U.S. patent application number 09/950374 was filed with the patent office on 2002-10-03 for materials and methods for treating oncological disease.
Invention is credited to Lawman, Michael J.P., Lawman, Patricia.
Application Number | 20020141981 09/950374 |
Document ID | / |
Family ID | 22101699 |
Filed Date | 2002-10-03 |
United States Patent
Application |
20020141981 |
Kind Code |
A1 |
Lawman, Michael J.P. ; et
al. |
October 3, 2002 |
Materials and methods for treating oncological disease
Abstract
Novel methods are disclosed for treating oncological disorders
in an individual or animal using a superantigen expressed in tumor
cells. A gene encoding a superantigen, such as an M-like protein of
group A streptococci, can be introduced into a tumor cell in order
to make the tumor cell more immunogenic in the host. Also
contemplated are methods wherein a cell expresses a superantigen or
superantigens, and immunogenic or immunostimulatory proteins, such
as foreign MHC, cytokines, porcine-derived hyperacute rejection
antigen, Mycobacterium-derived antigens, and the like. The subject
invention also pertains to cells transformed with polynucleotides
encoding a superantigen and foreign MHC antigen, cytokines, and
other immunogenic or immunostimulatory proteins. Transformed cells
according to the subject invention are then provided to an
individual or animal in need of treatment for an oncological
disorder. The immune response to tumor cells transformed according
to the present invention inhibits in vivo tumor growth and results
in subsequent tumor regression. The subject invention also pertains
to cell lines transformed with genes encoding a superantigen and,
optionally, a foreign Class II MHC antigen and/or a cytokine.
Inventors: |
Lawman, Michael J.P.; (Key
Largo, FL) ; Lawman, Patricia; (Key Largo,
FL) |
Correspondence
Address: |
SALIWANCHIK LLOYD & SALIWANCHIK
A PROFESSIONAL ASSOCIATION
2421 N.W. 41ST STREET
SUITE A-1
GAINESVILLE
FL
326066669
|
Family ID: |
22101699 |
Appl. No.: |
09/950374 |
Filed: |
September 10, 2001 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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09950374 |
Sep 10, 2001 |
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09394226 |
Sep 13, 1999 |
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09394226 |
Sep 13, 1999 |
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PCT/US99/00787 |
Jan 14, 1999 |
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60071497 |
Jan 14, 1998 |
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Current U.S.
Class: |
424/93.21 ;
424/155.1; 435/455; 514/44R |
Current CPC
Class: |
A61K 2039/5152 20130101;
A61K 39/092 20130101; A61K 38/164 20130101; A61K 39/0011 20130101;
A61K 2039/5156 20130101; A61K 48/00 20130101 |
Class at
Publication: |
424/93.21 ;
514/44; 424/155.1; 435/455 |
International
Class: |
A61K 048/00; A61K
039/395 |
Claims
1. A method for preventing or treating an oncological disease in a
human or domesticated animal, said method comprising introducing a
polynucleotide coding for a superantigen into a cell, wherein said
superantigen is expressed by said cell, and providing said
transformed cells to said human or animal, and wherein said
superantigen comprises an M-like protein.
2. The method according to claim 1, wherein said M-like protein is
the emmL-55 protein, or a fragment or variant thereof.
3. The method according to claim 1, said method further comprising
use of a traditional therapeutic treatment to treat said
oncological disorder.
4. The method according to claim 3, wherein said therapy is
selected from the group consisting of surgical resection of a
tumor, radiotherapy, chemotherapy, and antibody-directed antitumor
therapy.
5. The method according to claim 1, wherein said method further
comprises the use of a dendritic cell cancer vaccine.
6. The method according to claim 1, wherein said cells are
transformed in vivo in said human or animal.
7. The method according to claim 1, wherein said cells are
transformed in vitro.
8. The method according to claim 1, wherein the oncological
disorder to be treated is selected from the group consisting of
lynphomas, leukemias, carcinomas, sarcomas, brain tumors, gliomas,
glioblastomas, neuroblastomas, melanomas, hepatomas,
medulloblastomas and Wilm's tumors.
9. The method according to claim 1, wherein said polynucleotide
coding for said superantigen is introduced into said cell using a
delivery vector selected from the group consisting of adenovirus,
adeno-associated virus, retrovirus, pox virus, herpes virus,
plasmids, double-stranded nucleic acid and single-stranded nucleic
acid.
10. The method according to claim 1, wherein said polynucleotide
coding for said superantigen is introduced into said cell using a
liposome comprising said polynucleotide.
11. A method for preventing or treating an oncological disease in a
human or domesticated animal, said method comprising introducing a
polynucleotide coding for a first superantigen into a cell, and
further introducing into said cell a second polynucleotide coding
for at least one viral, bacterial or eukaryotic protein that is
immunogenic or immunostimulatory, wherein said first superantigen
and said viral, bacterial or eukaryotic protein is expressed by
said cell, and providing said transformed cells to said human or
animal, and wherein superantigen comprises an M-like protein.
12. The method according to claim 11, wherein said bacterial or
eukaryotic protein is selected from the group consisting of foreign
MHC antigens, cytokines, Mycobacterium-derived antigens,
porcine-derived hyperacute rejection antigens, and a second
superantigen.
13. The method according to claim 11, wherein said M-like protein
is the emmL-55 protein, or a fragment or variant thereof.
14. The method according to claim 11, said method further
comprising use of a traditional therapeutic treatment to treat said
oncological disorder.
15. The method according to claim 14, wherein said therapy is
selected from the group consisting of surgical resection of a
tumor, radiotherapy, chemotherapy, and antibody-directed antitumor
therapy.
16. The method according to claim 11, wherein said method further
comprises the use of a dendritic cell cancer vaccine.
17. The method according to claim 11, wherein said cells are
transformed in vivo in said human or animal.
18. The method according to claim 11, wherein said cells are
transformed in vitro.
19. The method according to claim 11, wherein the oncological
disorder to be treated is selected from the group consisting of
lymphomas, leukemias, carcinomas, sarcomas, brain tumors, gliomas,
glioblastomas, neuroblastomas, melanomas, hepatomas,
medulloblastomas and Wilm's tumors.
20. The method according to claim 11, wherein said polynucleotide
coding for said superantigen and/or said polynucleotide coding for
said viral, bacterial or eukaryotic protein is introduced into said
cell using a delivery vector selected from the group consisting of
adenovirus, adeno-associated virus, retrovirus, pox virus, herpes
virus, plasmids, double-stranded nucleic acid and single-stranded
nucleic acid.
21. The method according to claim 11, wherein said polynucleotide
coding for said superantigen and/or said polynucleotide coding for
said viral, bacterial or eukaryotic protein is introduced into said
cell using a liposome comprising said polynucleotide.
22. The method according to claim 12, wherein said cytokine is
selected from the group consisting of IL-1, IL-2, IL-3, IL-4,
TNF.alpha., IFN.alpha., IFN.beta., IFN.gamma., GM-CSF, MIP1.alpha.,
MIP1.gamma. and TGF.gamma..
23. The method according to claim 12, wherein said foreign MHC
antigen is selected from the group consisting of class I, class II
and class III.
24. A polynucleotide molecule, wherein said polynucleotide molecule
comprises a nucleotide sequence encoding an M-like protein and a
nucleotide sequence encoding a foreign MHC antigen.
25. A cell transformed with a polynucleotide molecule, wherein said
polynucleotide molecule comprises a nucleotide sequence encoding an
M-like protein and a nucleotide sequence encoding a foreign MHC
antigen.
Description
CROSS-REFERENCE TO A RELATED APPLICATIONS
[0001] This application is a continuation of U.S. application Ser.
No. 09/394,226, filed Sep. 13, 1999, which is a continuation of
International Application No. PCT/US99/00787, filed Jan. 14, 1999,
which claims priority from U.S. provisional application Serial No.
60/071,497, filed Jan. 14, 1998, now abandoned.
BACKGROUND OF THE INVENTION
[0002] While a place for somatic gene therapy in the treatment of
inherited single gene disorders is no longer disputed, the
potential of gene therapy for the treatment of malignancies may not
be readily apparent. Because most forms of cancer have been shown
to be complex, multifactorial, and multigenic in nature, there are
many conceptual and technical obstacles which must be overcome in
order to approach this disease at the genetic level. Yet, it is the
molecular nature of tumorigenesis, i.e., the activation of dominant
oncogenes and/or the inactivation of tumor suppressor genes, that
provides insight for such strategies in that these genetic events
represent novel targets for molecular therapy. Already, genetic
analysis is being used in diagnostic and prognostic predictions in
certain malignancies (e.g., amplification of erb-B2 in breast and
ovarian cancer; amplification of N-myc in neuroblastoma; and ras
mutations in adenocarcinoma of the lung).
