U.S. patent application number 09/985630 was filed with the patent office on 2003-01-30 for method of making therapeutic liposomal compositions.
Invention is credited to Boni, Larry, Kwak, Larry, Ochoa, Augusto C., Popescu, Mircea C..
Application Number | 20030021838 09/985630 |
Document ID | / |
Family ID | 21751949 |
Filed Date | 2003-01-30 |
United States Patent
Application |
20030021838 |
Kind Code |
A1 |
Popescu, Mircea C. ; et
al. |
January 30, 2003 |
METHOD OF MAKING THERAPEUTIC LIPOSOMAL COMPOSITIONS
Abstract
A vaccine comprising a liposome preparation including at least
one B-cell malignancy-associated antigen, IL-2, alone or in
combination with at least one other cytokine, and at least one type
of lipid molecule, is useful in a method of inducing humoral and
cellular immune responses against malignant B-cells in a
mammal.
Inventors: |
Popescu, Mircea C.;
(Plainsboro, NJ) ; Kwak, Larry; (Frederick,
MD) ; Ochoa, Augusto C.; (Frederick, MD) ;
Boni, Larry; (Monmouth Junction, NJ) |
Correspondence
Address: |
Michele M. Simkin
FOLEY & LARDNER
Washington Harbour
3000 K Street, N.W., Suite 500
Washington
DC
20007-5109
US
|
Family ID: |
21751949 |
Appl. No.: |
09/985630 |
Filed: |
November 5, 2001 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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09985630 |
Nov 5, 2001 |
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09125376 |
Oct 27, 1998 |
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6312718 |
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09125376 |
Oct 27, 1998 |
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PCT/US97/02351 |
Feb 13, 1997 |
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60011783 |
Feb 16, 1996 |
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Current U.S.
Class: |
424/450 ;
424/277.1; 424/85.2 |
Current CPC
Class: |
A61P 35/00 20180101;
A61P 35/02 20180101; A61K 39/395 20130101; A61K 2039/55511
20130101; A61K 9/127 20130101; A61K 39/395 20130101; A61K 38/20
20130101; A61K 2300/00 20130101; A61K 9/127 20130101; A61K 39/395
20130101 |
Class at
Publication: |
424/450 ;
424/277.1; 424/85.2 |
International
Class: |
A61K 039/00; A61K
038/20; A61K 009/127 |
Claims
What is claimed is:
1. A vaccine comprising a liposome preparation comprising a) at
least one B-cell malignancy-associated antigen; b) IL-2, alone or
in combination with at least one other cytokine; and c) at least
one type of lipid molecule.
2. A vaccine according to claim 1, wherein said antigen comprises
all or part of an antibody associated with or produced by a
malignant B-cell.
3. A vaccine according to claim 1, wherein said malignant B cell is
associated with lymphoma.
4. A vaccine according to claim 1, wherein said malignant B cell is
associated with chronic lymphocytic leukemia.
5. A vaccine according to claim 1, wherein said malignant B cell is
associated with multiple myeloma.
6. A vaccine according to claim 2, additionally comprising
tumor-associated antigen that is not an antibody or antibody
fragment.
7. A vaccine according to claim 6, wherein said tumor-associated
antigen is MUC-1, EBV antigen or an antigen associated with
Burkitt's lymphoma.
8. A vaccine according to claim 1, further comprising a B-cell
antigen produced by or associated with non-malignant B-cells.
9. A vaccine according to claim 8, wherein said B-cell antigen is a
class 1 or class 2 HLA antigen.
10. A vaccine according to claim 1, wherein said at least one other
cytokine is selected from the group consisting of M-CSF, GM-CSF,
and IFN-gamma.
11. A vaccine according to claim 1, wherein said lipid molecule is
selected from the group consisting of phospholipid, glycolipid,
cholesterol, and derivatives of said lipids.
12. A vaccine according to claim 1, further comprising a carrier
protein.
13. A vaccine according to claim 12, wherein said carrier protein
is albumin.
14. A vaccine according to claim 1, further comprising an
adjuvant.
15. A method for inducing humoral and cellular immune responses
against malignant B-cells in a mammal, comprising administering to
said mammal a vaccine according to any one of claims 1-14.
Description
BACKGROUND OF THE INVENTION
[0001] 1. Field of the Invention
[0002] The present invention relates to methods for inducing
humoral and cellular immune responses against malignant B cells. In
particular, this invention is directed to methods for producing an
integrated immunologic response against tumor cells using antigens
that are associated with a B-cell malignancy.
[0003] 2. Background
[0004] One of the major goals of immunotherapy is to harness a
patient's immune system against tumor cells or infectious
organisms. With regard to cancer therapy, the objective is to
direct the patient's immune system against tumor cells by targeting
antigens that are associated with tumor cells, but not normal
counterparts. These tumor associated antigens (TAAs) have been
difficult to identify. Certain tumor cells express antigens that
are normally not expressed, or expressed at very low levels, in
adult life, although they are present during fetal development. One
example of such oncofetal TAAs is .alpha.-fetoprotein, which is
expressed by liver cancer cells. Another oncofetal TAA is
carcinoembryonic antigen (CEA), which is expressed in most
adenocarcinomas of entodermally-derived digestive system epithelia,
as well as in breast tumor cells and non-small-cell lung cancer
cells. Thomas et al., Biochim. Biophys. Acta 1032: 177 (1990).
