U.S. patent application number 14/276056 was filed with the patent office on 2014-09-04 for hybrid cells for treating cancer patients.
This patent application is currently assigned to Orbis Health Solutions LLC. The applicant listed for this patent is Orbis Health Solutions LLC. Invention is credited to Thomas E. Wagner, Yanzhang Wei.
Application Number | 20140248316 14/276056 |
Document ID | / |
Family ID | 46331790 |
Filed Date | 2014-09-04 |
United States Patent
Application |
20140248316 |
Kind Code |
A1 |
Wagner; Thomas E. ; et
al. |
September 4, 2014 |
HYBRID CELLS FOR TREATING CANCER PATIENTS
Abstract
The present invention relates to cancer treatment compositions
and methods for treating a specific cancer patient population. In
particular, the application describes methods of treating a patient
with cancer, such as a neuroblastoma, with a hybrid cell
preparation.
Inventors: |
Wagner; Thomas E.;
(Greenville, SC) ; Wei; Yanzhang; (Greer,
SC) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Orbis Health Solutions LLC |
Greenville |
SC |
US |
|
|
Assignee: |
Orbis Health Solutions LLC
Greenville
SC
|
Family ID: |
46331790 |
Appl. No.: |
14/276056 |
Filed: |
May 13, 2014 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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12461760 |
Aug 24, 2009 |
8785186 |
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14276056 |
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11802566 |
May 23, 2007 |
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12461760 |
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11017733 |
Dec 22, 2004 |
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11802566 |
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09756293 |
Jan 9, 2001 |
6849451 |
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11017733 |
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60924611 |
May 22, 2007 |
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60175376 |
Jan 11, 2000 |
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Current U.S.
Class: |
424/277.1 ;
424/184.1; 435/346 |
Current CPC
Class: |
A61K 2039/5152 20130101;
A61K 39/0008 20130101; A61K 2039/80 20180801; A61K 35/15 20130101;
A61K 2035/122 20130101; A61K 35/74 20130101; A61P 37/06 20180101;
C12N 5/16 20130101; C12N 5/163 20130101; A61K 35/13 20130101; A61K
2039/55594 20130101; A61K 39/0011 20130101; A61P 35/00 20180101;
A61K 2039/545 20130101; A61K 2039/5154 20130101; A61K 35/74
20130101; A61K 2300/00 20130101; A61K 35/15 20130101; A61K 2300/00
20130101; A61K 35/13 20130101; A61K 2300/00 20130101 |
Class at
Publication: |
424/277.1 ;
424/184.1; 435/346 |
International
Class: |
A61K 39/00 20060101
A61K039/00 |
Claims
1.-24. (canceled)
25. A hybrid cell preparation for a vaccine composition, comprising
a hybrid of a normal cell that is a target cell to which immune
tolerance is desired and an antigen-presenting cell which lacks an
accessory factor required to generate a positive immune response,
wherein said hybrid cell is labeled with at least two different
markers, and wherein said preparation has been purified using the
markers and without antibiotic or metabolic selection.
26. The preparation according to claim 25, wherein said normal cell
is isolated from a transplant organ.
27. The preparation according to claim 25, wherein said markers are
selected from the group consisting of fluorescent dyes and
antibodies binding to differentially expressed surface markers.
28. The preparation according to claim 27, wherein said fluorescent
dyes are cyanine dyes.
29. The preparation according to claim 25, wherein said
antigen-presenting cell is an immature B cell.
30. The preparation according to claim 25, wherein said
antigen-presenting cell is a fibroblast cell.
31. A method for treating a subject having an autoimmune disease,
comprising: (i) preparing a hybrid cell preparation by a method
comprising (a) contacting a population of normal cells that are
target cells to which immune tolerance is desired with a first
marker, (b) contacting a population of antigen presenting cells
lacking an accessory factor with a second marker, (c) contacting
said normal cells and said antigen presenting cells lacking an
accessory factor under conditions that promote cell fusion, (d)
obtaining a resultant hybrid cell population stained with both the
first and second markers, and (e) purifying the resultant hybrid
cell population, wherein said method does not involve antibiotic or
metabolic selection; and (ii) administering the hybrid cell
preparation to said subject.
32. The method of claim 31, further comprising resuspending the
resultant hybrid cell preparation of step (i) in a pharmaceutically
acceptable carrier before administering to said subject.
33. A method of inducing an immune tolerance against a target cell
in a subject comprising: (i) preparing a hybrid cell preparation by
a method comprising (a) contacting a population of target cells
with a first marker; (b) contacting a population of antigen
presenting cells lacking an accessory factor with a second marker;
(c) contacting said target cells and said antigen presenting cells
lacking an accessory factor with one another under conditions that
promote cell fusion; (d) obtaining a resultant hybrid cell
population stained with both the first and second markers; and (e)
purifying the resultant hybrid cell population by cell sorting,
wherein said cell sorting does not involve antibiotic or metabolic
selection, and (ii) administering the hybrid cell preparation to
said subject.
34. The method of claim 33, further comprising resuspending the
resultant hybrid cell preparation of step (i) in a pharmaceutically
acceptable carrier before administering to said subject.
35. The method of claim 33, wherein the first marker is a
fluorescent dye, the second marker is a different fluorescent dye,
and the resultant hybrid cell population is purified by
fluorescence activated cell sorting.
36. The method of claim 33, wherein the target cell is isolated
from an organ for transplant in the subject.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation of U.S. patent
application Ser. No. 12/461,760, filed Aug. 24, 2009, which is a
continuation of U.S. patent application Ser. No. 11/802,566, filed
May 23, 2007, which claims priority to U.S. Provisional Patent
Application 60/924,611, filed May 22, 2007; which is also a
continuation-in-part of U.S. patent application Ser. No.
11/017,733, filed Dec. 22, 2004, which is a Divisional of U.S.
patent application Ser. No. 09/756,293, filed Jan. 9, 2001, now
U.S. Pat. No. 6,849,451, which claims priority from U.S.
Provisional Patent Application Ser. No. 60/175,376, filed Jan. 11,
2000. The entirety of these applications are incorporated herein by
reference.
BACKGROUND OF THE INVENTION
[0002] The present invention relates to hybrid cells and methods of
making and using hybrid cells. Perhaps the most substantial
practical application of hybrid cells is the production of
hybridomas, which are used to produce monoclonal antibodies. In
addition, they are used in a variety instances for research
purposes, but their broader application, for example, in a clinical
treatment setting has heretofore not been practical. These clinical
applications include the cellular vaccines for treating or
preventing cancer and other disorders, as well as preventing
transplant rejection. The present invention responds to such
deficiencies by providing methods and reagents that make the broad
applicability of hybrid cells a reality.
[0003] Recent advances in molecular immunology now make
immunotherapy a truly viable option for the treatment of patients
with cancer and metastatic disease. The past decade has seen the
approval and introduction of several immunotherapeutic strategies
for wide ranging use against several metastatic cancers, Parkinson
et al., in CANCER MEDICINE, 4.sup.th ed., pp. 1213-1226 (Holland et
al., eds. 1997). Perhaps the best known strategies include IL-2
therapy (Philip et al., Seminars in Oncology. 24(1 Suppl 4): S32-8,
1997 February) and tumor vaccines targeted against melanoma. Smith
et al., Int J Dermatol 1999; 38(7): 490-508. While these strategies
are efficacious against some tumors, their potency is limited
because they only enhance the already enfeebled ability of tumor
cells to present their "foreign" epitopes to CD8+ T-cells, and to
generate thereby a tumor-specific cytotoxic T lymphocyte (CTL)
response.
[0004] Autologous whole tumor cell-based vaccines were first used
for immunotherapy of malignant melanoma. Such whole tumor
cell-based vaccines are advantageous, because they contain large
numbers of antigens, which eliminate the need for targeting the
immune response against one antigen at a time. This is important
because currently there is little ability to identify specific
tumor-associated antigens (TAA) that are useful to induce immune
system-mediated tumor regression. Boon et al., Immunol Today 1997;
18:267-268. To date, however, autologous whole tumor cell-based
vaccines alone have shown only some isolated or marginal successes.
Smith et al., supra. As seen below, the marginal success of whole
tumor cell-based vaccines likely results from tumor cell mutations
that impair their ability to act as antigen presenting cells
("APCs").
[0005] Evidence from many tumor immunology laboratories
demonstrates that tumor cells persist in part because they have
selected a mutation which partially or completely destroys their
ability to act as APCs in the process of cytotoxic T lymphocyte CTL
generation. Stockert et al., J. Exp. Med. 1998; 187: 1349-1354;
Sahin et al., Proc. Natl Acad. Sci. USA 1995; 92:11810-11813;
Gabrilovich et al., Nature Med. 1996; 2:1096-1103; Ishida et al.,
J. Immunol. 1998; 161:4842-4851. These observations spurred
development of strategies that attempt to replace the tumor cell as
the APC, rather than trying to boost the tumor's enfeebled antigen
presenting process. The best candidate for such a replacement is
the dendritic cell ("DC").
[0006] DCs are "professional" antigen presenting cells that play a
vital role in stimulating immune responses. DCs not only can
activate naive CD4+ T helper cells but also stimulate unprimed CD8+
cytotoxic T lymphocytes. Steinman, R. M. Annu. Rev. Immunol. 1991;
9, 271-296; Macatonia, et al., J. Exp. Med. 1988; 169, 1255-1264;
Mehta-Damani et al., J. Immunol. 1994; 153, 996-1003; Porgador et
al., J. Exp. Med. 1995; 182, 255-260.
[0007] Because of these characteristics, DCs have been widely
studied as antigen presenting cells for cancer immunotherapy. DCs
can be loaded with tumor antigens by pulsing with whole tumor
antigens or tumor antigen peptides. (Young et al., J. Exp. Med.
1996; 183, 7-11; Mayordoma et al., Nat. Med. 1995; 1, 1297-1302;
Bakkar et al., Cancer Res. 1995; 55, 5330-5334; Flamand et al.,
Eur. J. Immunol. 1994; 24, 605-610; Gong et al., Gene Ther. 1997;
4, 1023-1028; Song et al., J. Exp. Med. 1997; 186, 1247-1256;
Specht et al. J. Exp. Med. 1997; 186, 1213-1256.)
[0008] Peptide- or tumor lysate-pulsed dendritic cells have been
used, for example, to vaccinate melanoma patients. (Rosenberg et
al., Nature Med 1998; 4: 321-327; Wallack et al., Cancer 1995;
75:34-42; Bystryn, Rec. Results Cancer Res. 1995; 139:337-348;
Mitchell et al., Semin. Oncol. 1998; 25: 623-635; Morton et al.,
Ann. N.Y. Acad. Sci. 1993; 690:120-134; Berd et al., Semin Oncol.
1998; 25:646-653; Berd et al., J. Clin. Oncol. 1997;
15:2359-2370.)
[0009] DCs loaded with tumor antigens are able to induce both
cellular and humoral, antigen-specific, anti-tumor immune
responses. (Shurin, M. R. Cancer Immunol. Immunother. 1996; 43,
158-164). This approach, however, is limited to application against
tumors expressing known tumor antigens. See, Haigh et al., Oncology
1999; 13, 1561-1573. It is worthless for those tumors with no
identified tumor antigen, like primary tumors from patients, which
constitute most real-world situations. Obviously alternative
strategies are needed.
[0010] An additional problem with antigen pulsing techniques is
that the antigen presenting system of an APC works more effectively
and efficiently when the protein/antigen is synthesized inside the
cell rather than outside the cell, a substantial drawback to using
antigen-pulsed cells. In an effort to avoid this problem, a number
of laboratories have attempted to use gene therapy to introduce
specific tumor antigens into dendritic cells. (Gong et al., 1997,
Gene Ther. 4, 1023-28; Song et al., 1997, J. Exp. Med. 186:
1247-56; and Specht et al., 1997, supra.). However, this gene
therapy approach is also fraught with many disadvantages including:
1) the limited ability to identify all of the important specific
tumor antigens, 2) the limited ability to map the genes of the
specific tumor antigens, 3) only one or a small number of the known
tumor antigen genes can be introduced into the dendritic cell and
4) the process is time-consuming and cumbersome.
[0011] On the other hand, fusions between DCs and tumor cells
represent an alternative way to produce effective tumor antigen
presenting cells by presenting the immune cells with all possible
tumor antigens. (Gong et al., Nat. Med. 1997; 3: 558-561; Wang et
al., J. Immunol. 1998; 161, 5516-5524; Lespagnard et al., Int. J.
