G. M. (1993) Immunology Today
14:107-111; Yamamura, M., et al. (1993) J. Clin. Invest.
91:1005-1010; Pisa, P., et al. (1992) Proc. Natl. Acad. Sci. USA
89:7708-7712; Fauci, A. S. (1988) Science 239:617-623).
Furthermore, certain autoimmune diseases have been shown to be
associated with a predominant Th1response. For example, patients
with rheumatoid arthritis have predominantly Th1 cells in synovial
tissue (Simon, A. K., et al. (1994) Proc. Natl. Acad. Sci. USA
91:8562-8566) and experimental autoimmune encephalomyelitis (EAE)
can be induced by autoreactive Th1 cells (Kuchroo, V. K., et al.
(1993) J. Immunol. 151:4371-4381).
[0006] Velupillai and Harn (Proc. Natl. Acad. Sci. USA (1994)
91:18-22) have shown that schistosome egg antigen (SEA), which
expresses the Lewis.sup.x antigen, and conjugates of the
Lewis.sup.x antigen, can stimulate IL-10 production by B cells from
Schistosoma mansoni infected mice, but not B cells from uninfected
mice, suggesting that during the course of S. mansoni infection,
the observed Th1to Th2 shift may results from IL-10 production by B
cells induced by SEA. This work, however, did not demonstrate
whether human immune cells (e.g., human immune cells in the absence
of S. mansoni infection) were responsive to Lewis
antigen-containing compounds, nor whether cell types other than B
cells, such as macrophages or T cells, could produce IL-10 in
response to stimulation with compounds comprising a Lewis antigen
in the absence of S. mansoni infection. Moreover, this work did not
demonstrate whether production other cytokines that regulate
development of Th1 and Th2 responses, such as IL-4, could be
stimulated
SUMMARY OF THE INVENTION
[0007] Given the role of either Th1 or Th2 cells in the development
or progression of many disease states, methods for influencing
whether a Th1 or Th2 response is mounted are desirable for a
variety of clinical situations. This invention provide methods for
modulating immune responses by modulating the interaction of immune
cells with a compound comprising a Lewis antigen such that
production by the immune cells of at least one cytokine that
regulates development of a Th1 or Th2 response is modulated. The
invention is based, at least in part, on the discovery that
stimulation of human immune cells, T cells or macrophages with
Lewis antigen-containing conjugates results in the production of
cytokines that regulate the development of Th1 or a Th2 response.
Moreover, it has now been discovered that human immune cells are
sensitive to stimulation by Lewis.sup.y antigen-containing
conjugates, that cells from human allergy patients and cancer
patients show responsiveness to Lewis antigens, that IL-4
production can be stimulated by Lewis antigen-containing conjugates
and that conjugates wherein the sugars represent approximately
20-24% of the conjugate by weight, or greater, are preferred for
stimulation. The immunomodulatory methods of the invention allow
for an immune response to be directed to either a Th1 or a Th2
response. The ability to influence the development of either a Th1
or a Th2 response using the immunomodulatory methods of the
invention is applicable to the treatment of a wide variety of
disorders, including cancer, infectious diseases (e.g., HIV and
tuberculosis), allergies and autoimmune diseases.
[0008] In one embodiment, the invention provides an
immunomodulatory method comprising contacting a human immune cell
with an agent that modulates interaction of a compound comprising a
Lewis antigen with the human immune cell such that production by
the human immune cell of at least one cytokine that regulates
development of a Th1 or Th2 response is modulated. The human immune
cell can be, for example, a T cell, a macrophage or a B cell. In
another embodiment, the invention provides an immunomodulatory
method comprising contacting a macrophage with an agent that
modulates interaction of a compound comprising a Lewis antigen with
the macrophage such that production by the macrophage of at least
one cytokine that regulates development of a Th1 or Th2 response is
modulated. In yet another embodiment, the invention provides an
immunomodulatory method comprising contacting a T cell with an
agent that modulates interaction of a compound comprising a Lewis
antigen with the T cell such that production by the T cell of at
least one cytokine that regulates development of a Th1 or Th2
response is modulated.
[0009] In one embodiment of the immunomodulatory methods of the
invention, production by immune cells of at least one cytokine
(preferably IL-10 or IL-4) that regulates development of a Th1 or
Th2 response is stimulated. In this embodiment, the agent with
which the immune cells are contacted preferably is a stimulatory
form of a compound comprising a Lewis antigen, such as a compound
comprising cross-linked (i.e., multivalent) Lewis.sup.y
oligosaccharides, Lewis.sup.x oligosaccharides, Lewis.sup.a
oligosaccharides or derivatives thereof (e.g., sulfated, sialylated
or sulfo-sialylated forms of these oligosaccharides). The
stimulatory compound can be, for example, a conjugate of the Lewis
antigen and a carrier molecule (e.g., human serum albumin or
polyacrylamide). For stimulating responses by human immune cells,
the agent preferably comprises a Lewis.sup.y oligosaccharide or a
derivative thereof.
[0010] In another embodiment of the immunomodulatory methods of the
invention, production by immune cells of at least one cytokine
(preferably IL-10 or IL-4) that regulates development of a Th1 or
Th2 response is inhibited. In this embodiment, the agent with which
the immune cells are contacted preferably is an inhibitory form of
a compound comprising a Lewis antigen, such as a soluble,
monovalent (i.e., non-crosslinked) form of a Lewis.sup.y
oligosaccharide, a Lewis.sup.x oligosaccharide, a Lewis.sup.a
oligosaccharide or a derivative thereof (e.g., sulfated, sialylated
or sulfo-sialylated forms of these oligosaccharides). For
inhibiting responses by human immune cells, the agent preferably
comprises a Lewis.sup.y oligosaccharide or a derivative
thereof.
[0011] The stimulatory or inhibitory compounds of the invention can
be contacted with immune cells in vitro to produce one or more
cytokines that regulate the development of a Th1 or Th2 response.
After in vitro stimulation, the immune cells can be administered to
a subject to influence whether a Th1 or a Th2 response predominates
in the subject. Alternatively, a stimulatory or inhibitory compound
of the invention can be administered to a subject such that
production of at least one cytokine that regulates development of a
Th1 or Th2 response is either stimulated or inhibited,
respectively, in the subject, thereby influencing whether a Th1 or
a Th2 response predominates in the subject. Accordingly, another
aspect of the invention pertains to pharmaceutical compositions
suitable for pharmaceutical administration. The pharmaceutical
compositions of the invention typically comprise a stimulatory or
inhibitory agent of the invention (e.g., a compound comprising a
Lewis antigen) and a pharmaceutically acceptable carrier. In one
embodiment, the composition is formulated to modulate responses by
human immune cells. In this embodiment, the active agent preferably
comprises a Lewis.sup.y oligosaccharide. In another embodiment, the
composition is formulated to modulate responses by macrophages. In
yet another embodiment, the composition is formulated to modulate
responses by T cells.
BRIEF DESCRIPTION OF THE DRAWINGS
[0012] FIG. 1A is a bar graph depicting the proliferation of
splenocytes from S. mansoni infected mice when stimulated with
either media alone, Con A, LPS. anti-IgM, SEA or LNFP III
conjugated to HSA.
[0013] FIG. 1B is a graph depicting the proliferation of
splenocytes from S. mansoni infected mice when stimulated with
increasing amounts of either Lewis.sup.a antigen, Lewis.sup.x
antigen, sialyl-Lewis.sup.x antigen or sialyl-Lewis.sup.a antigen,
each conjugated to polyacrylamide.
[0014] FIG. 1C is a bar graph depicting the proliferation of
splenocytes from uninfected mice when stimulated with either media
alone, Con A, LPS. anti-IgM, SEA or LNFP III conjugated to HSA.
[0015] FIG. 1D is a graph depicting the proliferation of
splenocytes from uninfected mice when stimulated with increasing
amounts of either Lewis.sup.a antigen, Lewis.sup.x antigen,
sialyl-Lewis.sup.x antigen or sialyl-Lewis.sup.a antigen, each
conjugated to polyacrylamide.
[0016] FIG. 2A is a bar graph depicting the production of IL-10by
splenocytes from S. mansoni infected mice when stimulated with
either media alone, Con A, LPS. anti-IgM, SEA or LNFP III
conjugated to HSA.
[0017] FIG. 2B is a graph depicting the production of IL-10 by
splenocytes from S. mansoni infected mice when stimulated with
increasing amounts of either Lewis.sup.a antigen, Lewis.sup.x
antigen, sialyl-Lewis.sup.x antigen or sialyl-Lewis.sup.a antigen,
each conjugated to polyacrylamide.
[0018] FIG. 2C is a bar graph depicting the production of IL-10 by
splenocytes from uninfected mice when stimulated with either media
alone, Con A, LPS. anti-IgM, SEA or LNFP III conjugated to HSA.