[0005] The administration of anti-idiotype antibodies (Ab2s)
mimicking TAAs represents a promising approach to cancer
immunotherapy. Goldenberg, Amer. J. Med. 94: 297 (1993). Ab2s are
antibodies directed against the variable regions of conventional
antibodies (Ab1). Certain Ab2s (termed "Ab2.beta.", "anti-idiotype"
or "internal-image" antibodies) can mimic the three-dimensional
structure of the nominal antigen, and thus Ab2 and antigen can bind
with the same regions of the Ab1-combining site. Jerne et al., EMBO
J. 1: 243 (1982); Losman et al., Int. J. Cancer 46: 310 (1990);
Losman et al., Proc. Nat'l Acad. Sci. USA 88: 3421 (1991); Losman
et al., Int. J. Cancer 56: 580 (1994). Individuals immunized with
Ab2.beta. can develop anti-anti-antibodies (Ab3), some of which can
bind the nominal antigen.
[0006] The antigen mimicry properties of anti-idiotype antibodies
have led to the use of Ab2.beta. as surrogate antigens (or idiotype
vaccines), when the nominal antigen is not readily available or
when the host is tolerant to the nominal antigen. In experimental
systems, immunization with Ab2.beta. mimicking certain TAA creates
specific immunity to the TAA and protect against subsequent tumor
growth. See, for example, Nepom et al., Proc. Nat'l Acad. Sci. USA
81: 2864 (1984); Raychaudhuri et al., J. Immunol. 139: 271 (1987).
Similarly, anti-idiotype vaccines have been developed against
infectious organisms, such as Streptococcus pneumoniae [McNamara et
al., Science 226: 1325 (1984)], hepatitus B virus [Kennedy et al.,
Science 223: 930 (1984)], Escherichia coli K13 [Stein et al., J.
Exp. Med. 160: 1001 (1984)], Schistosomiasis mansoni [Kresina et
al., J. Clin. Invest. 83: 912 (1989)], and Moloney murine sarcoma
virus [Powell et al., J. Immunol. 142: 1318 (1989)].
[0007] However, the usefulness of this approach is limited. Cancer
patients receiving an anti-TAA of animal origin will usually
produce antibodies to the Ab1 and these anti-immunoglobulin
antibodies include Ab2. Herlyn et al., J. Immunol. Methods 85: 27
(1985); Traub et al., Cancer Res. 48: 4002 (1988). The
anti-idiotype response also may include the generation of T cells
(T2). Fagerberg et al., Cancer Immunol. Immunother. 37: 264 (1993).
Moreover, Ab2 may subsequently induce a humoral and cellular
anti-anti-idiotypic response, Ab3 and T3, respectively, which may
recognize the same epitope as Ab1. Id. This is a problem because it
can reduce the effectiveness of the immune response.
[0008] Thus, an opportunity exists to provide an approach to
immunotherapy utilizing both humoral and cellular immune systems.
The present methods to provoke an integrated response against tumor
cells, particularly malignant B cells, is an initial result of this
approach.
SUMMARY OF THE INVENTION
[0009] Accordingly, it is an object of the present invention to
provide a vaccine and method of treatment by inducing humoral and
cellular immune responses against malignant B cells, in particular
lymphoma, chronic lymphocytic leukemia and multiple myeloma. The
vaccine comprises a liposomal preparation that incorporates at
least one B cell malignancy associated antigen, at least one
cytokine, and at least one type of lipid molecule. This combination
therefore provides a novel and more potent vaccine formulation for
B cell malignancies. The B-cell malignancy-associated antigen is
preferably derived from the patient to be treated and thus the
vaccine will be directed against the patient's malignant
B-cells.
[0010] Thus, in one embodiment, the invention provides a vaccine
comprising a liposome preparation comprising (1) at least one
B-cell malignancy-associated antigen; (2) IL-2, alone or in
combination with at least one other cytokine; and (3) at least one
type of lipid molecule.
[0011] In another embodiment, the B-cell malignancy-associated
antigen comprises all or part of an antibody associated with or
produced by a malignant B-cell. Such malignant B-cells include
those associated with lymphoma, chronic lymphocytic leukemia and
multiple myeloma. In a further embodiment, the vaccine of the
invention additionally comprises a tumor-associated antigen that is
not an antibody or antibody fragment. Examples of such additional
TAAs include, e.g., MUC-1, Epstein Barr Virus (EBV) antigen or an
antigen associated with Burkitt's lymphoma.
[0012] In an alternative embodiment, the vaccines of the invention
additionally comprise normal B-cell antigens such as HLA
antigens.
[0013] In another embodiment, the vaccine of the invention
additionally comprises a another cytokine; examples of additional
cytokines include M-CSF, GM-CSF and IFN-gamma.
[0014] The vaccines of the invention comprise at least one lipid
molecule selected from the group consisting of phospholipid,
cholesterol, and glycolipid and derivatives of these lipids. In a
further embodiment, the vaccines of the invention also comprise a
carrier protein, e.g., albumin.
[0015] In another embodiment, a method for inducing humoral and
cellular immune responses against malignant B-cells in a mammal is
provided, comprising administering to said mammal a vaccine
comprising a liposome preparation comprising (1) at least one
B-cell malignancy-associated antigen; (2) IL-2, alone or in
combination with at least one other cytokine; and (3) at least one
type of lipid molecule.
BRIEF DESCRIPTION OF THE DRAWINGS
[0016] FIGS. 1A, 1B and 1C show features of liposomes prepared
according to the invention.
