U.S. patent number 6,423,539 [Application Number 09/791,992] was granted by the patent office on 2002-07-23 for adjuvant treatment by in vivo activation of dendritic cells.
This patent grant is currently assigned to The Board of Trustees of the Leland Stanford Junior University. Invention is credited to Edgar G. Engleman, Lawrence H. Fong, Miriam Merad.
United States Patent |
6,423,539 |
Fong , et al. |
July 23, 2002 |
Adjuvant treatment by in vivo activation of dendritic cells
Abstract
The immunogenicity of an antigen is enhanced by increasing the
specific antigen presenting function of dendritic cells (DC) in a
mammalian host. The host is treated with a DC mobilization agent to
increase the number of circulating DC precursors. The host is then
given a local, injection of antigen in combination with a DC
activating agent. The activation step promotes recruitment and
maturation of the DC, along with antigen-specific activation and
migration from the tissues to lymphoid organs. These DC then
effectively interact with, and present processed antigen to, T
cells that are then able to respond to the antigen. In one aspect
of the invention, the antigen is a tumor antigen, and is used to
enhance the host immune response to tumor cells present in the
body.
Inventors: |
Fong; Lawrence H. (Menlo Park,
CA), Merad; Miriam (Palo Alto, CA), Engleman; Edgar
G. (Atherton, CA) |
Assignee: |
The Board of Trustees of the Leland
Stanford Junior University (Stanford, CA)
|
Family
ID: |
22678426 |
Appl.
No.: |
09/791,992 |
Filed: |
February 22, 2001 |
Current U.S.
Class: |
435/325; 435/326;
435/339; 530/350; 530/351 |
Current CPC
Class: |
A61K
39/39 (20130101); A61P 31/04 (20180101); A61P
35/00 (20180101); A61P 43/00 (20180101); A61P
31/10 (20180101); A61K 38/18 (20130101); A61P
31/12 (20180101); A61P 31/14 (20180101); A61P
33/00 (20180101); A61P 31/22 (20180101); A61P
31/06 (20180101); A61P 37/04 (20180101); A61K
38/18 (20130101); A61K 2300/00 (20130101); A61K
2039/55516 (20130101); A61K 2039/55561 (20130101); A61K
2039/55522 (20130101) |
Current International
Class: |
A61K
38/18 (20060101); A61K 39/39 (20060101); C12N
005/00 (); C12N 005/06 (); C07K 001/00 () |
Field of
Search: |
;435/325,326,339
;530/350,351 |
References Cited
[Referenced By]
U.S. Patent Documents
|
|
|
5976546 |
November 1999 |
Laus et al. |
5994126 |
November 1999 |
Steinman et al. |
|
Foreign Patent Documents
Other References
Hsu et al. (Jan. 1996), "Vaccination of Patients with B-Cell
Lymphoma Using Autologous Antigen-Pulsed Dendritic Cells." Nature
Medicine, vol. 2(1):52-58. .
Maraskovsky et al. (Nov. 1996), "Dramatic Increase in the Numbers
of Functionality Mature Dendritic Cells in Flt3 Ligand0Treated
Mice: Multiple Dendritic Cell Subpopulations Identified." J. Exp.
Med., vol. 187:1953-1962. .
Pulendran et al. (1997), "Deve;lopmental Pathways of Dendritic
Cells in Vivo." Journal of Immunology, vol. 159:2222-2231. .
Young et al. (Jan. 1996), "Dendritic Cells as Adjuvants for Class 1
Major Histocompatibility Complex-Restricted Antitumor Immunity." J.
Exp. Med., vol. 183:7-11..
|
Primary Examiner: Park; Hankyel T.
Attorney, Agent or Firm: Sherwood; Pamela J. Bozicevic,
Field & Francis LLP
Government Interests
GOVERNMENT SUPPORT
This application was made with Government support under contract
CA71725 awarded by the National Institutes of Health. The
Government has certain rights in this invention.
Parent Case Text
This application claims benefit to U.S. provisional application
Serial No. 60/184,810, filed Feb. 24, 2000.