[0003] At present, there are two general strategies for gene
therapy: gene augmentation and gene replacement. Gene augmentation
or gene addition is simply the introduction of foreign genetic
sequences into a cell. Usually this means the insertion of a normal
copy of a particular gene into a cell expressing a mutant form of
that gene. In many cases, the addition of functional genetic
information has been used successfully to restore a genetic
function in these defective cells. However, in other cases, the
addition of a normal gene is not sufficient to repair the
abnormality because this technique does not remove or correct the
resident, nonfunctional mutant gene. Furthermore, the random
insertion of foreign sequences into nonspecific sites of the genome
may result in mutagenic events such as insertional inactivation of
genes necessary for the viability of that cell, or uncontrolled
regulation of the transgene and/or flanking chromosomal sequences.
In these instances it will be necessary to modify specific gene
sequences by targeted gene replacement, i.e., site-specific
recombination of foreign DNA into targeted genomic sequences. Each
of these approaches have applications in gene therapy for
cancer.
[0004] The immune system has demonstrated the potential to play a
protective role in cancer. However, the vast majority of
malignancies arise in immunocompetent hosts. Although cellular
activity is normally regulated by the various protein kinases,
growth factors, growth factor receptors, and DNA binding proteins
encoded by proto-oncogenes, together with genes that can suppress
malignant transformation, such as the retinoblastoma and p53 genes,
if one or more of these genes is or becomes defective, it can
result in a clone with an abnormal pattern of growth control. The
fact that such a clone grows uncontrolled in an individual,
indicates that the immune system has either failed to recognize
tumor-specific antigens or has failed to effectively respond.
Transgenic immunotherapy, an important arm of somatic gene therapy
for cancer, aims at strengthening the immune surveillance of the
body.
[0005] It is known that the presence of cytokines at the site of a
tumor can drastically alter tumor/host relations. In some cases a
highly destructive and specific response to otherwise
nonimmunogenic tumors can be elicited by the insertion of genes
encoding cytokines (e.g., interleukin-2, interleukin-4,
interferon-.gamma., and tumor necrosis factor) into tumor cells
which are then used as "tumor vaccines." Anti-tumor responses can
also be enhanced by the transfection of these genes into cytotoxic
lymphocytes or macrophages. Autologous tumor-infiltrating
lymphocytes have been used successfully in such genetic
immunomodulation studies because of their inherent specificity for
the tumor, and their ability to home back to the tumor site when
reinfused into the patient. This approach has been termed "adoptive
immunotherapy." It is also possible to protect normal tissue by
stably transfecting normal bone marrow cells with cytokine genes
prior to chemotherapy, thereby achieving a more continuous effect
while obviating the need to infuse these drugs which have short
half-lives and produce systemic side effects when delivered
intravenously.
[0006] Despite the widespread use of chemotherapeutic agents for
the treatment of solid tumors, efficacy has been restricted by
their toxicity to normal cells. Transfection of normal stem cells
with transgenes conferring resistance to these agents would result
in cytotoxic drug-resistant cells and allow the administration of
more therapeutically significant doses. Another type of gene
therapy that is gaining momentum and which is now in clinical
trails is the use of "informational drugs." Antisense
oligonucleotides, small synthetic nuclease-resistant nucleotide
sequences complementary to specific RNA sequences, are perhaps the
best known example of this. By specifically binding and thereby
inhibiting transcription and/or translation of a single oncogene,
it may be possible to reverse clinical symptoms.
[0007] It is well established that T lymphocytes recognize two
different types of antigens, one being peptides derived from
conventional protein antigens and the other being superantigens.
The classical model for superantigen activity suggests that the
superantigens react in some ways like conventional antigens but
exhibit critical differences in others (Johnson et al., 1992).
Before a T helper cell can recognize conventional protein antigens,
these proteins must first undergo processing by macrophages or
other antigen presenting cells (APC). APCs then display the peptide
on the cell surface in combination with MHC. Unlike typical
antigens, however, superantigens bind MHC directly without uptake
and processing by APCs (Johnson et al., 1992).
[0008] Unlike conventional antigens, where recognition by T cells
involves both variable elements of the .alpha.and .beta. chains of
TCR, superantigen recognition depends primarily on the TCR V.beta.
region. Also, unlike ordinary antigens, superantigens bind to
specific V.beta. segments of TCR which are outside of the normal
antigen-binding groove. This binding occurs regardless of the
remaining structure of the TCR. These interactions lead to strong
V.beta.-specific T cell activation (Dellabona et al., 1990, and
Rust et al., 1990). Furthermore, because the number of different
types of V.beta. segments is small compared with the number of
.alpha., .beta. receptors, many more T cells are capable of
recognizing a particular superantigen than are able to identify a
specific antigen (Herman et al., 1991).
[0009] The classical model of superantigen activity has since been
revised. New observations suggest that there are additional
interactions between the TCR and MHC molecules during superantigen
engagement and this can have a significant impact on superantigen
specificity and function (Webb and Gascoigne, 1994). For example,
some studies show that it is not only V.beta., but also the .alpha.
chain of the TCR which plays a role in the recognition of
superantigens (Karp et al., 1990, and Panina et al., 1992).
Furthermore, the MHC molecule does not function as an inert
platform for superantigen presentation, but plays a role in T cell
activation by superantigens. Some studies indicate that individual
T cell clones are able to distinguish superantigens presented on
MHC molecules with different specificity. Some murine T cell
hybridomas can recognize superantigens in context of two different
MHC molecules (Mollick et al., 1991; Hartwig and Fleischer, 1993).
The revised model assumes that the molecular mechanism of T cell
stimulation is probably a multivalent cross-linking of the TCR with
MHC molecules. Additional adhesion molecules such as CD 2, LFA-1 or
CD 28, may play a role during T cell stimulation (Fleischer and
Hartwig, 1992, and Fraser et al., 1992). Another interesting
observation suggests that in spite of the requirement for MHC class
II molecules in T cell stimulation, there is evidence that
superantigens interact with TCRs directly, in the absence of class
II molecules. Binding to MHC II is not a prerequisite for T cell
activation as superantigen-mediated cytotoxicity has been found
against several class II-negative target cells. However, the
interaction of the superantigen with TCR is apparently of low
avidity and is usually insufficient to generate a full response
(Dohlsten et al., 1991, Herman et al., 1991, and Avery et al.,
1994).
[0010] Two general categories of superantigens have been described.
The soluble exotoxins produced by gram-positive bacteria such as
Staphylococcus aureus typify bacterially-derived superantigens and
are well known for their ability to cause food poisoning and
symptoms of shock. Viral superantigens have also been described. In
mice, prototypical viral superantigens are encoded by endogenous
mouse mammary tumor viruses (MMTVs). These viral superantigens
include Mls antigens and are strongly immunogenic for murine T
cells.
[0011] Because of their ability to stimulate strong T-cell
responses in vivo, superantigens have elicited wide interest.
M-like proteins of group A streptococci act as a key virulence
factor on the bacterial surface. M protein is defined by its
antiphagocytic function, whereas M-like proteins, while
structurally related to M protein, lack an established
antiphagocytic function. The emmL 55 gene, derived from the M
stereotype 55 group A streptococci isolate A928, has an amino acid
sequence typical of M-like proteins (Boyle et al., 1994, and Boyle
et al., 1995).
[0012] Bacterial superantigens, when covalently linked to mAbs
specific to the cell surface molecules of malignant, MHC II target
cells, can direct lysis of these cells (Dohlsten et al. 1991). It
has been demonstrated that conjugation between the superantigen
staphylococcal enterotoxin-A (SEA), and mAbs recognizing human
colon cancer enabled T cells to lyse colon carcinoma cells in
vitro. The use of Staphylococcal enterotoxins has been contemplated
for cancer vaccines (WO 95/00178).
[0013] Immunotherapeutic modalities for the treatment of
oncological disorders have been described by a number of scientific
investigators. The immunological rejection of tumors has been shown
in response to the transfection of tumor cells by such antigens
expressed by MHC genes (Hock et al., 1996) and Mycobacterium
(Menard et al., 1995). Research into immunotherapy for oncological
diseases using MHC antigens and the delivery thereof has been
described (EP 569678; WO 95/13092). Other antigens, bacterial and
viral, have also been used in combination with cytokine or other
immunomodulator gene expression and delivered by means of
adenovirus, retrovirus or plasmid vectors (WO 94/21808; WO
96/29093).
BRIEF SUMMARY OF THE INVENTION
[0014] The present invention concerns the use of superantigen, such
as M-like proteins, expressed by tumor cells as a method for
treating oncological disease in an individual or animal. In one
embodiment, the method comprises introducing a polynucleotide
coding for a superantigen, such as an M-like protein, into a cell
such as, for example, a tumor cell, wherein the superantigen is
expressed by the transformed cells, and introducing the transformed
cells into the patient to be treated. In an exemplified embodiment
of the invention, the M-like protein emmL 55 is employed. The emmL
55 gene encodes a polypeptide that has a sequence typical of an
M-like protein. In other embodiments, foreign major
histocompatibility complex (MHC) genes, such as class II genes,
and/or genes encoding cytokines can be inserted and expressed in
the cell transformed to express a superantigen.
[0015] In one embodiment, genes encoding an M-like protein and a
gene encoding a foreign class II MHC are introduced into and
expressed in a tumor cell. Polynucleotide molecules encoding
proteins contemplated by the subject invention can be used to
transform cells either in vitro or in vivo. If the transformation
is done in vitro, then the cells expressing the heterologous genes
are then implanted into a patient in need of oncological treatment.
