U.S. patent application number 09/816266 was filed with the patent office on 2001-08-09 for patient-specific white blood cell malignancy vaccine from membrane-proteoliposomes.
Invention is credited to Batenjany, Michael M., Boni, Lawrence, Popescu, Mircea C., Robb, Richard J..
Application Number | 20010012517 09/816266 |
Document ID | / |
Family ID | 22103016 |
Filed Date | 2001-08-09 |
United States Patent
Application |
20010012517 |
Kind Code |
A1 |
Popescu, Mircea C. ; et
al. |
August 9, 2001 |
Patient-specific white blood cell malignancy vaccine from
membrane-proteoliposomes
Abstract
Membrane-proteoliposome structures (MPs) are useful in preparing
patient-specific vaccines against specific white blood cell (WBC)
malignancies. The inventive MPs typically contain a membrane
component derived from a specific WBC. Other useful components
include immunostimulators and exogenous lipids. The resulting
vaccines are both patient- and malignancy-specific.
Inventors: |
Popescu, Mircea C.;
(Plainsboro, NJ) ; Boni, Lawrence; (Monmouth
Junction, NJ) ; Robb, Richard J.; (Princeton
Junction, NJ) ; Batenjany, Michael M.; (Hamilton,
NJ) |
Correspondence
Address: |
Bernhard D. Saxe
FOLEY & LARDNER
Washington Harbour
3000 K Street, N.W., Suite 500
Washington
DC
20007-5109
US
|
Family ID: |
22103016 |
Appl. No.: |
09/816266 |
Filed: |
March 26, 2001 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
09816266 |
Mar 26, 2001 |
|
|
|
09231650 |
Jan 15, 1999 |
|
|
|
6207170 |
|
|
|
|
60071702 |
Jan 16, 1998 |
|
|
|
Current U.S.
Class: |
424/277.1 ;
424/450 |
Current CPC
Class: |
A61K 2039/55572
20130101; A61K 39/39 20130101; A61P 35/02 20180101; A61K 39/0011
20130101; A61K 2039/55527 20130101; A61K 2039/55533 20130101; A61P
35/00 20180101; A61K 2039/55522 20130101; A61K 2039/5555
20130101 |
Class at
Publication: |
424/277.1 ;
424/450 |
International
Class: |
A61K 039/00; A61K
009/127 |
Claims
What is claimed is:
1. A patient-specific vaccine for treating white blood cell
malignancy, comprising a membrane-proteoliposome (MP) containing
plasma membrane from a malignant white blood cell.
2. A vaccine according to claim 1, wherein said malignant white
blood cell is a lymphoma cell.
3. A vaccine according to claim 1, wherein said malignant white
blood cell is a leukemia cell.
4. A vaccine according to claim 1, wherein said malignant white
blood cell is a myeloma cell.
5. A membrane-proteoliposome (MP), comprising integral membrane
from a malignant white blood cell, at least one immunostimulator
and an exogenous lipid.
6. An MP according to claim 5, wherein said membrane contains at
least one membrane component involved in immunity.
7. An MP according to claim 5, comprising at least two
immunostimulators.
8. An MP according to claim 6, wherein said component is selected
from the group consisting of a tumor-specific antigen, a major
histocompatability complex antigen and a costimulatory
molecule.
9. An MP according to claim 8, wherein said costimulatory molecule
is B7. 1 or B7.2.
10. An MP according to claim 5, wherein said immunostimulator is a
lymphokine.
11. An MP according to claim 10, wherein said lymphokine is
IL-2.
12. An MP according to claim 5, wherein said immunostimulator is an
interferon.
13. An MP according to claim 12, wherein said interferon is
IFN-.gamma..
14. An MP according to claim 5, wherein said immunostimulator is a
cytokine.
15. An MP according to claim 14, wherein said cytokine is GM-CSF or
M-CSF.
16. An MP according to claim 5, wherein said immunostimulator is an
adjuvant.
17. An MP according to claim 16, wherein said adjuvant is selected
from the group consisting of monophosphoryl lipid A, lipid A and
muramyl dipeptide (MDP) lipid conjugate.
18. An MP according to claim 5, wherein said lipid is a saturated
or unsaturated phospholipid or a glycolipid.
