U.S. patent application number 10/775942 was filed with the patent office on 2004-08-19 for cells having transferred proteins, and methods of use thereof.
Invention is credited to Chen, Aoshuang, Tykocinski, Mark L., Zheng, Guoxing.
Application Number | 20040161806 10/775942 |
Document ID | / |
Family ID | 23893419 |
Filed Date | 2004-08-19 |
United States Patent
Application |
20040161806 |
Kind Code |
A1 |
Tykocinski, Mark L. ; et
al. |
August 19, 2004 |
Cells having transferred proteins, and methods of use thereof
Abstract
Methods for transferring one or more proteins to a cell are
disclosed. The protein or proteins to be transferred are in the
form of a fusion protein, and contain at least one domain encoding
for a protein or peptide having trans signaling and/or adhesion
function. The fusion protein is transferred to a cell by binding to
a lipidated protein, which has been incorporated into the cell
membrane. Methods for using cells which have undergone protein
transfer according to the present methods are also disclosed. This
includes use in a cancer vaccine, use for treatment of cancer or
autoimmune disease, and use in determining costimulator threshold
levels.
Inventors: |
Tykocinski, Mark L.; (Merion
Station, PA) ; Chen, Aoshuang; (Wayne, PA) ;
Zheng, Guoxing; (Wayne, PA) |
Correspondence
Address: |
ECKERT SEAMANS CHERIN & MELLOTT
600 GRANT STREET
44TH FLOOR
PITTSBURGH
PA
15219
|
Family ID: |
23893419 |
Appl. No.: |
10/775942 |
Filed: |
February 10, 2004 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10775942 |
Feb 10, 2004 |
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09957056 |
Sep 20, 2001 |
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09957056 |
Sep 20, 2001 |
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09476828 |
Jan 3, 2000 |
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6316256 |
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Current U.S.
Class: |
435/7.21 ;
435/7.23 |
Current CPC
Class: |
A61K 2039/55516
20130101; C07K 2319/30 20130101; A61K 2039/5158 20130101; C07K
14/70532 20130101; C12N 5/0006 20130101; A61K 39/0011 20130101;
A61K 35/12 20130101; A61K 2039/6031 20130101; C07K 2319/00
20130101; C12N 2501/58 20130101; A61K 2039/80 20180801; C12N
2501/51 20130101; A61P 37/02 20180101; A61K 39/001129 20180801;
A61K 39/001166 20180801; C07K 14/70575 20130101; A61P 35/00
20180101 |
Class at
Publication: |
435/007.21 ;
435/007.23 |
International
Class: |
G01N 033/567; G01N
033/574 |
Goverment Interests
[0001] This work was supported in part by Grants R01 CA-74958 and
R01 A1-31044 from the National Institutes of Health.
Claims
What is claimed is:
1. A method for transferring a protein to a cell comprising:
coating the surface of said cell with a first protein, wherein said
first protein is a lipidated protein; and contacting said cell with
a second protein, wherein said second protein is a fusion protein
comprised of a first domain having affinity for said first protein
and a second domain of a peptide, protein, or derivative or
fragment thereof, having trans signaling and/or adhesion
function.
2. The method of claim 1, wherein either or both of said first
domain and said second domain is an extracellular domain.
3. The method of claim 1, wherein said second domain has
immunoregulatory function.
4. The method of claim 1, wherein the amount of protein transferred
to said cell is determined by the amount of second protein used in
said contacting step.
5. The method of claim 1, wherein said first protein is lipidated
with a C12-C22 lipid.
6. The method of claim 5, wherein said lipid is C16.
7. The method of claim 1, wherein said first protein is selected
from the group consisting of lipidated protein A and lipidated
protein G.
8. The method of claim 7, wherein said first protein is palmitated
protein A.
9. The method of claim 1, wherein said first domain is attached at
the amino terminus of said second protein.
10. The method of claim 1, wherein said first domain is attached at
the carboxyl terminus of said second protein.
11. The method of claim 1, wherein said second domain encodes a
portion of a type I membrane protein.
12. The method of claim 1, wherein said second domain encodes a
portion of a type II membrane protein.
13. The method of claim 1, wherein said second domain encodes a
costimulator.
14. The method of claim 1, wherein said second domain encodes a
coinhibitor.
15. The method of claim 13, wherein said costimulator is selected
from the group consisting of B7-1, B7-2, ICAM-1, ICAM-2, ICAM-3,
CD48, LFA-3, 4-1BB ligand, CD30 ligand, CD40 ligand, and heat
stable antigen.
16. The method of claim 15, wherein said second protein is
B7-1.cndot.Fc.gamma..sub.1.
17. The method of claim 14, wherein said coinhibitor is selected
from the group consisting of CD8, Fas ligand, and a single-chain Fv
derivative of immunoglobulin.
18. The method of claim 1, wherein said coated cell is contacted
with more than one type of second protein, and each type of second
protein is different.
19. The method of claim 18, wherein said second proteins are
introduced in a predetermined ratio.
20. The method of claim 1, wherein said coating step and said
contacting step take place in vivo.
21. The method of claim 1, wherein said coating step and said
contacting step take place in vitro.
22. The method of claim 18, further comprising the step of
injecting said contacted cells into a patient.
23. A cell produced according to the method of claim 1.
24. A method for determining costimulator activation thresholds in
T-cells comprising: a) coating the surface of a plurality of cells
with a first protein, wherein said first protein is a lipidated
protein; b) contacting said cells with a second protein, wherein
said second protein is a fusion protein comprised of a first domain
having affinity for said first protein and a second domain of a
costimulator; c) mixing the contacted cells of step b with T-cells;
and d) determining the level of T-cell proliferation.
25. The method of claim 21, further comprising the step of e)
determining cytokine secretion levels.
26. A method for treating a patient for an illness comprising:
coating the surface of a plurality of cells with a first protein,
wherein said first protein is a lipidated protein; and contacting
said plurality of cells with a second protein, wherein said second
protein is a fusion protein comprised of a first domain having
affinity for said first protein and a second domain of a peptide,
protein, or derivative or fragment thereof, having a trans
signaling or adhesion function specific for the treatment of the
illness; and administering an effective amount of said coated cells
to a patient.
27. The method of claim 26, wherein said illness is selected from
the group consisting of cancer, autoimmune diseases, and alloimmune
diseases.
28. The method of claim 27, wherein said illness is cancer and said
administration is by injection into a tumor.
29. The method of claim 26, wherein said cells are autologous.
30. The method of claim 26, wherein said cells are allogeneic.
31. The method of claim 30, wherein said cells are an allogeneic
cell line.
32. A method for treating a patient for an illness comprising:
transferring protein to a plurality of cells by administering to
said patient a first protein, which is a lipidated protein; and a
second protein, which is a fusion protein comprised of a first
domain having affinity for said first protein and a second domain
of a peptide, protein, or derivative or fragment thereof, having a
trans signaling or adhesion function specific for the treatment of
the illness; wherein an effective amount of cells within said
patient have fusion protein transferred thereto.