Polynucleotides encoding M-like protein and MHC class II can be
introduced into tumor cells using standard techniques known in the
art, such as by electroporation, targeted liposomes, viral vectors
and transfection with naked DNA. In vivo transformation of a
patients cells can be done using targeted liposomes, viral vectors,
direct injection with naked DNA and other methods known in the
art.
BRIEF DESCRIPTION OF THE DRAWINGS
[0016] FIG. 1 shows construction of the pSVK 3/emmL 55 expression
vector.
[0017] FIG. 2 shows construction of the pcDNA 3/emmL 55 expression
vector.
[0018] FIG. 3 shows a restriction map of the pSVK 3/emmL 55.
[0019] FIG. 4 shows a sequence of the pSVK 3/emmL 55 construct. The
sequence starts on the pSVK vector and shows the mutation of the
ATG start codon which shifts the transcription start site to the
second start codon at 761 bp causing deletion of 375 bp of the emmL
55 gene, but does not change the reading frame. The result is a
truncated form of the emmL 55 gene.
[0020] FIG. 5 shows a restriction map of the pcDNA 3/emmL 55.
[0021] FIGS. 6A-6D show morphology of untransfected or transfected
Neuro-2a cell:
[0022] FIG. 6A shows Neuro-2a without DNA;
[0023] FIG. 6B shows Neuro-2a transfected with MHC II (pcDV
1/.alpha. and pcDV 1/.beta.);
[0024] FIG. 6C shows Neuro-2a transfected with truncated emmL 55
(pSVK 3/emmL 55);
[0025] FIG. 6D shows Neuro-2a transfected with emmL 55 (PCDNA
3/emmL 55).
BRIEF DESCRIPTION OF THE SEQUENCES
[0026] SEQ ID NO. 1 shows the nucleotide sequence of a pSVK 3/emmL
55 construct according to the subject invention.
[0027] SEQ ID NO. 2 shows the nucleotide sequence of a truncated
pSVK 3/emmL 55 construct according to the subject invention.
[0028] SEQ ID NO. 3 shows the partial nucleotide sequence of pSVK
3/emmL 55 construct according to the subject invention.
[0029] SEQ ID NO. 4 shows an oligonucleotide primer used to amplify
an emmL 55 gene according to the subject invention.
[0030] SEQ ID NO. 5 shows an oligonucleotide primer used to amplify
an emmL 55 gene according to the subject invention.
[0031] SEQ ID NO. 6 shows an oligonucleotide primer used to
sequence an emmL 55 gene according to the subject invention.
DETAILED DISCLOSURE OF THE INVENTION
[0032] The present invention concerns novel methods for treating
persons or animals afflicted with oncological disorders, or
preventing oncological disorders in persons or animals predisposed
to such oncological disorders, such as solid tumors, soft tissue
tumors, leukemias, lymphomas and their various metastases and micro
metastases. The method comprises providing a patient in need of
treatment for an oncological disorder with cells, such as a tumor
cells, that has been transformed to express a superantigen protein.
In one embodiment, cells are treated so as to introduce a
polynucleotide encoding a superantigen protein that will be
expressed by the cells, and then providing a patient in need of
such treatment with the transformed cells of the invention. In a
preferred embodiment, the superantigen is an M-like protein.
Preferably, the M-like protein is emmL 55, or a fragment or variant
thereof. The expression of full length and truncated versions of
the emmL 55 protein in a cell are specifically exemplified.
[0033] The subject invention also concerns methods for treating or
preventing oncological disease in a human or animal that comprises
expressing a polynucleotide coding for a first superantigen in a
cell, and further expressing in the cell a second polynucleotide
coding for at least one viral, bacterial or eukaryotic protein that
is immunogenic or immunostimulatory and providing the human or
animal with the transformed cells. In one embodiment, the subject
method comprises expressing a superantigen, such as an M-like
protein, and a foreign MHC antigen, such as a class II antigen, in
a cell and providing a patient with the transformed cells that
express superantigen and foreign class II MHC antigen. In another
embodiment, the subject method comprises expressing a superantigen
and a cytokine on a cell and providing a patient with the
transformed cells expressing superantigen and cytokine. A further
embodiment of the subject method comprises expressing a
superantigen, a foreign MHC antigen and a cytokine in a transformed
cell and providing a patient with the transformed cell. Cytokines
useful in the subject method include interleukins, such as IL-1,
IL-2, IL-3, IL-4, TNF.alpha., IFN.alpha., IFN.beta., IFN.gamma.,
GM-CSF, MIP1.alpha., MIP1.beta. and TGF.beta., and any other
suitable cytokines capable of modulating immune response. The
expressed cytokines can be either retained on the cell surface or
secreted by the cell. In another embodiment of the present
invention, the method comprises expressing a superantigen and an
antigen or antigen complex, such as those from pigs (Sykes et al.,
1991) or Mycobacterium (EP 6571 68), which has been shown to be
highly immunogenic, that can induce an acute immune response to the
antigen in a xenotransplantation. Al so contemplated within the
scope of the subject invention are cells transformed to express
polynucleotides encoding agents that are chemotactic for immune
cells.
[0034] The methods of the subject invention can be used in
combination with other therapies that are useful in treating
oncological disorders. These include, for example, surgical
resection of the tumor, radiotherapy, chemotherapy, and antibody
directed anti-tumor therapy, such as tumor-specific antibodies
conjugated with toxins. Also contemplated for use in conjunction
with the subject invention are cancer therapies, such as dendritic
cell cancer vaccines (Banchereau, J., R. M. Steinman, 1998; Gilboa,
F. et al., 1998).
[0035] The tumor cells used with the subject method can be from the
person's or animal's own tumor cells to be treated. Also
contemplated for use in the subject invention are cells from
sources other than the patient to be treated. The cells can be
transformed with polynucleotide molecules encoding superantigens
(e.g., M-like proteins), foreign MHC antigens and/or cytokines
using standard techniques known in the art. Cells can be
transformed either in vivo or in vitro. If the transformation is
performed in vitro, then transformed cells expressing
polynucleotides according to the subject invention can be reinfused
back into the animal or person. For in vivo transformation, the
polynucleotides can be delivered to the cells, for example, using
targeted liposomes that harbor the polynucleotide molecules. Viral
vectors, such as adenovirus, adeno-associated virus, retrovirus,
pox virus, herpes virus, plasmids and nucleic acid, can also be
used for transforming cells with the polynucleotide molecules
encoding the polypeptides of the present invention. Cells can also
be transfected using naked DNA, i.e., transfection by direct
injection of a tumor with naked DNA encoding proteins useful in the
subject methods. Targeted liposomes, viral vectors and naked DNA
can also be used in vitro to transform cells.
[0036] The subject invention also concerns a cell transformed with
a polynucleotide molecule or molecules encoding a superantigen,
such as an M-like protein, and, optionally, a foreign MHC antigen,
such as class II antigen, and/or a cytokine. In a preferred
embodiment, the polynucleotide encodes an emmL 55 polypeptide, or a
fragment or variant thereof. Particularly preferred are truncated
versions of M-like proteins exemplified herein.
[0037] The materials and methods of the present invention can also
be employed in combination with cytokine or other immunomodulating
therapies. Also contemplated within the scope of the methods of the
present invention are cells transformed with other streptococcal
superantigens that are expressed on the surface of the cell in
conjunction with foreign class II MHC expression.
[0038] The subject invention also concerns truncated M-like
protein, and the polynucleotides that encode the truncated
proteins, wherein the truncated M-like protein exhibits greater
levels of expression compared to full length M-like protein when
expressed in transformed cells. In a preferred embodiment, the
M-like protein is emmL 55. In an exemplified embodiment, a
truncated emmL 55 protein comprises the sequence encoded by the
emmL 55 gene where the protein coding region starts at the second
in-frame ATG codon in the nucleotide sequence of the emmL 55 gene.
A truncated version of emmL 55 protein can be produced using the
plasmid pSVK3/emmL 55 described herein.
[0039] The superantigen, foreign MHC antigen, and cytokine proteins
that can be used in the present invention include not only those
proteins having the same amino acid sequence as found in nature,
including allelic variants, but also includes those variant
proteins having mutations such as conservative amino acid
substitutions, additions and deletions in the protein sequence, as
long as the variant protein retains biological or immunotherapeutic
activity.
[0040] Oncological disorders that can be treated using the methods
and compositions of the present invention include lymphomas;
leukemias; carcinomas of the bladder, breast, lung, cervix, colon,
kidney, liver, ovary, prostate, pancreas, cartilage, testis,
tongue, uterus and thyroid; sarcomas such as those of the pelvis,
rhabdomyo (muscle), bone and osteogenic; brain tumors; gliomas;
gliobastomas; neuroblastomas; melanoma; hepatomas; medulloblastoma;
and Wilm's Tumors.
[0041] As is well known in the art, the amino acid sequence of a
protein is determined by the nucleotide sequence of the DNA.