[0017] FIG. 2 shows survival rates of immunized and control mice
according to Example 2.
[0018] FIG. 3 shows survival rates of immunized and control mice
according to Example 3.
[0019] FIG. 4 shows survival rates of immunized and control mice
according to Example 5.
[0020] FIG. 5 shows anti-idiotype titers as a function of IL-2,
according to Example 6.
[0021] FIG. 6 shows T-cell proliferation as a function of vaccine
dose, according to Example 7.
[0022] FIG. 7 shows survival rates for immunized and control mice
according to Example 8.
DETAILED DESCRIPTION
[0023] The vaccine is composed of three categories of
molecules:
[0024] 1. At least one B-cell malignancy associated antigen. Such
an antigen is preferably an antibody, or a fragment of an
antibody.
[0025] 2. Cytokine, in the form of IL-2 alone or IL-2 plus one or
more different cytokines such as IL-2, M-CSF, GM-CSF or
IFN-gamma.
[0026] 3. At least one type of lipid molecule, in the form of one
or more phospholipids alone or in combination with one or more
different lipids such as cholesterol.
[0027] The vaccine structure comprises a microscopic vesicle
composed of lipid(s), cytokine(s) and at least one B-cell
malignancy-associated antigen. The vaccine of the invention may
also include an adjuvant or carrier protein, such as albumin.
[0028] 1. Definitions
[0029] An antigen is a substance that, upon introduction into a
vertebrate animal, stimulates the production of antibodies.
[0030] An idiotype is an antigenic determinant of the variable
region of an antibody.
[0031] A B-cell malignancy associated antigen is a molecule
produced by or associated with malignant B cells, but which is not
normally expressed, or is expressed at very low levels, by a
non-malignant B-cell. Examples of B-cell malignancy associated
antigens include antibodies, antibody fragments produced by
malignant B-cells, and other non-antibody antigens produced by or
associated with malignant B-cells. Antibody fragments according to
the invention normally comprise an idiotype.
[0032] A tumor cell associated antigen (TAA) is a molecule produced
by or associated with malignant cells, but is not normally
expressed, or expressed at very low levels, by a non-malignant
cell.
[0033] A lipid is any of a group of biochemicals which are variably
soluble in organic solvents, such as alcohol. Examples of lipids
include phospholipids, fats, waxes, and sterols, such as
cholesterol.
[0034] A vaccine is a material that is administered to a vertebrate
host to immunize the host against the same material. Typically, a
vaccine comprises material associated with a disease state, such as
viral infection, bacterial infection, and various malignancies.
[0035] 2. Production of Antigen
[0036] a. B-cell malignancy-associated antibodies and antibody
fragments
[0037] An antigen according to the present invention can be an
antibody molecule produced by the malignant B-cell or a fragment of
such an antibody. In lymphoma, the antibodies associated with B
cells typically contain a transmembrane domain. In chronic
lymphocytic leukemia, such antibodies also have a transmembrane
domain. In multiple myeloma, the malignant B-cells often secrete
fragments of antibodies.
[0038] In one embodiment, these antibodies will be derived from the
patient to be treated for B-cell malignancy. The antibodies can be
extracted from a sample of tissue containing malignant B-cells
which has been obtained from a patient with a B-cell malignancy.
Typically such a tissue sample will be taken from the lymph nodes
of the patient. In patients with multiple myeloma, antibodies can
be extracted from the patient's serum and urine. It is known in the
art that certain antibody light chain molecules are associated with
multiple myeloma. One example of such a protein is a Bence-Jones
protein. Using protein extraction and purification procedures well
known to those of skill in the art, the B-cell antibodies can be
isolated and purified. Such isolation and purification techniques
include affinity chromatography, for example with protein-A
sepharose, size exclusion chromatography and ion-exchange
chromatography. See, for example, CURRENT PROTOCOLS IN IMMUNOLOGY,
VOL 1, pages 2.7.1-2.7.12 (John Wiley & Sone 1991), METHODS IN
MOLECULAR BIOLOGY, VOL. 10, pages 79-104 (The Humana Press, Inc.
1992). It is also known in the art that three major idiotypes are
associated with chronic lymphocytic leukemia.
[0039] In another embodiment, the patient tissue sample containing
malignant B cells will be used to create monoclonal antibodies in
vitro. Typically, malignant tissue, containing malignant B-cells is
fused with a mouse cell line to produce a hybridoma cell line that
will produce a malignant B-cell-associated antibody. Techniques for
making monoclonal antibodies are well known to those of skill in
the art. See, for example, Kohler and Millstein, Nature 256: 495
(1975) and CURRENT PROTOCOLS IN IMMUNOLOGY, VOL 1, pages
2.5.1-2.6.7 (John Wiley & Sone 1991)
[0040] In one embodiment, the antigen will comprise a fragment of
an antibody produced by malignant B-cells. Normally, such a
fragment will comprise an idiotype associated with the malignant
B-cell. An antibody fragment according to the invention includes
(A) a "half antibody" molecule, i.e., a single heavy:light chain
pair, and (B) an enzymatically cleaved antibody fragment, such as
the univalent fragments Fab and Fab', the divalent fragment
F(ab').sub.2, and a single or double chain Fv fragment. An Fv
fragment of an antibody is made up of the variable region of the
heavy chain (Vh) of an antibody and the variable region of the
light chain of an antibody (Vl).