33. The method of claim 32, wherein said first protein and said
second protein are administered sequentially.
34. The method of claim 32 wherein said first protein and said
second protein are administered concurrently.
35. The method of claim 32, wherein said administration is by local
injection.
36. The method of claim 32, wherein said administration is by
systemic injection.
37. A cancer vaccine comprising: cells produced according to the
method of claim 1 in a suitable carrier.
38. The cancer vaccine of claim 37, wherein said first protein is
selected from the group consisting of lipidated protein A and
lipidated protein G.
39. The cancer vaccine of claim 37, wherein said first protein is
palmitated protein A.
40. The cancer vaccine of claim 37, wherein said first domain is
attached at the amino terminus of said second protein.
41. The cancer vaccine of claim 37, wherein said first domain is
attached at the carboxyl terminus of said second protein.
42. The cancer vaccine of claim 37, wherein said second domain
encodes a type I membrane protein.
43. The cancer vaccine of claim 37, wherein said second domain
encodes a type II membrane protein.
44. The cancer vaccine of claim 37, wherein said second domain
encodes a costimulator.
45. The cancer vaccine of claim 37, wherein said second domain
encodes a coinhibitor.
46. The cancer vaccine of claim 44, wherein said costimulator is
selected from the group consisting of B7-1, B7-2, ICAM-1, ICAM-2,
ICAM-3, CD48, LFA-3, 4-1BB ligand, CD30 ligand, CD40 ligand, and
heat stable antigen.
47. The cancer vaccine of claim 46, wherein said second protein is
B7-1.cndot.Fc.gamma..sub.1.
48. The cancer vaccine of claim 45, wherein said coinhibitor is
selected from the group consisting of CD8, Fas ligand and a single
chain Fv derivative of immunoglobulin.
49. The cancer vaccine of claim 37, wherein said vaccine comprises
more than one second protein.
50. The cancer vaccine of claim 37, wherein said vaccine comprises
more than one cell type, and each cell type has a different fusion
protein transferred thereto.
Description
FIELD OF THE INVENTION
[0002] The present invention relates to novel methods for
transferring one or more proteins to a cell. In addition to other
applications, the methodology is useful in the treatment of cancer
and autoimmune diseases, and for determining costimulator
activation thresholds and cooperative interactions among
costimulators.
BACKGROUND INFORMATION
[0003] T-cells, including cytotoxic T-lymphocytes (CTLs), are a
critical component of effective human immune responses to tumors,
viral infections and other infectious diseases. T-cells destroy
neoplastic or virally infected cells through recognition of
antigenic peptides presented by MHC class I molecules on the
surfaces of target cells. Activation of T-cells is dependent upon
coordinate signaling through antigen receptors and costimulator
receptors on T-cell surfaces. Many mechanisms contribute to the
escape of tumor cells and virally infected cells from immune
surveillance. One of the mechanisms is that these cells lack the
costimulatory molecules required for T-cell activation.
Immunotherapeutic strategies have been developed that are
predicated upon expressing costimulators on tumor cell, and other
antigen-presenting cell, surfaces.
[0004] Professional antigen-presenting cells (APCs), by virtue of
the surface costimulatory molecules, are geared towards potent
T-cell activation. APCs can be converted into deletional APCs, or
"artificial veto cells", by expressing coinhibitors at their
surfaces. This is discussed, for example, in U.S. Pat. Nos.
5,242,687; 5,601,828; and 5,623,056. Such coinhibitors bind to
coinhibitor receptors on cells, leading to T-cell inactivation.
[0005] One approach for expressing costimulators and coinhibitors
on APCs, such as tumor cells, is gene transfer. When used for APC
and tumor cell engineering, gene transfer techniques have
shortcomings. For example, APCs, including tumor cells, are often
poorly transfectable. In addition, transfection proceedings are
cumbersome and time-consuming. Furthermore, expressing more than a
costimulator (or coinhibitor) is difficult. These and other issues
have impeded the widespread application of gene therapy for APC and
tumor cell engineering.
[0006] Protein transfer offers a number of advantages over gene
transfer for engineering APCs and other cells. These advantages
include the ability to modify poorly transfectable cells (for
example, biopsy-derived tumor cells), the simplicity of expressing
multiple proteins on the same cell surface, and the relative ease
and rapidity of the procedure. The successful use of recombinant
GPI-modified costimulator and MHC protein derivatives for protein
transfer has been reported. (See, Brunschwig, et al. J. Immunol.,
155:5498 (1995); McHugh, et al; Proc. Natl. Acad. Sci. USA, 92:8059
(1995); and McHugh, et al. Cancer Res., 59:2433 (1999)). A
shortcoming of the GPI protein transfer strategy, however, resides
in scaling up the purification of GPI proteins from membranes of
transfected cells.
[0007] Kim and Peacock, J. Immunol. Methods, 158:57 (1993), report
the use of palmitate-conjugated protein A for coating cells with
artificial receptors which facilitate intercellular interactions.
More specifically, a method is reported for attaching an antibody
onto the surface of a cell using palmitated protein A. The article
does not teach use of a lipidated protein for attachment of
anything other than an antibody to a cell. As such, their modified
cells serve only as artificial receptors for antigens.
[0008] Phillips et al., Immunity, 5:163-172 (August, 1996) report
the preparation of a fusion protein using a CD8 leader segment, the
Fc domain, of immunoglobin and the ectodomain of a type II membrane
protein, CD94. The present transfer methods are applicable to both
type I and type II proteins and are neither taught nor suggested in
the article.
[0009] Darling, et al., Gene Therapy, 4(12):1350-60 (December 1997)
report the use of a biotin/avidin-based system for protein
transfer. This method involves biotinylation of the target cell,
attachment of an avidin group to the protein to be transferred, and
combining the biotinylated target cell and the avidin-tagged
protein This method has significant limitations, including its
dependence on covalent modifications that could perturb multiple
proteins on cell surfaces.
[0010] There remains a need, therefore, for methods of efficient
and quantitative transfer of proteins and peptides to cells. A
further need is to provide such methods in which immunoregulatory
molecules that retain their function can be attached to cells of
interest.
SUMMARY OF THE INVENTION
[0011] The present invention has met the above needs, by providing
methods for quantitative transfer of a domain having
trans-signaling and/or adhesion function onto a cell surface.
Typically, the domain will be the extracellular domain having one
or both of these functions. In a preferred embodiment, the
extracellular domain of an immuno-regulatory molecule is used. More
specifically, the present methods provide a two-step protein
transfer approach, which permits delivery of graded amounts of
proteins to a cell surface. The methods utilize a fusion protein
comprised of at least two domains, one of which preferably encodes
a molecule having immunoregulatory function. By adding the fusion
protein to cells coated with a lipidated protein, fine titration
of, for example, the immunoregulatory molecule's extracellular
domain is achieved.
[0012] The present protein transfer methods have wide application.