19. A MP according to claim 18, wherein said lipid is selected from
the group consisting of 1,2-dimyristoylphosphatidylcholine,
1,2-dipalmitoylphosphatidylcholine,
1,2-dimyristoylphosphatidylglycerol, cholesterol and combinations
thereof.
20. An MP according to claim 5, wherein said lipid forms a membrane
within which said integral membrane is patched.
21. An MP according to claim 5, wherein said lipid forms patches
within said integral membrane.
Description
BACKGROUND OF THE INVENTION
[0001] The present invention is directed to the production of novel
compositions, useful as vaccines for treating white blood cell
(WBC) malignancies. The invention relates to a liposomal,
patient-specific vaccine comprised of WBC membranes that may be
formulated by adding other lipids and/or immunostimulators, thereby
forming a novel membrane-proteoliposome (MP) structure.
[0002] Known vaccines typically utilize either purified antigen or
attenuated pathogen as the immunogen. However, attenuated vaccines
can actually cause the infection against which a person is being
immunized. On the other hand, purified antigens may not induce a
long-term immune response and sometimes induce no response at all.
In contrast to the short-term immune response obtained by direct
immunization with certain antigens, presentation of the antigen in
the presence of liposomes can induce a long-term response which is
essential for any effective vaccine.
[0003] Although typically formed from purified or partially
purified lipids, liposomes may also be formed, at least in part,
from cell membranes of malignant cells which contain potential
antigens. Due to the presence of membrane associated antigens,
these membrane-derived preparations may be used as
malignancy-specific vaccines. Indeed, some types of
membrane-derived preparations have been used as tumor specific
antigens (TSA) to treat melanomas and murine SL2 lymphosarcoma. See
Gershman et al., Vaccine Res. 3:83-92 (1994); Bergers et al., J.
Confr. Rel. 29:317-27 (1994); Bergers et al., J. Liposome Res.
6:339-35 (1996). In these cases, the production of vaccine suffered
from serious disadvantages. Namely, they required pooling culture
adapted cells to achieve large amounts of the desired cell
populations, use of whole .gamma.-irradiated tumor cells, detergent
solubilization or butanol for crude extraction of tumor-associated
antigens (TAA). See Gershman et al. (1994), supra; Abbas, et al.,
CELLULAR AND MOLECULAR IMMUNOLOGY, pp.372-73 (W. B. Saunders
Company, Philadelphia 1994); Bergers et al. (1994), supra; LeGrue
et al., J. Natl. Cancer Inst. 65:191-96 (1980). This approach,
moreover, is not patient-specific.
[0004] The art is also aware of some vaccines directed to certain B
cell malignancies. Typically, however, attempts at producing
vaccines for B-cell lymphoma have relied on the costly and time
consuming hybridoma technology. These methods depend on generating
a hybridoma able to produce the tumor-specific immunoglobulin (Ig)
in enough quantity to be then used as a vaccine. Kwak et al., Blood
76:2411-17 (1990); Kwak et al., N. Engl. J. Med. 327:1209-15
(1992). Known B-cell lymphoma vaccines employ Ig idiotype (Id) to
generate anti-idiotype antibodies to B-cells. Levy et al.,
PCT/US94/08601 (Feb. 23, 1995); Levy et al., U.S. Pat. No.
4,816,249 (1989). Similarly, known melanoma vaccines involved
harvesting cell surface antigens which are shed during culturing.
Bystryn, U.S. Pat. No. 5,635,188 (1997); Bystryn, U.S. Pat. No.
5,194,384 (1993); Bystryn, U.S. Pat. No. 5,030,621 (1991).
[0005] There is, therefore, an unmet need in the art for improved
liposome-based vaccines. A particular need exists for improved
vaccines against white blood cell malignancies.
SUMMARY OF THE INVENTION
[0006] Accordingly, it is an object of the invention to provide
novel vaccine compositions that overcome the above-identified and
other deficiencies in the art. According to this object of the
invention, membrane-proteoliposomes (MPs) are provided which are
malignancy-specific, patient-specific and are easily prepared.
Thus, in one embodiment, MPs are provided which comprise the cell
membrane of a white blood cell malignancy, at least one
immunostimulator and at least one lipid, where the lipid may be
added in the form of lipid powder, or preformed liposomes. Another
embodiment of the invention provides novel vaccine formulations
which comprise an MP comprising the cell membrane of a white blood
cell and may include at least one immunostimulator.