Because of the redundancy of the genetic code, i.e., a single amino
acid can be coded for by more than one coding nucleotide triplet
(codon). Accordingly, different nucleotide sequences can code for a
particular amino acid sequence. The amino acid sequences of the
proteins of the subject invention can be prepared by nucleotide
sequences other than the wild-type or native sequences.
Functionally equivalent nucleotide sequences encoding the amino
acid sequence of these proteins and fragments thereof can be
prepared by known synthetic procedures. Accordingly, the subject
invention includes such functionally equivalent nucleotide
sequences.
[0042] Thus, the scope of the subject invention includes not only
specific nucleotide sequences exemplified herein, but also all
equivalent nucleotide sequences coding for proteins of the
invention having substantially the same antigenic, immunogenic, or
therapeutic activity.
[0043] As used herein the term "foreign MHC antigen" refers to MHC
antigens that are distinct from the MHC antigens naturally
expressed on the cells of the person or animal. MHC antigens within
the scope of the invention include class I, class II and class III
antigens.
[0044] In an exemplified embodiment of the present invention, a
neuroblastoma cell line transformed to express a foreign MHC class
II antigen and an M-like protein was prepared and used to treat a
mouse strain in which the neuroblastoma arises. The clone Neuro-2a
was established from a spontaneous tumor of strain A albino mouse
in 1969. This tumor line, designated C1 300, arose spontaneously in
A/J mice and has been carried in this strain since. Neuro-2a
resembles human neuroblastoma in many respects and is commonly used
as an experimental model. Tumors quickly appear at the site of
inoculation after variable latency periods, which are dependent on
the numbers of cells inoculated (see Table 1). Concurrent physical
examination demonstrates soft, well vascularized tumors with blood
vessel transformation and secondary tumor formation. Histological
examination demonstrates that the primary sites of metastasis are
the lung and liver.
1TABLE 1 Tumorgenicity of neuroblastoma cell line Neuro-2a in
syngeneic A/J mice. Neuro-2a cells .times. 10.sup.6 Mice injected
Latency (days) % mice with tumor 3 5 7-13 100 1 5 8-20 100 0.5 5
10-20 80 0.1 5 14-28 80 0 5 0 0
[0045] The pSDV1 and pSVK3 vectors are useful for expression
studies in a wide variety of mammalian cell lines. They contain
sequences for efficient replication in E. coli, an mRNA splice site
and polyadenylation signal from SV40 for replication and expression
in eucaryotic cell line. The genes for Dw14.alpha. and .beta.
currently reside separately on pSDV1, a mammalian expression vector
originally designed by Okayama and Berg. These plasmids must be
cotransfected in order for the MHC class II-DR4 antigen to be
expressed on the cell surface. The use of these constructs acts as
a positive control in the transfection and expression in Neuro-2a
cells and in the suppression of tumor growth. Production of an
expression vector expressing the emmL 55 gene is based on the
amplification of the emmL 55 gene by PCR and generation of
restriction sites at the ends of the product which will allow
subsequent ligation to the multiple cloning site of the pSVK3
expression vector. The resulting PCR fragment was gel-purified and
subcloned into the pCRII vector. Restriction mapping confirmed that
several clones contained the correctly amplified product. A single
clone was used for further study. The restriction endonucleases
Hind III and EcoRI were used to excise the PCR product from the
pCRII cloning vector, gel-purified, and subcloned into pSVK3.
[0046] In order to evaluate the expression on the cell surface of
these antigens and to develop stably transfected Neuro-2a cell
lines, i.e., Neuro-2a cells expressing both emmL 55 and Dw14,
Neuro-2a cells were electroporated with plasmids containing either
the emmL 55 gene or the Dw14 .alpha. and .beta. genes as described
above. Expression of DW-14 and emmL 55 proteins was analyzed by
flow cytometry using monoclonal antibodies. Successfully
transfected cells were sorted by FACS and evaluated over time.
Within two hours of electroporation, greater than 80% of the
Neuro-2a cells cotransfected with Dw14 .alpha. and .beta. genes
expressed MHC class II-DR4 antigen on the surface. After 21 days,
50% of the cells still expressed this marker. Approximately 30% of
cells transfected with the emmL 55 construct expressed detectable
amounts of this antigen on their surface immediately following
electroporation, but none was detected after 7 days. Sequence
analysis revealed that the emmL 55 construct was missing the 5' ATG
codon along with other 5' nucleotides. This presumably occurred
during PCR amplification. Thus, it may be preferable to subclone
the emmL 55 gene directly from the original plasmid into another
expression vector containing a selectable marker as a means to keep
selective pressure on the cells to retain the plasmid.
MATERIALS AND METHODS
Mice
[0047] Adult A/J syngeneic female mice were purchased from The
Jackson Laboratory (Bar Harbor, Mass.). Mice used in this
experiment were seven weeks-old. All mice were housed in sterile
cages, fed with sterile food and water ad ubitum, and maintained in
a pathogen-free animal facility.
Cell Line
[0048] Neuro-2a (American Type Culture Collection, Rockville, Md.
USA), a subdlone of C1300 murine neuroblastoma was maintained in
culture on Iscove's Modified Dulbecco's Medium (IMDM) supplemented
with 2 mM L-glutamine, 10% (w/v) fetal bovine serum (FBS) (GIBCO
Laboratories, Grand Island, N.Y.), penicillin (100 units/ml) and
streptomycin (100 .mu.g/ml). The cells were incubated at 37.degree.
C. in a humidified incubator in an atmosphere 5% CO.sub.2, in 95%
air.
Tumor Cell Inoculation
[0049] The cells were prepared for inaculation by gently removing
them from 75 cm.sup.2-tissue culture flasks with a sterile cell
scraper. The cell suspension was harvested by centrifugation at
800.times.g and the resulting pellet resuspended in incomplete
IMDM. A/J mice were inoculated sub-cutaneously (sc) in the right
flank with Neuro-2a cells. Each set of five mice were injected with
the following cell concentrations: 3.times.10.sup.6,
1.times.10.sup.6, 5.times.10.sup.5, and 1.times.10.sup.5, in 0.2 ml
incomplete IMDM. The mice were examined for tumor development every
other day. A detailed physical examination was performed, by
dissecting the mice after 20 days. Tumors and other tissues were
removed from the mice and placed in 10% (w/v) neutral buffered
formalin for histological examination (Florida Hospital, Florida
Pathology Laboratory, Orlando, Fla.).
Amplification of emmL 55 by the Polymerase Chain Reaction (PCR)
[0050] The cDNA for emmL 55 was isolated from the parental vector,
pJLA 602, which was kindly provided by Dr. M. Boyle, (Medical
College of Ohio, 3000 Arlington Avenue, P.O. Box 10008, Toledo,
Ohio 43699). PCR assays were carried out in a 50 .mu.l format for
product preparation using the thermocycler Twin Block System
(Ericomp, San Diego, Calif.). Each PCR reaction contained the
following final concentration of reactants: 100 ng template DNA;
2.5 units Taq-Polymerase (Promega, Madison, Wis.); 1 mM of each
primer (Oligo, Wilsonville, Oreg.); 1.75 mM MgCl.sub.2 (Promega,
Madison, Wis.); 5 .mu.l of 10.times.PCR buffer (Promega, Madison,
Wis.), and 250 mM dNTPs (Pharmacia Biotech, Piscataway, N.Y.). For
cloning purposes, the primers contained 5' tags with either an Eco
RI or Xho I restriction site. The primers used to amplify the emmL
55 gene and for sequencing the amplified gene products are listed
in Table 2. Each assay was overlaid with 50.mu. of mineral oil and
denatured for 5 min. at 94.degree. C. The reaction mixture was
subjected to 35 cycles of 1 min. at 94.degree. C. followed by 1
min. at 60.degree. C., 1 min. at 72.degree. C. and 10 min. at
72.degree. C. PCR products for cloning were combined and
concentrated using
2TABLE 2 List of oligonucleotides for amplification and sequencing
of emmL 55. Oligonucleotide Description of Designation Sequence (5'
to 3') Target Site (a) oligonucleotides for amplification of the
emmL 55 coding sequence AS1 TAG AAT TCA TGG CTA AAA 5' end of emmL
55 ATA CCA CGA ATA G AS2 TTC TCG AGT TAG TTT TCT 3' end of emmL 55
TCT TTG CGT TTG AC (b) oligonucleotide utilized for sequencing the
amplified emmL 55 product AS3 CAG TTC CGC CCA TTC TTC 5' portion of
pSVK 3/emmL 55
[0051] a Microcon Concentrator-100 (Amicon, Beverly, Mass.). The
combined products were then applied to a 1% (w/v) agarose gel and
separated by electrophoresis at 50 V for 150 min. The resulting 1.6
kb product representing the amplified emmL 55 gene was extracted
from the gel and purified by QIAquick column (Qiagen, Chatsworth,
Calif.). The DNA was precipitated by incubating 0.1 volumes of 3 M
sodium acetate with 2.5 volume of ethanol at 70.degree. C. for 2
hours followed by centrifugation at 12, 000.times.g for 15 min.
