U.S. patent application number 11/909251 was filed with the patent office on 2009-08-27 for cancer vaccines and therapeutic methods.
This patent application is currently assigned to THE BOARD OF TRUSTEES OF THE UNIVERSITY OF ILLINOI. Invention is credited to Edward P. Cohen.
Application Number | 20090214494 11/909251 |
Document ID | / |
Family ID | 37054114 |
Filed Date | 2009-08-27 |
United States Patent
Application |
20090214494 |
Kind Code |
A1 |
Cohen; Edward P. |
August 27, 2009 |
Cancer Vaccines and Therapeutic Methods
Abstract
Compositions and methods of producing improved cancer vaccines
are described. In addition, methods of identifying tumor associated
antigens are also described.
Inventors: |
Cohen; Edward P.; (Chicago,
IL) |
Correspondence
Address: |
MCDONNELL BOEHNEN HULBERT & BERGHOFF LLP
300 S. WACKER DRIVE, 32ND FLOOR
CHICAGO
IL
60606
US
|
Assignee: |
THE BOARD OF TRUSTEES OF THE
UNIVERSITY OF ILLINOI
Urbana
IL
|
Family ID: |
37054114 |
Appl. No.: |
11/909251 |
Filed: |
March 28, 2006 |
PCT Filed: |
March 28, 2006 |
PCT NO: |
PCT/US06/11575 |
371 Date: |
May 2, 2009 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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60665985 |
Mar 29, 2005 |
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60697334 |
Jul 7, 2005 |
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60733663 |
Nov 4, 2005 |
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Current U.S.
Class: |
424/93.21 ;
435/29; 435/325; 435/375; 435/6.14 |
Current CPC
Class: |
A61K 38/208 20130101;
A61K 39/0011 20130101; A61K 48/00 20130101; A61K 38/2026 20130101;
A61K 2039/55522 20130101; A61K 35/12 20130101; A61K 38/193
20130101; A61K 39/39 20130101; A61K 2039/55527 20130101; A61K
2039/53 20130101; A61K 2039/5156 20130101; A61K 38/204 20130101;
A61K 2039/5154 20130101; A61K 38/2013 20130101; A61P 35/00
20180101; A61K 38/2013 20130101; A61K 2300/00 20130101; A61K
38/2026 20130101; A61K 2300/00 20130101; A61K 38/204 20130101; A61K
2300/00 20130101; A61K 38/208 20130101; A61K 2300/00 20130101; A61K
38/193 20130101; A61K 2300/00 20130101 |
Class at
Publication: |
424/93.21 ;
435/375; 435/29; 435/6; 435/325 |
International
Class: |
A61K 35/12 20060101
A61K035/12; C12N 5/00 20060101 C12N005/00; C12Q 1/02 20060101
C12Q001/02; C12Q 1/68 20060101 C12Q001/68; A61P 35/00 20060101
A61P035/00 |
Goverment Interests
STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH AND
DEVELOPMENT
[0002] This invention was made in part with Government support
under Grant Number R01-DEO13970-01A2 awarded by the National
Institute of Dental and Craniofacial Research (NIDCR). The
Government has certain rights in this invention
Claims
1. A composition comprising a chemotherapeutic agent and an
antigen-presenting cell modified to express an allogeneic
MHC-determinant, and wherein the cell has been transfected with
genomic DNA or c-DNA isolated from the tumor of a mammal.
2. The composition of claim 1, wherein the chemotherapeutic agent
is selected from the group consisting of paclitaxel, docetaxel,
vincristine, vinblastine, vinorelbine, irinotecan, topotecan,
etoposide, methotrexate, 5-fluorouracil, cyclophosphamide,
ifosphamide, melphalan, chlorambucil, BCNU, CCNU, decarbazine,
procarbazine, busulfan, thiotepa, daunorubicin, doxorubicin,
idarubicin, epirubicin, and mitoxantrone.
3. The composition of claim 1, wherein the chemotherapeutic agent
is paclitaxel.
4. The composition of claim 1, wherein the antigen-presenting cell
is further transfected with a nucleic acid molecule coding for at
least one cytokine.
5. The composition of claim 4, wherein the cytokine is selected
from the group consisting of IL-2, granulocyte macrophage colony
stimulating factor (GM-CSF), IL-4, IL-6, and IL-12.
6. The composition of claim 5, wherein the cytokine is IL-2.
7. The composition of claim 1, wherein the antigen-presenting cell
is selected from the group consisting of a fibroblast, a
macrophage, a B cell, and a dendritic cell.
8. The composition of claim 1, wherein the tumor is a solid tumor
or a hematological tumor.
9. The composition of claim 8, wherein the tumor is selected from
the group consisting of melanoma, lymphoma, plasmacytoma, sarcoma,
glioma, thymoma, leukemias, breast cancer, prostate cancer, colon
cancer, esophageal cancer, brain cancer, lung cancer, ovarian
cancer, cervical cancer and hepatoma.
10. The composition of claim 1, wherein the animal is a human
subject.
11. A method of treating a tumor in a mammal, comprising
administering to the mammal a chemotherapeutic agent and an
effective amount of an antigen-presenting cell modified to express
an allogeneic MHC-determinant, and wherein the cell has been
transfected with genomic DNA isolated from the tumor of the
mammal.
12. The method of claim 1, wherein the chemotherapeutic agent is
selected from the group consisting of paclitaxel, docetaxel,
vincristine, vinblastine, vinorelbine, irinotecan, topotecan,
etoposide, methotrexate, 5-fluorouracil, cyclophosphamide,
ifosphamide, melphalan, chlorambucil, BCNU, CCNU, decarbazine,
procarbazine, busulfan, thiotepa, daunorubicin, doxorubicin,
idarubicin, epirubicin, and mitoxantrone.
13. The method of claim 2, wherein the chemotherapeutic agent is
paclitaxel.
14. The method of claim 1, wherein the antigen-presenting cell is
further transfected with a nucleic acid molecule coding for at
least one cytokine.
15. The method of claim 4, wherein the cytokine is selected from
the group consisting of IL-2, granulocyte macrophage colony
stimulating factor (GM-CSF), IL-4, IL-6, and IL-12.
16. The method of claim 5, wherein the cytokine is IL-2.
17. The method of claim 1, wherein the antigen-presenting cell is
selected from the group consisting of a fibroblast, a macrophage, a
B cell, and a dendritic cell.
18. The method of claim 1, wherein the tumor is a solid tumor or a
hematological tumor.
19. The method of claim 8, wherein the tumor is selected from the
group consisting of melanoma, lymphoma, plasmacytoma, sarcoma,
glioma, thymoma, leukemias, breast cancer, prostate cancer, colon
cancer, esophageal cancer, brain cancer, lung cancer, ovarian
cancer, cervical cancer and hepatoma.
20. The method of claim 1, wherein the animal is a human
subject.
21. A method of treating a tumor in a mammal, comprising
administering to the mammal a chemotherapeutic agent and an
effective amount of an antigen-presenting cell that has been
transfected with genomic DNA or c-DNA isolated from the tumor of
the mammal.
22. A method of enriching a population of immunogenic cells capable
of inducing an immune response to a target cell in a patient,
comprising: (a) providing a population of immunogenic cells capable
of inducing an immune response to a target cell in a patient; (b)
incubating the immunogenic cells in growth medium; (c) either
before or after (b), diluting the suspension the immunogenic cells,
whereby an enriched population of immunogenic cells is produced;
and (d) optionally repeating (a)-(c).
23. The method of claim 22 wherein said enriched population of
immunogenic cells is polyclonal.
24. The method of claim 22 wherein said enriched population of
immunogenic cells is monoclonal.
25. The method of claim 22 wherein said population of immunogenic
cells are produced by a method comprising introducing DNA derived
from said target cell into a recipient cell.
26. The method of claim 25 wherein the recipient cell is syngeneic,
semi-allogeneic or allogeneic.
27. The method of claim 22 further comprising screening said
enriched population of immunogenic cells for the ability to induce
an immune response.
28. The method of claim 27 wherein said ability to induce an immune
response is the in vitro or in vivo stimulation of T cells.
29. A method of identifying a tumor associated antigen, comprising:
(a) providing a recipient cell and an immunogenic cell capable of
inducing an immune response to a tumor cell; and (b) comparing the
nucleic acid of the recipient cell and the immunogenic cell,
whereby a nucleic acid with increased levels in the immunogenic
cell encodes a tumor associated antigen.
30. A method of identifying a tumor associated antigen, comprising:
(a) providing a recipient cell and an immunogenic cell capable of
inducing an immune response to a tumor cell; and (b) comparing the
expressed proteins of the immunogenic cell and the recipient cell,
whereby a protein with increased expression in the immunogenic cell
is a tumor associated antigen.
31. A composition comprising an antigen-presenting cell modified to
express an allogeneic MHC-determinant, and wherein the cell has
been transfected with genomic DNA or c-DNA isolated from the tumor
of a mammal.
32. The composition of claim 31, wherein the antigen-presenting
cell is further transfected with a nucleic acid molecule coding for
at least one cytokine.
33. The composition of claim 31, wherein the cytokine is selected
from the group consisting of IL-2, granulocyte macrophage colony
stimulating factor (GM-CSF), IL-4, IL-6, and IL-12.
34. The composition of claim 31, wherein the cytokine is IL-2.
35. The composition of claim 31, wherein the antigen-presenting
cell is selected from the group consisting of a fibroblast, a
macrophage, a B cell, and a dendritic cell.
36. The composition of claim 31, wherein the tumor is a solid tumor
or a hematological tumor.
37. The composition of claim 31, wherein the tumor is selected from
the group consisting of melanoma, lymphoma, plasmacytoma, sarcoma,
glioma, thymoma, leukemias, breast cancer, prostate cancer, colon
cancer, esophageal cancer, brain cancer, lung cancer, ovarian
cancer, cervical cancer and hepatoma.
38. The composition of claim 31, wherein the animal is a human
subject.
Description
PRIORITY CLAIM
[0001] This application claims priority from U.S. provisional
patent applications 60/665,985 filed Mar. 29, 2005; 60/697,334
filed Jul. 7, 2005; and 60/733,663 filed Nov. 4, 2005; the
disclosures of which are incorporated herein in their entirety.
FIELD OF THE INVENTION
[0003] The present invention relates to vaccines and methods for
treating a disease, such as cancer. More specifically, the present
invention relates to immunogenic cells which act to stimulate and
induce an immunogenic response to an antigen, such as a tumor
associate antigen (TAA).
[0004] The present invention also concerns treatment of cancer
using a genomic DNA-based vaccine. In addition, the present
invention concerns the use of a genomic DNA-based vaccine in
combination with a chemotherapeutic agent for the treatment of
cancer.
DESCRIPTION OF RELATED ART
[0005] The frequency of cancer in humans has increased in the
developed world as the population has aged. For some types of
cancers and stages of disease at diagnosis, morbidity and mortality
rates have not improved significantly in recent years in spite of
extensive research. During the progression of cancer, tumor cells
become more and more independent of negative regulatory controls as
a result of mutated or dysregulated genes. The mutated or
overexpressed proteins of cancer cells may result in the cancer
cell becoming antigenecally distinct from normal cells. Such
proteins are referred to as tumor-associated antigens (TAAs), since
they may be recognized as foreign and may be attacked by a
patient's immune system (Sibille et al., J. Ex. Med. 172:35-45,
1990). Tumor associated antigens have been identified for a number
of tumors, including melanoma, breast adenocarcinoma, prostate
adenocarcinoma, esophageal cancer, lymphoma, and many others. Based
on such antigenic differences between malignant and non-malignant
cells, immunotherapy has been suggested as a reasonable means of
treating cancer.
[0006] Current immunotherapeutic approaches to cancer treatment
include cancer vaccines based on tumor cell lysates, apoptotic
tumor-cell bodies or defined antigens. To this point, cancer
vaccines have typically been weakly immunogenic. Thus, there is a
long-felt need in the art to identify new TAAs of sufficient
immunogenicity to serve as part of cancer vaccines.
[0007] The potential benefits of immunotherapy as an adjunct to
conventional forms of cancer treatment are under active
investigation (Yu B, Clin Cancer Res 2003; 9:285-94; Chang S Y, Int
J Cancer 2004; 111:86-95; Dsis M L, J Clin Oncol 2004; 22:1916-25;
Avigan D, Clin Cancer Res 2004; 10:4699-708). Activated cytotoxic T
lymphocytes (CTLs) capable of recognizing and destroying cancer
cells are generated in immunized mice, and patients. The immunity
is directed toward unique MHC class I restricted TAAs expressed by
the malignant cells (Boon T, Curr Opin in Immunol 2003; 15:129-130;
Banchereau J, Cancer Res 2001; 61:6451-8; Gajewski T F, Clin Cancer
Res 2001; 7: S895-S901; Marchand M, Eur J Cancer 2003;
39:70-7.).
[0008] Although experimental immunotherapy protocols in mice are
revealing the potential of this form of treatment, effective
vaccination strategies in cancer patients are wanting. One possible
explanation is that even though the immune system can adversely
affect diffuse and smaller tumors, it cannot effectively destroy
large, established neoplasms. An immunotherapeutic strategy that
would allow treatment at an early stage of the disease could have
significant benefits.
[0009] Efforts to increase immunogenicity of TAA-based vaccines
have ranged from using adjuvants or cytokines to genetically
modified tumor cells (Offringa et al., Curr. Opin. Immunol.
12:576-583, 2000). Additional types of modified cell lines for use
as a vaccine include transferring tumor DNA into highly immunogenic
cell lines (Whiteside et al., Proc Natl Acad Sci U.S.A. 99:9415-20,
2002).
[0010] Tumor cells are the richest source of tumor antigens.
Immunization with malignant cells modified to secrete
immune-augmenting cytokines such as IL-2 (Fearon E R, Cell 1990;
60:397-403; Cavallo F, Cancer Res 1993; 53:5067-70; Coinor J, J Exp
Med 1993; 177:1127-34.), GM-CSF (Dranoff G, Proc Natl Acad Sci
(USA) 1993; 90:3539-43.), IL-4 (Golumbek P T, Science 1991;
254:713-6.), IL-6 (Mullen C A, Cancer Res 1992; 52:6020-4.) and
IL-12 (Chen L, J Immunol 1997; 59:351-9; Tahara H, Cancer Res 1994;
54:182-9) resulted in rejection of the cytokine-secreting cells and
the induction of T cell mediated immunity toward the neoplastic
cells. In some instances, the induced immunity was sufficient to
prolong the lives of mice with established neoplasms. However, the
direct modification of cancer cells from a primary neoplasm is
technically challenging. It requires the establishment of a tumor
cell line, which cannot always be accomplished. This is especially
the case for breast cancer in patients.
SUMMARY OF THE INVENTION
[0011] The present invention is directed to compositions comprising
a combination of a chemotherapeutic agent and a genomic DNA-based
vaccine. The present invention is also directed to compositions
comprising a combination of a chemotherapeutic agent and a
c-DNA-based vaccine. The chemotherapeutic agent may be selected
from taxane, camptothecin, vinca alkaloid, antblacycline,
antibiotic, antimetabolite, platinum, or alkylating agent,
paclitaxel, docetaxel, vincristine, vinblastine, vinorelbine,
irinotecan, topotecan, etoposide, methotrexate, 5-fluorouracil,
cyclophosphamide, ifosphamide, melphalan, chlorambucil, BCNU, CCNU,
decarbazine, procarbazine, busulfan, thiotepa, daunorubicin,
doxorubicin, idarubicin, epirubicin, or mitoxantrone, as well as
other chemotherapeutic agents known to one of skill in the art. A
genomic DNA-based vaccine useful as compositions of the instant
invention comprises an antigen-presenting cell modified to express
an allogeneic MHC-determinant, and transfected with genomic DNA
isolated from a tumor of a mammal in need of cancer treatment.
[0012] A c-DNA-based vaccine useful as compositions of the instant
invention comprises an antigen-presenting cell modified to express
an allogeneic MHC-determinant, and transfected with c-DNA isolated
form a tumor of a mammal in need of cancer treatment. A c-DNA-based
vaccine and a genomic DNA-based vaccine of the invention can be
used either alone or in combination with a chemotherapeutic
agent.
[0013] Genomic DNA used in compositions of the instant invention
can be isolated from any neoplasm cancer, including melanoma,
lymphoma, plasmacytoma, sarcoma, glioma, thymoma, leukemias, breast
cancer; prostate cancer, colon cancer, esophageal cancer, brain
cancer, lung cancer, ovarian cancer, cervical cancer, hepatoma or
any other solid or hematological cancer cells.
[0014] C-DNA used in compositions of the instant invention can be
isolated from any neoplasm cancer, including melanoma, lymphoma,
plasmacytoma, sarcoma, glioma, thymoma, leukemias, breast cancer,
prostate cancer, colon cancer, esophageal cancer, brain cancer,
lung cancer, ovarian cancer, cervical cancer, hepatoma or any other
solid or hematological cancer cells.
[0015] Antigen-presenting cells used in compositions of the instant
invention can be further modified to express a cytokine. For
example, an antigen-presenting cell can be modified to express any
of the following cytokines: IL-2, granulocyte macrophage colony
stimulating factor (GM-CSF), IL-4, IL-6 or IL-12
[0016] The present invention is also related to a method of
enriching populations of immunogenic cells capable of inducing an
immune response to a target cell in a patient by providing a
population of immunogenic cells capable of inducing an immune
response to a target cell in a patient; incubating the immunogenic
cells in growth medium; diluting the immunogenic cells either
before or after incubating the immunogenic cells; and optionally
repeating these steps, whereby an enriched population of
immunogenic cells is produced. The enriched population of
immunogenic cells may be polyclonal or monoclonal. The population
of immunogenic cells may be produced by introducing DNA derived
from a cancer cell into a recipient cell that is syngeneic,
semi-allogeneic, or allogeneic.
[0017] The present invention is also related to a method of
screening immunogenic cells for the ability to induce an immune
response. The ability to induce an immune response may be the in
vitro or in vivo stimulation of T cells.
[0018] The present invention is also related to a method of
identifying a tumor associated antigen by providing a recipient
cell and an immunogenic cell capable of inducing an immune response
to a tumor cell and comparing nucleic acid of the recipient cell
and the immunogenic cell and identifying the nucleic acid with
increased expression in the immunogenic cell as the nucleic acid
encoding for a tumor associated antigen.
[0019] The present invention is also related to a method of
identifying a tumor associated antigen by providing a recipient
cell and an immunogenic cell capable of inducing an immune response
to a tumor cell and comparing the expressed proteins of the
immunogenic cell and the recipient cell and determining that the
protein with increased expression in the immunogenic cell as a
tumor associated antigen.
BRIEF DESCRIPTION OF THE DRAWINGS
[0020] FIG. 1 depicts the effect of the paclitaxel on the growth of
breast cancer cells in C3H/He mice.
[0021] FIG. 2 depicts expression of MHC class I
H-2K.sup.b-determinants by LM fibroblasts transduced with the
plasmid vector pBR327H-2K.sup.b.
[0022] FIG. 3 depicts that immunization with LM-IL-2K.sup.b/SB5b
cells inhibits the growth of SB5b breast cancer cells in C33H/He
mice.
[0023] FIG. 4 depicts immunity to breast cancer in C3H/He mice
receiving combined treatment with paclitaxel followed by
immunization with LM-IL-2Kb/SB5b cells.
[0024] FIG. 5 depicts survival of C3H/He mice with breast cancer
receiving combined therapy with paclitaxel and LM-IL-2K.sup.b/SB5b
cells.
[0025] FIG. 6 depicts immunity to breast cancer in C3H/He mice
receiving combined therapy with paclitaxel and LM-IL-2K.sup.b/SB5b
cells.