Cancer 1998; 76, 250-258; Rowse et al., Cancer Res. 1998; 58,
315-321). DCs have been fused with tumor cells and the fused cells
efficiently presented tumor antigens to the immune cells and
stimulated specific anti-tumor immune responses. (Gong et al.; Wang
et al.; Lespagnard et al., all supra).
[0012] These fusion schemes, however, rely on selectable markers
(gene products which render the cell resistant to specific cell
toxins or allow them to grow under certain metabolic conditions) in
each of the DCs and the tumor cells to isolate the resultant
hybrid. The rare cell fusion products are selected by long-term
culture in the presence of both cell toxins where only the fusion
product, containing both selectable markers, can survive. Since the
introduction and selection schemes using markers requires culture
and multiple cell division, they cannot be applied to dendritic
cells, because DCs are terminally differentiated, non-dividing
cells. Thus, it is no surprise that the previous fusion work relied
on well-defined tumor cell lines, bearing such a marker, and DC-
and tumor-specific conjugated antibodies, which limits the
usefulness of this strategy in cancer treatment.
[0013] In summary, the previous cancer-based fusion protocols have
the following limitations: 1) they require established tumor cell
lines which show specific marker(s); 2) they require both DC and
tumor cell specific antibodies to select the fused cells; 3) the
selection and expansion of the fused cells takes an impractical
amount of time.
[0014] The area of preventing transplant rejection using hybrids
are even less well-developed than cancer. In fact, no report of
such has been found.
[0015] Typical approaches to preventing transplant rejection
utilize non-selective immunosuppressive drugs that suppress the
entire immune system. Abbas et al., CELLULAR AND MOLECULAR
IMMUNOLOGY, pp. 347-350. Such approaches have the obvious
disadvantage of making the patient more susceptible to disorders
that otherwise could have been warded off by an intact immune
system.
[0016] It has been recognized that at least two interactions must
take place in order for an antigen presenting cell to activate a T
cell. These interactions are between an antigen-loaded major
histocompatibility (MHC) antigen and the T cell receptor, and
between certain accessory molecules and their cognate receptors on
the T cell. The best studied class of these accessory molecules is
B7 (B7.1 and B7.2), which interact with CD28 and CTLA4 on T cells.
Abbas et al., supra. Thus, disruption of either the MHC or the
accessory interaction should result in a non-response useful, for
example, in preventing transplant rejection.
[0017] In fact, disruption of B7 interaction not only prevents an
immune response, it induces permanent tolerance to any antigen
presented during the disruption. Wei et al., 1996, Stem Cells 14:
232-38. Thus, in the context of transplant rejection, blocking B7
should result in tolerance, preventing rejection. The problem with
such an approach, and the likely reason that it has not be
attempted clinically, is that tolerance would pertain to any
antigen presented during treatment, not just to transplant
antigens. In other words, if a patient were exposed to a pathogen
during the B7 disruption, the patient's immune system would be
rendered tolerant to the pathogen, permanently. This would prevent
the patient from warding off the pathogen, having perhaps lethal
consequences. Clearly, a more specific approach is needed.
[0018] A promising approach takes advantage of antigen presentation
by cells that lack accessory molecules, like B7. These cells
present antigen in the context of MHC, yet, because they lack the
accessory interactions required for activation, they induce
tolerance specific to the antigen presented. Thus, it is possible
to load these cells, which include immature (naive) B cells, with a
specific antigen, and induce antigen-specific anergy. As with the
cancer example described above, this antigen-by-antigen approach
does not have the general applicability needed for practical
clinical use. A methodology is needed which is applicable to any
transplant organ, irrespective of the immunogenic antigens the
organ displays.
[0019] Neuroblastoma is the most common extracranial solid tumor
and the most common tumor occurring during infancy. It also affects
young children, and is rarely found in children older than 10
years. Neuroblastoma is an embryonal malignancy of the sympathetic
nervous system arising from neuroblasts, which are pluripotent
sympathetic cells. In the developing embryo, these cells
invaginate, migrate along the neuraxis, and populate the
sympathetic ganglia, adrenal medulla, and other sites. The origin
and distribution of these cells during fetal development correlate
with the sites of primary disease presentation. The location of
tumors appears to vary also with the age of the patient. While most
neuroblastomas start in the abdomen, a few neuroblastomas develop
in the adrenal glands, abdominal ganglias, chest ganglias, neck,
spinal chord or the pelvis. Infants suffer more frequently from
thoracic and cervical tumors, whereas older children suffer more
frequently from abdominal tumors.
[0020] Age, stage, and some molecular defects in the tumor cells
are the prognostic factors used for risk assessment and treatment
strategy. The differences in outcome between patients with
neuroblastoma are striking Infants younger than 1 year have a good
prognosis, even in the presence of metastatic disease, whereas
older patients with metastatic disease fare poorly, even when
treated with aggressive therapy. Unfortunately, approximately
70-80% of patients older than 1 year suffer from metastatic
disease, usually to lymph nodes, liver, bone, and bone marrow.
Fewer than half of these patients are cured, even with the use of
high-dose therapy followed by autologous bone marrow or stem cell
rescue.
[0021] Treatments for neuroblastoma include surgery, chemotherapy,
and/or radiation therapy. Surgery is often used to try to remove as
much as possible of the tumor in combination with adjuvant
chemotherapy. Chemotherapy becomes the main treatment when the
cancer has spread too far to be completely removed by surgery. Most
common drugs used in chemotherapy include cyclophosphamide or
ifosfamide, cisplatin or carboplatin, vincristine, doxorubicin,
etoposide, teniposide and topotecan. A typical combination of drugs
commonly used consists of cyclophosphamide, doxorubicin, and
vincristine and is alternated with cisplatin plus etoposide. Common
side effects include nausea, vomiting, hair loss, mouth sores,
depression of the immune system, and bone marrow suppression. In
addition, ifosfamide and cyclophosphamide may produce bladder
inflammation and blood in the urine, and damage to the kidneys with
subsequent loss of salt and minerals in the urine. Cisplatin may
produce hearing loss or deafness, kidney damage, and severe and
delayed nausea. Doxorubicin (Adriamycin) may cause heart damage if
too much of the drug is given and can cause skin damage if the drug
should leak out of the vein during administration.
[0022] Accordingly, there is a need in the art for improved
treatment options for cancer patients, including neuroblastoma
patients, and the present invention satisfies that need. There is
also a need in the art for rapid methods for inducing and
suppressing specific immune responses to whole cells and specific
reagents for accomplishing these methods.
SUMMARY OF THE INVENTION
[0023] It is, therefore, an object of the invention to provide
solutions to the aforementioned deficiencies in the art.
[0024] Further to this object, the invention provides a kit useful
in preparing hybrid cells. In one aspect, the kit contains at least
two essentially endotoxin-free dyes and instructions for preparing
hybrid cells from reactant cells by a method that entails
contacting reactant cells with one of said dyes, respectively. In
another aspect, the kit contains at least two essentially
endotoxin-free dyes and an agent that promotes cell fusion. The
endotoxin-free dyes are preferably fluorescent dyes, such as
cyanine dyes.
[0025] Also according to this object of the invention, a hybrid
cell preparation is disclosed. In one embodiment, the preparation
contains a hybrid cell having no more than n-1 selectable markers,
where n represents the number of reactant cells used to form the
hybrid and the preparation is substantially free of non-hybrid
cells. In another embodiment, the preparation contains a hybrid of
a primary tumor cell and an antigen presenting cell. In still
another embodiment, the preparation contains a hybrid of a normal
cell and an antigen-presenting cell which lacks an accessory factor
required to generate a positive immune response. The normal cell
may be isolated from a transplant organ. An additional embodiment
is a preparation containing a hybrid between a pathogenic cell and
an antigen-presenting cell, like a cell from parasite. One hybrid
cell preparation is composed of a hybrid cell labeled with at least
two different dyes, that are preferably fluorescent, like cyanine
dyes. The hybrid cell my be derived from, for example, a dendritic
cell or an immature B cell.
[0026] Still another aspect of the invention provided in accord
with this object is a method of preparing a hybrid cell. One
embodiment of the inventive method entails bringing at least two
different cells into contact under conditions that promote cell
fusion, and then purifying the resultant hybrid without the need
for antibiotic or metabolic selection. In one aspect the method is
accomplished using fluorescent dyes, like cyanine dyes, and the
hybrids are isolated by fluorescence activated cell sorting. The
methods can involve fusing reactant cell, like a macrophage, a
dendritic cell, and an antigen presenting cell that lacks an
accessory factor required to generate a positive immune response,
with a second reactant cell, like a tumor cell, a pathogenic cell
and a normal cell. The method is preferably accomplished in less
than about 48 hours.
[0027] In still another aspect, the invention provides a method of
treating cancer, that involves providing an inventive hybrid cell
preparation that is derived reactant cell that is a cancer cell;
and administering the hybrid cell to a cancer patient. The method
may include adjunct treatment with a cytokine or lymphokine, like
interleukin-2.
[0028] In yet another aspect, the invention contemplates a method
of treating a disorder associated with the presence of a pathogenic
organism that involves providing an inventive hybrid cell
preparation that is derived from a cell isolated from said
pathogenic organism and administering the cell to a patient. Again,
the method may include adjunct treatment with a cytokine or
lymphokine, like interleukin-2.
[0029] Also in accord with the object of the invention, the
invention provides a method of inducing immune tolerance to an
antigen that entails providing an inventive hybrid cell preparation
that is derived from a cell that expresses an antigen against which
immune tolerance is sought and administering said preparation to a
patient. The cell against which immune tolerance is sought may be a
cell from a transplant organ, where the patient needs an organ
transplant.
[0030] Further to this object, the invention provides a method for
treating cancer in a patient, comprising co-administering to the
patient a composition that comprises a primary tumor cell fused to
a dendritic cell, and BCG (Mycobacterium bovis bacillus
Calmette-Guerin). The patient to be treated may have stage I, stage
II, stage III or stage IV cancer (staged according to the American
Joint Committee on Cancer AJCC Cancer Staging Manual, Sixth
Edition, Springer-Verlag New York, N.Y. 2002). Exemplary types of
cancer to be treated include, but are not limited to, breast,
prostate, bladder, colon, renal, ovarian, skin, lung and melanoma.
In one embodiment, the patient has neuroblastoma. In a further
embodiment, the method of treating cancer comprises treatment with
a population of hybrid cells derived from multiple cancer sites
within the patient, in conjunction with BCG therapy. In yet another
embodiment, prior to the hybrid cell-BCG combination therapy, the
patient has no evidence of disease following surgery, radiation
and/or chemotherapy.
[0031] In an additional embodiment, the present invention provides
a method for increasing survival of a cancer patient, such as a
late stage cancer patient, comprising administering to a patient a
composition that comprises a hybrid of a primary tumor cell and a
dendritic cell, in conjunction with BCG (Mycobacterium bovis
bacillus Calmette-Guerin) therapy. The patient to be treated may
have stage I, stage II, stage III or stage IV cancer. The patient
may also be rendered free of evidence of disease (no evidence of
disease patient or NED patient) following surgery, radiation and/or
chemotherapy. In one aspect of the invention, the cancer patient
has breast cancer, prostate cancer, bladder cancer, colon cancer,
renal cancer, ovarian cancer, skin cancer, lung cancer or melanoma.
In another embodiment, the patient has neuroblastoma.
[0032] In a further embodiment, the present invention provides a
method for stimulating an immune response in a cancer patient, such
as a late stage cancer patient, comprising administering to a
patient a composition that comprises a hybrid of a primary tumor
cell and a dendritic cell, in conjunction with BCG (Mycobacterium
bovis bacillus Calmette-Guerin) therapy. The patient to be treated
may have stage I, stage II, stage III or stage IV cancer. In one
aspect of the invention, the cancer patient has breast cancer,
prostate cancer, bladder cancer, colon cancer, renal cancer,
ovarian cancer, skin cancer, lung cancer or melanoma. In another
embodiment, the patient has neuroblastoma. In one aspect of the
invention, the patient has no evidence of disease following
surgery, radiation and/or chemotherapy.