[0007] It is yet another object of the invention to provide methods
for preparing MPs and vaccines which also overcome the deficiencies
in the art. According to this object of the invention, methods are
disclosed that do not rely on harvesting the vaccine antigen,
hybridoma production or other intermediate steps. The inventive
methods comprise formulating a vaccine directly from isolated
antigen-containing membranes from patients' own white blood cells
(WBCs), thus rendering them highly effective and
patient-specific.
BRIEF DESCRIPTION OF THE DRAWING
[0008] FIG. 1 exhibits the survival results after mice were
vaccinated with the MP formulation described in Example 5.
[0009] FIG. 2 demonstrates the patching seen in
membrane-proteoliposomes, where the patches consist of DMPC-rich
and WBC membrane-rich domains (see arrows).
DETAILED DESCRIPTION
[0010] Introduction
[0011] The instant invention provides membrane-proteoliposome
structures (MPs) that are useful in formulating patient-specific
vaccines for treating white blood cell (WBC) malignancies.
[0012] The inventors previously described a proteoliposomal vaccine
made of antigen idiotype (Id) and interleukin-2 (IL-2) proteins
within a liposomal structure (Popescu, et al., PCT/US97/02351). In
contrast to that earlier work, a novel liposomal structure is
disclosed herein, called a membrane-proteoliposome (MP), which
usually comprises phospholipid, integral membrane from a malignant
WBC and a potent immunostimulator. The present invention is based
in part on the discovery that membranes from WBC malignancies can
be fused with other components to form an effective vaccine against
the malignancy.
[0013] All WBCs, including polymorphonuclear cells (PMN), monocytes
and T- or B-lymphocytes, are subject to malignant transformation
leading to a spectrum of diseases. For example, B-cell malignancy
includes non-Hodgkin's lymphomas, chronic lymphocytic leukemia and
multiple myeloma. The present invention has relevance in treating
or preventing many such malignancies.
[0014] In preparing the inventive vaccines, WBC's can be obtained
directly from the patient to isolate the intact membranes, rich in
tumor-specific antigens (TSA) and tumor-associated antigens (TAA).
In general cells will be enucleated and their plasma membranes
separated from other components (e.g., mitochondria, lysosomes).
The plasma membranes typically are washed to remove cellular
contaminates, which may include cytoskeletal structures, and the
separation material. The plasma membrane suspensions may then be
exposed to mechanical size reduction, for example, by extrusion,
homogenization or other shearing methods. This will allow for
filtration through a sterilizing filter. The WBC membranes may also
be detergent solubilized, reconstituted with lipids of choice then
size reduced. In lieu of mechanical size reduction methods, the
isolated membranes may be sterilized by, for example,
.gamma.-irradiation.
[0015] The isolated malignant cell membranes, alone or in
combination with added lipids, can then be used to entrap an
immunomodulator. The extent of entrapment of immunomodulator,
immunogenicity and efficacy of the MP as a vaccine can be modulated
by the nature of the constitutive lipids. A thus optimized MP
formulation may then be used to vaccinate the patient against
his/her specific WBC malignancy.
[0016] The present invention is particularly useful in vaccinating
against non-Hodgkin's lymphomas. These lymphomas are characterized
by the expression of monotypic immunoglobulin (Ig) which can serve
as a tumor-specific antigen. In addition, these cells express
surface molecules involved in antigen presentation, such class I
and class II MHC molecules, and costimulation, such as adhesion
proteins and B7.1 and B7.2 (CD80 and CD86). In particular, the
presence of a class I MHC molecule in the inventive formulation
will potentially enhance the cytotoxic immune response against the
tumor. These characteristics make the B-cell lymphoma plasma
membrane an attractive candidate that can be used as a potentially
strong immunogenic tool in active specific immunotherapy.
