U.S. patent application number 12/767237 was filed with the patent office on 2010-08-26 for therapy-enhancing glucan.
This patent application is currently assigned to SLOAN-KETTERING INSTITUTE FOR CANCER RESEARCH. Invention is credited to Nai-Kong V. CHEUNG.
Application Number | 20100216743 12/767237 |
Document ID | / |
Family ID | 34216281 |
Filed Date | 2010-08-26 |
United States Patent
Application |
20100216743 |
Kind Code |
A1 |
CHEUNG; Nai-Kong V. |
August 26, 2010 |
THERAPY-ENHANCING GLUCAN
Abstract
This invention provides a method for introducing substances into
cells comprising contacting a composition comprising orally
administered beta-glucan with said cells. This invention also
provides a method for introducing substances into a subject
comprising administering to the subject an effective amount of the
above compositions. The substance which could be delivered orally
includes but is not limited to peptides, proteins, RNAs, DNAs,
chemotherapeutic agents, biologically active agents, plasmids, and
other small molecules and compounds. Finally, this invention
provides a composition comprising orally administered beta-glucan
capable of enhancing efficacy of IgM and different uses of the said
composition.
Inventors: |
CHEUNG; Nai-Kong V.; (New
York, NY) |
Correspondence
Address: |
LAW OFFICES OF ALBERT WAI-KIT CHAN, PLLC
141-07 20TH AVENUE, WORLD PLAZA, SUITE 604
WHITESTONE
NY
11357
US
|
Assignee: |
SLOAN-KETTERING INSTITUTE FOR
CANCER RESEARCH
New York
NY
|
Family ID: |
34216281 |
Appl. No.: |
12/767237 |
Filed: |
April 26, 2010 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
10565484 |
Jan 17, 2006 |
7704973 |
|
|
PCT/US2004/023099 |
Jul 16, 2004 |
|
|
|
12767237 |
|
|
|
|
10621027 |
Jul 16, 2003 |
7507724 |
|
|
10565484 |
|
|
|
|
Current U.S.
Class: |
514/54 ;
536/123.12 |
Current CPC
Class: |
A61K 39/44 20130101;
A61K 39/39 20130101; A61K 39/39541 20130101; A61K 31/739 20130101;
C08B 37/0024 20130101; A61K 39/39558 20130101; A61K 31/716
20130101; A61P 35/00 20180101; A61P 37/04 20180101; A61K 31/716
20130101; A61K 47/36 20130101; A61K 39/39 20130101; A61K 39/39541
20130101; A61K 39/44 20130101; A61K 39/39558 20130101; A61K 31/715
20130101; A61P 43/00 20180101; A61K 2300/00 20130101; A61K
2039/55583 20130101; A61K 2300/00 20130101; A61K 2300/00 20130101;
A61K 2300/00 20130101; A61K 2300/00 20130101 |
Class at
Publication: |
514/54 ;
536/123.12 |
International
Class: |
A61K 31/716 20060101
A61K031/716; C07H 3/06 20060101 C07H003/06; A61P 43/00 20060101
A61P043/00; A61P 35/00 20060101 A61P035/00 |
Claims
1. A composition for oral uptake of substance comprising an
appropriate amount of carbohydrates.
2. A composition for the oral delivery of one or more substances
comprising an effective amount of an orally administered
beta-glucan and one or more chemotherapeutic agents.
3-13. (canceled)
Description
[0001] This application is a continuation-in-part of U.S. Ser. No.
10/621,027, Filed Jul. 16, 2003, the contents of which are
incorporated in its entirety by reference here into this
application.
[0002] Throughout this application, various references are cited.
Disclosures of these publications in their entireties are hereby
incorporated by reference into this application to more fully
describe the state of the art to which this invention pertains.
BACKGROUND OF THE INVENTION
[0003] This disclosure relates to a method for introducing
substances into cells comprising contacting a composition
comprising orally administered beta-glucan with said cells. A
feature of this invention provides a method for introducing
substances into a subject comprising administering to the subject
an effective amount of the above compositions. The substance which
could be delivered orally includes but is not limited to peptides,
proteins, RNAs, DNAs, chemotherapeutic agents, biologically active
agents, and plasmids. Other small molecules and compounds may be
used as well. Another feature of the present invention is a
composition comprising orally administered beta-glucan capable of
enhancing efficacy of IgM antibodies.
[0004] Glucans derived from cell walls of yeasts, such as
Saccharomyces cervisiae or mutant yeast strains described in U.S.
Pat. No. 5,250,436, the disclosure of which is incorporated herein
in its entirety by reference, may be used in the above
compositions. Glucans having .beta.(1-3) and .beta.(1-6) linkages
may be prepared by the process described in U.S. Pat. Nos.
5,233,491 and 4,810,646, the disclosures of which are incorporated
herein in their entirety by reference. Soluble or aqueous glucans
which are suitable for oral administration may be produced by the
process described in U.S. Pat. Nos. 4,810,646 and 5,519,009, the
disclosures of which are incorporated herein in their entirety by
reference.
[0005] Beta-glucans have been tested for tumor therapy in mice for
nearly 40 years..sup.1,2 Several forms of mushroom derived
beta-glucans are used clinically to treat cancer in Japan,
including PSK (from Coriolus versicolor), Lentinan and
Schizophyllan. In randomized trials in Japan, PSK has moderately,
but significantly improved survival rates in some cancer trials:
after gastrectomy,.sup.3,4 colorectal surgery,.sup.5,6 and
esophagectomy.sup.7 to remove primary tumors. Results have been
less encouraging in breast cancer,.sup.8,9 and leukemia..sup.10
Schizophyllan has improved survival of patients with operable
gastric cancer,.sup.11 inoperable gastric cancer,.sup.12,13 and
cervical cancer..sup.14 Again, though survival differences between
groups were statistically significant, these improvements were
modest. While beta-glucans are not widely used by Western
oncologists, beta-glucan containing botanical medicines such as
Reishi and maitake.sup.15 are widely used by U.S. cancer patients
as alternative/complementary cancer therapies. These previous
studies that looked for a therapeutic effect of beta-glucan, did
not incorporate co-administration of therapeutic monoclonal
antibodies (MoAb) as part of the protocol. There is increasing
evidence that antibody is necessary to deposit iC3b which acts as a
potent opsonin of human tumors. When beta-glucan is administered
without co-administration of MoAb, its tumor cytotoxic effect
requires the presence of naturally-occurring antitumor antibodies
which can be quite variable among patients and even in experimental
mice.
[0006] Anti-tumor effect of beta-glucan when combined with cancer
specific antibodies was previously described. Previous studies have
shown that oral beta-glucans derived from barley or oats can
greatly enhance the anti-tumor activity of anti-tumor monoclonal
antibodies in xenograph models. See Therapy-Enhancing Glucan, Int'l
Application No. PCT/US02/01276, filed Jan. 15, 2002. Cheung et al.,
Oral (1-3), (1-4)-beta-glucan syngergizes with anti-ganglioside GD2
monoclonal antibody 3F8 in the therapy of neuroblastoma. Clin
Cancer Res. 2002;8:1217-1223. Cheung N K et al., Orally
administered beta-glucans enhance anti-tumor effects of monoclonal
antibodies. Cancer Immunol Immunother. 2002; 51:557-564. The phase
I clinical trial supports the prediction that barley beta-glucan
can enhance the antibody effect on metastatic cancer. As previously
noted, lentinan and laminarin, both (1.fwdarw.3),
(1.fwdarw.6)-.beta.-D-glucans, were not as effective as barley
glucan..sup.16 In addition, among the (1.fwdarw.3),
(1.fwdarw.4)-.beta.-D-glucans, small molecular weight preparations
and Lichenans were not as effective. The molecular size and the
fine structure of beta-glucan may have substantial influence on
their synergistic effect on antibodies towards tumors.