[0019] FIG. 2D is a graph depicting the production of IL-10 by
splenocytes from uninfected mice when stimulated with increasing
amounts of either Lewis.sup.a antigen, Lewis.sup.x antigen,
sialyl-Lewis.sup.x antigen or sialyl-Lewis.sup.a antigen, each
conjugated to polyacrylamide.
[0020] FIG. 3A is a bar graph depicting the production of IL-4 by
splenocytes from S. mansoni infected mice when stimulated with
either media alone, Con A, LPS. anti-IgM, SEA or LNFP III
conjugated to HSA.
[0021] FIG. 3B is a graph depicting the production of IL-4 by
splenocytes from S. mansoni infected mice when stimulated with
increasing amounts of either Lewis.sup.a antigen, Lewis.sup.x
antigen, sialyl-Lewis.sup.x antigen or sialyl-Lewis.sup.a antigen,
each conjugated to polyacrylamide.
[0022] FIG. 3C is a bar graph depicting the production of IL-4 by
splenocytes from uninfected mice when stimulated with either media
alone, Con A, LPS. anti-IgM, SEA or LNFP III conjugated to HSA.
[0023] FIG. 3D is a graph depicting the production of IL-4 by
splenocytes from uninfected mice when stimulated with increasing
amounts of either Lewis.sup.a antigen, Lewis.sup.x antigen,
sialyl-Lewis.sup.x antigen or sialyl-Lewis.sup.a antigen, each
conjugated to polyacrylamide.
DETAILED DESCRIPTION ON THE INVENTION
[0024] This invention provides immunomodulatory methods in which a
cell (e.g., a human immune cell, a macrophage or a T cell) is
contacted with an agent which modulates production by the cell of
one or more cytokines that regulate Th1 or Th2 responses. The
invention is based, at least in part, on the discovery that when
human immune cells, macrophages or T cells are stimulated with a
multivalent, crosslinked form of a Lewis antigen, the cells produce
one or more cytokines (such as IL-10and/or IL-4) that promote the
development of a Th2 response and inhibit the development of a
Th1response. Human immune cells that exhibit this responsiveness
include T cells from a pollen allergic individual and peripheral
blood mononuclear cells from individuals with colon or lung
carcinomas or B lymphoma. Moreover, it has now been discovered that
human immune cells preferentially respond to compounds comprising a
Lewis.sup.y oligosaccharides. Still further, it has now been
discovered that Lewis antigens conjugated to a carrier at a ligand
density such that the sugars represent approximately 20-24% of the
conjugate by weight are especially effective at stimulating
production of cytokines that promote the development of a Th2
response (e.g., IL-10 and IL-4), an in particular can stimulate
IL-4 production by splenocytes of uninfected subjects. A preferred
carrier for such stimulatory conjugates is polyacrylamide.
[0025] In contrast to these stimulatory methods, contact of the
cells with a monovalent, non-crosslinked form of the Lewis antigen
inhibits the production of cytokines that promote Th2 responses and
downregulate Th1 responses that normally are produced when the
cells encounter a stimulatory form of a Lewis antigen. Thus, the
methods of the invention allow for cytokine production either to be
stimulated or inhibited, depending on whether a stimulatory agent
or an inhibitory agent is used. Accordingly, the immunomodulatory
methods of the invention allow for an immune response to be
directed to either a Th1 or a Th2 response. The ability to
influence the development of either a Th1 or a Th2 response using
the immunomodulatory methods of the invention is applicable to the
treatment of a wide variety of disorders, including cancer,
infectious diseases (e.g., HIV and tuberculosis), allergies and
autoimmune diseases.
[0026] In order that the present invention may be more readily
understood, certain terms are first defined. Standard abbreviations
for sugars are used herein.
[0027] As used herein, the term "Lewis antigen" is intended to
include carbohydrates having as a core sequence either the lacto
type I structure {Gal(.beta.1-3)GlcNac} or the lacto type II
structure {Gal(.beta.1-4)GlcNac}, substituted with one or more
fucosyl residues. The Lewis antigen may comprise a single
substituted core sequence or a repetitive series of substituted
core sequences. Moreover, the core sequence may be present within a
larger sugar. Accordingly, a Lewis antigen-containing
oligosaccharide can be, for example, a trisaccharide, a
tetrasaccharide, a pentasaccharide, and so on. Types of Lewis
antigens include Lewis.sup.x, Lewis.sup.y, Lewis.sup.a and
Lewis.sup.b oligosaccharides and derivatives thereof. Synthetic
structural homologues of these carbohydrates that retain the
immunomodulatory capacity described herein are also intended to be
encompassed by the term "Lewis antigen".
[0028] As used herein, the term "Lewis.sup.x oligosaccharide"
refers to a lacto type II carbohydrate comprising the structure:
{Gal(.beta.1-4)[Fuc(.alpha.1-3)]GlcNac}.
[0029] As used herein, the term "Lewis.sup.y oligosaccharide"
refers to a lacto type II carbohydrate comprising the structure:
{Fuc(.alpha.1-2)Gal(.beta.1-4)[Fuc(.alpha.1-3)]GlcNac}.
[0030] As used herein, the term "Lewis.sup.a oligosaccharide"
refers to a lacto type I carbohydrate comprising the structure:
{Gal(.beta.1-3)[Fuc(.alpha.1-4)]GlcNac}.
[0031] As used herein, the term "Lewis.sup.b oligosaccharide"
refers to a lacto type I carbohydrate comprising the structure:
{Fuc(.alpha.1-2)Gal(.beta.1-3)[Fuc(.alpha.1-4)]GlcNac}.
[0032] As used herein, a "derivative" of a Lewis oligosaccharide
refers to a Lewis oligosaccharide having one or more additional
substituent groups. Examples of derivatives include terminally
sialylated forms of Lewis oligosaccharides (e.g.,
sialyl-Lewis.sup.x, sialyl-Lewis.sup.y, sialyl-Lewis.sup.a,
sialyl-Lewis.sup.b), sulfated forms of Lewis oligosaccharides and
sulfo-sialylated forms of Lewis oligosaccharides.
[0033] As used herein, the term "human immune cell" is intended to
include cells of the human immune cell which are capable of
producing cytokines that regulate the development of a Th1or Th2
response. Examples of human immune cells include human T cells,
human macrophages and human B cells.
[0034] As used herein, the term "macrophage" is intended to include
all cells within the macrophage lineage, including monocytes,
circulating macrophages, tissue macrophages, activated macrophages,
and the like, from a mammal (e.g., human or mouse).
[0035] As used herein, the term "T cell" (i.e., T lymphocyte) is
intended to include all cells within the T cell lineage, including
thymocytes, immature T cells, mature T cells and the like, from a
mammal (e.g., human or mouse).
[0036] As used herein, a "T helper type 2 response" (Th2 response)
refers to a response by CD4+ T cells that is characterized by the
production of one or more cytokines selected from IL-4, IL-5, IL-6
and IL-10, and that is associated with efficient B cell "help"
provided by the Th2 cells (e.g., enhanced IgG1 and/or IgE
production).
[0037] As used herein, a "T helper type 1 response" (Th1 response)
refers to a response by CD4+ T cells that is characterized by the
production of one or more cytokines selected from IL-2, IFN-g and
TNF-.beta., and that is associated with delayed type
hypersensitivity responses.
[0038] As used herein, the term "a cytokine that regulates
development of a Th1 or Th2 response" is intended to include
cytokines (produced by Th1 or Th2 cells or other cell types), that
have a positive or negative effect on the initiation and/or
progression of a Th1 or Th2 response, and especially those
cytokines that have reciprocal effects on the development of the
Th1 vs. Th2 subpopulations, in particular, cytokines that promote
the development of a Th2 response and downregulate the development
of a Th1 response. Preferred cytokines that are produced by the
methods of the invention are IL-10, IL-4 and prostaglandin E.sub.2
(PGE.sub.2). The most preferred cytokine produced by the methods of
the invention is IL-10.
[0039] As used herein, the term "development of a Th1 or Th2
response" is intended to include initiation of either a Th1 or Th2
response (e.g., commitment of T helper precursors to either the Th1
or Th2 lineage) and progression of either a Th1 or Th2 response
(e.g., further differentiation of cells to either the Th1 or Th2
phenotype and/or continued function of Th1 or Th2 cells during an
ongoing immune response). Thus, a cytokine(s) produced in
accordance with the methods of the invention may have an effect on
the initiation of a Th1 or Th2 response, the progression of a Th1
or Th2 response, or both.
[0040] As used herein, the various forms of the term "modulation"
are intended to include stimulation (e.g., increasing or
upregulating a particular response or activity) and inhibition
(e.g., decreasing or downregulating a particular response or
activity).
[0041] As used herein, the term "contacting" (i.e., contacting an
agent with a cell) is intended to include incubating the agent and
the cell together in vitro (e.g., adding the agent to cells in
culture) and administering the agent to a subject such that the
agent and cells of the subject are contacted in vivo.
[0042] Various aspects of the invention are described in further
detail in the following subsections.
