U.S. patent application number 11/004623 was filed with the patent office on 2005-08-11 for methods and compositions for the production of monoclonal antibodies.
Invention is credited to Paciotti, Giulio F., Tamarkin, Lawrence.
Application Number | 20050175583 11/004623 |
Document ID | / |
Family ID | 34748742 |
Filed Date | 2005-08-11 |
United States Patent
Application |
20050175583 |
Kind Code |
A1 |
Tamarkin, Lawrence ; et
al. |
August 11, 2005 |
Methods and compositions for the production of monoclonal
antibodies
Abstract
The present invention comprises compositions and methods for
making monoclonal antibodies. The present invention further
comprises vectors that replicate the immune system components,
particularly an antigen-presenting cell (APC) element of the immune
synapse. Additionally, the present invention may further comprise
synthetic T-cells.
Inventors: |
Tamarkin, Lawrence;
(Rockville, MD) ; Paciotti, Giulio F.; (Baltimore,
MD) |
Correspondence
Address: |
JOHN S. PRATT, ESQ
KILPATRICK STOCKTON, LLP
1100 PEACHTREE STREET
ATLANTA
GA
30309
US
|
Family ID: |
34748742 |
Appl. No.: |
11/004623 |
Filed: |
December 2, 2004 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
60526360 |
Dec 2, 2003 |
|
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|
Current U.S.
Class: |
424/85.2 ;
424/93.21; 514/1.5; 514/15.4; 514/16.6; 514/19.3; 514/19.6;
514/3.8; 514/4.3; 514/7.3 |
Current CPC
Class: |
A61P 11/00 20180101;
A61P 3/10 20180101; A61P 21/00 20180101; A61K 47/646 20170801; A61P
37/00 20180101; A61K 45/06 20130101; A61P 1/00 20180101; A61P 7/06
20180101; A61P 1/04 20180101; A61P 31/18 20180101; A61P 31/04
20180101; A61P 31/16 20180101; A61P 9/00 20180101; A61P 31/20
20180101; A61K 33/26 20130101; A61P 25/28 20180101; A61P 29/00
20180101; A61P 35/00 20180101; A61P 11/02 20180101; A61P 25/00
20180101; A61K 31/28 20130101; Y10S 530/811 20130101; A61P 7/00
20180101; A61K 33/06 20130101; A61P 1/16 20180101; A61P 19/00
20180101; A61P 19/02 20180101; A61P 27/02 20180101; A61K 33/38
20130101; A61K 47/6923 20170801; A61K 47/6929 20170801; A61P 17/00
20180101; C07K 16/241 20130101; A61K 33/243 20190101; A61P 37/08
20180101; A61K 33/242 20190101; A61P 31/22 20180101; A61P 13/12
20180101; B82Y 5/00 20130101; A61P 37/06 20180101; A61P 35/02
20180101 |
Class at
Publication: |
424/085.2 ;
514/006; 424/093.21 |
International
Class: |
A61K 038/16; A61K
048/00; A61K 039/395 |
Claims
What is claimed is:
1. A vector composition, comprising a synthetic APC, wherein the
synthetic APC comprises a colloidal metal, an antigen and a
component specific immunostimulating agent.
2. The vector composition of claim 1, wherein the component
specific immunostimulating agent comprises antigens, colloidal
metals, adjuvants, receptor molecules, nucleic acids, immunogenic
proteins, accessory cytokine/immunostimulators, pharmaceuticals,
chemotherapy agents or carriers.
3. The vector composition of claim 1, wherein the component
specific immunostimulating agent comprises IL-1, IL-2, IL-3, IL-4,
IL-5, IL-6, IL-7, IL-8, IL-10, IL-11, IL-12, IL-13, lipid A,
phospholipase A2, endotoxins, staphylococcal enterotoxin B, Type I
interferon, Type II interferon, Tumor Necrosis Factor
(TNF-.alpha.), Flt-3 ligand, Transforming Growth Factor
(TGF-.beta.), lymphotoxin, migration inhibition factor,
Granulocyte-Macrophage colony stimulating factor (CSF),
monocyte-macrophage CSF, Granulocyte CSF, Vascular epithelial
growth factor (VEGF), angiogenin, Transforming Growth Factor
(TGF-.alpha.), heat shock proteins, carbohydrate moieties of blood
groups, Rh factors, fibroblast growth factor, inflammatory and
immune regulatory proteins, nucleotides, DNA, RNA, mRNA, sense,
antisense, polynucleotides, cancer cell specific antigens, mutant
p53, tyrosinase, mucines, autoimmune antigens, immunotherapy drugs,
angiogenic or anti-angiogenic drugs.
4. The component specific immunostimulating agent of claim 3,
wherein the cancer cell specific antigen is MAGE, BAGE or MART.
5. The component specific immunostimulating agent of claim 3,
wherein the mucine is MUC-1, PSA or TSH.
6. The vector composition of claim 1, wherein the colloidal metal
comprises colloidal gold colloidal silver, colloidal iron,
colloidal aluminum, or colloidal platinum.
7. The vector composition of claim 1, further comprising a
pharmaceutically-acceptable component comprising excipients,
buffers or carriers.
8. The vector composition of claim 1, further comprising an
adjuvant, wherein the adjuvant comprises liposomes, emulsions,
microspheres, biodegradable polymers and polystyrene, alum, heat
killed M. butyricum and M. tuberculosis, Pertussis toxin and
Tetanus toxin, or LPS and Staphylococcal enterotoxin B.
9. The vector composition of claim 1, wherein the vector further
comprises a co-stimulatory protein.
10. The vector composition of claim 1, wherein the vector further
comprises a major histocompatibility complex.
11. The vector composition of claim 1, wherein the vector further
comprises a germinal center.
12. The vector composition of claim 1, wherein the vector further
comprises a structural protein.
13. The vector composition of claim 1, further comprising targeting
molecules, integrating molecules, PEG, derivatized PEG,
poly-L-lysine or derivatized poly-L-lysine, biotin-avidin,
bifunctional PEG, or bifunctional polymers.
14. The vector composition of claim 13, wherein the bifunctional
PEG comprises thiol and biotin.
15. The vector composition of claim 13, wherein the poly-L-lysine
comprises thiol and biotin.
16. The vector composition of claim 1, wherein the synthetic APC is
lyophilized and stored for a period of time prior to
administration.
17. A method for the production of monoclonal antibodies in vitro,
comprising: a) admixing or binding an antigen, a component specific
immunostimulating agent with a colloidal metal to form a synthetic
APC; and b) stimulating immune cells from a human in vitro to
activate the immune cells to produce a primary response to a
synthetic APC; wherein the synthetic APC comprises an antigen, a
component specific immunostimulating agent, and a colloidal metal;
and c) immortalizing the activated immune cells; and d) selecting a
monoclonal antibody producing cell.
18. The method of claim 17, further comprising a major
histocompatibility complex.
19. The method of claim 17, further comprising a germinal
center.
20. An antigen-specific, human-specific monoclonal antibody
comprised entirely of human protein, produced by a process of: a)
stimulating immune cells from a human in vitro to activate the
immune cells to produce a primary response to a synthetic APC;
wherein the synthetic APC comprises an antigen, a component
specific immunostimulating agent, and a colloidal metal; and b)
immortalizing the activated immune cells; and c) selecting a
monoclonal antibody producing cell.
21. A method for affecting an immune response, comprising,
administering a composition comprising at least one colloidal metal
particle, an antigen, and at least one component-specific
immunostimulating agent.
22. A method for enhancing vaccine efficacy comprising
administering a composition comprising at least one colloidal metal
particle, an antigen, and at least one component-specific
immunostimulating agent.
23. A method of treating disease and immune related dysfunctions
and pathologies, comprising, administering to a human or animal a
therapeutically effective amount of a vector composition comprising
a synthetic APC, wherein the synthetic APC comprises at least one
colloidal metal, an antigen and an component specific
immunostimulating agent.
24. The method of claim 23, wherein the colloidal metal particles
are of different sizes.
25. The method of claim 24, wherein administering the component
specific immunostimulating agent, the antigen, and the colloidal
metal particles of different sizes comprises simultaneously
administering the component specific immunostimulating agent, the
antigen and the colloidal metal particles of different sizes as a
single dose.
26. The method of claim 23, wherein the method is used to stimulate
or suppress immune components in the treatment of disease, wherein
the disease is cancer, solid tumor cancer, Crohn's disease, breast
cancer, Psoriasis, inflammatory bowel disease, adult respiratory
distress syndrome, allergies, rhinitis, eczema, urticaria,
anaphylaxis, transplant rejection, rheumatic diseases, systemic
lupus erthymatosus, rheumatoid arthritis, seronegative
spondyloarthritides, Sjogren's syndrome, systemic sclerosis,
polymyositis, dermatomyositis, Type I Diabetes Mellitus, Acquired
Immune Deficiency Syndrome, Hashimoto's thyroiditis, Graves'
disease, Addison's disease, polyendocrine autoimmune disease,
hepatitis, sclerosing cholangitis, primary biliary cirrhosis,
pernicious anemia, coeliac disease, antibody-mediated nephritis,
glomerulonephritis, Wegener's granulomatosis, microscopic
polyarteritis, polyarteritis nodosa, pemphigus, dermatitis
herpetiformis, psoriasis, vitiligo, multiple sclerosis,
encephalomyelitis, Guillain-Barre syndrome, Myasthenia Gravis,
Lambert-Eaton syndrome, sclera, episclera, uveitis, chronic
mucocutaneous candidiasis, Bruton's syndrome, transient
hypogammaglobulinemia of infancy, myeloma, X-linked hyper IgM
syndrome, Wiskott-Aldrich syndrome, ataxia telangiectasia,
autoimmune hemolytic anemia, autoimmune thrombocytopenia,
autoimrune neutropenia, Waldenstrom's macroglobulinemia,
amyloidosis, chronic lymphocytic leukemia, or non-Hodgkin's
lymphoma.
27. The method of claim 23, further comprising a major
histocompatibility complex.
28. A multi-particle self assembling synthetic APC, comprising at
least two colloidal metals, an antigen and at least one component
specific immunostimulating agent, wherein the multi-particle
synthetic APC is assembled using scaffolding molecules, and wherein
the scaffolding molecules comprises biotinylated spacer arms,
alkane linkers, protein, polyethylene glycol, derivatized PEG,
multi-arm PEG or thiolated poly-L-lysine.
29. The multi-particle self-assembling synthetic APC of claim 28,
wherein the colloidal metal particles are of different sizes.
30. The multi-particle self-assembling synthetic APC of claim 28,
further comprising targeting molecules and integrating
molecules.
31. The method of claim 28, further comprising a major
histocompatibility complex.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application claims priority to U.S. Provisional
Application No. 60/526,360 filed Dec. 2, 2003.
FIELD OF THE INVENTION
[0002] The present invention relates generally to immunology. The
present invention further relates to methods and compositions for
the production of monoclonal antibodies and in vitro methods for
production of such antibodies.
BACKGROUND OF THE INVENTION
[0003] The introduction of desired agents into specific target
cells has been a challenge to scientists for a long time. The
challenge of specific targeting of agents is to get an adequate
amount of the agent or the correct agent to the target cells of an
organism without providing too much exposure of the rest of the
organism. A very desired target for delivery of specific agents is
the immune system. The immune system is a complex response system
of the body that involves many different kinds of cells that have
differing activities. Activation of one portion of the immune
system usually causes a variety of responses due to unwanted
activation of other related portions of the system. Currently,
there are no satisfactory methods or compositions for producing a
specifically desired response by targeting the specific components
of the immune system.
[0004] The immune system is a complex interactive system of the
body that involves a wide variety of components, including cells,
and cellular factors, which interact with stimuli from both inside
the body and outside the body. Aside from its direct action, the
immune system's response is also influenced by other systems of the
body including the nervous, respiratory, circulatory, and digestive
systems.
[0005] One of the better-known aspects of the immune system is its
ability to respond to foreign antigens presented by invading
organisms, cellular changes within the body, or from vaccination.
Some of the first kinds of cells that respond to such activation of
the immune system are phagocytes and natural killer cells.
Phagocytes include among other cells, monocytes, macrophages, and
polymorphonuclear neutrophils. These cells generally bind to the
foreign antigen, internalize it and often times destroy it. They
also produce soluble molecules that mediate other immune responses,
such as inflammatory responses. Natural killer cells can recognize
and destroy certain virally-infected embryonic and tumor cells.
Other factors of the immune response include complement pathways,
which are capable of responding independently to foreign antigens
or acting in concert with cells or antibodies.
[0006] One of the aspects of the immune system that is important
for vaccination is the specific response of the immune system to a
particular pathogen or foreign antigen. Part of the response
includes the establishment of "memory" for that foreign antigen.
