U.S. patent application number 14/193498 was filed with the patent office on 2014-08-21 for targeted lipid-drug formulations for delivery of drugs to myeloid and lymphoid immune cells.
This patent application is currently assigned to Rodos Bio Target GmbH. The applicant listed for this patent is Rodos Bio Target GmbH. Invention is credited to Robert K. GIESELER, Guido Marquitan, Michael J. Scolaro, Sean M. Sullivan.
Application Number | 20140234400 14/193498 |
Document ID | / |
Family ID | 34381085 |
Filed Date | 2014-08-21 |
United States Patent
Application |
20140234400 |
Kind Code |
A1 |
GIESELER; Robert K. ; et
al. |
August 21, 2014 |
Targeted Lipid-Drug Formulations for Delivery of Drugs to Myeloid
and Lymphoid Immune Cells
Abstract
A method of preferentially delivering an active agent to an
immune cell, such as a myeloid progenitor cell, a dendritic cell, a
monocyte, a macrophage or a T-lymphocyte, or other cell type
restricted to a functional organ system or an anatomic entity, of a
mammalian subject by administering a lipid-drug complex to the
subject. The lipid-drug complex is comprised of an active agent,
such as a drug, and an outer surface with a targeting ligand that
binds a marker on the surface of the immune cell or other cell type
that is infected with or susceptible to infection with an
infectious agent. The other cell type that is infected with or
susceptible to infection with an infectious agent may belong to a
malignant tumor or a part of the immune system contributing to the
development, maintenance, or exacerbation of an autoimmune disease
or chronic inflammatory disease.
Inventors: |
GIESELER; Robert K.; (West
Hollywood, CA) ; Marquitan; Guido; (Los Angeles,
CA) ; Scolaro; Michael J.; (Los Angeles, CA) ;
Sullivan; Sean M.; (Gainesville, FL) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Rodos Bio Target GmbH |
Hanover |
|
DE |
|
|
Assignee: |
Rodos Bio Target GmbH
Hanover
DE
|
Family ID: |
34381085 |
Appl. No.: |
14/193498 |
Filed: |
February 28, 2014 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10943758 |
Sep 17, 2004 |
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14193498 |
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60503769 |
Sep 17, 2003 |
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60567376 |
Apr 30, 2004 |
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Current U.S.
Class: |
424/450 ;
424/144.1; 435/375 |
Current CPC
Class: |
A61K 39/085 20130101;
A61K 38/168 20130101; A61P 35/00 20180101; A61K 39/3955 20130101;
A61P 33/00 20180101; A61P 31/04 20180101; A61P 31/10 20180101; A61P
31/00 20180101; A61K 47/6913 20170801; A61P 31/12 20180101; A61K
36/185 20130101; A61K 39/395 20130101; A61P 37/02 20180101; A61K
9/127 20130101 |
Class at
Publication: |
424/450 ;
424/144.1; 435/375 |
International
Class: |
A61K 9/127 20060101
A61K009/127; A61K 38/16 20060101 A61K038/16; A61K 36/185 20060101
A61K036/185; A61K 39/395 20060101 A61K039/395; A61K 39/085 20060101
A61K039/085 |
Claims
1. A method of preferentially delivering a drug to an immune cell
being affected with, or susceptible to infection with, an
infectious agent, comprising: administering to a mammalian subject
a lipid-drug complex comprising: a) at least one drug; and b) a
lipid shell comprising on its outer surface targeting ligands that
specifically bind to the CD4 and CD45R0 marker combination
co-expressed on the surface of the immune cell, wherein said drug
is selected from the group consisting of an antiviral agent,
antibacterial agent, immunomodulatory agent and therapeutic
cytotoxic agent that is active in the immune cell targeted by the
targeting ligands and wherein the targeting ligands are monoclonal
antibodies or F(ab) or F(ab).sub.2 fragments thereof.
2. The method of claim 1, wherein the infectious agent is a virus,
a bacterium, a fungus, a protozoan, or a prion.
3. The method of claim 2, wherein the virus is selected from the
group consisting of HIV, HSV, EBV, CMV, Ebola and Marburg virus,
HAV, HBV, HCV and HPV.
4. The method of claim 1, wherein the lipid-drug complex is a
liposome-drug complex.
5. The method of claim 1, wherein the lipid-drug complex further
comprises one or more secondary drugs.
6. The method of claim 1, wherein administering is by a
subcutaneous, an intradermal, an intraperitoneal, an intravenous, a
transvascular, or a parenteral route.
7. The method of claim 1, wherein said immune cell is selected from
the group consisting of myeloid progenitor cells, monocytes,
dendritic cells, macrophages and T-lymphocytes.
8. The method of claim 7, wherein the dendritic cell is a myeloid
dendritic cell, a plasmacytoid dendritic cell, or a follicular
dendritic cell.
9. The method of claim 7, wherein the T lymphocytes are T-helper
cells or T-memory cells.
10. The method of claim 1, wherein the outer surface of the lipid
shell further comprises a Staphylococcus aureus protein A adapted
for specifically binding IgG.
11. The method of claim 10, wherein the targeting ligands are
monoclonal or polyclonal antibodies specifically bound by the
Staphylococcus aureus protein A.
12. The method of claim 1, wherein the drug is an expression vector
for dendritic cell-mediated vaccination.
13. The method of claim 1, wherein the drug is a natural
substance.
14. The method of claim 13, wherein the natural substance is
plant-derived and purified.
15. The method of claim 14, wherein the natural substance is
recombinantly produced.
16. The method of claim 13, wherein the natural substance is IDS-30
(Hox alpha) extract of the stinging nettle, rhizome-derived Urtica
dioica agglutinin (UDA) derived from Urtica dioica, or the
Myrianthus holstii lectin (MHL) derived from Myrianthus
holstii.
17. A method of preferentially targeting a mammalian immune cell
with a liposome, wherein said immune cell is an antigen-presenting
cell, comprising: administering to the immune cell a liposome,
wherein the liposome comprises: a) at least one active agent; and
b) a lipid shell comprising on its outer surface targeting ligands
that specifically bind to a CD4 and CD45R0 marker combination
co-expressed on the surface of the immune cell, or wherein said
active agent is an immunomodulatory agent that is active in an
immune cell targeted by the targeting ligands and wherein the
targeting ligands are monoclonal antibodies or F(ab) or F(ab).sub.2
fragments thereof.
18. The method of claim 17, wherein the immunomodulatory agent is
an immunosuppressant.
19. The method of claim 17, wherein the immunomodulatory agent is
an immunoactivating agent.
Description
[0001] This application is a continuation of U.S. patent
application Ser. No. 10/943,758, filed Sep. 17, 2004, which claims
priority under 35 U.S.C. 119 from U.S. provisional patent
application Nos. 60/503,769, filed Sep. 17, 2003, and 60/567,376,
filed Apr. 30, 2004, the entire disclosures of which are expressly
incorporated by reference herein.
BACKGROUND OF THE INVENTION
[0002] 1. Field of the Invention
[0003] The present invention relates to the medical arts, and in
particular, to targeted liposomal drug delivery.
[0004] 2. Discussion of the Related Art
[0005] Myeloid dendritic cells (My-DCs) belong to the most potent
group of professional antigen-presenting cells, with the unique
ability to induce primary cellular and humoral immune responses
(reviewed in Banchereau J, Paczesny S, Blanco P, Bennett L, Pascual
V, Fay J, Palucka A K, Dendritic cells: controllers of the immune
system and a new promise for immunotherapy, Ann N Y Acad Sci
987:180-7 [2003]). These cells, within the lymphoid organs and
structures, are also an important component of the HIV reservoir,
together with other major sanctuary populations, i.e. follicular
dendritic cells, macrophages, resting/memory T cells, and cells
within the central nervous system. (E.g., Schrager L K, D'Souza M
P, Cellular and anatomical reservoirs of HIV-1 in patients
receiving potent antiretroviral combination therapy, JAMA 280:67-71
[1998]). It is a key characteristic of reservoir cells that they
are compromised and exploited, but not killed, by HIV, thus leading
to a continuous infection of other immune and non-immune cells
within an infected person. (Gieseler R K, Marquitan G, Scolaro M J,
Cohen M D, Lessons from history: dysfunctional APCs, inherent
dangers of Sri and an important goal, as yet unmet, Trends Immunol.
2003; 24:11).
[0006] In-vitro generation of My-DCs has enabled comprehensive
phenotypic and functional characterization of the My-DCs and the
study of the ontogeny of these cells, which have been found to
share with macrophages an early common myeloid progenitor (Gieseler
R K, Rtiber R A, Kuhn R, Weber K, Osborn M, Peters J H, Dendritic
accessory cells derived from rat bone marrow precursors under
chemically defined conditions in vitro belong to the myeloid
lineage, Eur J Cell Biol 1991; 54:171-81; Peters J R, Xu H, Ruppert
J, Ostermeier D, Friedrichs D, Gieseler R K, Signals required for
differentiating dendritic cells from human monocytes in vitro, Adv
Exp Med Biol 1993; 329:275-80; Peters J H, Gieseler R, Thiele B,
Steinbach F, Dendritic cells: from ontogenetic orphans to
myelomonocytic descendants, Immunol Today 1996; 17:273-8; Gieseler
R, Heise D, Soruri A, Schwartz P, Peters J H, In-vitro
differentiation of mature dendritic cells from human blood
monocytes, Dev Immunol 1998; 6:25-39).
[0007] The discovery of the My-DC-specific intercellular adhesion
molecule 3-grabbing nonintegrin (DC-SIGN) in the year 2000 was a
milestone of immunologic research: DC-SIGN, one of several C-type
lectins, is both a distinctive key DC molecule and plays an
essential role in the capture and migratory transport of HIV.
Besides T-cell infection due to active virus production by My-DCs,
interaction of HIV and DC-SIGN eventually enables My-DCs to infect
in-trans cooperating T-helper cells. Also, variants of DC-SIGN are
expressed by macrophages (another major HIV-1 reservoir), as well
as by several mucosal and placental cell types (Soilleux, E J et
al. Constitutive and induced expression of DC-SIGN on dendritic
cell and macrophage subpopulations in situ and in vitro, J Leukoc
Biol 71:445-57 [2002]; Geijtenbeek, T B H et al., Marginal zone
macrophages express a murine homologue of DC-SIGN that captures
blood-borne antigens in vivo, Blood 100:2908-16 [2002]; Soilleux E
J et al., Placental expression of DC-SIGN may mediate intrauterine
vertical transmission of HIV, J Pathol. 195(5):586-92 [2001];
Soilleux E J, Coleman N, Transplacental transmission of HIV: a
potential role for HIV binding lectins, Int J Biochem Cell Biol.;
35(3):283-7 [2003]; Kammerer U et al., Unique appearance of
proliferating antigen-presenting cells expressing DC-SIGN (CD209)
in the decidua of early human pregnancy, Am J Pathol. 162(3):887-96
[2003]). These C-type lectins, therefore, qualify as major players
in the horizontal and vertical transmission of HIV within a given
individual (Geijtenbeek T B, van Kooyk Y, DC-SIGN: a novel HIV
receptor on DCs that mediates HIV-1 transmission, Curr Top
Microbiol Immunol 276:31-54 [2003]). In vivo, DC-SIGN is not only
expressed by myeloid DCs, but also by subpopulations of
macrophages, which are another main group of HIV reservoir cells
(Soilleux E J et al., Constitutive and induced expression of
DC-SIGN on dendritic cell and macrophage subpopulations in situ and
in vitro, J Leukoc Biol. 71(3):445-57 [2002]).
[0008] It is known that DC-SIGN is an endocytic adhesion
receptor.
[0009] First, DC-SIGN-attached particles are shuttled into the MHC
class II antigen processing and presentation pathway and are
accessed to the mechanism generating T-cell immunity (as desirable
in case of any viral infection), as well as B-cell immunity (as
supportive in the clearance of virus, by mechanisms secondary to
the generation of antibodies, such as Fc receptor-mediated
phagocytosis or, in case of cytotoxic antibodies,
complement-mediated lysis) (e.g., Schjetne K W et al., Mouse
C.sub.K-specific T cell clone indicates that DC-SIGN is an
efficient target for antibody-mediated delivery of T cell epitopes
for MHC class II presentation, Int Immunol 14(12):1423-30 [2002];
Engering, A et al., The dendritic cell-specific adhesion receptor
DC-SIGN internalizes antigen for presentation to T cells, J
Immunol. 168(5):2118-26 [2002]).
[0010] Second, Turville et al, demonstrated that Th-cell infection
by MyDCs with HIV-1 is a two-phased process that depends on the
DCs' developmental stage, including both directional transport of
virus to the immunological synapse, as well as active de-novo
synthesis of HIV-1 from proviral DNA (Turville S G, Santos J J,
Frank I et al. Immunodeficiency virus uptake, turnover, and
two-phase transfer in human dendritic cells, Blood; online
publication ahead of print: DOI 10.1182/blood-2003-09-3129 [2003]).
In addition, the important roles of DC-SIGN in the migratory
transport of virus by MyDCs (Geijtenbeek T B H, van Kooyk Y,
DC-SIGN: a novel HIV receptor on DCs that mediates HIV-1
transmission, Curr Top Microbiol Immunol; 276:31-54 [2003]) and in
the in-trans infection of Th cells (Geijtenbeek T B H, Kwon D S,
Torensma R et al. DC-SIGN, a dendritic cell-specific HIV-1-binding
protein that enhances trans-infection of T cells, Cell; 100:587-97
[2000]) very much support a pathogenetic key role for these cells.
Intriguingly, it has now been shown that passive transfer from
MyDCs to Th cells via DC-SIGN requires that HIV-1 is first
internalized into intracellular trypsin-resistant compartments
(McDonald D, Wu L, Bohks S M, KewalRamani V N, Unutmaz D, Hope T J,
Recruitment of HIV and its receptors to dendritic cell--T cell
junctions, Science; 300:1295-7 [2003]; Kwon D S, Gregorio G, Bitten
N, Hendrickson W A, Littman D R, DC-SIGN-mediated internalization
of HIV is required for trans-enhancement of T cell infection,
Immunity; 16:135-44 [2002]). Indeed, after infection with HIV-1,
intracytoplasmic compartments with accumulated infectious virus are
demonstrable in both immature and mature MyDCs (Frank I, Piatak M
Jr, Stoessel H, Romani N, Bonnyay D, Lifson J D, Pope M, Infectious
and whole inactivated simian immunodeficiency viruses interact
similarly with primate dendritic cells (DCs): differential
intracellular fate of virions in mature and immature DCs, J Virol;
76:2936-51 [2002]).
[0011] Highly Active Antiretroviral Therapy (HAART) has been shown
to be effective to reduce the plasma viral load to undetectable
levels in HIV-infected individuals and to markedly diminish the
number of HIV-1 RNA copies in secondary lymphoid tissues (Wong, J.
K. et al., Recovery of replication-competent HIV despite prolonged
suppression of plasma viremia, Science, 278: 1291-1295 [1997];
Cavert, W. et al., Kinetics of response in lymphoid tissues to
antiretroviral therapy of HIV-1 infection, Science
276(5314):960-964 [1997]). However, the capacity of HIV-1 to
establish latent infection allows viral particles to persist in
tissues despite immune responses and antiretroviral therapy (Gangne
J-F, Desormeaux A, Perron S, Tremblay M. J, Bergeron M. G, Targeted
delivery of indinavir to HIV-1 primary reservoirs with
immunoliposomes, Biochim Biophys Acta, 1558: 198-210 [2002]). It is
hypothesized that the susceptibility of dendritic cells to being
infected with HIV, together with their crucial immunologic
function, leads to the continuous spread of HIV. Therefore, it has
been suggested that targeting of anti-virals to these reservoir
cells is an important goal to achieve permanent reconstitution of
adaptive immunity (Gieseler R K, Marquitan G, Scolaro M J, Cohen M
D, Lessons from history: dysfunctional APCs, inherent dangers of
STI and an important goal, as yet unmet, Trends Immunol 24:11
[2003]).
[0012] Liposomes are a suitable vehicle for specifically delivering
encapsulated compounds to any given cell type, provided the
existence of an appropriate targeting structure. Because of its
highly restricted cellular expression, DC-SIGN qualifies as such a
targeting molecule. We have earlier shown inhibition of HIV
propagation in infected peripheral blood mononuclear leukocytes
after liposomal delivery of sense DNA directed towards the HIV 5'
tat splice acceptor site (Sullivan S M, Gieseler R K, Lenzner S,
Ruppert J, Gabrysiak T G, Peters J H, Cox G, Richer L, Martin W I,
Scolaro M J, Inhibition of human immunodeficiency virus-1
proliferation by liposome-encapsulated sense DNA to the 5' tat
splice acceptor site, Antisense Res Dev; 2:187-97 [1992]).