I. Immunomodulatory Agents
[0043] In the immunomodulatory methods of the invention, a cell
(e.g., a human immune cell, macrophage or T cell) is contacted with
an agent that modulates interaction of a compound comprising a
Lewis antigen with the cell such that production by the cell of at
least one cytokine that regulates development of a Th1 or Th2
response is modulated. Preferably, the agent itself comprises a
Lewis antigen, as described in further detail below. In one
embodiment, the agent is a "stimulatory agent", which stimulates
production by the cell of at least one cytokine that regulates
development of a Th1 or Th2 response. In another embodiment, the
agent is an "inhibitory agent", which inhibits production by the
cell of at least one cytokine that regulates development of a Th1
or Th2 response (i.e., the inhibitory agent can block the
production of cytokines that normally occurs when the cell
encounters a stimulatory form of a Lewis antigen).
[0044] A. Stimulatory Agents
[0045] The stimulatory agents of the invention stimulate production
by cells (e.g., human immune cells, macrophages or T cells) of at
least one cytokine that regulates development of a Th1 or Th2
response. In a preferred embodiment, the stimulatory agent is a
stimulatory form of a compound comprising a Lewis antigen. A
"stimulatory form of a compound comprising a Lewis antigen"
typically is one in which the carbohydrate structure is present in
a multivalent, crosslinked form. In a preferred embodiment, the
stimulatory form of a compound comprising a Lewis antigen is a
conjugate of a carrier molecule and multiple carbohydrate molecules
expressing a Lewis antigen. For example, carbohydrate molecules can
be conjugated to a protein carrier, such as a conjugate of human
serum albumin (HSA) and Lewis.sup.y oligosaccharides (referred to
herein as HSA-Le.sup.y). When a sugar-carrier protein conjugate is
to be administered to a subject, the carrier protein should be
selected such that an immunological reaction to the carrier protein
is not stimulated in the subject (e.g., a human carrier protein
should be used with a human subject, etc.). Alternative to a
carrier protein, multiple Lewis antigens can be conjugated to other
carrier molecules, such as a solid support, such as beads (e.g.,
polyacrylamide, agarose, sepharose, polystyrene and the like) or a
plate. The degree of stimulatory ability of the conjugate is
influenced by the density of sugars conjugated to the carrier (see
Example 4). Preferably, the sugar molecules comprise at least
10-25% of the conjugate by weight, more preferably at least 15-25%
of the conjugate by weight and even more preferably at least 20-25%
of the conjugate by weight. In a preferred embodiment, the
stimulatory form of a compound comprising a Lewis antigen is a
conjugate of multiple carbohydrate molecules expressing a Lewis
antigen and the carrier polyacrylamide. More preferably, the
polyacrylamide conjugates comprise 25 to 30 (or more)
sugars/conjugate, wherein the average molecular weight of the
conjugate is approximately 30 kD.
[0046] The Lewis antigens present in the conjugate can be, for
example, Lewis.sup.y, Lewis.sup.x, Lewis.sup.a or Lewis.sup.b
oligosaccharides, or derivatives thereof. For stimulation of human
cells, the stimulatory agent preferably comprises Lewis.sup.y
oligosaccharides or derivatives thereof. Within the stimulatory
agent, the Lewis antigen can be present within a larger
carbohydrate structure. For example, the carbohydrate portion of
the stimulatory agent can be lacto-N-fucopentaose III (LNFP-III),
which has the structure:
{Gal(.beta.1-4)[Fuc(.alpha.1-3)]GlcNac(.beta.1-3)Gal(.beta.1-4)Glc}
and comprises the Lewis.sup.x oligosaccharide, or
lacto-N-difucohexose I (LND), which has the structure:
{Fuc(.alpha.1-2)Gal(.beta.1-3)[Fuc(.alpha.1-4)]GlcNac(.beta.1-3)Gal(.beta-
.1-4)Glc} and comprises the Lewis.sup.boligosaccharide. Other
related carbohydrates comprising Lewis antigens that are suitable
for use in a stimulatory agent of the invention will be apparent to
those skilled in the art.
[0047] In addition to conjugates comprising Lewis
antigen-containing sugars described above, another form of a
stimulatory agent comprising a Lewis antigen is an isolated protein
that naturally expresses Lewis antigens in a form suitable for
stimulatory activity. One example of such a protein is schistosome
egg antigen (SEA), which expresses the Lewis.sup.x oligosaccharide.
Other proteins that have been reported to express Lewis antigens
include tumor-associated antigens (see e.g., Pauli, B. U., et al.
(1992) Trends in Glycoscience and Glycotechnology 4:405-414;
Hakomori, S-I. (1989) Adv. Cancer Res. 52:257-331) and HIV gp120
(Adachi, M., et al. (1988)J. Exp. Med 167:323-331).
[0048] Stimulatory agents for use in the methods of the invention
can be purchased commercially or can be purified or synthesized by
standard methods. Conjugates of Lewis antigen-containing sugars and
a carrier protein (e.g., HSA) are available from Accurate
Chemicals, Westbury, N.Y. Conjugates of Lewis antigen-containing
sugars and polyacrylamide are available from GlycoTech, Rockville,
Md. Schistosome egg antigen (SEA) can be purified from Schistosoma
mansoni eggs as described in Harn, D. H., et al. (1984) J. Exp.
Med. 159:1371-1387. Lewis antigen-containing sugars, or derivatives
thereof, can be conjugated to a carrier protein or solid support
(e.g., beads or a plate) by standard methods, for example using a
chemical cross-linking agent. A wide variety of bifunctional or
polyfunctional cross-linking reagents, both homo- and
heterofunctional, are known in the art and are commercially
available (e.g., Pierce Chemical Co., Rockford, Ill).
[0049] The ability of a stimulatory agent of the invention to
stimulate production by immune cells of at least one cytokine that
regulates a Th1 or Th2 response can be evaluated using an in vitro
culture system such as that described in the Examples. Cells (e.g.,
peripheral blood mononuclear cells) are cultured in the presence of
the stimulatory agent to be evaluated (e.g., at a concentration of
100 .mu.M for sugar conjugates) in a medium suitable for culture of
the chosen cells. After a period of time (e.g., 24-72 hours),
production of a cytokine that regulates development of a Th1or Th2
response is assessed by determining the level of the cytokine in
the culture supernatant. Preferably, the cytokine assayed is IL-10.
Additionally or alternatively, IL-4 and/or PGE.sub.2 levels can be
assessed. Cytokine levels in the culture supernatant can be
measured by standard methods, such as by an enzyme linked
immunosorbent assay (ELISA) utilizing a monoclonal antibody that
specifically binds the cytokine. An ELISA for measuring IL-10
levels is described further in Kullberg, M. C., et al (1992) J.
Immunol. 148:3264-3270. An ELISA kit for measuring PGE.sub.2 levels
is commercially available from Advanced Magnetics, Cambridge, Mass.
The ability of a stimulatory agent to stimulate cytokine production
is evidenced by a higher level of cytokine (e.g., IL-10) in the
supernatants of cells cultured in the presence of the stimulatory
agent compared to the level of cytokine in the supernatant of cells
cultured on the absence of the stimulatory agent.
[0050] B. Inhibitory Agents
[0051] The inhibitory agents of the invention inhibit production by
cells (e.g., T cells, macrophages or human immune cells) of at
least one cytokine that regulates development of a Th1 or Th2
response. More specifically, cytokine production by a cell that
normally occurs when the cell encounters a stimulatory form of a
Lewis antigen can be inhibited by contacting the cell with an
inhibitory agent of the invention (i.e., the inhibitory agents of
the invention act as blocking agents to inhibit cytokine production
that normally would result from interaction of the cell with a
stimulatory form of a Lewis antigen). In a preferred embodiment,
the inhibitory agent is an inhibitory form of a compound comprising
a Lewis antigen. A "inhibitory form of a compound comprising a
Lewis antigen" typically is one in which the carbohydrate structure
is present in a monovalent, non-crosslinked form. Preferred
inhibitory agents are soluble, "free" sugars comprising a Lewis
antigen. The Lewis antigen expressed by the inhibitory agent can
be, for example, a Lewis.sup.y, Lewis.sup.x, Lewis.sup.a or
Lewis.sup.b oligosaccharide (a single substituted core sequence or
a repetitive series of substituted core sequences) or derivatives
thereof. For inhibition of human cells, the inhibitory agent
preferably comprises a Lewis.sup.y oligosaccharide or a derivative
thereof. Within the inhibitory agent, the Lewis antigen can be
present within a larger carbohydrate structure. For example, the
carbohydrate portion of the inhibitory agent can be
lacto-N-fucopentaose III (LNFP-III), which has the structure:
{Gal(.beta.1-4)[Fuc(.alpha.1-3)]GlcNac(.beta.1-3)Gal(.beta.1-4)Glc}
and comprises the Lewis.sup.x oligosaccharide, or
lacto-N-difucohexose I (LND), which has the structure:
{Fuc(.alpha.1-2)Gal(.beta.1-3)[Fuc(.alpha.1-4)]GlcNac(.beta.1-3)Gal(.beta-
.1-4)Glc} and comprises the Lewis.sup.b oligosaccharide. Other
related carbohydrates comprising Lewis antigens that are suitable
for use in an inhibitory agent of the invention will be apparent to
those skilled in the art. Soluble, free sugars comprising a Lewis
antigen for use as inhibitory agents can be purchased commercially
or synthesized by standard methods (as described above).