[0041] In accordance with the present invention, fragments within
the invention can be obtained from an antibody by methods that
include digestion with proteases such as pepsin or papain and/or
cleavage of disulfide bonds by chemical reduction. For example,
antibody fragments can be produced by enzymatic cleavage of
antibodies with pepsin to provide a 5S fragment denoted
F(ab').sub.2. This fragment can be further cleaved using a thiol
reducing agent, and optionally a blocking group for the sulfhydryl
groups resulting from cleavage of disulfide linkages, to produce
3.5S Fab' monovalent fragments. Alternatively, an enzymatic
cleavage using pepsin produces two monovalent Fab fragments and an
Fc fragment directly. These methods are described, for example, by
Goldenberg, U.S. Pat. Nos. 4,036,945 and 4,331,647 and references
contained therein, which patents are incorporated herein in their
entireties by reference. Also, see Nisonoff et al., Arch Biochem.
Biophys. 89: 230 (1960); Porter, Biochem. J. 73: 119 (1959),
Edelman et al., in METHODS IN ENZYMOLOGY VOL. 1, page 422 (Academic
Press 1967), and Coligan at pages 2.8.1-2.8.10 and
2.10.-2.10.4.
[0042] Alternatively, antibody fragments encompassed by the present
invention can be synthesized using an automated peptide synthesizer
such as those supplied commercially by Applied Biosystems, Multiple
Peptide Systems and others, or they may be produced manually, using
techniques well known in the art. See Geysen et al., J. Immunol.
Methods 102: 259 (1978). Direct determination of the amino acid
sequences of the variable regions of the heavy and light chains of
the antibodies according to the invention can be carried out using
conventional techniques.
[0043] Proteolytic cleavage of an antibody can produce double chain
Fv fragments in which the Vh and Vl regions remain non-covalently
associated and retain antigen binding capacity. Double chain Fv
fragments also can be produced by recombinant expression methods
well known in the art. See Skerra et al., Science 240: 1038 (1988),
and King et al., Biochemical J. 290: 723 (1991). Briefly, the amino
acid sequence of the variable regions of the heavy and light chains
of antibodies according to the invention can be obtained by direct
amino acid sequencing using methods well known to those in the art.
From this amino acid sequence, synthetic genes can be designed
which code for these variable regions and they can both be inserted
into an expression vector. Two polypeptides can be expressed
simultaneously from a mammalian or bacterial host, resulting in
formation of an active Fv fragment.
[0044] An antigen of the present invention also can be a "single
chain antibody," a phrase used in this description to denote a
linear polypeptide that binds antigen with specificity and that
comprises variable or hypervariable regions from the heavy and
light chain chains of an antibody. Other single chain antibodies
according to the invention can be produced by conventional
methodology. The Vh and Vl regions of the Fv fragment can be
covalently joined and stabilized by the insertion of a disulfide
bond. See Glockshuber, et al., Biochemistry 1362 (1990).
Alternatively, the Vh and Vl regions can be joined by the insertion
of a peptide linker. A gene encoding the Vh, Vl and peptide linker
sequences can be constructed and expressed using a recombinant
expression vector. See Colcher, et al., J. Nat'l Cancer Inst. 82:
1191 (1990). Amino acid sequences comprising hypervariable regions
from the Vh and Vl antibody chains can also be constructed using
disulfide bonds or peptide linkers, as described herein.
[0045] Another form of an antibody fragment is a peptide
constituting a single complementarity-determining region (CDR). CDR
peptides, such as CDR3, ("minimal recognition units") can be
obtained by constructing and expressing genes encoding the CDR of
an antibody of interest. Such genes are prepared, for example, by
using the polymerase chain reaction to synthesize the variable
region from RNA of antibody-producing cells. See, for example,
Larrick et al., Methods: A Companion to Methods in Enzymology 2:
106 (1991).
[0046] b. Non-malignancy associated B-cell antigens
[0047] The vaccines of the invention can also comprise B-cell
antigens which are not specifically associated with malignant
B-cells ("non-malignancy associated B-cell antigens"). Examples of
these antigens are known in the art and include CD19, CD20, CD21,
CD22, CD23, CD25, CD5, and FMC7. Foon, K. Stem Cells 13(1):1-21
(1995). Also included in this group are class 1 and class 2 HLA
antigens (histocompatibility molecules). Class 1 HLA antigens are
also found on almost all other mammalian cells.
[0048] c. Other tumor-associated antigens (TAAs)
[0049] The vaccines of the invention can additionally comprise
other TAAs. Examples of such tumor-associated antigen are MUC-1,
EBV antigen and antigens associated with Burkitt's lymphoma.
[0050] 3. Preparation of Liposome
[0051] Liposomes are microscopic vesicles that consist of one or
more lipid bilayers surrounding aqueous compartments. See,
generally, Bakker-Woudenberg et al., Eur. J. Clin. Microbiol.
Infect. Dis. 12 (Suppl. 1): S61 (1993), and Kim, Drugs 46: 618
(1993). Liposomes are similar in composition to cellular membranes
and as a result, liposomes generally can be administered safely and
are biodegradable. Depending on the method of preparation,
liposomes may be unilamellar or multilamellar, and liposomes can
vary in size with diameters ranging from 0.02 .mu.m to greater than
10 .mu.m. A variety of agents can be encapsulated in liposomes:
hydrophobic agents partition in the bilayers and hydrophilic agents
partition within the inner aqueous space(s). See, for example,
Machy et al., LIPOSOMES IN CELL BIOLOGY AND PHARMACOLOGY (John
Libbey 1987), and Ostro et al., American J. Hosp. Pharm. 46: 1576
(1989).