[0013] Since the discovery in the 1960s that hydration of dry lipid
film forms enclosed spherical vesicles or liposomes that resemble
miniature cellular organelles with lipid bilayers, the potential
use of lipid-drug complexes as biodegradable or biocompatible drug
carriers to enhance the potency and reduce the toxicity of
therapeutics was recognized (e.g., Bangham A D, Liposomes: the
Babraham connection, Chem Phys Lipids 64:275-285 [1993]).
Lipid-drug complexes have long been seen as a potential way to
improve the Therapeutic Index (TI) of drugs by increasing their
localization to specific organs, tissues or cells. The TI is the
ratio between the median toxic dose (TD50) and the median effective
dose (ED50) of a particular drug. However, application of
lipid-drug complexes to drug delivery systems was not realized
until 30 years later. Only then were the first series of
liposome-based therapeutics approved for human use by the U.S. Food
and Drug Administration (FDA). Liposomes have been used as drug
carriers in pharmaceutical applications since the mid-1990s (Lian,
T. and Ho, R. J. Y., Trends and Developments in Liposome Drug
Delivery Systems, J. Pharm. Sci. 90(6):667-80 [2001]).
[0014] Although the lipid constituent can vary, many formulations
use synthetic products of natural phospholipid, mainly
phosphatidylcholine. Most of the liposome formulations approved for
human use contain phosphatidylcholine (neutral charge), with fatty
acyl chains of varying lengths and degrees of saturation, as a
major membrane building block. A fraction of cholesterol (.about.30
mol %) is often included in the lipid formulation to modulate
rigidity and to reduce serum-induced instability caused by the
binding of serum proteins to the liposome membrane.
[0015] Based on the head group composition of the lipid and the pH,
liposomes can bear a negative, neutral, or positive charge on their
surface. The nature and density of charge on the surface of the
liposomes influences stability, kinetics, and extent of
biodistribution, as well as interaction with and uptake of
liposomes by target cells. Liposomes with a neutral surface charge
have a lower tendency to be cleared by cells of the
reticuloendothelial system (RES) after systemic administration and
the highest tendency to aggregate. Although negatively charged
liposomes reduce aggregation and have increased stability in
suspension, their nonspecific cellular uptake is increased in vivo.
Negatively charged liposomes containing phosphatidylserine (PS) or
phosphatidylglycerol (PG) were observed to be endocytosed at a
faster rate and to a greater extent than neutral liposomes (Allen T
M, et al., Liposomes containing synthetic lipid derivatives of
poly(ethylene glycol) show prolonged circulation half-lives in
vivo, Biochim Biophys Acta 1066:29-36 [1991]; Lee R J, et al.,
Folate-mediated tumor cell targeting of liposome-entrapped
doxorubicin in vitro, Biochim Biophys. Acta 1233:134-144 [1995]).
Negative surface charge is recognized by a variety of receptors on
various cell types, including macrophages (Allen T M et al. [1991];
Lee R J, et al., Delivery of liposomes into cultured KB cells via
folate receptor-mediated endocytosis, J Biol Chem 269:3198-3204
[1994]).
[0016] Inclusion of some glycolipids, such as the ganglioside
GM.sub.1 or phosphotidylinositol (PI), inhibits uptake by
macrophages and RES cells and results in longer circulation times.
It has been suggested that a small amount of negatively charged
lipids stabilize neutral liposomes against an aggregation-dependent
uptake mechanism (Drummond D C, et al., Optimizing liposomes for
delivery of chemotherapeutic agents to solid tumors, Pharmacol Rev
51:691-743 [1999]). Positively charged (i.e. cationic) liposomes,
often used as a DNA condensation reagent for intracellular DNA
delivery in gene therapy, have a high tendency to interact with
serum proteins; this interaction results in enhanced uptake by the
RES and eventual clearance by lung, liver, or spleen. This
mechanism of RES clearance partly explains the low in vivo
transfection efficiency. Other factors, including DNA instability,
immune-mediated clearance, inflammatory response, and tissue
accessibility can also contribute to low transfection efficiency in
animals. In fact, high doses of positively charged liposomes have
been shown to produce varying degrees of tissue inflammation
(Scheule R K, et al., Basis of pulmonary toxicity associated with
cationic lipid-mediated gene transfer to the mammalian lung, Hum
Gene Ther 8:689-707 [1997]).
[0017] The surface of the liposome membrane can be modified to
reduce aggregation and avoid recognition by the RES using
hydrophilic polymers. This strategy is often referred to as surface
hydration or steric modification. Surface modification is often
done by incorporating gangliosides, such as GM.sub.1, or lipids
that are chemically conjugated to hygroscopic or hydrophilic
polymers, usually polyethyleneglycol (PEG). This technology is
similar to protein PEGylation. Instead of conjugating PEG to
therapeutic proteins such as adenosine deaminase (Alderase, for
treatment of severe combined immunodeficiency syndrome) to reduce
immune recognition and rapid clearance (Beauchamp C, et al.,
Properties of a novel PEG derivative of calf adenosine deaminase,
Adv Exp Med Biol 165:47-52 [1984]), PEG is conjugated to the
terminal amine of phosphatidylethanolamine. This added presence of
hydrophilic polymers on the liposome membrane surface provides an
additional surface hydration layer (Torchilin V P, Immunoliposomes
and PEGylated immunoliposomes: possible use of targeted delivery of
imaging agents, Immunomethods 4:244-258 [1994]). The resulting
liposomes can be recognized neither by macrophages nor the RES as
foreign particles, and thus escape phagocytic clearance. A number
of systematic studies have determined the optimum size of PEG
polymer and the density of the respective polymeric PEG lipid in
the Liposome membrane.
[0018] Early research has demonstrated that the liposome size
affects vesicle distribution and clearance after systemic
administration. The rate of liposome uptake by RES increases with
the size of the vesicles (Hwang K, Liposome pharmacokinetics, In:
Ostro M J, editor, Liposomes: from biophysics to therapeutics, New
York: Marcel Dekker, pp. 109-156 [1987]). Whereas RES uptake in
vivo can be saturated at high doses of liposomes or by predosing
with large quantities of control liposomes, this strategy may not
be practical for human use because of the adverse effects related
to sustained impairment of physiological functions of the RES. The
general trend for liposomes of similar composition is that an
increasing size results in enhanced uptake by the RES (Senior J, et
al., Tissue distribution of liposomes exhibiting long half-lives in
the circulation after intravenous injection, Biochim Biophys Acta
839:1-8 [1985]). Most recent investigations have used unilamellar
vesicles, 50-100 nm in size, for systemic drug delivery
applications. For example, the antifungal liposome product AmBisome
is formulated to the size specification of 45-80 nm to reduce RES
uptake. Serum protein binding is an important factor that affects
liposome size and increases the rate of clearance in vivo.
Complement activation by liposomes and opsonization depend on the
size of the liposomes (Devine D V, et al., Liposome-complement
interactions in rat serum: Implications for liposome survival
studies, Biochim Biophys Acta 1191:43-51 [1994]; Liu D, et al.,
Recognition and clearance of liposomes containing
phosphatidylserine are mediated by serum opsonin, Biochim Biophys
Acta 1235:140-146 [1995]). Even with the inclusion of PEG in the
liposome compositions to reduce serum protein binding to liposomes,
the upper size limit of long-circulation PEG-PE liposomes is
.about.200 nm. Due to biological constraints, development of long
circulating large (>500 nm) liposomes using steric stabilization
methods has not been successful. Hence, considerations of liposome
size and its control in manufacturing at an early stage of drug
development provide a means to optimize efficiency of liposome drug
delivery systems.
[0019] The exact mechanisms of biodistribution and disposition in
vivo vary depending on the lipid composition, size, charge, and
degree of surface hydration/steric hindrance. In addition, the
route of administration may also influence the in vivo disposition
of liposomes. Immediately after intravenous administration,
liposomes are usually coated with serum proteins and taken up by
cells of the RES and eventually eliminated. (Chonn A, et al.,
Association of blood proteins with large unilamellar liposomes in
vivo. Relation to circulation lifetimes, J Biol Chem
267:18759-18765 [1992]; Rao M, et al., Delivery of lipids and
liposomal proteins to the cytoplasm and Golgi of antigen presenting
cells, Adv Drug Deliv Rev 41:171-188 [2000]). Plasma proteins that
can interact with liposomes include albumin, lipoproteins (ie.,
high-density lipoprotein [HDL], low-density lipoprotein [LDL],
etc.) and cell-associated proteins. Some of these proteins (e.g.,
HDL) can remove phospholipids from the liposome bilayer, thereby
destabilizing the liposomes. This process may potentially lead to a
premature leakage or dissociation of drugs from liposomes.
[0020] One of the key properties that make liposomes an invaluable
drug delivery system is their ability to modulate the
pharmacokinetics of liposome-associated and encapsulated drugs
(Hwang K J, Padki M M, Chow D D, Essien H E, Lai J Y, Beaumier P L,
Uptake of small liposomes by non-reticuloendothelial tissues,
Biochim Biophys Acta; 901(1):88-96 [1987]; Allen T M, Hansen C,
Martin F, Redemann C, Yau-Young A, Liposomes containing synthetic
lipid derivatives of poly(ethylene glycol) show prolonged
circulation half-lives in vivo, Biochim Biophys Acta; 1066(1):29-36
[1991]; Allen T M, Austin G A, Charm A, Lin L, Lee K C, Uptake of
liposomes by cultured mouse bone marrow macrophages: influence of
liposome composition and size, Biochim Biophys Acta; 1061(1):56-64
[1991]; Hwang, K. [1987]; Allen T, et al., Pharmacokinetics of
long-circulating liposomes, Adv Drug Del Rev 16:267-284 [1995]).
Relative to the same drugs in aqueous solution, significant changes
in absorption, biodistribution, and clearance of
liposome-associated drug are apparent, resulting in dramatic
effects on both the efficacy and toxicity of the entrapped compound
(Gabizon A, Liposome circulation time and tumor targeting:
implications for cancer chemotherapy, Adv Drug Del Rev 16:285-294
[1995]; Bethune C, et al., Lipid association increases the potency
against primary medulloblastoma cells and systemic exposure of
1-(2-chloroethyl)-3-cyclohexyl-1-nitrosourea (CCNU) in rats, Pharm
Res 16:896-903 [1999]). However, therapeutic applications of
systemically administered liposomes have been limited by their
rapid clearance from the bloodstream and their uptake by the RES
(Alving C, et al., Complement-dependent phagocytosis of liposomes:
suppression by `stealth` lipids, J Liposome Res 2:383-395
[1992]).
[0021] As already mentioned, circulation time can be increased by
reducing the liposome size and modifying the surface/steric effect
with PEG derivatives. Also, liposomes with membranes engineered for
sufficient stability escaping clearance by the RES are now
available. Therefore, long-circulation liposomes that also
significantly reduce toxicological profiles of the respective drugs
can be used to maintain and extend plasma drug levels. Even though
only a small fraction of liposomes eventually accumulate at target
sites, prolonged circulation can indirectly enhance accumulation of
liposome-associated drugs to targeted tissues.
[0022] It is a desideratum to actively enhance targeting of
liposomes so as to direct them to the cell populations of interest
before substantial clearance by the RES occurs. For example,
immunoliposomes have been employed to target the erythrocyte
reservoirs of intracellular malarial parasites (Owais, M. et al.,
Chloroquine encapsulated in malaria-infected erythrocyte-specific
antibody-bearing liposomes effectively controls
chloroquine-resistant Plasmodium berghei infections in mice,
Antimicrob Agents Chemother 39(1):180-4 [1995]; Singh, A M et al.,
Use of specific polyclonal antibodies for site specific drug
targeting to malaria infected erythrocytes in vivo, Indian J
Biochem Biophys 30(6):411-3 [1993]).
[0023] It is also a desideratum to apply lipid-drug delivery
systems to the fight against the HIV/AIDS pandemic. More than 42
million people are estimated to be currently living with HIV/AIDS
(UNAIDS [2002; 2003]). This global figure has been projected to
increase considerably if no improved means of keeping this
infection at bay will be developed and introduced to the global
community (Morens D M, Folkers G K, Fauci A S, The challenge of
emerging and re-emerging infectious diseases, Nature; 430:242-9
[2004]).
[0024] Anti-HIV drugs, such as nucleoside analogs (e.g.,
dideoxynucleoside derivatives, including 3'-azido-3'-deoxythymidine
[AZT], ddC, and ddI), protease inhibitors, or phosphonoacids (e.g.,
phosphonoformic and phosphonoacetic acids), have previously been
lipid-derivatized or incorporated into liposomes (e.g., Hostetler,
K Y et al., Methods of treating viral infections using antiviral
liponucleotides, Ser. No. 09/846,398, US 2001/0033862; U.S. Pat.
No. 5,223,263; Hostetler, K Y et al., Lipid derivatives of
phosphonoacids for liposomal incorporation and method of use, U.S.
Pat. No. 5,194,654; Gagne J F et al., Targeted delivery of
indinavir to HIV-1 primary reservoirs with immunoliposomes, Biochim
Biophys Acta 1558(2):198-210 [February 2002]). Still, in one
report, subcutaneous injection of liposome-encapsulated ddI to
C57BL/6 mice, resulted in low accumulation of liposomes in lymph
nodes, compared to intravenous injection (Harvie, P et al.,
Lymphoid tissues targeting of liposome-encapsulated
2',3'-dideoxyinosine, AIDS 9(7):701-7 [1995]).
[0025] The use of specific vector molecules coupled to, or embedded
within, a liposome surface, has been described for enhanced
transmembrane delivery and uptake of liposome-encapsulated
compounds that otherwise are only insufficiently delivered into a
cell, or that are not efficiently delivered to a specifically
desirable intracellular organelle (reviewed in: Torchilin V P,
Lukyanov A N, Peptide and protein drug delivery to and into tumors:
challenges and solutions, Drug Discov Today 2003 Mar. 15;
8(6):259-66; Sehgal A, Delivering peptides and proteins to tumors,
Drug Discov. Today 8(14):619 [2003]; Koning G A, Storm G, Targeted
drug delivery systems for the intracellular delivery of
macromolecular drugs, Drug Discov Today 2003 Jun. 1; 8(11):482-3).
Such vectors molecules include so-called protein transduction
domains (PTDs), which are derived from various viruses or from
Drosophila antennapedia. Of special interest for application in HIV
disease are HIV Tat and its derivatives which act as PTDs (e.g.,
Schwarze, S. R., et al., In vivo protein transduction: delivery of
a biologically active protein into the mouse, Science 285:1569-72
[1999]).
[0026] Anti-HIV drugs have been encapsulated in the aqueous core of
immunoliposomes, which include on their external surfaces
antigen-specific targeting ligands (e.g., Bergeron, M G. et al.,
Targeting of infectious agents bearing host cell proteins, WO
00/66173 A3; Bergeron, M G. et al., Liposomes encapsulating
antiviral drugs, U.S. Pat. No. 5,773,027; Bergeron, M G. et al.,
Liposome formulations for treatment of viral diseases, WO 96/10399
A1; Gagne J F et al., Targeted delivery of indinavir to HIV-1
primary reservoirs with immunoliposomes, Biochim Biophys Acta
1558(2):198-210 [2002]; Dufresne I et al., Targeting lymph nodes
with liposomes bearing anti-HLA-DR Fab' fragments, Biochim Biophys
Acta 1421(2):284-94 [1999]; Bestman-Smith J et al., Sterically
stabilized liposomes bearing anti-HLA-DR antibodies for targeting
the primary cellular reservoirs of HIV-1 Biochim Biophys Acta
1468(1-2):161-74 [2000]; Bestman-Smith J et al., Targeting
cell-free HIV and virally-infected cells with anti-HLA-DR
immunoliposomes containing amphotericin B, AIDS 10; 14(16):2457-65
[2000]).
[0027] There are many examples of antibody-targeted liposomes in
animal models. Currently, there is also at least one
antibody-targeted liposome, termed DOXIL, evaluated clinically. By
employing a single-chain antibody that had been raised against
HER2/neu, it is targeted to certain types of breast cancer.
Developed by Papahadjopoulos and colleagues at UCSF, this
antibody-mediated targeting variant is currently being evaluated in
clinical trials at the National Cancer Institute (e.g., Park J W,
Hong K, Kirpotin D B, Colbem G, Shalaby R, Baselga J, Shao Y,
Nielsen U B, Marks J D, Moore D, Papahadjopoulos D, Benz C C,
Anti-HER2 Immunoliposomes: enhanced efficacy attributable to
targeted delivery, Clin Cancer Res. 2002 April; 8(4):1172-81
[2002]).