[0052] In addition to free sugars comprising Lewis antigens,
another form of an inhibitory agent is an antibody to a Lewis
antigen. Polyclonal antibodies, or more preferably monoclonal
antibodies (mAbs), that specifically bind a Lewis antigen (e.g., a
Lewis.sup.y, Lewis.sup.x, Lewis.sup.a or Lewis.sup.b
oligosaccharide, or derivatives thereof) can be used as inhibitory
agents. Monoclonal antibodies to Lewis antigens, or derivatives
thereof, are known in the art (e.g., mAbs to Le.sup.x, Le.sup.y,
sialyl-Le.sup.x or sialyl-Le.sup.y, from the Biomembrane Institute,
Seattle, Wash., as described in Martensson, S., et al. (1995) Hum.
Pathol. 26:735-739; and mAbs to Le.sup.a or sialyl-Le.sup.a, as
described in Kageshita, T., et al. (1995) Cancer Res.
55:1748-1751)
[0053] The ability of an inhibitory agent of the invention to
inhibit production by immune cells of at least one cytokine that
regulates a Th1 or Th2 response that normally occurs when the
immune cell encounters a stimulatory form of a Lewis antigen can be
evaluated using an in vitro culture system such as that described
in the Examples. Cells (e.g., peripheral blood mononuclear cells)
are cultured in the presence of both an inhibitory agent to be
evaluated and a stimulatory agent as described in the preceding
section (e.g., a sugar conjugate at a concentration of 100 .mu.M)
in a medium suitable for culture of the chosen cells. After a
period of time (e.g., 24-72 hours), production of a cytokine that
regulates development of a Th1 or Th2 response (e.g., IL-10 or
IL-4) is assessed by determining the level of the cytokine in the
culture supernatant as described in the preceding section. The
ability of an inhibitory agent to inhibit cytokine production is
evidenced by a lower level of cytokine in the supernatants of cells
cultured in the presence of both the inhibitory agent and the
stimulatory agent compared to the level of cytokine in the
supernatant of cells cultured only in the presence of the
stimulatory agent.
II. Pharmaceutical Compositions
[0054] Another aspect of the invention pertains to pharmaceutical
compositions of the stimulatory or inhibitory agents of the
invention. The pharmaceutical compositions of the invention
typically comprise a stimulatory or inhibitory agent of the
invention and a pharmaceutically acceptable carrier. As used herein
"pharmaceutically acceptable carrier" includes any and all
solvents, dispersion media, coatings, antibacterial and antifungal
agents, isotonic and absorption delaying agents, and the like that
are physiologically compatible. The type of carrier can be selected
based upon the intended route of administration. In various
embodiments, the carrier is suitable for intravenous,
intraperitoneal, subcutaneous, intramuscular, transdermal or oral
administration. In a preferred embodiment, the composition is
formulated such that it is suitable for intravenous administration.
Pharmaceutically acceptable carriers include sterile aqueous
solutions or dispersions and sterile powders for the extemporaneous
preparation of sterile injectable solutions or dispersion. The use
of such media and agents for pharmaceutically active substances is
well known in the art. Except insofar as any conventional media or
agent is incompatible with the active compound, use thereof in the
pharmaceutical compositions of the invention is contemplated.
Supplementary active compounds can also be incorporated into the
compositions.
[0055] Therapeutic compositions typically must be sterile and
stable under the conditions of manufacture and storage. The
composition can be formulated as a solution, microemulsion,
liposome, or other ordered structure suitable to high drug
concentration. The carrier can be a solvent or dispersion medium
containing, for example, water, ethanol, polyol (for example,
glycerol, propylene glycol, and liquid polyetheylene glycol, and
the like), and suitable mixtures thereof. The proper fluidity can
be maintained, for example, by the use of a coating such as
lecithin, by the maintenance of the required particle size in the
case of dispersion and by the use of surfactants. In many cases, it
will be preferable to include isotonic agents, for example, sugars,
polyalcohols such as manitol, sorbitol, or sodium chloride in the
composition. Prolonged absorption of the injectable compositions
can be brought about by including in the composition an agent which
delays absorption, for example, monostearate salts and gelatin.
Moreover, the modulators can be administered in a time release
formulation, for example in a composition which includes a slow
release polymer. The active compounds can be prepared with carriers
that will protect the compound against rapid release, such as a
controlled release formulation, including implants and
microencapsulated delivery systems. Biodegradable, biocompatible
polymers can be used, such as ethylene vinyl acetate,
polyanhydrides, polyglycolic acid, collagen, polyorthoesters,
polylactic acid and polylactic, polyglycolic copolymers (PLG). Many
methods for the preparation of such formulations are patented or
generally known to those skilled in the art.
[0056] Sterile injectable solutions can be prepared by
incorporating the active compound in the required amount in an
appropriate solvent with one or a combination of ingredients
enumerated above, as required, followed by filtered sterilization.
Generally, dispersions are prepared by incorporating the active
compound into a sterile vehicle which contains a basic dispersion
medium and the required other ingredients from those enumerated
above. In the case of sterile powders for the preparation of
sterile injectable solutions, the preferred methods of preparation
are vacuum drying and freeze-drying which yields a powder of the
active ingredient plus any additional desired ingredient from a
previously sterile-filtered solution thereof.
[0057] Depending on the route of administration, the agent may be
coated in a material to protect it from the action of enzymes,
acids and other natural conditions which may inactivate the agent.
For example, the agent can be administered to a subject in an
appropriate carrier or diluent co-administered with enzyme
inhibitors or in an appropriate carrier such as liposomes.
Pharmaceutically acceptable diluents include saline and aqueous
buffer solutions. Enzyme inhibitors include pancreatic trypsin
inhibitor, diisopropylfluorophosphate (DEP) and trasylol. Liposomes
include water-in-oil-in-water emulsions as well as conventional
liposomes (Strejan, et al., (1984) J. Neuroimmunol 7:27).
Dispersions can also be prepared in glycerol, liquid polyethylene
glycols, and mixtures thereof and in oils. Under ordinary
conditions of storage and use, these preparations may contain a
preservative to prevent the growth of microorganisms.
[0058] The active agent in the composition (i.e., a stimulatory or
inhibitory agent of the invention) preferably is formulated in the
composition in a therapeutically effective amount. A
"therapeutically effective amount" refers to an amount effective,
at dosages and for periods of time necessary, to achieve the
desired therapeutic result, such as the development or progression
of a Th2 or Th1 response to thereby influence the therapeutic
course of a particular disease state. A therapeutically effective
amount of an active agent may vary according to factors such as the
disease state, age, sex, and weight of the individual, and the
ability of the agent to elicit a desired response in the
individual. Dosage regimens may be adjusted to provide the optimum
therapeutic response. A therapeutically effective amount is also
one in which any toxic or detrimental effects of the agent are
outweighed by the therapeutically beneficial effects. In another
embodiment, the active agent is formulated in the composition in a
prophylactically effective amount. A "prophylactically effective
amount" refers to an amount effective, at dosages and for periods
of time necessary, to achieve the desired prophylactic result, for
example, influencing the development or progression of either a Th2
or Th1 response for prophylactic purposes. Typically, since a
prophylactic dose is used in subjects prior to or at an earlier
stage of disease, the prophylactically effective amount will be
less than the therapeutically effective amount.
[0059] A non-limiting range for a therapeutically or
prophylactically effective amounts of a stimulatory or inhibitory
agent of the invention is 0.01 nM-20 mM. It is to be noted that
dosage values may vary with the severity of the condition to be
alleviated. It is to be further understood that for any particular
subject, specific dosage regimens should be adjusted over time
according to the individual need and the professional judgment of
the person administering or supervising the administration of the
compositions, and that dosage ranges set forth herein are exemplary
only and are not intended to limit the scope or practice of the
claimed composition.
[0060] The amount of active compound in the composition may vary
according to factors such as the disease state, age, sex, and
weight of the individual. Dosage regimens may be adjusted to
provide the optimum therapeutic response. For example, a single
bolus may be administered, several divided doses may be
administered over time or the dose may be proportionally reduced or
increased as indicated by the exigencies of the therapeutic
situation. It is especially advantageous to formulate parenteral
compositions in dosage unit form for ease of administration and
uniformity of dosage. Dosage unit form as used herein refers to
physically discrete units suited as unitary dosages for the
mammalian subjects to be treated; each unit containing a
predetermined quantity of active compound calculated to produce the
desired therapeutic effect in association with the required
pharmaceutical carrier. The specification for the dosage unit forms
of the invention are dictated by and directly dependent on (a) the
unique characteristics of the active compound and the particular
therapeutic effect to be achieved, and (b) the limitations inherent
in the art of compounding such an active compound for the treatment
of sensitivity in individuals.