For example, the methods have been used to establish that
costimulator thresholds exist, and that the levels of surface
costimulator on APCs can dictate both the magnitude and the quality
of evoked T-cell responses. The present methods are also applicable
to the generation of cancer vaccines; these vaccines show
significant anti-tumor effects in vivo. Furthermore, the methods
can be used to generate artificial veto cells, expressing one or
more coinhibitors, that can be used to delete pathogenic T-cells.
Cells produced according to the present methods are therefore
useful in the treatment of cancer and also in the treatment of
autoimmune diseases. The methodologies described herein can also be
used in the establishment of animal models and for the study of
immunological issues regarding, for example, T-cell activation, use
of costimulators to override apoptotic signals, function of
coinhibitors versus costimulators, synergy of costimulators used in
the treatment of cancer, and use of coinhibitors in the treatment
of autoimmune diseases.
BRIEF DESCRIPTION OF THE FIGURES
[0013] FIG. 1, including 1A, 1B and 1C, demonstrates the efficacy
of coating cells with a lipidated protein, according to the methods
of Example 1 .
[0014] FIG. 2 provides the SDS-PAGE analysis of recombinant
B7-1.cndot.Fc.gamma..sub.1 prepared as described in Example 1.
[0015] FIG. 3 demonstrates that the present methods achieve fusion
protein transfer (FIG. 3A), in a quantitative manner (FIG. 3B), as
described in Example 1.
[0016] FIG. 4 demonstrates the stimulation of T-cell proliferation
in the presence of various proteins (as indicated) using either PHA
(FIG. 4A) or anti-CD3 mAb (FIG. 4B) as a first signal, as described
in Example 1.
[0017] FIG. 5 demonstrates B7-1 threshold concentrations for T-cell
proliferation using either PHA (FIG. 5A) or anti-CD3 mAb (FIG. 5B)
as a first signal, as described in Example 1.
[0018] FIG. 6 provides a comparison of B7 concentration thresholds
for IFN-.gamma. versus IL-2 production using either PHA (FIG. 6A)
or anti-CD3 mAb (FIG. 6B) as a first signal, as described in
Example 1.
[0019] FIG. 7 provides comparative single-cell analyses of B7-1
concentration thresholds for IFN-.gamma. versus IL-2, as described
in Example 1.
[0020] FIG. 8 shows the effect of reaction temperature during
protein transfer on the stability of transferred protein, as
described in Example 2.
[0021] FIG. 9 demonstrates the efficacy of the present cancer
vaccines in protecting a patient against a post-immunization tumor
challenge, as described in Example 3.
[0022] FIG. 10 demonstrates the efficacy of the present cancer
vaccines in treating a pre-existing tumor, as described in Example
4.
[0023] FIG. 11 demonstrates the efficacy of the present cancer
vaccines by intratumoral injection of costimulators, as described
in Example 5.
[0024] FIG. 12 shows the results of the JAM assay, as described in
Example 6.
DETAILED DESCRIPTION OF THE INVENTION
[0025] The present invention is directed to methods for
transferring one or more proteins to a cell, generally comprising
the steps of coating the surface of a cell with a first protein,
which is a lipidated protein, and contacting the coated cell with a
second protein, which is a fusion protein. The fusion protein is
comprised of a first domain having affinity for the lipidated
protein and a second domain of a peptide, protein, or derivative or
fragment thereof, having trans signaling and/or adhesion function.
Preferably, the extracellular domain is used, and the second domain
has immunoregulatory function. "Derivative", as used in reference
to peptides and proteins, refers to variants of peptides and
proteins such as analogues wherein, for example, one or more amino
acids within the peptide chain has been deleted, added or replaced
with an alternative amino acid. A "fragment" refers to a portion of
the amino acid sequence of a peptide or protein. It will be
understood that "derivatives" and "fragments" of peptides and
proteins retain the physiological function of the wild type peptide
or protein and thus are biologically active.
[0026] The present methods are applicable to any cell having a
lipid bilayer membrane. For example, any kind of a patient's
autologous cells can be used, harvested by any means known in the
art. Use of any allogeneic mammalian cell line is also within the
scope of the present invention. Examples of allogeneic cell lines
suitable for use in the present invention include, but are not
limited to, EL-4 cells (mouse thymoma cells), 293 cells (human
kidney cells), K562 cells (human leukemia cells), Daudi cells
(human B cell line) and JY cells (human B cell line). These cells
are commercially available from the American Type Culture
Collection, Manassas, Va. Non-commercially available cell lines are
also within the scope of the present invention.
[0027] Any protein that can be lipidated is suitable for use in the
present methods. Examples include, but are not limited to, protein
A and protein G, both of which are commercially available.
Similarly, any lipid can be used to prepare the lipidated protein.
Lipids having carbon chains between about 12 and 22 are preferred,
with a carbon chain of 16 (palmitate) being most preferred. The
length of the lipid chain can be varied based upon the needs and
desires of the user. It will be understood to those skilled in the
art that the lipidated portion of the first protein will become
attached to or incorporated into the phospholipid bilayer that
makes up the membrane of the cell, and that this is what is meant
by the phrase "coating the surface of the cell" as that phrase is
used herein.
[0028] The amount of lipidated protein used to coat the cell may
also vary based on the needs and desires of the user, and based on
the particular lipid and particular protein selected. Preferably,
enough lipidated protein is used to coat the entire cell This
amount will typically be at least about 30 micrograms of lipidated
protein for every 5.times.10.sup.6 cells.
[0029] Following coating of the cells, the cells are then contacted
with a second protein. The second protein is a fusion protein in
which two different domains have been fused, such as through
recombinant DNA technology standardly used in the art, to create a
single DNA sequence. The first domain can be attached at either the
amino terminus or the carboxyl terminus of the fusion protein. The
first domain encodes a peptide, protein, or derivative or fragment
thereof, what has affinity for the lipidated protein. Thus, the
protein used in the lipidated protein is ideally selected in
conjunction with the protein encoded by the first domain of the
fusion protein, so that proteins having affinity for one another
are used. Affinity between the proteins can be determined by
Biacore technology or other methods familiar in the art. Because of
this affinity, the fusion protein binds to the lipidated protein,
which has already been incorporated into the cell membrane. In this
manner, the fusion protein is transferred to the cell. A
particularly preferred combination uses a palmitated protein A and
a first domain encoding an Fc region. For example, the Fc region of
human immunoglobulin G1 (IgG1), designated Fc.gamma..sub.1, can be
used. Other suitable first domains in the fusion protein include
leucine zipper protein domains and single-chain Fv derivative
domains.
[0030] The second domain of the fusion protein encodes a peptide,
protein, or derivative or fragment thereof, having immunoregulatory
function and capable of trans signaling to a second cell. Examples
include, but are not limited to, costimulators and coinhibitors.
Any suitable costimulator can be used including but not limited to
B7-1, B7-2, CD48, ICAM-1, ICAM-2, ICAM-3, LFA-3, CD30 Ligand
(CD30L), CD40 Ligand (CD40L), 4-1BB Ligand (4-1BBL), and heat
stable antigen. Similarly, any suitable coinhibitor can be used
including, but not limited to, CD8, FasL and PP14.