[0028] Attempts at active targeting of lymphoid cell populations
with liposomes have met with some degree of success. Bestman-Smith
et al. (2000) reported that after subcutaneous injection of
immunoliposomes bearing anti-HLA-DR Fab' fragments into mice, there
was accumulation of the immunoliposomes in lymphoid tissues
(Bestman-Smith J et al., Targeting cell-free HIV and
virally-infected cells with anti-HLA-DR immunoliposomes containing
amphotericin B, AIDS 10; 14(16):2457-65 [2000]). Gagne J F et al.
[2002] reported that subcutaneous injections of
immunoliposome-encapsulated anti-HIV drugs resulted in an
accumulation of the drug in lymph nodes of injected mice with
relatively low toxicity, compared to administration of the free
drug; there was no significant difference reported in the ability
of anti-HLA-DR-targeted immunoliposomes containing indinavir to
inhibit HIV-1 replication in infected PM1 cells, compared to free
indinavir or non-targeted liposomal-indinavir complexes. Copland et
al. targeted the mannose receptors of monocyte-derived dendritic
cells (Mo-DCs) and reported that mannosylated liposomes were
preferentially bound and taken up by Mo-DCs at 37.degree. C.,
compared to non-mannosylated neutral liposomes and negatively
charged liposomes (Copland, M J et al., Liposomal delivery of
antigen to human dendritic cells, Vaccine 21:883-90 [2003]).
[0029] The present invention provides a liposomal delivery system
that facilitates the targeting of active agents, such as drugs,
immunomodulators, lectins or other plant-derived substances
specifically to myeloid cell populations of interest. The present
invention therefore addresses, inter alia, the need to target the
reservoirs of HIV, hepatitis C virus (HCV) in myeloid cells,
particularly dendritic cells and macrophages, as well as follicular
dendritic cells of myeloid origin, of persons infected with HIV and
those suffering from AIDS, or persons infected or co-infected with
HCV and those suffering from HCV-dependent pathologic alterations
of the liver. In addition, the present invention may allow for
indirect targeting of lymphoid cells, particularly T cells, upon
their physical interaction with myeloid cells. Moreover, the
present invention may allow for the specific elimination, or
down-modulation, of malignant tumor cells or immune cells mediating
autoimmunity; the enhancement of DC-dependent autologous tumor
immunization; the therapeutic down-regulation of autoimmune
diseases; or the DC-tropic stimulation of specific adaptive
immunity (both in terms of vaccination or treatment) against common
pathogens, or pathogens potentially employed as agents of
bioterrorism, for which there currently exists no efficient
protection. The present invention may also allow for
biotechnological advancement, such as, inter alia, by targeting DCs
for increasing the production of monoclonal antibodies, or by
allowing for the production of such immunoglobulins that cannot be
induced in the absence of inductive liposomal DC targeting.
SUMMARY OF THE INVENTION
[0030] The present invention relates to a method of preferentially,
or "actively," targeting and delivering an active agent, such as a
drug, to a mammalian immune cell, in vivo or in vitro.
[0031] In particular, the present invention is directed to a method
of preferentially targeting a liposome to a mammalian immune cell,
such as a myeloid progenitor cell, a monocyte, a dendritic cell, a
macrophage or a T-lymphocyte. The method involves administering to
the immune cell, in vitro or in vivo, a liposome comprising an
active agent and further comprising an outer surface that comprises
at least one targeting ligand that specifically binds a marker on
the surface of the immune cell, such as CD209 (DC-SIGN), CD45R0,
CD4, or HLA class II.
[0032] The present invention is also particularly directed to a
method of preferentially delivering a drug to an immune cell of a
mammalian subject, including a human. The targeted immune cells
include myeloid progenitor cells, monocytes, dendritic cells,
macrophages or T-lymphocytes. The method involves injecting into
the mammalian subject a lipid-drug complex, for example, but not
limited to a liposome that comprises the drug and further comprises
an outer surface comprising at least one targeting ligand that
specifically binds a marker on the surface of the immune cell, such
as, but not limited, to CD209 (DC-SIGN), the immune cell being
infected with, or susceptible to infection with, an infectious
agent, such as, but not limited to, human immunodeficiency virus,
types 1 and 2 (HIV-1; HIV-2).
[0033] The present invention is also directed to inventive targeted
liposomes. One embodiment of the targeted liposome comprises on its
external surface a targeting ligand that specifically binds CD209.
Another embodiment of the targeted liposome comprises on its
external surface a targeting ligand that specifically binds CD209
and a targeting ligand that specifically binds CD4. The inventive
targeted liposomes are useful for targeting immune cells, such as
dendritic cells.
[0034] The presence of HIV-1 in reservoir cells, e.g. dendritic
cells, leads to the continuous de-novo infection of naive T cells
within the lymphoid organs and tissues of an infected person. It
has been hypothesized that eradication of such sanctuary sites may
eventually eliminate HIV-1 from the individual. The present
invention provides a targeting system which, via targeting ligands
such as the dendritic cell-specific molecule DC-SIGN, delivers
chemical compounds directly into these cells. Thus, the present
invention is particularly, but not exclusively, of benefit for
delivering antiviral drugs, packaged in immunoliposomes, to
myeloid- and lymphoid-derived immune cells harboring HIV-1 or
HIV-2, such as the HIV reservoir in dendritic cells. Another
benefit of the present invention, by actively targeting immune
cells, is in providing vaccination strategies against HIV (e.g.,
Steinman R M, Granelli-Piperno A, Pope M, Trumpfheller C, Ignatius
R, Arrode G, Racz P, Tenner-Racz K, The interaction of
immunodeficiency viruses with dendritic cells, Curr Top Microbiol
Immunol 276:1-30 [2003]; Pope M, Dendritic cells as a conduit to
improve HIV vaccines, Curr Mol Med 3:229-42 [2003]). Additional
benefits provided by the present invention include utility in the
treatment of conditions involving abnormal proliferation of immune
cells, e.g., primary and metastatic lymphoid cancers (lymphomas and
leukemias), solid tumors or their post-surgical remnants, or
autoimmune diseases, including specifically targeting immune cells
in gene therapy applications. The present invention also provides a
way to target dendritic cells for facilitating the production of
anti-infective vaccines, anti-bioterrorism vaccines, anti-cancer
vaccines, or biotechnological and therapeutic tools such as
monoclonal antibodies.
[0035] The present invention is also directed to variations on the
inventive targeted delivery system. Any type of cell residing
within any kind of organ system (such as the endocrine or the
nervous systems), as well as any type of anatomic entity (such as
the urogenital or the respiratory tracts) can be targeted
selectively by the respective liposomal variant containing its
respective targeting ligand on the external surface and its active
agent of choice.
BRIEF DESCRIPTION OF THE FIGURES
[0036] FIG. 1 shows time-dependent targeting of calcein-labeled
liposomes to Mo-DCs mediated by DC-SIGN or other targeting ligands,
including bispecific combinations. The column entitled "Antigen
Expression" shows phenotypic expression of the respective
marker(s), as tested with the mAbs only. Detection was by flow
cytometry with a mAb-conjugated fluorescent dye,
fluorescein-5-iothiocyanate (FITC); the column "LS Binding/Uptake"
shows successful targeting and uptake, as evidenced by
intracellular delivery of a liposome-encapsulated fluorescent dye,
calcein.
[0037] FIG. 2 shows monospecific liposomal targeting with respect
to kinetics and efficacy. Mature MoDCs were generated according to
protocol described herein and investigated for uptake of different
constructs of targeted protein A liposomes furnished with mAbs
directed against CD4, CD14, CD45R0 or CD209. The MoDCs were
co-incubated with the liposomes for 1, 3 or 24 h and then harvested
and tested by flow cytometry. Control mAbs were used to detect
cellular surface expression of the respective antigens (column
headed "Marker Expression"). Empty curves indicate isotype
controls; shaded curves indicate test conditions. The two panels
bearing bold crosses show the highest mean fluorescence
intensities, indicating the highest rates of calcein uptake.
[0038] FIG. 3 illustrates liposomal targeting of DCs via two cell
markers (termed bispecific targeting), including time dependency of
the targeting efficacy over a 24-h period. Mature MoDCs were
generated according to protocol described herein and investigated
for uptake of different constructs of targeted Protein A liposomes
bearing 2-member combinations of anti-CD4, anti-CD45RO and
anti-CD209 mAbs. The MoDCs were co-incubated with the liposomes for
1, 3 or 24 h and then harvested and tested by flow cytometry.
Control mAbs were used to detect cellular surface expression of the
respective antigens (column headed "Marker Expression"). Empty
curves indicate isotype controls; shaded curves indicate test
conditions. FIG. 3A shows results for the combination of anti-CD4
plus anti-CD45RO targeting ligands. FIG. 3B shows results for the
combination of anti-CD4 plus anti-CD209 targeting ligands. FIG. 3C
shows results for the combination of anti-CD45RO plus anti-CD209
targeting ligands.
[0039] FIGS. 4A and 4B illustrate calculated values for targeting
and surface binding of immunoliposomes applied to MoDCs. Provided
in FIG. 4A and FIG. 4B are percentages of MoDCs expressing select
markers (FITC fluorescence; FIG. 4A shows arithmetic means and
upper extremes of n=2 independent experiments; FIG. 4B factors
derived from arithmetic means), and MoDCs targeted with
corresponding immunoliposomes (calcein fluorescence).
[0040] FIG. 5 shows surface binding vs. internalization of targeted
liposomes as determined by fluorescence microscopy. Original
magnifications: .times.1000 (panels 1 and 2) and .times.400 (panels
3-8).
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS
[0041] The present invention relates to a method of preferentially
delivering an active agent, such as a drug, to a mammalian immune
cell. In Some embodiments, delivery is in vitro, and in other
embodiments delivery of the active agent is in vivo.
[0042] The term "preferentially" refers to the fact that the lipid
drug complex, or the liposome, is delivered to the cell and the
active agent (e.g., the drug) is taken up by the cell, more
effectively than delivery and uptake of the agent using a
comparable lipid-drug complex, or liposome, having an outer surface
that does not comprise the at least one targeting ligand, in
contrast with the invention.
[0043] The targeted immune cells include myeloid progenitor cells,
monocytes, dendritic cells (DCs), macrophages, and
T-lymphocytes.
[0044] Monocytes are one of the types of cells produced by the
myeloid differentiation lineage of the bone marrow. It has been
shown that DCs can likewise be derived from monocytes or other
types of cells, i.e. mainly progenitor cells, generated within the
myeloid lineage (e.g., Peters J R, Ruhl S, Friedrichs D, Veiled
accessory cells deduced from monocytes, Immunobiology
176(1-2):154-66 [1987]; Gieseler R, Heise D, Soruri A, Schwartz P,
Peters J H, In-vitro differentiation of mature dendritic cells from
human blood monocytes, Dev. Immunol. 6(1-2):25-39 [1998]; Gieseler
R K H, Rober R-A, Kuhn R, Weber K, Osborn M, Peters J H, Dendritic
accessory cells derived from rat bone marrow precursors under
chemically defined conditions in vitro belong to the myeloid
lineage, Eur J Cell Biol 54(1):171-81 [1991]). As a consequence,
monocyte-derived dendritic cells (MoDCs) are a subset of MyDCs.
[0045] A dendritic cell includes a "myeloid dendritic cell"
(My-DC), i.e., a "myeloid lineage-derived DC", which includes a
monocyte-derived dendritic cell (Mo-DCs) as well as other DC types
such as, for example, promonocyte-derived dendritic cells. (e.g.,
Steinbach F, Gieseler R, Somri A, Krause B, Peters J H, Myeloid DCs
deduced from monocytes, In-vitro and in-vivo data support a
monocytic origin of DCs, Adv Exp Med Biol. 1997; 417:27-32 [1997]).
A dendritic cell also includes a "lymphoid dendritic cell" (Ly-DC),
ie., a "lymphoid lineage-derived DC"; the only type of DC presently
known to derive from the lymphoid lineage is the plasmacytoid
dendritic cell (pc-DC) (Facchetti F, Vermi W, Mason D, Colonna M,
The plasmacytoid monocyte/interferon producing cells, Virchows
Arch; 443(6):703-17. Epub 2003 Oct. 28 [2003]). A dendritic cell
also includes a follicular dendritic cell (FDC). It currently is
still controversial whether FDCs derive from the myeloid, the
lymphoid or a separate lineage; (Haberman A M, Shlomchik M J,
Reassessing the function of immune-complex retention by follicular
dendritic cells, Nat Rev Immunol; 3(9):757-64 [2003]). For an
overview of all types of dendritic cells, confer to Donaghy H,
Stebbing J, Patterson S, Antigen presentation and the role of
dendritic cells in HIV, Curr Opin Infect Dis; 17(1):1-6 [2004].
[0046] A macrophage denotes a cell class comprising various
organ-resident subtypes further including macrophages more typical
of lymphoid or of non-lymphoid organs and tissues (e.g., Barreda D
R, Hanington P C, Belosevic M, Regulation of myeloid development
and function by colony stimulating factors, Dev Comp Immunol 3;
28(5):509-54 [2004]).
[0047] A T-lymphocyte includes, but is not limited to, a T-helper
cell or a T-memory cell (Woodland D L, Dutton R W, Heterogeneity of
CD4.sup.+ and CD8.sup.+ T cells, Curr Opin Immunol; 15(3):336-42
[2003]).
[0048] In accordance with some in-vivo embodiments of the invention
a lipid-drug complex is injected into the mammalian subject, in
which the immune cell is present.
[0049] In some embodiments, the immune cell is infected with, or
susceptible to infection with, an infectious agent, such as a
virus, a bacterium, a fungus, a protozoan, or a prion. Examples of
viral infectious agents are HIV-1 and HIV-2 (including all their
clades), HSV, EBV, CMV, Ebola and Marburg virus, HAY, HBV, HCV and
HPV.
[0050] In some embodiments, the immune cell is, in the presence or
absence of infection, associated with the occurrence of an
organ-specific or a systemic autoimmune disease. Examples of such
diseases entities are Graves' disease; thyroid-associated
ophthalmopathy (a.k.a. Graves' ophthalmopathy; a.k.a. endocrine
ophthalmopathy); and multiple sclerosis (a.k.a. MS).
[0051] A "complex" is a mixture or adduct resulting from chemical
binding or bonding between and/or among its constituents or
components, including the lipid, drug, and other optional
components of the inventive lipid-drug complex Chemical binding or
bonding can have the nature of a covalent bond, ionic bond,
hydrogen bond, van der Waal's bond, hydrophobic bond, or any
combination of these bonding types linking the constituents of the
complex at any of their parts or moieties, of which a constituent
can have one or a multiplicity of moieties of various sorts. Not
every constituent of a complex needs to be bound to every other
constituent, but each constituent has at least one chemical bond
with at least one other constituent of the complex. In accordance
with the present invention, examples of lipid-drug complexes
include liposomes (lipid vesicles), or lipid-drug sheet disk
complexes. Lipid-conjugated drugs can also be a part of the
lipid-drug complex in accordance with the invention. However, drugs
can also be associated with a lipid or a lipid complex in the
absence of any type of chemical binding or bonding, such as is
provided in the case of liposomes encapsulating a soluble drug in
their aqueous interior space.
[0052] The lipid thug complex, e.g., the liposome, comprises an
active agent, such as a drug. For purposes of the present
invention, the drug is any drug known to be active against cellular
proliferation or active against an infectious agent of
interest.
[0053] The active agent, or drug, can be an anti-viral drug or
virostatic agent, such as, interferon, a nucleoside analog, or a
non-nucleoside anti-viral drug. Examples include anti-HIV drugs
(e.g., a HIV reverse protease inhibitor), such as indinavir (a.k.a.
Crixivan.RTM., Merck & Co., Inc., Rahway, N.J.; saquinavir
(N-tert-butyl-decahydro-2-[2(R)-hydroxy-4-phenyl-3(S)-[[N-(2-quinolylcarb-
onyl)-L-asparaginyl]amino]butyl]-(4aS,8aS)-isoquinoline-3(S)-carboxamide;
MW=670.86; a.k.a. Fortovase.RTM., Roche Laboratories, Inc., Nutley,
N.J.); or nelfinavir (i.e., nelfinavir mesylate, a.k.a.