[0052] Liposomes can adsorb to virtually any type of cell and then
slowly release the encapsulated agent. Alternatively, an absorbed
liposome may be endocytosed by cells that are phagocytic.
Endocytosis is followed by intralysosomal degradation of liposomal
lipids and release of the encapsulated agents. Scherphof et al.,
Ann. N.Y. Acad. Sci. 446: 368 (1985).
[0053] Among liposome vectors, cationic liposomes are the most
studied, due to their effectiveness in mediating mammalian cell
transfection in vitro. They are often used for delivery of nucleic
acids, but can be used for delivery of other therapeutics, be they
drugs or hormones.
[0054] Cationic lipids are not found in nature and can be
cytotoxic, as these complexes appear incompatible with the
physiological environment in vivo which is rich in anionic
molecules. Liposomes are preferentially phagocytosed into the
reticuloendothelial system. However, the reticuloendothelial system
can be circumvented by several methods including saturation with
large doses of liposome particles, or selective macrophage
inactivation by pharmacological means. Claassen et al., Biochim.
Biophys. Acta 802: 428 (1984). In addition, incorporation of
glycolipid- or polyethelene glycol-derivatised phospholipids into
liposome membranes has been shown to result in a significantly
reduced uptake by the reticuloendothelial system. Allen et al.,
Biochim. Biophys. Acta 1068: 133 (1991); Allen et al., Biochim.
Biohys. Acta 1150: 9 (1993).
[0055] Anionic liposomal vectors have also been examined. These
include pH sensitive liposomes which disrupt or fuse with the
endosomal membrane following endocytosis and endosome
acidification.
[0056] Liposome complexes are sometimes targeted to the cell type
or tissue of interest by the addition to the liposome preparation
of a ligand, usually a polypeptide, for which a corresponding
cellular receptor has been identified. An example of a cell
receptor that can be targeted is the folate receptor which has
recently been identified as a prominent tumor marker, especially in
ovarian carcinomas. KB cells are known to vastly overexpress the
folate receptor. Campbell et al. Cancer Res. 51: 6125-6132 (1991).
Yet other targeting ligands have been examined for liposome
targeting including transferrin, protein A, ApoE, P-glycoprotein,
.alpha..sub.2-macroglobin, insulin, asiolofetuin,
asialoorosomucoid, monoclonal antibodies with a variety of tissue
specificity, biotin, galactose or lactose containing haptens
(monovalent and tri-antennary), mannose, dinitrophenol, and vitamin
B12. The ligands are covalently conjugated to a lipid anchor in
either preformed liposomes or are incorporated during liposome
preparation. Lee and Low J. Biol. Chem. 269: 3198-3204 (1994) and
Lee and Low Biochim. Biophys. Acta 1233: 134-144 (1995).
[0057] Synthetic peptides are sometimes incorporated into
DNA/liposome complexes to enhance their activity, or to target them
to the nucleus. For example, in order to gain access to the
cytoplasm, the molecule to be delivered must overcome the plasma
membrane barrier. In nature, viral fusion peptides facilitate the
delivery into the cytoplasm by promoting viral membrane fusion with
the plasma membrane. For recent reviews on this subject see
Stegmann et al., Ann. Rev. Biophys. Chem. 18: 187-221 (1989). For
the influenza virus, the hemagglutinin (trimer) HA peptide
N-terminal segment (a hydrophobic helical sequence) is exposed due
to a conformational change induced by acidic pH in the endosomes
(pH 5-6), inserts into the target membrane, and mediates the fusion
between the virus and the target endosomal membrane. Weber et al.,
J. Biol. Chem. 269: 18353-58 (1994). Recently, several amphipathic
helix-forming oligopeptides have been designed to imitate the
behavior of the viral fusion peptide. See, for example, Haensler
and Szoka, Bioconj. Chem. 4: 372-79 (1993).
[0058] Cationic liposome preparations can be made by conventional
methodologies. See, for example, Felgner et al., Proc. Nat'l Acad.
Sci USA 84:7413 (1987); Schreier, J. of Liposome Res. 2:145 (1992);
Chang et al. (1988), supra. Commercial preparations, such as
Lipofectin.RTM. (Life Technologies, Inc., Gaithersburg, Md. USA),
also are available. For some recent reviews on methods employed see
Wassef et al., Immunomethods 4: 217-222 (1994) and Weiner, A. L.,
Immunomethods 4: 217-222 (1994).
[0059] It is possible to control the therapeutic availability of
the encapsulated agent by varying liposome size, the number of
bilayers, lipid composition, as well as the charge and surface
characteristics of the liposomes. The liposomal preparation could
contain one or more adjuvants. Furthermore, a carrier protein such
as serum albumin can be added.
[0060] 4. Delivery of the Liposome Preparation
[0061] In general, the dosage of administered liposome preparation
will vary depending upon such factors as the patient's age, weight,
height, sex, general medical condition and previous medical
history. Dose ranges for particular formulations can be determined
by using a suitable animal model.
[0062] Liposomes may be administered to a subject intravenously,
intraperitoneally, intrathecally, intramuscularly or
subcutaneously. See, for example, Kim, supra, Bakker-Woudenberg et
al. (1993), supra, Allen et al. (1993), supra, and Fielding et al.,
Clin. Pharmacokinetics 21: 155 (1991).