[0007] In Europe and USA beta-glucans especially from Bakers' yeast
have long been employed as feed additives for animals, as dietary
supplement for humans,.sup.17 in treatment of wounds,.sup.18 and as
an active ingredient in skin cream formulations. The basic
structural unit in beta-glucans is the .beta.(1.fwdarw.3)-linked
glucosyl units. Depending upon the source and method of isolation,
beta-glucans have various degrees of branching and of linkages in
the side chains. The frequency and hinge-structure of side chains
determines its immunomodulor effect. beta-glucans of fungal and
yeast origin are normally insoluble in water, but can be made
soluble either by acid hydrolysis or by derivatisation introducing
charged groups like -phosphate, -sulphate, -amine, -carboxymethyl
and so forth to the molecule..sup.19,20
[0008] Soluble glucan with the molecular structure where
(1.fwdarw.3)-.beta.-D-glucan units form the backbone with branches
made up of (1.fwdarw.3)-.beta.-D-glucan units positioned at
(1.fwdarw.6)-.beta.-D-glucan hinges was isolated from Baker's
yeast, Saccharomyces cerevisiae. High molecular weight fractions
were obtained and
##STR00001## [0009] tested for synergy with monoclonal antibodies
in tumor models. The anti-tumor effect of soluble yeast beta-glucan
was found to be comparable to the anti-tumor effect of soluble
barley beta-glucan, when combined with monoclonal antibodies
specific for human cancer as detailed below.
SUMMARY OF THE INVENTION
[0010] This invention provides a method for introducing substances
into cells comprising contacting a composition comprising orally
administered beta-glucan with said cells.
[0011] Another aspect of the present is a method for introducing
substances into a subject comprising administering to the subject
an effective amount of the above compositions. The substance which
could be delivered orally includes but is not limited to peptides,
proteins, RNAs, DNAs, chemotherapeutic agents, biologically active
agents, and plasmids. Other small molecules and compounds may be
used as well.
[0012] A further aspect of the present invention is a composition
comprising orally administered beta-glucan capable of enhancing
efficacy of IgM antibodies.
DETAILED DESCRIPTION OF THE FIGURES
[0013] FIG. 1. Barley (1.fwdarw.3), (1.fwdarw.4)-.beta.-D-glucan
plus antibody in the treatment of metastatic neuroblastoma in
patients. MIBG scan before and after treatment in a patient with
metastatic neuroblastoma refractory to multiple regimens of
chemotherapy. Patient received intravenous anti-GD2 antibody 3F8
(10 mg/m2/day) for a total of 10 days, plus oral barley beta-glucan
over the same time period. FIG. 1A shows baseline MIBG scan of
patient. Extensive osseous metastasis can be seen in the femora,
fibulae, pelvis, ribs, left scapula, right clavicle, humeri, skull
and spine. Heart, liver, stomach and colon uptakes are physiologic.
FIG. 1B shows MIBG scan of same patient 2 months later, following a
single cycle of therapy with 3F8 plus glucan. Areas of metastases
have significantly improved.
[0014] FIG. 2. Barley (1.fwdarw.3), (1.fwdarw.4)-.beta.-D-glucan
plus antibody in treatment of subcutaneous human lymphoma
xenografted in SCID mice. SCID mice with established subcutaneous
Daudi (n=9) (FIG. 2A), Hs445 (n=5) (FIG. 2B), EBV-derived LCL (n=9)
(FIG. 2C) and RPMI 6666 (n=10; data not shown) xenografts were
treated either with 200 ug intravenous rituximab twice weekly for 8
doses (.box-solid.),400 ug (1.fwdarw.3),
(1.fwdarw.4)-D-.beta.-glucan administered orally via intragastric
gavage daily for 29 days (.DELTA.) or a combination of rituximab
and (1.fwdarw.3), (1.fwdarw.4)-D-.beta.-glucan (x), or left
untreated (.diamond-solid.). Percentage tumor growth is plotted on
y-axis and days after treatment was commenced on x-axis. Error bars
represent SEM and have been shown only for rituximab alone and
combination groups. For all xenografts, only combination treatment
was associated with reduction in tumor growth. The reduction in
tumor growth per day in the group receiving beta-glucan in addition
to rituximab compared to rituximab alone was 2.0% (95% CI 1.3-2.7%;
p<0.0005) for Daudi, 0.8% for EBV-derived LCL (95% CI 0.4-1.2%;
p<=001), 2.2% for Hs445 (95% C.I. 1.2%-3.2%; p=0.0009), and 1.8%
for RPMI6666 (95% CI 1.0-2.7%; p<0.0002; data not shown)
xenografts.
[0015] FIG. 3. Barley (1.fwdarw.3), (1.fwdarw.4)-.beta.-D-glucan
plus antibody in treatment of disseminated human lymphoma
xenografted in SCID mice. 5.times.10.sup.6 Daudi (FIG. 3A) or Hs445
(FIG. 3B) cells in 100 .mu.l normal saline were injected
intravenously (IV) into SCID mice. Mice were treated either with
200 ug intravenous rituximab twice weekly for 8 doses (---) 400 ug
(1.fwdarw.3), (1.fwdarw.4)-D-.beta.-glucan administered orally via
intragastric gavage daily for 29 days ( . . . ) or a combination of
rituximab and (1.fwdarw.3), (1.fwdarw.4)-D-.beta.-glucan (), [0016]
or left untreated () [0017] commencing 10 days after tumor
implantation. Tumors grew systemically and mice became paralyzed
when tumor cells infiltrated the spinal canal, resulting in
hind-leg paralysis. Mice were sacrificed at onset of paralysis or
when animals lost 10% of their body weight. Kaplan-Maier survival
curves for the various groups are shown in FIGS. 2A (Daudi) and 2B
(Hs445). Mice treated with a combination of (1.fwdarw.3),
(1.fwdarw.4)-D-.beta.-glucan and rituximab had a significantly
increased survival when compared to all other treatment groups
(p<0.0005 for Daudi and p=0.001 for Hs445) or when compared to
rituximab alone (p<0.0005 for Daudi and p=0.01 for Hs445).
Median survival for mice with no treatment, rituximab alone, BG,
and rituximab+BG groups was 27,71,43 and 124 days respectively for
Daudi xenografts, and 12, 16, 31 and 243 days respectively for
Hs445 xenografts.
[0018] FIG. 4. Dose response of 3G6 (anti-GD2 IgM antibody) in the
presence of barley .beta.-glucan in the treatment of human
neuroblastoma. Two million LAN1 neuroblastoma cells were
xenografted subcutaneously in athymic Balb/c mice. Treatment
started in groups of 5 mice each, 2 weeks after tumor implantation
when visible tumors reached 0.7-0.8 cm diameter. 3G6 group (solid
squares) was treated with 200 ug of intravenous 3G6 injected
through the retroorbital plexus twice weekly (M and Th). 3G6+BG
group was treated with 200 ug i.v. 3G6 twice weekly plus oral
beta-glucan (BG) 400 ug daily by gavage for a total of 14-18 days.
3G6 was administered in 3 different doses (open triangle 8 ug per
dose, open square 40 ug, open circle 200 ug). BG group (solid
circles) received 400 ug oral beta-glucan alone. Tumor size was
measured from the first day of treatment, and the product of the
largest diameters expressed as percent of the size on day 0 of
treatment. Vertical bars represent standard errors, depicted in
only 4 groups for clarity. While BG alone and 3G6 alone showed no
anti-tumor effect, the BG+200 ug 3G6 group showed highly
significant tumor shrinkage and suppression which was 3G6
dose-dependent (p<0.05).
