U.S. patent application number 10/213939 was filed with the patent office on 2003-03-27 for in situ injection of antigen-presenting cells with genetically enhanced cytokine expression.
This patent application is currently assigned to University of Pittsburgh of the Commonwealth System of Higher Education. Invention is credited to Lotze, Michael T., Nishioka, Yasuhiko, Tahara, Hideaki.
Application Number | 20030060442 10/213939 |
Document ID | / |
Family ID | 22279913 |
Filed Date | 2003-03-27 |
United States Patent
Application |
20030060442 |
Kind Code |
A1 |
Tahara, Hideaki ; et
al. |
March 27, 2003 |
In situ injection of antigen-presenting cells with genetically
enhanced cytokine expression
Abstract
The use of professional antigen presenting cells genetically
modified to enhance expression of an immunostimulatory cytokine is
disclosed for the treatment of individuals having tumors or
infections. The genetically modified professional antigen
presenting cells are injected directly at or near the site of the
tumor or infection. Preferred professional antigen presenting cells
include dendritic cells, and preferred immunostimulatory cytokines
include interleukins such as IL-12.
Inventors: |
Tahara, Hideaki; (Tokyo,
JP) ; Lotze, Michael T.; (Pittsburgh, PA) ;
Nishioka, Yasuhiko; (Tokushima, JP) |
Correspondence
Address: |
TESTA, HURWITZ & THIBEAULT, LLP
HIGH STREET TOWER
125 HIGH STREET
BOSTON
MA
02110
US
|
Assignee: |
University of Pittsburgh of the
Commonwealth System of Higher Education
Pittsburgh
PA
|
Family ID: |
22279913 |
Appl. No.: |
10/213939 |
Filed: |
August 6, 2002 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10213939 |
Aug 6, 2002 |
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09395836 |
Sep 14, 1999 |
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6482405 |
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60100468 |
Sep 15, 1998 |
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Current U.S.
Class: |
514/44R ;
424/93.2; 424/93.21; 435/456 |
Current CPC
Class: |
A61K 2039/5156 20130101;
A61K 2039/5154 20130101; A61K 38/208 20130101; A61P 31/00 20180101;
A61K 2035/124 20130101; A61K 48/00 20130101; A61P 37/04 20180101;
A61P 35/00 20180101 |
Class at
Publication: |
514/44 ;
424/93.21; 424/93.2; 435/456 |
International
Class: |
A61K 048/00; C12N
015/867 |
Goverment Interests
[0001] This work was supported in part by Grant Number POI CA59371
from the National Cancer Institute of the National Institutes of
Health. The government has certain rights in the invention.
Claims
What is claimed is:
1. A method of treating an individual having an tumor comprising:
injecting an individual near a site of said infection or tumor with
an effective amount of genetically modified professional antigen
presenting cells, wherein said professional antigen presenting
cells have been genetically modified to enhance expression of an
immunostimulatory cytokine.
2. A method as in claim 1 wherein said professional antigen
presenting cells are dendritic cells.
3. A method as in claim 2 wherein said dendritic cells have been
genetically modified by transduction with a viral vector encoding
said cytokine.
4. A method as in claim 3 wherein said viral vector is a retroviral
vector.
5. A method as in claim 3 wherein said viral vector is selected
from the group consisting of adenoviral vectors and
adeno-associated viral vectors.
6. A method as in claim 4 wherein said dendritic cells have been
genetically modified by centrifuging said professional antigen
presenting cells with the supernatant of producer cells expressing
said retroviral vector encoding said cytokine.
7. A method as in claim 2 wherein said dendritic cells are selected
from the group consisting of CD34+-derived dendritic cells, bone
marrow-derived dendritic cells, monocyte-derived dendritic cells,
splenocyte derived dendritic cells, skin-derived dendritic cells,
follicular dendritic cells, and germinal center dendritic
cells.
8. A method as in claim 2 wherein said dendritic cells are
CD34+-derived dendritic cells cultured in the presence of at least
one factor selected from the group consisting of G-CSF, GM-CSF,
TNF-.alpha., IL-4, the Flt-3 ligand and the kit ligand.