Claims
What is claimed is:
1. A method of increasing the immune response in a mammalian host
to a target antigen in the absence of in vitro manipulation of
dendritic cells, the method comprising: administering to said
mammalian host a dose of a DC mobilization agent effective to
substantially increase the number of DC precursors present in the
periphery of said host; administering to said mammalian host a dose
of a DC activation agent effective to induce maturation of said DC
precursors in combination with said target antigen; wherein the
immune response of said mammalian host to said antigen is
increased.
2. The method of claim 1, wherein said DC mobilization agent is
Flt-3 ligand.
3. The method of claim 2, wherein said dose is effective in
increasing the number of DC precursors in the periphery by at least
2 fold.
4. The method of claim 3, wherein said increased number of DC
precursors is seen after one week.
5. The method of claim 1, wherein said DC activating agent is
administered after the number of DC precursors has increased at
least about 5 fold.
6. The method of claim 1, wherein said DC activating agent is an
immunostimulatory polynucleotide.
7. The method of claim 1, wherein said DC activating agent is
administered at a peripheral site.
8. The method of claim 7, wherein said administration is
sub-cutaneous.
9. The method of claim 8, wherein said antigen and said DC
activating agent are co-formulated.
10. The method of claim 8, wherein said antigen and said DC
activating agent are separately formulated.
11. The method of claim 1, wherein said target antigen is a tumor
antigen.
12. The method of claim 1, wherein said target antigen is a
bacterial antigen.
13. The method of claim 1, wherein said target antigen is a viral
antigen.
14. The method of claim 1, wherein said target antigen is in the
form of peptides, protein, or nucleic acids encoding peptides or
proteins.
15. The method of claim 1, wherein said mammalian host is a
human.
16. A method of increasing a T cell mediated immune response in a
mammalian host to a target antigen in the absence of in vitro
manipulation of dendritic cells, the method comprising:
administering to said mammalian host a dose of Flt-3 ligand
effective to substantially increase the number of DC precursors
present in the periphery of said host; and after the number of DC
precursors has increased at least about 5 fold in response to Flt-3
ligand, administering subcutaneously to said mammalian host a dose
of a DC activation agent effective to induce maturation of said DC
precursors in combination with said target antigen; wherein the
immune response of said mammalian host to said antigen is
increased.
17. The method of claim 16, wherein said DC activating agent is an
immunostimulatory polynucleotide.
18. The method of claim 17, wherein said target antigen is a tumor
antigen.
19. The method of claim 16, wherein said mammalian host is a human.
Description
While vaccination protocols have been some of the great medical
achievements in the last century, there are still conditions where
an effective immune response has been difficult to generate. For
example, human tumor immunotherapy has met with only limited
success. Among the reasons for this have been the limited
availability of tumor-associated antigens, and an inability to
deliver such antigens in a manner that renders them immunogenic.
Recent insights into the role of dendritic cells (DC) as the
pivotal antigen presenting cell for initiation of immune responses
may provide the basis for more effective immune responses,
particularly where conventional vaccination is inadequate.
The events whereby cells fragment antigens into peptides, and then
present these peptides in association with products of the major
histocompatibility complex, (MHC) are termed "antigen
presentation". The MHC is a region of highly polymorphic genes
whose products are expressed on the surfaces of a variety of cells.
T cells recognize foreign antigens bound to only one specific class
I or class II MHC molecule. The patterns of antigen association
with class I or class II MHC molecules determine which T cells are
stimulated.
T cells do not effectively respond to antigen unless the antigen is
processed and presented to them by the appropriate antigen
presenting cells (APC). The two major classes of antigen presenting
cells are DC and macrophages. DC are uniquely capable of processing
and presenting antigens to naive T cells. The efficacy of DC in
antigen presentation is widely acknowledged, but the clinical use
of these cells is hampered by the fact that there are very few in
any given organ. In human blood, for example, about 1% of the white
cells are DC. While DC can process foreign antigens into peptides
that immunologically active T cells can recognize, the low numbers
of DC makes their therapeutic use very difficult.