[0026] FIG. 7 shows survival statistics for C314 mice injected with
SB-1 breast cancer cells (isolated from a breast neoplasm that
arose spontaneously in a C3H/He mouse) and fibroblasts transfected
with DNA from the same neoplasm ( ), SB-1 tumor cells
(.box-solid.), cells modified to secrete I-2 (.DELTA.), cells
modified to secrete IL-2 and to express H-2K.sup.b determinants
(.largecircle.), fibroblasts transfected with DNA from SB 1 cells
(.sup..tangle-solidup.), and fibroblasts transfected with DNA from
EO771 cells (.quadrature.).
[0027] FIG. 8 shows an ex vivo anti-tumor response in three
different human T cell lines.
[0028] FIG. 9 shows survival (Panel A) and mean survival (Panel B)
of mice immunized with an anti-melanoma vaccine.
[0029] FIG. 10 depicts expression of H-2K.sup.b-determinants by LM
fibroblasts transduced with pBR327H-2K.sup.b, a plasmid vector
specifying H-2K.sup.b-determinants.
[0030] FIG. 11 is schematic of the strategy used to enrich
LM-IL-2K.sup.b/KLN cells for cells that induce immunity to KLN205
cells in DBA/2 mice.
[0031] FIG. 12 shows screening of various pools of
LM-IL-2K.sup.b/KLN cells for cells that induce immunity to KLN205
cells to the greatest (Immuno.sup.high) and least (Immuno.sup.low)
extent.
[0032] FIG. 13 shows comparison of The Immunogenic Properties of
Immuno.sup.high Pools of LM-IL-2K.sup.B/KLN cells after one, two or
three rounds of immune selection.
[0033] FIG. 14. Survival of tumor-bearing DBA/2 mice immunized with
cells from the Immuno.sup.high (3.degree.) pool of transfected
cells.
[0034] FIG. 15 shows that mAbs for CD8+ cells inhibit the cytotoxic
activity toward KLN205 cells in tumor-bearing DBA/2 mice immunized
with cells from the Immuno.sup.high (3.degree.) pool (sp
6-10-1).
[0035] FIG. 16 provides the size of DNA transfected into the
modified fibroblasts.
[0036] FIG. 17. C3H/He mice were in injected s.c. three times at
weekly intervals with 5.times.10.sup.6 modified LM fibroblasts
co-transfected with a cDNA expression library from SB5b cells
(LM-IL-2K.sup.b/cCSB5b).
[0037] FIG. 18 The Master Pool of transfected cells (nonselected
LM-IL-2K.sup.b/cSB5b cells) was divided into fifteen subpools. Each
subpool contained 1000 cells as the starting inoculum. The number
of cells in the pools was expanded and a portion was maintained
frozen/viable for later uses. The remaining portions from each
individual pools were used to immunize C3H/He mice. After
immunization, spleen cells were tested by both ELISPOT and
.sup.51Cr-release cytotoxicity assays for reactivity against the
SB5b breast cancer cells.
[0038] FIG. 19 Immunity to SB5b cells in mice immunized with cells
from subpool (SP) 6 exceeded that of mice immunized with cells from
any of the other pools, as determined by both ELISPOT and
.sup.51Cr-release cytotoxicity assays.
[0039] FIG. 20. Representative Elispot assay derived from the
spleen of mice immunized with immuno.sup.high (SP6-6) cells from
the second round of selection and immuno.sup.low (SP10-4) pools in
comparison with cells from the non-selected Master Pool
(LM-IL-2Kb/cSB5b).
[0040] FIG. 21. Immunity to breast cancer in mice immunized with
immuno.sup.high and immuno.sup.low sub-pools of transfected cells.
The mice were injected s.c. three times at weekly intervals with
5.times.10.sup.6 cells. One week afterward, the mice were injected
into the fat pad of the breast with 1.times.10.sup.5 SB5b breast
cancer cells.
[0041] FIG. 22. RT-PCR for MUC-1, a known breast cancer antigen,
was performed on extracts of the immuno.sup.high pool of
transfected cells. The highlighted sequence indicates the portion
of the molecule chosen for amplification. As controls, the same
procedure was followed except that extracts of immuno.sup.low pool
or non-enriched cell suspensions from the Master Pool were
substituted for extracts from the immuno.sup.high pool. As an
additional control, RT-PCR was performed to detect HER-2-neu.
[0042] FIG. 23. 1.times.10.sup.6 transfected fibroblasts from the
immuno.sup.high (SP 6-6) pool, and for comparison non-transfected
modified fibroblasts (LM-IL-2K.sup.b) and cells from the immunology
pool (SP 10-4), together with the SB5b breast cancer cells were
washed with PBS. The cells were then permeabilized with 1 ml of
Cytofix/Cytoperm.TM. (from BD Bioscience, San Diego, Calif.) at
4.degree. C. for 20 min in the dark followed by additional
washings. 1 ug of Muc-1 antibody (2 ul in 0.5 mg/ml) (anti-hamster
Muc-1; CT2) was added to stain each cell type for 30 min at
4.degree. C. After washing, FITC labeled anti-hamster IgG was added
for 30 min at 4.degree. C. After additional washes, the cells were
analyzed by flow cytometry.
[0043] FIG. 24. Comparison of gene expression in immuno.sup.high
and immuno.sup.low pools of transfected cells.
[0044] FIG. 25. Number of genes over-expressed in immuno.sup.high
and immuno.sup.low pools.
[0045] FIG. 26. Frequency of antigen-positive cells in
immuno.sup.high sub pools of transfected cells.
DETAILED DESCRIPTION
[0046] Before the present compounds, products and compositions and
methods are disclosed and described, it is to be understood that
the terminology used herein is for the purpose of describing
particular embodiments only and is not intended to be limiting. It
must be noted that, as used in the specification and the appended
claims, the singular forms "a," "an" and "the" include plural
referents unless the context clearly dictates otherwise.
[0047] As used herein, "administer" includes single or multiple
administrations.
[0048] As used herein, "allogeneic" refers to at least one class I
or class II MHC allele of a first cell coding for an HLA
specificity that is unmatched and immunologically incompatible with
respect to at least one class I or class II MHC allele of a second
cell.
[0049] As used herein, "semi-allogeneic" refers to at least one
class I or class II MHC determinant expressed by a first cell is
syngeneic with respect to a second cell and at least one class I or
class II MHC determinant expressed by the first cell is allogeneic
with respect to the second cell.
[0050] As used herein, "syngeneic" refers to an MHC allele coding
for an HLA specificity of a first cell that matches and is
immunologically compatible with a second cell.
[0051] As used herein, "treat" or "treating" when referring to
protection of an animal from a condition, means preventing,
suppressing, repressing, or eliminating the condition. Preventing
the condition involves administering a composition of the present
invention to an animal prior to onset of the condition Suppressing
the condition involves administering a composition of the present
invention to an animal after induction of the condition but before
its clinical appearance. Repressing the condition involves
administering a composition of the present invention to an animal
after clinical appearance of the condition such that the condition
is reduced or prevented from worsening. Eliminating the condition
involves administering a composition of the present invention to an
animal after clinical appearance of the condition such that the
animal no longer suffers the condition.
[0052] In mice, it was possible to treat breast cancer by
immunization with a vaccine prepared by transfer of sheared total
genomic DNA-fragments from various murine neoplasms, including
adenocarcinoma of the breast, into a highly immunogenic, mouse
fibroblast cell line (de Zoeten E, J Immunol 1999; 162:6934-41; Kim
T S, Cancer Res 1994; 54:2531-5; Sun T, Cancer Gene Therapy 1998;
5:110-8.). Because the transferred DNA was integrated and
replicated as the recipient cells divide, the vaccine could be
prepared from DNA derived from relatively small numbers of tumor
cells Sufficient DNA could be recovered from as few as 10 million
cancer cells. (A tumor of 4 mm contains an equivalent number of
cells.) The vaccine was readily prepared from primary neoplasms.
Furthermore, since the DNA was not fractionated before transfer, it
was likely that multiple mutant/dysregulated genes in the breast
cancer cells specifying an array of unidentifled weakly immunogenic
TAAs were expressed by the transfected cells.
[0053] Several groups reported that immunization of tumor-bearing
mice with the DNA-based vaccine alone was unable to successfully
control the growth of the highly aggressive breast cancer (de
Zoeten E, J Immunol 1999; 162:6934-41; Kim T S, Cancer Res 1994;
54:2531-5; Sun T, Cancer Gene Therapy 1998; 5:110-8.).
[0054] In an attempt to improve the therapeutic outcome for
patients with highly aggressive cancers, compositions and methods
of the instant invention were developed. The compositions include a
combination of a chemotherapeutic agent and immunization with a
DNA-based vaccine such as described in and made by methods
disclosed in U.S. Pat. Nos. 5,759,535 and 6,187,307 and U.S. patent
application Ser. No. 09/522,716; incorporated herein by reference.
The chemotherapeutic agent may be a taxane, camptothecin, vinca
alkaloid, anthracycline, antibiotic, antimetabolite, platinum, or
alkylating agent, and may be selected from the group consisting of
paclitaxel, docetaxel, vincristine, vinblastine, vinorelbine,
irinotecan, topotecan, etoposide, methotrexate, 5-fluorouracil,
cyclophosphamide, ifosphamide, melphalan, chlorambucil, BCNU, CCNU,
decarbazine, procarbazine, busulfan, thiotepa, daunorubicin,
doxorubicin, idarubicin, epirubicin, mitoxantrone, as well as other
chemotherapeutic agents known to one of skill in the art.
[0055] The present invention is also directed to compositions
comprising a chemotherapeutic agent and a vaccine derived from
genomic DNA taken from neoplasms and transfected into immunogenic
fibroblast cells or any other antigen-presenting cells for the
treatment of cancer. The present invention is also directed to
compositions comprising a chemotherapeutic agent and a vaccine
derived from a c-DNA obtained from neoplasms and transfected into
immunogenic fibroblast cells or any other antigen-presenting cells
for the treatment of cancer. According to methods of the invention,
the c-DNA can be obtained by any of the methods known to a person
skilled in the relevant art. These methods include, but not limited
to, Polymerase Chain Reaction (PCR) on RNA templates isolated from
a neoplasm with subsequent subcloning into a suitable vector as
well as PCR reactions on total genomic DNA isolated from a neoplasm
with subsequent subcloning into a suitable vector. Alternatively,
any other techniques for isolating a gene or a portion thereof from
a neoplasm can be used to obtain C-DNA of the invention. In some
instances, the isolated from a neoplasm c-DNA can be cloned into a
suitable vector such as a plasmid, bacteriophage, virus or an
artificial chromosome.
[0056] Antigen-presenting cells may be human fibroblasts derived
from donors who share identity with the cancer patient at one or
more MHC class I alleles. The fibroblasts may be modified to
provide immunologic specificity for cancer antigens expressed by
the patient's own neoplasm.
[0057] The present invention is also directed to methods of
preparation a DNA vaccine for the treatment of cancer. The vaccine
can be prepared from small amounts of tumor tissue. Preferably, the
vaccine is prepared using the patient's tumor tissue.
[0058] The present invention is further directed to immunization of
a patient with a DNA vaccine for the treatment of cancer.
Preferably, the treatment of immunization may occur in combination
with treatment by one or more chemotherapeutic agents.
[0059] The vaccine can be prepared by transfer of sheared genomic
DNA-fragments derived from an aggressive cancer into a highly
immunogenic mouse fibroblast cell line. This unique approach was an
application of classic studies indicating that the introduction of
high molecular weight total genomic DNA from one cell type into
another results in stable integration of the transferred DNA and
alteration of both the genotype and the phenotype of the cells that
incorporate the exogenous DNA (Hsu C, Nature 1984; 312:68-9;
Kavathas P, Proc Natl Acad Sci (USA) 1983; 80:524-8)<
[0060] Because the transferred DNA is integrated and replicated
when the recipient cells divide, the number of vaccine cells can be
expanded as required for multiple rounds of immunization. Thus, the
vaccine could be prepared by transfer of microgram amounts of total
genomic DNA derived from small quantities of tumor tissue.
[0061] Dendritic cells and fibroblasts are efficient antigen
presenting cells (Kundig T M, Science 1995; 268:1343-5; Buenafe A
C, J Neuroimmunol 2001; 112:12106-14; Wassenaar A, Clin Exp Immunol
1997; 110:277-84). However; other antigen presenting cells such as
B cells and macrophages, may also be used in the present
methods.
[0062] The fibroblasts may express MHC class I-determinants and
co-stimulatory molecules required for T cell activation. The use of
a fibroblast cell line enables the cells to be modified in advance
of DNA transfer to augment their immunogenic properties. The
fibroblasts can be modified to secrete a Th-1 cytokine (IL-2) and
to express foreign (allogeneic) MHC-determinants.
[0063] In addition to their important adjuvant properties, the
presence of allogeneic MHC determinants ensures that the vaccine of
the instant invention would be rejected. Thus, possible toxic
effects (a tumor derived from the vaccine itself or the appearance
of an autoimmune disease) are eliminated.
[0064] Human fibroblasts (derived from donors who share identity
with the cancer patient at one or more MHC class I alleles) may be
readily modified to provide immunologic specificity for cancer
antigens expressed by the patient's own neoplasm. The technique
allows the vaccine to be prepared from quite small amounts of tumor
tissue, providing an opportunity to treat patients at an early
stage of the disease. Immunization at an appropriate interval
following chemotherapy may result in an enhanced anti tumor immune
response.
[0065] Using the methods of the instant invention, tumor cells are
obtained from a patient; total or whole genomic DNA from the tumor
cells is then isolated by any of the methods for total genomic DNA
isolation known in the art. The DNA may then be fragmented,
preferably into 25 kb fragments. The vaccine may then be prepared
by transferring genomic DNA-fragments (25 kb) into fibroblast cells
by methods well known in the art, modified to enhance cells'
immunogenic properties.
[0066] While the genomic DNA vaccine is in preparation, the patient
may be undergoing chemotherapy with anti-cancer drugs such as
paclitaxel, docetaxel, vincristine, vinblastine, vinorelbine,
irinotecan, topotecan, etoposide, methotrexate, 5-fluorouracil,
cyclophosphamide, ifosphamide, melphalan, chlorambucil, BCNU, CCNU,
decarbazine, procarbazine, busulfan, thiotepa, daunorubicin,
doxomubicin, idarubicin, epirubicin, or mitoxantrone or any other
chemotherapeutic agent. At about 5 to 20 days after the
chemotherapy is completed and when the patient regains immune
competence, the patient receives an injection of genomic DNA
vaccine. The injection can be repeated preferably as many times as
needed. The patient may then be monitored for developing cellular
immunity against his or her cancer.
[0067] The present invention is also related to the discovery that
immunogenic cells expressing tumor associated antigens may be
expanded and enriched into more or less highly immunogenic
populations. The ability to expand immunogenic cells expressing
tumor associated antigens allows immunogenic cells to be prepared
using tumor DNA from a very small amount of tissue. According to
the methods of the invention, murine immunogenic cells expressing
tumor associated antigens may be enriched for different populations
of immunogenic cells with differences in antigenicity. By enriching
for immunogenic cells expressing tumor associated antigens, a more
effective vaccine may be prepared. In addition, a vaccine may be
prepared comprising immunogenic cells expressing a defined tumor
associated antigen or combinations of defined tumor antigens. The
ability to enrich immunogenic cells also allows for the
identification of particular tumor associated antigens.
[0068] The present invention is also directed to an immunogenic
cell expressing a tumor associated antigen. The immunogenic cell
may be prepared by transfecting a recipient cell with DNA derived
from a tumor cell, preferably the target tumor cell. The transfer
of tumor-derived DNA may alter the phenotype of the recipient cell
by expressing a tumor associated antigen. The tumor associated
antigen may induce an immune response in a patient to a target
tumor cell.
[0069] Tumor cells may be obtained from any source including, but
not limited to, a tumor cell line or from a patient to be treated
with the vaccine. The tumor cells may be selected for their
general, nonspecific, immune-augmenting properties, and the range
of expressed tumor associated antigens that characterize the tumor,
including antigens that may be present on only a small proportion
of the tumor cells.
[0070] The tumor cells may be obtained from tumors during surgery.
Alternatively, the tumor cells may be obtained from a biopsy as
described in Heo et al., Cancer Res. 49: 5167-5175, 1989. The tumor
cells may be obtained from primary tumors or from metastases.
[0071] The cancer cells may be obtained from any cancer type
including, but not limited to, carcinomas, melanoma, lymphoma,
plasmacytoma, sarcoma, glioma, thymoma, leukemias, breast cancer,
prostate cancer, colon cancer, esophageal cancer, brain cancer,
lung cancer, ovarian cancer, cervical cancer, or hepatoma. The
isolated tumor cells may be cultured and propagated using standard
techniques.
[0072] The tumor-derived DNA used to produce the immunogenic cell
may be derived from any form of nucleic acid of the tumor cell
including, but not limited to, total genomic DNA, cDNA and RNA. The
nucleic acid of the tumor cell may be isolated using standard
techniques. For methods of isolating nucleic acid, see Current
Protocols in Molecular Biology, Editors John Wiley & Sons,
2003. The tumor-derived DNA is then preferably mechanically sheared
or cut with an appropriate restriction enzyme to render high
molecular weight DNA fragments of preferably about 20-25 Kb.
[0073] The tumor-derived DNA may be used to transfect the recipient
cell using methods including, but not limited to, lipofection,
calcium phosphate, cationic liposome-mediated transfection,
electroporation, and ballistomagnetic gene delivery as described in
Wittig et al., Hum. Gene Ther. 12:267-278, 2001. Representative
methodologies for transfecting recipient cells are discussed in
Example 4.
[0074] Recipient cells may be co-transfected with sheared genomic
DNA isolated from tumor cells and plasmid DNA with a selectable
marker, such as an antibiotic-resistance marker. The selectable
marker allows for the selection of recipient cells that have taken
up the tumor-derived DNA. Representative examples of plasmids with
selectable markers include, but are not limited to hygromycin B
phosphotransferase, which confers resistance to hygromycin.
[0075] The recipient cell may be any cell that is capable of being
transformed with the tumor-derived DNA and capable of expressing
the genes encoded by the tumor-derived DNA. The recipient cell is
preferably an antigen presenting cell including, but not limited to
fibroblasts, macrophages, dendritic cells, B cells, monocytes,
marginal zone Kupffer cells, microglia, Langerhans' cells,
interdigitating dendritic cells, follicular dendritic cells, B
cells, and T cells. Antigen presenting cells preferably express
MHC, thereby allowing presentation of the tumor associated antigens
for inducing an immune response by binding with a T-cell with the
appropriate receptor. The recipient cell may be selected based on
the expression of defined MHC determinants.
[0076] The recipient cell may be isolated or derived from a patient
to be administered the vaccine. In such a case, the immunogenic
cells may be used to produce an autologous vaccine. The use of an
autologous vaccine may be advantageous to minimize the induction of
an immune response to antigens other than the tumor associated
antigens. The recipient cell may also be isolated or derived from a
source other than the patient to be administered the vaccine. In
such a case, the immunogenic cells may be used to produce a
heterologous vaccine, which may be syngeneic, semi-allogeneic, or
allogeneic. The use of a heterologous vaccine may be useful to
induce a stronger immune response to the tumor associated antigens
by using heterologous antigens as an adjuvant.
[0077] The use of semi-allogenic cells as recipient cells provides
a mechanism through the expression of syngeneic MHC determinants
for the direct presentation of tumor associated antigens to T
cells. The presence of allogeneic MHC determinants provides an
adjuvant stimulus to the immune system and ensures that the
vaccine, like any other foreign tissue graft, will be rejected.