Upon a secondary exposure, the memory function allows for a quicker
and generally greater response to the foreign antigen. Lymphocytes
in concert with other cells and factors play a major role in both
the memory function and the response.
[0007] Generally, it is thought that the response to antigens
involves both humoral responses and cellular responses. Humoral
immune responses are mediated by non-cellular factors that are
released by cells and which may or may not be found free in the
plasma or intracellular fluids. A major component of a humoral
response of the immune system is mediated by antibodies produced by
B lymphocytes. Cell-mediated immune responses result from the
interactions of cells, including antigen presenting cells and B
lymphocytes (B cells) and T lymphocytes (T cells).
[0008] One of the most widely employed aspects of the immune
response capabilities is the production of monoclonal antibodies.
The advent of monoclonal antibody (Mab) technology in the mid 1970s
provided a valuable new therapeutic and diagnostic tool. For the
first time, researchers and clinicians had access to unlimited
quantities of uniform antibodies capable of binding to a
predetermined antigenic site and having various immunological
effector functions. Currently, the techniques for production of
monoclonal antibodies are well known in the art.
[0009] These monoclonal antibodies are thought to hold great
promise in medicine and diagnostics. Unfortunately, the development
of therapeutic products based on these proteins has been limited
because of problems that are inherent in monoclonal antibody
therapy. For example, most monoclonal antibodies are mouse derived
and, thus, do not fix human complement well. They also lack other
important immunoglobulin functional characteristics when used in
humans.
[0010] The biggest drawback to the use of monoclonal antibodies is
the fact that nonhuman monoclonal antibodies are immunogenic when
injected into a human patient. After injection of a foreign
antibody, the immune response mounted by a patient can be quite
strong. The immune response causes the quick elimination of the
foreign antibody, essentially eliminating the antibody's
therapeutic utility after an initial treatment. Unfortunately, once
the immune system is primed to respond to foreign antibodies, later
treatments with the same or different nonhuman antibodies can be
ineffective or even dangerous.
[0011] Mice can be readily immunized with foreign antigens to
produce a broad spectrum of high affinity antibodies. However, the
introduction of murine antibodies into humans results in the
production of a human-anti-mouse antibody (HAMA) response due to
the presentation of a mouse antibody in the human body. Use of
murine antibodies in a patient is generally limited to a term of
days or weeks. Longer treatment periods may result in anaphylaxis.
Moreover, once HAMA has developed in a patient, it often prevents
the future use of murine antibodies for diagnostic or therapeutic
purposes.
[0012] To overcome the problem of HAMA response, researchers have
attempted several approaches to modify nonhuman antibodies, to make
them human-like. These approaches include mouse/human chimers,
humanization, and primatization. Early work in making more
human-like antibodies used combined rabbit and human antibodies.
The protein subunits of antibodies, rabbit Fab fragments and human
Fc fragments, were joined through protein disulfide bonds to form
new, artificial protein molecules or chimeric antibodies.
[0013] Recombinant molecular biological techniques have been used
to create chimeric antibodies. Recombinant DNA technology was used
to construct a gene fusion between DNA sequences encoding mouse
antibody variable light and heavy chain domains and human antibody
light chain (LC) and heavy chain (HC) constant domains to permit
expression of chimeric antibodies. These chimeric antibodies
contain a large number of nonhuman amino acid sequences and are
immunogenic to humans. Patients exposed to these chimeric
antibodies produce human-anti-chimera antibodies (HACA). HACA is
directed against the murine V region and can also be directed
against the novel V-region/C-region (constant region) junctions
present in recombinant chimeric antibodies.
[0014] To overcome some of the limitations presented by the
immunogenicity of chimeric antibodies, molecular biology techniques
are used to created humanized or reshaped antibodies. The DNA
sequences encoding the antigen binding portions or complementarity
determining regions (CDRs) of murine monoclonal antibodies are
grafted, by molecular means, on the DNA sequences encoding the
frameworks of human antibody heavy and light chains. The humanized
Mabs contain a larger percentage of human antibody sequences than
do chimeric Mabs. The end product, which comprises approximately
90% human antibody and 10% mouse antibody, contains a mouse
binding-site on a human antibody. It also contains certain amino
acid substitutions from the mouse Mab into the framework of the
humanized Mab in order to retain the correct shape, and thus,
binding affinity for the target antigen.
[0015] In practice, simply substituting murine CDRs for human CDRs
is not sufficient to generate efficacious humanized antibodies
retaining the specificity of the original murine antibody. There is
an additional requirement for the inclusion of a small number of
critical murine antibody residues in the human variable region. The
identity of these residues depends upon the structure of both the
original murine antibody and the acceptor human antibody. It is the
presence of these murine antibody residues that helps create a HACA
response in the patient, leading to rapid clearance of the
monoclonal antibodies and the fear of anaphylaxis.
[0016] Another technique, called resurfacing technology, is used
for humanizing mouse antibodies. Resurfacing involves replacing the
mouse antibody surface with a human antibody surface in a process
that is faster and more efficient than other humanization
techniques. This technique provides a method of redesigning murine
monoclonal antibodies to resemble human antibodies by humanizing
only those amino acids that are accessible at the surface of the
V-regions of the recombinant Fv. The resurfacing of murine
monoclonal antibodies may maintain the avidity of the original
mouse monoclonal antibody in the reshaped version, because the
natural framework-CDR interactions are retained. Again, these
antibodies suffer from the problem of being antigenic due to their
mouse origins.
[0017] Other technologies use primate, rather than mouse, sequences
to humanize Mabs. The rationale of this approach, called
primatization, is that most of the sequences in the primate
antibody variable region are indistinguishable from human
sequences. Primatized anti-CD4 Mabs for the treatment of rheumatoid
arthritis and severe asthma are being developed. However, these
Mabs are still foreign proteins to the immune system of the patient
and evoke an immune response.
[0018] In an effort to avoid the immune response to foreign
proteins, a variety of approaches are being developed to make human
Mabs that contain only human antibody components. One approach is
to isolate a human B cell clone that naturally makes antibody to
the desired antigen and to grow it in a trioma cell culture system.
Because human antibodies are made only against antigens that are
foreign to the host, none of the human B cells will make antibodies
against human antigens. Therefore, this approach is not useful to
produce Mabs against antigens that are human proteins.
[0019] Two other approaches to create human Mabs are phage display
and use of transgenic mice. Phage display technique takes advantage
of the ability of humans to make antibodies against any possible
structure. This technique uses the antibody genes from many
individual humans to create a large library of phage antibodies,
each displaying a functional antibody variable domain on its
surface. From this library, individual variable domains are
selected for their ability to bind to the desired antigen. The Mab
is created through molecular biology techniques by combining an
antibody variable domain having the desired binding characteristics
and a constant domain that best meets the potential human
therapeutic product. Again, this technique lacks antigen
specificity. The phage library cannot contain every binding region
for any and all desired antigens. It also may contain binding
regions, which lack specificity. Thus, this technique may require
considerable engineering to increase antibody affinities to useful
levels.
[0020] Transgenic mice are also being used to create "human"
antibodies. The transgenic mice are created by replacing mouse
immunoglobulin gene loci with human immunoglobulin loci. This
approach may provide advantages over phage display technologies
because it takes advantages of mouse in vivo affinity maturation
machinery.
[0021] All of the current technologies for producing human or
human-like Mabs are insufficient to provide a species-specific
antibody that is antigen specific for a described antigen. Chimeric
antibodies have the advantages of retaining the specificity of the
murine antibody and stimulating human Fc dependent complement
fixation and cell-mediated cytotoxicity. However, the murine
variable regions of these chimeric antibodies can still elicit a
HAMA response, thereby limiting the value of chimeric antibodies as
diagnostic and therapeutic agents.
[0022] Vaccines may be directed at any foreign antigen, whether
from another organism, a changed cell, or induced foreign
attributes in a normal "self" cell. The route of administration of
the foreign antigen can help determine the type of immune response
generated. For example, delivery of antigens to mucosal surfaces,
such as oral inoculation with live polio virus, stimulates the
immune system to produce an immune response at the mucosal surface.
Injection of antigen into muscle tissue often promotes the
production of a long lasting IgG response.
[0023] Vaccines may be generally divided into two types, whole and
subunit vaccines. Whole vaccines may be produced from viruses or
microorganisms which have been inactivated or attenuated or have
been killed. Live attenuated vaccines have the advantage of
mimicking the natural infection enough to trigger an immune
response similar to the response to the wild-type organism. Such
vaccines generally provide a high level of protection, especially
if administered by a natural route, and some may only require one
dose to confer immunity. Another advantage of some attenuated
vaccines is that they provide person-to-person passage among
members of the population. These advantages, however, are balanced
with several disadvantages. Some attenuated vaccines have a limited
shelf-life and cannot withstand storage in tropical environments.
There is also a possibility that the vaccine will revert to the
virulent wild-type of the organism, causing harmful, even
life-threatening, illness. The use of attenuated vaccines is
contraindicated in immunodeficient states, such as AIDS, and in
pregnancy.
[0024] Killed vaccines are safer in that they cannot revert to
virulence. They are generally more stable during transport and
storage and are acceptable for use in immunocompromised patients.
However, they are less effective than the live attenuated vaccines,
usually requiring more than one dose. Additionally, they do not
provide for person-to-person passage among members of the
population.
[0025] Production of subunit vaccines requires knowledge about the
epitopes of the microorganism or cells to which the vaccine should
be directed. Other considerations in designing subunit vaccines are
the size of the subunit and how well the subunit represents all of
the strains of the microorganism or cell. The current focus for
development of bacterial vaccines has shifted to the generation of
subunit vaccines because of the problems encountered in producing
whole bacterial vaccines and the side effects associated with their
use. Such vaccines include a typhoid vaccine based upon the Vi
capsular polysaccharide and the Hib vaccine to Haemophilus
influenzae.
[0026] Because of the safety concerns associated with the use of
attenuated vaccines and the low efficacy of killed vaccines, there
is a need in the art for compositions and methods that enhance
vaccine efficacy. There is also a need in the art for compositions
and methods of enhancing the immune system, which stimulate both
humoral and cell-mediated responses. There is a further need in the
art for the selective adjustment of an immune response and
manipulating the various components of the immune system to produce
a desired response. Additionally, there is a need for methods and
compositions that can accelerate and expand the immune response for
a more rapid activation response. There is an increased need for
the ability to vaccinate populations, of both humans and animals,
with vaccines that provide protection with just one dose.
[0027] What is needed are compositions and methods to target the
delivery of specific agents to only the target cells. Such
compositions and methods should be able to deliver therapeutic
agents to the target cells efficiently. What is also needed are
compositions and methods that can be used both in in vitro and in
vivo systems.
[0028] There is also a general need for compositions of monoclonal
antibodies and improved methods for producing them. There is a
particular need for methods for producing human antibodies having
affinity for a predetermined antigen. These human immunoglobulins
should be easily and economically produced in a manner suitable for
therapeutic and diagnostic formulation.
SUMMARY OF THE INVENTION
[0029] The present invention comprises compositions and methods for
making species-specific antigen-specific monoclonal antibodies,
preferably IgG monoclonal antibodies. The present invention further
comprises vectors that replicate elements of the immune system,
particularly the antigen-presenting cell (APC) element of the
immune synapse. A preferred vector optionally comprises binding an
antigen-loaded major histocompatibility (MHC) class II protein, the
co-stimulatory protein B7, and the structural protein intracellular
adhesion protein (I-CAM) onto the surface of colloidal metal
vectors. Such vectors replicate the 3-D orientation of the APC
(FIG. 3) generating a synthetic antigen-presenting cell (sAPC)
capable of activating CD4.sup.+ T-cells to mature the antibody
response of immunized B-cells.
[0030] The present invention further comprises vectors, including a
synthetic CD4.sup.+ T-cell (sTc), and a synthetic germinal center
(sGC). In one embodiment the synthetic CD4.sup.+ T-cell is
comprised of colloidal metal vectors bound with CD40 ligand and
cytokines. In another embodiment the synthetic germinal center is
comprised of colloidal metal vectors bound with B Lymphocyte
Stimulator; BlyS and CD30L/receptor system, that increase the
efficiency and specificity of B-cell antibody response to in vitro
immunization. While not wishing to be bound to any particular
theory, in one embodiment the physical juxtaposition of the antigen
with B-cell growth factors improves the uptake of the human TNF
antigen through the surface IgM antigen receptor and induces a more
robust B-cell response. Having these signals juxtaposed on the same
B-cell further improves the ability to elicit an antigen specific
B-cell response in vitro.
[0031] The present invention comprises methods of making the
synthetic immune component elements. Methods are taught herein for
making vector compositions that mimic the functionality of
components of the immune system. The present invention also
comprises methods of treatment of immune system-related diseases
and pathologies. Methods of vaccination are also included in the
present invention.