[0063] For purposes of therapy, antibodies or fragments are
administered to a mammal in a therapeutically effective amount. An
antibody preparation is said to be administered in a
"therapeutically effective amount" if the amount administered is
physiologically significant. An agent is physiologically
significant if its presence results in a detectable change in the
physiology of a recipient mammal. In particular, an antibody
preparation of the present invention is physiologically significant
if its presence invokes a humoral and/or cellular immune response
in the recipient mammal.
[0064] 5. Cytokines
[0065] The vaccines of the present invention comprise cytokines.
Examples of cytokines include the interferons (INFS) such as
INF-gamma, interleukins (ILs), M-CSF, GM-CSF, and tumor necrosis
factor. INF-.gamma. induces macrophages, as well as cell-surface
class II histocompatibility antigens on lymphoid cells and
monocytes. See, for example, Klegerman et al., "Lymphokines and
Monokines," in BIOTECHNOLOGY AND PHARMACY, Pezzuto et al. (eds.),
pages 53-70 (Chapman & Hall 1993), and Roitt et al.,
IMMUNOLOGY, 3rd Edition, pages 7.8-7.14 (Mosby 1993). IL-2 is a T
cell growth factor and a stimulator of natural killer cells and
tumor-reactive T cells. Id. Thus, INF-.gamma. and IL-2 are
preferred cytokines for the augmentation of the immune
response.
[0066] 6. Examples
[0067] Oncovax Materials:
[0068] Mouse antigen 38cId
[0069] DMPC: Survival Tech Lot RD 1426
[0070] MSA 25%): Biocell Laboratories, CAlot#4002160
[0071] IL-2: Survival Tech. Lot #RD 1534 @ 9.38 mg/ml)
[0072] OTx Buffer
[0073] PEG
[0074] To 50-200 mg of DMPC or DMPC/DMPG ata ratio of 4/1, the
following is added such that the final volume is between 0.4-1.0
ml:
[0075] 0.3-10 mg of antigen, i.e. 38cId
[0076] 0.0-7.times.10.sup.6 IU of IL-2
[0077] 0.0-12 mg mouse serum albumin
Example 1 (Freeze-thaw Procedure for Preparation of the
Vaccine)
[0078] Mix aqueous components. Add to the powdered lipid in a 5 mL
vial glass vial. Warm for 10 minutes in a 35-39.degree. C. water
bath. Vortex for 30 seconds. Bath sonicate at 25-45.degree. C. for
15 minutes. Freeze the vials in a dry ice/ethanol bath at
-80.degree. C. for 15 minutes. Thaw in a 35-39.degree. C. water
bath for 10 minutes. Repeat the vortexing, sonication, freezing and
thawing steps a total of three (3) times. Add aqueous buffer to
dilute as necessary. The sample may be washed by centrifugation.
Centrifuge at 12,000 rpm for 20 minutes. Remove supernatant and
wash two more times.
Example 2 (Sonication-fusion Procedure for Preparation of the
Vaccine)
[0079] Hydrate the lipid in aqueous buffer at a concentration of
100-300 mg/mL. Sonicate in a bath sonicator at 30-45.degree. C.
until clear. Sterile filter through a 0.2 micron filter. Add
antigen, IL-2 and serum albumin. Cool sample 4-15.degree. C. This
may be temperature cycled any number of times from -80.degree. C.
to 15.degree. C. as the low temperature to 23.degree. C. to
50.degree. C. as the high temperature. The sample may be diluted as
necessary, and washed by centrifugation as in Example 1.
Example 3 (PEG-fusion Procedure)
[0080] Hydrate the lipid in aqueous buffer at a concentration of
100-300 mg/mL. Sonicate in a bath sonicator at 30-45.degree. C.
until clear. Sterile filter through a 0.2 micron filter. Add
antigen, IL-2 and mouse serum albumin. Mix with an equal volume of
PEG solution of MW 1,000 on up to 20,000. The PEG solution should
be between 6% to 60% w/v. Following an incubation at 4.degree. C.
to 25.degree. C. for one to 24 hours, washing by centrifugation
will remove the PEG and unincorporated active ingredients.
Example 4 (Extrusion Procedure)
[0081] The sample from Examples 1-3 may be size-reduced by
extrusion through a 1.0, 0.4 and 0.2 micron polycarbonate filter.
The final size should be between 100-200 nm.
[0082] The vaccine products under this disclosure are called
OncoVAX. Each OncoVAX preparation as well as the (KLH-Id) control
was analyzed by determining the amount of antigen (Id), IL-2 and
lipid as well as the size of OncoVAX particles.
[0083] Final concentrations (ranges) of the structural components
of OncoVAX were as follows:
1TABLE 1 Structural Example Control Component #1 #2 #3 #4 (KLH-Id)
Lipid 20-60 3-30 20-60 20-60 none (mg/mL) Antigen Id 1-200 1-2,000
1-200 500-1,000 mcg/mL IL-2 (IU/mL) .times. 1-200 1-200 1-200 none
10.sup.4 Mean Size 2-4 1-2 0.1-0.3 Soluble (micron) (no
particle)
[0084] The following are examples of the characterization of
oncovax and the mouse studies exhibiting the antitumor immunity,
effective liposomal dose with respect to antigen and IL-2 content,
humoral and cellular responses elicited by liposomal vaccines, and
the effect on in vivo T cell depletion.