[0033] Both the foregoing general description and the following
brief description of the drawings and the detailed description are
exemplary and explanatory and are intended to provide further
explanation of the invention as claimed. Other objects, advantages,
and novel features will be readily apparent to those skilled in the
art from the following detailed description of the invention.
BRIEF DESCRIPTION OF THE DRAWINGS
[0034] FIG. 1 shows that hybrid cells according to the invention,
obtained from the fusion of dendritic cells with cancer cells, are
capable of generating a tumor-specific cytotoxic T cell response,
which is relevant to in vivo immunotherapy.
[0035] FIG. 2A shows FACS-detection of antigen-presentation markers
on control dendritic cells. A normal distribution is shown.
[0036] FIG. 2B shows FACS-detection of antigen-presentation markers
on dendritic/tumor cell hybrids. A normal distribution is shown, as
compared to the control dendritic cells, meaning that the hybrid
cells retain all of the markers necessary for antigen
presentation.
[0037] FIG. 3 is a flow diagram representing the process for
manufacturing autologous serum.
[0038] FIG. 4 is a flow diagram representing the tumor specimen
processing.
[0039] FIG. 5 is a flow diagram representing the process for the
generation of dendritic cells.
[0040] FIG. 6 is a flow diagram representing the procedure for
fluorescent dye staining of dendritic cells and tumor cells.
[0041] FIG. 7 is a flow diagram representing the cell fusion
procedure.
[0042] FIG. 8 is a flow diagram representing the steps for
dendritoma purification.
[0043] FIG. 9 is a flow diagram representing the procedure for
dendritoma cryopreservation.
[0044] FIG. 10 is a flow diagram showing the process for
pharmaceutical preparation and administration of the dendritoma
vaccine to a patient.
DETAILED DESCRIPTION OF THE INVENTION
[0045] In one aspect, the present invention provides a rapid and
efficient method of preparing hybrid cells that are useful in a
variety of clinical and non-clinical applications. The hybrid cells
are particularly useful in treatment regimes that invoke the immune
system to treat or prevent disease. For instance, in a preferred
embodiment, the inventive hybrid cells are used to treat cancer by
fusing a cancer cell to an antigen presenting cell. In another
embodiment, the inventive hybrid cells comprise a plasma cell and a
cancer cell which, like conventional hybridomas, are useful in
preparing monoclonal antibodies. In still another embodiment, the
present hybrid cells comprise an antigen presenting cell that lacks
an accessory component needed for an immunogenic response and a
cell from an organ destined for transplant in a patient. These
cells may be used to induce tolerance to the transplant cells,
thereby reducing the incidence of transplant rejection. Kits for
generating hybrid cells and for practicing the inventive methods
also are provided.
[0046] The present inventors unexpectedly discovered that hybrid
cells of primary tumor cells fused to dendritic cells are
exceptionally useful for treating a cancer patient, even a late
stage cancer patient, when administered in combination with BCG
(Mycobacterium bovis bacillus Calmette-Guerin).
[0047] Furthermore, even after cancer removal (e.g., tumor
resection) through surgery and/or radiation and/or chemotherapy,
NED patients are still at a risk for cancer recurrence, since it is
believed that cancer cells still exist in the body even though they
are not clinically detectable. Therefore, the present invention is
also directed to treating a cancer patient who has no clinical
evidence of disease with a hybrid cell composition in conjunction
with lipopolysaccharide therapy. Indeed, the inventors of this
application have surprisingly discovered that the treatment methods
of the present invention can decrease risk of cancer recurrence
and/or increase survival rate in NED patients.
Tumor Cells
[0048] The compositions of the present invention are prepared from
tumor cells obtained from surgically resected tumors. The cells are
extracted by dissociation, such as by enzymatic dissociation with
collagenase and DNase, by mechanical dissociation using a blender,
tweezers, mortar and pestle, scalpel blade, or by combination of
enzymatic dissociation with mechanical dissociation. Preferably,
the tumor cells are rendered replication incompetent by
conventional methods, such as irradiation at 5000 rads, prior to
fusion with the antigen presenting cells, such that they are
incapable of growth and division after administration to the
patient.
[0049] The tumor cells are derived from the same type of cancer as
that needs to be treated, and may be autologous or tissue-type
matched, or allogeneic. Preferably, the cells are autologous, e.g.,
are recognized as "self" by the immune system of the intended
recipient.
Antigen Presenting Cells
[0050] Antigen presenting cells can be prepared as described in the
Examples below. Preferably, the antigen presenting cells are
dendritic cells. Dendritic cells are isolated from the patient's
peripheral blood mononuclear cells (PBMC's) as described in Romani
et al. Generation of mature dendritic cells from human blood: an
improved method with special regard to clinical applicability. J
Immunol Methods. 196(2):137-151, 1996.
Method of Preparing Hybrid Cells
[0051] The hybrid cells of the present invention can be prepared by
a rapid, simple to use method that is applicable to fully
differentiated, non-dividing cells. The method comprises contacting
at least two different cells ("reactant cells") under conditions
that promote cell fusion, and then purifying the resultant hybrid
("product cell") without antibiotic or metabolic selection. In
general, the purification is accomplished in a relatively short
period of time, for example, in less than about 24 to 48 hours,
after exposure to conditions that promote cell fusion.
[0052] In an exemplary embodiment, the method is accomplished with
the aid of at least two different dyes, which can be fluorescent
dyes. Thus, the method may entail separately contacting, each with
a different dye, the two cell types to be fused. This pre-fusion
labeling marks each cell with a different dye, and permits
discrimination among each fusion parent cell and the hybrid fusion
product: the reactant cells (e.g., tumor cell and dendritic cell)
each are stained with one dye, and the product cells are stained
with both. This way, the hybrid fusion product may be separated
from the reactant cells, for example, by fluorescence activated
cell sorting (FACS), magnetic cell sorting, and other cell sorting
techniques that do not employ antibiotic or metabolic selection as
the means for sorting. The resulting hybrid cell retains the
antigen diversity of the tumor reactant cell.
[0053] Dyes useful according to the invention have the
characteristic of associating with a cell for a time sufficient to
detect them in such association. In addition, useful dyes do not
substantially diminish cell viability, with greater than about 50%
cell viability being preferred. Typically, they are fluorescent
dyes. One useful class of dyes comprises the so-called "cyanine"
dyes. Cyanine dyes come in a variety of types that fluoresce at
different wavelengths such that they can be individually or jointly
detected when associated with a cell. Some exemplary cyanine dyes
are found in Horan et al., U.S. Pat. No. 4,783,401 (1998), U.S.
Pat. No. 4,762,701 (1988) and U.S. Pat. No. 4,859,584 (1989), the
structures of which are hereby specifically incorporated by
reference.
[0054] Two particularly useful cyanine dyes are PKH26-GL and
PKH2-GL (Sigma Chemical Co.). These dyes are preferred because they
have been widely studied and used. For instance, they have been
used in animal studies in vivo for cell trafficking studies. Horan
et al., Nature 340: 167-168 (1989); Horan et al., Methods Cell
Biol. 33: 469-490 (1990); Michelson et al., Proc. Natl. Acad. Sci.
USA 93: 11877-11882 (1996). In laboratory animals these dyes have
been shown not to affect cell growth or function and not to migrate
from the cells stained with these dyes to other cells (Horan et
al., 1989). Thus, these dyes have low toxicity, which is a
desirable quality for in vivo applications.
[0055] Dyes employed in vivo in accordance with the present
invention should be free of endotoxin, as measured, for example, by
the Limulus amaebocyte (LAL) assay. Typically, when the measured
endotoxin level is less than about 1 ng/.mu.g dye, and preferably
less than about 0.1 ng/.mu.g dye, the dye is considered
"endotoxin-free."
[0056] More generally, the dyes are essentially pyrogen-free,
whether pyrogenicity is contributed by endotoxin or other pyrogens.
Thus, a dye is considered "essentially pyrogen-free" when the final
formulation of hybrid cells labeled with the dye (in a form to be
injected into a subject, for example) yields less than about 1
endotoxin unit (EU)/dose, but preferably less than about 0.1
EU/dose, and most preferably less than about 0.05 EU/dose. Toxicity
thresholds are informed by the fact that most in vivo methods
contemplated herein result in less than about 10.sup.-8 g of these
dyes, in association with cells, being introduced into a patient
when undertaking the inventive methods of treatment.
[0057] Conventional cyanine dye labeling methodologies require the
presence of cellular stabilizers (osmolarity-regulating agents),
like sugars (e.g., glucose or mannitol), amino acids and/or certain
Goods buffers. See, for example, Horan et al., U.S. Pat. No.
4,783,401 (1998). The inventors discovered that dimethyl sulfoxide
(DMSO) can substitute for such stabilizers. In particular, DMSO
diluted in a standard culture medium may be used as a solvent for
cyanine dyes, and it promotes efficient and stable uptake of dye
without substantial loss of cell viability. A generally useful
range of DMSO concentration is from about 10 to about 50%, but a
preferred range is from about 20 to about 40%. The invention
therefore also contemplates methods of labeling cells, and
corresponding kits, with cyanine dyes using DMSO in place of the
conventional stabilizers.
[0058] Once the reactant cells are labeled, they are put into
contact with one another, under conditions that promote fusion.
Such fusion-promoting conditions are well known to the artisan, and
typically involve the addition of an agent that promotes cell
fusion. These agents are thought to work by a molecular crowding
mechanism to concentrate cells to an extent that they are in close
enough proximity to cause fusion of cell membranes. While the
invention contemplates any agent that meets these characteristics,
exemplary useful agents are polymeric compounds, such as
polyethylene glycols. An effective amount of such an agent
generally will be from about 20% to about 80% (w/v). A preferred
range is from about 40% to about 60%, with about 50% being more
preferred.
[0059] Another suitable method for fusing the reactant cells is by
electrofusion, a technique known in the art. Electroporation is the
use of electrical fields to induce a reversible breakdown of the
cell's lipid bilayer membrane, causing temporary pore formation
through which various molecules such as proteins, peptides, DNA or
RNA may enter the cell. Electrofusion is the result of an intensive
electroporation with membrane breakdown of juxtaposed cells in an
inhomogeneous electrical field and consecutive fusion by mutual
resealing, Teissie et al., Biophys J 74: 1889-1898 (1998). Because
the size of the membrane pores is directly related to the strength
of the electric field, electrofusion settings are usually
characterized by higher field strengths (1000-1875V/cm) than
electroporation settings (250-1025V/cm), Shimizu et al, J
Immunother 27: 265-72 (2004); Kjaergaard et al., Cell Immunol 225:
65-74 (2003); Orentas et al., Cell Immunol 213: 4-12 (2001);
Meldrum et al., Biochem Biophys Res Comm 256: 235-239 (1999).
[0060] After hybrid cell formation, it is usually beneficial to
isolate them from the un-fused reactant cells. In the case of
cellular vaccines, for example, this purification substantially
increases the potency. Purification may be accomplished by
conventional FACS methodologies and the like.
[0061] The method explicitly contemplates hybrid cells of higher
order, which are fusions between more than two cells. In each case,
all that is needed is an additional dye that can serve as a marker
for selection of the higher-order hybrid. For example, three
different reactant cells labeled with three different dyes are used
to form a "tribred," and so on. Thus, as used herein, the term
"hybrid cell" contemplates fusions between two or more reactant
cells.
Kits of the Invention
[0062] The present invention also relates to kits for labeling
cells and for preparing hybrid cells. These kits are useful in
implementing the inventive method of preparing hybrid cells. A
labeling kit, for example, contains at least one dye, and may
contain DMSO and instructions for labeling. The inventive hybrid
cell preparation kit, comprises at least two essentially
endotoxin-free and/or pyrogen-free dyes and instructions for
preparing hybrid cells and/or it comprises an agent that promotes
cell fusion.
Hybrid Cell Preparations
[0063] The invention further contemplates a hybrid cell
preparation. In general, the preparation will be substantially free
of reactant cells (less than about 50% reactant cells, but
preferably less than about 10% to 25% reactant cells, and most
preferably less than about 5% reactant cells). The inventive hybrid
cells are prepared from reactant cells that may have a selectable
marker, but need not. In any event, at least one reactant cell
lacks such a marker. Thus, where n represents the number of
reactant cells, in most cases, n-1 will represent the maximum
number of selectable markers found in the hybrid cell. For example,
where two reactant cells fuse to form a hybrid, the hybrid will
contain no more than one selectable marker.