[0017] The present invention provides a proteoliposomal,
patient-specific vaccine for WBC malignancies that, in one
embodiment, is produced by entrapping a potent immunomodulator
together with malignant white blood cell membranes. The resulting
membrane-proteoliposome can be either (1) a cell-derived membrane
patched with at least one added membrane-forming lipid or (2) a
lipid membrane (e.g., a liposome) patched with cell-derived
membrane. By "patched" is meant that the resulting MP is
non-homogenous with respect to the component lipid sources. Thus,
contiguous portions of the MP will be essentially WBC
membrane-derived, while others will be derived essentially from the
added membrane-forming lipids. In three examples below, MP
formulations are described which contain membrane from a mouse
B-cell lymphoma (38C13), and which were used as effective vaccines
in a mouse model of non-Hodgkin's B-cell lymphoma.
[0018] Vaccine Compositions of the Invention
[0019] The vaccine compositions of the invention typically comprise
at least one membrane component of a malignant white blood cell.
Important membrane components specifically include components
involved in immunity. Components involved in immunity can include
any macromolecules, such as proteins, lipids and carbohydrates,
which are normally an integral part of, or simply associated with,
the cell membrane. Other organic and inorganic substances which are
similarly associated with the cell membrane also are included. Some
preferred components involved in immunity include tumor-specific
antigens (TSA), tumor associated antigens (TAA), major
histocompatability (MHC) antigens (class I and class II molecules)
and costimulatory molecules.
[0020] Costimulatory molecules are second signal immunostimulators
associated with T cell activation. Costimulatory molecules
typically are cell surface molecules which act in conjunction with
primary immune signals, i.e., antigen presented by MHC molecules,
to generate an immune response. Thus, acting in concert, primary
and secondary signal molecules facilitate antigen presentation by
antigen presenting cells (APC) to T cells. Examples of
costimulatory molecules include cellular adhesion molecules and
CD-40. Specific preferred costimulatory molecules include B7.1 and
B7.2.
[0021] Preferably, the membrane component takes the form of an
isolated plasma membrane (in whole or in part). The isolated plasma
membrane preferably is constituted of lipid which is
membrane-forming. Thus, all components normally integral to or
associated with the cell plasma membrane, including components
involved in immunity, typically are present. This preferred
membrane component will usually be isolated from a patient sought
to be vaccinated. Thus, the resultant vaccine comprising this
membrane component will be patient-specific and specific for the
WBC malignancy from which the membrane component is isolated. It is
envisioned that a vaccine formulated with a membrane component from
one patient will be useful in vaccinating another patient, given
similar antigenic determinants. Of course, it is also possible, due
to cross-reactivity or common antigenic determinants, that the
vaccine for one malignancy will prove useful in vaccinating against
another malignancy. Thus, as used herein, "patient-specific" refers
to the fact that the vaccine is derived from a particular patient
(it thus will be useful in treating that same patient), not that it
is useful only to treat the patient or the specific malignancy from
which it is derived. Although the patient will normally be human,
non-human animals may also be patients.
[0022] The inventive vaccine compositions can be made specific for
any white blood cell malignancy. The clinician will be familiar
with the various types of white blood cells and their malignancies.
Representative white blood cells include polymorphonuclear cells
(PMNs), monocytes, T-lymphocytes and B-lymphocytes. Some
representative white blood cell malignancies include lymphomas,
leukemias, and myelomas. Other white blood cell malignancies are
known in the art. Further examples of WBC malignancies are found in
McCance et al., PATHOPHYSIOLOGY: THE BIOLOGIC BASIS OF DISEASE IN
ADULTS AND CHILDREN, chapters 24 and 25, pp. 800-855 (The C.V.
Mosby Company 1990), which are hereby incorporated by
reference.
[0023] Some preferred vaccine compositions further comprise at
least one immunostimulator. Immunostimulators specifically include
any substance that can be used to modulate the immune response.
Especially useful immunostimulators are those which can be used to
stimulate the specific immune response to components involved in
immunity. Exemplary classes of such useful immunostimulators
include: lymphokines, such as IL-2, IL-4 and IL-6; interferons,
such as IFN-.gamma. and IFN-.alpha.; other cytokines, such as
GM-CSF and M-CSF; and adjuvants, such as Lipid A, monophosphoryl
lipid A (MPL), lipid A, or muramyl dipeptide (MDP).
Immunostimulators may be used alone or in any combination with one
another. Some compositions comprise at least two immunostimulators,
such as IL-2 and MPL or MDP, and other combinations of cytokines
with adjuvants.