[0085] The antigen concentration was determined by a sandwich ELISA
method where to unknown antigen bound to rabbit anti mouse IgM a
biotinylated rabbit anti mouse IgM was added. To this
streptavidin-Europium was added and the Europium fluorescence was
measured.
Example 5 (Characterization of a Typical Oncovax Preparation.)
[0086] Samples were rapidly frozen from room temperature between
copper planchets without cryoprotectant in liquid propane and
replicated in a Balzers freeze-fracture unit, and viewed on a
Philips 300 electron microscope. FIG. 5A reveals the multilamellar
liposomes formed. The mean size is roughly 3.0 microns, as
determined by single-particle optical sensing (SPOS). FIG. 5B
reveals a surface texture with numerous bulges and abrupt changes
in the ripple patterns. A smooth ripple pattern is characteristic
of DMPC liposomes at room temperature, as seen in FIG. 5C, the
control DMPC liposomes with mouse serum albumin. Bar=0.4
microns.
Example 6 (Immunity Test)
[0087] To determine whether liposomal formulation of Id could
produce the acquisition of protective antitumor immunity, ten
syngenic C3H/HEN mice per group were immunized i.p. with liposomal
Id or control Id preparations, or with 50 ug 38C13-derived Id in
PBS in a volume of 0.2 ml. Two weeks later the mice were challenged
with a lethal dose of 2.times.10.sup.3 38C13 cells. Statistical
comparisons of survival were made on the basis of nonparametric
mantel-log rank p values. Mice surviving greater than 90 days after
tumor challenge were euthanized and reported as long term
survivors. Immunization with liposomal Id demonstrated
significantly prolonged survival as well as protection (30%).
Example 7 (Optimization of Liposomal Vaccine Potency and Comparison
of Potency with KLH Conjugated Id Vaccine.)
[0088] Serial dilutions of input Id antigen were made to prepare
for liposomal vaccines which were otherwise identical. The actual
amounts of incorporated Id were determined for each vaccine after
preparation as outlined previously. Id-KLH was prepared by
gluteraldehyde conjugation at a 1:1 ratio of Id and KLH and the
dose of Id administered per animal as indicated in parenthesis. A
clear dose dependent effect on the induction of protective
antitumor immunity was observed, with mice receiving liposomal
vaccine formulations delivering 40, 10, and 2 ug Id per mouse
demonstrating significantly superior survival compared with
controls immunized with free Id. Mice immunized with liposomal
vaccines delivering 0.4 ug Id per mouse were not protected from
subsequent tumor challenge.
Example 8
[0089] The potency of a representative liposme vaccine containing
low amount of Id, compared with serial dilutions of ID-KLH in PBS
was examined. Previous studies have determined 50 ug Id in the
conjugate formulation to be the optimal dose. Ten mice per group
immunized with Id-KLH containing 50, 10, or 2 ug Id per mouse
demonstrated 40, 30, and 0 percent protection, respectively,
compared with nine mice immunized with a liposomal vaccine
containing 2 ug ID, which demonstrated 33% protection from
subsequent lethal dose tumor challenge (log rank p=0.007 compared
with Id-KLH 2 ug Id dose).
Example 9
[0090] The requirement for IL-2 as a component of the liposomal Id
vaccine formulation was investigated by preparing several
formulations with serial dilutions of input IL-2, holding the other
components constant. Mice immunized with the resulting liposomal
formulations, all containing a dose of 40 ug Id, were used to
immunize mice. Two weeks later all mice were challenged with
2.times.10.sup.3 38C13 cells from a single preparation of tumor and
followed for survival. The log rank p values refer to comparisons
against three Id groups. The survival patterns of these mice
following lethal dose tumor challenge shows a clear IL-2
dose-dependence on the induction of protective antitumor immunity.
Other experiments demonstrating the failure of liposomal Id
vaccines not containing IL-2 to induce any significant antitumor
immunity support the conclusion that IL-2 is a critical component
of the vaccine formulation, although liposomal vaccines containing
1/10 the amount of input IL-2 were capable on inducing significant
protective antitumor immunity (log rank p 0.004 vs. free Id).
Example 10
[0091] In an effort to investigate the cellular mechanism by which
liposomal Id vaccines promote the acquisition of protective
antitumor immunity, we first determined serum anti-idiotypic
antibody levels elicited by the various liposomal vaccine
formulations containing different doses of Id in the mice.
Individual serum samples were assayed for binding to Id-coated
microtitered plates in a direct ELISA. The specificity of the
antibody response for idiotype was demonstrated by the lack of
binding to control IgM proteins. Serum samples were collected from
five individual mice per group two weeks after immunization, just
prior to tumor challenge, and the mean anti-idiotypic antibody
levels are shown. A clear dose dependent effect of the
liposome-entrapped Id was apparent, with mean anti-idiotypic
antibody levels of 15, 7, 1, and 0.1 ug/ml detectable by ELISA.
This demonstration of humoral response specific for idiotype in the
three liposomal vaccine groups containing 2, 10, and 40 ug Id stood
out in stark contrast to free Id, which failed to induce any
detectable anti-idiotlypic antibody even in a single immunized
mouse. However, the mean levels of anti-idiotypic antibody elicited
by liposomal Id vaccines was considerably less than that elicited
by ID-KLH (55 ug/ml serum).