9. A method as in claim 1 wherein said cytokine is selected from
the group consisting of the interleukins (e.g., IL-1.alpha.,
IL-1.beta., IL-2, IL-3, IL-4, IL-6, IL-8, IL-9, IL-10, IL-12,
IL-18, IL-19, IL-20), the interferons (e.g., IFN-.alpha.,
IFN-.beta., IFN-.gamma.), tumor necrosis factor (TNF), transforming
growth factor-.beta. (TGF-.beta.), granulocyte colony stimulating
factors (G-CSF), macrophage colony stimulating factor (M-CSF),
granulocyte-macrophage colony stimulating factor (GM-CSF), the
Flt-3 ligand and the kit ligand.
10. A method as in claim 1 wherein said individual has a cancer
selected from the group consisting of melanomas, hepatomas,
adenocarcinomas, basal cell cancers, oral cancers, nasopharyngeal
cancers, laryngeal cancers, bladder cancers, head and neck cancers,
renal cell cancers, pancreatic cancers, pulmonary cancers, cervical
cancers, ovarian cancers, esophageal cancers, gastric cancers,
prostate cancers, testicular cancers, and breast cancers.
11. A method of treating an individual having an infection
comprising: injecting an individual near a site of said infection
or tumor with an effective amount of genetically modified
professional antigen presenting cells professional antigen
presenting cells, wherein said professional antigen presenting
cells have been genetically modified to enhance expression of an
immunostimulatory cytokine.
12. A method as in claim 11 wherein said professional antigen
presenting cells are dendritic cells.
13. A method as in claim 12 wherein said dendritic cells have been
genetically modified by transduction with a viral vector encoding
said cytokine.
14. A method as in claim 13 wherein said viral vector is a
retroviral vector.
15. A method as in claim 13 wherein said viral vector is selected
from the group consisting of adenoviral vectors and
adeno-associated viral vectors.
16. A method as in claim 14 wherein said dendritic cells have been
genetically modified by centrifuging said professional antigen
presenting cells with the supernatant of producer cells expressing
said retroviral vector encoding said cytokine.
17. A method as in claim 12 wherein said dendritic cells are
selected from the group consisting of CD34+-derived dendritic
cells, bone marrow-derived dendritic cells, monocyte-derived
dendritic cells, splenocyte derived dendritic cells, skin-derived
dendritic cells, follicular dendritic cells, and germinal center
dendritic cells.
18. A method as in claim 12 wherein said dendritic cells are
CD34+-derived dendritic cells cultured in the presence of at least
one factor selected from the group consisting of G-CSF, GM-CSF,
TNF-.alpha., IL-4, the Flt-3 ligand and the kit ligand.
19. A method as in claim 11 wherein said cytokine is selected from
the group consisting of the interleukins (e.g., IL-1.alpha.,
IL-1.beta., IL-2, IL-3, IL-4, IL-6, IL-8, IL-9, IL-10, IL-12,
IL-18, IL-19, IL-20), the interferons (e.g., IFN-.alpha.,
IFN-.beta., IFN-.gamma.), tumor necrosis factor (TNF), transforming
growth factor-.beta. (TGF-.beta.), granulocyte colony stimulating
factors (G-CSF), macrophage colony stimulating factor (M-CSF),
granulocyte-macrophage colony stimulating factor (GM-CSF), the
Flt-3 ligand and the kit ligand.
Description
FIELD OF THE INVENTION
[0002] The present invention is directed to the field of
immunology. In particular, the present invention is directed to the
use of genetically-modified professional antigen-presenting cells
to combat infections or tumors.
BACKGROUND OF THE INVENTION
[0003] Critical analysis of early events in the cellular immune
response to tumor and viral antigen have identified dendritic cells
as the major antigen-presenting cells (APCs) eliciting an effective
T cell response (Steinman (1991), Annu. Rev. Immunol. 9:271-296;
Macatonia et al. (1989), J. Exp. Med. 169:1255-1264). Dendritic
cells (DCs), appropriately activated, take up soluble antigen and
apoptotic bodies, migrate to the paracortical T cell-rich areas of
lymph nodes, and initiate a series of interactions leading to the
selection of antigen-specific T cells, and to the release of the DC
cytokines, interferon-.alpha. (IFN-.alpha.) and interleukin-12
(IL-12).