In recent years, the life cycle of DC has been elucidated. DC
precursors migrate from bone marrow and circulate in the blood to
specific sites in the body where they mature. This trafficking is
directed by expression of chemokine receptors and adhesion
molecules. Tissue resident DC include Langerhans cells in skin,
hepatic DC in the portal triads, mucosal DC and lung DC. Upon
exposure to antigen and activation signals, the DC are activated,
and leave tissues to migrate via the afferent lymphatics to the T
cell rich paracortex of the draining lymph nodes. The activated DC
then secrete chemokines and cytokines involved in T cell homing and
activation, and present processed antigen to T cells.
Mature DC have a distinct morphology characterized by the presence
of numerous membrane processes. These processes can take the form
of dendrites, pseudopods or veils. DC are also characterized by the
cell surface expression of large amounts of class II MHC antigens
and the absence of lineage markers, including CD14 (monocyte), CD3
(T cell), CD19, 20, 24 (B cell), CD56 (natural killer), and CD66b
(granulocyte). DC express a variety of adhesion and co-stimulatory
molecules, e.g. CD80 and CD86, and molecules that regulate
co-stimulation, such as CD40. The phenotype of DC varies with the
stage of maturation and activation, where expression of adhesion
molecules, MHC antigens and co-stimulatory molecules increases with
maturation. Antibodies that preferentially stain mature DC include
anti-CD83 and CMRF-44.
While methods have been described for the in vitro manipulation of
DC in order to enhance their immunologic function, such techniques
can be very expensive and labor intensive. The ability to enhance
DC antigen presentation in vivo (i.e. without in vitro culture)
would be of great clinical and experimental benefit.
Relevant Literature
Administration of Flt3 ligand to mice in vivo results in
preferential mobilization or release of DC precursors from the bone
marrow to the periphery and into lymphoid organs, and can increase
the number of circulating DC 10-30 fold (Maraskovsky et al. (1996)
J. Exp. Med. 184:1953-1962). It has been suggested that these cells
can then be removed for ex vivo manipulation and priming. Pulendran
et al. (1997) J. Immunol. 159:2222-2231 describe the expansion of
DC in animals treated with FL.
Hsu et al. (1996) Nat. Med. 2:52-58 describe vaccination of
patients with B-cell lymphoma using autologous DC that had been
removed from the patient, pulsed with antigen, and reinfused as an
autologous vaccine. Young and Inaba (1996) J. Exp. Med. 183:7-11
describe the use of DC are adjuvants for class I MHC-restricted
antitumor immunity.
U.S. Pat. No. 5,994,126, Steinman et a., issued Nov. 30, 1999
describes method for in vitro proliferation of DC precursors and
their use to produce immunogens. U.S. Pat. No. 5,976,546, Laus et
al., issued Nov. 2, 1999 describes immunostimulatory
compositions.
SUMMARY OF THE INVENTION
Methods are provided for enhancing the immunogenicity of an antigen
by increasing the specific antigen presenting function of DC in a
mammalian host. Prior to the immunization with an antigen, the host
is treated with a DC mobilization agent, e.g. Flt-3 ligand, GM-CSF,
G-CSF/FIt3L fusion protein, etc. This treatment effectively
increases the number of circulating DC precursors. The host is then
given a local, e.g. sub-cutaneous, intramuscular, etc., injection
of antigen in combination with a DC activating agent, e.g.
immunostimulatory DNA sequences, IL-1, alpha interferon, LPS,
endotoxin, CD40L, poly IC, etc. The activation step promotes
recruitment and maturation of the DC, along with antigen-specific
activation and migration from the tissues to lymphoid organs. These
DC then effectively interact with, and present processed antigen
to, T cells that are then able to respond to the antigen. The
methods of the invention are particularly useful in situations
where the host response to the antigen is sub-optimal, for example
in conditions of chronic infection, a lack of immune response to
tumor antigens, and the like. In one aspect of the invention, the
antigen is a tumor antigen, and is used to enhance the host immune
response to tumor cells present in the body.
BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1. ISS activate FL mobilized DC in vivo.
FIG. 2. ISS increase the immunogenicity of FL mobilized DC in
vivo.
FIG. 3. Studies of the antitumor effect of a treatment combining FL
and ISS.
FIG. 4. Treatment of preexisting tumors with the combination of FL
and ISS.
FIG. 5. Expansion of circulating blood DC following Flt3L
administration.
FIG. 6. Flt3L expanded human DC possess an immature phenotype.
FIG. 7. In vitro induction of CTL with human DC mobilized with FL
in vivo.
DESCRIPTION OF THE SPECIFIC EMBODIMENTS
A two step protocol is provided for the enhancement of T cell
mediated immune responses, in the absence of in vitro manipulation
of DC. The initial step of the method provides for the expansion
and mobilization in vivo of DC precursors, through administration
of a DC mobilizing agent.
After the host has responded to the DC expansion and mobilization,
usually from about 3 days to 2 weeks, there is an increased number
of DC precursors in the peripheral tissues, e.g. skin, muscle,
lungs, etc. These cells are not yet immunologically mature, but can
respond to DC activating agents, which agents include a variety of
immunostimulatory compounds. Of particular interest for this
purpose are immunostimulatory polynucleotide sequences. The DC
activating agent is preferably delivered directly to the peripheral
tissues.
The antigen of interest is delivered to the peripheral tissues
along with the DC activating agent, and may be given as a combined
formulation, or as separate formulations. The antigen may be
further provided in a booster dose, in combination with other
adjuvants as known in the art, etc.
The activation and antigenic stimulation in the peripheral tissues
activates the DC precursors to mature into functional DC, which are
then able to take-up and process the antigen of interest. On
maturation, the DC are competent to migrate to lymphatic organs,
particularly T cell rich regions of the lymph nodes, where T cell
activation occurs. Therefore, although the administration of
antigen and activating agent is localized, the resulting immune
response is not limited to that tissue.
Conditions of particular interest for use with the present methods
involve a lack of T cell mediated response to antigen, for example
chronic viral or bacterial infection, a lack of immune response to
tumor antigens, and the like. In one aspect of the invention, the
antigen is a tumor antigen, and is used to enhance the host immune
response to tumor cells present in the body.
Mammalian species that may require enhancement of T cell mediated
immune responses include canines and felines; equines; bovines;
ovines; etc. and primates, particularly humans. Animal models,
particularly small mammals, e.g. murine, lagomorpha, etc. may be
used for experimental investigations. Animal models of interest
include those involved with the immune response to infection and
tumors.
Methods
It is to be understood that this invention is not limited to the
particular methodology, protocols, cell lines, animal species or
genera, constructs, and reagents described, as such may vary. It is
also to be understood that the terminology used herein is for the
purpose of describing particular embodiments only, and is not
intended to limit the scope of the present invention which will be
limited only by the appended claims.
As used herein the singular forms "a", "and", and "the" include
plural referents unless the context clearly dictates otherwise.
Thus, for example, reference to "an immunization" includes a
plurality of such immunizations and reference to "the cell"
includes reference to one or more cells and equivalents thereof
known to those skilled in the art, and so forth. All technical and
scientific terms used herein have the same meaning as commonly
understood to one of ordinary skill in the art to which this
invention belongs unless clearly indicated otherwise.
DC mobilization agent, as used herein, refers to a compound,
particularly a naturally occurring protein or derivative thereof,
that acts on hematopoietic progenitor or stem cells to expand and
mobilize precursors of DC. During mobilization, the DC precursors
migrate from their tissue of origin, e.g. bone marrow, and move
into the peripheral blood and peripheral tissues. Some mobilization
agents will act broadly on the hematopoietic system, e.g. GM-CSF,
etc. In a preferred embodiment, the mobilization agent will
preferentially act to expand DC, e.g. by using Flt-3 ligand (FL) or
a fusion molecule containing FL and at least one other growth or
differentiation factor (e.g. G-CSF).