Viracept.RTM.; [3S-[2(2S*,
3S*),3a,4ab,8ab]]-N-(1,1-dimethylethyl)decahydro-2-[2-hydroxy-3-[(3-hydro-
xy-2-methylbenzoyl)amino]-4-(phenylthio)butyl]-3-isoquinolinecarboxamide
mono-methanesulfonate (salt), MW=663.90 [567.79 as the free base];
Agouron Pharmaceuticals, Inc., La Jolla, Calif.). Other examples of
antiviral drug include reverse transcriptase inhibitors, such as
tenofovir disoproxil fumarate (9-[(R)-2-[[bis
[[(isopropoxycarbonyl)oxy]methoxy]phosphinyl]methoxy]propyl]
adenine fumarate (1:1); MW=635.52; a.k.a. Viread.RTM., Gilead
Sciences, Foster City, Calif.). The anti-HIV drug can also be
HIV-specific small interfering RNA (siRNA), anti-sense or sense DNA
or RNA molecules.
[0054] In other embodiments, the active agent is an anticancer
drug, an antifungal drug, or an antibacterial drug. In other
embodiments, the active agent is an immunomodulatory agent (i.e.,
an immunoactivator, an immunogen, an immunosuppressant, or an
anti-inflammatory agent), such as cyclosporin, steroids and steroid
derivatives. Other examples of useful drugs, in accordance with the
invention, include therapeutic cytotoxic agents (e.g., cisplatin,
carboplatin, methotrexate, 5-fluorouracil, and amphotericin), naked
DNA expression vectors, therapeutic proteins, therapeutic
oligonucleotides or nucleotide analogs, interferons, cytokines, or
cytokine agonists or antagonists. Also useful as a drug is a
cytotoxic alkylating agent, such as, but not limited to, busulfan
(1,4-butanediol dimethanesulphonate; Myleran, Glaxo Wellcome),
chlorambucil, cyclophosphamide, melphalan, or ethyl ethanesulfonic
acid. Such drugs or agents are particularly useful in treating
conditions involving pathological proliferation of immune cells,
for example, lymphoid cancers or autoimmune diseases.
[0055] In other embodiments, the active agent is a natural
substance with therapeutic properties or benefits, such as
plant-derived substances in purified or recombinant form. Examples
of plant-derived substances include leaf extract IDS 30, rhizome
derived UDA lectin, and MHL.
[0056] The present invention contemplates the selective employment
of natural substances that have been long acknowledged for their
therapeutic properties and potentials in many cultures worldwide.
One of such plant-derived substances, salicylic acid, which is
found at varying concentrations in the bark of many trees, has
served as the starter substance for one of nowadays great remedies,
acetyl salicylic acid (ASS), or Aspirin, respectively. As to the
present invention, the stinging nettle (Urtica dioica) is a
prominent example from the numerous plants that have been known for
centuries to have great therapeutic benefits. Recent scientific
investigation concerning the action of some of the components of
Urtica dioica provides an opportunity for their targeted
application.
[0057] For example, MyDCs play an important role in the initiation
of rheumatoid arthritis (RA) which is an example for a disease
crossing the border between autoimmune and inflammatory conditions.
Broer and Behnke have shown that the Urtica dioica leaf extract IDS
30 (Hox-.alpha.), which has been recommended for adjuvant therapy
of RA, prevents the phenotypic/functional maturation of MyDCs;
diminishes the secretion of tumor necrosis factor-.alpha.; and
reduces the T cell-stimulating capacity of MyDCs, while it
dose-dependently increases the expression of chemokine receptor 5
and CD36 as well as the endocytic capacity of these cells. The
authors suggested that these effects of IDS 30 may contribute to
its therapeutic effect on T cell-mediated autoimmune/inflammatory
diseases such as RA (Broer J, Behnke B, Immunosuppressant effect of
IDS 30, a stinging nettle leaf extract, on myeloid dendritic cells
in vitro, J Rheumatol; 29(4):659-66 [2002]). It is reasonable to
assume that inhibition of the transcription factor NF-.kappa.B is
involved in this process (Riehemann K, Behnke B, Schulze-Osthoff K,
Plant extracts from stinging nettle (Urtica dioica), an
antirheumatic remedy, inhibit the proinflammatory transcription
factor NF-.kappa.B, FEBS Lett; 442(1):89-94 [1999]), so that this
extract or its active purified ingredients may inhibit a great
number of debilitating or life-threatening pathogenic conditions
that depend on the hyperactivation of NF-.kappa.B.
[0058] Lectins are another example of a natural substance that has
therapeutic properties and potentials. Lectins (i.e.,
carbohydrate-binding proteins with agglutinating properties) are
produced by a number of plants, mainly in their roots or rhizomes,
as vital components of their own immune systems. Shibuya et al.
first described the sugar-binding properties of the stinging nettle
lectin (Shibuya N, Goldstein U, Shafer J A, Peumans W J, Broekaert
W F, (Carbohydrate binding properties of the stinging nettle
(Urtica dioica) rhizome lectin, Arch Biochem Biophys; 249(1):215-24
[1986]). The (GlcNAc)n-specific lectin from the stinging nettle,
termed Urtica dioica agglutinin (UDA), has been shown to inhibit
HIV-1-, HIV-2-, CMV-, RSV-, and influenza A virus-induced
cytopathicity at an EC50 ranging from 0.3 to 9 .mu.g/ml as well as
syncytium formation between persistently HIV-1- and HIV-2-infected
HUT-78 cells and CD4.sup.+ Molt/4 (clone 8) cells (EC50: 0.2-2
.mu.g/ml). It has been suggested that UDA may act as a
virion/target cell fusion inhibitor (Balzarini J, Neyts J, Schols
D, Hosoya M, Van Damme B, Peumans W, De Clercq E. The
mannose-specific plant lectins from Cymbidium hybrid and Epipactis
helleborine and the (N-acetylglucosamine)n-specific plant lectin
from Urtica dioica are potent and selective inhibitors of human
immunodeficiency virus and cytomegalovirus replication in vitro.
Antiviral Res 18(2):191-207 [1992]). Such an action, if verified,
may relate to UDA's superantigen nature (Galelli A, Truffa-Bachi P,
Urtica dioica agglutinin A superantigenic lectin from stinging
nettle rhizome, J Immunol; 151(4):1821-31 [1993]).
[0059] Again, the rhizome-derived UDA lectin, in addition to the
leaf-derived IDS-30 extract, act therapeutically on certain
autoimmune diseases. This superantigen has been shown to induce a
rapid deletion of a large fraction of T-cell receptor
V.beta.8.3-expressing mature T-cells (Delcourt M, Penmans W J,
Wagner M C, Truffa-Bachi P, V.beta.-specific deletion of mature
thymocytes induced by the plant superantigen Urtica dioica
agglutinin, Cell Immunol; 168(2): 158-64 [1996]). In mice, this
activity has been demonstrated to prevent the development of
systemic lupus erythematosus, as UDA-treated animals did not
develop overt clinical signs of lupus and nephritis (Musette P,
Galelli A, Chabre H, Callard P, Peumans W, Truffa-Bachi P,
Kourilsky P, Gachelin G, Urtica dioica agglutinin, a
V.beta.8.3-specific superantigen, prevents the development of the
systemic lupus erythematosus-like pathology of MRL lpr/lpr mice,
Eur J Immunol; 26(8):1707-11 [1996]).
[0060] These are just two of several examples of Urtica
dioica-derived substances, as well as the constituents of many
other plants, that act therapeutically, either as single molecules,
or their oligomers, or in combination, on defined immune cells
(such as MyDCs). Pathologic conditions with which these substances
interfere include infectious, neoplastic, and autoimmune diseases.
The liposomal system described herein may be utilized to
specifically encapsulate such molecular plant components in
purified or recombinant form, and address cells that have been, or
will be, identified as their specific targets, so as to
dramatically increase their effect and harness their potential
while considerably reducing the risk of toxic side effects.
[0061] In addition, liposomes shuttled into intracellular
compartments, such as endosomes, may deliver lectins suitable to
agglutinate intracellularly stored pathogens (including HIV-1, HCV,
the Ebola virus, Mycobacterium tuberculosis, and others), so as to
generate large lectin-pathogen complexes that may, thus be
recognized by the infected cell and, subsequently, be degraded
enzymatically and/or pH-dependently. For example, one lectin that
is highly suitable for this purpose when addressing the HIV-1
reservoirs is the Myrianthus holstii lectin (MHL, a.k.a.
Myrianthin) which is obtained from the roots of the Tanzanian plant
Myrianthus holstii. MHL comprises several favorable
characteristics, namely agglutination of HIV-1; no toxicity for
greater than two orders of magnitude above the effective dosage in
50% of infected cells (EC.sub.50); and the lack of mitogenicity for
human leukocytes (Charan R D, Munro M H, O'Keefe B R, Sowder R C
II, McKee T C, Currens M J, Pannell L K, Boyd M R, Isolation and
characterization of Myrianthus holstii lectin, a potent HIV-1
inhibitory protein from the plant Myrianthus holstii, J Nat Prod
2000 August; 63(8):1170-4).
[0062] Compounds such as UDA, MHL and many others lectins or
agglutinins, respectively, may be encapsulated within liposomes, so
as to selectively unfold their properties within a given targeted
cell and, more specifically, inside a specified intracellular
compartment(s) of such a cell, or cell types.
[0063] Some embodiments of the inventive method of preferentially
targeting a mammalian immune cell with a liposome relate to
improved means of vaccination. In this case, active targeting of
dendritic cells, in accordance with the invention, is used for
vaccinating against cancer, or against a virus such as HIV. (E.g.,
Nair, S et al., Soluble proteins delivered to dendritic cells via
pH-sensitive liposomes induce primary cytotoxic T lymphocyte
responses in vitro, J. Exp. Med. 175(2):609-12 [1992]; Philip, R et
al., Transgene expression in dendritic cells to induce
antigen-specific cytotoxic T cells in healthy donors, Cancer Gene
Ther. 5(4):236-46 [1998]; Ludewig, B et al., Protective antiviral
cytotoxic T cell memory is most efficiently maintained by
restimulation via dendritic cells, J. Immunol. 163(4):1839-44
[1999]; Chikh, G and Schutze-Redelmeier, M P, Liposomal delivery of
CTL epitopes to dendritic cells, Biosci. Rep. 22(2):339-53 [2002];
Grunebach, F et al. Delivery of tumor-derived RNA for the induction
of cytotoxic T-lymphocytes, Gene Ther. 10(5):367-74 [2003]).
[0064] Targeting of dendritic cells in accordance with the
invention is also useful for improving vaccination strategies in
general via accessing intracellular endosomal MHC class I and/or
MHC class II antigen processing compartments. (E.g. Zhou F and
Huang L, Liposome-mediated cytoplasmic delivery of proteins: an
effective means of accessing the MHC class I-restricted antigen
presentation pathway, Immunomethods 1994; 4(3):229-35 [1994]; Owais
M et al., Use of liposomes as an immunopotentiating delivery
system: in perspective of vaccine development, Scand. J. Immunol.
54(1-2):125-32 [2001]; Mandal M and Lee K D, Listeriolysin
O-liposome-mediated cytosolic delivery of macromolecule antigen in
vivo: enhancement of antigen-specific cytotoxic T lymphocyte
frequency, activity, and tumor protection, Biochim. Biophys. Acta
1563(1-2):7-17 [2002]).
[0065] The inventive method of preferentially targeting a mammalian
immune cell with a liposome can also be used to target dendritic
cells for facilitating the production of monoclonal antibodies.
(See, e.g., Berry J D et al., Rapid monoclonal antibody generation
via dendritic cell targeting in vivo, Hybrid. Hybridomics
22(1):23-31 [2003]).
[0066] More than one drug can be incorporated by the lipid-drug
complex, or liposome, in accordance with the inventive method, such
that the lipid-drug complexes, e.g., liposomes, can incorporate a
first drug and a second drug, or more drugs, in combination, as
suits the particular needs of the practitioner. For example, useful
liposomes can comprise a combination of an anti-HIV drug and an
antifungal and/or antibacterial drug.
[0067] The present invention does not depend on any particular
chemical or biochemical mechanism by which the useful formulations
of lipid-drug complex, or liposome, are obtained or by which the
drug is released to target cells.
[0068] Useful techniques for making lipid-drug complexes, such as
liposomes, are known to the art (e.g., Sullivan S M, Gieseler R K
H, Lenzner S, Ruppert J, Gabrysiak T G, Peters J H, Cox G, Richer,
L, Martin, W J, and Scolaro, M J, Inhibition of human
immunodeficiency virus-1 proliferation by liposome-encapsulated
sense DNA to the 5' TAT splice acceptor site, Antisense Res Develop
2:187-197 [1992]; Laverman P, Boerman O C, Oyen W J G, Carstens F H
M, Storm G, In vivo applications of PEG liposomes: unexpected
observations, Crit Rev Ther Drug Carrier Syst 18(6):551-66 [2001];
Oussoren C, Storm G, Liposomes to target the lymphatics by
subcutaneous administration, Adv Drug Deliv Rev 50(1-2):143-56
[2001]; Bestman-Smith 3, Gourde P, Desormeaux A, Tremblay M J,
Bergeron M G, Sterically stabilized liposomes bearing anti-HLA-DR
antibodies for targeting the primary cellular reservoirs of HIV-1,
Biochim Biophys Acta 1468(1-2):161-74 [2000]; Bestman-Smith J,
Desormeaux A, Tremblay M J, Bergeron M G, Targeting cell free HIV
and virally-infected cells with anti-HLA-DR immunoliposomes
containing amphotericin B, AIDS 14(16):2457-65 [2000]; Mayer L D,
Hope M J, Cullis P R, Vesicles of variable sizes produced by a
rapid extrusion procedure, Biochim Biophys Acta 858: 161-168
[1986]; Kinman, L. et al., Lipid-drug associations enhanced HIV
protease inhibitor indinovir localization in lymphoid tissues and
viral load reduction: a proof of concept study in HIV-infected
macaques, J AIDS; 34:387-97 [2003]; Harvie P, Desormeaux A, Gagne
N, Tremblay M, Poulin L, Beauchamp D, Bergeron M G, Lymphoid
tissues targeting of liposome-encapsulated 2',3'-dideoxyinosine,
AIDS; 9:701-7 [1995]; U.S. Pat. No. 5,773,027; U.S. Pat. No.
5,223,263; WO 96/10399 A1).
[0069] Some useful methods of liposome preparation include
extrusion, homogenization, remote loading, and reversed-phase
evaporation. In extrusion, a lipid film composed of phospholipids
only, or in combination with cholesterol and/or other additives, is
formed by evaporating the organic solvent (such as chloroform) from
the lipid solution Hydrophobic drugs are added to the lipid
solution prior to solvent evaporation. For entrapment of water
soluble drugs, the dry lipid film is hydrated with and isotonic
aqueous solution containing the drug by agitation (ultrasound,
vortex, motorized stirrer, etc.). The lipid suspension is frozen
and thawed 3-4 times. The suspension is then passed through a
series of polycarbonate filters containing pores of a defined
diameter, such as 0.8 .mu.m, 0.4 .mu.m, 0.2 .mu.m, or 0.1 .mu.m.
For water soluble drugs, unencapsulated drugs are removed by gel
permeation column chromatography, dialysis or diafiltration. The
liposomes can be sterile-filtered (e.g., through a 0.22-.mu.m
filter).
[0070] A cryoprotectant, such as lactose, glucose, sucrose,
trehalose or maltose can be added to the sterile liposomes as long
as isotonicity is maintained. The liposomes can then be frozen and
lyophilized and stored indefinitely as a lyophilized cake (e.g.,
Mayer L D, Hope M J, Quills P R, Vesicles of variable sizes
produced by a rapid extrusion procedure, Biochim Biophys Acta 858:
161-168 [1986]; Tsvetkova N M et al., Effect of sugars on headgroup
mobility in freeze-dried dipalmitoylphosphatidylcholine bilayers:
solid-state 31P NMR and FTIR studies, Biophys J 75: 2947-2955
[1998]; Crowe J H, Oliver A E, Hoekstra F A, Crowe L M,
Stabilization of dry membranes by mixtures of hydroxyethyl starch
and glucose: the role of vitrification, Cryobiology 35: 20-30
[1997]; Sun W Q, Leopold A C, Crowe L M, Crowe J H, Stability of
dry liposomes in sugar glasses, Biophys J 70: 1769-1776
[1996]).
[0071] Homogenization is suited for large scale manufacture. The
lipid suspension is prepared as described above. Freeze and thaw
steps on a large scale may be a problem. The diameter of the
liposomes is reduced by shooting the lipid suspension as a stream
either at an oncoming stream of the same lipid suspension
(microfiuidization) or against a steel plate (gualinization). This
later technology has been used by the dairy industry for
homogenization of milk. Untrapped water-soluble drugs are removed
by diafiltration Hydrophobic drugs are completely entrapped and
there usually is no free drug to be removed (e.g., Paavola A,
Kilpelainen I, Yliruusi J, Rosenberg P, Controlled release
injectable liposomal gel of ibuprofen for epidural analgesia, Int J
Pharm 199: 85-93 [2000]; Zheng S, Zheng Y, Beissinger R L, Fresco
R, Liposome-encapsulated hemoglobin processing methods, Biomater
Artif Cells Immobilization Biotechnol 20: 355-364 [1992]).