[0061] A stimulatory or inhibitory agent of the invention can be
formulated into a pharmaceutical composition wherein the agent is
the only active compound therein. Alternatively, the pharmaceutical
composition can contain additional active compounds. For example,
two or more stimulatory or inhibitory agents may be used in
combination. Moreover, a stimulatory or inhibitory agent of the
invention can be combined with one or more other agents that have
immunomodulatory properties. For example, a stimulatory or
inhibitory agent may be combined with one or more cytokines or
adjuvants.
[0062] A pharmaceutical composition of the invention, comprising a
stimulatory or inhibitory agent of the invention, can be
administered to a subject to modulate immune responses (e.g., Th2
vs. Th1 responses) in the subject. As used herein, the term
"subject" is intended to include living organisms in which an
immune response can be elicited, e.g., mammals. Examples of
subjects include humans, dogs, cats, mice, rats, and transgenic
species thereof.
III. Modulation of Immune Responses
[0063] The invention provides immunomodulatory methods that can be
used to influence the development of a Th1 or a Th2 response. In
the methods of the invention, a cell (e.g., a human immune cell, a
macrophage or a T cell) is contacted with an agent that modulates
interaction of a compound comprising a Lewis antigen with the cell
such that production by the cell of at least one cytokine that
regulates development of a Th1 or Th2 response is modulated (i.e.,
stimulated or inhibited). The methods of the invention can be
practiced either in vitro or in vivo. For practicing the method of
the invention in vitro, cells can be obtained from a subject by
standard methods and incubated (i.e., cultured) in vitro with a
stimulatory or inhibitory agent of the invention to stimulate or
inhibit, respectively, the production of at least one cytokine that
regulates development of a Th1 or Th2 response. For example,
peripheral blood mononuclear cells (PBMCs) can be obtained from a
subject and isolated by density gradient centrifugation, e.g., with
Ficoll/Hypaque. Specific cell populations can be depleted or
enriched using standard methods. For example, monocytes/macrophages
can be isolated by adherence on plastic. T cells or B cells can be
enriched or depleted, for example, by positive and/or negative
selection using antibodies to T cell or B cell surface markers, for
example by incubating cells with a specific mouse monoclonal
antibody (mAb), followed by isolation of cells that bind the mAb
using anti-mouse-Ig coated magnetic beads. Monoclonal antibodies to
cell surface markers are commercially available.
[0064] When a stimulatory agent is used in vitro, resulting in
stimulation of the production of at least one cytokine that
regulates Th1 or Th2 responses, the cytokine(s) can be recovered
from the culture supernatant for further use. For example, the
culture supernatant, or a purified fraction thereof, can be applied
to T cells in culture to influence the development of Th1 or Th2
cells in vitro. Alternatively, the culture supernatant, or a
purified fraction thereof, can be administered to a subject to
influence the development of Th1 vs. Th2 responses in the subject.
Moreover, cells treated in vitro with either a stimulatory or
inhibitory agent can be administered to a subject to influence the
development of a Th1 vs. Th2 response in the subject. For
administration to a subject, it may be preferable to first remove
residual agents in the culture from the cells before administering
them to the subject. This can be done for example by a
Ficoll/Hypaque gradient centrifugation of the cells.
[0065] For practicing the methods of the invention in vivo, a
stimulatory or inhibitory agent is administered to a subject in a
pharmacologically acceptable carrier (as described in the previous
section) in amounts sufficient to modulate production of at least
one cytokine that regulates development of a Th1or Th2 response in
the subject. A preferred route of administration for the agent is
intravenous, although any route of administration suitable for
achieving the desired immunomodulatory effect is contemplated by
the invention.
[0066] The stimulatory methods of the invention (i.e., methods that
use a stimulatory agent) result in production of at least one
cytokine (preferably IL-10 or IL-4) which promotes development of a
Th2 response and/or inhibits development of a Th1 response.
Alternatively, the inhibitory methods of the invention (i.e.,
methods that use an inhibitory agent) inhibit the production of at
least one cytokine (preferably IL-10 or IL-4) which promotes
development of a Th2 response and/or inhibits development of a
Th1response. Thus, to apply a method of the invention to the
treatment of a disease condition wherein a Th1 response is
beneficial, an immunomodulatory method is selected that promotes
such a Th1response (while downregulating a Th2 response).
Alternatively, to apply a method of the invention to the treatment
of a disease condition wherein a Th2 response is beneficial, an
immunomodulatory method is selected that promotes such a Th2
response (while downregulating a Th1 response). Application of the
methods of the invention to the treatment of disease conditions may
result in cure of the condition, a decrease in the type or number
of symptoms associated with the condition, either in the long term
or short term (i.e., amelioration of the condition) or simply a
transient beneficial effect to the subject.
[0067] Numerous disease conditions associated with a predominant
Th1 or Th2-type response have been identified and could benefit
from modulation of the type of response mounted in the individual
suffering from the disease condition. Application of the
immunomodulatory methods of the invention to such diseases is
described in further detail below.
[0068] A. Cancer
[0069] The inhibitory methods of the invention can be used to
inhibit the production of cytokines that upregulate Th2 responses
and downregulate Th1 responses in cancer patients, thereby
promoting a Th1response in the patients to ameliorate the course of
the disease. The expression of Th2-promoting cytokines, in
particular IL-10, has been reported to be elevated in cancer
patients (see e.g., Yamamura, M., et al. (1993) J. Clin. Invest.
91:1005-1010; Pisa, P., et al. (1992) Proc. Natl. Acad Sci. USA
89:7708-7712). Moreover, malignant cells have been reported to
express tumor-associated antigens that comprise Lewis antigens (see
e.g., Pauli, B. U., et al. (1992) Trends in Glycoscience and
Glycotechnology 4:405-414; Hakomori, S-I. (1989) Adv. Cancer Res.
52:257-331; Martensson, S., et al. (1995) Hum. Pathol. 26:735-739;
Kageshita, T., et al. (1995) Cancer Res. 55:1748-1751; Ohta, S., et
al. (1995) Immunol. Letters 44:35-40). Still further, as
demonstrated herein in Examples 2 and 3, peripheral blood
mononuclear cells from patients with a variety of cancers (e.g.,
lung carcinoma, colon carcinoma or B lymphoma) respond to
stimulation with Lewis antigen-protein carrier conjugates by
producing IL10. Thus, in the natural setting, stimulatory forms of
Lewis antigens on tumor cells likely stimulate IL-10 production in
cancer patients, thereby shifting the immune response toward a Th2
response and away from a Th1response. This Th2 to Th1shift can be
counteracted by treating a cancer patient with an inhibitory agent
of the invention to inhibit IL-10 production in the patient and
promote a Th1 response.
[0070] A preferred inhibitory agent for inhibiting IL-10 production
in human subjects comprises an inhibitory form of a Lewis.sup.y
oligosaccharide or derivative thereof (e.g., soluble, free
Le.sup.y), although the particular Lewis antigen effective for
inhibiting IL-10 production in individual cancer patients may vary
for different patients and/or tumors (e.g., as shown in Examples 2
and 3, cells from two patients with B lymphoma and one patient with
colon carcinoma responded preferentially to Le.sup.y, whereas cells
from one patient with B lymphoma responded preferentially to
Le.sup.b and cells from one patient with lung carcinoma responded
to Le.sup.x). To determine which inhibitory agent is likely to be
most effective for a particular cancer patient, it may be necessary
first to determine which Lewis antigen is expressed on the tumor
cells of the patient (e.g., by reacting a sample of tumor cells in
vitro with a panel of mAbs that bind to different Lewis antigens)
and/or which Lewis antigen the cells from the cancer patient
preferentially respond to in vitro (e.g., by culturing PBMCs from
the cancer patient in vitro with various stimulatory forms of Lewis
antigens in an assay as described in the Examples). Based on these
in vitro tests, an appropriate inhibitory agent can be selected
that, when administered to the cancer patient, will block the
stimulation of IL-10 production that results when cells of the
cancer patient encounter Lewis antigens on tumor cells in vivo.
[0071] B. Infectious Disease
[0072] The inhibitory methods of the invention also can be used to
inhibit the production of cytokines that upregulate Th2 responses
and downregulate Th1 responses in patients with infectious
diseases, thereby promoting a Th1 response in the patients to
ameliorate the course of the disease. The expression of
Th2-promoting cytokines, in particular IL-10, has been reported to
increase during a variety of infections, including HIV infection,
tuberculosis, leishmaniasis, schistosomiasis, filarial nematode
infection and intestinal nematode infection (see e.g.; Shearer, G.