[0031] Significantly, the fusion protein of the present invention
can be either a type I or type II protein. Methods for transferring
a type II protein to a cell have never been reported. Because the
methods of the present invention are equally applicable to type I
and type II proteins, they provide a significant advance over the
art. Examples of type I membrane proteins include B7-1, B7-2 and
CD48; examples of type II membrane proteins include Fas ligand
(FasL or CD95L), CD40L, and 4-1BBL. For the type II proteins, the
first domains are fused at the carboxyl termini of the type II
proteins in order to preserve the functional ends of the molecules.
These lists are not exhaustive of the costimulators, coinhibitors
and other proteins that can be transferred according to the present
invention the lists reflect all forms of the various molecules
including, but not limited to, human and murine forms.
[0032] Another significant advance provided by the present methods
is that following transfer of the fusion protein to the cell, the
portion of the fusion protein having trans signaling function
retains this function. Thus, the cells prepared according to the
present methods are capable of eliciting an immune response by
binding to, and trans signaling through a counter-receptor on a
second cell.
[0033] In addition, more than one fusion protein can be used to
coat a single cell. In this manner, two, three, four, or more trans
signaling, for example, immunoregulatory proteins, can be
transferred to a cell. In the case of costimulators and
coinhibitors, combinations of such proteins can be chosen to have
the greatest immunological effect; combinations having additive or
even synergistic benefits can be selected and used according to the
present methods.
[0034] The present methods are further unique in that proteins can
be delivered to a cell's surface in a quantitative manner. As noted
above, it is preferred to use enough lipidated protein to fully
coat the cell. The amount of fusion protein that becomes
transferred to the cell is therefore determined by the amount of
fusion protein used to contact the coated cell; thus the amount of
fusion protein is the limiting or determinative factor. When using
more than one fusion protein, predetermined ratios of fusion
proteins can be used to contact the coated cell; protein will be
transferred to the cell in these approximate ratios.
[0035] The present methods can be effected either in vivo or in
vitro. In in vivo methods, the lipidated protein and the fusion
protein are injected directly into a patient. The injection can
occur sequentially (with the lipidated protein first) or
concurrently, with premixing of the lipidated protein and the
fusion protein(s). Injection can be localized, for example,
intra-tumoral, or systemic, for example, into a vessel. The present
methods, and cells produced thereby, in contrast to other
art-reported methods, have particularly high protein stability,
making the present method practical for in vivo application. In
vitro methods involve the extraction of cells from a patient, and
the subsequent coating and contacting of the cells; alternatively,
commercially obtained allogeneic cells can be used. In either case,
the treated cells can then be injected into a patient.
[0036] All of the above descriptions relating to cell type, first
proteins, second proteins, and in vivo, in vitro and other delivery
techniques apply equally to all embodiments of the invention
disclosed herein.
[0037] The present invention is further directed to methods for
determining costimulator activation thresholds in T-cells. These
methods generally comprise transferring one or more fusion proteins
to a cell, in the manner described above. The cells to which
protein has been transferred are then mixed with T-cells. T-cells
can be, for example, harvested from peripheral blood mononuclear
cells by methods known in the art. T-cell proliferation, if any,
can be measured, as can cytokine secretion levels according to
means known in the art, such as those described in the Example
section.
[0038] The present invention is further directed to methods for
treating an illness using the present protein transfer technology.
These methods generally comprise administering to a patient an
effective amount of the cells prepared in vitro according to the
method described above, or administering the proteins in vivo. The
method can be performed by either in vivo or in vitro protein
transfer of the fusion protein(s) to the target cells. For in vitro
methods, either extracted autologous cells or allogeneic cells are
coated with a lipidated protein and contacted with one or more
fusion proteins. An effective amount of these cells are then
administered to a patient. For in vivo methods, lipidated protein
and one or more fusion proteins are administered to a patient in an
amount sufficient to result in transfer of an effective amount of
fusion protein(s) to an effective amount of cells.
[0039] "Illness" as used herein refers to cancer and autoimmune and
alloimmune diseases, including but not limited to arthritis,
rheumatoid arthritis, asthma, graft-versus-host disease, organ
rejection, systemic lupus erythematosis, atopic allergy,
inflammatory bowel disease, multiple sclerosis and allergic
dermatitis. The methods are particularly applicable to treatment of
cancer, in that the lipidated protein and fusion protein(s) in the
in vivo methodology, or the coated and contacted cells in the in
vitro methodology, can be directly injected into one or more tumors
of the patient. "Patient" is used herein to refer to members of the
animal kingdom, including humans. The present methods are generally
applicable to patients capable of generating at least a minimal
immune response.
[0040] An effective amount of cells produced by the present protein
transfer methods should be used in the present treatment methods.
The effective amount is that amount of cells that will deliver the
amount of protein to a patient needed to bring about a desired
result. Generally, the desired result can be, for example,
stimulation of an immune response or suppression of an immune
response. In the case of cancer treatment, an effective amount
would be that amount which would protect a patient against tumor
growth or reduction, if not elimination, of tumors. In the case of
autoimmune or alloimmune disease, an effective amount would be that
amount which would alleviate if not eliminate one or more symptoms
of the autoimmune or alloimmune disease being treated. It will be
understood that the effective amount will vary from patient to
patient depending on such factors as the patient's size, the
condition of the patient's immune system, the patient's ability to
mount an immune response, and the type and severity of the illness.
The appropriate effective amount for each patient can be determined
by one skilled in the art, and will generally be at least about
10.sup.7 modified cells or 100 .mu.g Fc fusion protein
intratumorally.
[0041] The present invention is further directed to a cancer
vaccine comprising cells prepared according to the present protein
transfer methods contained in a suitable carrier. Any suitable
carrier can be used, provided compatibility problems do not arise.
Examples include PBS, and serum-free medium. The vaccine can
include a variety of fusion proteins; different cells each having a
different fusion protein, or cells having more than one fusion
protein attached thereto, can be used for example. Thus, a
"cocktail" of immunoregulatory proteins can be contained in the
present vaccines, and can be introduced to a patient according to
the present methods. The particular immunoregulatory proteins to
use in a cocktail can be determined by one skilled in the art based
upon such factors as the patient being treated and the type and
severity of the patient's illness. Different combinations could be
used to treat different types of tumors. The cocktail can be
pre-mixed and injected into a tumor bed, thereby leading to tumor
suppression. The vaccines have been found effective in both
pre-immunizing recipients against a subsequent tumor challenge and
in the treatment of established tumors.
EXAMPLES
[0042] The present examples are intended to illustrate the
invention and should not be construed as limiting the invention in
any way.
Example 1
[0043] The following example demonstrates a method for transferring
a B7-1.cndot.Fc.gamma..sub.1 fusion protein to a cell using
palmitated protein A.