[0072] Another method of drug entrapment is remote loading. The
drug to be entrapped must carry a charge. The degree of protonation
or deprotonation is controlled by the pK of the ionizable group. A
conjugate acid or base is trapped inside the liposomes. The
ionizable drug is added to the outside of the liposomes. The pH is
dropped such that the drug serves as a neutralizing salt of the
ionizable substance trapped inside the liposomes. Due to the change
in pH, the counter-ion to the entrapped ionizable molecule can
diffuse out of the liposomes. This creates a gradient with
sufficient energy to cause the drug to diffuse into the liposomes.
An example is the loading of doxorubicin into preformed
liposomes.
[0073] In reverse phase evaporation, a lipid film is solubilized in
diethylether to a final concentration of typically about 30 mM.
Typically, one part water with entrapped drug is added to three
parts ether lipid solution. Energy in the form of sonication is
applied forcing the suspension into a homogeneous emulsion. After a
stable emulsion has been formed (which does not separate when
resting for 1-3 h), the ether is removed by evaporation, typically
yielding liposomes with about a 200 nm diameter and a high trapping
efficiency.
[0074] Ethanol/calcium liposomes for DNA entrapment, typically
yielding liposomes 50 nm in diameter, are prepared by any of the
above methods (extrusion, homogenization, sonication). The
liposomes are mixed with plasmid DNA, or linear DNA fragments, plus
8 mM calcium chloride. Typically, ethanol is added to the
suspension to yield a concentration of about 40%. The ethanol is
removed by dialysis and the resultant liposomes are generally less
than 200 nm in diameter with about 75% of the DNA entrapped in the
liposomes.
[0075] For cellular targeting, in accordance with the present
invention, liposomes can be prepared by any of the above methods.
The Liposomes can contain a lipid to which proteins can be
crosslinked. Examples of these lipids are:
N-glutaryl-phosphatidylethnaolamine,
N-succinyl-phosphatidylethanolamine,
maleimido-phenyl-butyryl-phosphatidylethanolamine,
succinimidyl-acetylthioacetate-phosphatidylethanolamine,
SPDP-phosphatidylethnaolamine. The glutaryl and succinimidyl
phosphosphatidylethanolamine can be linked to a nucleophile, such
as an amine, using cyclocarbodiimide. The maleimido,
acetylthioacetate and SPDP phosphatidylethanolamines can be reacted
with thiols on the proteins, peptides or small molecular weight
ligands of <1000 g/mol. The protein can be derivatized to the
liposomes after formation. Underivatized protein can be removed by
gel permeation chromatography. Peptides and low molecular weight
ligands can be derivatized to the lipids and added to the organic
lipid solution prior to formation of the lipid film.
[0076] In accordance with the present invention, examples of useful
lipids include any vesicle-forming lipid, such as, but not limited
to, phospholipids, such as phosphatidylcholine (hereinafter
referred to as "PC"), both naturally occurring and synthetically
prepared phosphatidic acid (hereinafter referred to as "PA"),
lysophosphatidylcholine, phosphatidylserine (hereinafter referred
to as "PS"), phosphatidylethanolamine (hereinafter referred to as
"PE"), sphingolipids, phosphatidyglycerol (hereinafter referred to
as "PG"), spingomyelin, cardiolipin, glycolipids, gangliosides or
cerebrosides and the like used either singularly or intermixed such
as in soybean phospholipids (e.g., Asolectin, Associated
Concentrates). The PC, PG, PA and PB can be derived from purified
egg yolk and its hydrogenated derivatives.
[0077] Optionally, other lipids such as steroids, different
cholesterol isomers, aliphatic amines such as long-chained
aliphatic amines and carboxylic acids, long-chained sulfates, and
phosphates, diacetyl phosphate, butylated hydroxytoluene,
tocopherols, retinols and isoprenoid compounds can be intermixed
with the phospholipid components to confer certain desired and
known properties on the formed vesicles. In addition, synthetic
phospholipids containing either altered aliphatic portions such as
hydroxyl groups, branched carbon chains, cycloderivatives, aromatic
derivatives, ethers, amides, polyunsaturated derivatives,
halogenated derivatives or altered hydrophilic portions containing
carbohydrate, glycol, phosphate, phosphonate, quarternary amine,
sulfate, sulfonate, carboxy, amine, sulfhydryl or imidazole groups
and combinations of such groups can be either substituted or
intermixed with the above-mentioned phospholipids and used in
accordance with the invention. Some of these components are known
to increase liposomal membrane fluidity, thus entailing more
efficacious uptake, others are known to have a direct effect on,
e.g., tumor cells by affecting their differentiation potential. It
will be appreciated from the above that the chemical composition of
the lipid component prepared by the method of the invention can be
varied greatly without appreciable diminution of percentage drug
capture, although the size of a vesicle can be affected by the
lipid composition.
[0078] Saturated synthetic PC and PG, such as dipalmitoyl can also
be used. Other amphipathic lipids that can be used, advantageously
with PC, are gangliosides, globosides, fatty acids, stearylamine,
long-chained alcohols and the like. PEGylated lipids,
monoglycerides, diglycerides, triglycerides can also be included.
Acylated and diacylated phospholipids are also useful.
[0079] By way of further example, in some embodiments, useful
phospholipids include egg phosphatidylcholine ("EPC"),
dilauryloylphosphatidylcholine ("DLPC"),
dimyristoylphosphatidylcholine ("DOPC"),
dipalmitoylphosphatidylcholine ("DPPC"),
distearoylphosphatidylcholine ("DSPC"),
1-myristoyl-2-palmitoylphosphatidylcholine ("MPPC"),
1-palmitoyl-2-myristoyl phosphatidylcholine ("PMPC"),
1-palmitoyl-2-stearoyl phosphatidylcholine ("PSPC"),
1-stearoyl-2-palmitoyl phosphatidylcholine ("SPPC"),
dioleoylphosphatidylycholine ("DOPC"),
dilauryloylphosphatidylglycerol ("DLPG"),
dimyristoylphosphatidylglycerol ("DMPG"),
dipalmitoylphosphatidylglycerol ("DPPG"),
distearoylphosphatidylglycerol ("DSPG"), distearoyl sphingomyelin
("DSSP"), distearoylphophatidylethanolamine (DSPE),
dioleoylphosphatidylglycerol ("DOPG"), dimyristoyl phosphatidic
acid ("DMPA"), dipalmitoyl phosphatidic acid ("DPPA"), dimyristoyl
phosphatidylethanolamine ("DMPE"), dipalmitoyl
phosphatidylethanolamine ("DPPE"), dimyristoyl phosphatidylserine
("DMPS"), dipalmitoyl phosphatidylserine ("DPPS"), brain
phosphatidylserine ("BPS"), brain sphingomyelin ("BSP"), and
dipalmitoyl sphingomyelin. ("DPSP").
[0080] In one embodiment, phosphatidylcholine and cholesterol are
employed. However, any suitable molar ratio of non-steroidal lipid
to steroidal lipid (e.g., cholesterol) can optionally be employed
to promote the stability of a particular lipid-drug complex during
storage and/or delivery to a mammalian subject.
[0081] Mixing the drug and lipids can be by any useful known
technique, for example, by sonication, vortexing, extrusion,
microfluidization, homogenization, use of a detergent (later
removed, e.g., by dialysis). The drug and lipid are mixed at a
lipid-to-drug molar ratio of about 3:1 to about 100:1 or higher
which is especially useful for drugs that are relatively more
toxic, and more preferably of about 3:1 to about 10:1, and most
preferably of about 5:1 to about 7:1.
[0082] For some drugs, the use of an organic solvent can facilitate
the production of the lipid-drug complex, such as a liposome. After
mixing of the drug and lipids, the organic solvent is removed by
any suitable known means of removal, such as evaporating by vacuum,
or by the application of heat, for example by using a hair dryer or
oven, or hot ethanol injection (e.g., Deamer, U.S. Pat. No.
4,515,736), as long as the lipid and drug components are stable at
the temperature used Dialysis and/or chromatography, including
affinity chromatography, can also be employed to remove the organic
solvent. Hydrating the drug is performed with water or any
biocompatible aqueous buffer, e.g., phosphate-buffered saline,
HEPES, or TRIS, that maintains a physiologically balanced
osmolarity. Liposome rehydration can be accomplished simultaneously
by removing the organic solvent or, alternatively, can be delayed
until a more convenient time for using the liposomes (e.g.,
Papahadjopoulos et al., U.S. Pat. No. 4,235,871). The shelf life of
re-hydratable ("dry") liposomes is typically about 8 months to
about a year. This time span can be increased by
lyophilization.
[0083] In one embodiment, the lipid-drug complex is a unilamellar
liposome. Unilamellar liposomes provide the highest exposure of
drug to the exterior of the liposome, where it may interact with
the surfaces of target cells. However, multilamellar liposomes can
also be used in accordance with the present invention. The use of
PEGylated liposomes is also encompassed within the present
invention.
[0084] The lipid-drug complex further comprises an outer surface
comprising at least one targeting ligand that specifically binds a
marker on the surface of the immune cell. Examples of targeting
ligands include antibodies that specifically bind the marker of
interest, such as anti-CD209/DC-SIGN-specific antibodies, or
anti-CD4, anti-CD45R0, or anti-HLA class IL "Antibodies" include
whole antibodies as well as antibody fragments, with a specific
target-binding capability of interest, ie., antigen-specific or
hapten-specific targeting ligands. Antibody fragments include, for
example Fab, Fab', F(ab').sub.2, or F(v) fragments. Antibodies can
also be polyclonal or monoclonal antibodies. Antibodies also
include antigen-specific or hapten-specific targeting ligands
complexed with lipid-soluble linker moieties. In some embodiments,
antibodies are coupled to the lipid-drug complex, such as a
liposome-drug complex, via protein A of the Staphylococcus-aureus
type, or via protein G which is typical of some other bacterial
species.
[0085] Optionally, the lipid-drug complex further comprises one or
more biomembrane components that can further enhance the specific
(i.e., active) targeting ability, cytotoxicity, or other
therapeutic parameter of the liposome. Such biomembrane components
include a membrane-associated protein, an integral or transmembrane
protein (e.g., a glycophorin or a membrane channel), a lipoprotein,
a glycoprotein, a peptide toxin (e.g., bee toxin), a bacterial
lysin, a Staphylococcus aureus protein A, an antibody, a specific
surface receptor, or a surface receptor binding ligand. The use of
specific vector molecules coupled to, or embedded within a
liposomal surface, is also encompassed within the present invention
for enhanced transmembrane delivery and uptake of
liposome-encapsulated compounds that otherwise are only
insufficiently delivered to or into a cell, or that are not
efficiently delivered to a specifically desirable intracellular
organelle (e.g., as reviewed in: Torchilin V P, Lukyanov A N,
Peptide and protein drug delivery to and into tumors: challenges
and solutions, Drug Discov Today 2003 Mar. 15; 8(6):259-66; Sehgal
A, Delivering peptides and proteins to tumors, Drug Discov Today
2003 Jul. 15; 8(14):619; Koning G A, Storm G, Targeted drug
delivery systems for the intracellular delivery of macromolecular
drugs, Drug Discov Today 2003 Jun. 1; 8(11):482-3). Such vector
molecules can include so-called protein transduction domains (PTDs)
which are derived from various viruses or from Drosophila
antennapedia. For application in HIV disease, the HIV Tat protein,
or a derivative or fragment that acts as a PTD, is also useful
(e.g., Schwarze, S. R., et al., In vivo protein transduction:
delivery of a biologically active protein into the mouse, Science
285:1569-72 [1999]).
[0086] The lipid-drug complex, such as a liposome, is preferably,
but not necessarily, about 30 to about 150 nanometers in diameter,
and more preferably about 50 to about 80 nanometers in
diameter.
[0087] In accordance with the present invention, the lipid-drug
complexes can be preserved for later use by any known preservative
method, such as lyophilization (e.g., Crowe et al., U.S. Pat. No.
4,857,319). Typically, lyophilization or other useful
cryopreservation techniques involve the inclusion of a
cryopreservative agent, such as a disaccharide (e.g., trehalose,
maltose, lactose, glucose or sucrose).
[0088] The lipid-drug complex, e.g., a liposome, is administered to
a subject by any suitable means such as, for example by injection.
Administration and/or injection can be intrarterial, intravenous,
intrathecal, intraocular, intradermal, subcutaneous, intramuscular,
intraperitoneal, or by direct (e.g., stereotactic) injection into a
particular lymphoid tissue, or into a tumor or other lesion.
Introduction of the lipid-drug complex into lymphatic vessels,
preferably, is via subcutaneous or intramuscular injection.
[0089] In accordance with the present invention, "lymphoid tissue"
is a lymph node, such as an inguinal, mesenteric, ileocecal, or
axillary lymph node, or the spleen, thymus, or mucosal-associated
lymphoid tissue (e.g., in the lung, lamina propria of the of the
intestinal wall, Peyer's patches of the small intestine, or
lingual, palatine and pharyngeal tonsils, or Waldeyer's neck
ring).
[0090] Injection can also be by any non-intravenous method that
drains directly, or preferentially, into the lymphatic system as
opposed to the blood stream. Most preferred is subcutaneous
injection, typically employing a syringe needle gauge larger than
the lipid-drug complex. Intraperitoneal injection is also useful.
Typically, injection of the injectate volume (generally about 1-5
cm.sup.3) is into the subject's arm, leg, or belly, but any
convenient site can be chosen for subcutaneous injection. Because
drug subcutaneously administered, in accordance with some
embodiments of the present invention, enters the lymphatic system
prior to entering systemic blood circulation, benefits include
[0091] 1) Distribution throughout the lymphoid system and
localization in lymph nodes; and
[0092] 2) Avoiding or minimising of protein-mediated
destabilization of lipid-drug complexes.
[0093] Typically, in treating HIV/AIDS, the frequency of injection
is most preferably once per week, but more or less (e.g., monthly)
frequent injections can be given as appropriate.
[0094] Accordingly, the present invention facilitates a treatment
regimen that can involve a convenient weekly injection rather than
multiple drug doses daily, as practiced typically in current AIDS
treatment regimes. This feature may lead to improved patient
compliance with the full course of treatment for some individual
patients.
[0095] While the invention has been described with reference to its
preferred embodiments, it will be appreciated by those skilled in
this art that variations can be made departing from the precise
examples of the methods and compositions disclosed herein, which,
nonetheless, embody the invention.
EXAMPLES
Example 1
Materials and Methods
[0096] Preparation of Liposomes.
[0097] A 30 .mu.mol lipid film composed of DOPC/Chol/DOPE-MBP
(36.5:33.0:2.5 mol:mol:mol) was formed (cholesterol was purchased
from Calbiochem, San Diego, Calif., USA; and DOPE and DOPE-MPB were
from Avanti Polar Lipids, Alabaster, Ala., USA). Lipid films were
hydrated with 1 ml 50 mM calcein (Molecular Probes, Eugene, Oreg.,
USA) in PBS (pH 7M), sonicated in a bath sonicator (5 min) and
extruded .times.5 through a 0.1 .mu.m nucleopore filter (Avanti
Polar Lipids) using a hand-held extruder. Also, freeze-thaw cycles
can be employed. The mean liposome size was determined by
quasielectric light scattering with a Nicomp 380 ZLS Zeta-Potential
Particle Sizer (Particle Sizing Systems, Santa Barbara, Calif.,
USA), yielding an average diameter of 146.7.+-.31.0 nm.
[0098] Protein A Liposomes.
[0099] To be able to test the targeting ability of different
antibodies with a standardized liposome, immunoglobulin-molecules
were coupled to liposomes via protein A of Staphylococcus aureus.
Protein A is a bacterial cell wall component consisting of a single
polypeptide chain of molecular weight 42 kDa. Protein A has the
ability to specifically bind to the Fc region of immunoglobulin
molecules, especially IgG. One protein A molecule can bind at least
2 molecules of IgG simultaneously (Sjoquist J, Meloun B, Hjelm H,
Protein A isolated from Staphylococcus aureus after digestion with
lysostaphin, Eur J Biochem 29: 572-578 [1972]). Protein A bearing
liposomes were formed and their functionality in binding
antibody-molecules was tested. Targeting of DC-SIGN and other
membrane markers was achieved with Protein A liposomes
pre-incubated with established antibody concentrations of either of
several DC-SIGN-specific mAbs (all IgG1.kappa. isotype), or
irrelevant IgG1.kappa. control mAb (MOPC-21/P3), or anti-bodies
specific for the other membrane markers.