[0019] FIG. 5. Treatment of human neuroblastoma using 3G6 (anti-GD2
IgM antibody) in the presence of yeast (1.fwdarw.3),
(1.fwdarw.6)-.beta.-D-glucan. Two million LAN1 neuroblastoma cells
were xenografted subcutaneously in athymic Balb/c mice. Treatment
started in groups of 5 mice each, 2 weeks after tumor implantation
when visible tumors reached 0.7-0.8 cm diameter. 3G6 group (solid
squares) was treated with 200 ug of intravenous 3G6 injected
through the retroorbital plexus twice weekly (M and Th) for a total
of 5 doses. Particulate yeast glucan group (solid triangles)
received 400 ug oral particulate yeast glucan alone. 3G6+whole
yeast particles (open diamond) was treated with 200 ug iv 3G6 twice
weekly plus yeast particles 400 ug daily by gavage for a total of
14-18 days. 3G6+soluble yeast glucan group was treated with 200 ug
iv 3G6 twice weekly plus soluble yeast glucan 400 ug daily by
gavage for a total of 14-18days. 3G6+particulate yeast glucan group
was treated with 200 ug i.v. 3G6 twice weekly plus particulate
yeast glucan 400 ug daily by gavage for a total of 14-18 days.
Tumor size was measured from the first day of treatment, and the
product of the largest diameters expressed as percent of the size
on day 0 of treatment. Vertical bars represent standard errors,
depicted in only 4 groups for clarity. While glucan alone and 3G6
alone showed no anti-tumor effect, soluble and particulate yeast
glucan when combined with 3G6 group showed highly significant tumor
shrinkage and suppression (p<0.05).
[0020] FIG. 6. Treatment of human neuroblastoma using 3F8 (anti-GD2
IgG antibody) in the presence of barley and yeast .beta.-glucan.
Two million LAN1 neuroblastoma cells were xenografted
subcutaneously in athymic Balb/c mice. Treatment started in groups
of 5 mice each, 2 weeks after tumor implantation when visible
tumors reached 0.7-0.8 cm diameter. 3F8 group (solid diamonds) was
treated with 200 ug of intravenous 3F8 injected through the
retroorbital plexus twice weekly (M and Th) for a total of 5 doses.
Barley glucan group (solid squares) received 400 ug barely glucan
alone. 3F8+barley glucan group (open diamond) was treated with 200
ug i.v. 3F8 twice weekly plus barely glucan 400 ug daily by gavage
for a total of 14-18 days. 3F830 soluble yeast glucan group (open
squares) was treated with 200 ug iv 3F8 twice weekly plus soluble
yeast glucan 400 ug daily by gavage for a total of 14-18 days.
Tumor size was measured from the first day of treatment, and the
product of the largest diameters expressed as percent of the size
on day 0 of treatment. Vertical bars represent standard errors.
While glucan alone and 3F8 alone showed no anti-tumor effect,
barley and soluble yeast glucan when combined with 3F8 group showed
highly significant tumor shrinkage and suppression (p<0.05).
[0021] FIG. 7. Treatment of disseminating human lymphoma in SCID
mice using Rituxan and barley or yeast .beta.-glucan. 5.times.10e6
Daudi cells in 100 .mu.l normal saline were injected intravenously
(IV) into SCID mice. Tumors grew systemically and mice became
paralyzed when tumor cells infiltrated the spinal canal, resulting
in hind-leg paralysis. Mice were sacrificed at onset of paralysis
or when animals lost 10% of their body weight. Therapy was
initiated ten days after injection of tumor cells. 40 .mu.g
rituximab (Genentech, San Francisco, Calif.) was injected
intravenously twice weekly for a total of eight injections and 400
.mu.g glucan administered orally via intragastric gavage daily for
29 days. Mice were weighed weekly and observed clinically at least
once daily. Mice receiving rituxan plus barley glucan or rituxan
plus yeast soluble glucan have a highly significant prolonged
survival (p<0.05).
[0022] FIG. 8 illustrates the pEGP-C1 vector purchased from BD
Biosciences (Palo Alto, Calif.).
[0023] FIG. 9 shows glucan facilitates gene transfer into
monocytes.
[0024] FIG. 10 illustrates higher molecular weight .beta.-glucan
and gene transfer.
[0025] FIG. 11 illustrates presence of GFP mRNA in circulating
monocytes.
DETAILED DESCRIPTION OF THE INVENTION
[0026] This invention provides a composition for oral uptake of
substance comprising an appropriate amount of carbohydrates. In an
embodiment, the carbohydrate is glucan.
[0027] When administered orally, glucan is taken up by macrophages
and monocytes which carry these carbohydrates to the marrow and
reticuloendothelial system from where they are released, in an
appropriately processed form, onto myeloid cells including
neutrophils, and onto lymphoid cells including natural killer (NK)
cells. This processed glucan binds to CR3 on these neutrophils and
NK cells, activating them in tumor cytotoxicity in the presence of
tumor-specific antibodies.
[0028] Since macrophage and monocytes ingest glucan (whether
soluble, gel or particle) from the gut, glucan is a potential
conduit for gene therapy. Unlike proteins, DNA or plasmids are
relatively heat-stable, and can be easily incorporated into warm
soluble barley glucan which gels when cooled to room or body
temperature. When mice are fed these DNA-glucan complexes, reporter
genes can be detected in peripheral blood monocytes and macrophages
within days. More importantly these reporter genes are expressed in
these cells, a few days after ingestion of these DNA complexes.
These findings have potential biologic implications. Glucan and
similar carbohydrates may be conduits for DNA or plasmids to get
into the human body. Oral glucan may be a convenient vehicle for
correcting genetic defects of macrophages/monocytes, or
administering genetic vaccines.
[0029] As it can easily be appreciated by an ordinary skilled
artisan, other carbohydrates capable of functioning like glucan
could be identified and used in a similar fashion. One easy
screening for such carbohydrates can be established using glucan as
the positive control.
[0030] The glucan includes but is not limited to .beta.(1-3) and
.beta.(1-4) mixed linkage-glucan, and the glucan is of high
molecular weight. The glucan may also have .beta.(1-3) and
.beta.(1-6) linkages.
[0031] This invention also provides a method for introducing
substance into cells comprising contacting the above compositions
with said cells. One can use reporter genes or other markers to
assess the efficiency of the said introduction. Reporter genes or
markers are well known in the molecular biology field. In addition,
this invention provides a method for introducing substance into a
subject comprising administering to the subject an effective amount
of the above compositions.
[0032] This invention provides a composition for the oral delivery
of one or more substances comprising an effective amount of an
orally administered beta-glucan and one or more chemotherapeutic
agents.
[0033] In an embodiment, the glucan contains 1,3-1,6 or 1,3-1,4
mixed linkages, or a mixture of 1,3-1,6 and 1,3-1,4 mixed linkages.
In another embodiment, the glucan enhances the efficacy of
chemotherapeutic agents or anti-cancer antibodies.
[0034] In a further embodiment, the glucan is derived from grass,
plants, mushroom, yeast, barley, fungi, wheat or seaweed. The
glucan may be of high molecular weight. The molecular weight of the
glucan may be at least 10,000 Daltons.
[0035] In a further embodiment, the substance is a peptide,
protein, RNA, DNA, plasmid, or chemotherapeutic agent. As used
herein, chemotherapeutic agents include chemicals that combat
disease in the body of an animal or medications used to treat
various forms of cancer.
[0036] This invention provides a method for introducing substance
into cells comprising contacting the above-described composition
with said cells.
[0037] The substance which could be delivered orally includes but
is not limited to peptides, proteins, RNAs, DNAs, and plasmids.
Other small molecules and compounds may be used as well.
[0038] This invention provides a method for treating a subject
comprising administering to the subject an effective amount of the
above composition. In an embodiment, the method further comprises
the substance.
[0039] This invention provides a method for treating a subject with
genetic disorder comprising administering to the subject an
effective amount of the above-described composition and a substance
capable of correcting said genetic disorder. The substance includes
but is not limited to a peptide, protein, RNA, DNA, plasmid and
other small molecule and compound.