M. and Clerici, M. (1992) Prog. Chem. Immunol. 54:21-43; Clerici, M
and Shearer, G. M. (1993) Immunology Today 14:107-1 11; Fauci, A.
S. (1988) Science 239:617-623; Locksley, R. M. and Scott, P. (1992)
Immunoparasitology Today 1:A58-A61; Pearce, E. J., et al. (1991) J.
Exp. Med. 173:159-166; Grzych, J-M., et al. (1991) J. Immunol.
141:1322-1327; Kullberg, M. C., et al. (1992) J. Immunol.
148:3264-3270; Bancroft, A. J., et al. (1993) J. Immunol.
150:1395-1402; Pearlman, E., et al. (1993) Infect. Immun.
61:1105-1112; Else, K. J., et al. (1994) J. Exp. Med. 179:347-351)
and is associated with a Th1 to Th2 shift. Infected cells and/or
proteins from infectious agents have been reported to express Lewis
antigens. For example, the Lewis.sup.y oligosaccharide has been
reported to be expressed by HIV-infected cells (Adachi, M., et al.
(1988) J. Exp. Med. 167:323-331). Schistosome egg antigen expresses
the Lewis.sup.x oligosaccharide (Ko, A., et al. (1987) Proc. Natl.
Acad. Sci. USA 87:4159-4163). Liver cells in chronic active
hepatitis express the Lewis.sup.y oligosaccharide (Muguruma, M, et
al. (1994) Anatomic Pathology 102:176-181), while Kupffer cells
express sialyl oligomeric Lewis.sup.x (Okado, Y. and Tsuji, T.
(1990) Lancet 335:1302-1307). Thus, during the course of various
natural infection, stimulatory forms of Lewis antigens on infected
cells and/or expressed by proteins of the infectious agent likely
stimulate IL-10 production in the infected subject, thereby
shifting the immune response toward a Th2 response and away from a
Th1 response. This Th2 to Th1 shift can be counteracted by treating
the infected subject with an inhibitory agent of the invention to
inhibit IL-10 production in the infected subject and promote a Th1
response (similar to the approach described above for treating
cancer patients). The inhibitory agent that is likely to be most
effective for treatment of a particular infection can be determined
by approaches similar to those described above for selecting
inhibitory agents to treat cancer patient (for example, the type of
Lewis antigen expressed by infected cells and/or proteins of the
infectious agent can be determined and/or one can determine which
Lewis antigen the cells from the subject preferentially respond to
in vitro).
[0073] C. Allergies:
[0074] Allergies are mediated through IgE antibodies whose
production is regulated by the activity of Th2 cells and the
cytokines produced thereby. In allergic reactions, IL-4 is produced
by Th2 cells, which further stimulates production of IgE antibodies
and activation of cells that mediate allergic reactions, i.e., mast
cells and basophils. IL-4 also plays an important role in
eosinophil mediated inflammatory reactions. As demonstrated in
Example 2, T cells from a patient allergic to pollen respond to
stimulation with stimulatory forms of Lewis antigens by producing
IL-10. Production of IL-10 in allergic patients may promote a Th2
response and downregulates a Th1 response, thereby exacerbating the
allergic condition. An allergic subject can be treated with an
inhibitory agent of the invention to inhibit IL-10 production in
the subject and shift the immune response away from a Th2 response
and toward a Th1response as a means to alleviate the allergic
condition.
[0075] Allergic reactions may be systemic or local in nature,
depending on the route of entry of the allergen and the pattern of
deposition of IgE on mast cells or basophils. Thus, for treatment
of an allergic subject, an inhibitory agent of the invention can be
administered either systemically or locally. Moreover, it may be
beneficial to coadminister to the subject the allergen together
with the inhibitory agent to inhibit (e.g., desensitize) the
allergen-specific response.
D. Autoimmune Diseases:
[0076] The methods of the invention also can be used
therapeutically for treating autoimmune diseases which are
associated with a Th1- or Th2-type dysfunction. Many autoimmune
disorders are the result of inappropriate activation of T cells
that are reactive against self tissue and that promote the
production of cytokines and autoantibodies involved in the
pathology of the diseases. It has been shown that modulation of T
helper-type responses can either have a beneficial or detrimental
effect on an autoimmune disease. For example, in experimental
allergic encephalomyelitis (EAE), stimulation of a Th2-type
response by administration of IL-4 at the time of the induction of
the disease diminishes the intensity of the autoimmune disease
(Paul, W. E., et al. (1994) Cell 76:241-251). Furthermore, recovery
of the animals from the disease has been shown to be associated
with an increase in a Th2-type response as evidenced by an increase
of Th2-specific cytokines (Koury, S. J., et al. (1992) J. Exp. Med.
176:1355-1364). Moreover, T cells that can suppress EAE secrete
Th2-specific cytokines (Chen, C., et al. (1994) Immunity
1:147-154). Since stimulation of a Th2-type response in EAE has a
protective effect against the disease, stimulation of a Th2
response in subjects with multiple sclerosis (for which EAE is a
model) may be beneficial therapeutically.
[0077] Similarly, stimulation of a Th2-type response in type I
diabetes in mice provides a protective effect against the disease.
Indeed, treatment of NOD mice with IL-4 (which promotes a Th2
response) prevents or delays onset of type I diabetes that normally
develops in these mice (Rapoport, M. J., et al. (1993) J. Exp. Med.
178:87-99). Thus, stimulation of a Th2 response in a subject
suffering from or susceptible to diabetes may ameliorate the
effects of the disease or inhibit the onset of the disease.
[0078] Yet another autoimmune disease in which stimulation of a
Th2-type response may be beneficial is rheumatoid arthritis (RA).
Studies have shown that patients with rheumatoid arthritis have
predominantly Th1 cells in synovial tissue (Simon, A. K., et al.,
(1994) Proc. Natl. Acad. Sci. USA 91:8562-8566). By stimulating a
Th2 response in a subject with RA, the detrimental Th1 response can
be concomitantly downmodulated to thereby ameliorate the effects of
the disease.
[0079] To treat an autoimmune disease in which a Th2-type response
is beneficial to the course of the disease in the subject, a
stimulatory agent of the invention can be administered to the
subject in amounts sufficient to stimulate a Th2-type response. The
stimulatory agent can be used alone, or in combination with one or
more additional agents that promote Th2 responses (e.g.,
Th2-promoting cytokines, such as IL-4 or IL-10). Depending on the
disease, the stimulatory agent may be administered either
systemically or locally. For example in the case of rheumatoid
arthritis, the agent may be administered directly into the joints.
For systemic treatment, the stimulatory agent preferably is
administered intravenously. Alternative to direct administration of
the stimulatory agent to the subject, autoimmune diseases may be
treated by an ex vivo approach. In this case, immune cells (e.g., T
cells, macrophages and/or B cells) are obtained from a subject
having an autoimmune disease, cultured in vitro with a stimulatory
agent of the invention to stimulate production by the cells of one
or more cytokines that promote a Th2 response (e.g., IL-10),
followed by readministration of the cells to the subject.
[0080] In contrast to the autoimmune diseases described above in
which a Th2 response is desirable, other autoimmune diseases may be
ameliorated by a Th1 -type response. Such diseases can be treated
using an inhibitory agent of the invention (as described above for
cancer and infectious diseases). The treatment may be further
enhanced by administrating a Th1-promoting cytokine (e.g.,
IFN-.gamma.) to the subject in amounts sufficient to further
stimulate a Th1-type response.
[0081] The efficacy of agents for treating autoimmune diseases can
be tested in the above described animal models of human diseases
(e.g., EAE as a model of multiple sclerosis and the NOD mice as a
model for diabetes) or other well characterized animal models of
human autoimmune diseases. Such animal models include the
mrl/lpr/lpr mouse as a model for lupus erythematosus, murine
collagen-induced arthritis as a model for rheumatoid arthritis, and
murine experimental myasthenia gravis (see Paul ed., Fundamental
Immunology, Raven Press, New York, 1989, pp. 840-856). A modulatory
(i.e., stimulatory or inhibitory) agent of the invention is
administered to test animals and the course of the disease in the
test animals is then monitored by the standard methods for the
particular model being used. Effectiveness of the modulatory agent
is evidenced by amelioration of the disease condition in animals
treated with the agent as compared to untreated animals (or animals
treated with a control agent).