[0044] Palmitation of Protein A
[0045] Recombinant protein A (Calbiochem, La Jolla, Calif.) was
derivatized with the N-hydroxysuccinimide ester of palmitic acid
(Sigma, St. Louis, Mo.) as described by Kim and Peacock, J. Immunol
Methods, 158:57 (1993). Briefly, a stock solution of the
N-hydroxysuccinimide ester of palmitic acid was made, as was a
solution containing protein A in a concentration of about 1.5
mg/ml. The solutions were mixed in a ratio of about 10 .mu.g ester
per ml protein and incubated at room temperature with constant
mixing for about 18 h. The lipid-derivatized protein A was purified
as described by Huang, et al., J. Biol. Chem., 225:8015 (1980)
using a 30-ml Sephadex G-25 (Sigma) column. The protein product,
referred to herein as "pal-prot A", was quantitated using a
bicinchoninic acid kit (Bio-Rad, Richmond, Calif.), filter
sterilized, and stored at 4.degree. C. until use.
[0046] Membrane Incorporation of Pal-Prot A
[0047] Daudi EL-4, JY and K562 cells (3-7.times.10.sup.6/ml) were
separately resuspended in RPMI 1640 medium (BioWittaker,
Walkersville, Md.) after three washes with this same medium.
Varying concentrations of pal-prot A (or nonderivatized protein A
as negative control) were added to the cell suspension, and the
mixture was incubated at 4.degree. C. for 2 h with constant mixing.
To assess the incorporation of pal-prot A onto cell surfaces, cells
were washed twice in buffer (0.25% BSA/0.01% sodium azide/PBS) and
then incubated on ice for 1 h with 100 .mu.l of 100 .mu.g/ml
FITC-human IgG (Sigma) diluted with the same buffer. Cells were
washed twice in the buffer and analyzed on a FACStar.RTM. flow
cytometer (Becton Dickinson, Mountain View, Calif.).
[0048] In a first set of optimization experiments, efficient
incorporation of pal-prot A was documented in four cell lines (FIG.
1A) as detected with FITC-conjugated human IgG. As a negative
control, nonderivatized protein A lacked the capacity to bind to
the same cells. Data from the FACStar analysis was plotted as
arbitrary units of log10 fluorescence intensity versus number of
EL-4 cells; membrane incorporation was dose dependent and started
to plateau at about 33 .mu.g/ml pal-prot A, as shown in FIG. 1B.
EL-4 cells were incubated with 33 .mu.g/ml pal-prot A for the
indicated periods of time and processed as above; pal-prot A
incorporation was rapid, appearing immediately after addition to
the cells and reaching a plateau at .about.1 h, as shown in FIG.
1C. This data demonstrates that numerous different cell lines can
be used in the present protein transfer methods, and that the
lipidated protein was incorporated into the cell fairly
rapidly.
[0049] Preparation of Recombinant B7-1.cndot.Fc.gamma..sub.1
[0050] The expression plasmid pCDM8/B7Ig, encoding the complete
human B7-1 extracellular domain linked in-frame to the
Fc.gamma..sub.1, was obtained from the American Type Culture
Collection (Manassas, Va.). The sequence encoding
B7-1.cndot.Fc.gamma..sub.1 was mobilized from pCDM8/B7Ig by
digesting with XbaI, filling-in with Klenow fragment, and
subsequently digesting with HindIII. The mobilized fragment was
subcloned into the EBV episomal expression vector pREP7.beta.
(Invitrogen, San Diego, Calif.) with HindIII and filled-in BamHI
sites. The plasmid was transfected into 293 cells (human kidney
cell line; American Type Culture Collection), and hygromycin
B-resistant colonies were selected in serum-free UltraCulture
medium (BioWittaker) supplemented with 10 mM glutamine,
penicillin/streptomycin, and 200 .mu.g/ml hygromycin B. Secreted
B7-1.cndot.Fc.gamma..sub.1, was purified from conditioned medium by
protein A-agarose (Life Technologies, Germantown, Md.) affinity
chromatography and verified by SDS-PAGE. The protein was run on a
10% SDS-polyacrylamide gel and visualized with Coomassie blue as a
dominant single band of .about.80 kDA under both reducing (lane 2)
and nonreducing (lane 3) conditions as shown in FIG. 2. Its
identity was confirmed by ELISA, with a recombinant protein binding
strongly to the human B7-1 specific mAb, BB-1, but not to control
Ab (data not shown).
[0051] B7-1.cndot.Fc.gamma..sub.1 Protein Transfer
[0052] Cells precoated with pal-prot A were washed once and
resuspended in RPMI 1640 medium (3-7.times.1.sup.6 cells/ml).
pREP7B-transfected K562 cells (K562/REP7b) were serially incubated
with 33 .mu.g/ml protein A for 2 h, 33 .mu.g/ml Fc.gamma..sub.1
fusion protein for 1 h, and BB-1 as primary Ab and FITC-conjugated
goat anti-mouse IgG as secondary Ab. To monitor protein delivery,
10.sup.6 cells were washed twice with the same buffer as above,
incubated on ice for 1 h with 1 .mu.g of human B7-specific mAb BB-1
(PharMingen, San Diego, Calif.) in 100 .mu.l of buffer. Cells were
washed once and immunostained (on ice for 1 h) with 100 .mu.l of
1:100 diluted FITC-conjugated goat F(ab').sub.2 anti-mouse Ig
(Boehringer Mannheim, Indianapolis, Ind.) as secondary Ab. Cells
were washed once, resuspended in PBS, and analyzed on a FACStar
flow cytometer.
[0053] FIG. 3A shows that when K562 cells were precoated with
pal-prot A, secondarily applied B7-1.cndot.Fc.gamma..sub.1 attached
to the cell surface, as detected by immunostaining of the cells
with anti B7-1 BB-1 mAb and FITC-conjugated goat anti-mouse IgG.
When a control Fc fusion protein (CD28.cndot.Fc.gamma..sub.1) was
substituted for B7-1.cndot.Fc.gamma..sub.1, no BB-1 binding was
observed, substantiating BB-1 mAb's B7-1 specificity. When
underivatized protein A was substituted for pal-prot A, no BB-1
binding was observed, indicating the dependence of the lipid
anchoring for fusion protein attachment.
[0054] Quantitation of Exogenously Incorporated
B7-1.cndot.Fc.gamma..sub.1 at Cell Surfaces
[0055] Human B7-1.cndot.Fc.gamma..sub.1 was iodinated using
Iodo-beads (Pierce, Rockford, Ill.) according to the manufacturer's
protocol, and the labeled protein was purified on a Sephadex G-25
column (Pharmacia, Piscataway, N.J.). The specificity was adjusted
to 2.1.times.10.sup.6 cpm/.mu.g by addition of unlabeled
B7-1.cndot.Fc.gamma..sub.1. Protein transfer was performed as
described earlier, substituting the labeled protein. All
experiments were performed in duplicate. To control for nonspecific
binding, excess amounts of unlabeled human IgG (Sigma) were added
to specifically block the binding of B7-1.cndot.Fc.gamma..sub.1 to
protein A. After repeated washing, counts in cell pellets were
determined using a gamma counter (1272 Clinigamma; LKP Instruments,
Gaithersburg, Md.). Counts resulting from specific binding of
B7-1.cndot.Fc.gamma..sub.- 1 were calculated by subtracting
nonspecific counts obtained with human IgG. The average number of
molecules on a single cell was calculated according to the formula
A.times.B.sup.-1.times.C.sup.-1.times.N.sub.A, where A is the
determined radioactivity (cpm) in the cell pellet, B is the
specific activity of the labeled protein expressed as cpm/mol, C is
the number of cells in the cell pellet, and N.sub.A is Avogadro's
constant.