[0040] This invention provides a composition comprising an
effective amount of orally administered (1.fwdarw.3), (1.fwdarw.6)
beta-glucan capable of enhancing efficacy of IgM antibodies.
[0041] This invention provides a composition comprising an
effective amount of orally administered (1.fwdarw.3), (1.fwdarw.6)
beta-glucan capable of enhancing efficacy of antibodies. Glucans
derived from cell walls of yeasts, such as Saccharomyces cervisiae
or mutant yeast strains described in U.S. Pat. No. 5,250,436, the
disclosure of which is incorporated herein in its entirety by
reference, may be used in the above compositions. Glucans having
.beta.(1-3) and .beta.(1-6) linkages may be prepared by the process
described in U.S. Pat. Nos. 5,233,491 and 4,810,646, the
disclosures of which are incorporated herein in their entirety by
reference. Soluble or aqueous glucans which are suitable for oral
administration may be produced by the process described in U.S.
Pat. Nos. 4,810,646 and 5,519,009, the disclosures of which are
incorporated herein in their entirety by reference.
[0042] In an embodiment, the antibody is a monoclonal antibody, or
an antibody against cancer or tumor cells, which include but are
not limited to anti-CEA antibody, anti-CD20 antibodies, anti-CD25
antibodies, anti-CD22 antibodies, anti-HER2 antibodies,
anti-tenascin antibodies, MoAb M195, Dacluzimab, anti-TAG-72
antibodies, R24, Herceptin, Rituximab, 528, IgG, IgM, IgA, C225,
Epratuzumab, and MoAb 3F8. In another embodiment, the antibody is a
tumor-binding antibody.
[0043] Moreover, the antibody is capable of activating complement
and/or activating the antibody dependent cell-mediated
cytotoxicity. In another embodiment, the antibody modulates T-cell
or B-cell function.
[0044] In a further embodiment, the antibody is directed at the
epidermal growth factor receptor, a ganglioside, such as GD3 or
GD2.
[0045] In a further embodiment, the antibodies are effective
against cancers which include neuroblastoma, melanoma,
non-Hodgkin's lymphoma, Epstein-Barr related lymphoma, Hodgkin's
lymphoma, retinoblastoma, small cell lung cancer, brain tumors,
leukemia, epidermoid carcinoma, prostate cancer, renal cell
carcinoma, transitional cell carcinoma, breast cancer, ovarian
cancer, lung cancer, colon cancer, liver cancer, stomach cancer, or
other gastrointestinal cancers.
[0046] In a further embodiment, the above-described composition is
in a pharmaceutically acceptable carrier.
[0047] This invention provides a method for treating a subject
comprising administrating the above-described composition to a
subject.
[0048] This invention provides a composition comprising an
effective amount of orally administered (1.fwdarw.3), (1.fwdarw.6)
beta-glucan capable of enhancing efficacy of vaccines. In an
embodiment, the vaccine is against cancer or infectious agents,
such as bacteria, viruses, fungi, or parasites.
[0049] This invention provides a composition comprising an
effective amount of orally administered (1.fwdarw.3), (1.fwdarw.6)
beta-glucan capable of enhancing efficacy of natural antibodies or
infectious agents.
[0050] This invention provides a composition comprising an
effective amount of orally administered (1.fwdarw.3), (1.fwdarw.6)
beta-glucan capable of enhancing host immunity.
[0051] This invention provides a composition comprising an
effective amount of orally administered (1.fwdarw.3), (1.fwdarw.6)
beta-glucan capable of enhancing the action of an agent in
preventing tissue rejection. In an embodiment, the tissue is
transplanted tissue or transplanted organ or the host as in
graft-versus-host disease.
[0052] In an embodiment, the glucan of the above-described
composition has high molecular weight. The molecular weight of
glucan is at least 10,000 Daltons. In another embodiment, the
glucan is derived from barley, oat, mushroom, seaweed, fungi,
yeast, wheat or moss. In a further embodiment, the glucan is stable
to heat treatment.
[0053] In a further embodiment, above-describe composition is
stable after boiling for 3 hours. The effective dose of the
above-described composition is about >=25 mg/kg/day, five days a
week for a total of 2-4 weeks.
[0054] The invention will be better understood by reference to the
Experimental Details which follow, but those skilled in the art
will readily appreciate that the specific experiments detailed are
only illustrative, and are not meant to limit the invention as
described herein, which is defined by the claims which follow
thereafter.
Example I
[0055] Phase I Study of Barley .beta.-Glucan in Combination with
Anti-GD2 Antibody in Stage 4 Neuroblastoma.
[0056] A total of 24 patients were studied. These patients are all
children or adolescents with relapsed or refractory stage 4
neuroblastoma metastatic to bone, marrow or distant lymph nodes,
some with large soft tissue masses. Beta-glucan was well tolerated
with no dose-limiting toxicities. Anti-tumor responses were
recorded for marrow disease (histology, MIBG scans), soft tissue
tumors (CT), as well biochemical markers (urine VMA and HVA tumor
markers). One example of tumor response is shown in FIGS. 1A and
1B: .sup.131I-metaiodobenzylguanidine (MIBG) scans showing
near-complete resolution of extensive metastases after one
treatment cycle of 3F8 plus beta-glucan. These responses are
uncommon in patients with refractory or relapsed metastatic stage 4
NB treated with 3F8 alone or 3F8 in combination with cytokines. The
best response rate for 3F8 to date was in a Phase II trial of
combination 3F8 plus GMCSF where 7 of 33 (21%) children achieved
MIBG improvement. In contrast, 62% (13 of 21) evaluable patients on
3F8+beta-glucan had MIBG improvement, a near tripling of the
response rate (p=0.008 by X.sup.2). In addition, among 15 patients
with marrow disease, 5 achieved complete marrow remission (30%),
and 8 with stable disease in the marrow. [0057] (See FIG. 1)
Example II
[0058] Rituximab activates complement-mediated and
antibody-dependent cell-mediated cytotoxicities, and is effective
against B-cell lymphomas. Beta-glucans are naturally occurring
glucose polymers that bind to the lectin domain of CR3, a receptor
widely expressed among leukocytes, priming it for binding to iC3b
activated by antibodies. Barley-derived (1.fwdarw.3),
(1.fwdarw.4)-.beta.-D-glucan (BG), when administered orally (400
.mu.g per day.times.29 days), strongly synergized with
subtherapeutic doses of intravenous rituximab (200 .mu.g
twice/week.times.8 doses) in the therapy of CD20-positive human
lymphomas. Growth of established subcutaneous non-Hodgkin's
lymphoma (NHL) (Daudi and EBV-derived B-NHL) or Hodgkin's disease
(Hs445 or RPMI6666) xenografted in SCID mice was significantly
suppressed, when compared to mice treated with rituximab or BG
alone. Survival of mice with disseminated lymphoma (Daudi and
Hs445) was significantly increased. There was no weight loss or
clinical toxicity in treated animals. This therapeutic efficacy and
lack of toxicity of BG plus rituximab supports further
investigation into its clinical utility.
Introduction
[0059] The chimeric anti-CD20 antibody rituximab is being evaluated
in an increasing number of disorders. After clinical efficacy was
initially demonstrated against relapsed and refractory
follicular/low grade non-Hodgkin's lymphoma.sup.1, responses to
rituximab have been reported in other malignant and non-malignant
B-cell disorders.sup.2. Several mechanisms of action have been
proposed including activation of apoptotic pathways.sup.3,
elaboration of cytokines.sup.4, and elicitation of host
complement-dependent cytotoxicity (CDC) and antibody-dependent
cell-mediated cytotoxicity (ADCC).sup.5. Although many patients
with B-cell disorders respond to rituximab, remissions are often
transient.sup.6. More than 50% of lymphomas recurrent after
rituximab treatment failed to respond the second time.sup.7.