[0078] When the tumor associated antigen is expressed into the
cytoplasm, the recipient cell preferably expresses MHC Class I. MHC
Class I activates cytotoxic T cells and is expressed on most
nucleated cells. The recipient cell may also be fed to a secondary
recipient cell. In such a case, the recipient cell may first be
induced to undergo apoptosis, as described in (Whiteside et al.,
Proc Natl Acad Sci USA. 99:9415-20, 2002). By feeding the recipient
cell to the secondary recipient cell, the tumor associated antigens
expressed by the recipient cell may become localized in acid
vesicles of the secondary recipient cell. In such a case, the
secondary recipient cell preferably expresses MHC Class II. MHC
Class II activates helper T cells and is constitutively expressed
on cells including, but not limited to, B lymphocytes, dendritic
cells and thymic epithelial cells, but expression may be induced in
other cells by using activating factors, such as IFN-.gamma.. The
use of "recipient cell" herein is intended to also encompass
"secondary recipient cell," unless the context dictates
otherwise.
[0079] Representative examples of recipient cells include, but are
not limited to, dendritic cells, fibroblasts, bone marrow cells
(e.g., lymphocytes including B cells), adipocytes, muscle cells and
endothelial cells. Fibroblasts may be isolated from a patient to be
treated with a vaccine. Fibroblasts may also be isolated from
donors including, but not limited to, foreskin of circumcised
neonatals.
[0080] The recipient cell may be modified to secrete
immune-augmenting cytokines or to express co-stimulatory molecules.
The use of recipient cells secreting cytokines or expressing
co-stimulatory molecules may be used to increase the immune
response of a vaccine. Representative examples of such cytokines
include, but are not limited to, interleukin-1, interleukin-2,
interleukin-3, interleukin-4, interleukin-5, interleukin-6,
interleukin-7, interleukin-8, interleukin-9, interleukin-10,
interleukin-11, interleukin-12, interleukin 18, interferon-.alpha.,
interferon-.gamma., tumor necrosis factor, granulocyte macrophage
colony stimulating factor, and granulocyte colony stimulating
factor. The recipient cell may be modified to express a desired
cytokine using standard methods including, but not limited to,
those described in U.S. Pat. No. 6,187,307, the contents of which
are incorporated herein by reference.
[0081] The present invention is also related to methods of
screening for immunogenic cells capable of inducing an immune
response to a target cell in a patient. The immunogenic recipient
cells may induce an immune response to different tumor associated
antigens. As a result, screening for certain immunogenic recipient
cells may allow the production of a vaccine capable of inducing a
more robust or effective immune response against the target
cell.
[0082] Immunogenic recipient cells may be screened for the ability
to induce an immune response to tumor cells, preferably the tumor
cells that provided the tumor-derived DNA. The immunogenic cells
may be incubated with T-cells from a patient, or T-cells
representative of a patient. The stimulated T-cells may then be
incubated with a target antigen or target cell expressing a tumor
associated antigen. T-cells capable of responding to a particular
target antigen or target cell expressing a tumor associated antigen
may be identified by methods including, but not limited to,
.sup.S1Cr release assay or by ELISPOT, as described in Asai et al.,
Clin. Diagn. Lab. Immunol. 7: 145-154, 2000. Screening using
ELISPOT also allows for distinction of allo--from tumor-specific
responses by blocking with MHC class I or class II antibodies.
[0083] Immunogenic recipient cells may also be screened by the
ability to inhibit tumor formation. The immunogenic cells may be
administered to a test animal that has also been administered the
same tumor cells that provided the tumor DNA. After a sufficient
period of time for tumors to develop in control animals that do not
receive immunogenic recipient cells, the test animals are checked
for tumor formation, and the size of any tumors measured.
Representative examples of screening for immunogenic cells by the
ability to inhibit tumor formation are shown in Example 10 and
Example 12.
[0084] The present invention is also related to the enrichment of
immunogenic cells expressing a tumor associated antigen capable of
inducing an immune response. Not every transfected recipient cell
will be immunogenic. For example, not every recipient cell will be
transfected with DNA encoding a tumor associated antigen.
Alternatively, the tumor associated antigen may not be sufficiently
presented by the recipient cell to induce an immune response, or T
cells may be tolerant to a particular combination of MHC and bound
tumor associated peptide. By enriching for immunogenic cells
expressing a tumor associated antigen capable of inducing an immune
response, the immunogenic cells may be used to produce a more
effective vaccine. Enrichment of immunogenic cells also allows the
production of a vaccine using small amounts of starting material,
because the recipient cells may replicate the transferred DNA
encoding tumor associated antigens as they divide.
[0085] According to the present invention, populations of
immunogenic cells prepared by the methods of the present invention
may be enriched by providing a composition comprising a plurality
of immunogenic recipient cells. The immunogenic recipient cells may
be diluted into growth medium and allowed to expand. The
immunogenic cells may then be, in any order, divided into pools and
screened for the ability to induce an immune response. The steps of
dilution and expansion may be repeated as often as desired in order
to obtain a desired number of immunogenic recipient cells or to
obtain a desired clonal population of immunogenic recipient cells.
The present invention is also related to an enriched population of
immunogenic recipient cells. The enriched population of immunogenic
recipient cells may be polyclonal or monoclonal.
[0086] The present invention is also related to the identification
of tumor associated antigens. Immunogenic recipient cells capable
of inducing an immune response may be analyzed to determine the
tumor associated antigen giving rise to the immune response The
tumor associated antigen may be determined by comparing the genomic
DNA, mRNA or expressed proteins of the immunogenic recipient cell
to an untransfected recipient cell. The additional genomic DNA,
mRNA or expressed protein of the immunogenic recipient cell will
correlate to the tumor associated antigen. Differences in mRNA
levels may be determined by methods including, but not limited, the
use of Affymetrix Genome GeneChips as described in U.S. Pat. No.
6,344,316, the contents of which are incorporated by references
Differences in protein levels may be determined according to
methods of proteomics known in the art.
[0087] The present invention is also related to a vaccine
comprising enriched immunogenic recipient cells. The vaccine may be
more effective at inducing an immune response to a target tumor
cell than previously used vaccines. The immunogenic recipient cells
of the vaccine many polyclonal or monoclonal. A monoclonal vaccine
may be used to induce an immune response to a single tumor
associated antigen. A polyclonal vaccine may be used to induce an
immune response to multiple epitopes of one or more tumor
associated antigens. The polyclonal vaccine may be prepared by
pooling multiple clonal or polyclonal populations of enriched
recipient cells.
[0088] The vaccine may comprise a therapeutically acceptable
carrier. As used herein, a therapeutically acceptable carrier
includes any and all solvents, including water, dispersion media,
culture from cell media, isotonic agents and the like that aye
non-toxic to the host. Preferably, it is an aqueous isotonic
buffered solution. The use of such media and agents in therapeutic
compositions is well known in the art. Except insofar as any
conventional media or agent is incompatible with the immunogenic
recipient cells, the use of such conventional media or agent in the
vaccine is contemplated. Supplementary active ingredients can also
be incorporated into the vaccine.
[0089] The vaccine may be administered to an animal in need
thereof. The vaccine may be administered for inducing an immune
response in an animal in need of such response The animal may be
administered an immunologically effective amount of immunogenic
recipient cells. The precise amount of "an immunologically
effective amount" of immunogenic recipient cells may be determined
by a physician with consideration of individual differences in age,
weight, tumor size, extent of infection or metastasis, and
condition of the animal.
[0090] The vaccine may be administered to treat a cancer in an
animal. The cancers which may be treated by the vaccine include,
but are not limited to, melanoma, lymphoma, plasmocytoma, sarcoma,
glioma, thymoma, leukemias, breast cancer, prostate cancer, colon
cancer, esophageal cancer, brain cancer, lung cancer, ovary cancer,
cervical cancer, hepatoma, and other neoplasms known in the art,
such as those described by Shawler et al. (1997).
[0091] The immune response induced in the animal by administering
the vaccine may include cellular immune responses mediated
primarily by cytotoxic T cells, capable of killing tumor cells, as
well as humoral immune responses mediated primarily by helper T
cells, capable of activating B cells thus leading to antibody
production. A variety of techniques may be used for analyzing the
type of immune responses induced by the immunogenic recipient
cells, which are well described in the art; e.g., Coligan et al.
Current Protocols in Immunology, John Wiley & Sons Inc.
(1994).
[0092] The vaccine may be administered at a dosage of from about
1.times.10.sup.3 to about 5.times.10.sup.9 cells per
administration. The vaccine may be administered to an animal in any
convenient manner including, but not limited to, aerosol
inhalation, injection, ingestion, transfusion, implantation or
transplantation. Preferably, the vaccine is administered by
subcutaneous (s.c.), intraperitoneal (i.p.), intra-arterial (i.a.),
or intravenous (i.v.) injection.
[0093] Throughout this application, where publications are
referenced, the disclosures of these publications in their
entireties are hereby incorporated by reference into this
application in order to more fully describe the state of the art to
which this invention pertains.
[0094] When evaluating results of the treatment, the following
statistical analysis may be used. Kaplan-Meier log rank analyses
may be used and was used in the examples of this application to
determine the statistical differences between the survival of mice
in the various experimental and control groups. A p value less than
0.05 is considered significant. Student t test one-way Anova may be
used and was used in the examples of this application to determine
statistical difference between experimental and control groups in
the in vitro experiments.
[0095] The present invention has multiple aspects, illustrated by
the following non-limiting examples.
Example 1
Cytokine-Secretion by LM Mouse Fibroblasts
[0096] Among other advantages, the use of a fibroblast cell line as
the recipient of genomic DNA from the breast cancer cells enables
the recipient cells to be modified in advance of DNA-transfer to
augment their nonspecific immunogenic properties. In this instance,
the fibroblasts were modified to secrete IL-2 and to express
allogeneic MHC class I-determinants. IL-2 is a growth and
maturation factor for CTLs.
[0097] A replication-defective retroviral vector (pZipNeoSVIL-2)
was used to modify the fibroblasts used as DNA-recipients to
secrete IL-2 (pZipNeoSV-IL-2; from M.K.L. Collins, University
College, London, England) specifying human IL-2 was used for this
purpose. The IL-2-specifying vector was packaged in GP-1-env AM 12
cells (from A. Bank, Columbia University, New York, N.Y.). The
vector also included a neo.sup.r gene under control of the Moloney
leukemia virus long terminal repeat. The neo.sup.r gene conferred
resistance to the aminoglycoside antibiotic neomycin-derivative,
G418 (Gibco BRL), used for selection.
[0098] Virus-containing supernatants of GP-env AM12 cells
transduced with pZipNeoSV-IL-2 were added to the fibroblasts,
followed by overnight incubation at 37.degree. in growth medium
containing polybrene (Sigma; 5 mg/ml, final concentration). The
cells were maintained for 14 days in growth medium containing 400
.mu.g/ml G418 (Gibco BRL). One hundred percent of non-transduced
cells died in medium supplemented with G418 during this period.
Colonies of cells proliferating in the G418-containing growth
medium were pooled and maintained as modified cell lines for later
use in the experiments. An ELISA (BioSource, Camarillo, Calif.) was
used to determine the quantity of IL-2 secreted by the transduced
fibroblasts (LM-IL-2 cells) which indicated that 106
retrovirally-transduced cells formed 196 pg IL-2/ml/48 hrs.
[0099] The culture supernatants of LM fibroblasts transduced with
the IL-2 negative vector pZipNeoSV(X), like that of non-transduced
LM cells, failed to form detectable quantities of IL-2. Every third
passage, the transduced cells were placed in medium containing 600
.mu.g/ml G418. Under these circumstances, equivalent quantities of
IL-2 were detected in the culture supernatants of cells transduced
with pZipNeoSVIL-2 for more than six months of continuous culture.
The generation time of transduced and non-transduced fibroblasts,
approximately 24 hrs in each instance, were equivalent. The
introduction of DNA from the breast cancer cells into the
IL-2-secreting cells did not affect the quantity of IL-2-secreted
(these data are not presented).
Example 2
Modification of the Cytokine-Secreting Fibroblasts to Express
H-2K.sup.b-Class I-Determinants
[0100] Allogeneic class I-determinants are strong immune adjuvants
(Conte P F, Cancer 2004; 101:704-12; Hammerling G J, J Immunogen
1986; 13:15-157; Hui K M, J Immunol 1989; 143:3835-43;
Ostrand-Rosenberg S, J Immunol 1990; 144:4068-71). To further
augment their immunogenic properties, the fibroblasts were modified
to express MHC H-2K.sup.b-determinants, allogeneic in C3H/He mice.
A plasmid (pBR327H-2K.sup.b) (Biogen Research Corp., Cambridge,
Mass.) encoding H-2K.sup.b-determinants was used. Ten g of
pBR327H-2K.sup.b and 1 g of pBabePuro (from M. K. L. Collins), a
plasmid specifying a gene that confers resistance to puromycin,
were mixed with Lipofectin (Gibco BRL), and added to
1.times.10.sup.6 cytokine-secreting fibroblasts in 10 ml of DMEM,
without FBS. (A 10:1 ratio of tumor-DNA to plasmid DNA was used to
increase the likelihood that cells converted to
puromycin-resistance took up tumor-DNA as well.)
[0101] The IL-2-secreting fibroblasts were incubated under standard
cell culture conditions for 18 hr at 370 in growth medium. After
incubation, the cell cultures were divided and replated in complete
growth medium supplemented with 3.0 .mu.g/ml puromycin (Sigma; St.
Louis, Mo.), followed by incubation at 370 for 7 additional days.
The surviving colonies were pooled and maintained as a cell line
for later use (LM-IL-2K.sup.b cells). One hundred percent of
non-transduced cells maintained in growth medium containing
equivalent amounts of puromycin died during the seven-day period of
incubation.
[0102] Quantitative immunofluorescence staining with FITC-labeled
mAbs for mouse H-2K.sup.b determinants was used to measure
expression of the class I-determinants. The following protocol was
followed for the staining. The measurements were performed in an
Epic V flow cytofluorograph (Coulter Electronics, Hialeah, Fla.)
equipped with a multiparameter data-acquisition and display system
(MDADS). For the analysis, 0.1 mM EDTA in PBS was used to
disassociate the monolayer cultures from plastic cell culture
flasks. The cell-suspensions were washed with PBS containing 0.2%
sodium azide and 0.5% FBS. Afterward, FITC-conjugated H-2K.sup.k,
H-2K.sup.b, I-Ak, B7.1, B7.2 or ICAM-1 mAbs (Pharmingen, San Diego,
Calif.) were added to the cell-cultures, followed by incubation at
4.degree. for 1 hr. The cells were then washed with PBS containing
0.5% FBS and 0.2% sodium azide. Background staining was determined
by substituting FITC-conjugated IgG2a isotype serum (DAKO,
Carpenteia, Calif.) for the specific mAbs. One-parameter
fluorescence histograms were generated by analyzing at least
1.times.10.sup.4 cells in each instance.
[0103] As a control, aliquots of the puromycin-resistant cell
suspension were incubated with FITC-conjugated IgG2a isotype serum.
As an additional control, spleen cells from C57BL/6 mice (H-2 b)
were substituted for the transduced fibroblasts. After incubation,
the cells were washed and analyzed for fluorescent staining by flow
cytofluorometry. The dark-shaded area indicates transduced cells
stained with PE-conjugated anti-H-2K.sup.b mAbs. The light line
indicates transduced cells incubated with PE-conjugated isotype
serum. The dark line indicates spleen cells from C57BL/6 mice (H-2
b).
[0104] The results of procedure (FIG. 2) indicated that more than
99 percent of the transduced fibroblasts stained positively (mean
fluorescence index (MFI) at least ten fold greater than cells
stained with FITC-conjugated isotype serum, taken as background).
Under similar conditions, non-transduced fibroblasts or fibroblasts
incubated with FITC-conjugated isotype serum failed to stain. The
expression of H-2K.sup.b-determinants by the transduced cells was a
stable property. The staining intensity was essentially unchanged
after three months of continuous culture.
[0105] An analogous procedure was used to further characterize the
cells used as DNA-recipients. The modified fibroblasts were stained
with FITC-labeled mAbs for H-2Kk class I-determinants, FITC-labeled
I-Ak or with FITC-labeled mAbs for the co-stimulatory/cell adhesion
molecules B7.1, B7.2 and ICAM-1. The results indicated that 99
percent of the fibroblasts, derived from C3H/He mice, expressed
H-2Kk and B7.1 determinants constitutively (mean fluorescence index
five fold greater than the control (substitution of FITC-labeled
isotype serum for the mAbs, taken as background). Cells incubated
with FITC-labeled ICAM-1, B7.2 or I-Ak mAbs failed to stain above
background (these data are not presented.) The expression of MHC
class I-determinants and the co-stimulatory molecule by LM cells
was consistent with various reports indicating that fibroblasts,
like dendritic cells, are efficient antigen presenting cells Kundig
T M, Science 1995; 268, 1343-5; Buenafe A C, J Neuroimmunol 2001;
112:12106-14; Wassenaar A, Clin Exp Immunol 1997; 110:277-84;
Chesney J, Proc Natl Acad Sci (USA) 1997; 94:6307-12).
Example 3
Paclitaxel Inhibited the Growth of Breast Cancer Cells in C3H/He
Mice
[0106] Paclitaxel is a potent inhibitor of cell division (Ross J L,
Proc Natl Acad Sci (USA) 2004; 101:12910-5; Nettles J H, Science
2004; 305:866-9; Gaitanos T N, Cancer Res 2004; 64:5063-7). It
blocks cells in the G2/M phase of replication through its effect on
the formation and function of microtubules within the cell. To
determine if paclitaxel affected the growth of the breast cancer
cells used in the experiments described here, naive C3H/He mice
were injected into the fat pad of the breast with 1.times.10.sup.5
of the malignant cells (SB5b cells). Six days after injection of
the cancer cells, the mice received a single i.p. injection of
varying amounts of paclitaxel (range=05 to 2.25 mg/kg). The effect
of paclitaxel on the growth of SB5b cells was determined by
measurements of tumor volume at varying times afterward (FIG.
1).
[0107] Mean tumor volumes were determined by the equation 0.5
l.times.w.sup.2 where l=length and w=width. The dimensions of the
tumor were obtained with a dial caliper. There were three mice in
each group.
[0108] The results (FIG. 1) indicated that tumor growth occurred at
the injection site in each instance, including that of mice treated
with the highest dose of paclitaxel tested.
[0109] Paclitaxel is highly toxic. Since mounting an effective
immune response requires robust cell proliferation following
antigen administration, peripheral white blood counts were measured
at varying times after an injection of paclitaxel. The objective
was to administer the vaccine when the white blood count returned
at least to its pre injection value. The results indicated that six
days after a single injection of 2.25 mg/kg paclitaxel, the white
blood count had returned to pre-injection levels, consistent with a
full recovery from the toxic effects of the drug (these data are
not presented).
Example 4
Isolation of Total Genomic DNA from Cancer Cells and Preparation of
Vaccine
[0110] Eight to 10 week old pathogen-free C3H/HeJ female mice and
DBA/2 female mice (H-.sup.2d) were from the Jackson Laboratory (Bar
Harbor, Me.). The animals, between 10 to 14 weeks old when used in
the experiments, were maintained according to NIH Guidelines for
the Care and Use of Laboratory Animals. SB5b cells were a
short-term passage adenocarcinoma of the breast cell line derived
from a breast neoplasm that arose spontaneously in a C3H/He mouse
in our animal colony. B16 cells, a melanoma cell line of C57BL/6
origin, were obtained originally from I. Fidler (M.D. Anderson,
Houston, Tex.) The cells were maintained by serial passage in
histocompatible C3H/HeJ or C57BL6J mice respectively, or at
37.degree. in a humidified 7% CO.sub.2/air atmosphere in Dulbecco's
modified Eagle's medium (DMEM) (Gibco BRL, Grand Island, N.Y.)
supplemented with 10% heat inactivated fetal bovine serum (PBS)
(Sigma, St. Louis, Mo.) and antibiotics (Gibco BRL) (growth
medium).