[0056] As shown in FIG. 3B, when K562 cells were precoated with
excess amounts of pal-prot A (33 .mu.g/ml), surface levels of
B7-1.cndot.Fc.gamma..sub.1 were dependent on the concentrations of
applied B7-1.cndot.Fc.gamma..sub.1. Surface B7-1 epitope levels
started to plateau at 33 .mu.g/ml, and the epitope density was
similar to that on B7-1 transfected K562 cells (data not shown).
The average number of B7-1.cndot.Fc.gamma..sub.1 painted per cell
was determined using .sup.125I-labeled B7-1.cndot.Fc.gamma..sub.1.
Again, K562 cells incorporated increasing amounts of
B7-1.cndot.Fc.gamma..sub.1 as the reagent concentration was
increased during the painting process, as shown in Table 1.
1TABLE I Painting of B7-1 .multidot. Fc.gamma..sub.1 onto K562
cells B7-1 .multidot. Fc.gamma..sub.1 No. of B7-1 .multidot.
Fc.gamma..sub.1/cell.sup.b (.mu.g/ml).sup.a (mean .+-. SD) 0.033
460 .+-. 240 0.33 9,900 .+-. 1,200 3.3 92,000 .+-. 8,300 33 460,000
.+-. 34,000 .sup.aThe final concentration of B7-1 .multidot.
Fc.gamma..sub.1 present during the painting procedure. .sup.bValues
were determined as described in Materials and Methods. Specific
activity of .sup.125I-labeled B7-1 .multidot. Fc.gamma..sub.1 is
2.1 .times. 10.sup.6 cpm/.mu.g.
[0057] At the lowest concentration used (0.033 .mu.g/ml),
.about.460 molecules became anchored onto each K562 cell. At the
highest concentration used (33 .mu.g/ml), about 460,000
B7-1.cndot.Fc.gamma..sub.- 1 molecules became incorporated. Taken
together, these data establish that B7-1.cndot.Fc.gamma..sub.1 can
be applied to pal-prot A-coated cells in a quantitative
fashion.
[0058] Proliferation Assays
[0059] PBMC were isolated from fresh whole blood by Ficoll density
centrifugation. T-cells were purified by two rounds of treatment
with Lympho-kwik (One Lambda, Canoga Park, Calif.). T-cell purity
was verified by lack of a proliferative response to phytohemaglutin
("PHA") or [???] PMA in the absence of accessory cells. The human
CD3-specific mAb HIT3a (PharMingen) was bound to 96-well plates at
the indicated concentrations and used in this form to provide a
first activating signal to T-cells. Alternatively, PHA was used in
soluble form as a source of a first signal. K562 cells transfected
with the negative control vector pREP7.beta. (K562/pREP7.beta.)
were precoated with pal-prot A and secondarily coated with
B7-1.cndot.Fc.gamma..sub.1. For each proliferation assay,
1.times.10.sup.5 T-cells were incubated with 4.times.10.sup.4
B7-1.cndot.Fc.gamma..sub.1-coated and mitomycin C-treated
K562/REP.beta. cells for 60 h at 37.degree. C. Wells were pulsed
with 1 .mu.Ci [.sup.3H]thymidine for the last 16 h of the
incubation period. Cells were harvested and counted on a Betaplate
liquid scintillation counter.
[0060] In the proliferation assays, PHA and
B7-1.cndot.Fc.gamma..sub.1-coa- ted K562/REP.beta. cells (i.e.,
K562 cells stably transfected with the pREP7.beta. EBV episomal
expression vector) were used to provide first and second signals,
respectively, to T-cells. K562/REP7.beta. cells lack detectable
B7-1 (data not shown) and provide a suitable negative control for
experiments with K562/B7-1 transfected cells (i.e., K562 cells
stably transfected with a pREP7.beta. vector containing human B7-1
cDNA sequence). Surface B7-1 levels on K562/B7-1 transfected cells
and B7-1.cndot.Fc.gamma..sub.1-coated K562/REP7.beta. cells were
determined by immunostaining, and the mean fluorescence intensities
were 550 nm and 450 nm, respectively. As shown in FIG. 4A, in the
presence of suboptimal PHA concentrations (<0.5 .mu.g/ml),
B7-1.cndot.Fc.gamma..sub.1-coated K562/REP7.beta. cells, but not
K562/REP7.beta., significantly enhance T-cell proliferation. The
costimulatory effect was comparable to that achieved with K562/B7-1
transfected cells. The B7-1.cndot.Fc.gamma..sub.1- /pal-prot
A-dependence of the observed costimulation was verified by showing
that cells treated with a combination of (non-derivatized) protein
A and B7-1.cndot.Fc.gamma..sub.1, or with a combination of pal-prot
A and control CD8.cndot.Fc.gamma..sub.1, did not enhance T-cell
proliferation. In the presence of higher PHA concentrations (>1
.mu.g/ml), K562/REP7.beta. cells also costimulate T-cell
proliferation, although to a lesser extent than the B7-1 positive
cells.
[0061] To further confirm the costimulatory function of
cell-associated B7-1.cndot.Fc.gamma..sub.1, proliferation assays
were performed in which plate-bound anti-human CD3 mAb was
substituted for PHA as a more physiological first signal. In this
setting, in the presence of sub-optimal concentrations of anti-CD3
mAb (<10 .mu.g/ml) cell-associated B7-1.cndot.Fc.gamma..sub.1
costimulated even more effectively than native B7-1 expressed at
equivalent levels on transfected cells, as shown in FIG. 4B. Again,
CD8.cndot.Fc.gamma..sub.1, used as a negative control Fc fusion
protein, did not costimulate under the same conditions. Taken
together, these results establish that B7-1.cndot.Fc.gamma..sub.1,
tethered to membranes via pal-prot A, effectively costimulates
T-cell proliferation.
[0062] Effective depletion of accessory cells was documented in all
T-cell preparations by demonstrating the lack of response to PMA or
PHA in the absence of a source for costimulation. Points shown in
FIGS. 4A and B are the means and SEs of triplicate samples. The
data are representative of at least three independent experiments
with similar results.