[0100] Protein A was derivatized with
succinimidylacetyl-thioacetate (SATA, Pierce Biotechnology,
Rockford, Ill., USA) at a molar ratio of 10:1 SATA to protein in
PBS, pH 9.0 for 1 h. Unreacted SATA was removed from the protein A
using a Sephadex G-25 superfine spin column equilibrated with PBS
(pH=7.4). The thiol protecting group was removed by incubating the
derivatized protein A with 0.2 ml 0.5 M NH.sub.2OH (Sigma), 0.5 M
HEPES (pH=7.4) and 25 mM EDTA (Fisher) for 15 min. Reactants were
removed and buffer was changed using a second G-25 Sephadex spin
column equilibrated with PBS (pH=6.5). At the same time, the
calcein-containing liposomes were also centrifuged through a
Sephadex spin column equilibrated with PBS (pH=6.5) to remove
untrapped calcein. The derivatized protein A was immediately added
to the liposomes at a molar ratio of 100 lipid to protein After 2-h
incubation at RT, the liposome.times.protein A conjugate was
separated from free protein a using a sepharose CL-4B column
equilibrated with PBS. The number of thiols/protein A was verified
by their reaction with 2 mM 5,5'-dithio-bis(2-nitrobenzoic acid)
(Aldrich, Milwaukee, Wis., USA). As a measure for calcein
encapsulation efficiency and liposomal stability, the quenching (Q)
[%] of the pooled preparation in absence and presence of Triton
Tx-100 was determined according to:
Q = ( Pool + Tx - 100 ) OD 280 nm - ( Pool - Tx - 100 ) OD 280 nm (
Pool + Tx - 100 ) OD 280 nm .times. 100 [ % ] ( I )
##EQU00001##
Typically, Q.apprxeq.80% indicated that leakage of calcein was
insignificant.
[0101] Immunoliposomes and Antibodies.
[0102] Calcein-entrapping protein A liposomes were stored at
4.degree. C. in the dark and used for up to 3 months.
Immunoliposomes were prepared by incubation for 30 min at RT of
protein A liposomes with test monoclonal antibodies (mAb; see
below) or irrelevant negative control IgG (mAb MOPC-21/P3;
eBioscience, San Diego, Calif., USA); Reeves, J P et al.,
Anti-Leu3a induces combining site-related anti-idiotypic antibody
without inducing anti-HIV activity, AIDS Res Hum Retroviruses
7:55-63 [1991]) at a 5:1 molar ratio of mAb:protein A. The molar
ratio of lipid to protein A was approximately 1000. Unbound
antibody could be removed with magnetic Protein A beads (New
England Biolabs, Beverly, Mass., USA). However, no significant
effect on cell labeling was observed.
[0103] Monoclonal antibody binding to protein A liposomes was
tested by Ficoll flotation Specifically, antibodies were incubated
with liposomes (30 min, RT) at the mAb:lipid ratio used for cell
labeling. Polyclonal rabbit anti-mouse Ab.times.alkaline
phosphatase (AP) was added to the incubation. The mixture was made
from 20% ficoll 400 using a 30% Ficoll stock in PBS with a final
volume of 0.4 ml, transferred to a microfuge tube, and 0.4 ml of
10% ficoll/PBS was layered on top and subsequently added a 0.4-ml
layer of PBS. Tubes were centrifuged at 15,000 rpm for 15 min at
RT. The PBS/10% ficoll interface was assayed for AP activity.
Incubation with secondary Ab.times.AP yielded a 10-fold lower
activity than incubation with primary mAb and secondary antibody,
indicating that primary mAb had bound to protein A on the liposomes
(results not shown).
[0104] In order to identify an mAb ensuring maximal efficacy for
targeting of DC-SIGN, protein A liposomes were preincubated with
either of three different CD209-specific mAbs derived from clones
120507 (IgG2b), 120526 (IgG2a) (R&D Systems, Minneapolis,
Minn., USA) and DCN46 (IgG1.kappa.) (BD Biosciences, San Jose,
Calif., USA). Targeting with mAb 120507 turned out superior, and
the results described herein have exclusively been obtained with
this antibody. Further antibodies for phenotyping (employed as
primary mAbs) and for generating immunoliposomes were specific for
CD1a (BL6; Coulter Immunotech, Miami, Fla., USA), CD4 (SIM.4)
(NIH/McKesson; cf. Acknowledgments), CD14 (UCHM-1), CD45R0 (UCHL1)
and CD83 (HB15a17.11) (all from Serotec, Oxford, UK).
[0105] Cellular Binding/Uptake Studies.
[0106] Mature cells were harvested on day 7 of culture by pelleting
non-adherent veiled cells from the supernatants and detaching
weakly adherent cells with 1% EDTA in PBS for 30 min at 4.degree.
C.; strongly adherent cells were obtained by gently applying a cell
scraper (TPP). All fractions were pooled, washed with PBS and kept
in medium 80/20 plus 1% FBS on ice until used. For testing, cells
were plated in fresh culture medium with 1% FBS at a density of
2.times.10.sup.5 cells/well. To obtain the time-dependency of the
targeting to dendritic cells, the 2.times.10.sup.5 MoDCs per well
or onset in the same medium were incubated with liposomes at 50
.mu.M lipid at 37.degree. C. for 1, 3 and 24 hours or other times
and temperatures, as described hereinbelow. After incubation the
cells were washed three times with phosphate-buffered saline (PBS,
pH 7.2; without bivalent cations) and analysed by fluorescence
activated cell sorting (FACS; i.e., "flow cytometry," see below).
In all the experiments, the liposome-to-cell-ratio was
constant.
[0107] Flow Cytometry.
[0108] Flow cytometry can be employed: (1) to determine the
phenotypes of My-DCs and T-cells at different times throughout DC
differentiation and DC/T-cell co-culture (ie., mixed leukocyte
cultures or antigen-specific stimulation) with or without the DCs
being infected with select M- and/or T-tropic strains of HIV-1,
and/or treated with DC-SIGN-specific or control liposomes; and (2)
to determine co-delivery of calcein/drug(s) to infected My-DCs or,
more specifically, infected MoDCs. Labeled MoDCs were analyzed on a
Coulter Epics XL-MCL (Beckman Coulter, Fullerton, Calif.) flow
cytometer according to the manufacturer's instructions, immediately
after indirect staining with (i) primary mAbs and secondary
polyclonal IgG conjugated with fluorescein-5-isothiocyanate (FITC)
(eBioscience) (Gieseler, R et al., In-vitro differentiation of
mature dendritic cells from human blood monocytes, Dev. Immunol
6:25-39 [1998]), (ii) incubation with the respective
calcein-containing immunoliposomes, or (iii) negative controls.
Flow cytometry was performed; only gated cells were evaluated for
antigen expression, as well as for liposomal targeting and uptake
studies. Briefly, cells were gated via forward and side scatter dot
plotting to exclude debris. Histograms were established for gated
cells, as suitable for FITC and calcein, i.e. .lamda..sub.EX=488 nm
and .lamda..sub.EM=525 nm. Data were downloaded, and the
corresponding histograms for test samples and controls were
overlaid and analyzed with WinMDI 2.8 software (J. Trotter;
facs.scripps.edu). Targeting efficacy was determined directly after
incubating DCs (or, when employed, macrophages) with the respective
liposome/Protein A/mAb construct, or with liposomal negative
controls employing the irrelevant isotype control antibody
MOPC-21/P3. Results of negative controls employing protein A
liposomes not loaded with mAbs were identical to those obtained
with irrelevant control IgG. An influence via nonspecific uptake of
liposomes by MyDCs could thus be excluded.
[0109] Targeting Efficacy of Immunoliposomes.
[0110] To determine expression of a given marker by a specific mAb,
its efficient mean fluorescence intensity (.DELTA.MFI.sub.mAb) was
calculated as the difference of its measured MFI (MFI.sub.mAb) and
the MFI measured for negative control IgG (MFI.sub.Co-IgG),
i.e.
.DELTA.MFI.sub.mAb=MFI.sub.mAb-MFI.sub.Co-IgG (II)
and expressed as the percentage of MyDCs expressing this marker
(MyDC.sub.mAb.sup.+ [%]).
[0111] To determine the uptake of a given mAb-loaded immunoliposome
(ILS.sub.mAb), its efficient MFI (.DELTA.MFI.sub.ILS) resulted from
the difference of its measured MFI (MFI.sub.ILS-mAb) and the MFI
obtained for the immunoliposome negative control
(MFI.sub.ILS-Co-IgG), ie.
.DELTA.MFI.sub.ILS=MFI.sub.ILS-mAb-MFI.sub.ILS-Co-IgG (III)
[0112] thus providing the percentage of immunoliposome-positive
MyDCs (MyDC.sub.ILS.sup.+ [%]).
[0113] Marker expression and immunoliposomal binding and uptake do
not necessarily correlate. For instance, while clearly expressing a
given antigen when identified with a specific mAb, interaction of
the same antigen with the much larger immunoliposomes labeled with
the same mAb specificity may lead to shedding of the surface
marker, which will result in a loss of signal fluorescence. Based
on Equations (II) and (III), the immunoliposomal net targeting
efficacy (TE.sub.ILS) was thus determined as
TE ILS = MyDC ILS .times. 100 MyDC mAb . [ % ] ( IV )
##EQU00002##
wherein a result close to 100% indicates similar binding of an mAb
and its corresponding immunoliposome; a lower result indicates loss
of signal upon liposomal engagement; and a result clearly above
100% shows accumulation of liposomally delivered fluorophore, hence
suggesting active uptake of the respective type of immunoliposome.
Equation (IV) is easily transformed for the relative fluorescence
of immunoliposomes vs. fluorescently labeled mAbs (RF.sub.ILS),
RF ILS = MyDC ILS .times. 100 MyDC mAb - 100 [ % ] ( V )
##EQU00003##
wherein negative results indicate a loss, and positive results a
gain, in signal fluorescence.
[0114] Peripheral Blood Leukocytes (PBL).
[0115] Mononuclear leukocytes (MNLs) and/or T-cells were prepared
as described before (Gieseler, R, et al., In-vitro differentiation
of mature dendritic cells from human blood monocytes, Dev. Immunol.
6:25-39 [1998]). Briefly, MNLs were enriched from whole blood
diluted 1:1 with phosphate-buffered saline (PBS) without
Ca.sup.2+/Mg.sup.2+ (Cambrex, Walkersville, Md., USA) by density
gradient centrifugation over Lymphoprep (.rho.=1.077 g/cm.sup.3;
Nyegaard, Oslo, Norway). Buffy coats were harvested and pooled, and
residual platelets were removed by 3-4 washes with PBS. These
procedures involved several 10-min centrifugation steps at
260.times.g and 4 degrees C.
[0116] Magnetic-Activated Cell Separation (MACS) of Monocytes,
CD4.sup.+ and CD8.sup.+ T Cells.
[0117] Monocytes were isolated via negative magnetic-activated cell
separation (MACS; Miltenyi, Bergisch-Gladbach, Germany and Auburn,
Calif., USA) by removing CD3.sup.+, CD7.sup.+, CD19.sup.+,
CD45RA.sup.+, CD56.sup.+ and mIgE.sup.+ cells with mAb-coated
magnetic microbeads. Negative monocyte separation had been chosen
to avoid cell activation and was performed according to the
manufacturer's instructions. Briefly, the procedure involved 2
washes with PBS supplemented with 0.5% bovine serum albumin (BSA;
cell-culture grade, <0.1 ng/mg endotoxin; ICN, Irvine, Calif.,
USA) and 2 mM EDTA (Sigma, St. Louis, Mo., USA), and the washed
cells were passed through an LS magnetic microcolumn placed in a
defined magnetic field (Miltenyi), thus enriching the monocytes to
98.6-99.9% purity (range of n=3), as determined by flow cytometry
for CD14.
[0118] Differentiation of Myeloid Dendritic Cells.
[0119] Mature and immature MyDCs were generated from peripheral
blood monocytes. Briefly, monocytes were isolated by successive
density gradient centrifugation of PBS-diluted whole blood over
Lymphoprep (.rho.=1.077 g/cm.sup.3) (Nyegaard, Oslo, Norway) and,
successively, by negative magnetic cell separation (MACS), in
accordance with the manufacturer's instructions (Miltenyi).
Monocytes were then seeded at 1.times.10.sup.5/200 .mu.l in 96-well
microtiter plates (TPP, Trasadingen, Switzerland). According to two
generally accepted protocols, we differentiated two different
phenotypes of functionally competent DCs. Both protocols employed
granulocyte/macrophage colony-stimulating factor (GM-CSF), and
interleukin 4 (IL-4) as basic DC differentiation factors, thus
leading to an immature, antigen-capturing DC stage (Peters J H, Xu
H, Ruppert J, Ostermeier D, Friedrichs D & Gieseler R K,
Signals required for differentiating dendritic cells from human
monocytes in vitro, Adv Exp Med Biol; 329:275-80 [1993]; Ruppert J,
Schutt C, Ostermeier D & Peters J H, Down-regulation and
release of CD14 on human monocytes by IL-4 depends on the presence
of serum or GM-CSF, Adv Exp Med Biol; 329:281-6 [1993]).
[0120] Mature antigen-presenting DCs were then obtained by adding
tumor-necrosis factor (TNF)-.alpha., leading to a DC type able to
initiate both T-helper (Th)1- and Th2-dependent immunity (Caux C,
Dezutter-Dambuyant C, Schmitt D & Banchereau J, GM-CSF and
TNF-.alpha. cooperate in the generation of dendritic Langerhans
cells, Nature; 360:258-61 [1992]; Sallusto F & Lanzavecchia A,
Efficient presentation of soluble antigen by cultured human
dendritic cells is maintained by granulocyte/macrophage
colony-stimulating factor plus interleukin 4 and downregulated by
tumor necrosis factor alpha, J Exp Med; 179:1109-18 [1994];
Banchereau J & Steinman R M, Dendritic cells and the control of
immunity, Nature; 392:245-52 [1998]).
[0121] Alternatively, mature DCs were generated in presence of
interferon (IFN)-.gamma. (Gieseler R, Heise D, Soruri A, Schwartz P
& Peters J H, In-vitro differentiation of mature dendritic
cells from human blood monocytes, Develop Immunol; 6:25-39 [1998]).
Such DCs appear to primarily induce Th1 cells, thus activating
cytotoxic T-cells eliciting anti-tumor immunity (Soruri, A. et al.,
Specific autologous anti-melanoma T cell response in vitro using
monocyte-derived dendritic cells, Immunobiology; 198:527-38 [1998])
and, presumably, antiviral immune responses, due to MHC class
I-restricted antigen presentation. In most cases, DCs were
differentiated for 7 days. However, DCs were kept for up to 21 days
in select experiments. All differentiation factors were obtained
from Sigma (St. Louis, Mo., USA).
[0122] DC Harvesting and Liposome Incubation.
[0123] Harvested MyDCs and liposome preparations were incubated at
differing relative concentrations (depending on the experimental
context) for 3 hours at room temperature, followed by genotypic,
phenotypic and functional (PCR, flow cytometry, ELISA, mixed
leukocyte culture and stimulation for recall antigens) evaluation.
Mature non-adherent and adherent DCs were harvested on day 7.
[0124] First, the differentiation medium was collected,
centrifuged, and the pelleted DC fraction of non-adherent veiled
cells was harvested. Second, adherent DCs were detached from the
wells by incubating them with PBS/EDTA for 30 min at 4.degree. C.,
and by successively employing a rubber policeman. Detached adherent
DCs were pooled with the non-adherent fraction, adjusted to the
cell numbers and incubated with the liposome concentrations
indicated for each experiment.
[0125] As described above, myeloid dendritic cells obtained by
protocols employing TNF-.alpha. or IFN-.gamma., were analyzed
flow-cytrometically for expression of CD1a, CD4, CD14, CD40,
CD45RA, CD45R0, CD68, CD69, CD83, CD184, CD195, CD206, CD207,
CD208, and/or CD209 (i.e., DC-SIGN) with mouse anti-human
IgG1.kappa. mAbs (MOPC-21/P3 as control). Depending on whether only
one or two mAbs were employed, antigens were either stained
directly with FITC-, PE-, or PC5-labeled antibodies, or were
stained indirectly with unlabeled first mAbs plus secondary
polyclonal IgG.times.FITC (available from eBioscience).
[0126] MOPC-21/P3 was employed as the IgG1.kappa. isotype control.
Results served three purposes, i.e.