Finally, the DNA was resuspended in TE buffer (10 mM Tris, HCL and
1 mM EDTA, pH 8.0) and the yield was analyzed by electrophoresis
prior to ligation. Electrophoretic analysis of the PCR yield was
performed by applying 4 and 8 .mu.l of the product directly onto a
1% (w/v) agarose gel and compared with the DNA Mass Ladder from
Gibco (Grand Island, N.Y.).
Cloning of emmL 55 Gene into the pCR II Vector
[0052] The TA cloning kit (Invitrogen, San Diego, Calif.) uses the
pCR II vector and provides a quick, one-step cloning strategy for
the direct insertion of a PCR product into a plasmid vector. TA
cloning works by using a Taq polymerase nontemplate-dependent
activity, which adds a single deoxyadenosine (A) to the 3' ends of
PCR products and by using the pCR II, a linearized vector which has
3' deoxythmidine (T) residues, allowing efficient ligation.
[0053] The amount of PCR product needed to ligate with 50 ng (20
finoles) of pCR II vector was estimated using the formula below: 1
X ng PCR product = ( Y bp product ) ( 50 ng pCRII vector ) ( size
in bp of the pCRII vector : 3900 )
[0054] Two ligation reactions using the following final
concentrations were set up:
[0055] 1) for 1:1 (vector:product) reaction, 50 ng of pCR II
vector:20.51 ng of emmL 55 DNA were used;
[0056] 2) for 1:3 reaction, 50 ng of pCR II vector:61.53 ng of emmL
55 DNA were used.
[0057] 1 .mu.l of 10.times.ligation buffer, T4 DNA ligase (4.0
Weiss units) and H.sub.2O up to 10 .mu.l final volume were used for
each ligation reaction. Ligation reactions were incubated overnight
at 14.degree. C. and used for transformation of competent One Shot
INV.alpha.F' E. coli cells, provided in the One Shot competent cell
kit.
Transformation of One Shot INV.alpha.F' E. coli Competent Cells
[0058] Two .mu.l of 0.5 M .beta.-mercaptoethanol (.beta.-ME) were
added to 50 .mu.l of One Shot INV.alpha.F' competent cells and
mixed directly with 2 .mu.l of each ligation reaction, incubated on
ice for 30 min. and heat shocked at 42.degree. C. for 30 sec. After
transformation, the bacterial cells were grown in 450 .mu.l of SOC
medium (2% w/v tryptone, 0.5% w/v yeast extract, 10 mM NaCl, 2.5 mM
KCl, 10 mM MgCl.sub.2, 10 mM MgSO.sub.4 and 20 mM glucose) at
37.degree. C. for 1 hour with vigorous shaking. Fifty .mu.l and 200
.mu.l from each transformation vial were spread on Luria-Bertani
(LB), (10 g bacto tryptone, 5 g bacto yeast extract, 10 g NaCl, for
1 liter, pH 7) agar plates containing 50 .mu.g/ml of ampicillin and
40 .mu.g/ml X-Gal and incubated overnight. After incubation, plates
were shifted to 4.degree. C. for 24 hours to allow proper color
development.
Restriction Analysis of pCR II/emmL 55
[0059] Restriction analysis was used to determine the presence and
orientation of the emmL 55 insert. Blue-white screening of
bacterial clones was performed in order to obtain the clones
containing the amplified insert. White colonies were used for
cracking gel analysis. Briefly, cells from white colonies were
transferred into 5 ml of LB broth supplemented with 50 .mu.g/ml
ampicillin and grown overnight aerobically at 37.degree. C. Five
hundred .mu.l of bacterial culture were transferred into
microcentrifuge tubes and centrifuged for 1 min. at 1500.times.g.
Bacterial pellets were lysed in cracking buffer (1% (w/v) SDS, 2 mM
EDTA, 0.4 M sucrose, 0.05 M Tris HCL and 0.01% (w/v) Bromo Phenol
Blue) and bacterial lysates were run on a 1% (w/v) agarose gel.
Clones which had a 5.5 kb size plasmid were used for further
analysis. Preparation of the DNA for restriction analysis from One
Shot INV.alpha.F' cells was then performed using standard
procedures. The bacterial cells were lysed using the alkaline
mini-prep technique and the DNA was purified by phenol/chloroform
extraction (Sambrook et al., 1989). The recombinant DNA was
subsequently digested with the following restriction endonucleases:
Eco RI (12 units/.mu.l), Xho I (10 units/.mu.l) in combination
(Promega, Madison, Wis.).
Subcloning of emmL 55 into pSVK 3
[0060] To clone emmL 55 into the pSVK 3 plasmid (Pharmacia,
Piscataway, N.Y.), emmL 55 cDNA was excised from the pCRII/emmL 55
construct by Eco RI (12 units/.mu.l) and Xho I (10 units/.mu.l)
(Promega, Madison, Wis.) (FIG. 1). The digestion was carried out
overnight at 37.degree. C. The sample was run on a 1% (w/v) agarose
gel at 50 V for 180 min. and the 1.6 kb band was then extracted
from the gel and purified using a gel extraction kit (Qiagen,
Chatsworth, Calif.) according to the instructions of the supplier.
The pSVK3 plasmid (Pharmacia Biotech, Piscataway, N.Y.) was
digested with Eco RI (12 units/.mu.l) and Xho I (10 units/.mu.l) in
order to produce compatible ends needed for ligation with emmL 55.
Restriction enzymes were inactivated by incubation at 70.degree. C.
for 20 min. The amount of emmL 55 cDNA needed to ligate with 50 ng
of pSVK 3 vector (3919 bp) was estimated using Formula 1. Three
ligation reaction were set up using the following final
concentrations, for 1:1 (vector :insert) reaction 50 ng: 20.41 ng,
for 1:3, 50 ng: 61.24 ng and for 1:6, 50 ng: 122.48 ng of pSVK
3:emmL 55 cDNA, 1 .mu.l of 10.times.ligation buffer, T4 DNA ligase
(4.0 Weiss units) and H.sub.2O up to 10 .mu.l final volume for each
ligation reaction.
[0061] XL-1 Blue E. coli cells were transformed with the ligation
products using a standard heat shock procedure. One hundred .mu.l
of ice-cold transformation buffer TMF (10 mM Tris-HCL, 50 mM
CaCl.sub.2, 10 mM MgSO.sub.4.times.7 H.sub.2O, filter sterilized)
was added to 200 .mu.l of XL-1 Blue E. coli cells and mixed
directly with 2 .mu.l of each ligation reaction, incubated on ice
for 45 min. and heat shocked at 37.degree. C. for 2 min. After
transformation, the bacteria were left for 10 min. at room
temperature, then transferred into 500 .mu.l of LB broth and
incubated for 90 min. at 37.degree. C. Twenty five, 50, and 200
.mu.l from each transformation culture were spread on to LB plates
supplemented with ampicllin (50 .mu.g/ml) and tetracycline (10
.mu.g/ml) and then incubated overnight at 37.degree. C.
Restriction Map of pSVK 3/emmL 55 Construct
[0062] To determine the presence and orientation of the emmL 55
insert, bacterial colonies were isolated and grown overnight in LB
broth with 50 .mu.g/ml ampicillin. Four clones were chosen for
plasmid isolation and restriction endonuclease analysis. The
following endonucleases were used: Bam HI (10 units/.mu.l); Nhe I
(12 units/.mu.l); Xho I (10 units/.mu.l) and Eco RI (12
units/.mu.l); Hpa I (10 units/.mu.l) and Cla I (10 units/.mu.l);
Hpa I (10 units/.mu.l) and Nhe I (12 units/.mu.l). Clone number 2
was used for further map analysis. A restriction map was
constructed using Nhe I(12 units/.mu.l); Nde I (10 units/.mu.l);
Pvu II (10 units/.mu.l); Hind III (10 units/.mu.l); Pvu II (10
units/.mu.l) and Nhe I (12 units/.mu.l); Pvu II (10 units/.mu.l)
and Nde I (10 units/.mu.l); and Eco RI (12 units/.mu.l) and Xho I
(10 units/.mu.l).
Sequence Analysis of pSVK 3/emmL 55
[0063] DNA sequence analysis was performed using pSVK 3/emmL 55 as
a template. Sequencing was carried out using a sequenase version
1.0 DNA sequencing kit (Amersham, Arlington Heights, Ill.), a
C.B.S. SG-500-33 adjustable nucleic acid sequencer and the primers
listed in Table 2 according to the Sanger method of
dideoxy-mediated chain termination (Sanger et al., 1977).
Denaturation of the double-stranded template was carried out by
adding 0.1 volumes 2 M NaOH/2 mM EDTA and incubating 30 min. at
37.degree. C. The sample was neutralized by the addition of 0.1
volumes 3 M NaAc, pH 5.5, and the DNA was precipitated as above.