The dose of mobilizing agent will be effective to substantially
increase the number of DC precursors in peripheral tissues. The
increase in the number of DC precursors after the mobilization can
be quite high, usually by at least about 2 fold, more usually by at
least about 5 fold, and may be as high as a 20 or 75 fold increase.
DC precursors mobilized by Flt3L typically express CD4, MHC class
II, CD54, but lack expression of CD80, and may be identified by
these criteria.
The term "dendritic cell" refers to any member of a diverse
population of morphologically similar cell types found in lymphoid
or non-lymphoid tissues. These cells are characterized by their
distinctive morphology, high levels of surface MHC-class II
expression (Steinman, et al., Ann. Rev. Immunol. 9:271 (1991);
incorporated herein by reference for its description of such
cells).
The length of time required for expansion and mobilization is
usually at least about 3 or 4 days, more usually at least about 1
week, and can take about 10 days to 2 weeks for optimal expansion.
The length of time allotted for mobilization and expansion can be
predicted based on previous trials with the mobilizing agent at a
similar dose, or may be monitored individually by quantitating the
change in the number of DC precursors present in the peripheral
blood.
Various routes and regimens for delivery of the mobilization agent
may be used, as known and practiced in the art. For example, where
the mobilization agent is FL, the FL may be administered daily,
where the dose is from about 1 to 100 mg/kg body weight, more
usually from about 10 to about 50 mg/kg body weight, up to a
maximum dose of about 1 to 2 mg daily. Administration may be at a
localized site, e.g. sub-cutaneous, or systemic, e.g.
intraperitoneal, intravenous, etc.
Flt3 or Flk2 is a tyrosine kinase receptor structurally related to
macrophage colony-stimulating factor (CSF1) and to mast cell growth
factor receptor (c-kit). The FL is a growth factor that stimulates
the proliferation of certain hematopoietic progenitor cells. FL
mRNA is widely expressed in human tissues. The genetic sequence of
murine and human FL is described by Lyman et al. (1993) Cell 75:
1157-1167; and Lyman et al. (1994) Blood 83: 2795-2801 (Genbank
accession numbers L23636, and U03858, respectively).
For use in the subject methods, a native FL or modifications
thereof may be used. The FL sequence may be from any mammalian or
avian species, e.g. primate sp., particularly humans; rodents,
including mice, rats and hamsters; rabbits; equines, bovines,
canines, felines; etc. Of particular interest is the human protein.
Generally, for in vivo use the FL sequence will have the same
species of origin as the animal host.
The nucleic acid sequences encoding the human FL polypeptides may
be accessed from public databases as previously cited.
Identification of non-human Flt-3 ligands is accomplished by
conventional screening methods of DNA libraries or biological
samples for DNA sequences having a high degree of similarity to
known Flt-3 ligand sequences.
The sequence of the FL polypeptide may be altered in various ways
known in the art to generate targeted changes in sequence. The
polypeptide will usually be substantially similar to the sequences
provided herein, i.e. may differ by one more amino acids, but not
usually more than about ten amino acids. The sequence changes may
be substitutions, insertions or deletions. Scanning mutations that
systematically introduce alanine, or other residues, may be used to
determine key amino acids. Deletions may further include larger
changes, such as deletions of a domain or exon, providing for
active peptide fragments of the protein. Other modifications of
interest include epitope tagging, e.g. with the FLAG system, HA,
etc. Such alterations may be used to alter properties of the
protein, by affecting the stability, specificity, etc.
The protein may be joined to a wide variety of other oligopeptides
or proteins for a variety of purposes. By providing for expression
of the subject peptides, various post-expression modifications may
be achieved.
The FL for use in the subject methods may be produced from
eukaryotic or prokaryotic cells. Where the protein is produced by
prokaryotic cells, it may be further processed by unfolding, e.g.
heat denaturation, DTT reduction, etc. and may be further refolded,
using methods known in the art.
DC activating agent. Following the expansion and mobilization step,
the host periphery will have increased numbers of DC precursors.