[0111] KLN205 cells, a squamous carcinoma cell line derived from a
lung neoplasm that arose spontaneously in a DBA/2 mouse, were from
the American Type Culture Collection (ATCC), LM cells, a fibroblast
cell line of C3H/He mouse origin, were also from the ATCC. KLN205
cells were maintained by serial passage in histocompatible DBA/2
mice, or at 37.degree. in a humidified 7% CO2/air atmosphere in
DMEM (Gibco BRL, Grand Island, N.Y.) supplemented with 10% heat
inactivated fetal bovine serum (FBS) (Sigma, St. Louis, Mo.) and
antibiotics (Gibco BRL) (growth medium). LM Cells were maintained
in growth medium under the same conditions. mAbs for CD8+, CD4+ and
NK1.1 determinants were from Pharmingen, (San Diego, Calif.). Low
tox rabbit complement (C) was from Pel Freeze, (Rogers, Ark.).
[0112] A DNeasy isolation kit (Qiagen, Valencia, Calif.) was used
to obtain genomic DNA from the breast cancer cells or KLN205 cells,
according to the manufacturer's instructions. In brief,
1.times.10.sup.7 actively proliferating plastic adherent breast
cancer cells from in vitro culture were disassociated from the
plastic by treatment with EDTA (10.sup.-4 M). The cell suspension
was centrifuged at 300.times.g for 5 min. Afterward, the cell
pellet was re suspended in 200 .mu.l PBS followed by the addition
of 400 .mu.g RNase A and incubation at RT for 2 min. After
incubation, 12 mAU proteinase K and 2001 lysis buffer containing
guanidine HCl were added, followed by further incubation at
70.degree. for 10 min. Afterward, 200 .mu.l of 100% ethanol was
added. The extracted DNA was loaded onto the DNeasy spin column,
and eluted after two washes with buffer. The A.sub.260/A.sub.280
ratio of the isolated DNA was greater than 1.8 in each instance.
The molecular size of the extracted DNA was approximately 25 kb, as
determined by agarose gel electrophoresis. The same procedure was
followed to isolate DNA from B16 cells, a melanoma cell line.
[0113] The vaccine was prepared by transfer of sheared,
unfractionated total genomic DNA-fragments from SB5b breast cancer
cells or KLN205 cells into LM fibroblasts, which had been modified
to secrete IL-2 and to express H-2K.sup.b-determinants. The method
described by Wigler et al. (Wigler M, Proc Natl Acad Sci (USA)
1979; 76:1373-6) was used, as modified. In brief, 50 .mu.g of
sheared (25 kb) genomic DNA derived from approximately 1.times.107
breast cancer cells was mixed with 5 .mu.g pHyg (from L. Lau,
University of Illinois at Chicago), a plasmid that encoded the E.
Coli enzyme hygromycin B phosphotransferase gene, conferring
resistance to hygromycin B, used for selection. The sheared DNA and
pHyg were mixed with Lipofectin, according to the manufacturer's
instructions (Gibco BRL) and added to 1.times.10.sup.7 modified
fibroblasts divided 24 hrs previously into ten 100 mm plastic cell
culture plates. Eighteen his after the addition of the
DNA/Lipofectin mixture to the cells, the growth medium was replaced
with fresh growth medium containing sufficient quantities of
hygromycin (500 .mu.g/ml; Boehringer Mannheim, Indianapolis, Ind.)
to kill 100 percent of the non-transfected cells. For use as a
control, 5 .mu.g of pHyg alone mixed with Lipofectin was added to
an equivalent number of the modified fibroblasts. For use as a
control, the same procedure was followed except that DNA from B16
melanoma cells was substituted for DNA from SB5b cells. In each
instance, the cells were maintained for 14 days in growth medium
containing 500 .mu.g/ml hygromycin B. None of the non-transfected
cells maintained in the selection medium were viable by the end of
this period. The remaining colonies (at least 2.times.10.sup.4)
were pooled and maintained as cell lines for use in the experiments
(LM-IL-2Kb/SB5b cells and LM-IL-2Kb/B16 cells respectively).
[0114] Alternatively to lipofection, genomic DNA can be delivered
into recipient cells by other methods such as calcium phosphate
precipitation or ballistomagnetic gene delivery. If phosphate
precipitation was used, then tumor-derived DNA (10-100 .mu.g) was
diluted with water, mixed with 2M CaCl.sub.2 and 2.times. Hanks'
balanced salt solution by bubbling. The DNA solution was then added
to semiconfluent monolayers of recipient cells in transfection
medium (DMEM plus 10% FCS containing 25 .mu.M chloroquine; Sigma).
The cells were then incubated at 37.degree. C. in a humidified
atmosphere of 5% CO.sub.2 in air for 10 h before selection.
[0115] When ballistomagnetic gene delivery was used, gold particles
(0.8-16 .mu.m; ABCR, Karlsruhe, Germany) were coated with
tumor-derived DNA and superparamagnetic beads (65 nm; Miltenyi
Biotec, Auburn, Calif.) at the ratio of 1:3. The particles were
then propelled at 1,550 psi into recipient cells in 30-mm.sup.2
dishes using modified ballistic system (PDS-1000/He; Bio-Rad).
Immediately after DNA transfer, fresh medium was added and the
cells were incubated for 24 or 48 h before microscopic
examination.
Example 5
Immunity to Breast Cancer in Mice Immunized with
LM-IL-2K.sup.b/SB5b Cells
[0116] C3H/He mice are highly susceptible to the growth of SB5b
cells. The survival time of untreated mice injected into the fat
pad of the breast with as few as 1.times.10.sup.5 SB5b cells is
20-30 days.
[0117] To determine if the vaccine induced immunity to breast
cancer in tumor-free C3H/He mice, (inhibition of tumor growth and
survival), naive mice received a single s.c. injection of
5.times.10.sup.6 LM-IL-2Kb/SB5b cells Fourteen days later, the mice
were injected into the fat pad of the breast with 1.times.10.sup.5
SB5b cells. As a control, naive C3H/He mice were injected into the
fat pad of the breast with an equivalent number of SB5b cells. To
determine if paclitaxel augmented the vaccine's therapeutic effect,
the same protocol was followed except that the mice were injected
with the drug two days before the injection of the vaccine.
[0118] In FIG. 3(A), C3H/He mice (10 mice per group) received a
single i.p. injection of (2.25 mg/kg) paclitaxel. Two days later,
the mice were injected sock with 5.times.10.sup.6
LM-IL-2K.sup.b/SB5b cells (vaccine). Fourteen days afterward, the
mice were injected into the fat pad of the breast with
1.times.10.sup.5 SB5b cells. As controls, the mice were injected
according to the same schedule with an equivalent number of SB5b
tumor cells alone, with paclitaxel and SB5b cells, or with
LM-IL-2K.sup.b/SB5b cells and SB5b cells. The experiment was
terminated at day 63. Mean survival time.+-.SE: injected with SB5b
cells alone 20.+-.1 days; injected with paclitaxel and SB5b cells,
22.+-.1 days, injected with LM-IL2K.sup.b/SB5b cells 14 days before
the injection of SB5b cells, 30.+-.3 days, injected with paclitaxel
two days before the injection of LM-IL-2K.sup.b/SB5b cells,
followed by the injection of SB5b cells 14 days later, 35.+-.3
days* p<0.001 for the difference in survival of mice injected
with paclitaxel and LM-IL-2K.sup.b/SB5b cells or
LM-IL-2K.sup.b/SB5b cells alone followed by SB5b cells, and mice
injected with SB5b cells alone, or mice injected with paclitaxel
alone followed by SB5b cells.
[0119] In FIG. 3(B), C3H/He mice were injected i.p. with 2.25 mg/kg
paclitaxel. Two days afterward, the mice were injected s.c. with
5.times.10.sup.6 LM-IL-2K.sup.b/SB5b cells. Fourteen days later,
the mice were injected into fat pad of the breast with
1.times.10.sup.5 SB5b cells. As controls, the mice were injected
according to the same schedule with paclitaxel followed by SB5b
cells, with the vaccine alone followed by SB5b cells or with SB5b
cells alone. The experiment was terminated at day 38. Tumor volumes
were determined by 0.5 l.times.w.sup.2. Length and width were
obtained with the aid of a dial caliper.
[0120] The results (FIG. 3A) indicated that one hundred percent of
the animals in the control groups injected with SB5b cells alone or
with SB5b cells and paciltaxel alone died within 27 days. In
contrast, mice injected with the vaccine, followed by the injection
of SB5b cells survived for significantly longer periods than naive
mice in the control groups (p<0.001).
[0121] Two of 10 mice immunized with LM-IL-2Kb/SB5b cells, followed
by the challenging injection of SB5b cells, appeared to have
rejected the breast cancer cells. They survived indefinitely (more
than 62 days). Paclitaxel had no significant effect. The survival
of mice injected with paclitaxel, followed by immunization with
LM-IL-2Kb/SB5b cells before the injection of SB5b cells, was
essentially the same as that of mice injected with the vaccine
alone (FIG. 3A)<
[0122] Measurements of tumor growth in the preimmunized mice
injected with the breast cancer cells were consistent with the
vaccine's immunoprotective properties (FIG. 3B). Tumor growth was
inhibited both in mice immunized with the vaccine and in mice
injected with paclitaxel and the vaccine before the injection of
the breast cancer cells.
[0123] To further investigate the vaccine's immunogenic properties,
spleen cells from C3H/HeJ mice immunized with LM-IL-2Kb/SB5b cells
in vitro and were then tested in .sup.51Cr-release cytotoxicity
assays. For this test, spleen cell suspensions were obtained from
mice that had received a single subcutaneous injection of
5.times.106 LM-IL-2Kb/SB5b cells 14 days previously. After washing,
the cells were co-incubated under standard cell culture conditions
for 5 days with (mitomycin C-treated) SB5b cells. The incubation
medium consisted of RPMI-1640 medium (Gibco BRL) supplemented with
100 U/ml human IL-2, 10% FBS, 5.times.10.sup.-2 mmol
2-mercaptoethanol, 15 mmol HEPES, 0.5 mmol sodium pyruvate and
penicillin/streptomycin (Gibco). The ratio of spleen cells to
mitomycin-C-treated breast cancer cells during the co-incubation
was 30:1. At the end of the 5 day incubation period, the population
that failed to adhere to the plastic cell culture flasks was
collected and used as the source of effector cells for the
cytotoxicity determinations.
[0124] For the cytotoxicity assay, 5.times.10.sup.6 SB5b cells were
labeled with .sup.51Cr during a 1 hr incubation period at
37.degree. in growth medium containing 100 .mu.Ci Na.sup.2
51Cr.sub.04 (Amersham, Arlington Heights, Ill.). After three washes
with DMEM, 1.times.10.sup.4 51Cr-labeled cells were incubated for 4
hrs at 370 with the effector cell-population. The quantity of
isotope released was measured in a gamma counter (Beckman, Palo
Alto, Calif.). The percent specific cytolysis was calculated as:
Experimental .sup.51Cr release minus Spontaneous .sup.51Cr release
divided by Maximum .sup.51Cr release minus Spontaneous .sup.51Cr
release and multiplied by one hundred The spontaneous release of
.sup.51Cr was less than 15% of the total release in each
instance.
[0125] In FIG. 4(A), the same protocol as described in the legend
to FIG. 3A was followed. Spleen cells from mice injected with
paclitaxel and LM-IL-2K.sup.b/SB5b cells followed by the injection
of SB5b cells were co-incubated for 5 days with mitomycin C-treated
SB5b cells (spleen cell: breast cancer ratio=30:1). At the end of
the incubation, .sup.51Cr-labeled SB5b cells were added and the
specific cytotoxic activity was determined at varying E:T ratios in
a standard 4 hr .sup.51Cr-release assay.
[0126] *p<0.0005 for the specific release of the isotope from
SB5b cells co-incubated with spleen cells from mice injected with
paclitaxel, followed by LM-IL-2K.sup.b/SB5b cells and SB5b cells
[FIG. 4A-column d] relative to the release of isotope from SB5b
cells co-incubated with spleen cells from mice injected with
paclitaxel and SB5b cells alone [FIG. 4A-column b] or with SB5b
cells alone [FIG. 4A-column a]. **p<0.005 for the specific
release of isotope from SB5b cells co-incubated with spleen cells
from mice injected with LM-IL-2K.sup.b/SB5b cells and SB5b cells
[FIG. 4A-column c], relative to the release of isotope from SB5b
cells co-incubated with spleen cells from untreated mice injected
with SB5b cells alone [FIG. 4A-column a] or with spleen cells from
mice injected with paclitaxel and SB5b cells alone [FIG. 4A-column
b]. Difference in the specific release of isotope from SB5b cells
co-incubated with spleen cells from mice injected with paclitaxel
and LM-IL-2Kb/SB5b cells and SB5b cells [FIG. 4A-column d],
relative to the release of isotope from SB5b cells co-incubated
with spleen cells from mice injected with LM-IL-2K.sup.b/SB5b cells
[FIG. 4A-column c], not significant.
[0127] In FIG. 4(B), C3H/He mice received single i.p. injection of
(2.25 mg/kg) paclitaxel. Two days later, the mice were injected sac
with 5.times.10.sup.6 LM-IL-2K.sup.b/SB5b cells. Fourteen days
afterward, the mice were injected into the fat pad of the breast
with 1.times.10.sup.5 SB5b cells. Twelve days later, spleen cells
from the mice were co-incubated for 18 hr with SB5b cells (E:T
ratio=10:1) before they were analyzed in an ELISPOT-IFN-.gamma.
assay [FIG. 4B-column d]. As controls, spleen cells from untreated
mice injected with SB5b cells alone [FIG. 4B-column a] or mice
injected with paclitaxel followed by SB5b cells co-incubated with
SB5b cells [FIG. 4B-column b] were substituted for spleen cells
from mice injected with paclitaxel and LM-IL-2K.sup.b/SB5b cells
[FIG. 4B-column d]. As an additional control, spleen cells from the
immunized or non-immunized mice were incubated in medium alone
(control groups). *p<0.001 for the difference in number of spots
from mice injected with paclitaxel and LM-IL-2K.sup.b/SB5b cells
[FIG. 4B-column d] and mice in the control groups [FIG. 4B-column
a] and [FIG. 4B-column b] **p<0.001 for the difference in number
of spots in the group of mice injected with LM-IL-2K.sup.b/SB5b
cells followed by the injection of SB5b cells [FIG. 4B-column c]
and mice in the control groups [FIG. 4B-columns a] and [b]. Other
differences, not significant.
[0128] In FIG. 4(C), C3H/He mice (2/group) were injected i.p. with
2.25 mg/kg paclitaxel. Two days later, the mice were injected sac,
with 5.times.10.sup.6 LM-IL-2K.sup.b/SB5b cells. Sixty-three days
afterward, spleen cells from the immunized mice were co-incubated
for 5 days with (mitomycin-C-treated) SB5b cells, mAbs for CD8+ or
CD4+ cells and low tox rabbit complement (Pel Freeze, Rogers, Ark.)
were added to the pooled spleen cell suspensions 1 hr before the
cytotoxic activities toward .sup.51Cr-labeled SB5b cells were
determined (E:T=100:1) (FIG. 4C-column c). As controls, the same
protocol was followed except that the mice were injected with the
vaccine alone [FIG. 4B-column b] or the mice were not injected
(FIG. 4B-column a). Values represent means.+-.SD of triplicate
determinations. *p<0.05 for the difference between the specific
release of isotope in the groups treated with CD8+ mAbs and C and
groups treated with CD4+ mAbs and C.
[0129] The results (FIG. 4A) indicated that the specific release of
isotope from the labeled breast cancer cells was significantly
increased, relative to that of the control, untreated group
(p<0.005), Analogous results were obtained if the spleen cells
were tested in ELISPOT IFN-.gamma. assays (FIG. 4B). The number of
spots developing in spleen cell cultures from immunized mice
co-incubated with SB5b cells was significantly higher than that of
spleen cell cultures from control non-immunized mice (p<0.001).
An injection of paclitaxel before immunization had no significant
effect upon the cytotoxicity assay or the number of spots
developing in spleen cell cultures from mice injected with the
vaccine. Antibody inhibition studies indicated that prior treatment
of the spleen cell suspensions with CD8+ mAbs and C but not CD4+
mAbs and C significantly (p<0.005) inhibited the anti breast
cancer cytotoxicity responses (FIG. 4C). Co-administration of
paclitaxel had no significant effect upon either the cytotoxicity
response or the number of IFN-.gamma. spots in the ELISPOT
assays.
[0130] The ELISPOT assays were performed in the following way
Responder (R) T cells from the spleens of C3H/HeJ mice immunized
with the transfected cells were added into individual wells
(1.times.10.sup.6 cells per well in 0.2 ml growth medium) of
96-well ELISPOT IFN-.gamma. plates (B-D Pharmingen, ELISPOT Mouse
IFN-gamma Set (Cat #551083)) coated with 100 .mu.l of the capture
Ab (5 g/ml in PBS). Stimulator (S) SB5b breast cancer cells were
then added at an R:S ratio of 10:1. After incubation for 18 hr at
370, the cells were removed by washing with PBS-Tween (0.05%).
Detection antibodies (2 .mu.g/ml) were then added to each well. The
plates were incubated for 2 hrs at RT and the washing steps were
repeated.
[0131] Afterward, streptavidin-peroxidase (Streptavidin-HRP, 5
g/ml) was added to the individual wells and the plates were washed
four times with PBS-Tween and twice with PBS. One hundred .mu.l of
aminoethylcarbazole staining solution was added to each well to
develop the spots. The reaction was stopped after 4-6 min with
deionized water. The spots were counted by computer-assisted image
analysis (ImmunoSpot Series 2 analyzer: Cellular Technology
Limited, Cleveland, Ohio).
[0132] FIG. 7 provides survival statistics for C3H mice injected
with SB-1 breast cancer cells. These data are in agreement with
data of FIG. 3 discussed above.
[0133] Taken together the data presented in this Example show that
immunity to the breast cancer was generated in C3H/HeJ mice
immunized with a vaccine prepared by transfection of modified
fibroblasts with genomic DNA-fragments from the breast cancer cells
Our prior experience (Sun T, Cancer Gene Therapy 1998; 5.110-8;
deZoeten E, Gene Therapy 2002; 9:1163-72; de Zoeten E F, J Immunol
1998; 160:2915-22; Cohen E P. Trends in Molecular Medicine 2001;
7:175-8) indicated that tumor immunity failed to develop in mice
immunized with nontransfected fibroblasts, or mice immunized with
fibroblasts transfected with DNA from a heterologous tumor.
Example 6
Treatment of Breast Cancer with a Combination of Paclitaxel and
Genomic DNA-Based Vaccine LM-IL-2K.sup.b/SB5b
[0134] The therapeutic effects of paclitaxel administered in
combination with the DNA-based vaccine were investigated in mice
with breast neoplasms derived from SB5b cells. Cancers were first
established in the fat pad of the breast of C3H/He mice following a
single injection of 1.times.10.sup.5 SB5b cells. Six days later,
when the average tumor was approximately 3 mm, the mice received a
single s.c. injection of 2.25 mg/kg paclitaxel. Six days afterward,
the mice received the first of three sec, injections at weekly
intervals of 5.times.10.sup.6 LM-IL-2Kb/SB5b cells. As controls,
the mice were injected into the fat pad with an equivalent number
of SB5b breast cancer cells alone, with SB5b cells followed by a
single injection of paclitaxel alone or with paclitaxel followed by
the vaccine (FIG. 5(A)).