The sample was redissolved in 7 .mu.l distilled water. The
annealing reaction was carried out by heating the DNA mixture (0.5
.mu.g/.mu.l) with the primers (1 mM) and 2 .mu.l of the reaction
buffer at 65.degree. C. for 2 min. After incubation, the reaction
was slowly cooled to 35.degree. C. for 15-30 min. To the ice-cold
annealed DNA, the following labeling reagents were added: 0.1 M
DTT; 0.1 M, 2 .mu.l diluted labeling mix; .sup.35S dATP (0.5
.mu.l); and Klenow sequencing polymerase (2 .mu.l). The labeling
reaction was carried out by incubating the reagents at room
temperature for 2-5 min. The reactions were terminated by adding
the labeling reaction (3.5 .mu.l) to the prewarmed A, T, G, C,
termination mixtures (2.5 .mu.l) and incubating at 37.degree. C.
for 5 min and quenched by adding 4 .mu.l of stop solution. Before
loading on a 6% (w/v) sequencing gel, the samples were heated for 2
min. at 75.degree. C. The gel was run at constant power of 35-40 W.
After fixing the gel with 10% (w/v) methanol and 10% (w/v) acetic
acid, the gel was transferred to 3 MM Whatman paper and placed
under vacuum in a dryer for 40 min. at 80.degree. C. The gel was
then exposed to X-ray film (Kodak) at room temperature for 24 hours
and subsequently developed in Kodak-M35A X-OMAT Processor.
Subcloning of emmL 55 cDNA into pcDNA 3
[0064] The cDNA for emmL 55 was isolated from the parental vector,
pJLA 602, which was kindly provided by Dr. M. Boyle. Isolation and
preparation of the parental plasmid from E. coli DH5.alpha. was
performed using standard techniques (Sambrook et al., 1989). The
emmL 55 cDNA for was excised from the pJLA 602 by restriction
endonuclease digestion with Bam HI (10 units/.mu.l) and Eco RI (12
units/.mu.l) (FIG. 2). DNA purification was by electrophoresis
elution using a QIAquick gel extraction kit (Qiagen, Chatsworth,
Calif.). The emmL 55 cDNA was ligated into the pcDNA 3 expression
vector (Invitrogen, San Diego, Calif.) which was prepared by double
digestion with Bam HI (10 units/.mu.l) and Eco RI(12 units/.mu.l)
as above, to reveal the necessary restriction sites.
[0065] E. coli JM109 high efficiency competent cells (Promega,
Madison, Wis.) were transformed with the ligation reaction products
which were prepared as follows: The amount of emmL 55 cDNA was
estimated using Formula 1. Three ligation reactions were performed
for 1:1 (vector:insert) 15.2 ng of emmL 55, for 1:3 45.6 ng of emmL
55.50 ng of pcDNA 3 (5400 bp), T4 DNA ligase (4.0 Weiss units) and
H.sub.2O up to 10 .mu.l final volume for each ligation reaction.
Reactions were incubated overnight at 14.degree. C. and used for
transformation of E. coli JM109 high efficiency competent cells.
One hundred .mu.l of bacterial cells were mixed with 2 .mu.l of
each ligation reaction. After gentle mixing, cells and DNA were
incubated on ice for 2 min. and transferred to 42.degree. C. for 1
min. Cultures were then incubated in 1 .mu.l LB broth for 45 min.
at 37.degree. C. with shaking. Twenty, 50, and 200 .mu.l of each
culture were spread over the surface of LB agar plates supplemented
with 50 .mu.g/ml of ampicillin and incubated overnight. Cracking
gel analysis was performed and three clones carrying 7,000 bp
inserts were studied further.
Restriction Map of pcDNA 3/emmL 55
[0066] Preparation of pcDNA 3/emmL 55 DNA for restriction map
analysis was performed by the mini-prep alkaline lysis method
(Sambrook et al., 1989). Purified DNA from the three selected
clones was analyzed by restriction endonuclease digestion with Pst
I (10 units/.mu.l), Hind III (10 units/.mu.l) and Xba I (10
units/.mu.l) and agarose gel electrophoresis. A single clone was
chosen for the restriction map of pcDNA 3/emmL 55 and was digested
with: Nhe I (12 units./.mu.l); Eco RV (20 units/.mu.l); Bam HI (10
units/.mu.l) and Eco RI (12 units/.mu.l); Nde I (10 units/.mu.l);
Xba I (10 units/.mu.l); Pst I (10 units/.mu.l); and Hind III (10
units/.mu.l).
Growth Curve of Neuro-2a
[0067] Neuro-2a cells were harvested by trypsinization at
37.degree. C. for 5 min. and centrifugation at 800.times.g for 10
min. at 25.degree. C. Following enumeration by trypan blue
exclusion, the cells were serially diluted (6.4.times.10.sup.5,
3.2.times.10.sup.5, 1.6.times.10.sup.5, 8.times.10.sup.4,
4.times.10.sup.4, 2.times.10.sup.4, 1.times.10.sup.4,
5.times.10.sup.5, 2.5.times.10.sup.3, and 1.2.times.10.sup.3),
plated in quadruplicate onto 96 well plates and incubated at
37.degree. C., in a humidified atmosphere of 5% CO.sub.2 in 95% air
for 2, 4, 6, or 8 days. Cell proliferation was measured by the
Alamar Blue assay (Alamar Bioscience, Sacramento, Calif.). This
assay incorporates a fluorometric/colometric growth indicator which
detects metabolic activity. This indicator both fluoresces and
changes color in response to a chemical reduction of the growth
media resulting from cell growth. Alamar Blue regent was added
aseptically to each well in an amount equal to 10% of the cell
culture volume (20 .mu.l) and incubated with for 3 hours under cell
growth conditions. Absorbency was measured on a Ceres UV 900 HD
(Bio Tek Instruments) at a wavelength of 570 nm. Background
adsorbance (600 nm) was subtracted prior to tabulating the
data.
Inhibition Assay of Neuro-2a with G418
[0068] In order to determine the concentration of G418 (Gibco
Laboratories, Grand Island, N.Y.) needed for selection of
transfected cells, the following experiment was performed. Two cell
concentrations (2.times.10.sup.4 and 1.6.times.10.sup.5) were
chosen and plated onto 96 well plates and G418 at five
concentrations (100, 200, 300, 400, and 500 .mu.g/ml) was added.
All dilutions were plated in quadruplicate. Replicate plates were
incubated at 37.degree. C. in a humidified atmosphere of 5%
CO.sub.2 on air for 2, 4, 6, 8 or 10 days. Survival was assessed
using the Alamar Blue assay as previously described.
Gene Transfer of pcDV 1/.alpha. and pcDV 1/.alpha. into Neuro-2a by
Electroporation
[0069] The MHC II cDNA (DR4, DW 14) was generously provided in the
Okayama and Berg expression vector, pCDV 1 (Okayama et al, 1987),
by Peter K. Gregersen M.D. (North Shore University Hospital,
Cornell University Medical College, Manhasset, N.Y.). pcDV
1/.alpha. and pcDV 1/.beta. constructs were used for cotransfection
of Neuro-2a cells with the MHC II, DR 4 gene. The .alpha. gene
encodes the .alpha. domain of MHC and the .beta. gene encodes for
.beta. domain of MHC. The cells were harvested and washed twice
with incomplete IMDM. For each electroporation reaction,
2.times.10.sup.5/ml of Neuro-2a cells were used. Cell suspension
samples of 250 .mu.l were mixed with 250 .mu.l (0.1 .mu.g/.mu.l)
each pcDV 1/.alpha. and pcDV 1/.beta. DNA in a Gene Pulser Cuvette.
The DNA samples were resuspended prior to mixing in 2.times.Hank's
balanced salts buffer (Hbs) (1.4 mM Na.sub.2HPO.sub.4, 10 mM KCl,
12 mM glucose, 275 mM NaCl and 40 mM HEPES, pH 7.2).
Electroporation was carried out under three conditions: 1000 V, 21
.mu.F; 450 V, 500 .mu.F; and 300 V, 900 .mu.F. After
electroporation, the cells were placed on ice for 10 min. then
transferred to 6 well plates. The cells were incubated in complete
IMDM at 37.degree. C., under 5% CO.sub.2. After 24 hours, 3 ml
spent medium was replaced with 4 ml fresh medium and the cells were
incubated for an additional 48 hours. Gene expression was measured
by flow cytometry after 48 hours and after 18 days.
[0070] In order to obtain stably transfected cells Neuro-2a cells
were electroporated with 20 .mu.g each of pSG1NEOpA, pcDV 1/.alpha.
and pcDV 1/.beta. at 260 V and 1050 .mu.F. The cells were plated
immediately in 5 ml of IMDM and incubated at 37.degree. C. in 5%
CO.sub.2. After 48 hours, 3 ml spent medium was replaced with fresh
3 ml IMDM and G418 was added to final concentration of 500
.mu.g/ml. G418 was replaced every five days.
Gene Transfer of pSVK 3/emmL 55 into Neuro-2a by
Electroporation
[0071] pSVK 3/emmL 55 was transfected into Neuro-2a cells by
electroporation. The Neuro-2a cells were prepared for gene transfer
as previously described except that the number of cells used for
each reaction was 2.times.10.sup.6/ml. pSVK 3/emmL 55 was
linearized before transfection with Bam HI (10 units/.mu.l). Twenty
.mu.g DNA was resuspended in H.sub.2O and used for each
electroporation reaction. Electroporation was carried under three
different conditions: 220 V, 1050 .mu.F; 260 V, 1050 .mu.F; and 300
V, 1050 .mu.F. Following electroporation, the cells were plated on
the 9 wells culture plates and 5 ml of complete IMDM was added.
