U.S. patent application number 09/791992 was filed with the patent office on 2001-11-01 for adjuvant treatment by in vivo activation of dendritic cells.
Invention is credited to Engleman, Edgar G., Fong, Lawrence H., Merad, Miriam.
Application Number | 20010036462 09/791992 |
Document ID | / |
Family ID | 22678426 |
Filed Date | 2001-11-01 |
United States Patent
Application |
20010036462 |
Kind Code |
A1 |
Fong, Lawrence H. ; et
al. |
November 1, 2001 |
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) |
Correspondence
Address: |
Pamela J. Sherwood
BOZICEVIC, FIELD & FRANCIS LLP
200 Middlefield Road, Suite 200
Menlo Park
CA
94025
US
|
Family ID: |
22678426 |
Appl. No.: |
09/791992 |
Filed: |
February 22, 2001 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
60184810 |
Feb 24, 2000 |
|
|
|
Current U.S.
Class: |
424/204.1 ;
424/234.1; 424/277.1 |
Current CPC
Class: |
A61K 38/18 20130101;
A61P 37/04 20180101; A61P 31/12 20180101; A61K 2039/55522 20130101;
A61P 35/00 20180101; A61P 31/10 20180101; A61P 31/04 20180101; A61K
38/18 20130101; A61K 2300/00 20130101; A61P 43/00 20180101; A61K
39/39 20130101; A61P 33/00 20180101; A61K 2039/55516 20130101; A61P
31/14 20180101; A61P 31/22 20180101; A61K 2039/55561 20130101; A61P
31/06 20180101 |
Class at
Publication: |
424/204.1 ;
424/277.1; 424/234.1 |
International
Class: |
A61K 039/12; A61K
039/02; A61K 039/00 |
Claims
What is claimed is:
1. A method of increasing the immune response in a mammalian host
to a target antigen, the method comprising: administering a dose of
a DC mobilization agent effective to substantially increase the
number of DC precursors present in the periphery of said host;
administering a dose of a DC activation agent effective to induce
maturation of said DC precursors in combination with said target
antigen.
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.
Description
[0001] 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.
[0002] 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.
[0003] 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.
[0004] 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.
[0005] 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.
[0006] 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.
[0007] Relevant Literature
[0008] 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.
[0009] 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.
[0010] U.S. Pat. No. 5,994,126, Steinman et al., 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
[0011] 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/Flt3L 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
antigenspecific 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
[0012] FIG. 1. ISS activate FL mobilized DC in vivo.
[0013] FIG. 2. ISS increase the immunogenicity of FL mobilized DC
in vivo.
[0014] FIG. 3. Studies of the antitumor effect of a treatment
combining FL and ISS.
[0015] FIG. 4. Treatment of preexisting tumors with the combination
of FL and ISS.
[0016] FIG. 5. Expansion of circulating blood DC following Flt3L
administration.
[0017] FIG. 6. Flt3L expanded human DC possess an immature
phenotype.
[0018] FIG. 7. In vitro induction of CTL with human DC mobilized
with FL in vivo.
DESCRIPTION OF THE SPECIFIC EMBODIMENTS
[0019] 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.
[0020] 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.
[0021] 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.
[0022] 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.
[0023] 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.
[0024] 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
[0025] 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.
[0026] 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.
[0027] 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).
[0028] 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.
[0029] 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).
[0030] 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.
[0031] 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.
[0032] 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).
[0033] 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.
[0034] 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.
[0035] 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.
[0036] 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.
[0037] 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.
[0038] 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.
[0039] 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.
[0040] 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)
[0041] 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 a. (1999) Vaccine 17(1):19-25; Hasan et
al. (1999) J Immunol Methods 229(1-2):1-22; and others.
[0042] 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.1X.sub.2CGX.sub.3X.sub.43', 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).
[0043] 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.
[0044] 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.
[0045] 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.
[0046] 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.
[0047] 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.
[0048] 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.
[0049] 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.
[0050] 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.
[0051] 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.
[0052] 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.
[0053] 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
[0054] 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
[0055] 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.
[0056] 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.
[0057] 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 a greater than 60 days after follow-up. The data is shown
in FIG. 3.
[0058] 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.
[0059] 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.
[0060] An expansion of circulating blood DC was found following
FIt3L 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.
[0061] 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.
[0062] 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.51Cr 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.
[0063] 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.
[0064] 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.
[0065] 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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