[0135] The experiment was terminated 47 days after injection of the
breast cancer cells. Mean survival time standard error (SE) is
presented in FIG. 5(A): Mice injected with SB5b cells alone 27.+-.2
days; mice injected with paclitaxel alone, 26.+-.2 days, mice
injected with LM-IL2K.sup.b/SB5b cells, 27.+-.2 days; mice injected
with paclitaxel and LM-IL-2K.sup.b/SB5b cells, 42.+-.3 days.
[0136] *p<0.01 for the difference in survival of mice receiving
the combined therapy and mice in any of the other groups.
[0137] In FIG. 5(B), the same protocol as described in (FIG. 5A)
was followed. Tumor volumes were determined by the equation 0.5
l.times.w.sup.2. Length and width were determined with a dial
caliper.
[0138] In FIG. 5(C), C3H/He mice (10/group) were injected with SB5b
cells followed by paclitaxel and LM-IL-2K.sup.b/SB5b cells,
according to the schedule described in (A). Additional controls
included mice receiving paclitaxel alone, mice injected with (non
transfected) LM-IL-2K.sup.b cells, mice injected with
LM-IL2K.sup.b/SB5b cells alone, and mice injected with paclitaxel
and LM-IL2K.sup.b/B16 cells.
[0139] *p<0.01 for the difference in survival of mice receiving
the combined therapy and mice in any of the other groups.
[0140] In FIG. 5(D), the same protocol as described in (C) was
followed. Tumor volumes were determined by the equation 0.5
l.times.w.sup.2. Length and width were determined with a dial
caliper.
[0141] As indicated (FIG. 5A), mice with established breast
neoplasms that received the combination of paclitaxel followed by
immunization with the transfected fibroblasts survived
significantly (p<0.01) longer than mice in any of the other
groups. The survival of mice with breast cancer treated with
paclitaxel alone, or by immunotherapy alone, was not significantly
different than that of untreated mice with breast cancer.
[0142] To determine if the therapeutic effects of the vaccine were
specific, the experiment was repeated to include treatment of mice
with breast cancer with a vaccine prepared by transfer of
DNA-fragments from B16 melanoma cells into the modified fibroblasts
(LM-IL-2Kb/B 16 cells). As indicated (FIG. 5C), the survival of
mice with breast cancer treated with a combination of paclitaxel
and LM-IL-2Kb/B16 cells was not significantly different than that
of untreated mice or mice treated with paclitaxel alone
Immunization with non-transfected modified fibroblasts
(LM-IL-2K.sup.b cells) had no significant therapeutic effect.
Measurements of tumor growth in mice with breast cancer treated by
the combined therapy were consistent with therapeutic outcome.
Tumor growth was delayed in mice receiving the combined therapy,
relative to that of mice in any of the other groups (FIGS. 5B and
5D).
[0143] The results of two independent spleen cell assays designed
to detect the presence of CTLs reactive with SB5b cells were
consistent with the enhanced survival of mice receiving the
combined therapy. Mice with breast cancer were treated according to
the same protocol with paclitaxel, followed by immunization with
LM-IL-2Kb/SB5b cells. Seven days after the last injection of the
vaccine, spleen cells from the immunized, tumor-bearing mice were
tested in .sup.51Cr-release cytotoxicity assays (FIG. 6(A)).
[0144] C3H/He mice (2 per group) were injected i.p. with 2.25 mg/kg
paclitaxel. Six days later, the mice received the first of two s.c.
injections at weekly intervals of 5.times.10.sup.6
LM-IL-2K.sup.b/SB5b cells. One week after the second injection,
aliquots of a suspension of spleen cells from the immunized mice
were tested in a standard .sup.51Cr-release assay for the presence
of CTLs reactive with SB5b cells at three different E:T ratios. As
controls, the same protocol was followed except that the mice were
injected with SB5b cells alone, with SB5b cells and paclitaxel
alone, with SB5b cells and (non transfected) LM-IL-2K.sup.b cells,
with SB5b cells and LM-IL-2K b/SB5b cells, or with paclitaxel and
LM-IL-2K.sup.b/B16 cells. P<0.001 for the specific release of
isotope in the group receiving the combined therapy and that of any
of the other groups.
[0145] FIG. 6(B) shows results of ELISPOT IFN-.gamma. assays in
which C3H/He mice (2 per group) were injected i.p. with 2.25 mg/kg
paclitaxel. Two days later, the mice received the first of two s.c.
injections at weekly intervals of 5.times.10.sup.6
LM-IL-2K.sup.b/SB5b cells. One week after the second injection,
aliquots of a suspension of spleen cells from the immunized mice
were divided into two populations. One population was co-incubated
for 18 hr with SB5b cells (E:T ratio=10:1). One population was
incubated for the same period without SB5b cells. At the end of the
incubation, the cells were analyzed by ELISPOT IFN-.gamma. assays.
As controls, the same protocol was followed except that the mice
were injected with an equivalent amount of paclitaxel alone, with
equivalent numbers of (non transfected) LM-IL-2K.sup.b cells alone,
with LM-IL-2K.sup.b/SB5b cells alone, with paclitaxel and
LM-IL-2K.sup.b/B16 cells alone or the mice were injected with SB5b
cells alone. *p<0.01 for the difference in the number of spots
in the group injected with paclitaxel and LM-IL-2K.sup.b/SB5b cells
co-incubated with SB5b cells and in any of the other groups.
[0146] FIG. 6(C) shows results of .sup.51Cr-release cytotoxicity
assays in the presence of CD4+, CD8+ or NK1.1 antibodies. The same
protocol described in FIG. 6A was followed except that antibodies
for CD4+, CD8+ or NK1.1 determinants and C were added before the
cytotoxicity assays were performed. P<0.001 for the differences
in percent specific lysis of SB5b cells in the presence and absence
of CD8+ and NK1.1 antibodies in the group injected with paclitaxel
and LM-IL2K b/SB5b cells.
[0147] As shown in FIG. 6A, the percent specific lysis from the
group of mice receiving the combined therapy was significantly
(p<001) higher than that of any of the other groups including
spleen cells from mice immunized with the vaccine alone or mice
immunized with paclitaxel and LM-IL-2Kb/B16 cells (p<001).
Analogous results were obtained in ELISPOT IFN-.gamma. assays. The
highest number of spots was obtained if the spleen cells were from
mice receiving the combined therapy (FIG. 6B). To determine the
classes of cells mediating resistance to the tumor, mAbs for NK1.1,
CD8+ or CD4+ determinants were added to the spleen cell suspensions
before the 51 Cr-release cytotoxicity assays were performed. As
shown in FIG. 6C, the greatest inhibitory responses were in the
groups treated with NK1.1, CD8+ mAbs.
[0148] In conclusion, data presented in Example 6 show that combing
the administration of paclitaxel with immunotherapy with a unique
DNA-based vaccine successfully prolonged the survival of mice with
breast cancer.
Example 7
Isolation of Dendritic Cells
[0149] Peripheral blood mononuclear cells (PBMC) were obtained from
a donor and were separated from other blood components by Ficoll
separation. PBMCs were then suspended in AIM-V medium (10.sup.7/ml)
and incubated for 1 h at 37.degree. C. in T75 flasks (Falcon/Becton
Dickinson). Plastic-adherent cells were then cultured in AIM-V
medium supplemented with 1,000 units/ml of IL-4 and 1,000 units/ml
of granulocyte macrophage colony-stimulating factor for 6 days at
37.degree. C./5% CO.sub.2 in air. The DCs were then harvested on
day 6 in cold Hanks' solution (Life Technologies), washed, and
resuspended at a concentration of 2.times.10.sup.6 cells per ml in
AIM-V medium.
Example 8
Ex vivo Anti-Tumor Response of Human DC Recipient Cells
[0150] PCI-13/IL-2 cells, a transformed human cell line modified to
secrete IL-2 were transfected in a manner similar to Example 4 with
genomic DNA obtained from, an HLA-A2.sup.+ squamous carcinoma cell
line. The transfected PCI-13/IL-2 cells were subjected to
UVB-induced apoptosis then fed to dendritic cells isolated from an
HLA-A2.sup.+ donor, which was then incubated with peripheral blood
mononuclear cells from the same donor as the dendritic cells. Three
T-cell lines were generated in 14-day cultures (T1, T2 and T3).
[0151] Each T-cell line was stimulated with a range of tumor cell
targets in ELISPOT assays, as described in (Asai et al., Clin.
Diagn. Lab. Immunol 7: 145-154, 2000). Briefly, responder (R) T
cells (1.times.10.sup.3 to 5.times.10.sup.3) were plated in 96-well
plates with nitrocellulose membrane inserts (Millipore) coated with
50 .mu.l of the capture antibody (10 .mu.g/ml in 1.times.PBS, clone
MAB1-D1K; MABTECH, Stockholm). Stimulator (S) cells were then added
at the R:S ratio of 20:1 Stimulator cells were transformed
PCI13/IL-2 cells, PCI-1 cells, OSC-19 cells and HR (HLA-A2.sup.+
gastric carcinoma).
[0152] After a 24-h incubation, the cells were removed by washing
the plates 6 times with 0.05% (wt/vol) Tween-20 in PBS (Fisher
Scientific). The detection antibody (2 .mu.g/ml, clone Mab7-B6-1;
MABTECH) was added to each well. The plates were incubated for 2 h
and the washing steps were repeated. After a 1-h incubation with
avidin-peroxidase (Vectastain Elite Standard ABC kit; Vector
Laboratories), the plates were washed. Aliquots (100 .mu.l) of
aminoethylcarbazole staining solution (Sigma) were added to each
well to develop the spots. The reaction was stopped after 4-6 min
by washing with water. The spots were counted with
computer-assisted image analysis (ELISPOT 4.14.3 Zeiss).
[0153] FIG. 8A shows that T1 cells responded to PCI-1, the DNA
donor, and to PCI-13/IL-2 cells. Each of these responses was
inhibited (p<0.01; see asterisks) by anti-MHC Abs (anti-HLA-A2
antibodies). FIG. 8B shows that T2 cells responded only to
PCI-13/IL-2 cells, with the response being blocked by anti-HLA-2
antibodies. FIG. 8C shows that T3 cells recognized OSC-19, the DNA
donor, and PCI-13/IL-2 cells, with each of these responses being
inhibited by anti-class I MHC Abs (anti-HLA-A2 antibodies). The
stimulated T cells recognize the tumor cells from which the DNA was
obtained to transfect the recipient cells. This shows that tumor
epitopes encoded in tumor-DNA were expressed by the recipient cells
and were able to induce tumor-specific T cells.
Example 9
Expansion and Enrichment of Immunogenic Recipient Cells Expressing
Tumor Associated Antigens
[0154] LM fibroblasts modified to secrete IL-2 and to express
H-2K.sup.b determinants (as an allo stimulus) were transfected with
genomic DNA from KMN205 cells, a squamous carcinoma cell line. The
transfected cells were divided into ten pools, each pool consisting
of 1.times.10.sup.3 cells. The cell number in each pool was allowed
to increase and cells from each pool were then tested for their
capacity to induce an anti-tumor, immune response.
[0155] C3H mice (syngeneic with KLN205 cells) were injected s.c.
two times at weekly intervals with 5.times.10.sup.6 transfected
cells from each pool. Six days after the last injection, T cells
from the spleens of the immunized mice were tested in a standard
.sup.51Cr-release assay for cytolytic activity toward KLN205 cells.
The anti-tumor activity of the cells from pool number 9 greatly
exceeded those of the other pools. Furthermore, cells from pool
number 6 were without significant anti-tumor activity. These
results indicate that dilution of the immunogenic cells may provide
pools of cells of differing immunogenic properties.
Example 10
Production of Immunogenic Dendritic Cells from Mice
[0156] Dendritic cells, which are to be used as recipient cells,
are generated from C57BL/6 mice, as described by Sallusto and
Lanzavecchia (Sallusto & Lanzavecchia, J. Exp. Med. 179:
1109-1118, 1994) with modification. Briefly, PBMC isolated from
mice suspended in AIM-V medium are incubated for 1 h at 37.degree.
C. in T75 flasks (Falcon/Becton Dickinson). Plastic-adherent cells
are cultured in AIM-V medium supplemented with 1,000 units/ml of
IL-4 and 1,000 units/ml of granulocyte macrophage
colony-stimulating factor for 6 days at 37.degree. C./5% CO2 in
air.
[0157] The dendritic cells are then transfected with DNA derived
from a syngeneic breast neoplasm arising in MTag mice, in a manner
similar to that described in Example 3, MTag mice are transgenic
for the polyomavirus middle T antigen under control of the mouse
mammary tumor virus promoter/enhancer. The mice develop breast
cancer by 12 weeks of age. The tumors develop in the epithelium of
the breast and metastasize to regional lymph nodes, among other
organs and tissues.
[0158] The transfected dendritic cells are then grown in culture in
the presence of GM-CSF, CD40L and LPS to stimulate the dendritic
cells to undergo maturation, as indicated by the increase in
expression of class I and II MHC-determinants and co-stimulatory
molecules. The functional status of the transfected dendritic cells
is determined by their capacity to induce allogeneic T cell
proliferation in mixed leukocyte reactions, in a manner similar to
that described in Example 8.
Example 11
Testing of Immunogenic Dendritic Cells from Mice
[0159] The ability of the transfected dendritic cells to act as a
vaccine is determined by measuring the time to first appearance of
tumor and time of survival of immunized mice injected with varying
numbers (range 1.times.0.sup.3 to 5.times.10.sup.5) of the same
breast cancer cells that provided the donor DNA. These results are
compared to those of syngeneic mice injected with an equivalent
number of breast cancer cells alone. Additional controls include
mice injected with dendritic cells transfected with DNA from normal
(non-neoplastic) liver cells, with dendritic cells transfected with
DNA from an unrelated tumor (melanoma) or with non-DNA-transfected
dendritic cells.
Example 12
Determination of the Proportion of Transgenic Cells that Express
Tumor Associated Antigens
[0160] In order to define the proportion of immunogenic dendritic
cells from that express tumor associated antigens, limiting
dilution assays are performed in combination with the application
of Poisson statistics. Varying numbers (range=5.times.10.sup.3 to
1.times.10.sup.5) of transfected dendritic cells are distributed to
20 replicate wells at each cell number. After further cell
proliferation, naive C57BL/6 mice are immunized, with cells derived
from individual wells. Three mice receive three injections of
5.times.10.sup.6 cells at weekly intervals from each pool. One week
after the last injection, the mice are challenged with an s.c.
injection of 5.times.10.sup.3 the same breast cancer cells that
provided the donor DNA. Tumor growth and rates of survival of mice
in each treatment group are determined Inhibition of tumor growth
is taken as an indication that the original well contained cells
that expressed tumor associated antigen characteristic of breast
cancer cells. Poisson statistics is used to determine the
proportion of the transfected cells that induced immunity to the
tumor. The results indicate that a vaccine enriched for transfected
dendritic cells that express tumor associated antigens is likely to
be more therapeutically effective than non-enriched cells.
Example 13
Enrichment of Immunogenic Dendritic Cells from Mice Expressing
Tumor Associated Antigens
[0161] In order to enrich dendritic recipient cells that express
tumor associated antigens, dendritic cells transfected are divided
into ten pools. Each of the ten pools is added to culture medium
AIM-V to expand the cells of each pool Aliquots from each pool are
use to immunize C57BL/6 mice three times at weekly intervals Spleen
cells are then isolated one week later from the immunized mice and
incubated for 24 hours with the donor breast cancer cells. The
samples are then tested for T cell immunity to the tumor cells
using ELISPOT, in a manner similar to that described in Example 8.
Those pools leading to T cell immunity are then divided into ten
new pools and the steps of expansion, immunization and T cell
immunity testing are repeated.
Example 14
Identification of Genes Encoding Tumor Associated Antigens
[0162] Clonal immunogenic recipient cells are produced by repeated
serial dilutions. Differentially expressed genes encoding tumor
associated genes are identified by using an Affymetrix GeneChip
Murine Genome U74 Set, which is a three Gene Chip.RTM. probe array
capable of interrogating approximately 36,000 full-length mouse
genes and EST clusters from the UniGene database.
[0163] RNA is isolation and purified from transfected and
non-transfected recipient cells using the Rneasy Mini Kit.
Biotin-labeled cRNA probes are prepared using the standard
GeneChip.RTM. eukaryotic target labeling protocol (Affymetrix,
Santa Clara, Calif.). The biotin-labeled cRNA probes generated from
transfected and non-transfected recipient cells are then hybridized
to separate oligonucleotide arrays, followed by binding to
streptavidin-conjugated fluorescent marker. Detection of bound
probe is achieved following laser excitation of the fluorescent
marker and scanning of the resultant emission spectra using a
scanning confocal laser microscope. The relative signal is measured
for the transfected recipient cells at each oligomer (representing
a single gene), and compared to the normalized signal obtained with
a labeled cRNA from the non-transfected recipient cells.
Example 15
Identification of Expressed Tumor Associated Antigens
[0164] We prepared cDNA expression libraries from SB5b cells, an
Aden carcinoma of the breast that arose spontaneously in a C3H/He
mouse in our animal colony. The libraries were constructed with a
Lambda Zap vector, using a cDNA library kit (Stratagene). In brief
cDNAs greater than 0.5 kb were selected by size fractionation via
gel filtration and directionally cloned into a pBK-CMV vector with
an EcoRI restriction site on the 5' end and an XhoI site on the 3'
end. The cDNA expression libraries yielded approximately
4.times.10.sup.5 pfu/ug DNA with an individual cDNA insert. The
size the cDNA transfected into the modified fibroblasts was between
0.5-7.0 kb (FIG. 16).
[0165] The library was co transfected into LM cells, a fibroblast
cell line of C3H/He mouse origin, along with pHyg, a plasmid
specifying resistance to hygromycin, used for selection (Ratio of
cDNA: pHyg=10:1.) After selection in medium containing sufficient
quantities of hygromycin to kill one hundred percent of
non-transfected cells, the surviving colonies (at least 2.times.04)
were pooled and maintained as a cell line for use in the
experiments. To augment the recipient cells' nonspecific
immunogenic properties, the fibroblasts were modified before
DNA-transfer to secrete IL-2 and to express allogeneic MHC class
I-determinants (H-2K.sup.b).
[0166] Since only a small proportion of the transfected cell
population would be expected to have incorporated genes specifying
TAA, we devised a new strategy to enrich the population for
TAA-positive cells. We accomplished this by dividing the
transfected cell-population into small pools (we used 96 well
plates), and then expanding the cell number from each pool in
vitro. We reasoned that if the starting inoculums were small
(1.times.10.sup.3 cells), then the number of cells in each of the
individual pools that expressed TAA would not be the same. After
allowing the cell number to increase, and maintaining an aliquot of
the expanded cell-suspension frozen/viable (for later use), cells
from the expanded individual pools could then be tested in
syngeneic C3H/He mice for their immunogenic properties against
breast cancer. In this way, pools that induced breast cancer
immunity to the greatest extent (immuno.sup.high) could be
identified and the frozen cells from that pool could be
reestablished in culture Immuno.sup.high cells would indicate the
presence of a higher number of cells in the initial inoculum that
expressed TAA. Further rounds of distribution of cells from the
immuno.sup.high pools, (and for comparison, with pools that
stimulated immunity to the least extent, immuno.sup.low pools) and
the identification of pools that stimulated immunity to the
greatest extent would lead to a progressive increase in the
proportion of cells that expressed TAA. Our preliminary data,
described below, support the validity of this approach. After
sufficient rounds of enrichment, by comparing microarrays from
immuno.sup.high and immuno.sup.low pools, candidate genes
specifying breast cancer antigens can be identified. Cloned
candidate genes can then be inserted into an expression vector and
introduced into the fibroblast cell line. Verification of the
immunogenic properties of the candidate gene can be accomplished by
the induction of a therapeutic anti breast cancer immune response
in immunized syngeneic mice susceptible to the tumor.