[0024] Other preferred vaccine compositions comprise lipids other
than those present in the cell membrane component (i.e., from an
exogenous source). These "exogenous" lipids may be from natural or
synthetic sources. Preferred lipids include phospholipids,
glycolipids, and especially saturated phospholipids. Saturated
phospholipids include 1,2-dimyristoylphosphatidylcholine (DMPC),
1,2-dipalmitoylphosphatidylcho- line (DPPC),
1,2-dimyristoylphosphatidylglycerol (DMPG). Other useful lipids
include cholesterol and derivatives thereof. Of course combinations
of these and other lipids are also useful.
[0025] Methods for Preparing the Inventive Vaccines
[0026] Preparing an inventive vaccine involves first isolating WBC
membrane components, free of other cellular components. One such
example is provided below as Example 4. According to a preferred
embodiment, isolated membranes typically are combined with other
lipids and/or immunostimulators to form MPs. There are many
liposome-forming methods known in the art and any of these standard
methods may be employed in preparing the present MP.
[0027] In one exemplary method MPs can be prepared containing IL-2
as an immunostimulator. The IL-2 is mixed with the WBC membranes
and entrapped by freeze/thawing from -70.degree. C. to 37.degree.
C., followed by brief vortexing and short bath sonication (30
seconds). The preparation can include the addition of a suitable
lipid powder at 50 to 300 mg/mL (final). Thus, using this method
MPs can be formed independent of exogenous lipids or from WBC
membrane components mixed with exogenous lipids.
[0028] MPs can also be prepared containing only WBC membrane
components, which may be fused with preexisting liposomes which
comprise exogenous lipids. A WBC membrane suspension is lyophilized
then hydrated with a suitable liquid, for example, water, normal
saline solution (NSS) or a suitable buffer. The hydration liquid
may contain an immunomodulator. Thus, MPs comprising WBC membrane
components are formed. In addition, pre-formed multilamellar
vesicles (MLVs), composed of suitable exogenous liposome-competent
lipids, may be mixed with the WBC membrane suspension prior to
lyophilization. The MLVs may contain a pre-incorporated
immunomodulator, in which case the lyophilized preparation is
hydrated with a suitable liquid, which may contain at least one
immunomodulator. Thus, in any method for preparing MPs, any
combination of multiple immunostimulators may be incorporated at
any suitable point in the process.
[0029] In another method, the WBC membrane suspension is size
reduced by extrusion, homogenization or other shearing methods to
form small unilamellar vesicles (SUVs). The SUVs are then
lyophilized and hydrated with a suitable liquid, optionally
containing an immunomodulator. SUVs prepared from exogenous lipids
also may be added prior to the lyophilization step. In addition,
the immunomodulator may be mixed with the SUV's prior to
lyophilization. In any event, whenever the lyophilized preparation
is hydrated, the liquid may contain an immunomodulator.
[0030] In yet another method, a WBC membrane suspension is added to
MLVs comprising exogenous lipids. The resulting mixture is
lyophilized and hydrated with water, NSS or buffer followed by size
reduction (extrusion, homogenization or other shearing methods) to
form SUV's. An immunomodulator may be added and the mixture is
allowed to fuse overnight.
[0031] Another method involves adding a WBC membrane suspension to
MLVs comprising exogenous lipids. The mixture is lyophilized and
hydrated with a suitable liquid. The resulting suspension is size
reduced by extrusion, homogenization or other shearing methods to
form SUVs. The SUVs are lyophilized and hydrated with a suitable
liquid, optionally containing an immunomodulator.
[0032] Also, the WBC membrane suspension may be size reduced by
extrusion, homogenization or other shearing methods to form SUVs.
An immunomodulator is added to the SUVs and the mixture is then
allowed to fuse overnight.
[0033] Vaccines may also be formulated with a pharmaceutically
acceptable excipient. Such excipients are well known in the art,
but typically should be physiologically tolerable and inert or
enhancing with respect to the vaccine properties of the inventive
compositions. When using an excipient, it may be added at any point
in formulating the vaccine or it may be admixed with the completed
vaccine composition.
[0034] Vaccines may be formulated for multiple routes of
administration. Specifically preferred routes include
intramuscular, percutaneous, subcutaneous, or intradermal
injection, aerosol, oral or by a combination of these routes, at
one time, or in a plurality of unit dosages. Administration of
vaccines is well known and ultimately will depend upon the
particular formulation and the judgement of the attending
physician.