[0004] It has previously been demonstrated that administration of
DCs pulsed with synthetic tumor-associated peptides serve as
effective therapeutic antitumor vaccines, inducing an effective
antitumor immune response in vitro and following adoptive transfer
in mice (Mayordomo et al. (1995), Nature Med. 1:1297-1302; Zitvogel
et al. (1996), J. Exp. Med. 183:87-97; Porgador and Gilboa (1995),
J. Exp. Med. 182:255-260; Porgador et al. (1996), J. Immunol.
156:2918-2926). However, T cell-defined epitopes have been
identified only for a limited number of human tumor types. Several
approaches to overcome this problem including pulsing DCs with
acid-eluted bulk tumor peptides Zitvogel et al. (1996), tumor
extracts and RNA (Flamand et al. (1994), Eur. J. Immunol.
24:605-610; Ashley et al. (1997), J. Exp. Med. 186:1177-1182;
Boczkowski et al. (1996), J. Exp. Med. 184:465-472) or fusion of
tumor with DC (Gong et al. (1997) Nature Med. 3:558-561) have been
employed for DC-based vaccination strategies against tumors. Even
though these approaches will allow treatment of tumors for which
tumor associated antigen is not well characterized, there are still
significant problems, particularly in the preparation of clinical
samples from human solid cancers.
[0005] IL-12 is a heterodimeric cytokine produced by DCs,
macrophages, polymorphonuclear leukocytes and keratinocytes (Lamont
and Adorini (1996), Immunol. Today 17:214-217). IL-12 enhances
natural killer (NK) cell and cytotoxic T lymphocyte (CTL)
activities, plays a key role in the induction of Th1 immune
responses including IFN-.gamma. production, and has
IFN-.gamma./interferon-inducible protein 10 (IP-10)-dependent
antiangiogenic effects (Lamont and Adorini (1996), supra; Voest et
al. (1995), J. Natl. Cancer Inst. 87:581-586; Sgadari et al.
(1996), Blood 87:3877-3882). DCs are capable of producing IL-12
after ligation of CD40 and class II molecules, presumably only
following interaction with T cells, and IL-12 delivery in
conjunction with DCs enhances CTL response in vitro (Heufler et al.
(1996), Eur. J. Immunol. 26:659-668; Koch et al. (1996), J. Exp.
Med. 184:741-746; Bhardwaj et al. (1996), J. Clin. Invest.
98:715-722).
[0006] There have been reports of potent antitumor effects of IL-12
in a vaccination model with IL-12 gene-modified tumor cells, as
well as with systemic administration of IL-12 protein (Brunda et
al. (1993), J. Exp. Med. 178:1223-1230; Nastala et al. (1994), J.
Immunol. 153:1697-1706; Tahara et al. (1995), J. Immunol.
154:6466-6474; Martinotti et al. (1995), Eur. J. Immunol.
25:137-146). Direct injection of IL-12-transduced fibroblasts also
effectively eliminated established tumors with concomitant
induction of effective systemic immunity (Zitvogel et al. (1995),
J. Immunol. 155:1393-1403). Based on these results, an initial
clinical trial of IL-12 gene therapy has been completed using
autologous fibroblasts in the context of a phase I study (Tahara et
al. (1997), Proc. Am. Soc. Clin. Oncol. 16:439a). Partial responses
were observed in patients with melanoma, breast cancer, and head
and neck tumors persisting for up to two years.
SUMMARY OF THE INVENTION
[0007] The present invention depends, in part, upon the discovery
that professional antigen-presenting cells (APCs) which have been
genetically modified to enhance expression an immunostimulatory
cytokine, may be directly injected into or near the site of an
infection or tumor to induce a specific immunological response
against antigens associated with the infection or tumor without
pre-loading or pulsing the APCs with the antigens. In particular,
it has been found that dendritic cells (DCs), and preferably bone
marrow-derived dendritic cells (BM-DCs) or CD34+-derived dendritic
cells (CD34+-DCs), which have been genetically modified to enhance
expression an immunostimulatory cytokine, preferably interleukin-12
(IL-12), may be injected into or near the site of an infection or
tumor to induce a specific immune response against antigens
associated with the site of injection.