Example 11
[0092] Because the magnitude of the anti-idiotyic antibody response
did not correlate entirely with the relative levels of protection
induced by liposomal Id vaccines and the Id-KLH, we also examined
evidence for idiotype-specific T cell activation. Splenocytes
obtained from 2-3 mice per group which had been immunized i.p. as
indicated two weeks earlier were pooled and enriched by T cells
passed over nylon wool and placed in 96 well bottomed microliter
plates with Id at various concentrations (200 ul, 2.times.10.sup.5
cells/well). Irradiated (2000 rads) spleen cells from normal
syngenic mice were also added to splenocyte cultures
(2.times.10.sup.5) as a source of antigen presenting cells.
Cultures were maintained at 37.degree. C., 5% CO.sub.2 for 5-7
days, and 18-24 hours before harvesting 1 uCi [.sup.3H-thymidine]
(2 Ci/mmol, New England Nuclear Research Products, Boston, Mass.)
in 50 ul medium to each well. Incorporated radioactivity was
measured in an LKB 1205 beta plate liquid scintillation counter.
All determinations were performed in quadruplicate and the data are
presented as the mean CPM plus standard error of the mean. Splenic
T cells obtained from mice given a single immunization with a
liposomal Id vaccine, empty liposomes, free Id, or Id-KLH two weeks
earlier were assayed in vitro for proliferative responses to
various doses of Id. The representative experiment demonstrates a
significant T cell proliferative response to Id but not among
groups primed with empty liposomes or free Id. These results are
also particularly revealing, because such evidence of T cell
activation has never been observed after immunization with Id-KLH
and was not observed with an Id-cytokine fusion protein.
Example 12
[0093] To definitively establish the role of idiotype-specific T
cells in the effector phase of induced protective antitumor
immunity, we tested the effect of T cell subset depletion in vivo
in immunized mice. Mice were immunized with a single liposomal Id
vaccine preparation two weeks after immunization, eight mice per
group were randomly assigned to receive treatment with depleting
mAb specific for either CD4+ ((GK1.5, ammonium sulfate purified
assightese from the BRMP pre-clinical repository, Frederick, Md.),
CD8+ T cells (53.6-72, ammonium sulfate purified assightese from
the BRMP pre-clinical repository, Frederick, Md.); a combination of
the two antibodies, or with normal rat IgG (Sigma, St.Louis, Mo.)
every other day for three doses, just prior to challenge with a
single preparation of tumor. Three weeks after immunization, all
mice were challenged i.p. with 2.times.10.sup.3 38c13 cells from a
single preparation of tumor and were followed for survival.
Depletion of lymphocyte subsets was assessed one and two weeks
after final treatment by flow cytometric analysis of spleen cells
from normal mice treated with monoclonal antibodies in parallel.
For both timepoints of analysis, greater than 95% depletion of the
appropriate subset was achieved with normal levels of the other
subsets. As shown, depletion of either CD4+ or CD8+T cells among
immunized mice was associated with marked reduction of protective
antitumor immunity (log rank p=0.012 for either group vs. liposomal
id immunized, normal rat Ig-treated mice). Although mAb treated
groups were not significantly different compared with control mice
immunized with free Id (log rank p=0.09 and 0.16, respectively, vs.
free Id). Combined treatment with anti-CD4 and anti-CD8 mAbs did
not result in further abrogation of protection (log rank p=0.10 vs.
free Id). Thus, it appears clear that there is an absolute
requirement for both CD4+ and CD8+ effector T cells in liposomal Id
vaccine induced protective antitumor immunity.
Example 13
[0094] As an initial step towards testing liposomal Id immunization
against previously established tumors we performed experiments in
which tumor challenge was performed first, followed by vaccination
later the swne day. For these studies we modified an existing
protocol vaccination against subcutaneous 38C13 tumors which
required a non-curative dose of cyclophophamide (CTX) chemotherapy
on day ten to retard the growth of this virulent tumor. Mice were
injected with 10.sup.4 tumor cells subcutaneously in the flank and
then randomly assigned to immunization with liposomal Id, liposomal
control Id vaccines, or PBS i.p. later the same day. The
subcutaneous route of tumor inoculation was used because of the
availability of tumor size monitoring as a surrogate endpoint for
survival, and by day ten all mice lo developed macroscopic,
palpable tumor masses of approximately 1 cm diameter. CTX
administration (75 mg/kg i.p.) was associated with complete
disappearance of tumors which was uniformly transient in all
control mice but durable in a modest but significant proportion of
mice immunized with liposomal Id vaccines (log rank p=0.01 for
pooled liposomal Id vs. control groups).
2TABLE 2 Therapeutic effect of Lip Id vaccines against a large s.c.
tumor inoculum. No. Survivors/ Exp. Immunogen Total no. mice 1 Lipo
Id (10 .mu.g) 2/10 Lipo control Id (10 .mu.g) 0/5 PBS 0/5 2 Lipo Id
(20 .mu.g) 3/10 PBS 0/10
[0095] C3H mice were injected with 10.sup.4 38C13 tumor cells s.c.
and then immunized i.p. as indicated later the same day (day 0).
All groups received CTX 75 mg/kg i.p. on day 10. Mice surviving
>60 days without tumor relapse were apparently cured. Although
the foregoing refers to particular embodiments, it will be
understood that the present invention is not so limited. It will
occur to those of ordinary skill in the art that various
modifications may be made to the disclosed embodiments and that
such modifications are intended to be within the scope of the
present invention.
* * * * *