[0064] The phrase "selectable marker" is used here in its
conventional sense, to refer to an antibiotic resistance or a
metabolic marker, such as hypoxanthine phosphoribosyl transferase
(HPRT), and the like. Selectable markers are endogenously produced,
and do not include exogenously added materials, like dyes.
[0065] In one embodiment, the inventive hybrid cell preparation
comprises a tumor cell and an antigen presenting cell (APC) as
reactants. Such hybrids may be used as cellular vaccines to induce
an immune response against a disease, such as cancer. The tumor
cell may be of any type, including the major cancers, like breast,
prostate, ovarian, skin, lung, and the like. The APC preferably is
a professional APC, like a macrophage or a dendritic cell. Due to
their superior antigen presentation capabilities, dendritic cells
are more preferred. Both syngeneic and allogeneic fusions are
contemplated as the inventors have discovered using a mouse model
that both work equally well.
[0066] An additional embodied hybrid comprises a pathogenic cell
and an APC. These hybrids also are useful as cellular vaccines.
Again, antigen presenting cells, and dendritic cells, in
particular, are favored. The pathogenic cell, on the other hand,
may be of virtually any type. For example, it may be a bacterial
cell (Helicobacter, etc.) that has had its cell wall removed. The
pathogenic cell may be a fungal cell, like Candida, Cryptococcus,
Aspergillus and Alternaria.
[0067] The pathogenic cell also may be a parasitic cell from, for
example, trypanosomal parasites, amoebic parasites, miscellaneous
protozoans, nematodes, trematodes and cestodes. Exemplary genera
include: Plasmodium; Leishmania; Trypanosoma; Entamoeba; Naeglaria;
Acanthamoeba; Dientamoeba; Toxoplasma; Pneumocystis; Babesia;
Isospora; Cryptosporidium; Cyclospora; Giardia; Balantidium;
Blastocystis; Microsporidia; Sarcocystis; Wuchereria; Brugia;
Onchocerca; Loa; Tetrapetalonema; Mansonella; Dirofilaria; Ascaris
(roundworm); Necator (hookworm); Ancylostoma (hookworm);
Strongyloides (threadworm); Enterobius (pinworm); Trichuris
(whipworm); Trichostrongylus; Capillaria; Trichinella; Anasakis;
Pseudoterranova; Dracunculus; Schistosoma; Clonorchis; Paragonimus;
Opisthorchis; Fasciola; Metagonimus; Heterophyes; Fasciolopis;
Taenia; Hymenolepis; Diphyllobothrium; Spirometra; and
Echinococcus.
[0068] In another embodiment, the inventive hybrid cell preparation
comprises a target cell against which immune tolerance is desired
and an antigen presenting cell that lacks an accessory factor
needed for an immunogenic response. Typically these APCs lack B7
(e.g., B7.1 or B7.2); exemplary cells are naive, immature B cells
and fibroblasts, but any cell capable of presenting antigen (having
MHC molecules), yet lacking an accessory molecule, will suffice. In
the case of B7, specific antibodies are known, and the artisan will
be well apprised of methods to ascertain whether any particular
cell type lacks B7. Naive B cells are preferred because they
express high levels of MHC molecules and all the adhesive molecules
known in the art to be necessary for efficient cell-cell
contact.
[0069] In any event, the resultant hybrids have the ability to
present antigen to the immune system, since they bear class I and
class II MHC molecules, yet they will not have the ability to
activate the immune system, since they do not have the necessary
accessory markers, like B7 (CD28 or FLTA4 ligands). Thus, instead
of inducing an immune response, these hybrids will induce apoptotic
clearance, thereby rendering the immune system tolerant to the
target cell antigens presented by these hybrids. Such immune cell
hybrids are useful in treating autoimmune disorders like transplant
rejection.
[0070] The APC preferably is a professional APC, like a macrophage
or a dendritic cell. Due to their superior antigen presentation
capabilities, dendritic cells are preferred. Both autologous and
allogeneic fusions are contemplated. Ultimately, the antigen
diversity of the starting tumor cell population is maintained in
the resultant hybrid cell population.
[0071] The inventive hybrid cell preparation may be made using a
combination of dyes, as detailed above. Thus, the inventive hybrid
cell may be labeled with at least two different dyes. These dyes
are preferably fluorescent and, again, cyanine dyes are favored.
Alternatively, hybrid cells may be prepared, for example, using
cell surface markers differentially expressed on the reactant cells
and corresponding antibodies to them. The antibodies may be used to
pan sequentially for each marker.
BCG Preparation
[0072] The invention further contemplates a BCG (Mycobacterium
bovis bacillus Calmette-Guerin) preparation, which is an
inactivated form of the bacterium Mycobacterium bovis. Safe and
effective BCG preparations are commercially available.
Methods of Treatment
[0073] Multiple factors determine the survival rate of a patient
with cancer, including tumor size and thickness, extension, lymph
node status, and metastatic status. The patient's prognosis is also
affected by other factors, such as age of the patient and
health-related factors. Cancer staging is important for identifying
appropriate treatment options for a particular cancer and
individual. In 1998, the American Joint Committee on Cancer (AJCC),
in collaboration with the National Cancer Institute Surveillance,
Epidemiology and End Results Program (NCI-SEER); Centers for
Disease Control and Prevention National Program of Cancer
Registries (CDC/NPCR); National Cancer Registrars Association
(NCRA); North American Association of Central Cancer Registries
(NAACCR); and American College of Surgeons (ACOS) Commission on
Cancer (CoC), began addressing the discrepancies in staging
guidelines among the three major staging systems used in the United
States, and developing a unified system between the tumor-lymph
nodes-metastasis (TNM) staging system of the AJCC and the SEER
Summary Staging System. The Collaborative Staging System is based
on and compatible with the terminology and staging used in the
sixth edition of the AJCC Cancer Staging Manual, published in
2002.
[0074] The AJCC TNM staging system uses three basic characteristics
of cancer that are then grouped into stage categories to determine
an overall degree of severity for the patient's cancer. "T"
describes the size and the extent of the primary tumor. The T
component is accompanied by a number 1-4 that further identifies
the size and local spread of the tumor. A higher number indicates
either a larger tumor or one that has a greater effect on the
surrounding tissues. "N" describes the absence or presence and
extent of regional lymph node metastasis, the number of nodes
involved and their size. A number from 0 to 2 indicates the level
of lymph node involvement; and a higher number indicates a more
severe condition. "M" describes the absence or presence of distant
metastasis. The stage categories range from Stage 0 through Stage
IV, with the lower staging number corresponding to a less severe
cancer. This simplified staging method helps determining the best
course of treatment and provides an indication of the patient's
prognosis.
[0075] The SEER Extent of Disease (EOD) coding system is a
five-field, 10 digit system that provides information on the tumor
size, extension of the primary tumor, regional lymph node
involvement, number of pathologically reviewed regional lymph nodes
that are positive, and number of pathologically examined regional
lymph nodes.
[0076] The present inventors surprisingly discovered that hybrid
cells, in combination with BCG therapy, are exceptionally useful in
treating a cancer patient, even a late stage cancer patient.
[0077] In one aspect of the invention, the patient has a solid
tumor cancer, such as renal cancer, ovarian cancer, lung cancer,
breast cancer, prostate cancer, bladder cancer, colon cancer, or
skin cancer, such as melanoma. In another aspect of the invention,
the patient has neuroblastoma. In yet another aspect of the
invention, the patient has stage II neuroblastoma (stage IIA or
IIB), stage III neuroblastoma or metastatic stage IV neuroblastoma
(staged according the International Neuroblastoma Staging System,
INSS).
[0078] Removal of all identifiable cancer growths, with multiple
resections at multiple sites of disease, if necessary, may render
the patient free of evidence of disease (NED) even at a late stage,
such as stage IV. Thus, in this patient population, cancer removal
followed by hybrid cells treatment in conjunction with BCG therapy,
can decrease the risk of cancer recurrence and increase patient
survival rate. This method improves the likelihood of a favorable
long-term prognosis in patients with stage I-NED, stage II-NED,
stage III-NED, and even stage IV-NED cancer. The cancer can be any
solid tumor cancer, including but not limited to, renal cancer,
ovarian cancer, lung cancer, breast cancer, prostate cancer,
bladder cancer, colon cancer, skin cancer, such as melanoma, or
neuroblastoma. Accordingly, the hybrid cell composition of the
invention can be administered to a patient who has no clinical
evidence of disease (NED) as a result of, for example, surgery
(including surgical resection of tumor), and/or radiation therapy
and/or chemotherapy (including antimetabolites, alkylating agents,
immunomodulatory agents, various natural products (e.g., vinca
alkaloids, epipodophyllotoxins, antibiotics, and amino
acid-depleting enzymes), antibodies, hormones and hormone
antagonists), regardless of the stage of cancer.
[0079] The method of treatment according to the present invention
comprises co-administering to a cancer patient a composition
comprising a non-proliferative hybrid between a first reactant cell
and a second reactant cell, typically an antigen-presenting cell,
and BCG. For the purposes of the present invention, the term
"patient" denotes an animal. In a preferred aspect of the
invention, the patient is a mammal. In the most preferred aspect of
the invention, the mammal is a human.
[0080] The first reactant cell is one against which an immune
response is sought, such as a primary tumor cell. Preferably, the
treatment method involves co-administering a composition comprising
a hybrid cell prepared by fusing a first reactant cell isolated
from a patient, such as a tumor cell, with an antigen presenting
cell, such as a dendritic cell. In a preferred embodiment, the
hybrid cell is prepared by fusing a tumor cell from a neuroblastoma
isolated from a patient having neuroblastoma, with a dendritic cell
obtained from the peripheral blood of the patient. Preferably, the
tumor reactant cells are lethally irradiated prior to fusion.
Irradiation of the tumor cells does not prevent efficient
presentation of the tumor antigen(s) by the resultant hybrid cell.
Both autologous and allogeneic fusions are contemplated. The
non-proliferative hybrid cell thus obtained from the fusion of the
tumor cells with their dendritic cells are called dendritomas. A
pharmaceutically-acceptable dendritoma vaccine, comprising the
hybrid cells thus produced and an excipient, such as phosphate
buffered saline (PBS), is then co-administered subcutaneously,
preferably near a lymph node, to a cancer patient, in conjunction
with the BCG preparation.
[0081] In one embodiment, the dendritoma vaccine and the BCG
preparation are not mixed but are co-administered. For the purposes
of the present invention, the terms "co-administering" and
"co-administration" refer to a process by which a BCG preparation
according to the invention is administered to the cancer patient
subcutaneously, simultaneously or sequentially with respect to the
administration of the dendritoma vaccine. The term "simultaneous"
denotes the simultaneous administration of the dendritoma vaccine
and the lipopolysaccharide to the patient. In a preferred
embodiment, administration of the BCG preparation to the patient is
sequential to the administration of the dendritoma vaccine, i.e.,
the BCG is preferably administered after the dendritoma vaccine,
such as about 5 minutes, about 10 minutes, about 15 minutes, about
20 minutes, about 25 minutes, about 30 minutes, or about 45 minutes
after administration of the dendritoma vaccine. In this context,
the term "about" refers to .+-.1 minute.
[0082] In another embodiment, the dendritoma vaccine and BCG are
formulated together and administered to the cancer patient.
Vaccine Formulations
[0083] The dendritoma vaccine compositions of the invention are
administered in a mixture or in combination with a
pharmaceutically-acceptable carrier, such as phosphate buffered
saline (PBS). After the initial vaccination, the patient may be
revaccinated every 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6
weeks, 7 weeks, 8 weeks, 9 weeks, 10 weeks, 11 weeks, 12 weeks, 13
weeks, 14 weeks, 15 weeks, 16 weeks, 17 weeks, 18 weeks, 19 weeks,
20 weeks, 21 weeks, 22 weeks, 23 weeks, or 24 weeks, but preferably
every 4-6 weeks. For example, the patient may be revaccinated every
4 weeks, receiving a maximum of 6 vaccinations, depending on the
total number of dendritomas obtained. Dosages of the dendritoma
vaccine may be determined by the artisan skilled in the art and may
be based on the patient's clinical condition, as well as potency of
the vaccine material, use and type of vaccine adjuvant or
formulation, how different the vaccine is from the host, route and
schedule of administration, immune status of the recipient, body
weight, etc. In a preferred embodiment of the invention, the
composition comprises a dendritoma vaccine comprising at least
about 100,000 dendritomas in a pharmaceutically acceptable carrier
or diluent, such as, but not limited to, phosphate buffered saline
(PBS). The dendritomas may be administered by subcutaneous
injection into an area of lymph nodes in the axilla or inguinal
area of the patient. The injection site may be rotated to avoid
injection in the same lymph node bed on two consecutive
administrations.