BRIEF DESCRIPTION OF THE FIGURES
[0032] FIG. 1 provides a schematic representation of the immune
synapse.
[0033] FIG. 2 provides a schematic representation of the
differentiation of primary antibody response by activated
CD4.sup.+T-cell.
[0034] FIG. 3A provides a schematic of the colloidal gold synthetic
antigen-presenting cell. FIG. 3B provides a schematic of the
colloidal gold synthetic T-Cell. FIG. 3C provides a schematic of
the colloidal gold synthetic germinal center.
[0035] FIG. 4 provides a schematic representation of the inability
of a single particle sAPC to form a functional immune synapse.
[0036] FIG. 5 provides a schematic representation of the generation
of a multiple particle colloidal gold sAPC.
[0037] FIG. 6 provides a graph depicting the binding multiple
cytokines to the same particle of colloidal gold.
[0038] FIG. 7 is a series of photographs of EGF streptavidin gold
that was targeted to macrophages (FIG. 7A), dendritic cells (FIG.
7B) and B-Cells (FIG. 7C).
[0039] FIG. 8 provides a graph of the immunoreactivity of cells in
response to various stimuli in vitro.
[0040] FIG. 9A provides a schematic of the self-assembly of
colloidal gold particle on the solid support of an EIA plate. 1=EIA
plate; 2=Murine Mab against human TNF; 3=human TNF (blue box); 4=32
nm colloidal gold bound with streptavidin an TNF; 5=biotinylated
BSA; 6=17 nm streptavidin colloidal gold; 7=biotinylated human
IL-6; 8=alkaline phosphatase conjugated rabbit anti-human IL-6.
[0041] FIG. 9B provides a schematic of self-assembly of colloidal
gold particles bound with either IL-1 or TNF on a four-arm
PEG-thiol backbone (Sun Bio, Inc.).
[0042] FIG. 10A provides a graph of the immunoreactivity signal
generated by the particle in FIG. 9A.
[0043] FIG. 10B provides a graph of the immunoreactivity signal
generated by the particle in FIG. 9B.
[0044] FIG. 11 provides a schematic representation of the colloidal
gold/TNF binding apparatus
[0045] FIG. 12 provides a graph of the effect of ionic strength on
the stability of the colloidal gold TNF vector after
lyophilization.
[0046] FIG. 13 provides a schematic representation of a model for
TNF binding to colloidal gold in low ionic strength solutions.
[0047] FIG. 14 provides a schematic representation of a model for
TNF binding to colloidal gold at high ionic strength solutions.
DETAILED DESCRIPTION OF THE INVENTION
[0048] The present invention may be understood more readily by
reference to the following detailed description of specific
embodiments included herein. Although the present invention has
been described with reference to specific details of certain
embodiments, thereof, it is not intended that such details should
be regarded as limitations upon the scope of the invention. The
entire text of the references mentioned herein are hereby
incorporated in their entireties by reference, including U.S.
Provisional Application Ser. No. 60/526,360.
[0049] The present invention comprises methods and compositions for
generating antigen specific, species-specific IgG monoclonal
antibodies. The present invention comprises methods and
compositions comprising naturally occurring and/or synthetic
vectors that replicate the antigen-presenting cell (APC), T cell
and germinal center elements of the humoral immune response.
[0050] The present invention comprises vectors that mimic any of
the elements or stages of the immune response. The immune response
is initiated by the recognition of foreign antigens by various
kinds of cells, principally macrophages or other antigen presenting
cells. This leads to activation of lymphocytes, in particular, the
lymphocytes that specifically recognize that particular foreign
antigen and results in the development of the immune response, and
possibly, elimination of the foreign antigen. Overlaying the immune
response directed at elimination of the foreign antigen are complex
interactions that lead to helper functions, stimulator functions,
suppresser functions and other responses. The power of the immune
system's responses must be carefully controlled at multiple sites
for stimulation and suppression or the response will either not
occur, over respond, or not cease after elimination.
[0051] The recognition phase of response to foreign antigens
consists of the binding of foreign antigens to specific receptors
on immune cells. These receptors generally exist prior to antigen
exposure. Recognition can also include interaction with the antigen
by macrophage-like cells or by recognition by factors within serum
or bodily fluids.
[0052] In the activation phase, lymphocytes undergo at least two
major changes. They proliferate, leading to expansion of the clones
of antigen-specific lymphocytes and amplification of the response,
and the progeny of antigen-stimulated lymphocytes differentiate
either into effector cells or into memory cells that survive, ready
to respond to re-exposure to the antigen. There are numerous
amplification mechanisms that enhance this response.
[0053] In the effector phase, activated lymphocytes perform the
functions that may lead to elimination of the antigen or
establishment of the vaccine response. Such functions include
cellular responses, such as regulatory, helper, stimulator,
suppressor or memory functions. Many effector functions require the
combined participation of cells and cellular factors. For instance,
antibodies bind to foreign antigens and enhance their phagocytosis
by blood neutrophils and mononuclear phagocytes. Complement
pathways are activated and may participate in the lysis and
phagocytosis of microbes in addition to triggering other body
responses, such as fever.
[0054] In the immune response to antigens, immune cells interact
with each other by direct cell-to-cell contact or indirect
cell-to-cell (factor mediated) communication. For example,
interactions between T cells, macrophages, dendritic cells, and B
cells are necessary for an effective immune response.
Antigen-presenting cells (APC) activate B and T cells by presenting
them B and T cells with processed antigens and other activation
signals. Activated T cells help control immune responses and
participate in the removal of foreign organisms. Helper T cells
cause cells to become better effector cells, such as helping
cytotoxic T cell precursors to develop into killer cells, helping B
cells make antibodies, and helping increase functions of other
cells like macrophages. Activated B cells divide and produce
antigen specific antibodies and memory B cells. The cells involved
in the immune response also secrete cellular factors or cytokines,
which enhance the functions of phagocytes, stimulate inflammatory
responses and affect a variety of cells.
[0055] The reactions of these cells also involve feedback loops.
Macrophages and other mononuclear phagocytes, or APCs, actively
phagocytose antigens for presentation to B and T cells and such
activity can be enhanced by lymphocytic cellular factors.
Macrophages also produce cytokines that, among other activities,
stimulate T cell proliferation and differentiation, recruit other
inflammatory cells, especially neutrophils, and are responsible for
many of the systemic effects of inflammation, such as fever. One
such cytokine, called interleukin-12, is especially important for
the development of cell-mediated immunity.
[0056] Dendritic cells are also APCs, which initiate an immune
response. There are a number of different types of dendritic cells,
including lymphoid dendritic cells and Langerhans cells of the
skin. They can be found throughout the body and particularly in the
spleen, lymph nodes, tonsils, Peyer's patches, and thymus. They are
irregularly shaped cells, which continuously extend and contract
dendritic (tree-like) processes. One of their roles in the immune
system is to induce and regulate B and T cell activation and
differentiation. They are potent accessory cells for the
development of cytotoxic T cells, antibody formation by B cells,
and some polyclonal responses like oxidative mitogenesis. They also
stimulate T cells to release the cytokine interleukin-2.
[0057] An important arm of vaccination is the response to antigens
that is provided by B lymphocytes or B cells. B cells represent
about 5 to 15% of the circulating lymphocytes. B cells produce
immunoglobulins, IgG, IgM, IgA, IgD, and IgE, which may be released
into body fluids, secreted with attached proteins or be inserted
into the surface membrane of the B cell. Such immobilized
immunoglobulins act as specific antigen receptors. In responding to
antigen, these immunoglobulin receptors are crosslinked at a
specific site on the B cell. This process, which is known as
capping, is followed by internalization and degradation of the
immunoglobulin. In APCs, which may include B cells, antigen
fragments are combined with the MHC and ultimately expressed on the
surface of the APC.
[0058] The B plasma cells produce and secrete antibody molecules
that can bind foreign proteins, polysaccharides, lipids, or other
chemicals in extracellular or cell-associated forms. The antibodies
produced by a single plasma cell are specific for one antigen. The
secreted antibodies bind the antigen and trigger the mechanisms
that facilitate their destruction.
[0059] In 1975, Kohler and Milstein (Kohler, G., and Milstein, C.,
Nature (London). 1975. volume 256: pp-495) described a method for
fusing antibody-producing B cells isolated from the spleens of
immunized mice with aggressively proliferating mouse myeloma cells.
This resultant hybrid cell, a hybridoma, possesses the
characteristics of both parental cells. It produces and secretes
large amounts of antibody during its continued growth and
proliferation. Through a series of systematic cellular dilutions,
genetically singular hybridoma cells can be isolated that produce
an antibody of singular specificity, a monoclonal antibody
(Mab).
[0060] The most common procedures require that the production of
monoclonal antibodies start with the immunization of an animal.
Antigen, draining into a local lymph node or spleen, activates
naive B-cells to produce IgM antibodies. These activated B cells
are then presented with antigen-activated CD4.sup.+ T cells to
induce class switching. Class switching is characterized by a
change in the production of antibody type from IgMs to IgGs (Kuby,
J., Immunology Third Edition 1997. eds Allen D., pp-205-213).
Antibody secreting B cell lymphocytes are isolated from the lymph
node or spleen of the immunized animal, and are fused with
species-specific myeloma cells. The fused cells are then allowed to
grow to produce antigen specific IgG antibodies. During the
screening process, positive fusion clones are selected for their
therapeutic potential.
[0061] Mice can be readily immunized with foreign antigens to
produce a broad spectrum of high affinity antibodies. However, the
introduction of murine antibodies into humans results in the
production of a human-anti-mouse antibody (HAMA) response due to
the presentation of a foreign protein in the body. Use of murine
antibodies in a patient is generally limited to a term of days or
weeks. Moreover, once HAMA has developed in a patient, it often
prevents the future use of murine antibodies for other diagnostic
or therapeutic purposes.
[0062] The early success of this technology in animals prompted
scientists in the 1980's to extend this concept and attempt to
produce human monoclonal antibodies. However, extrapolation from
animal to human was fraught with difficulties. The first hurdle was
the lack of antigen specific B cells. Standard monoclonal antibody
procedure requires that these cells be harvested from an animal
that had been immunized, a method not generally applicable to
humans. This problem is further compounded by (i) the fact that
there is no ready source of activated B cells, (ii) the paucity of
immune competent B cells present in peripheral blood, and (iii) the
inability to obtain either lymph nodes or spleens from human
subjects. These factors prompted the development of a variety in
vitro strategies to produce human monoclonal antibodies. Although
initial results showed great promise, the inability to completely
reconstruct the sequence of events of the in vivo antibody response
ultimately caused the technology to fail and this technical
approach has been essentially abandoned.
[0063] The first barrier to in vitro antibody production is the
relatively low conversion rate of naive human B cell lymphocytes to
activated B cells. In the past resolving this challenge proved
difficult even when recall antigens, such as Tetanus toxin (Butler
et. al., J. Immuol. 1983. volume 130: pp-165), were used to induce
a primary antibody response from human peripheral blood B cell
lymphocytes. The present invention comprises methods for making
vectors that activate pathways that lead to antibody generation.
The present invention also comprises compositions of naturally
occurring or synthetic vectors. Such vectors comprise colloidal
gold platforms with multiple B cell ligands associated.
[0064] Numerous examples of cross-linking of receptor/ligand pairs
to potentiate biologic responses have been described (Carroll, K.,
Prosser, E., and Kennedy, R. Hybridoma 1991. 10: 229-239). The
present invention comprises vectors of colloidal metal that
increase the efficiency and specificity of B cell antibody response
to in vitro immunization. Though not wishing to be bound by any
particular theory, it is believed that the physical juxtaposition
of the antigen with B cell growth factors improves the uptake of
the antigen through the surface IgM antigen receptor and induces a
more robust B cell response. There is also improved antigen
processing and presentation. Having these signals juxtaposed on the
same B cell improves the ability to elicit an antigen specific B
cell response in vitro.
[0065] In one embodiment, the component-specific immunostimulating
molecule and/or MHC protein and/or the antigen may be bound
directly to the colloidal metal platform or may be bound to the
colloidal metal platform through members of a binding group. Such
binding groups may comprise free sulfhydryl or pyridyl groups
present on, or synthetically added to the immune component. A
preferred embodiment of the present invention comprises a colloidal
metal as a platform that is capable of binding a member of a
binding group to which a component-specific immunostimulating
agent, or a MHC protein or an antigen are bound to create a
synthetic APC. In an alternatively preferred embodiment, the
binding group is streptavidin/biotin and the component-specific
immunostimulating agent is a cytokine. Embodiments of the present
invention may also comprise binding the antigen, or the MHC protein
or the component-specific immunostimulating agent in a less
specific method, without the use of binding partners, such as by
using polycations or proteins. As such, the present invention
contemplates the use of interacting molecules such as polycationic
elements known to those skilled in the art including, but not
limited to, polylysine, protamine sulfate, histones or
asialoglycoproteins.