Mechanisms of resistance to rituximab are as yet unclear, and may
include paucity or loss of target antigen.sup.8, pharmacokinetic
variations among individual patients, FcR polymorphism.sup.9,
resistance to complement activity.sup.10, or inherent gene
expression of the lymphoma.sup.11.
[0060] beta-glucans are complex polymers of glucose with affinity
for the lectin site of the CR3 receptor on leucocytes.sup.12. With
bound beta-glucan, CR3 (CD11b) is primed to engage iC3b fragments
deposited on cells by complement-activating antibodies. This
receptor mediates the diapedesis of leukocytes through the
endothelium and stimulates phagocytosis, degranulation and tumor
cytotoxicity. Many fungi present beta-glucan or beta-glucan-like
CR3 binding ligands on their cell surface. Hence, when iC3b
deposition occurs, both CD11b and lectin sites become engaged, and
phagocytosis and respiratory burst is triggered.sup.13. In
contrast, tumor cells lack such molecules, and even when coated
with iC3b do not generally activate CR3 and cannot activate
leucocytes. Soluble forms of beta-glucan bind to lectin sites and
prime both phagocytic and NK cells to kill iC3b-coated tumor
targets.sup.14.
[0061] (1.fwdarw.3), (1.fwdarw.4)-D-.beta.-glucan (BG), a soluble,
barley-derived beta-glucan has advantages over previously studied
(1.fwdarw.3), (1.fwdarw.6)-.beta.-glucans, particularly efficacy
when administered orally and a good safety profile.sup.15. In vivo
synergism between BG and the complement-fixing antibody 3F8 against
human neuroblastoma xenografts.sup.15,16was recently demonstrated.
The synergism between BG and rituximab against lymphoma is now
reported.
Study Design
Cell Lines:
[0062] Human Burkitt's lymphoma cell line, Daudi, and Hodgkin's
disease (HD) cell lines Hs445 and RPMI 6666 were purchased from
American Type Culture Collection (Rockville, Md.). Human EBV-BLCL
were established using previously described methods.sup.17.
Mice:
[0063] Fox Chase ICR SCID mice (Taconic, White Plains, N.Y.) were
maintained under institutionally approved guidelines and
protocols.
Tumor Models:
[0064] Subcutaneous tumors were established by injecting
5.times.10.sup.6 cells suspended in 0.1 ml of Matrigel
(Becton-Dickinson, Franklin Lakes, N.J.) into mice flanks. Tumor
dimensions were measured two to three times a week and tumor size
was calculated as the product of the two largest diameters. Mice
were sacrificed when maximum tumor dimension exceeded 20 mm. A
disseminated tumor model was established in SCID mice as previously
described.sup.18. Briefly, 5.times.10.sup.6 Daudi or Hs445 cells in
100 .mu.l normal saline were injected intravenously into SCID mice.
Tumors grew systemically and mice became paralyzed when tumor cells
infiltrated the spinal cord, resulting in hind-leg paralysis. Mice
were sacrificed at onset of paralysis or when animals lost 10% of
their body weight.
Treatment Regimens:
[0065] For mice with subcutaneous tumors, therapy was initiated
after tumors were established (7-8 mm diameter). For the
disseminated tumor model, therapy was initiated ten days after
injection of tumor cells. Groups of at least five mice per
treatment regimen received either rituximab, BG, neither or both.
200 .mu.g rituximab (Genentech, San Francisco, Calif.) was injected
intravenously twice weekly for a total of eight injections and 400
.mu.g BG (Sigma, St. Louis, Mo.) administered orally via
intragastric gavage daily for 29 days. Animals were weighed weekly
and observed clinically at least once daily.
Statistical Analysis:
[0066] Tumor growth was calculated by fitting a regression slope
for each individual mouse to log transformed values of tumor size.
Slopes were compared between groups using t-tests using a
previously described method for censored observations.sup.19.
Survival in mice with disseminated disease was compared using
Kaplan-Meier analysis and proportion of deaths was compared by
Fisher's exact X2 test. Analyses were conducted using STATA 7
(Stata Corporation, College Station, Tex.).
Results and Discussion
[0067] In all subcutaneous xenograft models, significant reduction
in tumor growth was noted in mice treated with a combination of
rituximab and BG. Mice treated with rituximab alone showed a modest
reduction in tumor growth, while those treated with BG alone or
left untreated had unabated tumor growth (FIGS. 1A, 1B, 1C). All
tumors except for those treated with combination therapy grew
beyond 20 mm size and mice had to be sacrificed. Mice on
combination treatment had persistent tumor suppression even after
treatment was stopped. In a multivariable linear model of tumor
growth rate, using dummy variables for treatment, the interaction
between BG and rituximab was positive and significant,
demonstrating synergism.
[0068] For disseminated xenografts, there was a significant
difference in survival between the combination and control groups
for both NHL and HD models (p<0.005, by log-rank) (FIG. 2). 5/38
mice and 2/8 mice with disseminated Daudi and Hs445 tumors
respectively treated with combination BG and rituximab were
surviving >12 months after therapy was discontinued suggesting
complete eradication of disease. In contrast, 0/29 and 0/8 mice
receiving rituximab alone in respective groups survived (15% vs. 0%
survival; X2=0.01). There was no significant weight loss or other
clinically apparent adverse effects. That BG is absorbed can be
inferred from the fact that it could be detected intracellularly
within fixed and permeabilized peripheral blood leucocytes by
immunofluorescence (data not shown).
[0069] In these studies, synergism between BG and rituximab was
highly significant irrespective of the type of CD20-positive
lymphoma. Improved responses in Daudi xenografts as compared to
Hs445 may be attributable to higher CD20 expression in the former
(Mean geometric fluorescence channel for Daudi 241 compared to 184
for Hs445). When tumors that progressed were examined for CD20
expression by immunofluorescence studies of single cell suspensions
or indirect immunohistochemistry of frozen sections, no significant
difference was noted between groups treated with rituximab, BG
alone or rituximab+BG (data not shown), indicating that treatment
with rituximab+BG was not associated with loss of CD20.
[0070] Synergism between other complement-activating monoclonal
antibodies and BG.sup.15,16 were previously demonstrated. The
current data extend this observation to rituximab. CDC is
considered an important mechanism for rituximab cytotoxicity.
Rodent complement is not inhibited efficiently by human complement
regulatory proteins (mCRP). Therefore CDC can be an effective
anti-tumor mechanism in xenograft models. However in a study, at
sub-therapeutic doses of antibody, rituximab-mediated ADCC and CDC
were not sufficient to effect tumor cell killing. Since BG has no
direct effect on ADCC.sup.20, this synergy is most likely a result
of iC3b-mediated tumor cytotoxicity. Lymphoma cells express mCRP
including CD46, CD55, and CD59.sup.10,21. However, iC3b-mediated
cytotoxicity is unaffected by the presence of CD59 which affects
only MAC-mediated complement cytotoxicity.sup.22. Furthermore, in
human breast carcinoma tumors, deposition of iC3b has been
demonstrated despite the presence of mCRP.sup.23 indicating that
unlike their inhibitory effect on MAC, effect on iC3b-mediated
tumor cytotoxicity is not absolute.
[0071] If this synergistic effect can be safely reproduced in
humans, iC3b-mediated cytotoxicity may be a potential strategy to
overcome rituximab resistance in patients with B-cell malignancies.
Since neither T nor B cells are required for this synergistic
effect, BG may have a potential role even in immunocompromised
lymphoma patients. Furthermore, in patients with autoimmune
disorders, B-cell depletion may be enhanced with this non-toxic
oral therapy. Conversely, beta-glucans can enhance release of
cytokines such as TNF-.alpha. and IL-6.sup.24, and because the
acute toxicities of rituximab are also related to cytokine release
secondary to complement activation.sup.25, there is a potential of
increased toxicity when BG and rituximab are used in combination.