[0167] The first step was to determine the specificity of the
immune response in C3H/He mice immunized with modified fibroblasts
transfected with a cDNA library from breast cancer cells (SB5b).
The results are presented in FIG. 17.
[0168] As additional controls, the same protocol was followed
except that the mice were immunized with non transfected modified
fibroblasts (LMK.sup.b), with IL-2-secreting non transfected
fibroblasts (LM-IL-2K.sup.b), or with LM-IL-2K.sup.b cells
transfected with a cDNA expression library from B16F1 melanoma
cells (LM-IL-2K.sup.b/cB16F1), a non cross reactive neoplasm.
P<0.01 for differences in specific isotope release from SB5b
cells in mice immunized with LM-IL-2K.sup.b/SB5b cells versus any
of the other groups. The results shown in FIG. 17 indicate that the
anti-tumor immune response in mice immunized with
LM-IL-2K.sup.b/cSB5b was specific for SB5b cells.
[0169] Mice immunized with a vaccine prepared by cDNA-transfected
cells from B16F1 melanoma failed to develop immunity to the breast
cancer cells.
[0170] We next identified pools of transfected cells that
stimulated immunity to SB5b breast cancer cells to the greatest
(immuno.sup.high) and least (immuno.sup.low) extent. The schema is
presented in FIG. 18.
[0171] As indicated (FIG. 19, upper portion) immunity to SB5b cells
in mice immunized with cells from subpool (SP) 6 exceeded that of
mice immunized with cells from any of the other pools, as
determined by both ELISPOT and .sup.51Cr-release cytotoxicity
assays. Subpool 6 was designated immuno.sup.high. Cells from pool
10 stimulated immunity to SB5b cells to the least extent. It was
designated immuno.sup.low. Frozen/viable cells from each of these
pools were recovered Small aliquots (1.times.10.sup.3) were
distributed in individual wells of a 96 well plate, and the
procedure was repeated for a second round of immunoselection.
[0172] The results (FIG. 19, lower portion) indicate that by the
second round of selection, the cytotoxic activity toward SB5b cells
in mice immunized with cells from the immuno.sup.high pools was
significantly (P<0.001) greater than that of cells from the
immuno.sup.low pools.
[0173] A representative Elispot assay derived from the spleen cells
of mice immunized with immuno.sup.high (SP6-6) cells from the
second round of selection and immuno.sup.low (SP10-4) pools, and,
for comparison with cells from the non selected Master Pool
(LM-IL-2Kb/cSB5b) is presented in the FIG. 20. Specifically, C3H/He
mice were injected s.c. with 5.times.10.sup.6 cells from the
immuno.sup.high pool SP 6-6. One week after the last injection,
spleen cells from the immunized mice were serially ten fold diluted
and then tested in an ELISPOT assay for the presence of spleen
cells reactive with SB5b cells. As control, the same procedure was
followed except that cells from the immuno.sup.low pool SP 10-4 or
from the non-selected Master Pool were substituted for cells from
the immuno.sup.high pool. The right hand figure indicates the
number of spleen cells from mice immunized with cells from the
immuno.sup.high the immuno.sup.low or the Master Pool required to
achieve one-half the maximum number of spots.
[0174] ELISPOT assays were performed as follows: Responder (R)
T-cells (1.times.10.sup.3 to 5.times.10.sup.3) from spleen cell
cultures incubated 7 to 10 days with the transfected cells are
plated in wells of 96-well plates with nitrocellulose membrane
inserts (Millipore, Bedford, Mass.) coated with 50 .mu.L of the
capture antibody (10 .mu.g/ml in 1.times.PBS; clone MABI-DIK;
(Mabtech, Nacka, Sweden). Stimulator (S) cells (SB5b cells) are
then added at the R:S ratio of 20:1. After 24 hr incubation, cells
are removed by washing. The detection antibody (2 .mu.g/ml) is
added to each well. The plates are incubated for 2 hrs and the
washing steps are repeated. Following 1 hr incubation with
avidin-peroxidase, the plates are washed Aliquots of (100 .mu.L) of
aminoethylcarbazole staining solution are added to each well to
develop the spots. The reaction is stopped after 4-6 min with
water. The spots are counted using computer-assisted image analysis
(Zeiss ELISPOT 4.14.3. Jena, Germany). When experimental values are
significantly different from the mean number of spots against
non-pulsed cells (background values), as determined by a two-tailed
Wilcoxon's rank sum test, the background values are subtracted from
the experimental values. The coefficient of variation (CV) for the
assay has been determined to be <15% (n=50). This strategy is
expected to enhance the therapeutic benefits of the vaccine by
enriching the cell-population for transfected cells that express
TAA that characterize the breast cancer. The studies will further
optimize the therapeutic effects in mice with established breast
neoplasms.
[0175] The immunogenic properties of cells from the immuno.sup.high
and immuno.sup.low pools were then tested for their
immunoprotective properties in C3H/He mice, highly susceptible to
the growth of the breast cancer cells. The results are presented in
FIG. 21. Specifically, Cells from the immuno.sup.high and
immuno.sup.low pools were tested for their immunogenic properties
in C3H/He mice, susceptible to the growth of the breast cancer
cells. The mice were injected s.c. three times at weekly intervals
with 5.times.10.sup.6 cells. One week afterward, the mice were
injected into the fat pad of the breast with 1.times.10.sup.5 SB5b
breast cancer cells.
[0176] The results shown in FIG. 21 indicate that the survival of
mice with breast cancer immunized with the immuno.sup.high pool
(SP6-6) (from the second round) exceeded that of mice immunized
with cells from any of the other pools including mice immunized
with cells from the master pool LM-IL-2K.sup.b/SB5b. (P<0.01).
Mice with breast cancer immunized with cells from the
immuno.sup.low pool (SP10-4) or with cells transfected with a cDNA
library from B16F1 melanoma cells (LM-IL-2K.sup.b/B16F1) failed to
survive significantly longer than tumor-bearing mice injected with
saline.
Example 16
Detection of Muc-1, a Known Breast Cancer Antigen, in Cells from
the Immuno.sup.high Pool of cDNA-Transfected Cells
[0177] RT-PCR was used to determine if cells from the
immuno.sup.high pool specified Muc-1, a known breast cancer
antigen. The results, presented in the FIG. 22, revealed the
presence of Muc-1 both in SB5b breast cancer cells and in cells
from the immuno.sup.high pool. Muc-1 was not detected by this
method in cells from the immuno.sup.low pool. An analogous
procedure was used to detect the presence of HER-2/neu. As
indicated in FIG. 22, HER-2/neu was detected in SB5b cells, but not
in either the immuno.sup.high or the immuno.sup.low pool.
[0178] A monoclonal antibody for the cytoplasmic domain of Muc-1
was used to determine the relative staining intensity of cells from
the immuno.sup.high and immuno.sup.low pools of transfected cells.
The results are presented in the FIG. 23 which shows that the
staining intensity of cells in the immuno.sup.high pool (SP 6-6)
was significantly (P<0.01) higher than that of cells from the
immuno.sup.low pool (SP 10-4). Non-transfected cells
(LM-IL-2K.sup.b) cells failed to stain. The staining intensity of
SB5b breast cancer cells, the source of cDNA used to transfect
LM-IL-2K.sup.b cells, exceeded that of any of the other
cell-types.
Example 17
Strategy for Identification Genes Specifying Therapeutic Breast
Cancer Antigens
[0179] An oligonucleotide-based Affymetrix Mouse Genome 430 2.0
GeneChip Array can be used for identification of the antigens.
Cells are collected from five experimental groups: initial master
pool of transfected cells, selected sub pools of immuno.sup.high
and immuno.sup.low cells, breast cancer cells alone and non
transfected fibroblasts. Each experimental group is technically
replicated three times starting from 3 separate labeling reactions
per, RNA sample. Total cellular RNA is isolated with the use of
RNeasy columns (Qiagen) according to the manufacturer's protocol.
All labeling reactions and hybridizations are carried out according
to the standard GeneChip.RTM. eukaryotic target labeling protocol
(Affymetrix). Briefly, 1-5 .mu.g total cellular RNA per sample is
used to synthesize the double-stranded cDNA, which then will be
transcribed in vitro in the presence of biotinylated dNTPs.
Biotinylated target cRNA will be fragmented and brought tip in
hybridization mix. Successful labeling of all the samples (a
minimum of 15 .mu.g of IVT product per sample) is followed by the
test array hybridizations Test hybridizations is performed with the
use of "Test3" arrays (Affymetrix) to ensure quality of the
biotinylated target (Test3 allay contains probe sets corresponding
to commonly expressed genes from the human, mouse, rat, and yeast
genomes along with prokaryotic control genes.) Successful test
hybridizations indicating efficient cRNA amplification and strong
target hybridization activity is followed by actual experimental
hybridizations Hybridizations are followed by binding to
streptavidin-conjugated fluorescent marker. Detection of bound
probe will be achieved following laser excitation of the
fluorescent market and scanning of the resultant emission spectra
using a scanning confocal laser microscope.
[0180] Data acquisition is performed using Affymetrix GeneChip
Operating Software Package. Collected hybridization images is
subjected to quality control to remove from analysis arrays failed
to meet criteria both suggested by Affymetrix and developed
internally. These quality requirements include: low Q-noise (1-10);
low background (less than a 100); sample dependant percent of
probes, detected as present (20-50% for mammals); 3'/5' ratio of no
more than 3; hybridization efficiency defined by intensities
detected for the spike control probe sets (preferably higher than
2,000 fluorescent units); minimal deviation of scaling factors for
the whole set of arrays to be analyzed.
[0181] Expression profiles of the cells derived from each selected
sub pool (immuno.sup.high and immuno.sup.low) are compared to the
transcriptional profile of the initial super pool of transfected
cells. Hybridization intensity values collected from all
experimental samples are subjected to background correction,
normalization, and statistical significance analysis with the use
of S+ArrayAnalyser statistical software package. Identified
statistically significant differentially expressed transcripts will
be initially annotated with the use of the NetAffx Analysis Center
(http://www.affymetrix.com) according to the most up to date
version of Gene Ontology Database (http://www.geneontologys.org/).
More advanced functional annotation is performed with the use of
the PathwayAssist software package.
[0182] Changes in the transcript expression levels as detected by
Affymetrix experiments are verified by real-time PCR. Reactions are
performed in a Pelkin-Elmer/Aplied Biosystems ABI PRISM 770
Sequence Detection system. Primers are designed for each selected
gene, based on the sequence information available through the
Affymetrix NetAffx web-based resource (http://www.affymetrix.com).
Primer design is performed using Primer Express 15 software. The
primers are validated, standardized, and used on cDNA prepared from
test samples.
[0183] As shown in FIG. 24, the primary objective is to use the
enrichment strategy to identify genes specifying therapeutic breast
cancer antigens Microarrays were performed on immuno.sup.high and
immuno.sup.low cell pools to detect differences in gene expression
between to two pools.
[0184] Data in FIG. 25 compare differences in gene expression in
immuno.sup.high pools and immuno.sup.low pools as well as the
non-transfected modified fibroblasts used as recipients of cDNA
from the breast cancer cells (SB5b). One hundred forty one
identifiable genes were over expressed in cells from the
immuno.sup.high pool of transfected cells.
[0185] Ontologic classification of genes over-expressed by
immune.sup.high cells and several candidate genes chosen for
further study are presented in the following Table,
TABLE-US-00001 GenBank # Gene name Ratio Receptor activity NM
030721 G-protein-couple receptor 84 2.4 NM 46590 Olfactory receptor
Mor1 2.3 NM 009107 Retinoid receptor X gamma 2.0 RNA/DNA binding NM
011247 Retinoblastoma binding protein 6 2.2 NM 011585 Cytotoxic
granule-associated RNA-binding protein 2.4 NM 011549 Transcription
factor EB 2.1 Metabolism NM 139305 Carbonic anhydrase 9 5.4 NM
007955 Protein tyrosine phosphatase receptor type V 2.3 A1593846
MAPKK5 3.4 Cell constituent NM 00729 Procollagen, type XI, alpha 1
2.1 Others AA120189 Aurora kinase 3.4 BG074447 Rap guanine
nucleotide exchange factor 1 3.3 CANDIDATE GENES AK006529 Mus
musculus adult male testis NM 013825 Lymphocyte antigen 75 NM
019643 Teratocarcinoma expressed, serine rich NM 011247
Retinoblastoma binding protein 6 Legend: Candidate genes are those
chosen initially for further study to verify their
immunotherapeutic properties.
Example 18
Immunotherapy of Breast Cancer with Cellular Vaccines that Express
Defined Breast Cancer Antigens
[0186] Treatment protocols will be used to test the
immunotherapeutic properties of fibroblasts modified to express
identified TAA in mice with established breast cancer. Tumors will
be established in syngeneic C3H/He or BALB/c mice as appropriate
susceptible to the growth of the syngeneic breast cancer cells. The
mice will be treated with a cDNA-based vaccine that expresses
defined TAA. We will use cells transfected with cDNA specifying TAA
that were found by ex vivo analyses (ELISPOT and .sup.51Cr-release
cytotoxicity assays) to stimulate immunity to the breast cancer to
the greatest extent.
[0187] On day 0, breast cancer cells are injected into the fat pad
of the breast. The number of tumor cells to be used in these
experiments will be determined by the results of the prior studies.
If, for example, the treatment deterred growth of 2.times.10.sup.6
cells and prevented the growth of 1.times.10.sup.6 cells,
1.times.10.sup.6 cells would be used. Thus, as the size of the
tumor progressed after implantation, we can determine the point at
which the burden exceeds the therapy.
[0188] On days 1, 2, 5, 10 and 15 after injection of the breast
cancel cells, the mice will be immunized by s.c. injections of the
vaccine. After four immunizations, two animals from each group will
be sacrificed. Spleen cell suspensions will be tested in both
ELISPOT and .sup.51Cr-release cytotoxicity assays from labeled
breast cancer cells, used as targets. As a control, naive mice will
be euthanized and their spleen cells will be tested in the same
manner. This will provide the "before treatment" data since the
mice are inbred. Survival will be measured in days post treatment.
Our preliminary data and prior experience indicate that the
treatment of mice with smaller tumor burdens will be successful. It
is also expected that by allowing the tumor to increase in size, to
grow unchecked for a number of days, the burden will eventually
become be too large to be purged by the immune system.
Nevertheless, even if no group remains tumor free, there will be a
difference in the time to first appearance of the tumor mass in the
treated group, and in overall survival.
[0189] The increased survival of mice in the groups receiving
therapy should correlate with the delay in time until the
measurable tumor first appears. These experiments will provide
greater insight into the capacity of treatment with vaccines that
express defined antigens to affect existing tumors and the maximum
size of the tumor burden that can be successfully treated by
immunization with the vaccines. Control groups including mice
treated with cDNA transfected cells from melanoma will included to
determine the specificity of the response. As an additional
control, the mice will be treated according to the same protocol
with non-transfected fibroblasts. Untreated animals injected with
the breast cancer cells alone will form the base for evaluation of
the therapeutic response. We plan to test at least ten individual,
defined TAA by this approach. Our expectation is that the
immunotherapeutic properties of each of the TAA will not be the
same.
Example 19
Immunotherapy of Breast Cancer with Cellular Vaccines that Express
Multiple Defined Breast Cancer Antigens
[0190] As noted previously, the metastatic spread and
aggressiveness of the growth of cancer cells in the patient results
from the varied genotype of cells within the malignant cell
population Numerous random mutations generate subpopulations of
cancer cells that are capable of invasion and metastasis. Others
changes lead to the appearance of cancer cells that able to resist
drugs commonly used for chemotherapy.
[0191] It is likely that multiple altered and dysregulated genes
specifying weakly immunogenic TAA are present in cancer cells.
Immunization with a vaccine, therefore, that expresses multiple TAA
may be more successful in eliminating a greater proportion of the
malignant cell population than a vaccine that expresses a single
TAA. Although the major thrust of this proposal is the
identification of therapeutic breast cancer TAA, we will determine
the immunotherapeutic properties of vaccines that express multiple
defined TAA. In Section 2.2, we described vaccines that expressed
multiple TAA. We will compare the immunotherapeutic properties of
vaccines that express multiple defined TAA with those that express
a single TAA, using the following the protocol outlined above.
These experiments will determine the relative immunotherapeutic
properties of single epitope vaccines with multiple epitope
vaccines. One hundred percent of the transfected cells are expected
to express the defined antigens chosen for study.
Example 20
Identification of the Cell Types Activated for Immunity to Breast
Cancer in Mice Immunized with cDNA Transfected Cells that Express
Defined TAA
[0192] The cell types mediating the rejection of neoplasms in mice
with breast cancer treated with cDNA-transfected cells that express
defined TAA have not been defined. Conceivably, different TAA
stimulate different classes of immune-effector cells. To
investigate this question, naive syngeneic mice will first be
depleted of specific T cell subsets by i.p. injections of anti CD4
monoclonal antibody (GK1.5 rat hybridoma), anti CD8 monoclonal
antibody (83-23-5 mouse hybridoma) or NK/LAK antibody (asialo GM1).
The depleted mice will then be injected into the fat pad of the
breast with syngeneic breast cancer cells, followed by immunization
with the vaccines that express defined TAA.
[0193] The extent of T cell-depletion will be determined by FACS
analysis, (Our prior experience indicates that more than 99 percent
of the relevant T cell subtype can be depleted from the mice by
this approach.) C3H/He mice will be injected into the fat pad of
the breast with 5.times.10.sup.4 syngeneic SB5b cells. The tumors
will be allowed to grow to approximately 5 mm.sup.3 before
beginning treatment with the vaccine. As controls, additional naive
mice are injected with an irrelevant, isotype-specific monoclonal
antibody according to the same schedule, or with an equivalent
numbers of breast cancer cells alone. The survival of mice in the
groups injected with breast cancer alone and the irrelevant
monoclonal antibody form the reference against which the effect of
cell depletion is measured. We analyze the cell types mediating
resistance to the breast cancer for ten defined breast cancer
TAA.
[0194] This approach defines the cell types mediating tumor
rejection in mice immunized with vaccines that express defined TAA.
It further outlines the parameters of the therapeutic benefits of
the vaccines in the treatment of mice with breast cancer. It
characterizes the immunologic underpinning responsible for the
vaccines' beneficial effects.
Example 21
Creation of a Hierarchy of Breast Cancer Antigens Based on their
Relative Immunotherapeutic Properties
[0195] In his classic paper, Gilboa described four categories of
tumor antigens. The antigens were divided into "patient specific
(incidental mutated gene products)," "tumor-specific" (mutated
related to the oncogenic process)," "tissue restricted," (e.g.,
MACE) and "Others," that included differentiation antigens such as
gp100. We wish to apply the strategy outlined in this proposal to
create a hierarchy of TAA that stimulate immunity to breast cancer.
The hierarchy is based upon the antigen stimulates immunity to
breast cancer to the greatest and to the least extent We determine
the relative immunogenic properties of each prototype TAA by both
ex vivo (ELISPOT and .sup.51Cr-release assays) and in vivo studies
designed to determine the vaccines' immunotherapeutic properties in
tumor-bearing mice. Our long-term objective is to develop a
vaccination strategy that can be of benefit to breast cancer
patients Our expectation is that not all breast cancer TAA will be
equally efficacious in promoting breast cancer immunity. The
results of this important investigation will enable us to describe
the basic characteristics of the desired TAA. It will provide a
guide for the vaccines that can be used most effectively in the
clinical studies to follow.