[0127] (a) To verify that the cells differentiated in vitro
exhibited genuine DC phenotypes,
[0128] (b) To define their phenotypic and interindividual
differences, and
[0129] (c) To compare the expression of a given marker with the
histogram pattern displayed after incubation with liposomes
targeted by the same antibody.
[0130] Prior to DC targeting, and for each test onset, 20 .mu.l
anti-CD209 (DC-SIGN) and/or other antibody at working dilution were
incubated with 30 .mu.l liposomes on a rotator for 1 h at RT.
Aliquots of cell suspension of at least 5.times.10.sup.4 DCs (or,
when employed, macrophages) were incubated with liposomes under
saturating conditions for 3 h at RT under continuous agitation, and
then examined by flow cytometry. (Tested conditions were 1 h, 3 h
and 24 h. The most reliable and reproducible results were obtained
by 3-h co-incubation.).
[0131] HIV Strains.
[0132] HIV strains were obtained from the NIH Repository (Rockville
Pike, Bethesda, Md.), ie., M-(R5-)tropic Ada-M and Ba-L; and
T-(X4-)tropic HXB3, Lai, Lai/IIIB and HTLV-IIIB. HIV strains were
tested for their "tissue-culture 50% infective dosage" (TCID50)
according to protocols known to the art. According to the TCID50
results, viral supernatants were diluted, aliquoted and frozen at
-80.degree. C. until employed for infection at different
dose-infection kinetics.
[0133] Cryostorage of T Cells.
[0134] Separated CD4.sup.+ or CD8.sup.+ T cells, complete T cells,
or total lymphocytes (comprising T and B cells) were stored
individually or as pools from two to four donors (for allogeneic
stimulation) at -80.degree. C. or -196.degree. C., according to
methods known to the art. Such cells are thawed when needed for
autologous or allogeneic mixed leukocyte cultures, or for recall
antigen tests.
[0135] Liposomes and Antiviral Drugs.
[0136] For primary experimental purposes, liposomes were
surface-labeled with Protein A so as to exchangeably bind
antibodies specific for different antigens. These liposomes were
entrapping calcein as a fluorescent tracer dye. To find a suitable
drug targeting system, a range of single or combined drugs
interfering with HIV propagation (e.g., Viread.RTM. [tenofovir],
Retrovir.RTM. [AZT], Epivir.RTM. [3-TC], Zerit.RTM. [d4T],
Videx.RTM. [didanosine], Emtriva.RTM. [emtricitabine], Sustiva.RTM.
[efavirenz], Viramun.RTM. [nevirapine], Rescriptor.RTM.
[delavirdine], Norvir.RTM. [ritonavir], Agenerase.RTM.
[amprenavir], Hivid.RTM. [ddC], lopinavir, Kaletra.RTM.
[lopinavir+ritonavir], Viracept.RTM. [nelfinavir], Crixivan.RTM.
[indinovir sulfate], Fortovase.RTM. [saquinavir], Invirase.RTM.
[saquinavir mesylate] and/or Atazanavir.RTM.), as well as other
drugs that are still in the experimental phase of therapeutic
research, can be employed to obtain proof of anti-HIV efficacy.
[0137] ELISA for HIV p24 Core Antigen.
[0138] Supernatants can be tested according to the manufacturer's
instructions for presence of p24 by a commercially available ELISA
(Abbott Laboratories).
[0139] Quantitative Polymerase Chain Reaction (qPCR) for HIV.
[0140] The degree of integration of HIV proviral DNA into
dendritic-cell host DNA can be determined by using nested primer
pairs (nested semi-qPCR) for HIV proviral sequences, such as the
following:
TABLE-US-00001 Outer Primers: (SEQ ID NO: 1)
5'-agt-ggg-ggg-aca-tca-agc-agc-cat-gca-aat-3' // (SEQ ID NO: 2)
5'-tca-tct-ggc-ctg-gtg-caa-3' // Inner Primers: (SEQ ID NO: 3)
5'-cag-ctt-aga-gac-cat-caa-tga-gga-agc-5g-3' (5-FAM) //;
this is a LUX-primer, labeled with 5-carboxyfluorescein, i.e.,
5-FAM; "5"=5-FAM).
TABLE-US-00002 (SEQ ID NO: 4) 5'-ggt-gca-ata-ggc-cct-gca-t-3'
//.
Isolation of DNA can be accomplished according to manufacturer's
instructions ("Easy-DNA-Kit", in protocol #3 "Small Amounts of
Cells, Tissues, or Plant Leaves", Invitrogen). The PCR reaction
mixture typically includes the following: Buffer (5 .mu.l of
10.times.PCR Rxn Buffer, Invitrogen); MgCl.sub.2 (3 .mu.l of 50 mM
MgCl.sub.2, Invitrogen); dNTP (1 .mu.l of mixture of dATP, dCTP,
dGTP, dTTP: 10 .mu.M, each); Outer Primer (SEQ ID NO:1; 1 .mu.l of
10 pmol/.mu.l); Outer Primer (SEQ ID NO:2; 1 .mu.l of 10
pmol/.mu.l); Taq (0.2 .mu.l of 5 Units/.mu.l, Platinum Taq DNA
Polymerase, Invitrogen); double distilled water (37 .mu.l); DNA
sample (2 .mu.l). One standard thermal cycling profile was the
following: 5 min at 95.degree. C.; (20 s at 95.degree. C.; 30 s at
55.degree. C.; 30 s at 72.degree. C.).times.25; 2 min at 72.degree.
C.; hold at 4.degree. C. PCR is generally repeated using two
microliters of amplified DNA transferred from the first reaction in
fresh PCR reaction mixture, except using the inner primers (SEQ ID
NO:3 and SEQ ID NO:4) instead of the outer primers, and employing a
different thermal cycling profile: 5 min at 95.degree. C.; (20 s at
95.degree. C.; 30 s at 55.degree. C.; 30 s at 72.degree.
C.).times.35; 2 min at 72.degree. C. (melting curve 95.degree. C.
down to 55.degree. C. in steps of 0.5.degree. C.).
[0141] In a given sample, DNA quantification can be achieved by
comparison with a serial dilution of a DNA sample of known quantity
of HIV proviral DNA. To allow quantifying HIV proviral DNA from
samples with different contents of total cellular DNA (e.g., from
dendritic cells), a Multiplex-PCR can be performed. Briefly, a
second nested PCR can be performed in the same reaction, with a LUX
primer labeled with
6-carboxy-4',5'-dichloro-2',7'-dimethoxyfluorescein succinimidyl
ester, for a human chromosome sequence (genome equivalent). This
permits quantification of the total DNA content per sample. Numbers
of proviral copies per human genome equivalent can be calculated
from such data.
Example 2
Active Targeting of Immune Cells with Monospecific or Bispecific
Immunoliposomes
[0142] Peripheral blood mononuclear cells (PBMNCs) were evaluated
according to their size (forward scatter) and granularity (side
scatter) and thus were gated as naive T and B cells; activated
T-cells and B-cells; and monocytes, including a small proportion of
blood dendritic cells (data not shown). Cultured monocyte-derived
dendritic cells (MoDCs) were tested for expression of markers
delineating their developmental stage (maturity), as well as for DC
subtype markers. The DCs expressed markers typical for skin and
mucosal DC phenotypes that are considered to play a key role in HIV
infection. When being infected via the mucosal route, mucosal DCs
are the first immune cell type to be directly infected by HIV (and
integrate its genetic information as proviral DNA) and/or harvest
HIV on their surface by DC-SIGN and/or take up HIV by any of
various mechanisms to retain it in intracytoplasmic compartments
(e.g., endosomes, fused phago-endosomes, or phagolysosomes). Such
cells then migrate to regional and local lymph nodes where passing
on HIV to other cell types, most prominently T-helper cells (i.e.,
"CD4 cells") as well as other reservoir cells, including the next
generation of lymph node-settling DCs. In considering all this, the
DCs generated in our in-vitro system thus provide an ideal model
for testing the presumptive targeting efficacy for such cells in
vivo.
[0143] MoDCs matured by 7-day culture with GM-CSF, IL-4 and
subsequent TNF-.alpha. were tested by flow cytometry for expression
of markers generally expressed by DCs or subpopulations thereof.
Apart from DC-SIGN (CD-209), we chose markers delineating mature
DCs in vitro and in vivo (CD40, CD45R0, CD83), as well as dendritic
Langerhans cells of the epidermis (CD1a) and the intestinal (CD4)
and nasal mucosa (CD14). Phenotyping thus served (i) for verifying
MoDCs generated in vitro as mature; (ii) for proving strong
expression of DC-SIGN (CD209) as the pre-conceived target for
immunoliposomal compound delivery to MyDCs; (iii) for pinpointing
further potential target antigens conforming to the requirement of
consistent high expression; and (iv) for determining whether the
generated MoDCs expressed CD1a and/or CD14 as potential targeting
structures expressed by epidermal and mucosal Langerhans cells in
vivo.
[0144] Relative mean fluorescence intensities (.DELTA.MFI) of test
conditions vs. negative controls (n=3) characterized the phenotypic
profile of mature MoDCs as CD1a.sup.+++, CD4.sup..+-.,
CD14.sup..+-. to +++, CD40.sup.++ to +++, CD45R0.sup.+ to +++,
CD83.sup.+ and CD209.sup.+++ [with: (-), test antibody congruent
with negative control; (.+-.), .DELTA.MFI peak .ltoreq..times.5
above negative control; (+), .DELTA.MFI peak .ltoreq..times.10
above negative control; and (+++), .DELTA.MFI
peak.times..gtoreq.250 negative control]. Of all markers tested,
expression of CD14 varied most considerably among the donors. In
contrast, DC-SIGN (CD209) and CD1a (a Langerhans-cell marker)
consistently revealed high expression in all donors examined.
[0145] FIG. 1 shows targeting of calcein-labeled liposomes to MoDCs
mediated by DC-SIGN or other targeting ligands. Mature MoDCs were
generated in vitro for 7 days. Liposomes were incubated with either
one or two monoclonal antibodies (mAbs) specific for key markers
expressed by MoDCs, so as to obtain monospecific liposomes (for
CD1a, CD83, or CD209) or bispecific liposomes (for CD1a+CD83,
CD1a+CD209, or CD 83+CD209) (Zhou L J, Tedder T F, CD14.sup.+ blood
monocytes can differentiate into functionally mature CD83.sup.+
dendritic cells, Proc Natl Acad Sci USA; 93(6):2588-92 [1996];
Gieseler R, Heise D, Soruri A, Schwartz P, Peters J H, In-vitro
differentiation of mature dendritic cells from human blood
monocytes, Develop Immunol.; 6(1-2):25-39 [1998]. Geijtenbeek T B,
Torensma R, van Vliet S J, van Duijnhoven G C, Adema G J, van Kooyk
Y, Figdor C G, Identification of DC-SIGN, a novel dendritic
cell-specific ICAM-3 receptor that supports primary immune
responses, Cell; 100(5):575-85 [2000]; Geijtenbeek T B, Kwon D S,
Torensma R, van Vliet S J, van Duijnhoven G C, Middel J,
Cornelissen I L, Nottet H S, KewalRamani V N, Littman D R, Figdor C
G, van Kooyk Y, DC-SIGN, a dendritic cell-specific HIV-1-binding
protein that enhances trans-infection of T cells, Cell;
100(5):587-97 [2000]).
[0146] Best results were obtained when mature MoDCs were incubated
with liposomes for 3 h at 37.degree. C. under continuous gentle
agitation. Employing the above-described protocol, further
targeting variants now included CD1a and CD83 as potential targets
expressed by Langerhans cells in the surface-forming tissues
(Teunissen M B M, Dynamic nature and function of epidermal
Langerhans cells in vivo and in vitro: a review, with emphasis on
human Langerhans cells, Histochem. J. 24:697-716 [1992]), as well
as mature intralymphoid MyDCs (Zhou L J, Tedder T F, CD14.sup.+
blood monocytes can differentiate into functionally mature
CD83.sup.+ dendritic cells, Proc Natl Acad Sci USA 93:2588-92
[1996]; Gieseler R et al., In-vitro differentiation of mature
dendritic cells from human blood monocytes, Dev Immunol 1998;
6:25-39 [1998]). The CD1a and CD83 markers turned out to be
comparatively unlikely targeting structures. In contrast, targeting
of DC-SIGN again showed high liposomal binding and uptake of the
fluorochrome (FIG. 2).
[0147] Monoclonal antibodies (mAbs) and mAb-labeled immunoliposomes
tested were specific for CD4, CD45R0 and CD209 (DC-SIGN).
Experiments showed the most favorable incubation time for mature
MyDCs with immunoliposomes and investigated whether incubation with
either one or two types of immunoliposomes (the latter at half the
concentrations employed upon single targeting) might offer a
decisive advantage. Binding of specific mAbs visualized with
FITC-labeled secondary antibody (left-hand column) revealed the
degree of antigen (Ag) expression. Mature MyDCs generated from the
same donors were incubated for 1, 3 or 24 h with immunoliposomes at
37.degree. C. [a preliminary experiment had proven 37.degree. C.
superior to 4.degree. C. or RT (not shown)] (FIG. 1, right column).
Flow-cytometric histograms for phenotyping or targeting (shaded
curves) and negative controls (empty curves) revealed the best
signal-to-noise ratio, most consistent uptake and highest
reproducibility for 3-h incubation. Most intense staining was found
for anti-CD209 and, secondarily, anti-CD45R0. Combination of both
conditions had no substantial advantage over mono-specific
targeting of DC-SIGN. As shown in FIG. 1, most efficacious
targeting and delivery of liposomal contents was achieved with
monospecific liposomal targeting of CD209 (DC-SIGN). When the
targeting efficacies of mAbs only and LS-coupled mAbs were
compared, it was apparent that liposomal delivery led to increased
(intra)cellular fluorescence. It was shown that liposomal delivery
of calcein led to a right shift compared to antibody-conjugated
FITC.
[0148] FIG. 2 shows monospecific liposomal targeting with respect
to kinetics and efficacy. In contrast to previous experiments (see,
FIG. 1) where cells had been incubated with liposomes for 2 hrs
before harvesting and measuring, we here investigated the time
kinetics of liposomal uptake, i.e. uptake of calcein at a number of
time points over a 24-hour period. Although the MoDCs expressed
CD14 over a broad range of membrane densities (cf. left hand
graph), this phenotypic pattern was not reflected after targeting.
In contrast, CD209 (DC-SIGN) targeting again revealed the highest
rate of uptake; also, the patterns of antigen expression (left-hand
graph) and targeting efficacy (3-h graph) were very similar. This
implies that upon binding of CD209-targeted liposomes,
DC-SIGN-liposome complexes apparently are almost completely
internalized, thus delivering the liposomal content to
intracellular compartments. This conclusion is consistent with one
main function known for the CD209 receptor, i.e. uptake of larger
infectious particles over a broad range of sizes including
antigens, HIV, Candida albicans, and Leishmania amastigotes (e.g.,
Engering A, Geijtenbeek T B H, van Vliet S J, Wijers M, van Liempt
B, Demaurex N, Lanzavecchia A, Fransen J, Figdor C G, Piguet V, van
Kooyk Y., The dendritic cell-specific adhesion receptor DC-SIGN
internalizes antigen for presentation to T cells, J Immunol.
168(5):2118-26 [2002]; Kwon D S, Gregorio G, Bitton N, Hendrickson
W A, Littman D R, DC-SIGN-mediated internalization of HIV is
required for trans-enhancement of T cell infection, Immunity
16(1):135-44 [2002]; Cambi A, Gijzen K, de Vries J M, Torensma R,
Joosten B, Adema G J, Netea M G, Kullberg B J, Romani L, Figdor C
G, The C-type lectin DC-SIGN (CD209) is an antigen-uptake receptor
for Candida albicans on dendritic cells, Eur J Immunol. 33(2):532-8
[2003]; Colmenares M, Puig-Kroger A, Pello O M, Corbi A L, Rivas L,
Dendritic cell (DC)-specific intercellular adhesion molecule 3
(ICAM-3-)grabbing nonintegrin (DC-SIGN, CD209), a C-type surface
lectin in human DCs, is a receptor for Leishmania amastigotes, J
Biol Chem. 277(39):36766-69 [2002]).