[0008] Therefore, in one aspect, the present invention provides
methods for treating an individual having an infection or tumor
comprising injecting the individual near or at the site of the
infection or tumor with an effective amount of professional antigen
presenting cells (APCs) which have been genetically modified to
enhance the expression of an immunostimulatory cytokine.
[0009] In preferred embodiments, the genetically modified APCs are
professional antigen presenting cells, and most preferably the
PAPCs are dendritic cells selected from the group consisting of
CD34+-derived dendritic cells, bone marrow-derived dendritic cells,
monocyte-derived dendritic cells, splenocyte derived dendritic
cells, skin-derived dendritic cells, follicular dendritic cells,
and germinal center dendritic cells. In particularly preferred
embodiments, the dendritic cells are CD34+-derived dendritic cells
cultured in the presence of at least one factor selected from the
group consisting of G-CSF, GM-CSF, TNF-.alpha., IL-4, the Flt-3
ligand and the kit ligand.
[0010] In addition, in preferred embodiments, the immunostimulatory
cytokine is selected from the group consisting of the interleukins
(e.g., IL-1.alpha., IL-1.beta., IL-2, IL-3, IL-4, IL-6, IL-8, IL-9,
IL-10, IL-12, IL-18, IL-19, IL-20), the interferons (e.g.,
IFN-.alpha., IFN-.beta., IFN-.gamma.), tumor necrosis factor (TNF),
transforming growth factor-.beta. (TGF-.beta.), granulocyte colony
stimulating factor (G-CSF), macrophage colony stimulating factor
(M-CSF), granulocyte-macrophage colony stimulating factor (GM-CSF),
the Flt-3 ligand and the kit ligand.
[0011] Further, in preferred embodiments, the APCs have been
genetically modified by transduction with a viral vector encoding
the immunostimulatory said cytokine and, most preferably, with a
retroviral vector. In other embodiments, however, the APCs may be
genetically modified with adenoviral vectors or adeno-associated
viral vectors, or by lipofection, ballistic injection, or other
means of genetic modification known in the art.
[0012] Further, in any of the foregoing embodiments, the individual
which is treated may suffer from a cancer selected from the group
consisting of melanomas, hepatomas, adenocarcinomas, basal cell
cancers, oral cancers, nasopharyngeal cancers, laryngeal cancers,
bladder cancers, head and neck cancers, renal cell cancers,
pancreatic cancers, pulmonary cancers, cervical cancers, ovarian
cancers, esophageal cancers, gastric cancers, prostate cancers,
testicular cancers, breast cancers, or other solid tumors.
Alternatively, the individual may suffer from a refractory
infection.
BRIEF DESCRIPTION OF THE DRAWINGS
[0013] FIG. 1 shows the time course of IL-12 production by
IL-12-gene modified BM-DCs. (A) After transduction on day 4,
supernatant in DC culture with GM-CSF and IL-4 was collected daily
and assayed in an IL-12 ELISA. (B) Day 6 BM-DCs transduced with
IL-12 gene were harvested, washed twice, and recultured at 10.sup.6
cells/ml to evaluate IL-12 production. Open circles indicate
non-transduced cells, open triangles indicate DFG-hCD80-neo
transduced cells, and solid circles indicate DFG-mIL-12 transduced
cells.
[0014] FIG. 2 shows the changes in area of established (A) MCA205,
(B) B16, and (C) D122 tumors after injection of HBSS (open
circles), 10.sup.6 Zeo-transduced (triangles) and IL-12-transduced
(solid circles) BM-DCs into the tumors. Data are presented as
mean.+-.SE.
[0015] FIG. 3 shows the correlation between IL-12 production by
genetically modified DCs and the effect of intratumoral injection
of these DCs on the area of MCA205 tumors.
[0016] FIG. 4 shows the effect on the growth of established MCA205
tumors of injection of HBSS (open circles), 10.sup.6 Zeo-transduced
(triangles), IL-12-transduced BM-DCs (squares) or IL-12-transduced
syngeneic fibroblasts (solid circles). Data are presented as
mean+SE.