[0063] Concentration-Dependence of Cell-Associated
B7-1.cndot.Fc.gamma..su- b.1's Costimulatory Activity
[0064] With an effective costimulator protein transfer method in
hand, quantitative aspects of B7-1 costimulation were evaluated. To
this end, T-cell proliferation assays were performed using
K562/REP7.beta. cells painted with variable concentrations of
B7-1.cndot.Fc.gamma..sub.1. The concentration dependence of
B7-1.cndot.Fc.gamma..sub.1-mediated costimulation could be readily
demonstrated when a fixed suboptimal concentration of PHA (0.25 or
0.5 .mu.g/ml) was used as a source of first signal, as shown in
FIG. 5A. For example, in the presence of 0.5 .mu.g/ml PHA, T-cell
proliferation was observed once a threshold
B7-1.cndot.Fc.gamma..sub.1 concentration (0.1 .mu.g/ml) was
reached, and the level of proliferation continued to rise with
increasing B7-1.cndot.Fc.gamma..sub.1 concentrations until reaching
a plateau at .about.3.3 .mu.g/ml. In the presence of a lower
concentration of PHA (0.25 .mu.g/ml), T-cell proliferation was
observed when a higher threshold B7-1 concentration (1 .mu.g/ml)
was reached, indicating that costimulator thresholds can be
modulated by the strength of the first signal.
[0065] Similar results were obtained when anti-human CD3 mAb was
used as a source of first signal instead of PHA, as shown in FIG.
5B. Again, in the presence of a fixed suboptimal concentration of
plate-bound anti-CD3 mAb (0.37 or 1.1 .mu.g/ml), costimulation was
observed only after a threshold B7-1.cndot.Fc.gamma..sub.1
concentration was reached, and a further dose-dependent increase in
proliferation was also seen. Hence, in the presence of a suboptimal
first signal (whether PHA or anti-CD3 mAb), a threshold B7 level is
required for T-cells to proliferate and the extent of T-cell
proliferation is dictated by the costimulator level.
[0066] ELISA Measurement of Secreted Cytokines and Determination of
a Hierarchy of B7-1 Costimulator Thresholds for Distinct Cytokine
Responses
[0067] A total of 10.sup.6 T-cells was incubated with
5.times.10.sup.5 processed K562/REP7.beta. cells
(B7-1.cndot.Fc.gamma..sub.1 positive or negative) in 48-well plates
using either plate-bound HIT3a or PHA as a source of first signal.
Supernatants were collected after 48 h, and ELISAs for human
IFN-.gamma. and IL-2 were performed using a commercial ELISA kit
according to manufacturer's protocol (Genzyme, Cambridge, Mass.).
More specifically, ELISA was used to measure T-cell cytokine
secretion in response to varying painted B7-1.cndot.Fc.gamma..sub.1
concentrations and fixed suboptimal primary stimulus
concentrations. At a fixed PHA dose, the B7-1.cndot.Fc.gamma..sub.1
concentrations eliciting minimal and maximal cytokine responses
differed for IFN-.gamma. and IL-2 with the general hierarchy being
IFN-.gamma.<IL-2, as shown in FIG. 6A. A similar hierarchy for
the cytokine responses was observed when anti-CD3 mAb (3.3
.mu.g/ml) was used as a source of first signal as shown in FIG. 6B.
For instance, at a B7-1.cndot.Fc.gamma..sub.1 concentration of 0.33
.mu.g/ml, IFN-.gamma. output was 60% of the maximal response,
whereas IL-2 output showed no increase above basal levels (FIG.
6B). This observed IFN-.gamma.>IL-2 hierarchy for B7-1
costimulator thresholds matches the order described for TCR
activation thresholds. Having documented that B7-1 levels can
modulate the extent of T-cell proliferative responses, it was then
determined that B7-1 levels can also dictate the quality of immune
responses by altering the ratios of cytokines produced by activated
T-cells.
[0068] Analysis of Intracellular Cytokine Production and Evaluation
of Evidence for Hierarchical Costimulator Thresholds for Cytokine
Responses at the Single-Cell Level
[0069] To substantiate the ELISA findings with bulk T-cell
populations, multiparameter flow cytometric analyses were performed
to assess intracellular IFN-.gamma. and IL-2 levels within
individual cells. A total of 10.sup.6 T-cells was incubated with
5.times.10.sup.5 B7-1.cndot.Fc.gamma..sub.1-coated K562/REP7.beta.
cells in 48-well plates for 48 h. Again, either plate-bound HIT3a
or PHA was used as a source of first signal. Monesin (Sigma) was
added to a final concentration of 3 .mu.M, and the mixture was
incubated for an additional 6 h to accumulate cytokine within the
cells. Cells were then collected, fixed by incubating them in 100
.mu.l of fixation solution [4% paraformaldehyde/PBS (pH 7.4] on ice
for 20 min. and then washed twice with staining buffer (0.1%
saponin/1% heat-inactivated FCS/0.1% sodium azide/Dulbecco's PBS).
Immunostaining for intracellular cytokines was performed by
incubating the cells on ice for 1 h with 100 .mu.l of the staining
buffer containing 0.5 .mu.g of FITC-anti-IFN-.gamma. and 0.5 .mu.g
of PE-anti-IL-2 Abs (PharMingen). Cells were subsequently washed
once with staining buffer without saponin. T-cells were gated using
forward light scatter/side light scatter parameters, and
2-5.times.10.sup.4 cells were analyzed in each run.
[0070] At low B7-1.cndot.Fc.gamma..sub.1 concentrations, the T-cell
response was dominated by IFN-.gamma.-only producers; however, at
higher B7-1.cndot.Fc.gamma..sub.1 concentrations, substantial
numbers of IFN-.gamma. and IL-2 double producers emerged (FIG. 7).
Relatively few IL-2 only producers were observed, even at the
highest B7-1.cndot.Fc.gamma..sub.1 concentrations. These findings
are consistent with the bulk T-cell cytokine response data, showing
that an IFN-.beta. response requires less B7-1 costimulators than
does an IL-2 response.
Example 2
[0071] The effect of temperature on membrane-incorporated protein A
was studied; the transferred protein must remain cell-bound in vivo
in order to prime T-cells, which requires stable engagement of
costimulators for at least several hours. It was determined that
the reaction temperature at which a lipidated protein is
transferred to the cell membrane has a major impact on long-term
retention of the protein on the membrane. Protein transfer
reactions were performed at 4.degree. C., 25.degree. C. or
37.degree. C.; palmitated protein A was transferred onto K562
cells. An hB7-1.cndot.Fc was .sup.125I-labeled and transferred to
the protein A-coated cells in the manner described in Example 1. To
prevent interference of endocytosis likely to occur at temperatures
above 4.degree. C., the cells were treated with the metabolic
inhibitors sodium azide and 2-deoxyglucose prior to the transfer
reaction.
[0072] To determine the long-term retention of the transferred
protein on the cell membrane, the cells were thoroughly washed to
remove unincorporated proteins, and subsequently incubated in
suspension for up to three days at 37.degree. C. in DMEM medium
containing 10% fetal calf serum. At several intervals, aliquots of
the suspension were taken and cells were pelleted. The amount of
radioactive label remaining in the cell pellet was compared to the
total amount of radioactive counts in the aliquot. The ratio
between the two was calculated as the relative portion of the
transferred protein still retained on the cell membrane. As
depicted in FIG. 8, there is direct relationship between a higher
protein transfer reaction temperature and a better long-term
retention rate. More importantly, by raising the transfer
temperature from 4.degree. C. to 37.degree. C., the transferred
proteins can remain membrane-bound at the physiological temperature
of 37.degree. C. for three days without significant loss (after the
initial six hours).