[0066] The members of the binding pair comprise any such binding
pairs known to those skilled in the art, including but not limited
to, antibody-antigen pairs, enzyme-substrate pairs; receptor-ligand
pairs; and streptavidin-biotin. Novel binding partners may be
specifically designed. An essential element of the binding partners
is the specific binding between one of the binding pair with the
other member of the binding pair, such that the binding partners
are capable of being joined specifically. Another desired element
of the binding members is that each member is capable of binding or
being bound to either an integrating molecule or a targeting
molecule.
[0067] The compositions of the invention comprise a colloidal
metal, an antigen, and a component specific immunostimulating
agent. Alternatively, compositions of the invention comprise a
colloidal metal, a MHC protein, an antigen, and a component
specific immunostimulating agent. The component specific
immunostimulating agent may comprise biologically active agents
that can be used in therapeutic applications or the component
specific immunostimulating agent may be useful in detection
methods. In additional embodiments, one or more component specific
immunostimulating agents are admixed, associated with or bound
directly or indirectly to the colloidal metal. Admixing,
associating and binding includes covalent and ionic bonds and other
weaker or stronger associations that allow for long term or short
term association of the derivatized-PEG or the derivatized
poly-1-lysine, component specific immunostimulating agents, and
other components with each other and with the colloidal metal
particles.
[0068] In yet another embodiment, the compositions may also
comprise one or more targeting molecules admixed, associated or
bound to the colloidal metal. The targeting molecule can be bound
directly or indirectly to the metal particle. Indirect binding
includes binding through molecules such as polylysines or other
integrating molecules or any association with a molecule that binds
to both the targeting molecule and either the metal sol or another
molecule bound to the metal sol.
[0069] Of particular interest are detection agents such as dyes or
radioactive materials that can be used for visualizing or detecting
the sequestered colloidal metal vectors. Fluorescent,
chemiluminescent, heat sensitive, opaque, beads, magnetic and
vibrational materials are also contemplated for use as detectable
agents that are associated or bound to colloidal metals in the
compositions of the present invention.
[0070] Generation of a primary antibody response from naive human B
cells in vitro represents only the first step in the in vitro
reconstruction of the human antibody response. The primary antibody
response from immunized human B cells results in the secretion of
IgM antibodies. A second class of lymphoid cells, known as antigen
presenting cells (APCs), also internalizes the antigen. Once
internalized these cells process the protein antigen into
fragments, which are then expressed on the cell's surface bound to
one of two major histocompatibility complexes (MHCs). These cells
are important for antibody class switching.
[0071] A current theory of immune system responses is herein
presented. The present invention is not limited to the mechanisms
described herein, but can function in multiple methods, not limited
by any particular theory described herein. Depending on the
microenvironment, APCs expressing antigen bound to class II MHC
molecules activate one of two subsets of CD4.sup.+ T cells. These
cells, also known as helper T cells, perform the necessary
accessory functions to facilitate the cellular or the humoral
(antibody) immune response. T.sub.H1 CD4.sup.+ cells facilitate the
cellular immune response, while the T.sub.H2 subset of CD4.sup.+
cells interact with IgM secreting B cells to initiate the process
of class switching.
[0072] The activation of CD4.sup.+ T.sub.H2 T-cells by the APC
occurs with the formation of a bicellular cleft known as the immune
synapse (Wulfing C, Sumen C, Sjaastad M D, Wu L C, Dustin M L,
Davis M M. Nat Immunol 2002. 31: 42-7). The formation of the immune
synapse involves interaction and rearrangement of signaling and
structural ligands on the APC with their respective receptors on
the T cell to form a three-dimensional (3-D) bridge that allows
contact and signaling between these two cells (FIG. 1). Antigen
signaling between the APC and the T cell occurs through the binding
of the MHC/antigen complex with the T cell receptor complex, while
the structural integrity of the immune synapse is maintained by the
interaction of ICAM (intracellular adhesion molecule), LFA-3, and
CD72 on the APC with LFA-1, CD2, and CD5 receptors on T cells,
respectively. The successful formation of the immune synapse causes
the CD4.sup.+ T cell to express a B cell stimulatory molecule known
as CD40 ligand.
[0073] The formation of the immune synapse may signal the T cell to
become active or inactive (anergic). Which response is initiated is
dependent on the strength of the co-stimulatory signals provided by
the B7 molecule on the APC to the T cell. The B7 molecule may
interact with either B7 receptor molecule on the T cell, CD28 or
CTLA4. These B7 receptors differ with respect to their density on
the surface of the T cell as well as their affinity for the B7
molecule. CD28 has a lower affinity for B7 than CTLA4, but is
present at a much higher density on the surface of the T cell. The
binding of B7 to the CD28 receptor sends an activation signal to
the T cell, while the binding of B7 by CTLA4 induces T cell anergy
(Kuby, J., Immunology Third Edition 1997. eds Allen D., pp.
213-218). Thus, presenting excess B7 in the immune synapse will
ensure that the T cells will be activated. The activated
CD4.sup.+/CD40.sup.+ T cell forms a synapse with an IgM secreting B
cell. The interaction of CD40 ligand on the T cell with the CD40
receptor on the B cell causes the IgM secreting B cell to undergo
class switching to produce IgGs (FIG. 2).
[0074] The present invention comprises methods of making sAPCs
capable of activating CD4+ T cells, and synthetic CD4.sup.+ T-cells
(sTc) and synthetic germinal centers (sGC) able to mature the
antibody response of immunized B cells or immortalized B cells. The
compositions of the present invention comprise colloidal metal
vectors capable of activating T cells and vectors that cause the
maturation of immunized or immortalized B cells. For example, a
vector may have an antigen-loaded major histocompatibility (MHC)
class II protein, the co-stimulatory protein B7, and the structural
protein intracellular adhesion molecule (ICAM) associated with the
surface of colloidal metal vectors. This vector replicates the 3-D
orientation of the APC (FIG. 3) and functions as a synthetic
antigen-presenting cell (sAPC) capable of activating CD4.sup.+ T
cells to mature the antibody response of immunized B cells. One
embodiment of the sAPC comprises all of the components on a single
particle of colloidal metal. Another embodiment of the sAPC
comprises the constituent proteins of the immune synapse bound on
separate particles of colloidal gold that self-assemble in vitro to
form the sAPC.
[0075] The methods and compositions of the present invention
comprising synthetic antigen-presenting cells (sAPC) comprise
compositions that are readily available and can be "pulled out of
the refrigerator" and used to manipulate the human antibody
response. Thus the present invention comprises methods of treatment
of diseases and immune related dysfunctions and pathologies. The
colloidal metal compositions provide control over the variables
that are responsible for initiating, maintaining and regulating the
immune response (either down-regulating or up-regulating), such as
particle size, the amount of protein bound per particle, the
flexibility of protein movement on the particle, as well as the 3-D
assembly of the particles, ensures reproducible control of the
sAPC.
[0076] The vector compositions of the present invention can be used
in in vitro production of monoclonal antibodies. Such monoclonal
antibodies can be used in methods of treatment for multiple
diseases. The vector compositions of the present invention can also
be used in making improved vaccine compositions.
[0077] In vaccine therapy, compositions of synthetic immunogens
specifically designed to stimulate both the cellular and humoral
responses of the human immune system are used. By creating specific
synthetic cellular immune elements for the presentation of the
antigen and stimulation of specific cells, a more predictable and
efficient vaccine response is enabled.
[0078] The present invention comprises combination vaccines and DNA
vaccines. An example of a combination vaccine is the Bordetella
pertussis toxin and its surface fimbrial hemaglutinin. In DNA
vaccination, the patient is administered nucleic acids encoding a
protein antigen that is then transcribed, translated and expressed
in some form to produce strong, long-lived humoral and
cell-mediated immune responses to the antigen.
[0079] The immune response created by vaccines can be
non-specifically enhanced by the use of adjuvants. These are a
heterogeneous group of compounds or carrier components, such as
liposomes, emulsions or microspheres, with several different
mechanisms of action. Methods of the present invention comprise use
of vaccines for protection against disease, and to treat
cancer.
[0080] Many diseases, in addition to cancer, are mediated by the
immune system and the present invention comprises methods of
treatment of such diseases by the administration of an effective
amount of a composition comprising a colloidal metal vector that is
capable of stimulating the immune system and its components. The
diseases include, Crohn's disease, psoriasis, inflammatory bowel
disease, adult respiratory distress syndrome, allergies, eczema,
rhinitis, urticaria, anaphylaxis, transplant rejection, such as
kidney, heart, pancreas, lung, bone, and liver transplants;
rheumatic diseases, systemic lupus erthematosus, rheumatoid
arthritis, seronegative spondylarthritides, sjogren's syndrome,
systemic sclerosis, polymyositis, dermatomyositis, type 1 diabetes
mellitus, acquired immune deficiency syndrome, hand foot and mouth
disease, Hashimoto's thyroiditis, Graves's disease, Addison's
disease, polyendocrine autoimmune disease, hepatitis, sclerosing
cholangitis, primary biliary cirrhosis, pernicious anemia, coeliac
disease, antibody-mediated nephritis, glomerulonephritis, Wegener's
granulomatosis, microscopic polyarteritis, polyarteritis nodosa,
pemphigus, dermatitis herpetiformis, vitiligo, multiple sclerosis,
encephalomyelitis, Guillain-Barre syndrome, myasthenia gravis,
Lambert-Eaton syndrome, sclera, episclera, uveitis, chronic
mucocutaneous candidiasis, Bruton's syndrome, transient
hypogammaglobulinemia of infancy, myeloma, X-linked hyper IgM
syndrome, Wiskott-Aldrich syndrome, ataxia telangiectasia,
autoimmune hemolytic anemia, autoimmune thrombocytopenia,
autoimmune neutropenia, Waldenstrom's macroglobulinemia,
amyloidosis, chronic lymphocytic leukemia, and non-Hodgkin's
lymphoma.
[0081] The present methods enhance vaccine effectiveness by
targeting specific immune components for activation. Compositions
comprising component-specific immunostimulating agents in
association with colloidal metal and antigen are used. Examples of
diseases for which vaccines are currently available include, but
are not limited to, cholera, diphtheria, Haemophilus, hepatitis A,
hepatitis B, influenza, measles, meningitis, mumps, pertussis,
small pox, pneumococcal pneumonia, polio, rabies, rubella, tetanus,
tuberculosis, typhoid, Varicella-zoster, whooping cough, and yellow
fever.
[0082] The combination of route of administration and the vectors
used to deliver the antigen to the immune system is a powerful tool
in designing the desired immune response. The present invention
comprises methods and compositions comprising various vectors or
vectors in association with delivery agents, such as liposomes,
microcapsules, or microspheres that can provide long-term release
of immune stimulating vector compositions. These delivery systems
act like internal depots for holding the vector and slowly
releasing it for immune system activation. For example, a liposome
may be filled with a composition comprising a vector comprising an
antigen and component-specific immunostimulating agents associated
with colloidal metal.
[0083] The antigen/component-specific immunostimulating agent/metal
complex is slowly released from the liposome and is recognized by
the immune system as foreign and the specific component to which
the component-specific immunostimulating agent is directed
activates the immune system. The cascade of immune response is
activated more quickly by the presence of the component-specific
immunostimulating agent and the immune response is generated more
quickly and more specifically.
[0084] Other methods and compositions contemplated in the present
invention include using antigen/component-specific
immunostimulating agent/colloidal metal complexes in which the
colloidal metal particles have different sizes. Sequential
administration of component-specific immunostimulating agents may
be accomplished in a one-dose administration by the use of these
differently sized colloidal metal particles. One dose would include
four independent component-specific immunostimulating agents
complexed to an antigen and each with a differently sized colloidal
metal particle. Thus, simultaneous administration would provide
sequential activation of the immune components to yield a more
effective vaccine and more protection for the population. Other
types of such single dose administration with sequential activation
could be provided by combinations of differently sized colloidal
metal particles and liposomes or liposomes filled with differently
sized colloidal metal particles.
[0085] Use of such a vaccination system as described above is very
important in providing vaccines that can be administered in one
dose. One dose administration is important in treating animal
populations such as livestock or wild populations of animals. One
dose administration is vital in treatment of populations that are
resistant to healthcare such as the poor, homeless, rural residents
or persons in developing countries that have inadequate health
care. Many persons, in all countries, do not have access to
preventive types of health care, such as vaccination. The
re-emergence of infectious diseases, such as tuberculosis, has
increased the demand for vaccines that can be given once and still
provide long-lasting, effective protection. The compositions and
methods of the present invention provide such effective
protection.