Example 22
Analysis by Limiting Dilution to Determine the Proportion of
Transfected Fibroblasts that Express Breast Cancer Antigens
[0196] It is likely that a subpopulation of cells incorporated
therapeutically relevant genes that specified antigens associated
with the breast cancer cells We will use an assay based on limiting
dilution and the application of Poisson statistics to determine the
proportion of the transfected cell-population that induced the anti
tumor response (Poisson distribution is a statistical function that
describes how objects are distributed at random. For instance, when
different numbers of transfected cells are distributed into a
series of culture wells, some wells will receive no TAA-positive
cells, some will receive one TAA-positive cell, some two, and so
on. From the Poisson distribution it is known that there is on
average one TAA-positive cell per well when the frequency of
negative wells is 37%.). Varying numbers (range=5.times.10.sup.3 to
1.times.10.sup.5) of immuno.sup.high and immuno.sup.low cells are
distributed to 20 replicate wells at each cell number.
[0197] The plates are incubated for five days at 37.degree. C.
under standard cell culture conditions, to allow the cells to
proliferate. Afterward, cells from individual wells are transferred
to culture flasks. After further cell proliferation, naive C3H/He
mice are immunized with cells derived from individual wells. The
mice receive three injections at weekly intervals of
5.times.10.sup.6 cells. There are three mice in each group. One
week after the last injection, the mice are challenged by an
injection of 5.times.10.sup.3 SB5b cells. Inhibition of tumor
growth and the induction of spleen cell-mediated immunity to the
breast cancer cells can be used as an indication of the relative
proportion of cells in the transfected cell population that
expressed TAA.
[0198] As shown in FIG. 26, the analysis indicates that
approximately 1 in 10,000 cells in the immuno.sup.high pool SP6-6
expressed breast cancer TAA. This assay defines the proportion of
transfected cells that express "therapeutically relevant" TAA
(promote tumor regression) that characterize the breast cancer
cells. The studies determine the fundamental basis of the
therapeutic effects in mice with breast cancer. It tests the
hypothesis that a vaccine composed mainly of DNA transfectants that
express tumor antigens is likely to be more therapeutically
effective than one containing DNA transfectants that express few
relevant tumor antigens.
Example 23
Testing Vaccines that Express Defined Tumor Antigens for Toxicity,
Including the Possible Generation of Systemic or Organ Specific
Autoimmune Disease in Vaccinated Animals
[0199] No evidence of toxic effects in mice injected with DNA-based
vaccines was detected in our previous experiments. The animals
lived their anticipated normal, expected life spans without
evidence of disease. As described, the vaccine, which expresses
allogeneic determinants, like other allografts was rejected. In
this study, vaccines are prepared that express defined TAA. One
hundred percent of the transfected cells express the defined TAA
chosen for analysis. Conceivably, immunization with a strongly
potent vaccine can result in the generation of autoimmunity in the
breast, and elsewhere. We can carry out additional experiments to
investigate this important question. To determine if mice immunized
with the DNA-based vaccines that express defined TAA develop
autoimmune disease, we can carry out the following assays: for
signs of generalized autoimmunity, we can prepare H and E sections
of skin, brain, thyroid, heart, liver, kidney, breast, lung,
stomach, and ovary. The microscopic sections can be examined for
signs of inflammation, as characterized primarily by mononuclear
cell infiltrates. The presence of immune complexes can be
determined by standard immunohistochemical staining. To detect the
presence of autoantibody to a diffuse antigen, we can assay for the
presence of anti nuclear antibody (ANA) as well as antibodies to
desmoglein 3, expressed by keratinocytes in the skin. To detect the
presence of autoantibody to an organ specific antigen, we can
perform assays for antibodies for thyroid peroxidase,
thyroglobulin, and thyrotropin receptor. We can carry out these
assays in animals immunized for each of the vaccines investigated
in this study. In each instance, the animals are maintained through
their anticipated life spans in the event delayed toxic effects
appear.
[0200] The lack of autoantibodies or cellular infiltrates is strong
evidence that the vaccine is not toxic and does not induce
autoimmune disease.
[0201] It is likely that those breast cancer antigens that have
been identified represent only a small proportion of the total
array of TAA within the tumor cell population. Until now, the
identification of breast cancer antigens has depended upon their
relative over expression and altered molecular characteristics in
tumor cells, when compared to non-malignant cells from the
tumor-bearing host. In this innovative approach, we describe a new
method for the identification of breast TAA. Our expectation is
that anti tumor immune responses following immunization with
vaccines that specify defined, highly immunogenic breast cancer
antigens may exceed those of vaccines prepared from unfractionated
tumors. This approach can become an important adjunct to
conventional therapy in the treatment of breast cancer
patients.
Example 24
Cytokine-Secretion by Lm Mouse Fibroblasts Transduced with
pZipNeoSVIL-2, a Retroviral Vector Specifying IL-2
[0202] Among other advantages, the use of a fibroblast cell line as
the recipient of DNA from the SCC enables the recipient cells to be
conveniently modified in advance of DNA-transfer to augment their
nonspecific immunogenic properties. In this instance, the
fibroblasts, of C3H/He mouse origin, were modified to secrete IL-2
and to express additional allogeneic MHC class I-determinants
(described, below). Allogeneic MHC class I-determinants are strong
immune adjuvants and ensure that the vaccine will be rejected
(Ostrand-Rosenberg S, J Immunol 1990; 144:4068-71; Fearon E R,
Cancer Res 1988; 48: 2975-80; Nabel G J, Proc Natl Acad Sci USA
1996; 93: 15388-93; DcBniyne L. Cancer Immunol Immunother 1996;
43:180-9).
[0203] A replication-defective retroviral vector (pZipNeoSVIL-2)
was used to modify the cells to secrete IL-2. The vector specified
the gene for human IL-2 along with a gene (neo.sup.r), which
conferred resistance to the neomycin analog G418 (Like mouse IL-2,
human IL-2 stimulates the proliferation and maturation of mouse T
cells.) After selection in growth medium containing sufficient
quantities of G418 to kill one hundred percent of non-transduced
cells (600 .mu.g/1 ml), the surviving colonies were pooled and
maintained as a cell line (LM-IL-2 cells). An analysis by ELISA of
the culture supernatants of LM-IL-2 cells indicated that 10.sup.6
retrovirally-transduced cells formed 196 pg IL-2/ml/10.sup.6
cells/48 hrs. The culture supernatants of LM fibroblasts transduced
with the IL-2 negative vector pZipNeoSV (X).sub.7 like that of
non-transduced LM cells, failed to form detectable quantities of
IL-2. Every third passage, the transduced cells were placed in
medium containing 600 .mu.g/ml G418. Under these circumstances,
equivalent quantities of IL-2 were detected in the culture
supernatants of cells transduced with pZipNeoSVIL-2 for more than
six months of continuous culture. The generation time of transduced
and non-transduced fibroblasts, approximately 24 hrs in each
instance, were equivalent. The introduction of genomic
DNA-fragments from the SCC into the IL-2-secreting cells did not
affect the quantity of IL-2-secreted (these data are not
presented).
Example 25
Modification of LM Fibroblasts to Express Allogeneic MHC Class I
(H-2K.sup.b)-Determinants
[0204] The SCC used in the study originated in DBA/2 mice
(H-2.sup.d), H-2K.sup.b-determinants are allogeneic in this mouse
strain. To further augment their immunogenic properties, the
IL-2-secreting fibroblasts (of C3H/He mouse origin (H-2.sup.k) were
also modified to express IL-2K.sup.b-determinants A plasmid,
pBR327H-2K.sup.b, specifying H-2K.sup.b-determinants was used for
this purpose. LM-IL-2 cells were co-transfected with
pBR327H-2K.sup.b DNA along with the vector pBabePuro (confers
resistance to puromycin), used for selection. A 10:1 ratio of
pBR327H-2K.sup.b to pBabePuro was used to ensure that the cells
that took up pBabePuro DNA incorporated pBR327H-2K.sup.b DNA as
well. After selection in medium containing sufficient quantities of
puromycin to kill one hundred percent of non-transduced cells, the
surviving colonies were pooled and maintained as cell line
(LM-IL-2K.sup.b cells).
[0205] Quantitative immunofluorescence measurements with PE-labeled
mAbs for mouse H-2K.sup.b determinants were used to measure
expression of the class I-determinants. As a control, aliquots of
the puromycin-resistant cell suspension were incubated with
PE-conjugated IgG2a isotype Ig.
[0206] 1.times.10.sup.6 LM fibroblasts transduced with the plasmid
pBR327H-2K.sup.b (LM-IL-2K.sup.b cells) suspended in 100 ul PBS
were incubated for 1 hr at 37.degree. with PE-conjugated
H-2K.sup.b, H-2K.sup.k B7.1 (CD 80) or I-A mAbs. As controls, the
same procedure was followed except that (non-transduced) LM cells,
LM-IL-2K.sup.b cells transfected with DNA-fragments from KLN205
cells (LM-IL-2K.sup.b/KLN, Master pool)) or LM-IL-2K.sup.b/KLN
cells from sub pools after three rounds (3.degree.) of immune
selection were substituted for LM-IL-2K.sup.b cells. As an
additional control, PE-conjugated IgG2a isotype Ig was substituted
for the mAbs. After incubation, the cells were washed and analyzed
for fluorescent staining by flow cytofluorometry. Dark-shaded area:
Cells stained with PE-conjugated anti-H-2K.sup.b, H-2K.sup.k, B7.1
or I-A mAbs. Light line: Cells stained with PE-conjugated isotype
Ig.
[0207] The results (FIG. 10) indicated that more than 99 percent of
the transduced fibroblasts stained positively (MFI at least ten
fold greater than cells stained with PE-conjugated isotype Ig,
taken as background). Under similar conditions, non-transduced LM
cells (of C3H/He mouse origin, H-2.sup.k) or fibroblasts stained
with PE-conjugated isotype Ig failed to stain above background. The
introduction of DNA from KLN205 cells into the transduced
fibroblasts did not affect the intensity of immunofluorescent
staining. The expression of H-2K.sup.b-determinants by the
transduced cells was a stable property. The staining intensity was
essentially unchanged after three months of continuous culture.
[0208] An analogous procedure was used to further characterize the
cells used as DNA-recipients. The modified fibroblasts were stained
with PE-labeled mAbs for H-2K.sup.k class I-determinants or with
PE-labeled mAbs for the co-stimulatory molecule B7.1 or I-A class
II MHC determinants. The results indicated that the fibroblasts
expressed H-2K.sup.k determinants constitutively (MFI 10.9.+-.1.1).
Both transduced and non-transduced LM cells also expressed B7.1,
but not I-A determinants (MFIs 4.6.+-.07, 0.9.+-.0.8 and 1.8.+-.0.7
respectively). The expression of MHC class I-determinants and the
co-stimulatory molecule by LM cells was consistent with various
reports indicating that fibroblasts, like dendritic cells, are
efficient antigen presenting cells (Alberg A J, J Clin Oncol 2005;
23:3175-85; Morse Mass., Cancer Res 2005; 65:553-61; Hirschowitz E
A, J Clin Oncol. 2004; 22:2808-15; Raez L E, J Clin Oncol 2004;
22:2800-7).
Example 26
Strategy for the Enrichment of the Cellular Vaccine for Cells that
Induce Immunity to Squamous Carcinoma in DBA/2 Mice and
Identification of Highly Immunogenic (Immuno.sup.high) Pools of
Transfected Cells
[0209] A cellular vaccine for SCC was prepared by transfer of 25 kb
DNA-fragments from KLN205 cells into LM-IL-2K.sup.b cells. A novel
enrichment strategy was devised since only a small proportion of
the transfected cell population would be expected to induce the
anti tumor immune response in the immunized mice. The strategy,
outlined in FIG. 11, was designed to enrich the transfected
cell-population for cells that induced immunity to the SCC. LM-IL-2
cells were transfected with sheared DNA-fragments from KLN205
cells, along with a plasmid (pHyg) conferring resistance to
hygromycin B, used for selection, as described. After selection,
small aliquots (1.times.10.sup.3) of the transfected cells were
added to each of ten wells of a 96 well plate. The cells were
cultured under standard conditions. A portion of the expanded cell
population was maintained frozen/viable. The remaining portion was
used to immunize DBA/2 mice. Spleen cells from mice immunized with
cells from the individual pools were then tested by
.sup.51Cr-release cytotoxicity and ELISPOT IFN-.gamma. assays for
their immunogenic properties against KLN205 cells. The objective
was to identify the pool that stimulated immunity to KLN205 cells
to the greatest (Immuno.sup.high) and least (Immuno.sup.low)
extent. Frozen/viable cells from the Immuno.sup.high and the
Immuno.sup.low pools were reestablished in culture. Small aliquots
(1.times.10.sup.3) of cells from each of these pools were then
added to each of ten wells of a 96 well plate and the process was
repeated for two additional rounds of immune selection. Master
pool=LM-IL-2K b/KLN cells before immune selection.
Sp-6-10=LM-IL-2K.sup.b/KLN cells from subpool 6 after two rounds of
immune selection. Sp-6-10=LM-IL-2K.sup.b/KLN cells from subpool 6
after three rounds of immune selection.
[0210] Small aliquots of the transfected cell-population were added
to individual wells of a 96 well plate. We reasoned that if the
starting inoculums were sufficiently small, then some pools would
contain greater numbers of highly immunogenic cells than others
Pools containing greater numbers of immunogenic cells could be
identified by their heightened immunogenic properties against
KLN205 cells in immunized DBA/2 mice. To test this strategy, we
added 1.times.10.sup.3 transfected cells to each of ten wells of a
96 well cell culture plate. As the cell number increased, cells
from individual pools were transferred to progressively larger cell
culture plates, and then flasks. Alter the number of cells from
individual wells had increased to about 5.times.10.sup.7, a portion
of the expanded cell population from each pool was collected and
maintained frozen/viable. The remaining portion was used to
immunize naive DBA/2 mice. After immunization, two independent
means (ELISPOT-IFN-.gamma. and .sup.51Cr-release cytotoxicity
assays) were used to identify pools that stimulated spleen
cell-mediated immunity toward KLN205 cells to the greatest
(immuno.sup.high) and least (immuno.sup.low) extent.
[0211] As shown in FIG. 12A, DBA/2 mice received two s.c.
injections at weekly intervals of 4.times.10.sup.6 cells from
individual pools of LM-IL-2K.sup.b/KLN cells. One week after the
second injection, spleen cells from mice immunized with cells from
the individual pools were co-incubated for 5 days with (mitomycin
C-treated) KLN205 cells (E:T ratio=30:1). Afterward,
.sup.51Cr-labeled KLN205 cells were added and the specific
cytotoxic activity was determined in a standard 4 hr .sup.51
Cr-release assay.
[0212] The same procedure as described in (12A) was followed for
FIG. 12B, except that the spleen cells were co-incubated for 18 hi
with (mitomycin C-treated) KLN205 cells (E:T ratio=10:1) before
they were analyzed in ELISPOT-IFN-.gamma. assays. As controls,
spleen cells from naive mice were substituted for spleen cells mice
immunized with the transfected cells.
[0213] Immuno.sup.high (2.degree.)=Pool selected for further
analysis after two rounds of immune selection. Immuno.sup.low
(2.degree.)=Pool selected for further analysis after, two rounds of
immune selection. Immuno.sup.high (3.degree.)=Pool selected for
further analysis after three rounds of immune selection.
Immuno.sup.low (30)=Pool selected for further analysis after three
rounds of immune selection. The results (FIG. 12) indicated that
after the first round of selection the immunogenic properties of
transfected cells derived from each pool were not the same. The
immunogenic properties of transfected cells from subpool (sp)
6-10-1 exceeded those of any of the other pools (sp
6-10-1=immuno.sup.high). In a similar manner, cells from sp 9-6-2
stimulated immunity to KLN205 cells to the least extent (sp
9-6-2=immuno.sup.low). Frozen/viable cells from each of these pools
were recovered and the procedure was repeated for two additional
rounds of immuntoselection, using 1.times.10.sup.3 transfected
cells as the starting inoculums in each instance.
[0214] The strategy resulted in a progressive increase in the
immunogenic properties of the cells from the immuno.sup.high pools
(FIG. 13).
[0215] As seen in FIG. 13A, DBA/2 mice were injected s.c. two times
at weekly intervals with 4.times.10.sup.6 cells from the
Immuno.sup.high subpool (sp) 6-10-1, taken after three rounds
(3.degree.) of immune selection. One week later, spleen cells from
the immunized mice were co-incubated for 5 days with (mitomycin
C-treated) KLN205 cells (E:T ratio 30:1). At the end of the
incubation, .sup.51Cr-labeled KLN205 cells were added and the
specific cytotoxic activity toward the labeled cells was determined
in a standard 4 hr .sup.51Cr-release assay. For comparison, the
same procedure was followed except that cells taken after one
(1.degree.) or two (2.degree.) rounds of immune selection were
substituted for cells taken after three rounds (3.degree.) of
immune selection. As controls, cells from the non-selected Master
Pool or from non-transfected LM-IL-2K.sup.b cells were substituted
for transfected cells from the third round of immune selection. As
an additional control, cells from the Immuno.sup.low pool after
three rounds of selection were substituted for cells from the
Immuno.sup.high pools.
[0216] p<0.005 for the specific release of isotope from KLN205
cells co-incubated with spleen cells from mice immunized with cells
from the Immuno.sup.high (3.degree.) pool and spleen cells from
mice immunized with cells from the Immuno.sup.high (2.degree.) or
Immuno.sup.high (1.degree.) pools or with cells from the (non
selected) Master Pool p<0.001 for the specific release of
isotope from KLN205 cells co-incubated with spleen cells from mice
immunized with cells from the Immuno.sup.high (3.degree.) pool and
the spleen cells from mice immunized with cells from the
Immuno.sup.low (3.degree.) pool or mice immunized with
non-transfected cells.
[0217] The same procedure as described in FIG. 13A, was followed in
FIG. 13B, except that spleen cells from mice immunized with cells
from the various pools were co-incubated for 18 hr with
(mitomycin-C-treated) KLN205 cells (E:T ratio 10:1) before they
were analyzed in ELISPOT-IFN-.gamma. assays, p<0.001 for the
number of spots developing in the group co-incubated with spleen
cells from mice immunized with cells from the Immuno.sup.high
(3.degree.) pool and cells from any of the other pools.
[0218] By the second round of immune selection, as determined by
.sup.51Cr-release cytotoxicity and ELISPOT-IFN-.gamma. assays, the
immunogenic properties toward KLN205 cells in mice immunized with
cells from the immuno.sup.high pool sp 6-10 were clearly
(p<0.001) greater than those of cells from the initial non
selected (Master Pool) or from the immuno.sup.low pool sp 9-6. By
the third round, the immunogenic properties of cells from the
immuno.sup.high pool were higher than cells from any of the other
pools. Cells from the immuno.sup.low pools failed to stimulate
immunity to KLN205 cells in DBA/2 mice, and presumably contained an
insufficient number of immunogenic cells. The expression of MHC
class I-determinants of cells from the immuno.sup.high and
immuno.sup.low pools of transfected cells were equivalent (FIG.
10).
Example 27
Tumor Growth was Inhibited and Survival was Prolonged in DBA/2 Mice
Immunized with Cells from the Immuno.sup.high Pool of Transfected
Cells
[0219] To determine if the immunogenic properties of the
immuno.sup.high pool of transfected cells, as revealed by in vitro
measurements, could be extended to mice with SCC, DBA/2 mice were
injected s.c. two times at weekly intervals with 4.times.10.sup.6
cells from the immuno.sup.high pool (3.degree.). One week later,
the mice received a single challenging s.c. injection of
1.times.10.sup.6 KLN205 cells. As controls, the same procedure was
followed except that cells from the immuno.sup.high pool after the
first (1.degree.) or second round (2.degree.) of immunoselection
were substituted for cells from the immuno.sup.high pool
(3.degree.). As additional controls, cells from the nonselected
Master Pool or from the immuno.sup.low subpool (sp 9-6-2) were
substituted for cells from the immuno.sup.high pool.