These cells are not highly active antigen presenting cells, but can
be induced to mature into APC. The maturation process is stimulated
by a combination of DC activating agent, and the antigen of
interest.
The presence of DC precursors in the periphery indicates that that
the most effective route for delivering the activating agent is
through a local delivery, particularly dermal, sub-cutaneous and
intramuscular administration (see U.S. Pat. No. 5,830,877, Carson
et al., issued Nov. 3, 1998). Generally the antigen and the DC
activating agent will be delivered to the same site, and may be
co-formulated, e.g. mixed together, coadministered, conjugated
together, etc.; or formulated separately, depending on the
requirements of the specific agents.
A number of DC activating agents are known in the art, including
LPS and endotoxins in small doses, alpha interferons, interleukin-1
(see Boraschi et al. (1999)
Methods 19(1):108-13), modified tumor necrosis factor, CD40 ligand,
poly IC, etc. Of particular interest is the use of
immunostimulatory polynucleotide sequences (ISS), which have been
shown to be highly effective in the activation of DC, and other
antigen presenting cells. The use of these sequences is known in
the art, for examples see Bauer et al. (1999) Immunology
97(4):699-705; Klinman et al (1999) Vaccine 17(1):19-25; Hasan et
al. (1999) J Immunol Methods 229(1-2):1-22; and others.
An "immunostimulatory oligonucleotide" refers to an oligonucleotide
that contains a cytosine/guanine dinucleotide sequence and
stimulates maturation and activation of DC. An immunostimulatory
oligonucleotide of interest may be between 2 to 100 base pairs in
size and typically contain a consensus mitogenic CpG motif
represented by the formula: 5'X.sub.1 X.sub.2 CGX.sub.3 X.sub.4 3',
where C and G are unmethylated, X.sub.1, X.sub.2, X.sub.3 and
X.sub.4 are nucleotides and a GCG trinucleotide sequence is not
present at or near the 5' and 3' termini (see U.S. Pat. No.
6,008,200, Krieg et al., issued Dec. 28, 1999, herein incorporated
by reference).
Preferably the immunostimulatory oligonucleotides range between 8
to 40 base pairs in size. In addition, the immunostimulatory
oligonucleotides are preferably stabilized oligonucleotides,
particularly preferred are phosphorothioate stabilized
oligonucleotides. In one embodiment, X.sub.1 X.sub.2 is the
dinucleotide GpA. In another embodiment, X.sub.3 X.sub.4 is the
dinucleotide TpC or TpT.
The dose and protocol for delivery of the DC activating agent will
vary with the specific agent that is selected. Typically one or
more doses are administered. One particular advantage of the use of
ISS in the methods of the invention is that ISS exert
immunomodulatory activity even at relatively low dosages. Although
the dosage used will vary depending on the clinical goals to be
achieved, a suitable dosage range is one which provides from about
1 Fg to about 1,000 Fg or about 10,000 Fg of ISS in a single
dosage. Alternatively, a target dosage of ISS can be considered to
be about 1-10 FM in a sample of host blood drawn within the first
24-48 hours after administration of ISS. Based on current studies,
ISS are believed to have little or no toxicity at these dosage
levels.
Concurrent with the administration of a DC activating agent,
antigen is provided in one or more doses. Preferably the initial
dose of antigen is given at the same site as the DC activating
agent. Subsequent doses may be given at the same or a different
site, and may utilize other adjuvants as desired.
Antigens of interest include polypeptides and other immunogenic
biomolecules, which may be isolated or derived from natural
sources, produced by recombinant methods, etc., as known in the
art. Alternatively complex antigens may be used, for example cell
lysates, virus which may be inactivated, bacterial cells or
fractions derived therefrom, and the like.
The formulations are useful when used in conjunction with vaccines
such as, but not limited to, those for treating chronic bacterial
infections, e.g. tuberculosis, etc.; chronic viral infections such
as those associated with herpesvirus, lentivirus and retrovirus,
etc. Antigens of interest may also include allergens, e.g. for the
conversion of a Th2 to a Th1 type response. The antigens which may
be incorporated into the present formulations include viral,
prokaryotic and eukaryotic antigens, including but not limited to
antigens derived from bacteria, fungi, protozoans, parasites and
tumor cells.