[0086] The term "colloidal metal," as used herein, includes any
water-insoluble metal particle or metallic compound as well as
colloids of non-metal origin such as colloidal carbon dispersed in
liquid or water (a hydrosol). Examples of colloidal metals, which
can be used in the present invention include, but are not limited
to, metals in groups IIA, IB, IIB and IIIB of the periodic table,
as well as the transition metals, especially those of group VIII.
Preferred metals include gold, silver, aluminum, ruthenium, zinc,
iron, nickel and calcium. Other suitable metals may also include
the following in all of their various oxidation states: lithium,
sodium, magnesium, potassium, scandium, titanium, vanadium,
chromium, manganese, cobalt, copper, gallium, strontium, niobium,
molybdenum, palladium, indium, tin, tungsten, rhenium, platinum,
and gadolinium. The metals are preferably provided in ionic form
(preferably derived from an appropriate metal compound), for
example, the Al.sup.3+, Ru.sup.3+, Zn.sup.2+, Fe.sup.3+, Ni.sup.2+
and Ca.sup.2+ ions. A preferred metal is silver, particularly in a
sodium borate buffer, having the concentration of between
approximately 0.1% and 0.001%, and most preferably as approximately
a 0.01% solution. Another preferred metal is gold, particularly in
the form of Au.sup.3+. An especially preferred form of colloidal
gold is HAuCl4 (OmniCorp, South Plainfield, N.J.). The color of
such a colloidal silver solution is yellow and the colloidal
particles may range from 1 to 100 nanometers. Such metal ions may
be present in the complex alone or with other inorganic ions.
[0087] Any antigen may be used in the present invention. Examples
of antigens useful in the present invention include, but are not
limited to, Interleukin-1 ("IL-1"), Interleukin-2 ("IL-2"),
Interleukin-3 ("IL-3"), Interleukin-4 ("IL-4"), Interleukin-5
("IL-5"), Interleukin-6 ("IL-6"), Interleukin-7 ("IL-7"),
Interleukin-8 ("IL-8"), Interleukin-10 ("IL-10"), Interleukin-11
("IL-11"), Interleukin-12 ("IL-12"), Interleukin-13 ("IL-13"),
lipid A, phospholipase A2, endotoxins, staphylococcal enterotoxin
B, Pertussis toxin, Tetanus toxin and other toxins, Type I
Interferon, Type II Interferon, Tumor Necrosis Factor (TNF-.alpha.
or b), Transforming Growth Factor-.beta. ("TGF-.beta."),
Lymphotoxin, Migration Inhibition Factor, Granulocyte-Macrophage
Colony-Stimulating Factor ("CSF"), Monocyte-Macrophage CSF,
Granulocyte CSF, vascular epithelial growth factor ("VEGF"),
Angiogenin, transforming growth factor ("TGF-.alpha."), heat shock
proteins, Epidermal growth factor ("EGF"), carbohydrate moieties of
blood groups, Rh factors, fibroblast growth factor, and other
inflammatory and immune regulatory proteins, nucleotides, DNA, RNA,
mRNA, sense, antisense, cancer cell specific antigens; such as
MART, MAGE, BAGE, and heat shock proteins (HSPs); mutant p53;
tyrosinase; mucines, such as Muc-1, PSA, TSH, autoimmune antigens;
immunotherapy drugs, such as AZT; and angiogenic and
anti-angiogenic drugs, such as angiostatin, endostatin, basic
fibroblast growth factor, and vascular endothelial growth factor,
prostate specific antigen and thyroid stimulating hormone.
[0088] The component-specific immunostimulating agent may be any
molecule or compound which effects the immune system, for example,
any molecule that increases the APC's ability to stimulate the B
cell's production of antibodies. Examples of component-specific
immunostimulating agents include, but are not limited to, antigens,
colloidal metals, adjuvants, receptor molecules, nucleic acids,
immunogenic proteins, and accessory cytokine/immuostimulators,
pharmaceuticals, chemotherapy agents, and carriers.
[0089] Any type of pharmaceutical agent can be employed in the
present invention. For example, anti-inflammatory agents such as
steroids and nonsteroidal anti-inflammatory agents, soluble
receptors, antibiotics, analgesic, COX-2 inhibitors.
Chemotherapeutic agents of particular interest include the
following non-limiting examples, taxol, paclitaxel, taxanes,
vinblastin, vincristine, doxorubicin, acyclovir, cisplatin,
methotrexate, mithramycin and tacrine.
[0090] These component-specific immunostimulating agents may be
employed separately, or in combinations. They may be employed in a
free state or in complexes, such as in combination with a colloidal
metal.
[0091] Examples of component-specific immunostimulating agents
useful in the present invention include, but are not limited to,
Interleukin-1 ("IL-1"), Interleukin-2 ("IL-2"), Interleukin-3
("IL-3"), Interleukin-4 ("IL-4"), Interleukin-5 ("IL-5"),
Interleukin-6 ("IL-6"), Interleukin-7 ("IL-7"), Interleukin-8
("IL-8"), Interleukin-10 ("IL-10"), Interleukin-11 ("IL-11"),
Interleukin-12 ("IL-12"), Interleukin-13 ("IL-13"), lipid A,
phospholipase A2, endotoxins, staphylococcal enterotoxin B and
other toxins, Type I Interferon, Type II Interferon, Tumor Necrosis
Factor ("TNF-.alpha."), Flt-3 ligand, Transforming Growth
Factor-.beta. ("TGF-.beta.")Lymphotoxin, Migration Inhibition
Factor, Granulocyte-Macrophage Colony-Stimulating Factor ("CSF"),
Monocyte-Macrophage CSF, Granulocyte CSF, vascular epithelial
growth factor ("VEGF"), Angiogenin, transforming growth factor
("TGF-.alpha."), heat shock proteins, carbohydrate moieties of
blood groups, Rh factors, fibroblast growth factor, and other
inflammatory and immune regulatory proteins, nucleotides, DNA, RNA,
mRNA, sense, antisense, cancer cell specific antigens; such as
MART, MAGE, BAGE, and heat shock proteins (HSPs); mutant p53;
tyrosinase; autoimmune antigens; immunotherapy drugs, such as AZT;
and angiogenic and anti-angiogenic drugs, such as angiostatin,
endostatin, basic fibroblast growth factor, vascular endothelial
growth factor (VEGF) and prostate specific antigen and thyroid
stimulating hormone.
[0092] Adjuvants useful in the invention include, but are not
limited to, heat killed M. Butyricum and M. Tuberculosis.
Nonlimiting examples of nucleotides are DNA, RNA, mRNA, sense, and
antisense. Examples of immunogenic proteins include, but are not
limited to, KLH (Keyhole Limpet Cyanin), thyroglobulin, and fusion
proteins, which have adjuvant and antigen moieties encoded in the
gene.
[0093] Component-specific immunostimulating agents may be delivered
in their nucleic acid form, using known gene therapy methods, and
produce their effect after translation. Additional elements for
activation of immune components, such as antigens, could be
delivered simultaneously or sequentially so that the cellularly
translated component-specific immunostimulating agents and
externally added elements work in concert to specifically target
the immune response.
[0094] An especially preferred embodiment provides methods for
activation of the immune response using vector compositions
comprising agents comprised of a specific antigen in combination
with a component-specific immunostimulating agent. Such methods are
effective and can be used in in vitro or in vivo. As used herein,
component-specific immunostimulating agent means an agent that is
specific for a component of the immune system, such as a B or T
cell, and that is capable of affecting that component, so that the
component has an activity in the immune response. The
component-specific immunostimulating agent may be capable of
affecting several different components of the immune system, and
this capability may be employed in the methods and compositions of
the present invention. The agent may be naturally occurring or can
be generated or modified through molecular biological techniques or
protein receptor manipulations.
[0095] The activation of the component in the immune response may
result in a stimulation or suppression of other components of the
immune response, leading to an overall stimulation or suppression
of the immune response. For ease of expression, stimulation of
immune components is described herein, but it is understood that
all responses of immune components are contemplated by the term
stimulation, including but not limited to stimulation, suppression,
rejection and feedback activities.
[0096] The immune component that is affected may have multiple
activities, leading to both suppression and stimulation or
initiation or suppression of feedback mechanisms. The present
invention is not to be limited by the examples of immune responses
detailed herein, but contemplates component-specific effects in all
aspects of the immune system.
[0097] The activation of each of the components of the immune
system may be simultaneous, sequential, or any combination thereof.
In one embodiment of a method of the present invention, multiple
component-specific immunostimulating agents are administered
simultaneously. In this method, the immune system is simultaneously
stimulated with multiple separate preparations, each containing a
vector composition comprising a component-specific
immunostimulating agent. Preferably, the vector composition
comprises the component-specific immunostimulating agent associated
with the colloidal metal. More preferably, the composition
comprises the component-specific immunostimulating agent associated
with the colloidal metal of one sized particle or of different
sized particles and an antigen. Most preferably, the composition
comprises the component-specific immunostimulating agent associated
with the colloidal metal of one sized particle or of differently
sized particles, an antigen and PEG or PEG derivatives, preferably
thiol-PEG (PEG(SH).sub.n), or derivatized poly-1-lysine, preferably
poly-1-lysine thiol (PLL(SH).sub.n).
[0098] Component-specific immunostimulating agents provide a
specific stimulatory, up regulation, effect on individual immune
components. For example, Interleukin-10 (IL-10) specifically
stimulates macrophages, while TNF-.alpha. (Tumor Necrosis Factor
alpha) and Flt-3 ligand specifically stimulate dendritic cells.
Heat killed Mycobacterium butyricum and Interleukin-6 (IL-6) are
specific stimulators of B cells, and Interleukin-2 (IL-2) is a
specific stimulator of T cells. Vector compositions comprising such
component-specific immunostimulating agents provide for specific
activation of macrophages, dendritic cells, B cells and T cells,
respectively. For example, macrophages are activated when a vector
composition comprising the component-specific immunostimulating
agent IL-1.beta. is administered. A preferred composition is
IL-1.beta. in association with colloidal metal, and a most
preferred composition is IL-1.beta. in association with colloidal
metal and an antigen to provide a specific macrophage response to
that antigen. Vector compositions can further comprise targeting
molecules, integrating molecules, PEGs or derivatized PEGs.
[0099] Many elements of the immune response may be necessary for an
effective immune response to an antigen. An embodiment of a method
of simultaneous stimulation is to administer four separate
preparations of compositions of component-specific
immunostimulating agents comprising 1) IL-1.beta. for macrophages,
2) TNF-.alpha. and Flt-3 ligand for dendritic cells, 3) IL-6 for B
cells, and 4) IL-2 for T cells. Each component-specific
immunostimulating agent vector composition may be administered by
any route known to those skilled in the art, and may use the same
route or different routes, depending on the immune response
desired.
[0100] In another embodiment of the methods and compositions of the
present invention, the individual immune components are activated
sequentially. For example, this sequential activation can be
divided into two phases, a primer phase and an immunization phase.
The primer phase comprises stimulating APCs, preferably macrophages
and dendritic cells, while the immunization phase comprises
stimulating lymphocytes, preferably B cells and T cells. Within
each of the two phases, activation of the individual immune
components may be simultaneous or sequential. For sequential
activation, a preferred method of activation is administration of
vector compositions that cause activation of macrophages followed
by dendritic cells, followed by B cells, followed by T cells. A
most preferred method is a combined sequential activation
comprising the administration of vector compositions which cause
simultaneous activation of the macrophages and dendritic cells,
followed by the simultaneous activation of B cells and T cells.
This is an example of methods and compositions of multiple
component-specific immunostimulating agents to initiate several
pathways of the immune system.
[0101] One method of binding an agent to metal sols comprises the
following steps, though for clarity purposes only, the methods
disclosed refer to binding a single agent, TNF, to a metal sol,
colloidal gold. An apparatus was used that allows interaction
between the particles in the colloidal gold sol and TNF in a
protein solution. A schematic representation of the apparatus is
shown in FIG. 11. This apparatus maximizes the interaction of
unbound colloidal gold particles with the protein to be bound, TNF,
by reducing the mixing chamber to a small volume. This apparatus
enables the interaction of large volumes of gold sols with large
volumes of TNF to occur in the small volume of a T connector. In
contrast, adding a small volume of protein to a large volume of
colloidal gold particles is not a preferred method to ensure
uniform protein binding to the gold particles. Nor is the opposite
method of adding small volumes of colloidal gold to a large volume
of protein. Physically, the colloidal gold particles and the
protein, TNF are forced into a T-connector by a single peristaltic
pump that draws the colloidal gold particles and the TNF protein
from two large reservoirs. To further ensure proper mixing, an
in-line mixer is placed immediately down stream of the T-connector.