[0149] FIG. 4A (left panel) and FIG. 4B (left panel) show
calculated values of targeting and surface binding of monospecific
immunoliposomes applied to MoDCs; the results depicted are
representative of at least three independent experiments. Provided
in FIG. 4A are percentages of MoDCs expressing select markers (FITC
fluorescence), and MoDCs targeted with corresponding
immunoliposomes (calcein fluorescence). As FITC and calcein
concentrations were equimolar in all mAb or liposome conditions,
the immunoliposomal net targeting efficacy (TE.sub.ILS) and
relative fluorescence of immunoliposomes vs. mAbs (RF.sub.ILS)
could be determined (equations IV and V; FIG. 4B). Of all variants
tested, mono-specific immunoliposomes targeting DC-SIGN revealed
the highest TE.sub.ILS and were the only preparation showing a
positive RF.sub.ILS value (indicating liposomal accumulation on or
within targeted MoDCs).
[0150] The liposomal targeting efficacy of CD209-coupled liposomes
was 83.31% (FIG. 4A, left panel), and the respective LS
Binding/Uptake graph in FIG. 1 demonstrates for all cells a right
shift (shaded curve), relative to the control peak (open curve).
This indicates that 100% of the cells had been efficiently
targeted, even when only faintly expressing DC-SIGN. Second, at
first sight, combinations of anti-DC-SIGN liposomes with anti-CD1a
or anti-CD83 liposomes (bispecific liposomes) did not effect
increased uptake. However, in combinatorial onsets, antibody
concentrations were only half of those employed when targeting with
one antibody only. Therefore, further investigations were warranted
to determine whether bispecific targeting might, indeed, enhance
the targeting efficacy, when compared to monospecific tareting.
[0151] FIG. 3 illustrates liposomal targeting of DCs via two cell
markers (termed "bispecific targeting"), including time dependency
of the targeting efficacy over a 24-h period. Bispecific targeting
was carried out with all 2-member combinations, or permutations, of
CD4, CD45R0 and CD209. As in FIG. 2, best results were, here again,
obtained upon 3-h incubation of cells with targeted liposomes.
[0152] FIG. 3A shows results for the combination of anti-CD4 plus
anti-CD45R0 targeting ligands. Irrespective of the incubation time,
when compared to the experiment shown in FIG. 2, a subtractive
effect on liposomal uptake was obtained. Combination of anti-CD4-
and anti-CD45R0-specific targeting, therefore, did not appear to
support enhanced uptake by a double-positive cell subset, e.g. the
resting T-memory cell population residing in lymphoid organs. A
similar result was observed for liposomes bearing the combination
of anti-CD209 plus anti-CD45R0 targeting ligands (FIG. 3C).
[0153] FIG. 3B shows results for the combination of anti-CD4 plus
anti-CD209 targeting ligands. When compared to the experiment shown
in FIG. 2, there was an additive effect on liposomal uptake as a
result of the combination of targeting ligands. Of note, the
abscissa in FIG. 3B shows liposomal uptake as a logarithmic
increase in fluorescence. Therefore, the improvement of uptake by
combined targeting of CD4 and CD209 was at least by a factor of 2
and thus, in accordance with the invention, liposomal targeting
dendritic cells employing a combination of anti-CD4 and anti-CD209
targeting ligands can be a useful option, for example, in treating
HIV disease. Adipocytes, another HIV reservoir, can also be
targeted by targeting via CD4 and CD45 (e.g., Hazan, U. et al.,
Human adipose cells express CD4, CXCR4, and CCR5 receptors: a new
target cell type for the immunodeficiency virus-1? FASEB J. 16,
1254-1256 [2002; Erratum in: FASEB J. 16:4 (2000); Kannisto, K. et
al., Expression of adipogenic transcription factors, peroxisome
proliferator-activated receptor .gamma. co-activator 1, IL-6 and
CD45 in subcutaneous adipose tissue in lipodystrophy associated
with highly active antiretroviral therapy, AIDS 17, 1753-1762
[2003]).
[0154] At half-saturating concentration in the bi-specific onsets,
targeting for CD209 seemed to compensate for much of the lacking
targeting efficacy of the CD1a- or CD83-directed variants (B;
bi-specific: solid-lined bars). However, comparison between the
mono-specific and bi-specific onsets for CD209 (FIG. 4A) revealed
that, even at half-saturating conditions, all cells expressing
DC-SIGN had been labeled, which resulted in a TE.sub.ILS of 107.46%
for CD1a/CD209 and a TE.sub.ILS of 112.17% for CD83/CD209 (FIG. 4B;
bi-specific: dashed bars; approximations due to the saturating
anti-CD209 reference condition in FIG. 4A). Both the results for
saturating and non-saturating CD209-specific liposomes suggest for
MyDCs a limiting uptake kinetic at TE.sub.ILS about 110%.
[0155] In FIG. 4B (right panel), a net targeting efficiency with a
positive (+) value indicates that the percentage of cells targeted
efficaciously was higher than the percentage recognized by antibody
only; negative (-) values indicate less efficient targeting than
with antibody; a value of -100% indicates that no cells at all have
been targeted. All values refer to 3-hour co-incubation of cells
and targeted liposomes. The three best targeting conditions were
CD209>CD83+CD209>CD1a+CD209. Targeting efficacy for
bispecific immunoliposomes targeting CD4+CD45R0 was 58.54%;
targeting efficacy for bispecific immunoliposomes targeting
CD4+CD209 was 68.74%; targeting efficacy for bispecific
immunoliposomes targeting CD45R0+CD209 was 62.21%.
[0156] The data presented herein indicate that a DC-SIGN-targeted
system can target different HIV reservoir populations, i.e.,
myeloid dendritic cells and macrophage subsets, for delivering
HIV-inhibiting compounds of any or all types currently known. In
accordance with the present invention, these reservoir populations
can be targeted for integrating DC-SIGN-attached viruses for
successive generation of immunity as well as to remove virus from
the cells' surfaces, and mother-to-child virus transfer during
pregnancy can be prevented. DC-SIGN is strongly expressed by
mucosal and skin types of dendritic cells in humans and macaques.
(Geijtenbeek, T B et al., DC-SIGN: a novel HIV receptor on DCs that
mediates HIV-1 transmission, Curr Top Microbiol Immunol. 2003;
276:31-54 [2003]; Yu Kimata M T et al., Capture and transfer of
simian immunodeficiency virus by macaque dendritic cells is
enhanced by DC-SIGN, J Virol. 76(23):11827-36 [2002]). Thus,
treating HIV-infected individuals with DC-SIGN-targeted liposomes,
in accordance with the present invention, offers the benefit of
actively targeting the first cell population infected and affected
in the etiology of HIV disease.
[0157] DC-SIGN is further expressed by dendritic and other cells
located within certain placental anatomic structures. (E.g.,
Soilleux E J et al, Placental expression of DC-SIGN may mediate
intrauterine vertical transmission of HIV, J Pathol. 195(5):586-92
[2001]; Soilleux E J, Coleman N, Transplacental transmission of
HIV: a potential role for HIV binding lectins, Int J Biochem Cell
Biol. 2003 March; 35(3):283-7 [2003]; Kammerer U et al., Unique
appearance of proliferating antigen presenting cells expressing
DC-SIGN (CD209) in the decidua of early human pregnancy, Am J
Pathol. 162(3):887-96 [2003]). Thus, if administered by
intravenous, subcutaneous or direct in-utero application, the
inventive method offers the benefit of targeting those cells that
apparently play a major role in mother-to-child HIV transfer, also
termed vertical transmission.
Example 3
Fluorescence-Microscopic Uptake Studies
[0158] After infection with HIV-1, intracytoplasmic compartments
with accumulated infectious virus are demonstrable in both immature
and mature MyDCs (Frank, I et al., Infectious and whole inactivated
simian immunodeficiency viruses interact similarly with primate
dendritic cells (DCs): differential intracellular fate of virions
in mature and immature DCs, J Virol 76:2936-51 [2002]). Therefore
for comparison, immature or mature MoDCs were incubated for 3, 4 or
5 h at 37.degree. C. with anti-CD209-labeled liposomes (each at
n=3). The cells were then harvested as described above and gently
pipetted to dissociate homotypic clusters (as controlled by phase
microscopy). Pelleted single cells were successively dissolved in
100 .mu.l of ProLong antifade mounting medium to which was added 5
.mu.M of the positively charged AT-binding DNA dye,
4',6-diamidino-2-phenylindole (DAPI) (both from Molecular Probes,
Eugene, Oreg., USA). Fifty .mu.l of each preparation were
transferred to poly-L-lysine-coated slides (Labscientific,
Livingston, N.Y., USA), cover-slipped, sealed and kept in the dark
for at least 15 min before being viewed. Sifting through about 100
cells per preparation, MoDCs were then screened with a Zeiss
Axioskop microscope (Carl Zeiss, Thornwood, N.Y., USA) for surface
and intracellular fluorescence of calcein (green) and DNA/nuclei
(blue). Photomicrographic tomographies of MyDCs displaying
representative staining were performed at 0.5-.mu.m steps,
achieving 27-35 serial sections per cell (thus implying a range in
diameter for MyDCs of 13.5-17.5 .mu.m at n=12). Digital photography
was carried out with an ORCA-1 CCD camera (Hamamatsu, Bridgewater,
N.J., USA). Photographic processing, merging of green and blue
fluorescence, as well as microtomography linking to generate film
clips covering MyDCs in optical depth was performed with the
Northern Elite V.6.0 software package (Empix Imaging, Cheek Towaga,
N.Y., USA). Dead cells were excluded from the evaluation by nuclear
staining with propidium iodide as well as by their extremely bright
nuclear DAPI staining. Immunoliposomes carrying mAb MOPC-21/P3 were
taken as negative controls; positive controls employed anti-CD209
mAb.times.FITC.
[0159] FIG. 5 illustrates surface binding vs. internalization of
targeted liposomes determined by fluorescence microscopy as
described above. For discerning intracellular from outshining
membrane fluorescence, we then, at steps of 0.5 .mu.m, photographed
27 to 35 microtomographies per MoDC body. After 3-h incubation with
CD209-specific liposomes (corresponding to the CD209 condition in
FIG. 4B), green calcein labeling was seen only on the cell surface
and was mainly confined to larger DC-SIGN-rich lipid rafts (FIG. 5,
panel 1; depicting the median optical section). An overlay of all
serial sections of the same cell reveals another superimposed
DC-SIGN lipid raft in the lower foreground, and some scattered
fluorescence corresponding to the size of liposomes (FIG. 5, panel
2). However, after 5-h incubation, liposomally entrapped calcein
had been completely internalized. In all of about 100 MoDCs
examined per condition, the cells revealed both diffuse and
concentrated areas of intracellular fluorescence (FIG. 5, panels
3-8). Importantly, areas displaying much lower fluorescence
intensity (FIG. 5, panel 3; arrowhead) were always identified as
nuclei, clearly proving intracellular delivery of the tracer
compound (FIG. 5, panel 4; depicting the cell shown in FIG. 5,
panel 3, merged with blue nuclear DAPI staining). Occasionally,
some liposomal binding was still seen in CD209-rich surface rafts
(FIG. 5, panel 5; arrow) while most calcein was internalized (FIG.
5, panel 5; arrowhead). At this time, compartments highly enriched
in calcein were seen in all MoDCs (FIG. 5, panel 6; arrowhead), and
about one quarter of them revealed prominent perinuclear
fluorescence (FIG. 5, panel 7). Depending on the amount of uptake,
this area sometimes covered a large portion of the extranuclear
space (FIG. 5, panel 8). While results obtained with immature MyDCs
incubated under the same conditions were essentially identical,
intracellular liposome/calcein uptake was seen already after 4-h
incubation (not separately shown). Quenching of extracellular
fluorescence with trypan blue completely blocked out fluorescence
when cells had been incubated for 3 h, but had no effect after 5-h
incubation, thereby confirming the results depicted in FIG. 5.
[0160] Negative controls did not show surface binding or uptake,
while positive controls were very rapidly bound and internalized
(not shown). When adding DC-SIGN-specific FITC-conjugated mAb to
lipopolysaccharide-matured human MyDCs, Schjetne et al. have shown
that it is located extracellularly 15 min later, and
intracellularly after 45 min (Schjetne K W et al., A mouse
C.sub.K-specific T cell clone indicates that DC-SIGN is an
efficient target for antibody-mediated delivery of T cell epitopes
for MHC class II presentation, Int Immunol 14:1423-30 [2002]).
Employing DCs generated by a slightly different protocol, the
results in our positive control with FITC-labeled anti-DC-SIGN mAb
were similar.
[0161] In contrast, intracellular uptake of the larger, targeted
liposomes took longer, up to 5 hours, depending on the MoDCs' stage
of maturity. While these results imply that the size of
DC-SIGN-bound particles inversely correlates with the time required
for cellular uptake, the size of the liposomes employed herein
(with an average diameter of about 150 nm) does not preclude their
uptake. Therefore, by replacing the tracer compound with suitable
drugs, these liposomes, in accordance with the invention, are
valuable DC-specific targeting vehicles. This reasoning is further
supported by the consistently high surface expression of CD209
(DC-SIGN) with, for example, at least 1.times.10.sup.5 molecules
per immature MoDC, thus furnishing a very reliable target (Baribaud
F et al., Quantitative expression and virus transmission analysis
of DC-SIGN on monocyte-derived dendritic cells, J Virol 76:9135-42
[2002]). Importantly, the targeting efficacy we demonstrated was
achieved in the presence of mannan- or mannose-binding lectin (MBL)
which very likely--as a liver-derived substance (Downing, I et al.,
Immature dendritic cells possess a sugar-sensitive receptor for
human mannan-binding lectin, Immunology 109:360-4
[2003])--constitutes a component of the small amount of fetal
bovine serum employed during culture and incubation. In any event,
it has recently been shown that MBL is even autologously secreted
by immature human MoDCs (Downing I et al., Immature dendritic cells
possess a sugar-sensitive receptor for human mannan-binding lectin,
Immunology 2003; 109:360-4 [2003]). Furthermore, MBL, via its own
C-type lectin domain, can prevent HIV-1 from binding to DC-SIGN
(Spear G T et al., Inhibition of DC-SIGN-mediated trans infection
of T cells by mannose-binding lectin, Immunology 2003; 110:80-5
[2003]). Therefore, soluble MBL (and perhaps other unidentified
molecules displaying similar characteristics) did not prevent the
inventive DC-SIGN-specific liposomes from interacting with the
membrane-bound C-type lectin.
[0162] By employing a liposomally entrapped tracer, calcein, we
flow-cytometrically and mathematically demonstrated a superior
targeting efficacy for DC-SIGN, as compared with select other MyDC
markers (CD1a, CD4, CD45R0, CD83). Fluorescence microscopy further
revealed time-dependent surface binding and intracellular uptake of
DC-SIGN-specific liposomes by both immature and mature MyDCs. The
net targeting efficacy we found for DC-SIGN-specific
immunoliposomes, as well as the fluoromicrographic uptake studies,
clearly reveal efficient binding, internalization and intracellular
compound delivery. We have shown that DC-SIGN-targeted
immunoliposomes (i.e., including targeting ligand that specifically
binds CD209) deliver their contents both to immature and mature
MyDCs, and that, in addition to cytoplasmatic distribution, their
contents strongly accumulate in discrete intracellular compartments
(FIG. 5), or endosomes, respectively. These observations, together
with the fact that HIV-1 and the liposomes administered are
comparable in size, enable the inventive delivery system to reach
exactly the same compartments where highly infectious HIV-1 is
stored and rescued from any systemic attack until being released to
infect further Th cells. Suitable immunoliposomally delivered
agents, in accordance with the present invention, will thus reach
an important sanctuary that is not as yet addressed by any
therapeutic strategy. Another important benefit is that, due to the
fact that these liposomes are retained on the surface of MyDCs for
prolonged times, Th cells interacting with DCs within lymphoid
organs and tissues in the course of antigen-specific stimulation
can also be reached therapeutically by this strategy (Gieseler R K,
Marquitan G, Hahn M J, Perdon L A, Driessen W H P, Sullivan S M,
Scolaro M J, DC-SIGN-specific liposomal targeting and selective
intracellular compound delivery to human myeloid dendritic cells:
implications for HIV disease, Scand J Immunol; 59:415-24 [2004];
Marquitan G, Gieseler R K, Driessen W H P, Perdon L A, Hahn M J,
Wader T, Sullivan S M, Scolaro M J, Intracellular compound delivery
to human monocyte-derived dendritic cells by immunoliposomal
targeting of the C-type lectin DC-SIGN. MACS & MORE, in press
[2004]).
Sequence CWU 1
1
4130DNAArtificial SequencePrimer 1agtgggggga catcaagcag ccatgcaaat
30218DNAArtificial SequencePrimer 2tcatctggcc tggtgcaa
18328DNAArtificial SequencePrimer; Labeled with
5-carboxyfluorescein 3cagcttagag accatcaatg aggaagcg
28419DNAArtificial SequencePrimer 4ggtgcaatag gccctgcat 19
* * * * *