[0035] Vaccine formulations can be maintained as a suspension, or
they may be lyophilized and hydrated later to generate a useable
vaccine.
EXAMPLES
Example 1
[0036] Isolation of Plasma Cell Membranes
[0037] This example demonstrates a method for isolating WBC plasma
membranes. Frozen (-70.degree. C.) 38C13 murine B-lymphoma cells
were quick-thawed at 37.degree. C., suspended at 2.times. 10.sup.8
cells/mL in homogenization buffer (HB). The composition of HB was
0.25 M sucrose, 10 mM Tris/HCl, 1 mM MgCl.sub.2, 1 mM KCl,
phenylmethylsulfonyl fluoride (PMSF, 2 mM, final),
trypsin-chymotrypsin inhibitor (200 .mu.g/mL, final), DNase (10
.mu.g/mL, final) and RNase (10 .mu.g/mL, final), pH 7.3. The 38C13
cells were enucleated in a hand-held Dounce homogenizer (20-30
passes while on ice). The slurry was spun at 500.times.g (10
minutes, 4.degree. C.) and the supernatant collected. The pellet
was resuspended in HB and the homogenization and centrifugation
steps repeated until the pellet, as judged by light microscopy, was
essentially free of intact cells (.about.95% nuclei only). The
pooled supernatants were layered on a discontinuous sucrose
gradient (p=1.11, 1.18 and 1.25 g/mL) and spun at 28K.times.g (30
minutes, 4.degree. C.). The plasma membrane-rich region was
collected (p=1.11/1.18 interface), diluted two-fold with NSS and
spun 28K.times.g (1 hour, 4.degree. C.). The pellet was again
diluted two-fold with the NSS and respun as above. The washed
membranes were resuspended in a minimal amount of NSS and stored at
4.degree. C.
Example 2
[0038] Preparation of Membrane Proteoliposomes
[0039] This example demonstrates a method of formulating a vaccine
from isolated WBC membranes. Experimental vaccine MB-RM-1A was
formulated as follows: DMPC powder (1 g), 4 mL of the isolated
38C13 membranes (225 .mu.g/mL IgM) in NSS and 160 ul of IL-2
(1.25.times.10.sup.8 IU/mL) were placed in a 5 mL sterile glass
vial, immediately vortexed, heated to 37.degree. C. for 15 minutes
in a water bath, then sonicated at 37.degree. C. for 30 seconds in
a bath sonicator. This suspension was subjected to three
freeze/thaw cycles as follows:
[0040] 1) Freezing at -70.degree. C. (dry ice/methanol bath) for 15
minutes
[0041] 2) Thawing at 37.degree. C. (water bath) for 15 minutes
[0042] 3) Vortexing briefly
[0043] 4) Sonicating for 30 seconds in a bath sonicator at
37.degree. C.
[0044] The preparation was adjusted to a total volume of 5 mL with
NSS and stored at -70.degree. C.
Example 3
[0045] Comparative Vaccine Effectiveness
[0046] This example demonstrates the effectiveness of exemplary
vaccines produced according to the invention, and particularly the
freeze/thaw method of Example 2. The results presented below, and
depicted in FIG. 1, show that the vaccinated mice survived a lethal
B-cell lymphoma challenge.
[0047] As seen in the following Table, the exemplary freeze-thaw MP
vaccine, MB-RM-1A, effectively protected 85% of the mice challenged
with a lethal dose of 38C13 lymphoma cells. By comparison, as
summarized below, the survival associated with control vaccine
formulations was lower.
1 TABLE Vaccine Description Percent Survival MB-RM-1A MP vaccine 85
4C5-Id Non-specific antigen 0 38C13-Id Control vaccine 10
38C13-Id-KLH Control vaccine 40 OV XIV-2 Liposomal vaccine 50
[0048] The foregoing sample designations correspond to the
following: 4C5-Id is a non-specific antigen control; 38C13-Id is a
vaccine consisting of soluble 38C13 immunoglobulin; 38C13-Id-KLH is
a vaccine consisting of a conjugate between keyhole limpet
hemocyanin and soluble 38C13; OV XIV-2 is a liposomal vaccine
containing soluble 38C13-Id and IL-2. See Kwak et al. J. Immunol.