The mixer vigorously mixes the colloidal gold particles with TNF,
both of which are flowing through the connector at a preferable
flow rate of approximately 1 L/min.
[0102] Prior to mixing with the agent, the pH of the gold sol is
adjusted to pH 8-9 using 1 N NaOH. A preferred method for adjusting
the pH of the gold sol uses 100 mM TRIS to adjust the pH of the
colloidal gold sol to pH 6. Highly purified, lyophilized
recombinant human TNF is reconstituted. A preferred method for
diluting TNF is in water that has been adjusted to pH 6 with 100 mM
TRIS. Before adding either the sol or TNF to their respective
reservoirs, the tubing connecting the containers to the T-connector
is clamped shut. Equal volumes of colloidal gold sol and TNF
solution are added to the appropriate reservoirs. Preferred
concentrations of the active agent in solution range from
approximately 0.01 to 15 .mu.g/ml, and can be altered depending on
the desired ratio of the agent to metal sol particles. Preferred
concentrations of TNF in the solution range from 0.5 to 4 .mu.g/ml
and the most preferred concentration of TNF for the TNF-colloidal
gold composition is 0.5 .mu.g/ml.
[0103] Once the solutions are properly loaded into their respective
reservoirs, the peristaltic pump is turned on, drawing the agent
solution and the colloidal gold solution into the T-connector,
through the in-line mixer, through the peristaltic pump and into a
collection flask. The mixed solution is stirred in the collection
flask for an additional hour of incubation.
[0104] In compositions comprising PEG, whether derivatized or not,
the methods for making such compositions comprise the following
steps, though for clarity purposes only, the methods disclosed
refer to adding PEG thiol to a metal sol composition. Any PEG,
derivatized PEG composition or any sized PEG compositions or
compositions comprising several different PEGs, can be made using
the following steps. Following the 15-minute incubation, a thiol
derivatized polyethylene glycol (PEG) solution is added to the
colloidal gold/TNF sol. The present invention contemplates use of
any sized PEG with any derivative group, though preferred
derivatized PEGs include mPEG-OPSS/2,000, mPEG-OPSS/5,000,
mPEG-OPSS/10,000, mPEG-OPSS/12,000, mPEG-OPSS/20,000,
mPEG-OP(SS).sub.2/2,000, mPEG-OP(SS).sub.2/3,400;
mPEG-OP(SS).sub.2/8,000- , mPEG-OP(SS).sub.2/10,000,
thiol-PEG-thiol/2,000, mPEG-thiol 5,000, and MPEG thiol 10,000,
mPEG thiol 12,000, mPEG thiol 20,000 (Sun-BIO Inc.). A preferred
PEG is mPEG-thiol 5000 at a concentration of 150 .mu.g/ml in water,
pH 5-8. Thus, a 10% v/v of the PEG solution is added to the
colloidal gold-TNF solution. The gold/TNF/PEG solution is incubated
for an additional 15 minutes.
[0105] In a preferred method, the TNF and PEG-THIOL moiety
simultaneously binds to the colloidal gold nanoparticle. In this
method the pH of the colloidal gold nanoparticles is adjusted to
6.0 using 100 mM TRIS Base. Similarly the pH of water is adjusted
to 6.0 using the 100 mM TRIS solution. Into the latter solution TNF
and PEG-THIOL (20,000) are diluted to a final concentration of 5
and 15 ug/ml, respectively. Both the colloidal gold nanoparticles
and TNF/PEG-THIOL solutions are loaded into their respective
reservoirs and bound through the T-connector and in-line mixer
using a peristaltic pump to draw each solution through the
T-connector. After binding for 15 minutes Human Serum Albumin (200
.mu.g/ml in H.sub.2O) is added to the colloidal gold/TNF/PEG-THIOL
solution and incubated for an additional 15 minutes.
[0106] The colloidal gold/TNF/PEG solution is subsequently
ultrafiltered through a 50-100 K MWCO diafiltration cartridge. The
50-100 K retentate and permeate are measured for TNF concentration
by ELISA to determine the amount of TNF bound to the gold
particles.
[0107] After diafiltration, cryoprotectants, such as a compositions
of mannitol, 20 mg/ml; and/or human serum albumin, 5 mg/ml, are
added and the samples frozen at -80.degree. C. The samples are
lyophilized to dryness and sealed under a vacuum, subsequently
reconstituted and analyzed for the amount of free and colloidal
gold bound TNF present in the reconstituted samples.
[0108] The compositions of the present invention can be
administered to in vitro and in vivo systems. In vivo
administration may include direct application to the target cells
or such routes of administration, including but not limited to
formulations suitable for oral, rectal, transdermal, ophthalmic,
(including intravitreal or intracameral) nasal, topical (including
buccal and sublingual), vaginal or parenteral (including
subcutaneous, intramuscular, intravenous, intradermal,
intratracheal, and epidural) administration. A preferred method
comprising administering, via oral or injection routes, an
effective amount of a composition comprising vectors of the present
invention.
[0109] The formulations may conveniently be presented in unit
dosage form and may be prepared by conventional pharmaceutical
techniques. Pharmaceutical formulation compositions are made by
bringing into association the metal sol vectors and the
pharmaceutical carrier(s) or excipient(s). In general, the
formulations are prepared by uniformly and intimately bringing into
association the compositions with liquid carriers or finely divided
solid carriers or both, and then, if necessary, shaping the
product.
[0110] This invention is further illustrated by the following
examples, which are not to be construed in any way as imposing
limitations upon the scope thereof. On the contrary, it is to be
clearly understood that resort may be had to various other
embodiments, modifications, and equivalents thereof which, after
reading the description herein, may suggest themselves to those
skilled in the art without departing from the spirit of the present
invention and/or the scope of the appended claims.
EXAMPLES
Example 1
Manufacture of Colloidal Gold
[0111] Colloidal gold sols are manufactured using the reaction
described by Frens and Horisberger (Frens, G. Nature Phys. Sci.
1972, 241, 20-22, and Horsiberger, M. Biol. Cellulaire. 1979. 36:
253-258). In this reaction ionic gold, in the form of HAuCl.sub.4,
is reduced to nanoparticles of Au.sup.0 by the addition of sodium
citrate. Typically, 2.5 ml of a 4% chloroauric acid (in water)
solution is added to 1 L of deionized water. The solution is
vigorously stirred and heated to a rolling boil. The reduction
reaction is initiated by the addition of a 1% sodium citrate
solution. The size of the particle is controlled by the amount of
citrate added to the reaction. For example, 17, 32, and 64 nm
particles are formed by the addition of 40, 15, and 7.5 ml of the
citrate solution, respectively. After the addition of citrate, the
solution is allowed to boil and mix for an additional 45 minutes.
Upon cooling, the sol is filtered through a 0.22 .mu.m
sterilization filter and stored at room temperature until used.
[0112] The production of colloidal gold sols has been scaled-up
from 1.0 L to 10 L. UV-VIS wavelength scans, dynamic light
scattering, and differential centrifugation techniques are used to
check these particles for average particle size and homogeneity.
Manufactured particles have a mean particle size that routinely
measures within 10% of their predicted size and exhibit a
poly-dispersity measure of 1.03-1.12 or less.
Example 2
Increasing the Number of Immune Competent B Cells
[0113] To increase the number of immune competent B cells for
immunization, MHC class II restricted-surface IgM.sup.+/sIgD.sup.+
human B cells are isolated from units of whole blood or buffy
coats. Magnetic beads coated with anti-IgM, anti-IgD and anti-CD19
antibodies separate the B cell populations. Treating
sIgM.sup.+/sIgD.sup.- immature B cells with the cytokine
interleukin-7 is used to recruit additional B cells (Sudo, T., Ito,
M., Ogawa, Y., Iizuka, M., Kodoma, H., Kunisasa, T., Hayashi, S.
C., Ogawa, M., Sakai, K., Nishikawa, S., Nishkawa, S. C. J. Exp.
Med. 1989. 170: 333-338). This treatment has been shown to mature
these B cells as signaled by the phenotype conversion of
sIgM.sup.+/sIgD.sup.- B cells to sIgM.sup.+/sIgD.sup.+ B cells.
These isolated cells are purified to near homogeneity using FACS
separation.
[0114] Conjugating TNF to carriers such as KLH or thyroglobulin
(see discussion below) enhances the antigenicity of human TNF.
TNF:KLH antigen is bound to the surface of colloidal gold particles
which contain a B cell targeting/activating agent such as
interleukin-6 (IL-6). IL-6 is a cytokine known to stimulate the
synthesis of antibodies from immunized B cells. Having both
moieties on the same particle of gold, ensures that B cells receive
the KLH:TNF antigen signal as well as the IL-6 signal to activate
the antibody response.
Example 3
Differentiation of the Primary Antibody Response
[0115] Critical to the production of a therapeutic antibody is the
process of class switching. The primary antibody response from
immunized human B cells results in the secretion of IgM antibodies.
A second class of lymphoid cells, known as antigen presenting cells
(APCs), also internalizes the antigen. Once internalized these
cells process the protein antigen into fragments, which are then
expressed on the cell's surface bound to one of two major
histocompatibility complexes (MHCs).
[0116] Depending on the microenvironment, APCs expressing antigen
bound to class II MHC molecules activate one of two subsets of
CD4.sup.+ T cells. These cells, also known as helper T cells,
perform the necessary accessory functions to facilitate the
cellular or the humoral (antibody) immune response. T.sub.H1
CD4.sup.+ cells facilitate the cellular immune response, while the
T.sub.H2 subset of CD4.sup.+ cells interact with IgM secreting B
cells to initiate the process of class switching.
[0117] The activation of CD4.sup.+ T.sub.H2 T cells by the APC
occurs with the formation of a bicellular cleft known as the immune
synapse. The formation of the immune synapse involves interaction
and rearrangement of signaling and structural ligands on the APC
with their respective receptors on the T cell to form a
three-dimensional (3-D) bridge that allows contact and signaling
between these two cells (FIG. 1). Antigen signaling between the APC
and the T cell occurs through the binding of the MHC/antigen
complex with the T cell receptor complex, while the structural
integrity of the immune synapse is maintained by the interaction of
ICAM, LFA-3, and CD72 on the APC with LFA-1, CD2, and CD5 receptors
on T cells, respectively. The successful formation of the immune
synapse causes the CD4.sup.+ T cell to express a B cell stimulatory
molecule known as CD40 ligand.
[0118] The formation of the immune synapse may signal the T cell to
become active or inactive (anergic). Which response is initiated is
dependent on the strength of the co-stimulatory signals provided by
the B7 molecule on the APC to the T cell. The B7 molecule may
interact with either B7 receptor molecule on the T cell, CD28 or
CTLA.sub.4. These B7 receptors differ with respect to their density
on the surface of the T cell as well as their affinity for the B7
molecule. CD28 has a lower affinity for B7 than CTLA.sub.4, but is
present at a much higher density on the surface of the T-cell. The
binding of B7 to the CD28 receptor sends an activation signal to
the T cell while the binding of B7 by CTLA.sub.4 induces T cell
anergy (Kuby, J., Immunology Third Edition 1997. eds Allen D.,
pp-213-218). Thus presenting excess B7 in the immune synapse will
ensure that the T cells will be activated.
[0119] In this process immunized human B cells undergo
rearrangement of the immunoglobulin genes to produce highly
specific high affinity IgG antibodies.
[0120] This vector is initially assembled from MHC, B7, and ICAM
proteins onto the surface of colloidal gold particles. The
presentation of the immune synapse is in the 3-D orientation to
allow this vector to successfully trigger CD4.sup.+T-cells to
express CD40 ligand in an MHC-restricted fashion.
[0121] This process is also optionally initiated by using the sTc
that expresses CD40 Ligand in combination with various cytokines
and the synthetic germinal center whose multiple molecules signal
the affinity maturation critical to a therapeutic mAb.
Example 4
Creation of sAPC/sTc/sGC with Spacer Arms
[0122] This sAPC is built on streptavidin colloidal gold particles
that are used to bind biotinylated forms of the MHC, B7, and ICAM
proteins. This single particle sAPC has a greater degree of
flexibility, since the constituent proteins are bound to the
colloidal gold particle indirectly through biotinylated spacer arms
that form a biotin-avidin bridge. Similarly, the sTc and sGCs may
be generated using a similar strategy for tethering their
respective components to the colloidal metal.