[0017] FIG. 5 shows the effect on the growth of established MCA205
tumors of repeated injections of HBSS (open circles), 10.sup.6
IL-12-transduced BM-DCs (solid circles) or IL-12-transduced
syngeneic fibroblasts (triangles). Data are presented as
mean.+-.SE.
[0018] FIG. 6 shows (A) the tumor-specific CTL activity of
splenocytes from mice treated with HBSS (open circles),
Zeo-(triangles) and IL-12-(squares) transduced BM-DCs and
IL-12-transduced fibroblasts (solid circles) against MCA205, and
(B) CTL activity by splenocytes from mice injected with
IL-12-transduced BM-DCs against MCA205 (open circles), YAC-1
(triangles) and syngeneic fibroblasts (squares).
[0019] FIG. 7 shows the effect of intratumoral injection of HBSS
(open circles), Zeo-transduced (triangles) and IL-12-transduced
(solid circles) BM-DCs on the area of (A) injected and (B)
non-injected, contralateral MCA205 or B16 tumors. Data are
presented as mean.perp.SE.
DETAILED DESCRIPTION OF THE INVENTION
[0020] Definitions
[0021] In order to more clearly and concisely point out and
describe the subject matter which applicants consider as the
invention, the following definitions are provided for certain terms
used in the written description and appended claims.
[0022] As used herein, the term "antigen-presenting cell" or the
abbreviation "APC" means a cell that can process a protein antigen,
break it into peptides, and present it in conjunction with MHC
molecules on the cell surface, where it may interact with
appropriate T cell receptors. The term antigen presenting cell, as
used herein, is intended to encompass both professional and
non-professional antigen presenting cells.
[0023] As used herein, the term "professional antigen-presenting
cell" or the abbreviation "PAPC" means an antigen presenting cell
with highly efficient immunostimulatory capacity. PAPCs display
antigenic peptide fragments in association with the proper class of
MHC molecules and also bear costimulatory surface molecules.
Different classes of PAPCs include Langherhans' cells,
interdigitating cells (IDCs), follicular dendritic cells (FDCs),
germinal center dendritic cells (GCDCs), B cells, and
macrophages.
[0024] As used herein with respect to the APCs of the invention, a
"genetically modified" APC means an APC into which, or into an
ancestor or precursor of which, has been introduced an exogenous
nucleic acid which is transcribed and translated to produce
molecules encoded by the introduced nucleic acid, or which
integrates into the genome of an APC and enhances the transcription
and/or translation of endogenous nucleic acid sequences encoding an
immunostimulatory cytokine. The term "genetically modified" may be
used herein to embrace any method of introducing exogenous nucleic
acids including, but not limited to, transformation, transduction,
transfection, and the like.
[0025] As used herein, a coding sequence and a regulatory region
are said to be "operably joined" when they are covalently linked in
such a way as to place the expression or transcription of the
coding sequence under the influence or control of the regulatory
region. If it is desired that the coding sequences be translated
into a functional protein, two DNA sequences are said to be
operably joined if induction of promoter function results in the
transcription of the coding sequence and if the nature of the
linkage between the two DNA sequences does not (1) result in the
introduction of a frame-shift mutation, (2) interfere with the
ability of the regulatory region to direct the transcription of the
coding sequences, or (3) interfere with the ability of the
corresponding RNA transcript to be translated into a protein. Thus,
a regulatory region would be operably joined to a coding sequence
if the regulatory region were capable of effecting transcription of
that DNA sequence such that the resulting transcript might be
translated into the desired protein or polypeptide.
[0026] As used herein with respect to the genetically modified APCs
of the invention, the term "enhance expression" of an
immunostimulatory cytokine means to increase the levels of
transcription and/or translation of nucleic acid sequences encoding
the cytokine.