Carefully designed phase I studies are necessary in order to define
the safety and efficacy in developing BG as an adjunct to rituximab
therapy in the treatment of B-cell disorders and in antibody-based
therapies of other cancers.
References for Example II
[0072] 1. Maloney D G, Liles T M, Czerwinski D K, Waldichuk C,
Rosenberg J, Grillo-Lopez A, Levy R. Phase I clinical trial using
escalating single-dose infusion of chimeric anti-CD20 monoclonal
antibody (IDEC-C2B8) in patients with recurrent B-cell lymphoma.
Blood. 1994;84:2457-2466 [0073] 2. Cheson B D. Rituximab: clinical
development and future directions. Expert Opin Biol Ther.
2002;2:97-110 [0074] 3. Alas S, Emmanouilides C, Bonavida B.
Inhibition of interleukin 10 by Rituximab results in
Down-regulation of Bcl-2 and sensitization of B-cell Non-Hodgkin's
lymphoma to apoptosis. Clin Cancer Res. 2001;7:709-723 [0075] 4.
Chow K U, Sommerlad W D, Boehrer S, Schneider B, Seipelt G, Rummel
M J, Hoelzer D, Mitrou P S, Weidmann E. Anti-CD20 antibody
(IDEC-C2B8, rituximab) enhances efficacy of cytotoxic drugs on
neoplastic lymphocytes in vitro: role of cytokines, complement, and
caspases. Haematologica. 2002;87:33-43 [0076] 5. Reff M E, Carner
K, Chambers K S, Chinn P C, Leonard J E, Raab R, Newman R A, Hanna
N, Anderson D R. Depletion of B cells in vivo by a chimeric mouse
human monoclonal antibody to CD20. Blood. 1994;83:435-445 [0077] 6.
McLaughlin P, Grillo-Lopez A J, Kink B K, Levy R, Czuczman M S,
Williams M E, Heyman M R, Bence-Bruckler I, White C A, Cabanillas
F, Jain V, Ho A D, Lister J, Wey K, Shen D, Dallaire B K. Rituximab
chimeric anti-CD20 monoclonal antibody therapy for relapsed
indolent lymphoma: half of patients respond to four-dose treatment
program. J Clin Oncol. 1998;16:2825-2833 [0078] 7. Davis T A,
Grillo-Lopez A J, White C A, McLaughlin P, Czuczman M S, Link B K,
Maloney D G, Weaver R L, Rosenberg J, Levy R. Rituximab anti-CD20
monoclonal antibody therapy in non-Hodgkin's lymphoma: safety and
efficacy of re-treatment. J Clin Oncol. 2000;18:3135-3143 [0079] 8.
Davis T A, Czerwinski D K, Levy R. Therapy of B-cell lymphoma with
anti-CD20 antibodies can result in the loss of CD20 antigen
expression. Clin Cancer Res. 1999;5:611-615 [0080] 9. Cartron G,
Dacheux L, Salles G, Solal-Celigny P, Bardos P, Colombat P, Watier
H. Therapeutic activity of humanized anti-CD20 monoclonal antibody
and polymorphism in IgG Fc receptor FcgammaRIIIa gene. Blood.
2002;99:754-758 [0081] 10. Golay J, Zaffaroni L, Vaccari T, Lazzari
M, Borleri G M, Bernasconi S, Tedesco F, Rambaldi A, Introna M.
Biologic response of B lymphoma cells to anti-CD20 monoclonal
antibody rituximab in vitro: CD55 and CD59 regulate
complement-mediated cell lysis. Blood. 2000;95:3900-3908 [0082] 11.
Bohen S P, Troyanskaya O G, Alter O, Warnke R, Botstein D, Brown P
O, Levy R. Variation in gene expression patterns in follicular
lymphoma and the response to rituximab. Proc Natl Acad Sci USA.
2003;100:1926-1930 [0083] 12. Bohn J A, BeMiller J N.
(1-3)-B-D-Glucans as biological response modifiers:a review of
structure-functional activity relationships. Carbohydr Polymers.
1995;28:3-14 [0084] 13. Ross G D, Cain J A, Myones B L, Newman S L,
Lachmann P J. Specificity of membrane complement receptor type
three (CR3) for beta-glucans. Complement Inflamm. 1987;4:61-74
[0085] 14. Xia Y, Vetvicka V, Yan J, Hanikyrova M, Mayadas T, Ross
G D. The beta-glucan-binding lectin site of mouse CR3 (CD11b/CD18)
and its function in generating a primed state of the receptor that
mediates cytotoxic activation in response to iC3b-opsonized target
cells. J Immunol. 1999;162:2281-2290 [0086] 15. Cheung N K, Modak
S. Oral (1-3),(1-4)-beta-glucan syngergizes with anti-ganglioside
GD2 monoclonal antibody 3F8 in the therapy of neuroblastoma. Clin
Cancer Res. 2002;8:1217-1223 [0087] 16. Cheung N K, Modak S,
Vickers A, Knuckles B. Orally administered beta-glucans enhance
anti-tumor effects of monoclonal antibodies. Cancer Immunol
Immunother. 2002;51:557-564 [0088] 17. Koehne G, Gallardo H F,
Sadelain M, O'Reilly R J. Rapid selection of antigen-specific T
lymphocytes by retroviral transduction. Blood. 2000;96:109-117
[0089] 18. Wei B R, Ghetie M A, Vitetta E S. The combined use of an
immunotoxin and a radioimmunoconjugate to treat disseminated human
B-cell lymphoma in immunodeficient mice. Clin Cancer Res.
2000;6:631-642 [0090] 19. Vardi Y, Ying Z, Zhang C-H. Two-sample
tests for growth curves under dependent right censoring.
Biometrika. 2001;88:949-960 [0091] 20. Yan J, Vetvicka V, Xia Y,
Coxon A, Carroll M C, Mayadas T N, Ross G D. B-glucan a "Specific"
biologic response modifier that uses antibodies to target tumors
for cytotoxic recognition by leukocyte complement receptor type 3
(CD11b/CD18). J Immunol. 1999;163:3045-3052 [0092] 21. Treon S P,
Mitsiades C, Mitsiades N, Young G, Doss D, Schlossman R, Anderson K
C. Tumor cell expression of CD59 is associated with resistance to
CD20 serotherapy in patients with B-cell malignancies. J
Immunother. 2001;24:263-271 [0093] 22. Jurianz K, Ziegler S,
Garcia-Schuler H, Kraus S, Bohana-Kashtan O, Fishelson Z,
Kirschfink M. Complement resistance of tumor cells: basal and
induced mechanisms. Mol Immunol. 1999;36:929-939 [0094] 23.
Vetvicka V, Thornton B P, Wieman T J, Ross G D. Targeting of
natural killer cells to mammary carcinoma via naturally occurring
tumor cell-bound iC3b and beta-glucan-primed CR3 (CD11b/CD18). J
Immunol. 1997;159:599-605 [0095] 24. Adachi Y, Okazaki M, Ohno N,
Yadomae T. Enhancement of cytokine production by macrophages
stimulated with (1->3)-beta-D-glucan, grifolan (GRN), isolated
from Grifola frondosa. Biol Pharm Bull. 1994;17:1554-1560 [0096]
25. Van der Kolk L E, Grillo-Lopez A J, Baars J W, Hack C E, van
Oers M H. Complement activation plays a key role in the
side-effects of rituximab treatment. Br J Haematol.