[0082] Non-limiting examples of autoimmune diseases and disorders
having an autoimmune component that may be treated according to the
invention include diabetes mellitus, arthritis (including
rheumatoid arthritis, juvenile rheumatoid arthritis,
osteoarthritis, psoriatic arthritis), multiple sclerosis,
myasthenia gravis, systemic lupus erythematosis, autoimmune
thyroiditis, dermatitis (including atopic dermatitis and eczematous
dermatitis), psoriasis, Sjogren's Syndrome, including
keratoconjunctivitis sicca secondary to Sjogren's Syndrome,
alopecia areata, allergic responses due to arthropod bite
reactions, Crohn's disease, aphthous ulcer, iritis, conjunctivitis,
keratoconjunctivitis, ulcerative colitis, asthma, allergic asthma,
cutaneous lupus erythematosus, scleroderma, vaginitis, proctitis,
drug eruptions, leprosy reversal reactions, erythema nodosum
leprosum, autoimmune uveitis, allergic encephalomyelitis, acute
necrotizing hemorrhagic encephalopathy, idiopathic bilateral
progressive sensorineural hearing loss, aplastic anemia, pure red
cell anemia, idiopathic thrombocytopenia, polychondritis, Wegener's
granulomatosis, chronic active hepatitis, Stevens-Johnson syndrome,
idiopathic sprue, lichen planus, Crohn's disease, Graves
ophthalmopathy, sarcoidosis, primary biliary cirrhosis, uveitis
posterior, and interstitial lung fibrosis.
[0083] E. Inflammatory Bowel Disease in Infants
[0084] The methods and compositions of the invention also can be
used to downregulate inflammatory immune responses in the bowel in
newborn infants. The largest biological source for Lewis-antigen
containing-oligosaccharides is human breast milk and breast fed
babies have a lower incidence of diarrhea and colitis/inflammatory
bowel disease. Commercially available formula was tested for the
presence of Lewis antigen-containing sugars by ELISA and was found
not to contain detectable amounts of these sugars, nor did the
ingredients list these sugars as additives. Nonfat dry milk was
listed as an additive to the formula but when non-fat dry milk was
tested for the presence of Lewis antigen-containing sugars by
ELISA, again none were detected. Thus, Lewis antigen-containing
compounds as described herein can be added to commercial baby
formulas and administered to infants orally to inhibit diarrhea and
colitis/inflammatory bowel disease. Accordingly, the invention
provides an infant formula comprising one or more Lewis
antigen-containing compounds as an additive.
[0085] In addition to the foregoing disease situations, the
immunomodulatory methods of the invention also are useful for other
purposes. For example, the stimulatory methods of the invention
(i.e., methods using a stimulatory agent) can be used to stimulate
production of Th2-promoting cytokines (such as IL-10 or IL-4) in
vitro for commercial production of these cytokines (e.g., cells can
be cultured with a stimulatory agent in vitro to stimulate IL-10 or
IL-4 production and the IL-10 or IL-4 can be recovered from the
culture supernatant, further purified if necessary, and packaged
for commercial use).
[0086] Furthermore, the immunomodulatory methods of the invention
can be applied to vaccinations to promote either a Th1 or a Th2
response to an antigen of interest in a subject. That is, the
agents of the invention can serve as adjuvants to direct an immune
response to a vaccine either to a Th1 response or a Th2 response.
For example, to stimulate an antibody response to an antigen of
interest (i.e., for vaccination purposes), the antigen and a
stimulatory agent of the invention can be coadministered to a
subject to promote a Th2 response to the antigen in the subject,
since Th2 responses provide efficient B cell help and promote IgG1
production. Alternatively, to promote a cellular immune response to
an antigen of interest, the antigen and an inhibitory agent of the
invention can be coadministered to a subject to promote a Th1
response to the antigen in a subject, since Th1 responses favor the
development of cell-mediated immune responses (e.g., delayed
hypersensitivity responses). The antigen of interest and the
modulatory agent can be formulated together into a single
pharmaceutical composition or in separate compositions. In a
preferred embodiment, the antigen of interest and the modulatory
agent are administered simultaneously to the subject.
Alternatively, in certain situations it may be desirable to
administer the antigen first and then the modulatory agent or vice
versa (for example, in the case of an antigen that naturally evokes
a Th1response, it may be beneficial to first administer the antigen
alone to stimulate a Th1 response and then administer a stimulatory
agent, alone or together with a boost of antigen, to shift the
immune response to a Th2 response).
[0087] This invention is further illustrated by the following
examples which should not be construed as limiting. The contents of
all references, patents and published patent applications cited
throughout this application are hereby incorporated by
reference.
EXAMPLE 1
[0088] In this example, the ability of macrophages to produce IL-10
upon stimulation with various oligosaccharide conjugates was
examined. Granuloma macrophages were prepared from C57BL/6 mice as
described in Flores Villanueva, P.O., et al. (1994) J. Immunol.
153:5190-5199 and Flores Villanueva, P.O., et al. (1994) J.
Immunol. 152:1847-1855. Two million cells in 1 ml of DMEM, 10 % FCS
were incubated with either no stimulant (i.e., medium alone; a
negative control), lipopolysaccharide (LPS; at 20 .mu.g/ml; a
positive control), schistosome egg antigen (SEA; at 10 .mu.g/ml),
various sugars conjugated to human serum albumin (at 100 .mu.g/ml)
or human serum albumin alone (HSA; at 100 .mu.g/ml; a negative
control). The sugar conjugates tested were lacto-N-fucopentaose III
(LNFP-III), lacto-N-neotetraose (LNT) and Lewis.sup.y (Le.sup.y).
The oligosaccharide conjugates were obtained from Accurate
Chemicals, Westbury, N.Y.
[0089] At various time points (24, 48 and 72 hours), the level of
IL-10 in the supernatant was determined by two-site ELISA as
described by Kullberg, et al. (J. Immunol. (1982) 148:3264-3270).
Briefly, polystyrene microtiter plates (Costar) were coated with
IL-10-specific monoclonal antibodies (clone SXC-1; DNAX Corp.).
Culture supernatants were incubated on the antibody-coated plates
and then probed with biotinylated antibody. Serial dilutions of
recombinant mouse IL-10 (PharMingen) were assayed simultaneously to
construct a standard curve for relative IL-10 concentration. To
detect the biotinylated antibody bound to IL-10, avidin-peroxidase
conjugate (Sigma Chemical Corp., St. Louis, Mo.) was diluted in
phosphate buffered saline (PBS)/10% fetal calf serum and added to
the wells for 30 minutes at room temperature. The plates were
washed with PBS/0.5% Tween 20 (Fisher), and then
tetramethylbenzidine substrate (Kirkegaard & Perry
Laboratories) was added; the reaction was stopped with 0.4 M
phosphoric acid and read at 450 nm in a UvMax Reader (Molecular
Devices).
[0090] The results are shown below in Table I. TABLE-US-00001 TABLE
I IL-10 Secretion by Granuloma Macrophages IL-10 LEVEL (PG/ML)
STIMULANTS 24 HR 48 HR 72 HR Medium 898 1510 871 LPS 7864 6559 4555
SEA 2805 2282 2835 HSA-LNFP-III 1967 1682 1211 HSA-LNT 1038 1298
912 HSA-Le.sup.y 3860 3955 3076 HSA 1247 1214 1089
[0091] The greatest response was observed with the Lewis.sup.y
conjugate as the stimulant. A less vigorous response was seen with
SEA and the LNFP-III conjugate, both of which expresses the
Lewis.sup.x antigen. No response was seen with the LNT conjugate.
The results shown in Table I indicate that granuloma macrophages
can produce IL-10 upon stimulation with compounds comprising a
Lewis antigen, in particular a Lewis.sup.y antigen.
EXAMPLE 2
[0092] In this example, the ability of peripheral blood mononuclear
cells (PBMCs) from humans suffering from various disorders to
produce IL-10 upon stimulation with various oligosaccharide
conjugates was examined. Human PBMCs were obtained from: 1) an
individual who was allergic to pollen; 2) an individual suffering
from lung carcinoma and 3) an individual suffering from colon
carcinoma. One million PBMC in RPMI-1640 medium containing 10%
human AB serum were incubated with either no stimulant (i.e.,
medium alone; a negative control), lipopolysaccharide (LPS; at 20
.mu.g/ml; a positive control), various sugars conjugated to human
serum albumin (at 100 .mu.g/ml) or human serum albumin alone (HSA;
at 100 .mu.g/ml; a negative control). The sugar conjugates tested
were lacto-N-fucopentaose I (LNFP-I), lacto-N-fucopentaose III
(LNFP-III), lacto-N-neotetraose (LNT) and Lewis.sup.y
(Le.sup.y).
[0093] After 24 hours, the level of IL-10 (in pg/ml) in the
supernatant was determined by two-site ELISA as described by in
Example 1, using an anti-human IL-10 monoclonal antibody obtained
from PharMingen. Additionally, cellular proliferation was measured
by standard .sup.3H-thymidine uptake at 90 hours.