Potential tumor antigens for immunotherapy include tumor specific
antigens, e.g. immunoglobulin idiotypes and T cell antigen
receptors; oncogenes, such as p21/ras, p53, p210/bcr-abl fusion
product; etc.; developmental antigens, e.g. MART-1/Melan A; MAGE-1,
MAGE-3; GAGE family; telomerase; etc.; viral antigens, e.g. human
papilloma virus, Epstein Barr virus, etc.; tissue specific
self-antigens, e.g. tyrosinase; gp100; prostatic acid phosphatase,
prostate specific antigen, prostate specific membrane antigen;
thyroglobulin, .alpha.-fetoprotein; etc.; and over-expressed self
antigens, e.g. her-2/neu; carcinoembryonic antigen, muc-1, and the
like.
Tumor cell derived protein extracts or RNA may be used as a source
of antigen, in order to provide multiple antigens and increase the
probability of inducing immunity to more than one tumor associated
antigen. Although the target antigens are initially undefined, the
immunogen can be later identified.
As an alternative to injecting antigen along with the DC activating
agent, endogenous tissues expressing antigen can be used as an
endogenous source of antigen. For example, tumors that express a
tumor antigen maybe injected with the DC activating agent in
conjunction with DC expansion to serve as the source to tumor
antigen. The DC activating agent would serve to recruit and
activated DC within the tumor where they would be capable of taking
up tumor derived antigen.
A number of antigens expressed on normal tissues as well as tumors
are useful as immunotherapy targets, and have been shown to
stimulate T cell responses when the antigens are presented by
DC.
Antigenic formulations will typically contain from about 0.1 .mu.g
to 1000 .mu.g, more preferably 1 .mu.g to 100 .mu.g, of the
selected antigen. The antigen composition may additionally contain
biological buffers, excipients, preservatives, and the like.
The antigen is administered to the host in the manner conventional
for the particular immunogen, generally as a single unit dose of an
antigen in buffered saline, combined with the adjuvant formulation,
where booster doses, typically one to several weeks later, may
additionally be delivered enterally or parenterally, e.g.,
subcutaneously, intramuscularly, intradermally, intravenously,
intraarterially, intraperitoneally, intranasally, orally, etc.
Subcutaneous or intramuscular injection is, however, preferred.
EXPERIMENTAL
The following examples are put forth so as to provide those of
ordinary skill in the art with a complete disclosure and
description of how to make and use the subject invention, and are
not intended to limit the scope of what is regarded as the
invention. Efforts have been made to insure accuracy with respect
to the numbers used (e.g. amounts, temperature, concentrations,
etc.) but some experimental errors and deviations should be allowed
for. Unless otherwise indicated, parts are parts by weight,
molecular weight is weight average molecular weight, temperature is
in degrees centigrade; and pressure is at or near atmospheric.
Example 1
In animal studies, it was found that immunostimulatory nucleic acid
sequences were are to activate dendritic cell precursors that had
been mobilized in vivo. The results are shown in FIG. 1. Mice
received 9 days of FL followed by 1 s.c injection of ISS, ODN or
PBS 4 days later DC were collected from the LN and analyzed by flow
cytometry. (A) DC were defined as MHC class II positive and CD11c
positive cells. Mature DC are distinguished from immature DC by
their high surface expression levels of MHC class II, CD86, CD40,
and CD62L. The ratio of mature vs. immature DC were calculated for
each group of mice. (B) The mean channel fluorescence range of
mature vs. immature DC is demonstrated.
The treatment also increased the immunogenicity of FL mobilized DC
in vivo. In order to analyze the immunogenicity induced by the
association of ISS and FL, FL treated mice were injected s.c. with
ISS, ODN or PBS mixed with ovalbumin into the foot pad. The
draining LN were collected 1 week later and T cells were tested for
their ability to proliferate in presence of antigen. As shown in
FIG. 2, ISS dramatically increases T cell priming.