[0027] As used herein, the term "immunostimulatory cytokine" means
a soluble molecule which mediates interactions amongst immune
system cells and, in particular, causes an activation or increase
in an immune response against an antigenic peptide presented by an
APC. Particularly intended are immunostimulatory cytokines selected
from the group consisting of the interleukins (e.g., IL-1.alpha.,
IL-1.beta., IL-2, IL-3, IL-4, IL-6, IL-7, IL-8, IL-9, IL-10, IL-12,
IL-18, IL-19, IL-20), the interferons (e.g., IFN-.alpha.,
IFN-.beta., IFN-.gamma.), tumor necrosis factor (e.g.,
TNF-.alpha.), transforming growth factor-.beta. (TGF-.beta.),
granulocyte colony stimulating factors (G-CSF), macrophage colony
stimulating factor (M-CSF), granulocyte-macrophage colony
stimulating factor (GM-CSF), the Flt-3 ligand and the kit ligand.
As used herein, references to any of these cytokines are intended
to embrace the human homologs and any other mammalian homologs
having activity in humans substantially similar to the human
protein.
[0028] I. Methods of Treatment
[0029] The present invention derives, in part, from the discovery
that antigen-presenting cells (APCs), which have been genetically
modified to enhance expression an immunostimulatory cytokine, may
be directly injected into or near the site of an infection or tumor
to induce a specific immunological response against antigens
associated with the infection or tumor without pre-loading or
pulsing the APCs with the antigens. In particular, it has been
found that professional antigen presenting cells, such as dendritic
cells (DCs), and more preferably bone marrow-derived dendritic
cells (BM-DCs) or CD34+-derived dendritic cells (CD34+-DCs), which
have been genetically modified to enhance expression an
immunostimulatory cytokine, preferably interleukin-12 (IL-12), may
be injected into or near the site of an infection or tumor to
induce a specific immune response against antigens associated with
the site of injection. Significantly, in experiments involving
injection into tumors, it has been found that professional antigen
presenting cells, such as DCs engineered to constitutively express
IL-12 can cause tumor regression, both at the site of injection and
at distant sites, and can induce tumor-associated antigen (TAA)
specific Th1 cell responses in both regional lymph nodes and the
spleen.
[0030] Thus, in general, the present invention provides methods of
treatment for subjects, particularly human subjects, having an
infection or tumor, in which APCs, preferably professional antigen
presenting cells, genetically modified to enhance expression an
immunostimulatory cytokine are injected into or near the site of
the infection or tumor. Thus, the methods comprise the steps
obtaining a sample of APCs, genetically modifying the APCs to
enhance expression an immunostimulatory cytokine, and injecting the
genetically modified APCs, preferably professional antigen
presenting cells, into or near the site of an infection or tumor.
Before or after genetically modifying the APCs, and before
injecting them, they may be clonally expanded by standard
techniques of cell culture which are well known in the art. If the
APCs are to be autologous, the steps of obtaining and modifying the
cells, as well as optionally clonally expanding the cells, is
preferably performed as close as possible to the time of injection.
If, however, heterologous but syngeneic APCs are to be used, the
cells may be obtained and genetically modified far in advance of
injection, and may be maintained indefinitely prior to use.
[0031] Subjects for treatment with the methods of the present
invention include cancer patients and subjects with refractory
infections. Because it is preferred that the genetically-modified
APCs, preferably professional antigen presenting cells, are
injected directly into the site of a tumor or infection, it is
expected that the present methods will be most useful in subjects
having at least one physically well-defined tumors or a physically
well-defined site of an infection, rather than a diffuse, highly
metastasized cancer or diffuse infection. On the other hand, as
shown in the examples below, injection into one site at which a
tumor (or infection) is present, can lead to the development of a
systemic immune response. Therefore, the methods of the present
invention may be effective in treating diffuse, highly metastasized
cancers or diffuse infections if at least one site of a tumor or
infection can be identified at which the APCs, preferably
professional antigen presenting cells, can be injected and can
effectively load tumor-associated or infection-associated
antigens.
[0032] In currently preferred embodiments, the methods of the
present invention are used to treat patients having solid tumors,
into which the genetically modified APCs, preferably professional
antigen presenting cells, of the invention may be directly
injected. Appropriate solid tumors may include melanomas,
hepatomas, adenocarcinomas, basal cell cancers, oral cancers,
nasopharyngeal cancers, laryngeal cancers, bladder cancers, head
and neck cancers, renal cell cancers, pancreatic cancers, pulmonary
cancers, cervical cancers, ovarian cancers, esophageal cancers,
gastric cancers, prostate cancers, testicular cancers, and breast
cancers. For many of these cancers, an association between DC
infiltration and prognosis has been established.