[0220] FIG. 15A shows results of the cytotoxicity tests. DBA/2 mice
were injected s.c. with 1.times.10.sup.6 KLN205 cells. Six days
later, the mice received the first of two injections at weekly
intervals of 4.times.10.sup.6 cells from the Immuno.sup.high
(3.degree.) pool (6-10-1). One week later, spleen cells from the
immunized tumor-bearing mice were co-incubated for 5 days with
(mitomycin-C-treated) KLN205 cells. At the end of the incubation,
.sup.51Cr-labeled KLN205 cells were added at varying E:T ratios and
the percent specific lysis were determined. As controls, the same
procedure was followed except that the mice were immunized with
(non transfected) LM-IL-2K.sup.b cells, with LM-IL-2K.sup.b/B16
cells, with cells from the Immuno.sup.low pool sp-9-6-2
(3.degree.), or with cells from the (non selected) Master Pool
(LM-IL-2K.sup.b/KLN). As addition controls, the mice were injected
with KLN205 cells alone or the mice were not injected (naive).
p<0.001 for the specific release of isotope from KLN205 cells
co-incubated with spleen cells from mice immunized with cells from
the Immuno.sup.high (3.degree.) pool (sp-6-10-1) and KLN205 cells
co-incubated with spleen cells from mice immunized with cells from
any of the other groups excepting mice immunized with cells from
the Master pool (LM-IL-2K.sup.b/KLN). p<0.05 for the specific
release of isotope from KLN205 co-incubated with spleen cells from
mice injected with cells from the Immuno.sup.high (3.degree.) pool
(sp-6-10-1) and mice immunized with cells from the Master pool.
[0221] FIG. 15B presents results of antibody inhibition procedure.
The same procedure described in 6A was followed except that mAbs
for CD4+, CD8+ or NK1.1 determinants, plus C, were added to the
mixed cell cultures one hr before the cytotoxicity determinations
were performed.
[0222] The same protocol described in 15A was followed in FIG. 15C,
except that spleen cells from the immunized mice were co-incubated
for 18 hr with KLN205 cells (E:T ratio=10:1) before they were
analyzed in ELISPOT-IFN-.gamma. assays. As controls, spleen cells
from mice injected with (non-transfected) LM-IL-2K.sup.b cells,
LM-IL-2K.sup.b/B16 cells, or spleen cells from non-immunized mice
were substituted for spleen cells from mice immunized with cells
from the Immuno.sup.high (3.degree.) pool. The ELISPOT plates from
both stimulated (incubated with KLN205 cells) and unstimulated
(incubated without KLN205 cells) cultures are presented.
[0223] FIG. 15D presents results of determination of the number of
spots presented in 15C. p<0.005 for the difference in the number
of spots in the group of mice immunized with cells from the
Immuno.sup.high (3.degree.) pool sp 6-10-1 and mice immunized with
cells from the Immuno.sup.low (3.degree.) pool sp 9-6-2 or with
cells from the (non selected) Master pool. p<0.001 for the
difference in the number of spots in the group of mice immunized
with cells from the Immuno.sup.high (3.degree.) pool sp 6-10-1 and
mice immunized with non transfected LM-IL-2K.sup.b cells, with
LM-IL-2K.sup.b/B16 cells, or mice injected with KLN205 cells
alone.
[0224] The results (FIG. 15A) indicate that mice immunized with
cells from the immuno.sup.high pools followed by the challenging
injection of KLN205 cells survived significantly longer
(p<0.001) than mice immunized with cells from any of the control
groups Eight of ten mice in the group immunized with cells from the
immuno.sup.high pool (3.degree.) (sp6-10-1) survived more than 80
days, without evidence of disease. Lesser immunogenic properties
were detected if the mice were immunized with cells after the first
or second round of immunoselection Six of 10 mice immunized with
cells from immuno.sup.high pool after two rounds of immune
selection followed by the injection of KLN205 cells survived more
than 80 days, without evidence of disease. Fewer numbers of mice
immunized with cells from the immuno.sup.low pool after the first
round of selection or with cells from the non-selected Master pool
survived more than 80 days None of the mice immunized with cells
from the immuno.sup.low pool or mice injected with PBS before the
injection of KLN205 cells survived longer than 65 days. (p<0.001
for survival of mice immunized with immuno.sup.high pool
(3.degree.) versus mice immunized with cells from the
immuno.sup.low pool or mice injected with PBS.)
[0225] Measurements of tumor growth in mice immunized with cells
from the immuno.sup.high pool were consistent with survival. The
greatest inhibition of tumor growth was in mice immunized with
cells from the immuno.sup.high pool (sp 6-10-1) after three rounds
of immune selection.
[0226] Thus, by successive rounds of immunoselection, the
immunogenic properties of the transfected cell populations
increased, as determined by both in vitro and in vivo
measurements.
Example 28
Treatment of Mice with Established SCC by Immunization with Cells
from the Immuno.sup.high Pool of Transfected Cells
[0227] The immunotherapeutic properties of transfected cells from
the immuno.sup.high pool were also investigated in tumor-bearing
DBA/2 mice, highly susceptible to the growth of KLN205 cells.
[0228] As shown in FIG. 14A, DBA/2 mice were injected s.c. two
times at weekly intervals with 4.times.10.sup.6 cells from the
Immuno.sup.high (3.degree.) pool (sp-6-10-1 (3.degree.)). One week
after the last immunization, the mice were injected s.c. with
1.times.10.sup.6 KLN205 cells. As controls, cells from the
Immuno.sup.high (1.degree.) pool (sp-6), the Immuno.sup.high
(2.degree.) pool (sp-6-10), from the non-selected Master Pool
(LM-IL-2K.sup.b/KLN) or non-transfected (LM-IL-2K.sup.b) cells were
substituted for cells from the Immuno.sup.high (3.degree.) pool. As
an additional control, the mice were injected with PBS before they
were injected with KLN205 cells. Tumor volumes were determined by
the formula 0.5 length.times.width.sup.2. (Length and width were
determined with a dial caliper.) Mean survival time.+-.standard
error (SE): Mice immunized with KLN205 cells alone, 44.+-.3.5 days;
mice immunized with LM-IL-2K.sup.b cells (Master pool) 74.+-.5.8
days; mice immunized with cells from the Immuno.sup.high
(1.degree.) pool, 66.+-.9.0 days; mice immunized with cells from
the Immuno.sup.high (2.degree.) pool, 88.+-.10.5 days; mice
immunized with cells from the Immuno.sup.high (3.degree.) pool,
99.+-.14.5 days; mice immunized with cells from the Immuno.sup.low
(3.degree.) pool, 52.+-.2.7 days p<0.001 for the difference in
survival of mice immunized with cells from the Immuno.sup.high
(3.degree.) pool and any of the other groups except mice immunized
with cells from the Immuno.sup.high (2.degree.) pool where p for
the difference in survival of mice immunized with cells from the
two pools was p<0.01.
[0229] For therapeutic treatment, DBA/2 mice were first injected
s.c. with 1.times.10.sup.6 KLN205 cells (FIG. 14B). Six days later;
the tumor-bearing mice received the first of two weekly s.c.
injections 4.times.10.sup.6 cells from the Immuno.sup.high
(3.degree.) pool (sp6-10-1). As controls, the same procedure was
followed except that cells from the Immuno.sup.low (3.degree.) pool
(sp-9-6-2), LM-IL-2K.sup.b/KLN cells from the Master Pool
(LM-IL-2K.sup.b/KLN, Mp), non-transfected LM-IL-2K.sup.b cells or
LM-IL-2K.sup.b cells transfected with DNA from B16 melanoma cells
(LM-IL-2K.sup.b/B16) were substituted for cells from the
Immuno.sup.high sp-6-10-1 (3.degree.) pool. Tumor volumes were
determined by the equation 0.5 l.times.w.sup.2. Mean survival
time.+-.standard error (SE): Mice injected with KLN205 cells alone,
33.+-.7.7 days; mice immunized with LM-IL-2K.sup.b cells, 34.+-.8.7
days, mice immunized with LM-IL-2K.sup.b/B16 cells, 35.2.+-.5.3
days; mice immunized with cells from the Immuno.sup.low (sp-9-6-2)
(3.degree.) pool, 36.1.+-.7.1 days; mice immunized with
LM-IL-2K.sup.b/KLN cells (Mp), 39.+-.7.4 days; mice immunized with
cells from the Immuno.sup.high (sp-6-10-1) (3.degree.) pool,
50.+-.6.9 days. p<0.01 for the difference in survival of mice
immunized with cells from the Immuno.sup.high (3.degree.) pool (sp
6-10-1) and any of the other groups.
[0230] Tumors were first established in immunocompetent naive DBA/2
mice by an s.c. injection of KLN205 cells. One week later, when the
tumor at the injection site reached a size of approximately 3-5 mm,
the mice received the first of two s.c. injections at weekly
intervals of 4.times.10.sup.6 cells from the immuno.sup.high pool
(3.degree.) (sp 6-10-1). As controls, the same protocol was
followed except that the mice were injected with cells from the
immuno.sup.low pool (sp 9-6-2), with cells from the (non selected).
Master pool, with non-transfected modified fibroblasts
(LM-IL-2K.sup.b cells), with PBS, or, as a specificity control,
with LM-IL-2K.sup.b cells transfected with DNA-fragments from B16
melanoma cells (LM-IL-2K.sup.b/B16).
[0231] As indicated, (FIG. 14B), tumor-bearing mice treated solely
by immunization with cells from the immuno.sup.high pool
(3.degree.) survived significantly (p<0.01) longer than
tumor-bearing mice treated by immunization with cells from any of
the other pools. The survival of tumor-bearing mice immunized with
cells from the immuno.sup.low pool or with cells transfected with
DNA from the melanoma cells were not significantly different than
those of tumor-bearing mice injected with PBS. Measurements of
tumor growth in mice treated with the various cell constructs were
consistent with the heightened immunotherapeutic properties of the
immuno.sup.high pool (FIG. 14B).
Example 29
CD8+ T Cells Mediated Immunity Toward KLN25 Cells in Tumor-Bearing
DBA/2 Mice Immunized with Transfected Cells from the
Immuno.sup.high Pool (Sp 6-10-1 (3.degree.)
[0232] MAbs were used to determine the classes of cells mediating
resistance to SCC in tumor-bearing DBA/2 mice immunized with the
transfected cells. As a first step, mice with established (3-5 mm)
neoplasms received the first of two weekly s.c. injections of
4.times.10.sup.6 transfected cells from the immuno.sup.high pool
(3.degree.) (sp 6-10-1). One week later, spleen cells from the
immunized tumor-bearing mice were analyzed for the presence of
cytotoxic cells, at varying effector: target (E:T) ratios. As
controls, the tumor-bearing mice were injected with cells from the
non selected Master Pool, with cells from the immuno.sup.low pool
(sp-9-6-2 (30) or with non transfected LM-IL-2K.sup.b cells. As
indicated (FIG. 15A), the cytotoxic reactions of greatest magnitude
were in mice immunized with cells from the immuno.sup.high pool
(3.degree.). Lesser responses were present in mice immunized with
the non-selected Master Pool (p<0.001). The responses in mice
immunized with cells form the immuno.sup.low pool or with
non-transfected cells were not significantly different than those
of tumor-bearing mice injected with PBS. Analogous results were
obtained if the analyses were performed by ELISPOT-IFN-.gamma.
assays (FIG. 15C).
[0233] The effect of monoclonal antibodies for CD8+, CD4+ and NK1.1
cells on the cytotoxicity reactions were next used to determine the
cell types activated for immunity to KLN205 cells in tumor-bearing
mice immunized with cells from the immuno.sup.high pool
(3.degree.). The results (FIG. 15B) indicated that the addition of
CD8+ antibodies and Complement (C) to the spleen cell suspensions
inhibited the cytotoxic reaction toward KLN205 cells to the
greatest extent. Lesser effects were observed if CD4+ or NK1.1 mAbs
were added. As a specificity control, tumor-bearing DBA/2 mice were
immunized with LM-IL-2K.sup.b cells transfected with DNA from B16
melanoma cells (LM-IL-2K.sup.b/B16). An analysis of the spleen
cell-mediated immunity toward KLN205 cells in these mice failed to
indicate the presence of immunity toward KLN205 cells.
[0234] Various clinical trials are in progress, designed to test
immune-based therapies (Morse M A, Cancer Res 2005; 65:553-61;
Hirschowitz E A, J Clin Oncol. 2004; 22:2808-15; Raez L E, J Clin
Oncol 2004; 22.2800-7; Chang G C, Cancer 2005; 103:763-71). In lung
cancer, determinants such as survivin (Xiang R, Cancer Res 2005;
65:553-61), p185 (HER-2/neu) (Akita K H, Jpn J. Cancer Res 2002;
93:1007-12), epidermal growth factor (Gonzalez G, Ann Oncol 2003;
14:461-6), p53 (Wang T, Lung Cancer 2001; 34:363-74) among others
(Chang G C, Cancer 2005; 103; 763-71; Hirschowitz E A, J Clin Oncol
2004; 22:2808-15) were identified as potential targets of
immune-mediated attack. It is likely that these are only a few of a
potentially large number of TAA. Cancer cells are notoriously
genetically unstable (Peltomaki P, Cancer Res 199.3; 53: 5853-5;
Gonzalez-Zulueta M, Cancer Res 1993; 53: 5620-3; Risinger J I,
Cancer Res 1993; 53: 5100-3; Han H-J, Cancer Res 1993; 53: 5087-9;
Bavoux C, Cancer Res 2005; 65:325-30; Talcahashi Y, Mol Carcinog
2005; 42:150-8).
[0235] An additional important advantage was that the vaccine could
be prepared from microgram amounts of amounts of tumor tissue.
Forty .mu.g of DNA, derived from approximately 10.sup.7 cells, (4
mm tumor) was sufficient. As the transferred DNA spontaneously
integrates into the genome of the recipient cells, and is
replicated as the cells divide, the number of vaccine cells could
be readily expanded for multiple rounds of therapy. The ability to
prepare an effective vaccine from such small neoplasms provides an
opportunity to prepare vaccines from patients with minimal disease
after conventional therapy.
[0236] However, only an undefined, small proportion of the
transfected cells was expected to have incorporated DNA-fragments
specifying TAA. Several lines of evidence indicated that the
strategy designed to enrich the transfected cell population for
highly immunogenic cells resulted in an increase in the vaccine's
immunotherapeutic properties. Both cytotoxicity and
ELISPOT-IFN-.gamma. assays revealed a progressive increase in the
immunogenic properties of the selected cell pools. By the third
round of immune-selection, the percent specific lysis of KLN205
cells from mice immunized with cells from the immuno.sup.high pool
was more than three fold greater than that of mice immunized with
cells from the non-selected (Master) pool.
[0237] Analogous results were obtained if the anti tumor immune
responses were tested by ELISPOT-IFN-.gamma. assays Furthermore,
tumor-bearing mice treated solely by immunization with cells from
the immuno.sup.high pool (3.degree.) survived significantly longer
than mice in various control groups, including mice treated by
immunization with fibroblasts transfected with DNA from B16F1
cells, a melanoma cell line. It is conceivable that the immunogenic
properties of the vaccine could be further enhanced by additional
rounds of immune selection. As the transfected cells failed to
express syngeneic MHC class I-determinants, cross priming may have
been responsible for the induction of immunity to the SCC (Donnelly
J J, J Immunol 2005; 175:633-9; Bohnenkamp H R, Cell Immunol 2004;
231:112-25).
[0238] The cells in the highly immunogenic pool expressed an array
of undefined antigens associated with the squamous carcinoma cells.
TAA expressed by the transfected cells were not identified. The
identification of TAA expressed by the patient's neoplasm may not
be required to generate a vaccine that can be used effectively in
patient therapy. Nevertheless, strategies disclosed herein permit
the identification of such antigens.
[0239] The strategy reported here raises the possibility that an
analogous approach can be used to generate a vaccine of enhanced
effectiveness that can become part of the overall management of
patients with non small cell lung cancer and other histologic types
of cancer as well.
Sequence CWU 1
1
112235DNAMus musculus 1ccacctcaca cacggagcgc cagccttgag tttgttttct
agccccttcc cgcctgttca 60ccaccaccat gaccccgggc attcgggctc ctttcttcct
gctgctactt ctagcaagtc 120taaaaggttt tcttgccctt ccaagtgagg
aaaacagtgt cacctcatct caggacacca 180gcagttcctt agcatcgact
accactccag tccacagcag caactcagac ccagccacca 240gacctccagg
ggactccacc agctctccag tccagagtag cacctcttct ccagccacca
300gagctcctga agactctacc agtactgcag tcctcagtgg cacctcctcc
ccagccacca 360cagctccagt gaactccgcc agctctccag tagcccatgg
tgacacctct tccccagcca 420ctagcctttc aaaagactcc aacagctctc
cagtagtcca cagtggcacc tcttcagctc 480cggccaccac agctccagtg
gattccacca gctctccagt agtccacggt ggtacctcgt 540ccccagccac
cagccctcca ggggactcca ccagctctcc agaccatagt agcacctctt
600ctccagccac cagagctccc gaagactcta ccagtactgc agtcctcagt
ggcacctcct 660ccccagccac cacagctcca gtggactcca ccagctctcc
agtagcccat gatgacacct 720cttccccagc cactagcctt tcagaagact
ccgccagctc tccagtagcc cacggtggca 780cctcttctcc agccaccagc
cctctaaggg actccaccag ttctccagtc cacagtagtg 840cctccatcca
aaacatcaag actacatcag acttagctag cactccagac cacaatggca
900cctcagtcac aactaccagc tctgcactgg gctcagccac cagtccagac
cacagtggta 960cctcaactac aactaacagc tctgaatcag tcttggccac
cactccagtt tacagtagca 1020tgccattctc tactaccaaa gtgacgtcag
gctcagctat cattccagac cacaatggct 1080cctcggtgct acctaccagt
tctgtgttgg gctcagctac cagtctagtc tataatacct 1140ctgcaatagc
tacaactcca gtcagcaatg gcactcagcc ttcagtgcca agtcaatacc
1200ctgtttctcc taccatggcc accacctcca gccacagcac tattgccagc
agctcttact 1260atagcacagt accattttct accttctcca gtaacagttc
accccagttg tctgttgggg 1320tctccttctt cttcttgtct ttttacattc
aaaaccaccc atttaattct tctctggaag 1380accccagctc caactactac
caagaactga agaggaacat ttctggattg tttctgcaga 1440tttttaacgg
agattttctg gggatctcta gcatcaagtt caggtcaggc tccgtggtgg
1500tagaatcgac tgtggttttc cgggagggta cttttagtgc ctctgacgtg
aagtcacagc 1560ttatacagca taagaaggag gcagatgact ataatctgac
tatttcagaa gtcaaagtga 1620atgagatgca gttccctccc tctgcccagt
cccggccggg ggtaccaggc tggggcattg 1680ccctgctggt gctggtctgt
attttggttg ctttggctat cgtctatttc cttgccctgg 1740cagtgtgcca
gtgccgccga aagagctatg ggcagctgga catctttcca acccaggaca
1800cctaccatcc tatgagtgaa taccctacct accacactca cggacgctac
gtgccccctg 1860gcagtaccaa gcgtagcccc tatgaggagg tttcggcagg
taatggcagt agcagtctct 1920cttataccaa cccagctgtg gtgaccactt
ctgccaactt gtaggagcaa gtcaccccac 1980ccacttgggg cagctttggc
ggtctgctcc ctcagtggtc actgccagac ccctgcactc 2040tgatctgggc
tggtgagcca ggacttctgg taggctgttc atgccctttg tcaagcgcct
2100caactacgta agcctggtga agcccagccc tgccctgggg gacactgggg
cagttagtgg 2160tggctctcag aaggactggc ctggaaaact ggagacaggg
atgggaaccc aaacatagct 2220gaataaaaga tggcc 2235
* * * * *
References