Example 5
Self-Assembling APCs/sTcs/sGCs
[0123] Self-assembling synthetic APCs are developed. Binding each
APC protein to a different colloidal gold particle creates a
complex matrix of immune synapse proteins. To direct the assembly
of this sAPC, site directed molecular scaffolds are made to better
orient the various particles in 3-D. Shown in FIG. 5 is a
representation of this self-assembling sAPC. The formulation of
each particle subunit allows for a single particle to bind multiple
reagents. For illustration purposes the MHC class II molecule is
bound to a 32 nm colloidal gold particle that is also bound with
streptavidin. The remaining two subunits of the sAPC, the B7 and
ICAM, are bound to 17 nm particles. Like the MHC particle the ICAM
subunit contains streptavidin-docking sites. To assemble this
particle biotinylated human serum albumin is used to join the ICAM
and MHC particles together. To complete the assembly of the vector,
dithiolated polyethylene glycol is used to link the MHC and B7
particles together.
[0124] In this model, the formation of the immune synapse occurs
through T-cell receptor/membrane rearrangements. This vector may
also be bound to a solid support stage such as an EIA plate. These
scaffolds allow both colloidal gold-targeted antigens and sAPCs
present in the same matrix. As a result, upon immunization of the
naive B-cell the sAPC may activate the CD4 cell to express CD40
ligand and as a result induce class switching.
[0125] By changing the binding partner to CD40L/cytokine or
BLYS/CD30L the self-assembling synthetic T cells or synthetic
germinal centers are generated.
Example 6
Binding of Proteins to Colloidal Gold Particles
[0126] The binding of proteins to colloidal gold particles is
influenced by the pH of the colloidal gold sol and protein
solutions. At an optimal pH, proteins bind to the surface of
colloidal gold particles and prevent their precipitation by salts.
Salt-induced precipitation of the colloidal gold is easily
documented by the changes in the color of the sol from red to
black. The pH binding optimum is determined for each protein
described, including the MHC, B7, ICAM, IL-6 and the KLH:TNF
antigen. As an example, the procedure described below outlines the
method for binding the MHC molecule to the colloidal gold
particles. A similar procedure will be used to determine the
binding conditions for each of the other proteins
[0127] The pH binding optimum for MHC binding to colloidal gold is
determined by adjusting the pH of 1 ml aliquots of colloidal gold
from 4-11 with 1N NaOH. 100 .mu.l aliquots from each of the gold
solutions are placed into micro-centrifuge tubes and incubated for
30 minutes with 1 ng of the MHC protein. 100 .mu.l of a 10% NaCl
solution is then added to each tube. The pH binding optimum is
defined as the pH that allows the MHC protein to bind to the
colloidal gold particles, while preventing salt-induced
precipitation.
[0128] In addition to determining the pH binding optimum, a
saturation binding analysis is performed for each protein. For this
test the pH of the colloidal gold particles will be adjusted to the
pH binding optimum as described above. Subsequently increasing
amounts (0.025-5 ng of protein) of the MHC protein is added to the
100 .mu.l aliquots of colloidal gold. After binding for 30 minutes,
the various aliquots are centrifuged at 10,000 rpms to separate
free from colloidal gold bound protein. The supernatant and
colloidal gold pellets are analyzed for the relative amount of MHC
protein present in each fraction.
Example 7
Quantification of the Mass of the MHC Protein Bound
[0129] To quantify the mass of the MHC protein bound per particle
of gold, quantitative EIAs are developed for the measurement of the
MHC and B7 proteins. EIAs for ICAM are already commercially
available. The MHC and B7 proteins are quantitatively measured by
developing a competitive binding EIA for each protein. Commercially
available antibodies to B7 and MHC proteins (both antibodies are
available from Research Diagnostics, Inc.) are coated onto EIA
plates using a carbonate/bicarbonate buffer at pH 9.6. MHC and B7
reference standards are generated to provide a dose range of 1.56
ng/ml to 500 ng/ml. These standards are added to the EIA plate
containing specific antibodies for either the MHC or B7 protein.
The colloidal gold bound samples are added to other designated
wells in the EIA plate.
[0130] The concentration of the various proteins is determined by
establishing a competitive binding reaction between the protein
present in the sample or standard and a biotinylated form of the
molecule for antibody sites. The biotinylated ligand is detected
with streptavidin alkaline phosphatase. Upon the addition of
substrate, an inverse relationship is generated between the mass of
analyte present in the sample and the amount of color
developed.
Example 8
Binding Multiple Proteins to the Same Particle
[0131] To increase the efficiency and specificity of the in vitro
immunization multiple chemically distinct proteins need to be bound
onto the surface of a single colloidal gold particle. The binding
of three different protein cytokines (IL-1, IL-6 and TNF) to the
same particle of colloidal gold is demonstrated. Each cytokine
binds to colloidal gold at a specific pH.
[0132] As demonstrated above, it was determined that IL-1 bound to
colloidal gold at a pH between 6 and 8 while TNF and IL-6 bound at
a pH of 8 and 11, respectively. A solution containing 0.25 ng/ml of
the three cytokines in water was mixed with a colloidal gold sol at
pH 8. A sample was removed and the pH of the remaining solution was
adjusted to 11. Prior to each pH change additional samples were
collected. The two samples were centrifuged and the resultant
pellets of colloidal gold were re-suspended in PBS.
[0133] To demonstrate the presence of all three cytokines on the
same particle of gold the various pellets were added to an EIA
plate that was coated with a monoclonal antibody to TNF. After
binding, the plate was washed and designated wells were incubated
with either an alkaline phosphatase conjugated rabbit anti IL-1,
IL-6, or TNF. After a wash, substrate was added to each well to
initiate color development. The data presented in FIG. 6 show that
due to the overlap in pH binding optimum both IL-1 and TNF were
present on the particle at pH 8. However, very little IL-6 signal
could be detected. Increasing the pH 11 allowed IL-6 to bind to
these particles.
Example 9
Targeting of Chimeric Vectors to Specific Cells
[0134] EGF and streptavidin were bound to the same 32 nm particle
of colloidal gold. The sample was divided into three aliquots for
the binding of secondary/targeting molecules. One sample was bound
with biotinylated IL-1, another biotinylated GM-CSF, and the third
with biotinylated IL-6. After binding the biotinylated ligands, the
samples were centrifuged to remove any free reagents and the
colloidal gold pellets were added to Ficoll-separated human white
blood cells. After 8 days in culture the uptake of the various
colloidal gold vectors was documented by digital photography.
[0135] EGF streptavidin gold was targeted to macrophages (FIG. 7A),
dendritic cells (FIG. 7B) and B-Cells (FIG. 7C) by using
biotinylated IL-1, biotinylated GM-CSF, or biotinylated IL-6 for
targeting. The black staining (highlighted by the red arrows) in
each of the figures represents the uptake of the various colloidal
gold vectors.
[0136] As can be seen in FIG. 7, the various colloidal
gold/cytokine chimeras differentially targeted the various cellular
elements of the immune system. The black staining (highlighted by
the arrows) represents the colloidal gold particles, which have
been internalized and aggregated by the various immune cells. These
data indicate that IL-1 targeted the colloidal gold EGF to
macrophages, while GM-CSF targeted the chimera to dendritic cells,
and IL-6 targeted the vector to B cells.
Example 10
Immunization of Human Lymphocytes
[0137] These vectors can be used to generate a primary immune
response from isolated lymphocytes. White blood cells were
collected from whole blood by density centrifugation. These cells
were treated with a thyroglobulin conjugated TNF/IL-6 colloidal
gold vector. The cells received pulses of the colloidal gold vector
every 2 days for a total of eight days. After the final pulse, the
cells were cultured for another 5 days. The supernatants were
collected and tested for the presence of human anti-human TNF
(IgM/IgD and IgG combination) antibodies using a direct EIA. As can
be seen in FIG. 8, the chimeric cAU thyroglobulin TNF had the
highest immunodensity.
Example 11
Immunization of Human B-Cells and Dendritic Cells for Class II MHC
Expression
[0138] Two different approaches to increase the efficiency of in
vitro human lymphocyte immunization are used. First, coupling TNF
to immunogenic carriers, such as Thyroglobulin, Keyhole Limpet
Hemocyanin or Murine Serum Albumin enhances TNF's s immunogenicity.
Carrier:TNF conjugations are performed using standard EDC/NHS and
gluteraldehyde methods. Second, coupling them to particles of
colloidal gold, containing cell-specific targeting agents increases
the specificity of these antigens. To target the delivery of the
antigen to B cells the carrier:antigen complex is bound to
particles of colloidal gold particles containing IL-6. To target
the delivery of the carrier antigen to dendritic cells the
carrier:antigen complex is bound to colloidal gold particles
containing GM-CSF.
[0139] These vectors are initially used to immunize naive MHC
restricted human B cells and dendritic cells for the generation of
the class II MHC antigen. These same vectors are used at a later
time to induce the primary antibody response from a new or
replicate set of naive B cells. The immunization scheme involves
the sequential immunization of B cells and dendritic cells with the
various vectors. As a result the B cells and dendritic cells see
the carrier once and the TNF antigen three times.
Example 12
Generation of Class II MHC Protein by B Cells
[0140] To cause human B-cells to produce class II MHC protein,
10.sup.6 surface IgM.sup.+/IgD.sup.+ human B-cells are plated in
24-well plates and cultured in 1.5 ml of AIM V media. Twenty four
hours after plating, the cells are pulsed with the THYRO:TNF
antigen bound to an IL-6 targeted colloidal gold vector. Two days
later the cells are pulsed with the KLH:TNF carrier targeted by the
IL-6 vector. After an additional two days in culture the cells are
immunized with the third carrier:TNF antigen, MSA:TNF. The cells
are incubated for an additional three to seven days and tested for
the presence of Class II MHC expression by FACS analysis.
Alternatively, the cells may be simultaneously pulsed with the
colloidal gold antigens.
[0141] A similar procedure is used to pulse dendritic cells to
express the MHC class II protein. These cells are immunized with
the TNF:carrier antigen bound to GM-CSF targeted colloidal gold
vectors. Dendritic cell precursors are isolated from peripheral
blood using magnetic beads coated with anti-CD34. These cells are
expanded in vitro by incubating them in AIM V serum free media
supplemented with 1000 ng/ml of GM-CSF and 100 ng/ml of IL-4. Upon
their maturation, confirmed by FACS analysis for the detection of
CD1a and empty class II MHC molecules, the cells are differentiated
into mature dendritic cell with a 10 ng/ml pulse of TNF. These
mature dendritic cells are immunized with the GM-CSF targeted
colloidal gold TNF antigen. Antigen loaded MHC class II protein
complexes are detected by FACS or in situ analysis of the
biotinylated antigen peptide detected with a streptavidin
conjugated phycoerythrin (Research Diagnostic Inc.) detection
system.
Example 13
Method Development for the Isolation of the MHC Class II
Antigen
[0142] The method for the isolation of the MHC uses
"generic"-non-MHC compatible blood samples. These MHC molecules are
used to define the pH and saturation optima for the protein on
colloidal gold particles. Once defined, these methods are adapted
to purify antigen loaded MHC from immunized MHC restricted blood
pools.
[0143] The isolation of generic and antigen loaded human class II
MHC is done using the method described by Sette (Sette et al., J.
Immunol. 1992. 148: 844). Briefly the buffy coats from non-HLA
matched human whole blood are frozen at a minimum density of
10.sup.8 cells/ml and sonicated to disrupt the cells. These cells
are suspended in a buffer of 50 mM TRIS-HCl, pH 8.5 with 2% Renex,
150 mM NaCl, 5 mM EDTA and 2 mM PMSF. Large particulates including
the nuclei are removed by centrifugation (10000.times.g for 20
minutes). The cell lysate is then fractionated on an affinity
column made by binding murine antibodies to the human class II MHC
molecule (Research Diagnostics Inc.) to protein A/G sepharose
beads. The lysate is passed through the column at least 5 times to
maximize the binding of the MHC protein to the immobilized
antibody. The column is washed with 10 column volumes of a buffer
containing 10 mM TRIS-HCI pH 8.0/0.1% Renex followed by an
additional wash of 5 column volumes of PBS with 1%
n-ocytlglucoside. The MHC class II protein is eluted from the
column using a buffer of 50 mM diethylamine in saline with 1%
n-ocytlglucoside at a pH 11.5. Upon elution each fraction is
immediately neutralized with the addition of 2 M glycine, pH 2.0.
The fractions containing the MHC II molecules are aliquoted and
lyophilized in 25 .mu.g aliquots.
Example 14
Generation of Human B7.1 Molecule
[0144] The human co-stimulatory molecule B7.1 is made by
recombinant DNA technology. The gene is supplied as part of a
commercially available transient expression vector system (In
Vivogen Inc.). The construct is provided with the appropriate
restriction sites allowing for the separation of the active gene
from the plasmid construct. The human B-7.1 gene is isolated from
the pORF host plasmid using the restriction enzyme NcoI and NheI.