To determine whether the combined treatment of FL+ISS would be able
to vaccinate against syngeneic tumors, mice were injected ip with
FL for 10 day period. Groups of mice received FL alone or 1 s.c.
injection of ISS or ODN in association with FL. All the groups were
immunized with ovalbumin. One week later the mice were injected
s.c. with tumor cells from B16 melanoma transduced with ovalbumin
gene. The group treated with FL+ISS did not develop any tumors
while the other groups developed tumors at different time points.
The ISS dramatically increased the antitumor effect of FL, and ODN
did not induce any antitumor response. 6 weeks after the first
immunization the mice were challenged with the same tumor cells.
Only one out of 5 mice treated with FL+ISS developed small tumors,
while the other 4 mice did not develop any tumors and survived
greater than 60 days after follow-up. The data is shown in FIG.
3.
Preexisting tumors could also be treated with the combination of FL
and ISS. To determine whether combined treatment of FL+ISS would be
able to vaccinate animals bearing tumor, mice were initially
injected s.c. with B16 melanoma transduced with ovalbumin gene. 5
days later, mice started on 10 daily injections of FL ip. On the
final day of FL treatment, mice were immunized with ovalbumin with
or without ISS. By six weeks following tumor challenge, all control
mice had developed tumors, while only 40% of the mice in the FL+ISS
group developed tumors, shown in FIG. 4.
Clinical studies were also performed to determine the effect of the
activation methods in human patients. It was shown that
administration of FL to patients with advanced cancer mobilizes DC
precursors into the blood, resulting in an increase of circulating
DC of 20 fold on average.
An expansion of circulating blood DC was found following Flt3L
administration. PBMC from patients with advanced cancer were
assessed either before or following 10 days of Flt3L
administration. DC were characterized phenotypically by expression
of HLA DR without expression of lineage markers (CD3, 14, 19, 56).
Patients developed a significant increase in circulating blood DC
precursors as assessed by flow cytometry, shown in FIG. 5.
The Flt3L expanded human DC possessed an immature phenotype, shown
in FIG. 6. DC were gated on their expression of HLA DR and lack of
lineage markers CD3, 14, 19, 56. Flt3L expanded DC express
intermediate levels of CD4 and CD54 more homogeneously by
comparison with unmobilized DC. Flt3L expanded DC, however, lack
surface expression of CD86 and CD40 compared with unmobilized DC.
Results are from one patient and are representative of three
patients studied.
These in vivo mobilized cells were shown to be capable of inducing
CTL in vitro. To assess the ability of FL mobilized DC to prime CD8
cytotoxic T lymphocytes (CTL) in vitro, DC precursors were purified
with immunomagnetic beads following FL mobilization in vivo.
Purified DC were then cultured overnight with the target peptide
Cap-1D alone or in the presence of activating agents ISS,
adenovirus (Adeno), or CD40L. Purified DC were then irradiated and
cultured with purified autologous CD8 T cells. Subsequent CTL
cultures where then assess by 4 hour .sup.51 Cr release assay for
their ability to kill target cell line T2 with or without the
target peptide Cap-1D. Induction of antigen specific CTL were only
seen in CTL cultures using ISS activated DC as the stimulating
antigen presenting cell, shown in FIG. 7.
It is apparent from the above results that an effective stimulation
of immune response can be gained by mobilizing dendritic cell
precursors, following by activation and antigenic stimulation. The
methods result in a tumor specific immune response against new, and
pre-existing tumors.
All publications and patent applications cited in this
specification are herein incorporated by reference as if each
individual publication or patent application were specifically and
individually indicated to be incorporated by reference.
Although the foregoing invention has been described in some detail
by way of illustration and example for purposes of clarity of
understanding, it will be readily apparent to those of ordinary
skill in the art in light of the teachings of this invention that
certain changes and modifications may be made thereto without
departing from the spirit or scope of the appended claims.
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