Gene expression was measured by flow cytometry after 72 hours and
11 days.
[0072] In order to obtain stably transfected cells Neuro-2a cells
were electroporated with 20 .mu.g each of pSG1NEOpA and pSVK 3/emmL
55 at 260 V and 1050 .mu.F. Cells were cultured under conditions as
described for gene transfer of pcDV 1/.alpha. and pcDV
1/.beta..
Gene Transfer of pcDNA 3/emmL 55 into Neuro-2a by
Electroporation
[0073] Twenty .mu.g of pcDNA 3/emmL 55 was electroporated into
Neuro-2a cells at concentration of 2.times.10.sup.6 cells/ml at 260
V and 1050 .mu.F. The cells were plated immediately in 5 ml IMDM
and incubated at 37.degree. C. in 5% CO.sub.2 in air. After 48
hours, 3 ml spent medium was replaced with fresh 3 ml IEDM and G418
was added to final concentration of 500 .mu.g/ml. G418 was replaced
every five days. The cells was analyzed for expression of emmL 55
by flow cytometry after 21 days.
Analysis of Gene Expression by Glow Cytometry
[0074] For flow cytometric analysis, the transfected Neuro-2a cells
were prepared by gently removing them from the tissue culture
flasks with a sterile cell scraper. The cells were washed and
collected by centrifugation at 800.times.g, resuspended in
incomplete IMDM to concentration 1.times.10.sup.6 cells/ml. The
cells were then incubated for one hour with the primary antibodies
at a dilution of 1:500 of antibody:cells. For MHC II expression the
primary antibody (anti -MHC-DR-FITC) was purchased from Becton
Dickinson (San Jose, Calif.). After washing, fluorescence of the
antibody-exposed cells was analyzed by an Epics Elite ESP (Coulter
Electronics, Hialeah, Fla.).
[0075] Anti-M-like protein antibody (polyclonal .alpha. II o and
monoclonal 8 F-10; 25 C; and 15 .beta.3) were provided by Dr. M.
Boyle (Boyle et al., 1994, and Boyle et al., 1995). After staining
with primary antibody the cells were washed three times in
incomplete medium then incubated in the presence of a Avidin FITC
conjugate for polyclonal .alpha. II o or with mouse IgG-FITC
(Becton Dickinson San Jose, Calif.) for the monoclonals at 1:1000
(secondary conjugate:cells) at 4.degree. C. in the dark for 30 min.
The exposed cells were washed three times, resuspended in a volume
of 0.5 ml of incomplete IMDM, then analyzed by flow cytometry.
Immunoflourescence background was measured by comparing the
staining of untransfected Neuro-2a with both the primary and
secondary conjugate and secondary conjugate alone.
[0076] Following are examples which illustrate materials and
procedures for practicing the invention. These examples are
intended for illustrative purposes only and should not be construed
as limiting.
EXAMPLE 1
Tumorgenicity of Neuro-2a in A/J Syngeneic Mice
[0077] Neuroblastoma cells were highly tumorigenic in A/J sygeneic
mice. Visible tumors appeared at the site of inoculation after a
variable latency period. The duration of latency was dependent upon
the number of cells injected (Table 1). Injection of
3.times.10.sup.6 and 1.times.10.sup.6 cells caused tumor formation
in 100% of the mice tested, and in these cases, the latency period
was 7-20 days. Eighty percent of the mice injected with
5.times.10.sup.5 and 1.times.10.sup.5 cells developed tumors within
10-28 days. Once the tumors appear, they grew rapidly and their
size was not dependent on number of cells originally injected.
[0078] The tumors grew under the skin, invaded the surrounding
muscle tissue and approached 75% of the weight of the mouse.
Resected tumors were soft and well vascularized; angiogenesis of
blood vessels was evident. Additionally, in some mice secondary
tumor formation was observed. The overwhelming size of the
malignant tissue caused necrosis in some of the tumors examined,
but on physical examination, there were no neoplasms apparent in
other tissues (lung, liver, spleen, bone marrow). Histological
examination, however, showed that the metastasis had occurred with
microscopic, neoplastic foci being present in the lung and the
liver, but not in the spleen or bone marrow.
EXAMPLE 2
Construction and Analysis of the pCR II/emmL 55
[0079] In order to subclone the emmL 55 gene into pSVK 3, emmL 55
was amplified from the parental vector, pJLA 602 and restriction
sites necessary for subcloning were added to the ends of the
amplified product. Agarose gel electrophoresis of the emmL 55 PCR
product showed that the size of amplified gene was 1.6 kb and the
amount of the amplified product was 8 ng per 1 .mu.l of the TE
buffer.
[0080] One Shot INFV.alpha.F' E coli. competent bacterial cells
were then transformed with pCR II/emmL 55. Bacterial clones were
screened for the presence of the recombinant construct by
white/blue selection. One clone showed the expected size 5.5kb and
was chosen for restriction analysis. The pCR II/emmL 55 construct,
when digested with Eco RI and, Xho I restriction enzymes, released
the emmL 55 gene. Agarose gel electrophoresis of the digested
product show the expected band pattern, 1.6 kb for emmL 55 and 3.9
kb for the pCR II vector. The amplified emmL 55 gene was used for
further cloning into the pSVK 3 expression vector.
EXAMPLE 3
Construction and Analysis of pSVK 3/emmL 55
[0081] From the 20 analyzed bacterial clones, transformed with the
pSVK 3/emmL 55, four clones with 5.5 kb size were selected and
digested with restriction enzymes. Three out of four clones
transformed with pSVK 3/emmL 55 exhibited the expected banding
pattern and one was used for the subsequent analysis. pSVK 3/emmiL
55 when digested with Nhe I and with Nde I generated a 5.5 kb band
as expected. Digestion with Pvu II yielded a 2 kb and a 3.5 kb band
and digestion with Hind III generated two bands, 0.9 kb and 4.6 kb.
Double digestion with Pvu II and Nhe I resulted in three bands of
approximately 1 kb, 2 kb and 5.5 kb pairs and digestion with Pvu II
and Nde I generated three bands, 0.77 kb, 1.2 kb, and 3.5 kb (FIG.
3). Restriction analysis of pSVK 3/emmL 55 revealed that the
recombinant construct had the expected size (5.5 kb) and the
expected map.
[0082] In order to confirm that the correct product was amplified,
sequence analysis was performed. The partial sequence of the emmL
55 gene and the pSVK 3 vector was determined and is presented in
FIG. 4. The analyzed gene exhibited the typical sequence of emmL
55. However, because of a mutation in the sequence at the site of
the first start codon the transcription start site was shifted to
the second ATG codon at 761 bp position of the emmL 55 gene and the
expressed recombinant protein was truncated (FIG. 4, the second
start codon is at 761 bp site of pSVK 3/emmL 55.
EXAMPLE 4
Construction and Analysis of the pcDNA 3/emmL 55
[0083] FIG. 5 shows results from the restriction analysis of pcDNA
3/emmL 55. The digestion of three clones with Hind II and Pst I in
separate reactions yielded the expected band sizes. One of these
clones was analyzed by the subsequent restriction analysis. The
recombinant plasmid was linearized by Eco RV, which recognizes a
site in pcDNA 3 and by Nhe I, which recognizes a site in emmL 55.
Digestion with Bam HI and Eco RI yielded a 1.6 kb and a 5.4 kb
fragment. Nde I recognizes a site the expression vector backbone
and in the emmL 55 gene, generating two bands (1.3 kb and 5.8 kb).
There are two Pst I sites present in pcDNA 3 and restriction with
this enzyme generated two fragments (1.4 kb and 5.7 kb bands). Hind
III generates three bands 351 bp, 912 bp and 5818 bp. The
restriction digests of the pcDNA 3/emmL 55 construct and agarose
gel electrophoresis confirm the identity of this plasmid. It was
concluded that the recombinant plasmid could be used for the
transfection of the Neuro-2a cells.
EXAMPLE 5
Growth Characteristics of Neuro-2a
[0084] Cell proliferation assays were performed to estimate the
growth characteristics of the Neuro-2a cells needed for subsequent
experiments. Ten starting cell concentrations were continuously
incubated over an 8 day period with cell numbers being determined
every other day. Cell proliferation was measured using Alamar Blue.
Results were expressed as a mean of Alamar Blue adsorbance of four
cultures plus or minus standard error. The lower cell
concentrations, up to 1.times.10.sup.4 cells/ml showed standard
growth characteristics. In higher concentrations, between
2.times.10.sup.4 and 4.times.10.sup.4 cells/ml, cells grew
exponentially for the first four days then reached stationary phase
and started to die. Exponential growth was observed in
concentrations between 8.times.10.sup.4 and 1.6.times.10.sup.5
cells/ml for the first two days and the viable count decreased
slowly in the following days. Two cell concentrations:
2.times.10.sup.4 and 1.6.times.10.sup.5 cells/ml were chosen for
use in for the drug sensitivity assay.