160: 3637-3641 (1998); Popescu et al. PCT/US97/02351.
Example 4
[0049] Comparative Vaccine Effectiveness
[0050] Preparation. A frozen (-70.degree. C.) pellet of 38C13 cells
was quickly thawed at 37.degree. C. in a water bath, then placed on
ice. All subsequent treatments, unless otherwise specified were
carried out on ice at 4.degree. C. The pellet was washed with (5)
volumes of ice cold PBS at 1K.times.g.times.10 minutes. The pellet
was resuspended in ice-cold NSS containing 2 mM
phenylmethylsulfonyl fluoride (PMSF), and 100 .mu.g/mL each of
DNAse and RNAse. The cells were enucleated by emulsification
through a 22 gauge needle (50 passes) and by using a hand held
Dounce homogenizer (50 passes). The suspension was spun to pellet
the nuclei at 330.times.g.times.10 minutes and the supernatant was
layered on a Nycodenz gradient (p=1.22) and spun at
60K.times.g.times.1 hour. WBC plasma membranes were collected at
the water/Nycodenz interface and washed with (7) volumes of NSS at
60K.times.g.times.30 minutes. The pelleted membranes were
resuspended in NSS. A portion of these membranes was used to
formulate MCFC9803 using the freeze/thaw method as described in
Example 2. The rest of the washed cell membranes were homogenized
at .about.22K psi (15-20 passes) and used to formulate preparations
MCFA9803 and MCFB9803. The following describes the experimental
design of formulations MCFA9803, MCFB9803 and MCFC9803:
[0051] MCFA9803:
[0052] a) Homogenized WBC membranes were added to DMPC SUVs and
IL-2.
[0053] b) The mixture was allowed to coalesce into MLVs by
overnight incubation at 19.degree. C. (Boni et al., PCT Application
based on U.S. Ser. No. 60/060,606 "Multilamellar Coalescence
Vesicles (MLCV) Containing Biologically Active Compounds").
[0054] MCFB9803:
[0055] a) Homogenized WBC membranes were added to DMPC SUVs and
lyophilized.
[0056] b) MLVs were formed upon reconstitution with buffer
containing IL-2.
[0057] MCFC9803:
[0058] a) DMPC (powder) and IL-2 were added to a suspension of WBC
cell membranes.
[0059] b) MLVs were formed by the freeze-thaw method detailed in
Example 2.
[0060] Survival data. FIG. 1 shows the effectiveness of MP vaccines
produced according to the invention, and particularly shows
survival data after formulation using three different methods for
preparation. The results show that the vaccinated mice survived a
lethal B-cell lymphoma challenge. The exemplary MP vaccines,
MCFA9803 and MCFC9803, effectively protected 78% ({fraction (7/9)})
and 90% ({fraction (9/10)}), respectively, of the mice challenged
with a lethal dose of 38C13 lymphoma cells. Addition of IL-2 after
lyophilization and during reconstitution was less effective
(MCFB9803, 20% survival). However, all the formulations offer some
protection in this model of lymphoma.
Example 5
[0061] Proteoliposome Characterization
[0062] This example demonstrates one alternate method of
formulating a vaccine from isolated WBC membranes. SUVs (0.5 mL at
200 mg/mL) prepared from DMPC, WBC membranes (0.5 mL) and IL-2 (19
.mu.l at 1.07.times.10.sup.8 IU/mL) are combined and the mixture is
lyophilized. Upon reconstitution with 0.5 mL distilled water 59% of
the IL-2 is recovered of which 100% is incorporated in the
membrane-proteoliposomes and 84% of the IgM is recovered of which
80% is incorporated in the membrane-proteoliposomes. The mean
membrane-proteoliposome size is 2.8 microns. Freeze-fracture
electron microscopy shown in FIG. 2 reveals
membrane-proteoliposomes with the characteristic ripple phase DMPC
liposomes mixed with WBC membranes containing intramembranous
particles. The two distinct domains in one membrane-proteoliposome
define the "patching" of WBC membranes with DMPC lipids.
[0063] The foregoing discussion and examples are presented merely
for illustrative purposes and are not meant to be limiting. Thus,
one skilled in the art will readily recognize additional
embodiments within the scope of the invention that are not
specifically exemplified.
* * * * *