[0084] In one embodiment, the BCG and dendritoma vaccine are
formulated together. In another embodiment, the BCG preparation of
the invention is administered subcutaneously within about 5
minutes, 10 minutes, 15 minutes, 20 minutes, 25 minutes, 30
minutes, or 45 minutes of the administration after each dose of the
dendritoma vaccine. The dosage of BCG administered after the
initial vaccination may be determined by the artisan skilled in the
art based upon the detection of changes in the immunodeficiency
panel and in the number of T cells expressing interferon .gamma.
(IFN-.gamma.) in the patient, or the reaction to the TB skin test
administered to the patient prior to administration, as depicted in
Table 1 below:
TABLE-US-00001 TABLE 1 Dosage of BCG After Initial Vaccination - TB
skin test result Dermatologic reaction BCG dose Negative None
1,000,000 Units Positive <10 mm In Duration .sup. 250,000 Units
Positive .gtoreq.10 mm In Duration, 20,000 Units
[0085] BCG preparations for second administration to the patient
comprise at least about 10,000 units, preferably at least about
20,000 units, more preferably at least about 100,000 units, still
more preferably at least about 250,000 units, even more preferably
at least about 500,000 units, and most preferably at least about
1,000,000 units, depending on whether the initial injection
produces ulceration at the injection site, in which case the BCG
dose is reduced to 50% of the initial dose. BCG preparations for
subsequent administrations may comprise from about 0.0 to about
1,000,000 units, and may be determined by the artisan skilled in
the art based on the patient's reaction to the first and second
administration of the BCG preparation. If the initial BCG
administration produces ulceration at the injection site, and
ulceration at the site of injection occurs after the dosage of BCG
is decreased by 50% for the second administration, the dendritoma
vaccine is administered alone, without BCG, on all subsequent
injections.
Clinical Response
[0086] The patient's response to the methods of treatment of the
present invention may be evaluated according to standard criteria
based on target lesions and non-target lesions. Target lesions are
defined as all measurable lesions up to a maximum of 10 lesions
representative of all involved organs, and are measured and
recorded at baseline. Non-target lesions are all other lesions, or
sites of disease, recorded, but not measured, at baseline as
present or absent. In the evaluation of target lesions, a complete
response (CR) indicates complete disappearance of all target
lesions; a partial response (PR) indicates at least about 30%
reduction in the sum of the longest diameter (LD) target lesions
taking as reference the baseline LD; a stable disease (SD)
indicates neither sufficient shrinkage in target lesions to qualify
for partial response, nor sufficient increase in target lesions to
qualify for progressive disease (PD), when considering the smallest
sum LD recorded since the beginning of treatment; and a progressive
disease (PD) indicates at least a 20% increase in the sum of the LD
of target lesions when considering the smallest sum LD recorded
since the beginning of treatment, or the appearance of one or more
new lesions. In the evaluation of non-target lesions, a complete
response indicates the disappearance of all non-target lesions; a
non-complete response (non-CR) or non-progressive disease (non-PD)
indicates the persistence of one or more non-target lesions; and a
progressive disease (PD) indicates the appearance of one or more
new lesions or the unequivocal progression of existing non-target
lesions.
[0087] The best overall response is the best response recorded for
the start of the treatment until disease progression/recurrence
(taking as reference for the progressive disease the smallest
measurements recorded since the treatment started). The patient's
best response assignment will depend on the achievement of both
measurement and confirmation. The evaluation of the best overall
response is summarized in Table 2.
TABLE-US-00002 TABLE 2 Evaluation of best overall response* Target
New Overall Lesions Non-Target Lesions Lesions Response CR CR No CR
CR Incomplete No PR response/SD PR Non-PD No PR SD Non-PD No SD PD
Any Yes or No PD Any PD Yes or No PD Any Any Yes PD *CR, complete
response; PR, partial response; SD, stable disease; PD, progressive
disease.
[0088] To be assigned the status of partial response or complete
response, the changes in tumor measurement must be confirmed by
repeat assessments performed .gtoreq.4 weeks after the criteria for
the response are first met. If the patient has no evidence of
disease at Day 0, time to recurrence will be evaluated and any
recurrence will be classified as progression of disease.
Preferably, upon treatment with the dendritoma vaccine and BCG
therapy of the present invention, the patient has at least a
partial response, and most preferably the patient has complete
response to the therapy.
[0089] Although the patient may have a "mixed response," the
treatment method may nevertheless still be considered effective. A
treatment regimen is considered "effective" if it can prolong the
time to relapse or increase a patient's overall survival beyond the
patient's expected age of survival, given the stage and type of
cancer immediately prior to the treatment according to the present
invention. For example, the method of treatment of the present
invention is useful in increasing survival of a patient having late
stage cancer by at least about 1 month, at least about 2 months, at
least about 3 months, at least about 4 months, at least about 5
months, at least about 6 months, at least about 7 months, at least
about 8 months, at least about 9 months, at least about 10 months,
at least about 11 months, at least about 12 months, at least about
1 year, at least about 1.5 years, at least about 2 years, at least
about 2.5 years, at least about 3 years, at least about 3.5 years,
at least about 4 years, at least about 4.5 years, at least about 5
years, at least about 6 years, at least about 7 years, at least
about 8 years, at least about 9 years, at least about 10 years,
etc.
[0090] The foregoing detailed description and the following
examples are offered for illustrative purposes and are not meant to
be limiting. The artisan will recognize that there are additional
embodiments that fall within the invention, but are not described
with particularity. All references identified herein, including
U.S. patents, are hereby expressly incorporated by reference.
EXAMPLES
Example 1
Animal Studies
[0091] This example demonstrates the preparation of certain hybrids
between cancer cells and dendritic cells, called dendritomas. These
hybrids were used as a cellular vaccine to prevent cancer in a
murine metastatic cancer model system.
[0092] To prepare dendritic cells from bone marrow, the appropriate
number of female C57BL/6J mice to support later dendritoma
injections (two mice for every one mouse to be injected) were
sacrificed. The femur and tibia of both hind legs were removed from
each mouse. Bone marrow was flushed out of the bones using a
syringe containing RPMI 1640 with 25 mM Hepes (Gibco BRL). The
media containing the bone marrow was filtered through a 40 .mu.m
cell strainer into a 50 ml conical centrifuge tube. The bone marrow
cells were pelleted by centrifugation at 1500 rpm for five minutes.
After removing the supernatant, the tube was gently tapped to
loosen the cell pellet. Red blood cell lysis was achieved by adding
5 ml/mouse of ACK Lysing Solution (0.15 M NH.sub.4Cl, 1 mM
KHCO.sub.3, 0.1 mM Na.sub.2EDTA, pH 7.3) and incubating at room
temperature for five minutes. The cells were pelleted by
centrifugation at 1500 rpm for five minutes. The supernatant was
removed, and the cells were gently resuspended in 10 ml/mouse of
complete DC media (RPMI 1640, 10% fetal bovine serum (FBS), 100
.mu.g/ml gentamicin, 10 ng/ml GM-CSF, 10 ng/ml IL-4). The cells
were plated into two wells/mouse of a six well tissue culture
plate. After incubating the cultures overnight at 37.degree. C., 5%
CO.sub.2, the floating cells were removed from each culture.
Adherent cells were washed twice with 1.times. Phosphate Buffered
Saline (PBS). Each well of cells was fed 5 ml of complete DC media.
The cultures were incubated for 48 hours at 37.degree. C., 5%
CO.sub.2. The dendritic cells were harvested from the 6 well plate
by removing the supernatant containing the cells to a 15 ml conical
centrifuge tube. Each well was washed twice with 3 ml of
1.times.PBS. The cells were lightly trypsinized by adding 1 ml of
0.25% Trypsin/EDTA (Gibco BRL) to each well. After rocking the
plate to cover the entire surface, the trypsin solution was quickly
removed from the plate. The plate was lightly tapped to remove any
loosely attaching cells. These cells were resuspended in 2 ml of
complete DC media and added to the 15 ml tube. The cells were
pelleted by centrifugation at 1500 rpm for five minutes. After
resuspending the cells in 10 ml of complete DC media, a cell count
was taken.
[0093] B16F0 murine melanoma cells were obtained from the ATCC
(CRL-6322) and cultured using standard tissue culture techniques.
When the cells were ready for use, they were trypsinized using
0.25% Trypsin/EDTA. After taking a cell count the number of cells
needed for experimentation were pelleted by centrifugation at 1500
rpm for five minutes. The remaining cells were cultured for later
use.
[0094] For general cell membrane labeling of murine dendritic cells
and B16F0 melanoma cells, a commercial fluorescent cell linker kit
was used. The dendritic cells were labeled fluorescent green using
Sigma stock number PKH2-GL; the B16F0 melanoma cells were labeled
fluorescent red using Sigma stock number PKH26-GL. The staining
procedure was performed at 25.degree. C. The cells to be stained
were washed with serum-free media. The cell suspension was
centrifuged at 400 g for five minutes to obtain a loose pellet.
Supernatant was removed leaving less than 25 .mu.l of medium on the
pellet. The pellet was resuspended by tapping the tube, and 1 ml of
Diluent A or C for green or red staining respectively was added to
resuspend the cells. Immediately prior to staining,
4.times.10.sup.-6 molar dyes (2.times.) were prepared with Diluent
A or C in polypropylene tubes. To minimize ethanol effects, the
amount of dye added was less than 1% of the individual sample
volume. The cells in the diluent were rapidly added into 1 ml of
2.times. dye. The cells and dye were immediately mixed by gentle
pipetting. The mixture was then incubated at 25.degree. C. for five
minutes. The staining process was stopped by adding an equal volume
of FBS and incubating for one minute. The stained cells were
diluted with equal volume of complete culture medium. Stained cells
were removed from the staining solution by centrifuging at 400 g
for 10 minutes. After a total of three washes, the cells were
resuspended in complete medium at a proper concentration.
Efficiency of staining was monitored by fluorescent microscopy.
[0095] Prior to the fusion process, the red fluorescently stained
B16F0 murine melanoma cells were irradiated with 5,000 rads. Murine
dendritic cells and B16F0 melanoma cells were fused together by
mixing the two cell types at a 1:1 ratio in a 50 ml conical
centrifuge tube. The tube was filled with serum-free RPMI 1640 with
25 mM Hepes. The cell mixture was centrifuged at 1500 rpm for five
minutes at room temperature. During the fusion process, all
solutions as well as the tube in which the fusion was performed
were kept at 37.degree. C. using double-beaker water baths. The
supernatant from the mixed cell pellet was aspirated and discarded.
Using a 1 ml serological pipet, 1 ml of prewarmed 50% PEG/DMSO
(Sigma), which contained 50% PEG and 10% DMSO in PBS (Ca.sup.++-
and Mg.sup.++-free), was added to the mixed cell pellet
drop-by-drop over one minute, stirring the cells with the pipet tip
after each drop. The mixture was stirred for an additional minute
with the pipet.
[0096] Using a clean 2 ml serological pipet, 2 ml of prewarmed
serum free RPMI 1640 with 25 mM Hepes was added to the cell mixture
drop-by-drop over two minutes, stirring after each drop. With a 10
ml serological pipet, 7 ml of prewarmed serum free RPMI 1640 with
25 mM Hepes was added drop-by-drop over two to three minutes. The
cells were pelleted by centrifugation at 1500 rpm for five minutes
at room temperature. The supernatant was discarded, and the tube
was placed back into the beaker water bath. With a clean 10 ml
serological pipet, the cell pellet was resuspended in 10 ml of
complete DC media by forcefully discharging about 3 ml of media
onto the pellet and then gently adding the remaining media. The
resuspended cells were put into a T75 tissue culture flask. The
instant dendritomas (fused dendritic cells with melanoma cells)
were incubated overnight at 37.degree. C., 5% CO.sub.2. A drop of
the cells was placed on a slide and evaluated by fluorescent
microscopy to ensure the occurrence of fusion.
[0097] The instant dendritomas were removed from the tissue culture
flask by saving the supernatant containing the cells as well by
lightly trypsinizing the adherent cells as previously described.