2001;115:807-811
Example III
[0097] Barley .beta.-Glucan Extract Synergizes with IgM
Antibodies
[0098] Natural IgM antibody from human serum when administered i.v.
was cytotoxic for human neuroblastoma (NB) cells effecting growth
arrest of subcutaneous solid human NB xenografts in nude rats. (1,
2) IgM was taken up by the tumors with massive perivascular
complement activation and accumulation of granulocytes after 24
hours. (3) In metastatic NB model, IgM antibody was effective in
eliminating tumors in 90% of the mice. (4) The absence of this
anti-NB IgM antibody during infancy and among NB patients (of any
age), and its prevalence after 12 months of age has raised the
hypothesis that natural IgM antibodies could play a role as an
immunological control mechanism against NB.( 5) 3G6 is an anti-GD2
mouse IgM monoclonal antibody (MoAb). Within 48 hours after i.v.
injection of biotinylated 3G6, subcutaneous NB xenografts showed
membrane staining of tumor cells. Although 3G6 had lower mean
fluorescence (53.+-.19 fluorescent channel units, n=7 mice) when
compared to 3F8, an IgG MoAb (149.+-.44, n=7), 3G6 plus beta-glucan
was effective against sc human NB (p<0.05), with a dose response
curve (FIG. 4) comparable to that of 3F8. (6) These findings were
consistent with those using human natural anti-NB IgM. (1, 2) These
data support the idea that beta-glucan can enhance not just IgG
inducing vaccines, but also IgM inducing vaccines.
References for Example III
[0099] 1. David K, Ollert M W, Juhl H, et al: Growth arrest of
solid human neuroblastoma xenografts in nude rats by natural IgM
from healthy humans. Nat Med 2:686-9, 1996 [0100] 2. Ollert M W,
David K, Schmitt C, et al: Normal human serum contains a natural
IgM antibody cytotoxic for human neuroblastoma cells. Proc Natl
Acad Sci USA 93:4498-503, 1996 [0101] 3. Ollert M W, David K,
Vollmert C, et al: Mechanisms of in vivo anti-neuroblastoma
activity of human natural IgM. Eur J Cancer 33:1942-8, 1997 [0102]
4. Engler S, Thiel C, Forster K, et al: A novel metastatic animal
model reflecting the clinical appearance of human neuroblastoma:
growth arrest of orthotopic tumors by natural, cytotoxic human
immunoglobulin M antibodies. Cancer Res 61:2968-73, 2001 [0103] 5.
Erttmann R, Schmitt C, Ollert M W, et al: Naturally occurring
humoral cytotoxicity against neuroblastoma (NB) cells in healthy
persons and NB patients. Pediatr Hematol Oncol 13:545-8, 1996
[0104] 6. Cheung N, Modak S: Oral (1-3),(1-4)-beta-glucan
syngergizes with anti-ganglioside GD2 monoclonal antibody 3F8 in
the therapy of neuroblastoma. Clin Cancer Res 8:1217-1223, 2002
Example IV
(1.fwdarw.3), (1.fwdarw.6) .beta.-Glucan Derived From Baker's Yeast
(Derived From Saccharomyces Cerevisiae) is Also Effective in
Enhancing Antibody Therapy of Cancer
[0105] LAN-1 tumor cells were planted (2.times.10.sup.6 cells) in
100 .mu.l of Matrigel (Sigma) subcutaneously. Tumor dimensions were
measured two to three times a week with vernier calipers, and tumor
size was calculated as the product of the two largest perpendicular
diameters. All treatment studies started in groups of 4-5 mice when
tumor diameters reached 0.7 to 0.8 cm. Mice received antibody (3F8
or 3G6) treatment (200 ug per day) i.v. (by tail vein injection)
twice weekly.times.5 doses and oral beta-glucan (400 ug per day) by
intragastric injection every day for a total 14-18 days. (See FIGS.
5 and 6)
[0106] Glucans derived from cell walls of yeasts, such as
Saccharomyces cervisiae or mutant yeast strains described in U.S.
Pat. No. 5,250,436, the disclosure of which is incorporated herein
in its entirety by reference, may be used in the above
compositions. Glucans having .beta.(1-3) and .beta.(1-6) linkages
may be prepared by the process described in U.S. Pat. Nos.
5,233,491 and 4,810,646, the disclosures of which are incorporated
herein in their entirety by reference. Soluble or aqueous glucans
which are suitable for oral administration may be produced by the
process described in U.S. Pat. Nos. 4,810,646 and 5,519,009, the
disclosures of which are incorporated herein in their entirety by
reference. Beta-glucans such as the Soluble beta-1,3/1,6 glucan or
SBG manufactured by Biotec Pharmacon (Norway) may also be used.
[0107] In similar experiments a subcutaneous lymphoma model was
studied. Here 5.times.10.sup.6 cells suspended in 0.1 ml of
Matrigel (Becton-Dickinson, Franklin Lakes, N.J.) were planted into
mice flanks. Tumor dimensions were measured two to three times a
week and tumor size was calculated as product of the two largest
diameters. Mice were sacrificed when maximum tumor dimension
exceeded 20 mm. 200 .mu.g rituximab (Genentech, San Francisco,
Calif.) was injected intravenously twice weekly for a total of
eight injections and 400 .mu.g glucan administered orally via
intragastric gavage daily for 29 days. Mice were weighed weekly and
observed clinically at least once daily. The rate of tumor response
and the percent of mice achieving complete remissions were
comparable between barley glucan and yeast glucan. These series of
subcutaneous tumor models showed that soluble yeast (1.fwdarw.3),
(1.fwdarw.6) beta-glucan of large molecular weight (>10,000
Daltons) is equally potent as barley (1.fwdarw.3), (1.fwdarw.4)
beta-glucan. In addition, the source and physical form of yeast
glucan can make substantial differences.
[0108] Metastatic lymphoma model was also studied. A model of
disseminated tumors was established in SCID mice as previously
described. (1) Briefly, 5.times.10.sup.6 Daudi cells in 100 .mu.l
normal saline were injected intravenously (i.v.) into SCID mice.
Tumors grew systemically and mice became paralyzed when tumor cells
infiltrated the spinal canal, resulting in hind-leg paralysis. Mice
were sacrificed at onset of paralysis or when animals lost 10% of
their body weight. Therapy was initiated ten days after injection
of tumor cells. 40 .mu.g rituximab (Genentech, San Francisco,
Calif.) was injected intravenously twice weekly for a total of
eight injections and 400 .mu.g glucan administered orally via
intragastric gavage daily for 29 days. Mice were weighed weekly and
observed clinically at least once daily. (See FIG. 7)
[0109] Again both barley glucan and yeast glucan showed comparable
effect when combined with Rituxan. Neither barely glucan nor yeast
glucan has any effect on survival when used alone (data not
shown).
References for Example IV
[0110] 1. Wei B R, Ghetie M A, Vitetta E S: The combined use of an
immunotoxin and a radioimmunoconjugate to treat disseminated human
B-cell lymphoma in immunodeficient mice. Clin Cancer Res 6:631-642,
2000
Example V
[0111] Mechanism by Which Orally Administered .beta.-Glucans
Function with Anti-Tumor Monoclonal Antibodies to Mediate Tumor
Regression.(1)
[0112] Using syngeneic tumor (GD2+RMA-S) in wild type (WT) C57Bl/6
mice versus either CR3-deficient (CD11b -/-) or C3-deficient (C3
-/-) C57Bl/6 mice, MoAb alone elicited no tumor regression, whereas
combining the i.v. anti-GD2 MoAb with oral barley or yeast
beta-glucan elicited significant regression in WT but not in
CR3-deficient mice. Moreover, the combined treatment with i.v. MoAb
and oral beta-glucans produced 60-100% tumor-free survivors in WT
mice, but only 0-20% survival in the CR3-deficient mice. These
experiments demonstrated a near absolute requirement for leukocyte
CR3 for the anti-tumor effect, especially when oral barley
beta-glucan was given with anti-tumor MoAb. A therapy protocol
comparing WT to C3-deficient mice similarly showed that oral
beta-glucan therapy required serum C3. When barley beta-glucan and
yeast beta-glucan were labeled with fluorescein (BG-F and YG-F) and
given to mice by intragastric injection, the trafficking of
beta-glucan was followed. Within three days of daily oral
administration of BG-F or YG-F, macrophages in the spleen and lymph
nodes contained fluorescein-labeled beta-glucan. After 4 d, YG-F
and BG-F were also observed in macrophages in bone marrow. When the
uptake of YG-F and BG-F by WT versus CR3-deficient mice was
compared, no differences were apparent in either the percentage of
macrophages containing ingested beta-glucan-F or the amount of
beta-glucan-F per cell. Thus, the uptake of barley and yeast
beta-glucan by gastrointestinal macrophages does not require CR3
and is likely mediated instead by Dectin-1. (2) Macrophages in
vitro and in the marrow were able to degrade large molecules of
barley or yeast beta-glucan into smaller biologically-active
fragments of beta-glucan that are then released.