This double digestion results in the formation of two linearized
pieces of DNA. One of the gene fragments consists of the B-7.1 gene
(893 bp) while the other fragment (3210 bp) constitutes the
accessory genes of the p-ORF plasmid. The gene fragments are
fractionated on a 1% agarose gel and visualized by ethidium bromide
staining. The bands are cut from the gel and purified using
QuiaQuick gel extraction resin. The purified linearized gene is
inserted into a baculovirus expression system (CloneTech Inc.)
under the control of the strong CMV promoter. The baculovirus
incorporated genes are transfected into the SF9 insect cell line
according to the manufacturers specifications and conditions.
10.sup.6 B7 transfected NOS cells will be expanded in bioreactors.
The incubation media and cell lysates are processed by affinity
chromatography using a murine monoclonal antibody against the human
B7.1 protein (Research Diagnostics Inc.) previously immobilized to
a protein A/G sepharose column.
Example 15
Generation of the Synthetic Antigen Presenting Cell: The Single
Particle sAPC
[0145] To mature the primary antibody response the sAPC capable
must induce the CD4 T-cell/B-cell interactions that result in
antibody class switching. The first sAPC is developed by binding
the proteins of the immune synapse on a single particle of
colloidal gold. This vector as well as one built on a streptavidn
colloidal gold core are tested for their ability to activate
CD4.sup.+ T-cells.
[0146] Once the components of the immune synapse are isolated and
purified to homogeneity they are bound to colloidal gold particles
to develop the single particle sAPC. Two strategies are used to
develop these APCs. The first strategy involves the direct binding
of the components of the immune synapse (i.e., the peptide loaded
MHC, B7 and ICAM molecules) to particles of colloidal gold. While
not wishing to be bound, it is believed that each ml of gold will
bind 250 ng of each protein/ml of colloidal gold sol.
[0147] The first scaffold was assembled on the surface of an EIA
plate. The materials include an EIA plate coated with a monoclonal
antibody to human TNF; a 32 nm TNF/streptavidin colloidal gold
chimera; biotinylated BSA: a 17 nm streptavidin colloidal gold
vector; biotinylated human IL-6, Rabbit anti human IL-6 conjugated
to alkaline phosphatase. The various components were assembled into
a scaffold as depicted in FIG. 9A. The control for this study
simply was the 32 nm particle without the TNF docking site upon
which the scaffold was built. As presented in FIG. 10A a strong
signal was generated when all of the molecular bricks of the
scaffold were present. By merely omitting the TNF docking site the
scaffold did not form and as a result no signal was generated.
[0148] The direct binding of the immune synapse proteins to a
single particle of colloidal gold results in a rigid orientation of
the proteins on the surface of the particle. To increase the
flexibility of movement for these proteins on sAPC an alternative
single particle sAPC is developed. This single particle sAPC is
developed on a streptavidin colloidal gold platform that binds
biotinylated forms of the MHC, B7, and ICAM proteins. The proteins
are bound to the streptavidin gold particle through the biotin
residue that is linked to the protein through a spacer arm.
[0149] The proteins are biotinylated using several biotinylating
reagents such as NHS-Biotin (Pierce Chemical Co.). This reagent
places a 1.35 nm spacer arm between the protein and biotin
moieties. Alternatively, NHS-LC-LC-Biotin is used to biotinylate
the proteins. This agents place a 3.05 nm spacer arm between the
protein and biotin residue. Such a spacer arm facilitates movement
of the proteins to promote ligand binding. This added flexibility
improves the ability of the proteins to achieve a proper 3-D
orientation and to form a functional immune synapse with the
CD4.sup.+T-cell.
Example 16
Generation of a Self-Assembling sAPC
[0150] The multiparticle sAPC will have the flexibility of self
oreintation during immune synapse formation. The flexibility is a
direct result of assembling the moieties used to join the particles
together. Linkers can be alkane, protein, and polyethylene glycol
(PEG) to allow for the greatest vector functionality.
[0151] The second scaffold (shown in FIG. 9B) was assembled using a
four-arm Polyethylene glycol (10,000 MW) backbone containing four
terminal free thiols. This linker was used to join individual
particles of colloidal gold bound with either IL-1 or TNF. After
linkage the preparation was centrifuged and assayed for both
proteins using an EIA plate coated only with an IL-1 monoclonal
antibody. After binding the plate was washed and detected using
enzyme linked IL-1 or TNF polyclonal antibodies. Similarly the
vector described in FIG. 9B generated a signal (FIG. 10B) for both
proteins only in the presence of the linker. Without the linker
only background color was observed.
[0152] To further increase the flexibility of the sAPC the
component proteins are assembled on different particles of
colloidal gold. These particles are assembled into a scaffolding
system to generate a sAPC capable of inducing CD4.sup.+ T-cell
activation. The multi-particle sAPC may be used in solution or as
is shown in FIGS. 9A and 9B to provide a solid support on an EIA
plate.
[0153] The MHC, B7 and ICAM proteins are bound to different
particles of colloidal gold as previously described. The particles
are physically joined by a variety of scaffolding molecules. The
function of the "joining" molecules is to provide greater
flexibility of the individual particles of colloidal gold in the
formation of the immune synapse. This flexibility occurs whether
the sAPC is provided as an independent particle or as part of a
matrix bound to a solid surface.
[0154] The first additive consists of modified di-thiol alkane
moieties. The function of alkane di-thiol binds, through the
formation of a thiol-gold bond, the individual particles of
colloidal gold together. These moieties have been used to build
self-assembling gold structures on the surface of glass slides in
the development of biosensors (Mirkin, C. A., Letsinger, R., Mucic,
R. C., and Storhoff, J. J. Nature. 1996. 382 607-609). The thiol
group allows the binding of the alkane moiety directly to the
surface of the colloidal gold particle. Examples of the
commercially available alkane thiol reagents include: 1,5 pentane
di-thiol, 1,6 hexane di-thiol, and decane di-thiol (Sigma Chemical
Company).
[0155] As an alternative to the alkane di-thiols various sizes of
2, 3 and 4-arm poly-ethylene glycol (SunBio, Walnut Creek, Calif.)
are also used. Each arm of these polymers has a free thiol group,
which is used to bind the individual particles of colloidal gold
through the formation of a gold-thiol bound. These reagents provide
the added advantage of complete solubility in water.
[0156] Binding multiple protein moieties of the immune synapse to
either single or multiple particles of colloidal gold enables the
generation of a synthetic antigen-presenting cell (sAPC) capable of
driving the cellular events that cause class switching in immunize
human B-cells.
Example 17
Stimulation of CD4.sup.+T-Cells by sAPC to Express CD40 Ligand
[0157] Single particles and self-assembling sAPCs are tested for
their ability to induce the expression of CD40 ligand from MHC
restricted CD4.sup.+ T-cells. Subsequently, 0.1 to 10 ug of antigen
loaded MHC (present on the sAPC) are added to 10.sup.6 class II
restricted CD4.sup.+ T cells growing in AIM V media. The
stimulation occurs in the presence of IL-4 and IL-10, which drives
the production of the T.sub.H2 subset of CD4.sup.+ T cells. After
4, 12 and 24 hours of sAPC stimulation the CD4 cells are collected
and stained with a FITC labeled mouse anti human CD40 ligand
antibody and analyzed by FACS.
[0158] During the activation of the CD4.sup.+ T cells a new set
(i.e., cells not used for the isolation of the MHC) of MHC
restricted B cell lymphocytes are immunized as was previously
described to undergo the production of antigen specific IgM
antibodies. MHC restricted B cells are immunized using the targeted
TNF antigens previously described. Upon the detection of antigen
specific IgMs and CD40 ligand production from their respective
cells the activate CD4.sup.+ T cells are added to IgM secreting B
cells. Class switching is monitored by the detection
human-anti-human TNF IgGs. IgG positive clones are fused with the
K6H6/B5 mouse human heteromyeloma cell line as described below.
Example 18
Antibody Detection and Immortalization of B Cells
[0159] All of the cells from positive wells are combined,
centrifuged once, washed with PBS and combined with
2.times.10.sup.6 mouse/human heteromyeloma K6H6/B5 cells. The
heteromyeloma cell line, K6H6/B5 (available through the ATCC), is
an ideal fusion partner for these human lymphocytes because these
cancer cells are non-secretors of antibody and are available with
no patent restrictions. The human and myeloma cells are fused using
standard fusion protocols with PEG. Successfully fused cells are
selected using traditional HAT/HT selection protocols. A direct
ELISA is used to test growing clones for the production of TNF
specific human IgG antibody. Those clones that show antigen
recognition are scaled-up in T-75 flasks, at which point all clones
are cryopreserved and their supernatants tested for neutralizing
antibody activity as described below.
Example 19
Neutralization of TNF Biologic Activity
[0160] The ability of the TNF antibodies to neutralize the biologic
activity of TNF is tested using the well-characterized WEHI 164
bioassay. Briefly, TNF dose-dependently inhibits the in vitro
proliferation of these cells. For this bioassay 5000 WEHI cells are
plated in 24-well tissue culture clusters. TNF (15.6 pg/ml to 500
pg/ml) is added to designated wells in the plate. To determine the
ability of the human monoclonal antibodies to neutralize the action
of TNF an identical standard dose range of TNF standards is made in
the presence of 1 .mu.g of each of the TNF monoclonal antibodies.
The cells with the various treatments are cultured for 5 days and
cell number is determined using a Coulter Counter.
Example 20
Effect of Ionic Strength on the Lyophilization Stability of
Colloidal Gold Bound TNF
[0161] The colloidal gold binding apparatus, shown in FIG. 11, was
used to bind TNF to colloidal gold nanoparticles as previously
described. After binding, 30K PEG-Thiol was added to the solution
at 50 .mu.g/ml in deionized water, pH 9.
[0162] To test the effect of ionic strength on the stability of the
TNF-colloidal gold bond various amounts of salt (in the form of
1.times. normal phosphate buffered saline; PBS) were added to the
container holding the TNF solution. Final concentrations of PBS
varied from 0 to 0.325% of normal PBS. After binding and
diafiltration, cryoprotectants (mannitol, 20 mg/ml; human serum
albumin, 5 mg/ml) were added to the samples. The samples were
subsequently aliquoted into 1 ml samples and frozen at -80.degree.
C. After freezing the samples were lyophilized to dryness and
sealed under a vacuum.
[0163] Subsequently the samples were reconstituted with 1 ml of
deionized water and diluted ten-fold in a 1% PEG-1450/water
solution. The samples were centrifuged to separate colloidal gold
bound TNF from free TNF. Both the colloidal gold pellets and
supernatants were analyzed for TNF concentrations by EIA. The data
from these studies are presented in Table I.
1TABLE 1 Release Profile of Lyophilized Colloidal Gold TNF
Manufactured in the Absence of Salt Percent of Total TNF Colloidal
Gold Pellet 68 Supernatant 32
[0164] Table I shows that 32% of the TNF is released from the
vector following lyophilization. In repetitive studies we observed
that as much as 50% of the protein is released after
lyophilization.
Example 21
Effect of Increasing Ionic Strength on the Stability of a
Lyophilized Colloidal Gold-TNF Drug
[0165] The solution of TNF, which was previously diluted in a 3 mM
TRIS solution to a final concentration of TNF of 0.5 .mu.g/ml, was
modified by adding 0.25.times. solution (77.25 milli-osmol/kg) of
normal phosphate buffered saline. The solution was bound as was
described above. After binding, 30K PEG-Thiol and the
cryoprotectants described above were added and the samples frozen
at -80.degree. C. The samples were lyophilized as described above,
subsequently reconstituted and analyzed for the amount of free and
colloidal gold bound TNF present in the reconstituted samples. The
data from this study are presented in FIG. 12.
[0166] As can be seen in FIG. 2, increasing the ionic strength
significantly improves the stability of the vector during
lyophilization. The salting effect was dose dependent. As shown in
Table II as the amount of salt added to the TNF is decreased more
of the protein is released after lyophilization.
2TABLE II Effect of Salt Concentration on TNF Release Following
Vector Lyophilization Salt Concentration 0 4.5 19.3 46.5 77.25
(milli-osmol/kg) Percent Released 40 30 10 6 6
[0167] Binding TNF in the absence of salt results in a portion of
the TNF being bound in the ionic double layer rather than directly
on the gold particle (FIG. 13). During lyophilization water
solvating the ionic layer is lost and thus upon reconstitution this
vector released the portion of TNF bound in the ion cloud. By
increasing the ionic strength through the addition of salt the
ionic layer shrinks/collapses (FIG. 14) and allows for all of the
TNF to bind directly to the particles' surface. After
lyophilization, this preparation has all of the TNF bound to the
particles' surface.
[0168] All patents, publications and abstracts cited above are
incorporated herein by reference in their entirety. It should be
understood that the foregoing relates only to preferred embodiments
of the present invention and that numerous modifications or
alterations may be made therein without departing from the spirit
and the scope of the present invention as defined in the following
claims.
* * * * *