The cells were pelleted by centrifugation at 1500 rpm for five
minutes. The cell pellet was resuspended in 2 ml of 1.times.PBS and
put into a sterile, polystyrene, round bottom, 12.times.75 mm
Falcon tube. After centrifuging the cells at 1500 rpm for five
minutes, they were resuspended in 1 ml of 1.times.PBS. The instant
dendritomas were sorted out based on dual green and red
fluorescence using a FACS Caliber (Becton Dickinson), using
standard methods.
[0098] The sorted cells were pelleted by centrifugation at 2000 rpm
for 30 minutes. After removing the supernatant, the cells were
resuspended at a concentration of 50,000 cells/0.5 ml 1.times.PBS.
A drop of the cells was placed on a slide and evaluated by
fluorescent microscopy to ensure the general purity of the
sort.
[0099] Three days prior to the fusion process, female C57BL/6J mice
were challenged with 0.75.times.10.sup.6 B16F0 melanoma cells in
0.4 ml 1.times.PBS by intravenous injection. Once the instant
dendritomas were pelleted and resuspended, each mouse was injected
intravenously with 50,000 cells. The mice were monitored up to four
weeks for pulmonary metastasis.
[0100] At the end of four weeks, the mice were sacrificed and the
metastases were counted. Each of the four control animals, which
were not treated with the instant dendritomas, had greater that 50
tumors. On the other hand, only one of the treated animals had
measurable metastases. These data indicate that the hybrid cells
are effective in treating cancer in a proven animal model system.
The data are compiled in the following Table 3.
TABLE-US-00003 TABLE 3 Number of Metastases in Control and Treated
Mice Number of Metastases GROUP (including tumors at non-lung
sites) Control A >50 B >50 C >50 D >50 Experimental A 0
B 3 C 0 D 0
Example 2
Method of Producing Hybrid Cells
A. Collection and Processing of Autologous Serum
[0101] Blood is collected from the patient into a dry blood
collection bag. Autologous serum is used for culture of the
patient's dendritic cells and cryopreservation of the patient's
tumor cells and dendritomas. Serum is isolated using standard
procedures from 200 ml of peripheral blood collected without
anticoagulants. The blood is allowed to clot at room temperature
and then put in the refrigerator overnight. The serum is then
removed from the clotted blood and centrifuged. The supernatant is
filtered through a 0.22 .mu.m filter. The serum is then inactivated
by incubating at 56.degree. C. for 30 minutes, aliquoted into 15 ml
tubes, labeled and stored at -80.degree. C. A small amount of the
serum sample is also used for quality control testing. This
autologous serum comprises 10% by volume of the culture media used
for the culture of the patient's dendritic cells and the freezing
medium for cryopreservation of tumor cells and dendritomas. If
needed, additional serum may be obtained during additional
collection procedures. A flow diagram representing the process for
manufacturing autologous serum is shown in FIG. 3.
B. Tumor Cell Preparation from Surgically Excised Tissue
[0102] Tumor specimens are collected from patients undergoing
biopsy or other appropriate procedure and further processed.
Quality control evaluation of the tumor samples includes label
verification and visual inspection of the container.
[0103] A tumor section is obtained at the time of biopsy or
excisional resection before the specimen is sent to the pathology
laboratory. After separating fat and necrotic tissue away from the
tumor tissue, the tumor is cut into small pieces and put into a T75
flask. Twenty milliliters of digestion medium (RPMI-1640, 0.5 U/ml
collagenase, 50 .mu.g/ml Pulmozyme) are added to the flask. This
solution is rocked for 1-4 hours at 37.degree. C. The cell
suspension is then collected and filtered through a 40 .mu.m cell
strainer (Falcon Cat#2340). The cells are pelleted at 500 g for 5
minutes at room temperature. 15 to 40 ml of ACK lysing solution
(Cambrex) is added to the cell pellet and incubated for 5 minutes
at room temperature to lyse the red blood cells.
[0104] For the tumor cell skin test, 8 million tumor cells are
collected, washed, resuspended in 7.2 ml medium (80% RPMI-1640 and
10% autologous serum), and irradiated with 10,000 rads. After
irradiation and the addition of 0.8 ml DMSO, the cells are
aliquoted (1.times.10.sup.6 cells/vial) and cryopreserved. At the
same time, a sample is taken for lot-release testing. At the time
of skin testing, the tumor cells are thawed and transferred to a 1
cc syringe for intradermal injection. The remaining tumor cells are
immediately cryopreserved using a similar procedure, but with
higher concentrations (10-50 million cells/ml). A quality control
sample is collected before the cryopreservation. If the number of
tumor cells is insufficient to manufacture the dendritoma vaccine,
additional tissue may be obtained, if possible, or patient may be
withdrawn from study. FIG. 4 provides an overview of the tumor
specimen processing.
C. Dendritic Cell Generation
[0105] Dendritic cells are generated from the patient's peripheral
blood monocytes (PBMCs). Three hundred milliliters of sodium
heparinized peripheral blood are collected from the patient and
diluted 1:1 with 1.times.PBS. Then, 120 ml of the diluted blood are
layered over 85 ml of room temperature Ficoll-Paque Plus in six 230
ml centrifuge bottles, and centrifuged at 500 g for 30 minutes at
room temperature. The PBMC layers are removed from the Ficoll
gradients, and placed into clean 250 ml centrifuge tubes. Four
volumes of 1.times.PBS are added and the tubes are inverted to mix.
The PBMCs are then centrifuged at 500 g at room temperature for 10
minutes. Ten ml of 1.times.PBS are added into each tube, and the
cells are resuspended and pooled into 1 tube. The PBMCs are again
centrifuged at 500 g at room temperature for 10 minutes. The cells
are resuspended in 20 to 40 ml of ACK lysing solution (Cambrex) and
incubated at room temperature for 5 minutes. The cells are then
centrifuged again. The PBMCs are resuspended in 50 ml of RPMI
1640+25 mM Hepes media. The cells are then placed onto 100 mm
tissue culture plates, swirled, and incubated at 37.degree. C. for
3 to 4 hours. The non-adherent cells are then removed. Ten ml of
1.times.PBS are added, the plate is swirled, and the PBS is
removed. Afterwards, 10 ml of complete DC media (RPMI 1640+10%
autologous patient serum+800 U/ml GM-CSF+1000 U/ml IL-4+100 m/ml
gentamicin) is added to each plate. The plates are then incubated
at 37.degree. C., 5% CO.sub.2 for 7-10 days to generate dendritic
cells. FIG. 5 provides an overview of the process generating
dendritic cells.
Example 3
Method of Manufacturing the Dendritoma Vaccine
[0106] The manufacturing of the dendritoma vaccine can be divided
into several phases: A. fluorescent dye staining; B. fusion of
dendritic cells and tumor cells; C. purification; and D. final
product formulation. These stages are shown below.
A. Fluorescent Dye Staining of Dendritic Cells and Tumor Cells
[0107] The fluorescent dyes used in dendritoma production are
PKH26-GL and PKH2-GL from Sigma. These dyes have been used in
animal studies in vivo for cell trafficking studies. In laboratory
animals these dyes have been shown not to affect cell growth or
function and not to migrate from stained cells to other cells. In
the clinical investigation, less than 10.sup.-14 g of these dyes
associated with the dendritoma vaccines are introduced into the
patient.
[0108] Dendritic cells generated from patient's blood are stained
fluorescent green and tumor cells from the same patient are stained
fluorescent red. After irradiation, the red tumor cells are fused
with the green dendritic cells using PEG. Hybrid formation by cell
fusion using PEG as fusing agent is routine procedure. The
procedure outlined below is a variation of the one reported by
Prado et al. for PEG mediated fusion of somatic cells in
monolayers. The hybrid cells which retain both the character of the
tumor cells as well as the ability of the dendritic cells to act as
an effective antigen presenting cell (APC), are then purified by
FACS according to their unique fluorescent color. FIG. 6 shows a
flow diagram representing the procedure for fluorescent dye
staining of dendritic cells and tumor cells.
B. Dendritic Cells and Tumor Cells Fusion
[0109] The green dendritic cells are mixed with the red tumor cells
irradiated with a single dose of 5000 rads sufficient to render the
cells replication incompetent at ratios of 1:1 to 5:1 or 1:1 to 1:5
in a 50 ml conical centrifuge tube. The tube is filled with
serum-free RPMI 1640 medium. The cell mixture is centrifuged for 5
minutes at 500 g. While the cells are being centrifuged, two
37.degree. C. double-beaker water baths are prepared in the laminar
flow hood by placing a 250-ml beaker containing 150 ml of
37.degree. C. water into a 600-ml beaker containing 150 ml of
37.degree. C. water. Tubes of prewarmed 50% PEG/DMSO solution and
serum-free RPMI 1640+Hepes solution are placed into one of the
37.degree. C. water baths in the hood. The supernatant from the
cell mixture is then aspirated and discarded.
[0110] Cell fusion is then performed at 37.degree. C. by placing
the tube containing the mixed-cell pellet in one of the
double-beaker water baths in the laminar flow hood. One ml of
prewarmed 50% PEG is added to the mixed-cell pellet drop-by-drop
over one minute, stirring the cells with the pipette tip after each
drop. The mixture is then stirred for an additional minute. Using a
clean pipette, 2 ml of prewarmed RPMI 1640+HEPES is added to the
cell mixture drop-by-drop over 2 minutes, stirring after each drop.
With a 10 ml pipette, 7 ml of prewarmed RPMI 1640+HEPES is added
drop-by-drop over 2 to 3 minutes. This mixture is then centrifuged
for five minutes at 500 g. While the cells are in the centrifuge,
the double beakers are rewarmed to 37.degree. C. and placed in the
hood. Prewarmed, complete DC media containing 10% autologous serum
is placed in the beaker water bath. The supernatant from the fusion
mixture is then discarded and the tube is placed in the
double-beaker water bath. With a pipette, 10 to 20 ml of prewarmed
complete DC media containing 100 ng/ml TNF.alpha. are forcefully
discharged onto the cell pellet and placed in a T75 flask. This
cell mixture is incubated overnight in a humidifier at 37.degree.
C., 5% CO.sub.2 incubator. FIG. 7 provides an overview of the cell
fusion procedure.
C. Dendritoma Purification
[0111] Following the fusion step, the fused cells are purified as
shown in FIG. 8.
D. Final Product Formulation and Filling
[0112] Purified dendritomas are re-suspended in medium (80%
RPMI-1640 and 10% autologous serum) at a concentration of 200,000
cells/0.9 ml medium and irradiated with 10,000 rads. Following
irradiation, 10% DMSO is added and cells are aliquoted into 2 ml
freezing vials (1 ml/vial). The last vial may contain less than 1
ml. The vials are cryopreserved at .ltoreq.-150.degree. C. in the
liquid phase of a liquid nitrogen freezer designed to prevent
contamination. All samples for release testing are obtained prior
to cryopreservation. 2.0 ml cryovials are manufactured by Sarsted.
These vials are transparent and in a conical shape. They are 10.8
mm in diameter and 46 mm in length, made of polypropylene and
include an external thread format. The vials are sterile.
Example 4
Preparation of the Dendritoma Vaccine for Patient
Administration
[0113] The dendritoma vaccine vials are thawed at 37.degree. C.,
formulated for patient administration and placed into labeled
syringes. The content of the syringe is then administered
subcutaneously to the patient. FIG. 10 provides an overview of the
preparation and administration of the dendritoma vaccine.
[0114] Skin test tumor cells are thawed, formulated and placed into
syringes like the dendritoma vaccine. The content of the syringe is
then administered intradermally to the patient.
Example 5
Induction of Cancer Cell Specific Cytotoxic T Cell Response by the
Dendritoma Vaccine
[0115] This example demonstrates that the inventive hybrid cells
induce a cancer cell-specific cytotoxic T cell response.
A. Cytotoxic T Cells
[0116] CD8+, cytotoxic T cells (CTLs) were prepared by the
following method. Peripheral blood mononuclear cells (PBMC's) were
isolated from whole blood by obtaining 40 ml of peripheral blood
from the patient in preservative-free or sodium heparin tubes and
10 ml in ACD tubes. The blood was diluted 1:1 with 1.times.PBS.
Eight ml of the diluted blood was layered over 4 ml of room
temperature Ficoll-Paque Plus in 15 ml conical centrifuge tubes.