[0113] To determine if the soluble beta-glucan-F released by
macrophages had indeed been taken up by bone marrow granulocytes,
groups of WT or CR3-deficient mice that had been given YG-F or BG-F
for 10 days were injected i.p. with thioglycolate medium to elicit
the marginated pool of bone marrow granulocytes into the peritoneal
cavity. Only WT granulocytes were able to pick up the YG-F and BG-F
released from macrophages. These data suggest a sequential
ingestion of beta-glucan by gastrointestinal macrophages that
shuttle the beta-glucan to the bone marrow where soluble
degradation fragments are released and taken up by granulocytes via
membrane CR3. When peritoneal granulocytes were isolated from WT
and CR3-deficient mice that had been given oral beta-glucan, only
WT granulocytes were able to kill iC3b-coated tumor cells in vitro.
These experiments show that bone marrow granulocytes and tissue
macrophages acquire membrane CR3-bound soluble beta-glucan from
gastrointestinal macrophages, and that this bound beta-glucan
primes the CR3 of both granulocytes and macrophages so that when
they are recruited to a site of inflammation they are able to kill
iC3b-coated tumor cells.
References for Example V
[0114] 1. Hong F, Yan J, Baran J T, et al: Mechanism by which
orally administered beta(1,3)-glucans function with anti-tumor
monoclonal antibodies to mediate tumor regression and tumor-free
survival. J Exp Med, 2004 [0115] 2. Herre J, Gordon S, Brown G D:
Dectin-1 and its role in the recognition of beta-glucans by
macrophages. Mol Immunol 40:869-76, 2004
Example VI
Soluble .beta.-Glucan Can be Used as a Conduit for Plasmids.
[0116] The major obstacles for the delivery of DNA, RNA and
proteins orally are the acidic and proteolytic environment of the
stomach, and limited uptake of proteins by the GALT. It is believed
that M cells within the Peyer's patches and phagocytes are the
predominant vehicles for uptake of microparticulates. However,
nanoparticles may also access GALT via a paracellular mechanism
.sup.1,2 and by transcytosis..sup.3 In either case, particle uptake
observed can be improved using particles with mucoadhesive
properties or affinity for receptors on cells. Many polymers have
been used to fabricate nanoparticles are mucoadhesive. Among them
are alginate, carrageenans, and pectin. Although these materials
were often used as the core polymers in nanoparticulates, no
specific receptor has been identified for these polymers and the
efficiency of uptake remains suboptimal. Dectin-1 is now known to
be a universal receptor for .beta.-glucan, and is found in many
human tissues including monocytes and phagocytes. The gelling
properties of high molecular weight .beta.-glucan allows RNA, DNA
and proteins to be embedded. Since sugars are highly resistant to
acid conditions and enzymes, proteins, RNA and DNA remain protected
during their passage through the gastrointestinal tract. Through
the high affinity Dectin-1 receptor for .beta.-glucan, these
substances can be introduced into the phagocytes as potential
vehicles to the rest of the body.
[0117] The pEGP-C1 vector (See FIG. 8) was purchased from BD
Biosciences (Palo Alto, Calif.) and prepared according to
manufacturers' instructions. pEGFP-C1 encodes a red-shifted variant
of wild-type GFP (1-3) which has been optimized for brighter
fluorescence and higher expression in mammalian cells. (Excitation
maximum=488 nm; emission maximum=507 nm.) The vector backbone also
contains an SV40 origin for replication in mammalian cells only if
they express the SV40 T-antigen. A bacterial promoter upstream of
this cassette expresses kanamycin resistance in E. coli. The
pEGFP-C1 backbone also provides a pUC origin of replication for
propagation in E. coli and an f1 origin for single-stranded DNA
production.
[0118] Mice were fed with 50 .mu.g pEGFP-c1 plasmid mixed into 400
.mu.g beta-glucan (.about.200,000 Daltons) in 100 .mu.l saline by
oral gavage while control mice were given plasmid alone. Oral
feeding was done for 3 consecutive days (days 1, 2 and 3). 50 .mu.l
blood taken from tail vein were analysed by FCAS analysis after
lysis of RBC and the % of GFP-expressing cells in the monocyte
population were recorded. The mean ratio of % green cells in glucan
verus no glucan groups (n=4-9 mice per group) is presented in FIG.
9. Throughout the 14 days of the experiment, % green monocytes in
the no-glucan group remained stable at background levels. On the
other hand, after day 1 of oral gavage, there was a consistent
higher % of circulating green monocytes, which peaked around day 8.
Since the GFP is not normally found in mouse monocytes, the
presence of green cells is consistent with GFP protein expression
following entry of the plasmid into the monocytes which circulate
in the blood.
[0119] The experiment was repeated using barley .beta.-glucan of
higher molecular weight (.about.350,000 Daltons) with better
gelling properties. In FIG. 10, similar kinetics was seen, with a
higher percent of green cells that persisted from day 8 through day
11 (n=4 mice per group).
[0120] Presence of GFP mRNA was tested using quantitative
reverse-transcription PCR analysis. Mice were fed with 50 .mu.g
pEGFP-c1 plasmid mixed into 400 .mu.g high molecular weight
(.about.350,000 Daltons) beta-glucan in 100 .mu.l saline by oral
gavage while control mice were given plasmid alone. 50 .mu.l
peripheral blood was used to extract total RNA, reverse transcribed
and quantitative real-time PCR was performed using a modification
of the method previously described..sup.4 The house keeping gene
mouse GAPDH is used as internal control. Transcript level is
calculated using a known GFP and GAPDH standard. Transcript units
are calculated separately for GFP and GAPDH and results as a ratio
of GFP over GAPDH. In FIG. 11, the mean RNA level (GFP/GAPDH) is
expressed as a ratio of glucan versus no glucan groups (n=4 mice
per group). GFP mRNA was detected up to day 10.
References for Example VI
[0121] 1. Damge C, Aprahamian M, Marchais H, et al: Intestinal
absorption of PLAGA microspheres in the rat. J Anat 189 (Pt
3):491-501, 1996 [0122] 2. Jani P, Halbert G W, Langridge J, et al:
Nanoparticle uptake by the rat gastrointestinal mucosa:
quantitation and particle size dependency. J Pharm Pharmacol
42:821-6, 1990 [0123] 3. Florence A T: The oral absorption of
micro- and nanoparticulates: neither exceptional nor unusual. Pharm
Res 14:259-66, 1997 [0124] 4. Cheung I Y, Lo Piccolo M S, Collins
N, et al: Quantitation of GD2 synthase mRNA by real-time reverse
transcription-polymerase chain reaction: utility in bone marrow
purging of neuroblastoma by anti-GD2 antibody 3F8. Cancer
94:3042-8, 2002
Sequence CWU 1
1
1194DNAArtificial SequenceEGFP Portion of pEGFP-C1 Vector
1TACAAGTCCG GACTCAGATC TCGAGCTCAA GCTTCGAATT CTGCAGTCGA CGGTACCGCG
60GGCCCGGGAT CCACCGGATC TAGATAACTG ATCA 94
* * * * *