[0094] The results are shown below in Table II. TABLE-US-00002
TABLE II Responses of Human Cells from Allergic or Cancer Patients
to Various Sugar Conjugates POLLEN ALLERGIC LUNG CARCINOMA COLON
CARCINOMA STIMULANT CPM IL-10 CPM IL-10 CPM IL-10 Medium 649 160
143 1200 30 0 LPS 1946 906 232 1400 222 8221 HSA-LNFP-I 490 432
3452 10000 423 347 HSA-LNFP-III 1456 442 137 1000 270 202 HSA-LNT
1133 220 141 1000 186 304 HSA-Le.sup.y 5960 736 458 1200 934 689
HSA 460 166 152 1200 141 0
[0095] The greatest response by cells of the pollen allergic
individual was observed with the Lewis.sup.y conjugate as the
stimulant. Less vigorous responses were seen with the LNFP-I and
LNFP-III conjugates. A minimal response was seen with the LNT
conjugate. The greatest response by cells of the individual
suffering from colon carcinoma also was observed with the
Lewis.sup.y conjugate as the stimulant. Less vigorous responses
were seen with the LNFP-I, LNFP-III and LNT conjugates. For the
individual with lung carcinoma, a vigorous response was observed
only with the LNFP-I conjugate. The results shown in Table II
indicate that human PBMCs from individuals suffering from various
disorders (e.g., allergies or cancers) can produce IL-10 upon
stimulation with compounds comprising a Lewis antigen.
EXAMPLE 3
[0096] In this example, the ability of peripheral blood mononuclear
cells (PBMCs) from three humans with B lymphomas to produce IL-10
upon stimulation with various oligosaccharide conjugates was
examined. One million PBMC in RPMI-1640 medium containing 10% human
AB serum were incubated with either no stimulant (i.e., medium
alone; a negative control), lipopolysaccharide (LPS; at 20
.mu.g/ml; a positive control), various sugars conjugated to human
serum albumin (at 100 .mu.g/ml) or human serum albumin alone (HSA;
at 100 .mu.g/ml; a negative control). The sugar conjugates tested
were lacto-N-fucopentaose I (LNFP-I), lacto-N-fucopentaose III
(LNFP-III), lacto-N-neotetraose (LNT), Lewis.sup.y (Le.sup.y) and
lacto-N-difucohexose I (LND).
[0097] After 24 hours, the level of IL-10 (in pg/ml) in the
supernatant was determined by two-site ELISA as described by in
Example 2. Additionally, cellular proliferation was measured by
standard .sup.3H-thymidine uptake at 90 hours.
[0098] The results are shown below in Table III. TABLE-US-00003
TABLE III Responses of Human PBMC from B Lymphoma Patients to
Various Sugar Conjugates B-LYMPHOMA-1 B-LYMPHOMA-2 B-LYMPHOMA-3
STIMULANT CPM IL-10 CPM IL-10 CPM IL-10 Medium 444 814 288 0 1710 0
LPS 80424 6053 1084 201 3424 1049 HSA-LNFP-I 889 2422 970 0 4504 0
HSA-LNFP-III 459 4386 1009 0 3425 64 HSA-LNT 444 1081 731 0 3530 39
HSA-Le.sup.y 6211 6786 1099 2 5838 210 HSA-LND 1214 3521 502 307
4324 0 HSA 248 945 660 0 3149 0
[0099] The greatest response by cells of the B-lymphoma-1 patient
was observed with the Lewis.sup.y conjugate as the stimulant. Less
vigorous responses were seen with the LNFP-I, LNFP-III and LND
conjugates. Similarly, the greatest response by cells of the
B-lymphoma-3 patient was observed with the Lewis.sup.y conjugate as
the stimulant. Less vigorous responses were seen with the LNFP-III
and LNT conjugates. For the B-lymphoma-2 patient, a vigorous
response was observed only with the LND conjugate, which expresses
the Lewis.sup.b oligosaccharide. The results shown in Table III
indicate that human PBMCs from individuals suffering from B
lymphomas can produce IL-10 upon stimulation with compounds
comprising a Lewis antigen.
EXAMPLE 4
[0100] In this example, the ability of splenocytes from uninfected
mice or mice infected with S. mansonito produce IL-10 or IL-4 was
examined upon stimulation of the cells with various oligosaccharide
conjugates having different sugar densities. In particular, the
stimulatory ability of LNFP III-HSA (which comprises the
Lewis.sup.x antigen) was compared to conjugates of Lewis.sup.a
antigen, Lewis.sup.x antigen, sialyl-Lewis.sup.x antigen or
sialyl-Lewis.sup.a antigen to a polyacrylamide (PAA) carrier
molecule. The PAA conjugates (obtained from GlycoTech, Rockville,
Md.) have a ligand density approximately 5-6 fold higher than that
of the HSA conjugate. Within the LNFP III-HSA conjugate,
approximately 10-12 sugar molecules/HSA molecule are present. The
total conjugate weight is 67 kD, of which the LNFP III molecules
comprise approximately 3.5-4.0% of the total weight. Thus, when 50
.mu.g/ml of LNFP III-HSA conjugate is used for stimulation, the
actual concentration of sugar used is between 1.5 and 2.0 .mu.g/ml.
Within the polyacrylarnide conjugates, approximately 25-30 sugar
molecules/PAA molecule are present. The total conjugate is
approximately 30 kD, of which the Lewis antigens comprise
approximately 20-24% of the total weight. Thus, when 50 .mu.g/ml of
Lewis antigen-PAA conjugate is used for stimulation, the actual
concentration of sugar used is approximately 10-12 .mu.g/ml. e
[0101] In these experiments unfractionated splenocytes isolated
from 7-week S. mansoni-infected CBA/J mice or uninfected mice were
used. The splenocytes were incubated either with media alone, Con A
(50 .mu.g/ml), LPS (50 .mu.g/ml), anti-IgM (25 .mu.g/ml) (all as
controls), SEA (5-10 .mu.g/ml), HSA-LNFP III (50 .mu.g/ml),
Lewis.sup.a -PAA (2, 10 or 50 .mu.g/ml), Lewis.sup.x-PAA (2, 10 or
50 .mu.g/ml), sialyl-Lewis.sup.x-PAA (2, 10 or 50 .mu.g/ml) or
sialyl-Lewis.sup.a-PAA (2, 10 or 50 .mu.g/ml).
[0102] Proliferation of the splenocytes was assessed by standard
tritiated thymidine uptake. The results are shown in FIGS. 1A, 1B,
1C and 1D. Lewis.sup.a, Lewis.sup.x, sialyl-Lewis.sup.x and
sialyl-Lewis.sup.a, each conjugated to PAA, did not stimulate
proliferation of the splenocytes from either infected or uninfected
mice.
[0103] The level of IL-10 production was assessed as described in
Example 1. The results are shown in FIGS. 2A, 2B, 2C and 2D.
Lewis.sup.a, Lewis.sup.x, sialyl-Lewis.sup.x and
sialyl-Lewis.sup.a, each conjugated to PAA, were able to stimulate
IL-10 production by splenocytes from infected mice and were
substantially more effective at stimulating IL-10 production than
LNFP III-HSA (compare FIG. 2A and FIG. 2B). For example, 50
.mu.g/ml of LNFP III-HSA stimulated the production of approximately
1000 pg/ml of IL-10 (see FIG. 2A), whereas the same amount of
Lewis.sup.x-PAA and sialyl-Lewis.sup.a-PAA stimulated production of
approximately 17,500 pg/ml of IL-10 and the same amount of
Lewis.sup.a-PAA and sialyl-Lewis.sup.x-PAA stimulated production of
approximately 7,500 pg/ml of IL-10 (see FIG. 2B). Lewis.sup.x-PAA
was also able to stimulated IL-10 production by splenocytes from
uninfected mice (e.g., production of approximately 600 pg/ml of
IL-10 was stimulated by 50 .mu.g/ml of Lewis.sup.x-PAA, whereas the
same amount of LNFP III-HSA did not stimulate IL-10 production by
splenocytes from uninfected mice; compare FIGS. 2C and 2D).
[0104] The level of IL-4 production in the cultures was assessed by
a standard ELISA using an anti-mouse IL-4 monoclonal antibody
obtained from PharMingen. The results are shown in FIGS. 3A, 3B, 3C
and 3D. The Lewis.sup.x-PAA was effective at stimulating IL-4
production by splenocytes from both uninfected and infected mice
(see FIGS. 3B and 3D). Sialyl-Lewis.sup.a-PAA stimulated lower
amounts of IL-4 production in splenocytes from infected mice (see
FIG. 3B), as did LNFP III-HSA (see FIG. 3A), whereas these
conjugates did not stimulate IL-4 production by splenocytes from
uninfected mice (see FIGS. 3C and 3D).
[0105] These results demonstrate that Lewis antigen-containing
sugar conjugates, in particular those with a high sugar density,
can stimulate IL-10 and IL-4 production by naive splenocytes (i.e.,
splenocytes from uninfected mice). In particular, IL-4 production
was greater by stimulated naive splenocytes than stimulated
splenocytes from infected mice. This IL-4 production likely is not
by B cells. Rather this IL-4 production is likely by T cells and/or
other non-T, non-B cells (e.g., mast cells, basophils and/or
eosinophils).
EQUIVALENTS
[0106] Those skilled in the art will recognize, or be able to
ascertain using no more than routine experimentation, many
equivalents to the specific embodiments of the invention described
herein. Such equivalents are intended to be encompassed by the
following claims.
* * * * *