Example 3
[0073] C3H/HeN mice, purchased from Harlen (USA), Indianapolis,
were immunized with a cell vaccine generated from the T-50 cell
line, obtained from Avranham Hochberg, Hadassah University
Hospital. The vaccine was prepared following the procedure
generally outlined in Example 1, using palmitated protein A and
mB7-1.cndot.Fc, m4=1BBL.cndot.Fc, and hCD40L.cndot.Fc fusion
proteins. Basically, the cells were coated with the lipidated
protein A at 37.degree. C. at a ratio of 40 .mu.g protein A per
40.times.10.sup.6 cells. The cells were then incubated at 4.degree.
with an equal mixture of the three fusion proteins at a ratio of 20
.mu.g total protein per 4.times.10.sup.7 cells. The cell vaccine
was injected into the mice subcutaneously at a dose of 10.sup.6
cells per injection. The injections were given once a week and
continued for three weeks. One week after the last injection, the
animals were challenged with 10.sup.6 wild-type T-50 tumor cells,
injected intradermally on the rear flank. As FIG. 9 shows, the cell
vaccine improved the survival rate of the immunized animals. In
FIG. 9: open circle, an untreated control group (n=6); square,
another control group that received a control vaccine generated by
protein A transfer (n=5); closed circle, the test group that
received a cell vaccine generated by protein transfer with immune
costimulatory proteins B7-1, 4-1BBL, and CD40L in complex with
protein A (n=6).
Example 4
[0074] DBA/2J mice were purchased from The Jackson Laboratory,
Maine. The animals were inoculated intradermally with a lethal dose
of L5178Y-R tumor cells and given subcutaneous injections of a cell
vaccine as a treatment on days 5, 6, and 7 after the tumor
inoculation. The same cell vaccine in Example 3 was used here, at a
dose of 10.sup.6 cells per injection. FIG. 10 shows that the cell
vaccine improved the survival rate of the treated animals. In FIG.
10: open circle, an untreated control group (n=8); square, another
control group that received a control vaccine generated by protein
A transfer (n=8); closed circle, the test group that received the
cell vaccine generated by protein transfer with the immune
costimulatory fusion proteins in complex with lipidate protein A
(n=8).
Example 5
[0075] A vaccine was formed with palmitated protein A and
FasL.cndot.Fc, B7-1.cndot.Fc, 4-1BBL.cndot.Fc and CD40L.cndot.Fc
fusion proteins by mixing them in vitro at three parts of lipidated
protein A and one part of each of the fusion proteins. The protein
mixture was then injected intratumorally at 4 .mu.g of total
protein per tumor site. The vaccine was subsequently injected
directly into a tumor; the immune costimulatory proteins in the
vaccine modified the immunogenic property of tumor cells in
situ.
[0076] DBA mice were inoculated with a lethal dose of L5178Y-R
tumor cells. As tumor mass grew to about 50 mm.sup.2 in size, the
cancer vaccine was injected directly into the tumor site. The
vaccines were pre-assembled with palmitated protein A, which
confers the ability to anchor costimulators on the tumor cells in
situ according to the present methods. As shown in FIG. 11, the
survival of the mice treated with the indirectly lipidated
costimulators was significantly prolonged. In FIG. 11: open circle,
an untreated control group (n=6); square, another control group
that were injected with lipidated protein A alone (n=7); closed
circle, the test group that were injected with the immune
costimulatory fusion proteins in complex with lipidate protein A
(n=7).
Example 6
[0077] To generate a fusion protein for FasL, a coding sequence for
a human Fc.gamma..sub.1 domain, obtained from ATCC, was fused at
the N-terminus of the coding sequence for the extracellular domain
of FasL following the fusion strategy reported in Immunity, 5:163,
1996. The purified fusion protein was fully functional, as
determined by a standard killing assay when loaded on protein
A-coated cells. FasL is a cell surface protein that binds to
another protein, Fas, found on the surface of other cells, for
example, activated T-cells. When FasL binds to Fas, the cells
expressing Fas undergo apoptosis. Significantly, the FasL.cndot.Fc
fusion protein, after being transferred onto the cell surface
through the lipidated protein A, retained its apoptotic
activity.
[0078] More specifically to determine whether the Fc-hFasL fusion
protein was functional after anchoring onto cell surfaces, a
standard JAM assay is performed. The effector cells were CHO cells
that were painted with palmitated protein A (pal-prot A) and
subsequently with Fc fusion protein. The target cells were Jurkat
cells that constitutively express Fas and thus are susceptible to
Fas/FasL-mediated apoptosis. A standard JAM assay was performed,
according to the protocol described by P. Matzinger (J. Immunol.
Methods, 145:185-192,1991). Briefly, 2.times.10.sup.4
3H-thymidine-labeled target Jurkat cells were co-incubated with
2.times.10.sup.5 CHO cells (from ATCC) that were pre-coated with
pal-prot A as previously described in Example 1, and subsequently
painted with 30 .mu.g/ml of Fc-hFasL fusion protein or control
fusion protein The cells were co-cultured in 200 .mu.l of RPMI-10
in a 96-well plate for 18 hours at 37.degree. C. in a humidified
incubator at 5% CO.sub.2. To harvest the JAM test, the cells and
their medium were aspirated onto fiber glass filters using a
harvester (as used in Example 1 for the proliferation assays). %
specific killing was calculated as follows: (S-E)/S.times.100,
where S=spontaneous release without effector cell, E=experimental
release in the presence of effector cells The results are
summarized in FIG. 12. When CHO cells that were painted with
pal-prot A and Fc-hFasL were used as effector cells, significant
killing was observed. As negative controls, when CHO cells were
painted with pal-prot A and control Fc fusion protein (hCD80-Fc or
hCD28-Fc), no specific killing was observed. The surface
anchorage-dependence of Fc-hFasL was demonstrated by the negative
control where the CHO cells were painted with unpalmitated protein
A and Fc-FasL. The specificity of the killing was demonstrated by
complete blockade of the killing by FasL neutralizing mAb NOK-1,
whereas the control Ab did not block the killing.
[0079] In summary, these results demonstrate that Fc-hFasL, after
being transferred onto cell surface through pal-prot A, retains its
function to elicit apoptosis in Fas-positive Jurkat cells.
[0080] The above examples demonstrate the efficacy of the present
methods. Through the use of lipidated proteins, fusion proteins can
be transferred to cells both ex vivo and in situ. Significantly,
these fusion proteins retain their immunoregulatory function after
transfer. The examples demonstrate this retained function against
post-imitation challenge and against pre-existing tumors. The
methods were demonstrated as being effective both in vivo and in
vitro.
[0081] Whereas particular embodiments of this invention have been
described above for purposes of illustration, it will be evident to
those skilled in the art that numerous variations of the details of
the present invention may be made without departing from the
invention as defined in the appended claims.
* * * * *