EXAMPLE 6
Drug Sensitivity Assay
[0085] A drug sensitivity assay was performed to establish the
concentration of G418 needed for killing Neuro-2a cells. Cell death
was measured by the Alamar Blue assay. The growth of Neuro-2a cells
(2.times.10.sup.4 cells/ml) was efficiently suppressed by 500
.mu.g/ml of G418. This concentration of the toxic agent caused
growth inhibition of up to 78% after 2 days and the viability of
cells dropped to zero after 6 days. Growth of Neuro-2a was also
inhibited at concentrations of 400 and 300 .mu.g/ml, while
concentrations of 100 and 200 .mu.g/ml of G418 had minimal effect
on cell growth.
[0086] Similar patterns of the growth inhibition by G418 were noted
when cell concentration was 1.6.times.10.sup.5 cells/ml. In this
experiment the most efficient inhibition of cell growth was
observed when 500 .mu.g/ml of drug was used. From these results it
was concluded that 500 .mu.g/ml is an effective amount of G418 for
selecting stably transfected Neuro-2a cells.
EXAMPLE 7
Morphological Characteristics of Stable Transfected Cell Lines
[0087] Introduction of the vectors into cells changed the cell
morphology (FIG. 6). The transfected cells selected by G418 (panels
B, C, and D) grew in characteristic clumps, whereas the
untransfected neuroblastoma cells formed an even monolayer (panel
A). This finding can not be explained by the presence of G418 in
the medium since in previous experiments (inhibition assay)
Neuro-2a did not show any morphological changes in the presence of
drug.
EXAMPLE 8
Expression of MHC Class II Antigen by Neuro-2a Cells
[0088] Using flow cytometry, it was found that the recombinant
antigen, DR 4, DW 14 (MHC II) was successfully expressed by
Neuro-2a cells. Forty eight hours after electroporation, up to
84.8% of the cells were able to express the recombinant antigen.
The expression of the DR 4, DW14 antigen was still present 18 days
after electroporation. Twenty nine to 48.2% of the Neuro-2a cells
were able to bind mAbs against DR 4, DW 14 after 18 days.
[0089] It was also observed that the level of DR 4 expression was
dependent on the electroporation conditions. In a second
independent experiment, up to 55.1% of Neuro-2a cells was able to
express the recombinant antigen 48 hours after electroporation
(Table 3). The highest level of fluorescence was observed in cells
transfected at 1000 V and 21 .mu.F. Fifty three % of cells
expressed MHC II-DR 4 antigen when they were electroporated at 50
V, 500 .mu.F and 33.2% of cells expressed the antigen when they
were transfected at 300 V and 900 .mu.F.
3TABLE 3 Expression of MHC II-DR 4 and truncated emmL 55 by
Neuro-2a cells Electroporation 220 V 260 V 300 V 300 V 450 V 1000 V
conditions 1050 .mu.F 1050 .mu.F 1050 .mu.F 900 .mu.F 500 .mu.F 21
.mu.F Expression of NA NA NA 33.2% 53.2% 55.1% MHC II-DR4
Expression of 7% 35% 20.4% NA NA NA truncated emmL 55
EXAMPLE 9
Expression of Truncated emmL 55 Gene in Neuro-2a Cells
[0090] Flow cytometry was used to determine the efficiency of
transfection of Neuro-2a cells with the pSVK 3/emmL 55 construct.
The cells demonstrated moderate expression of the truncated emmL 55
gene (Table 3). Seventy two hours after 35% of the cells exhibited
the immunofluorescence using polyclonal antibodies when
electroporated at 260 V and 1050 .mu.F transfection, 20.4% at 300 V
and 1050 .mu.F and 7% at 220 V and 1050 .mu.F. Eleven % of Neuro-2a
cell was able to express the recombinant protein 11 days after
electroporation.
EXAMPLE 10
Stable Expression of Full Length emmL 55. Truncated emmL 55 and MHC
II in Neuro-2a Cells
[0091] The murine neuroblastoma cell line was transfected with the
pcDNA 3/emmL 55 construct containing the complete emmL 55 gene. The
surface expression of the transfected genes was confirmed by flow
cytometry using the 8 F-10 mAb. Twenty one days after
electroporation, 10% of the transfected Neuro-2a cells kept under
G418 selection were able to bind the 8 F-10 antibody.
[0092] For stable expression, Neuro-2a cells were cotransfected
with pSG1NeopA and pSVK 3/emmL 55 plasmid at 260 V and 1050 .mu.F
and kept in culture with G418 for several weeks. Since these
conditions were the most efficient in previous electroporations,
they were used then for the electroporation of Neuro-2a with the
pcDV 1/.alpha. pcDV 1/.beta. and pSG1NeopA. Stably transfected
cells were kept in culture under G418 selection for several
weeks.
[0093] It should be understood that the examples and embodiments
described herein are for illustrative purposes only and that
various modifications or changes in light thereof will be suggested
to persons skilled in the art and are to be included within the
spirit and purview of this application and the scope of the
appended claims.
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Sequence CWU 1
1
6 1 850 DNA Streptococcus 1 ctgtggaatg tgtgtcagtt agggtgtgga
aagtccccag gctccccagc aggcagaagt 60 atgcaaagca tgcatctcaa
ttagtcagca accaggtgtg gaaagtcccc aggctcccca 120 gcaggcagaa
gtatgcaaag catgcatctc aattagtcag caaccatagt cccgccccta 180
actccgccca tcccgcccct aactccgccc agttccgccc attctccgcc ccatggctga
240 ctaatttttt ttatttatgc agaggccgag gccgcctcgg cctctgagct
attccagaag 300 tagtgaggag gcttttttgg aggcctaggc ttttgcaaaa
agctatcgaa ttaatacgac 360 tcattatagg gagatcgaat tcggcwtggc
taaaaatacc acgaatagac ackattcgct 420 tagaaaatta aaaacaggaa
cggcttcagt agcagtagct ttgactgttt ttgggacagg 480 actggtagca
gggcagacag taaaagcaaa ccaaacagaa ccatctcaga ccaataacag 540
attatatcaa gaaagacaac gtttacagga tttaaaaagt aagtttcaag acctgaaaaa
600 tcgttcagag ggatacattc agcaatacta cgacgaagaa aagaacagtg
gaagtaactc 660 taactggtac gcaacctact taaaagaatt aaatgacgaa
tttgaacaag cttataatga 720 acttagtggt gatggtgtaa aaaaattagc
tgcaagtttg atggaagaaa gagtcgcttt 780 aagagacgaa atcgatcaga
ttatgaaaat atcagaagaa ttaaaaaata agctgagagc 840 aacagaagaa 850 2
847 DNA Streptococcus 2 ctgtggaatg tgtgtcagtt agggtgtgga aagtccccag
gctccccagc aggcagaagt 60 atgcaaagca tgcatctcaa ttagtcagca
accaggtgtg gaaagtcccc aggctcccca 120 gcaggcagaa gtatgcaaag
catgcatctc aattagtcag caaccatagt cccgccccta 180 actccgccca
tcccgcccct aactccgccc agttccgccc attctccgcc ccatggctga 240
ctaatttttt ttatttatgc agaggccgag gccgcctcgg cctctgagct attccagaag
300 tagtgaggag gcttttttgg aggcctaggc ttttgcaaaa agctatcgaa
ttaatacgac 360 tcattatagg gagatcgaat tcatggctaa aaataccacg
aatagacacg attcgcttag 420 aaaattaaaa acaggaacgg cttcagtagc
agtagctttg actgtttttg ggacaggact 480 ggtagcaggg cagacagtaa
aagcaaacca aacagaacca tctcagacca ataacagatt 540 atatcaagaa
agacaacgtt tacaggattt aaaaagtaag tttcaagacc tgaaaaatcg 600
ttcagaggga tacattcagc aatactacga cgaagaaaag aacagtggaa gtaactctaa
660 ctggtacgca acctacttaa aagaattaaa tgacgaattt gaacaagctt
ataatgaact 720 tagtggtgat ggtgtaaaaa aattagctgc aagtttgatg
gaagaaagag tcgctttaag 780 agacgaaatc gatcagatta tgaaaatatc
agaagaatta aaaaataagc tgagagcaac 840 agaagaa 847 3 178 DNA
Streptococcus 3 tttgcaaaaa gctatcgaat taatacgact cattataggg
agatcgaatt cggcttggct 60 aaaaatacca cgaatagaca ctattcgctt
agaaaattaa aaacaggaac ggcttcagta 120 gcagtagctt tgactgtttt
tgggacagga ctggtagcag ggcagacagt aaaagcaa 178 4 31 DNA
Streptococcus 4 tagaattcat ggctaaaaat accacgaata g 31 5 32 DNA
Streptococcus 5 cagtttgcgt ttcttctttt gattgagctc tt 32 6 18 DNA
Streptococcus 6 cagttccgcc cattcttc 18
* * * * *