The Ficoll gradients were centrifuged at 400 g at room temperature
for 40 minutes. Using a Pasteur pipet, the PBMC layers were
carefully removed from the Ficoll gradients and put into a sterile
15 ml centrifuge tube. Four volumes of 1.times.PBS were added to
the tube and inverted several times to mix thoroughly. The PBMC's
were centrifuged at 100 g at room temperature for 10 minutes. After
removal of the supernatant, 10 ml of 1.times.PBS was added to the
cells and inverted to mix. The PBMC's were pelleted by
centrifugation at 100 g at room temperature for 10 minutes and
resuspended in complete lymphocyte media (RPMI 1640, 10% FBS, 100
.mu.g/ml gentamicin).
[0117] PBMC's were isolated from patients in preservative-free or
sodium heparinized blood. They were subjected to the same panning
technique as previously described except that anti-CD4 antibody was
used to coat the plate. Prior to panning the PBMC's were enriched
for T lymphocytes by passing them through a nylon wool column. This
was done by packing 0.5 g of teased nylon wool into a 10 ml syringe
which has a stopcock attached to the tip. The column was washed
twice at 37.degree. C. with RPMI 1640 with 10% FBS. The stopcock
was closed and incubated at 37.degree. C. for one hour. After
draining the media from the column to the top of the wool, the
PBMC's were added to the column (up to 2.times.10.sup.8 in 2 ml of
media). The stopcock was opened and the media was drained until the
cell volume had entered the packed wool. After closing the
stopcock, additional media was added to cover the top of the wool.
The column was incubated for one hour at 37.degree. C. The
nonadherent T cells were collected by two media washes. After this
T cell enrichment, the T lymphocytes were panned using the anti-CD4
coated plate. T cells that were not bound by the CD4 antibody were
recovered and assumed to be CD8+ cells (cytotoxic T lymphocytes).
This was confirmed by FACS analysis.
[0118] To have constant re-stimulators for tumor cell specific
CTLs, the PBMC's isolated from the ACD blood were immortalized by
Epstein-Barr virus (EBV) transformation. This was accomplished by
resuspending the PBMC's at a concentration of 1.times.10.sup.6
cells/ml in complete lymphocyte media. To this, 1 ml of EBV
supernatant and 0.2 ml of phytohemagglutinin were added. The cell
mixture was cultured in a T25 tissue culture flask at 37.degree.
C., 5% CO.sub.2.
B. Stimulation of Cytotoxic T Cells
[0119] The instant dendritomas obtained from the FACS sort were
mixed with the enriched, panned CD8+ T lymphocytes in a 1:10 ratio.
The CD8+ cells to be used were pelleted by centrifugation at 1500
rpm for five minutes and resuspended in 1 ml of medium containing
RPMI 1640, 10% FBS, 1000 U/ml IL-6, 5 ng/ml IL-12, and 10 U/ml
IL-2. This was added to the instant dendritomas plated after the
sort. This culture was incubated at 37.degree. C., 5% CO.sub.2 for
one week. During that week the cells were refed with the same
media.
[0120] After one week, the primed CD8+ T cells (CTLs) were
restimulated with irradiated EBV-transformed lymphocytes that were
pulsed with tumor lysate. Tumor cells, which had been previously
cultured, were subjected to four freeze thaw cycles to lyse the
cells. To obtain the lysate containing tumor antigens, the lysed
cells were centrifuged at 600 g for ten minutes. The supernatant
was collected and centrifuged at 13,000 g for one hour. The
supernatant containing the lysate of tumor antigens was collected.
To restimulate the CTLs a viable cell count was taken using trypan
blue exclusion. Once the viable cell number was determined the same
number of EBV transformed lymphocytes were pulsed with the tumor
lysate by incubating the lysate with the lymphocytes at 37.degree.
C., 5% CO.sub.2 for one hour. The pulsed lymphocytes were
irradiated with 5,000 rads and then mixed with the CTLs in media
containing RPMI 1640, 10% FBS, 10 U/ml IL-la, 5 U/ml IL-2, 50 U/ml
IL-4, 125 U/ml IL-6, and 30 U/ml IL-7. The culture was incubated at
37.degree. C., 5% CO.sub.2 and refed every two days. This
re-stimulation was performed at 7 and 14 days after initial
priming.
[0121] Each day the CTLs were refed, the supernatant that was
removed was stored at -20.degree. C. When feasible, an
Interferon-gamma (IFN-.gamma.) assay was performed using an OptEIA
Human IFN-.gamma. Kit (PharMingen). The protocol was performed
exactly according to the manufacturer's directions. The assay was
read using a Benchmark Microplate Reader (BioRad).
[0122] To determine if the instant dendritomas stimulated a tumor
cell specific CTL response, a CTL assay was performed using the
cultured tumor cells as target cells. Fifty thousand tumor cells
were harvested and pelleted in a 15 ml conical centrifuge tube by
centrifugation at 200 g for five minutes. The supernatant was
discarded leaving 0.1 ml of medium on the pellet. The cells were
gently resuspended in the remaining medium. The tumor cells were
then labeled with .sup.51Cr by adding 0.1 ml of 1 mCi/ml .sup.51Cr
solution and 10 .mu.l FBS and mixing gently. This mixture was
incubated by loosening the cap of the tube and placing at
37.degree. C., 5% CO.sub.2 for one hour. After the incubation, the
labeled tumor cells were washed twice with 14 ml of RPMI 1640 and
resuspended at a concentration of 5.times.10.sup.4 cells/ml in
complete lymphocyte media.
[0123] The CTL effector cells were plated in 4 wells of a round
bottom 96 well tissue culture plate at concentrations that equaled
100:1, 30:1, 10:1, and 3:1 effector to target cell ratios. Five
thousand labeled target cells were added to the wells containing
the effector cells as well as two additional wells for natural and
maximum release controls. The cells were mixed and centrifuged at
200 g for 30 seconds. The plate was then incubated at 37.degree.
C., 5% CO.sub.2 for four hours. Thirty minutes prior to the end of
the incubation, 0.1 ml of Triton X-100 was added to the maximum
release control well. At the end of the incubation, the cells were
centrifuged in the plate at 200 g for five minutes. 0.1 ml of each
supernatant was added to liquid scintillation counter vials
containing 5 ml of scintillation cocktail. The amount of .sup.51Cr
release was measured using a LS6500 Multi-purpose Scintillation
Counter (Beckman).
[0124] The CTL assay results showed that as the ratio of hybrid
cell-primed CTLs to tumor cells increased, the release of the
isotope increased, indicating a positive correlation between the
number of CTLs and tumor killing. Greater than 50% killing was
observed at a 100:1 effector:target ratio. On the other hand, there
was no such correlation with control T cells that were not primed
with the inventive hybrid cells. Even at a ratio of 100:1, the
control T cells did not lyse more tumor cells than at lower ratios.
These results demonstrated that the CTLs generated using our hybrid
antigen presenting cells are fully functional and tumor cell
specific. The results are depicted in FIG. 1.
Example 6
Dendritoma Characterization
[0125] This example provides further characterization of the
reactant cells and the dendritomas described in the Examples
above.
[0126] Fluorescent microscopic analysis showed that 100% of the
stained cells were successfully labeled. To test whether the dye
can interstain between the two different type of cells, green DCs
and red tumor cells were mixed together and incubated overnight.
Fluorescent microscopic examination showed there was no
interstaining Immediate examination of the fusion product
demonstrated that the green DCs and the red tumor cells were fused
together and after an overnight recovery, the fused cells showed
both colors. The double colored cells (approximately 10% of the
total cells), instant dendritomas, were then purified by FACS
sorting. More than 95% of the sorted cells were double colored
fused cells.
[0127] Instant dendritomas express all the molecules necessary for
antigen presentation. FACS analysis showed that instant dendritomas
express the molecules required for antigen presentation, such as
MHC class I and II and co-stimulating molecules CD80 (B7.1) and
CD86 (B7.2). The data are depicted in FIG. 2. "Isotype" is the
negative control; HLA-A,B,C is MHC class I and HLA-DR is MHC class
II. Under a microscope, moreover, instant dendritomas also have
those dark granules that melanoma tumor cells have.
[0128] For FIG. 2, human DCs from peripheral blood were stained
with the green dye and tumor cells were stained with the red dye,
respectively, and fused, using the above protocol. After overnight
incubation, the cells were equally divided into 4 groups. They were
then stained with Cy-Chrome conjugated antibodies by incubating the
cells with the antibodies (1 million cells/microgram antibody,
Becton/Dickinson) on ice for 30 min. The different groups were as
follows: anti-human HLA-A,B,C [group I]; anti-human HLA-DR [group
II]; anti-human CD80 [group III]; and anti-human CD86 [group IV].
The un-bound antibodies were removed by two washes and the cell
pellet was re-suspended in 0.5 ml of staining buffer (PBS
containing 0.1% BSA and 0.1% sodium azide). Three-color analysis
was performed by FACS, using CellQuest software. Control human DCs
were also stained with the same antibodies in the same way.
Example 7
Clinical Protocol
[0129] This example provides an exemplary clinical protocol for
treating human cancer patients with the inventive hybrid cell
preparations in conjunction with BCG therapy. This regimen is
useful, for example, to treat neuroblastoma patients with a
dendritoma vaccine in combination with BCG therapy, prepared
according to the inventive methodologies.
A. Blood Draws for Dendritic Cells and Serum Preparation
[0130] For culture of the patient's dendritic cells and
cryopreservation of the patient's tumor cells and dendritomas, the
patient's own autologous serum is used, however, commercially
available serum free dendritic cell media may also be used. Serum
is isolated using standard procedures from 100 mL of peripheral
blood collected without anti-coagulants. In order to generate the
dendritoma vaccine, dendritic cells are harvested from the patient.
Three hundred milliliters of sodium heparinized peripheral blood is
collected from the patient.
[0131] Additional blood draws for dendritic cells and/or serum are
required for the generation of the vaccine. Two or 3 blood draws
may be necessary to obtain an adequate number of dendritic cells.
Additional autologous serum may also need to be isolated. However,
additional blood draws for dendritic cells and serum are determined
by the patient's hemoglobin level assessed after each blood draw.
Blood is drawn from the patient if the hemoglobin level is greater
than or equal to 8.5 mg/dL. If the hemoglobin level is below 8.5
mg/dL, the patient will receive packed red blood cells prior to
having the next blood draw to increase the hemoglobin level.
B. Dendritic Cell Generation
[0132] Mature dendritic cells are generated from the patient's
peripheral blood monocytes (PBMCs). In one embodiment, the
dendritic cells are generated by leukapheresis, which is the
selective separation and removal of leukocytes from withdrawn
blood, the remainder of the blood is then retransfused into the
donor. In another embodiment, 300 mls of peripheral blood are
obtained from the patient in preservative free or sodium heparin
tubes. Briefly, the blood is diluted 1:1 with 1.times.PBS. Then, 8
mls of the diluted blood are layered over 4 mls of room temperature
Ficoll-Paque Plus in a 15 ml centrifuge tube, and centrifuged at 40
g for 40 minutes. The PBMC layer is removed from the Ficoll
gradient, and placed into a clean 250 ml centrifuge tube. 4 volumes
of 1.times.PBS are added and the tube is inverted to mix. The
PBMC's are then centrifuged at 100 g at room temperature for 10
minutes. 10 ml of 1.times.PBS are added, and the cells are mixed by
inverting the tube. The PBMC's are again centrifuged at 100 g at
room temperature for 10 minutes. The PBMC's are resuspended in 5 ml
complete DC medium (RPMI 1640+10% human serum+800 U/ml GM-CSF+1000
U/ml IL-4). Then the dendritic cells/precursors are optionally
panned using anti-CD14 coated plates. 2.times.10.sup.8 PBMC's are
placed onto the anti-CD14 coated plate and swirled. They are left
to incubate at room temperature for 30 minutes. The non-adherent
cells are then removed. 10 ml of 1.times.PBS are added, the plate
is swirled, and the PBS is removed. This PBS washing is repeated
for a total of four times, pipetting at the same place each time.
Afterwards, 10 ml of complete DC media is added to the plate. They
are then incubated at 37.degree. C., 5% CO.sub.2 for 5-10 days to
generate dendritic cells.
C. Tumor Cells
[0133] A tumor section is obtained at the time of biopsy or
excisional resection. The tumor cells are cultured using the
following technique. After separating fat and necrotic tissue away
from the tumor tissue (1-5 grams), the tumor is cut into small
chunks and put into a T 75 flask. This solution will rock for 1
hour at 37.degree. C., and then 15 ml of complete media (DMEM+10%
human serum+gentamicin) are added. The chunks are then removed and
put into a clean T75 flask. This flask is left at 37.degree. C. in
5% CO2 overnight. Then the cell suspension/typsin/complete media is
centrifuged at 1000 g for 5 minutes. These cells are resuspended in
15 ml of complete media and cultured in a T75 flask at 37.degree.
C. in 5% CO2 for 24 hours. After overnight incubation in the
absence of media, 20 ml of complete media are added to the flask
with chunks, and this solution is left for two days at 37.degree.
C. in 5% CO2. The chunks are removed, and the adherent cells are
cultured. The tumor cells used for dendritic fusion result from
both cultures.
D. Hybrid Formation
[0134] The next step comprises the fusion of tumor cells and
dendritic cells received from the patient. Hybrid formation by cell
fusion became routine after the introduction of the use of
polyethylene glycol as a fusing agent. The procedure outlined below
is a variation of the one reported by Prado et al., 1989 FEBS
Lett., 259: 149-52, for the PEG-mediated fusion of somatic cells in
monolayers.
[0135] First, the tumor cells are exposed to a single dose of 5000
rads, sufficient to kill all of the cells. Then, the dendritic
cells are stained green using the PKH2-GL fluorescent dye (Sigma),
and the tumor cells are stained red using the PKH26 fluorescent dye
(Sigma). The staining procedure is performed at 25.degree. C.,
using a slight modification of the Sigma procedure. The cells to be
stained are washed with serum-free media. The cell suspension is
centrifuged at 400 g for five minutes to obtain a loose pellet, and
the supernatant fraction is removed. The pellet is resuspended by
tapping the centrifuge tube, and 1 ml of diluent (20% DMSO in
serum-free RPMI) is added to resuspend the cells. Immediately prior
to staining, 4.times.10.sup.-6 molar dyes (2.times.) were prepared
with diluent in polypropylene tubes. The cells in the diluent are
rapidly added into 1 ml of 2.times. dye, and the mixture is
immediately mixed by gentle pipetting. The mixture is then
incubated at 25.degree. C. for five minutes. The staining process
is stopped by adding an equal volume of 10% human serum, which may
be the patient's own serum, and incubating for one minute. The
stained cells are diluted with equal volume of complete culture
medium. Stained cells are removed from the staining solution by
centrifuging at 400 g for 10 minutes.
[0136] The green dendritic cells are mixed with the red tumor cells
at a 1:1 ratio in a 50-ml conical centrifuge tube. The tube is
filled with complete serum-free DMEM. The cell mixture is
centrifuged for 5 minutes at 500 g. While the cells are being
centrifuged, three 37.degree. C. double-beaker water baths are
prepared in the laminar flow hood by placing a 400-ml beaker
containing 100 ml of 37.degree. C. water into a 600-ml beaker
containing 75 to 100 ml of 37.degree. C. water. Tubes of prewarmed
50% PEG solution and complete serum-free DMEM are placed into two
of the 37.degree. C. water baths in the hood. Then, the supernatant
from the cell mixture is aspirated and discarded. The cell fusion
is performed at 37.degree. C. by placing the tube containing the
mixed-cell pellet in on of the double-beaker water baths in the
laminar flow hood. Then, 1 ml of prewarmed 50% PEG is added to the
mixed-cell pellet drop-by-drop over one minute, stirring the cells
with the pipette tip after each drop. The mixture is then stirred
for an additional minute.
[0137] Using a clean pipette, 1 ml of pre-warmed RPMI+HEPES is
added to the cell mixture drop-by-drop over one minute, stirring
after each drop. This step is repeated once with an additional 1 ml
of prewarmed RPMI+HEPES solution. With a 10-ml pipette, 7 ml of
prewarmed RPMI+HEPES is added drop-by-drop over 2 to 3 minutes.
This mixture is then centrifuged for five minutes at 500 g. While
the cells are in the centrifuge, the water baths are rewarmed to
37.degree. C. and placed in the hood. Prewarmed complete DC media
is placed in the beaker water bath. Then the supernatant from the
mixture is discarded; the tube is placed in the beaker water bath.
With a pipette, 10 ml of prewarmed complete DC media are forcefully
discharged onto the cell pellet and placed in a T75 flask. This is
incubated overnight in a humidified 37.degree. C., 5% CO2
incubator.
[0138] The next day, the cells are analyzed on a FACS Caliber
fluorescence activated cell sorter using the CELLQuest software
(Becton/Dickenson), which will sort the fusion cells with both the
green and red dye. These fusion cells, dendritomas, are then
resuspended in 1 ml of NS (Normal Saline) and injected into the
patient.
E. The Dendritoma Vaccine
[0139] The vaccine consists of at least 100,000 (or more)
irradiated tumor cells fused to dendritic cells i.e. dendritomas. A
minimum dose of 100,000 dendritomas is administered during each
vaccination. These dendritomas are resuspended in 1 ml of normal
saline (NS) and injected subcutaneously into an area of lymph nodes
in the axilla or inguinal area of the patient. The injection site
is rotated to avoid injection in the same lymph node bed on two
consecutive administrations. After the initial vaccination, the
patient is revaccinated every 6 weeks. The patient may receive a
maximum of 6 vaccinations, depending on the total number of
dendritomas obtained.
[0140] The BCG preparation is administered subcutaneously within 10
minutes of the administration after each dose of the dendritoma
vaccine. The dosage of BCG (Mycobacterium bovis bacillus
Calmette-Guerin) to be administered after the initial vaccination
with the dendritoma vaccine is based upon the patient's reaction to
the TB skin test administered prior to BCG administration.
Depending on the patient's reaction to the TB skin test, the BCG
composition consists of at least 20,000 units, preferably at least
250,000 units, and more preferably at least 1,000,000 units of BCG
(see Table 1 above). The BCG therapy is administered subcutaneously
within 10 minutes of the administration after each dose of the
dendritoma vaccine.
[0141] Following administration of the second dendritoma vaccine,
within 10 minutes of vaccination, the patient is administered a
second BCG preparation. Preferably, the BCG preparation is
administered at the same dosage as the first BCG preparation,
unless the initial injection produced ulceration at the injection
site. If ulceration occurs at the injection site after the initial
administration, half the dose (50% of the BCG dose) of the initial
vaccination is used.
[0142] Subsequent administration of BCG preparation is based on the
patient's reaction to previous BCG administration. Preferably, the
BCG preparation is administered at the same dosage as the first BCG
preparation, unless the initial injection produced ulceration at
the injection site. If ulceration occurs at the injection site
after the dosage of BCG is reduced by 50%, then BCG is not
administered and the dendritoma vaccine is administered alone on
all subsequent injections.
[0143] Interleukin 2 (e.g., Aldesleukin) also may optionally be
given in a low-dose regimen. When used, IL-2 is administered by
subcutaneous injection in a dosage of 18 million units daily for 5
days beginning on the day of vaccination.
Example 8
Clinical Trials
[0144] Patient 1 with metastatic stage IV neuroblastoma (staged
according the International Neuroblastoma Staging System INSS)
exhibited paraplegia secondary to tumor around the spinal cord. The
primary tumor was suprarenal with erosion through the spinal
column. The cancer was spread to distant sites, including the
patient's bone marrow, liver and lumbar vertebrae. The tumor was
not n-myc amplified. The patient had 7 cycles of chemotherapy and
after completing therapy underwent surgery to remove the bulk tumor
on the right suprarenal area. Bone marrow aspirate and biopsy
showed persistent neuroblastoma. CT scans of chest, abdomen and
pelvis showed persistent disease in the liver and lumbar vertebrae.
The patient was not a candidate for bone marrow transplant because
of persistent disease in the bone marrow.
[0145] Patient 1's tumor cells, after irradiation, were fused with
their dendritic cells to produce a non-proliferative hybrid cell,
also known as a dendritoma. The dendritomas were obtained and
counted before administration and the patient was vaccinated with
the dendritoma vaccine approximately every six weeks following the
initial vaccination according to the following scheme:
TABLE-US-00004 Administration Time Line CFU of BCG (Post initial
treatment) Dendritoma Dose administered Initial Treatment -0 weeks
275,000 cells 1,000,000 Second Treatment -6 weeks 275,000 cells
1,000,000 Third Treatment -12 weeks 210,000 cells 1,000,000 Fourth
Treatment - 18 weeks 200,000 cells 500,000 Fifth Treatment - 24
weeks 200.000 cells 0
[0146] The dendritoma vaccine was injected subcutaneously into an
area of lymph nodes in the axilla or inguinal area of the
patient.
[0147] BCG was administered to patient 1 subcutaneously into an
area of lymph nodes in the axilla or inguinal area of the patient
within 10 minutes of the first four dendritoma doses. The BCG used
in this study is commercially available and is manufactured by
Organon Teknika Corporation (Durham, N.C.). All BCG injections
where administered within 10 minutes of the dendritoma vaccine.
[0148] The injection sites for the dendritoma vaccine and the BCG
preparation can be rotated to avoid injection in the same lymph
node bed on two consecutive administrations, by alternating
anterior and posterior injections with lateral and medial
injections.
[0149] The dendritoma vaccine and the BCG preparations were not
mixed and the BCG dosage was determined based upon the patient's
reaction to the TB skin test administered prior to the study entry.
Administration of the BCG dosage followed the following scheme:
TABLE-US-00005 TB Skin Test Result Dermatologic Reaction BCG Dose
Negative None 1,000,000 units Positive <10 mm in duration .sup.
250,000 units Positive .gtoreq.10 mm in duration, 20,000 units
erythema
[0150] BCG is measured in colony forming unit (CFU). For example,
20,000 BCG units on a culture plate will produce 20,000 bacterial
colonies.
[0151] In this case, the TB skin test results for Patient 1 were
negative, and the initial BCG dose administered with the dendritoma
vaccine to Patient 1 was therefore 1,000,000 CFU.
[0152] For the second vaccination, the same BCG dose as the initial
vaccination is used, unless the initial injection produces
ulceration at the injection site. If ulceration occurs, half the
dose (50% of the BCG dose) of the initial vaccination is used. In
this case, Patient 1 was administered a BCG dose of 1,000,000 CFU
for the second vaccination.
[0153] For subsequent vaccinations, the same BCG dose as the
initial vaccination is used, unless the initial injection produces
ulceration at the injection site. If ulceration occurs after the
dosage of BCG is decreased by 50%, then BCG is not administered
again and the dendritoma vaccine is given without the BCG on all
subsequent injections. If, however, no ulceration occurs after the
dosage of BCG is decreased by 50%, then BCG is administered again
using the same decreased dosage. In this case, ulceration occurred
in Patient 1 after the dosage of BCG was decreased by 50%, so the
fifth vaccine was administered without concomitant BCG.
[0154] Patient 1 showed great improvement at the end of the
treatment with the dendritoma vaccine and the BCG preparation
according to the invention. As a result of the treatment, the
progression of the neuroblastoma in the patient has considerably
slowed and the patient's life expectancy has dramatically
increased.
[0155] Table 4 and Table 5 below summarize the studies performed
prior, during and after the initial vaccination (Table 2), and
before and after the second vaccination (Table 3).
TABLE-US-00006 TABLE 4 The schedule of time and events prior to
study entry, during, and following the initial vaccination
##STR00001## .sup.#Within 2 days .sup.##Within +/- 7 days
.sup.$SAEs will be monitored from the first vaccination until six
weeks after the last vaccination. *Every 8 to 10 weeks
TABLE-US-00007 TABLE 5 The schedule of time and events prior to and
after revaccination ##STR00002## *Also the 6-week follow-up
.sup.#Within 2 days .sup.##Within +/- 7 days .sup.$SAEs will be
monitored from the first vaccination until six weeks after the last
vaccination. .sup..dagger.Every 8 to 10 weeks
.sup..dagger..dagger.Performed annually
[0156] It will be apparent to those skilled in the art that various
modifications and variations can be made in the methods and
compositions of the present invention without departing from the
spirit or scope of the invention. Thus, it is intended that the
present invention cover the modification and variations of the
invention provided they come within the scope of the appended
claims and their equivalents.
* * * * *