[0033] The genetically modified APCs of the invention may be
injected using standard sterile techniques for subcutaneous,
intradermal, transdermal, intramuscular, intraperitoneal or other
forms of injection. The cells may be administered in a
physiologically acceptable solution or buffer and may be
administered in combination with other agents, particularly
cytokines, which may promote the ability of the APCs to survive,
load antigen, traffic to the draining lymph nodes or spleen, and
present antigen to activate an immune response. The number of cells
to be introduced depends upon a number of factors, including the
number of sites to be injected, the number of injections which are
to be performed over time, the size of the tumor or infectious
lesion, and the nature of the tumor or infection. Although the
number of cells to be used will vary with such factors, it is
presently expected that 10.sup.4-10.sup.8, preferably
10.sup.5-10.sup.7, cells will be injected per site per
treatment.
[0034] Details and currently preferred embodiments relating to the
choice and isolation of APCs, choice of immunostimulatory
cytokines, and genetic modification of the APCs, are described
separately below.
[0035] II. Choice and Isolation of APCs
[0036] The present invention employs antigen presenting cells APCs,
preferably professional antigen presenting cells, most preferably,
dendritic cells, which have been genetically modified to enhance
expression of an immunostimulatory cytokine.
[0037] Preferably, the original source of the APCs is the subject
to be treated, such that the APCs are autologous. Allogeneic APCs,
obtained from other individuals, may also be employed in the
present invention, but preferably the APCs are derived from
histocompatible or syngeneic individuals so as to provide proper
MHC presentation to the cognate, antigen-specific T cell receptors
of the subject. In addition, genetically engineered animals, such
as mice or pigs, may be created which express human or humanized
MHC proteins, and optionally co-stimulatory molecules, and may be
used as a renewable source of APCs capable of proper MHC
presentation to the cognate, antigen-specific T cell receptors of
the subject.
[0038] Preferred PAPCs are dendritic cells, and particularly
CD34+-derived DCs (CD34+-DCs) harvested from mobilized peripheral
blood, and bone marrow-derived dendritic cells (BM-DCs) harvested
from bone marrow. Other DCs which may be useful in the invention
include monocyte-derived DCs harvested from blood, CD34+-DCs
harvested from bone marrow, splenocyte derived DCs harvested from
the spleen, skin-derived DCs, follicular dendritic cells (FDCs),
and germinal center dendritic cells (GCDCs). Methods of isolating
these dendritic cells from the tissues in which they arise and or
localize are well known in the art.
[0039] For example, methods for isolating BM-DCs are described in
Inaba et al. (1992), J. Exp. Med. 176:1693-1702. Alternatively,
CD34+ progenitor cells may be obtained from human umbilical cord or
adult blood, and may be stimulated with cytokines to differentiate
into dendritic cells (see, for example, Caux et al. (1996) J. Exp.
Med. 184:695-706; Romani et al. (1994), J. Exp. Med. 180:83-93).
The effectiveness of the Flt-3 ligand in generating dendritic cells
is described in, for example, Shurin et al. (1997), Cell Immunol.
179:174-184. BM-DCs or CD34+-DCs cultured with GM-CSF and IL-4 for
several (e.g., 5 days) are particularly preferred. TNF-.alpha. and
the kit ligand have also been shown to be effective in to increase
the yield of DCs grown in culture (see, e.g., Mayordomo et al.
(1997), Stem Cells 15:94-103, and references cited therein), and
may be used to obtain the DCs of the present invention. A majority
of such DCs may display the immature phenotype as determined by
flow cytometry and MLR assay in accordance with previous reports
(Pierre et al. (1997), Nature 388:787-792; Inaba et al. (1993), J.
Exp. Med. 178:479-488). DCs have antigen-capturing and processing
as well as trafficking abilities only during their immature phase
(Pierre et al. (1997), Nature 388:787-792; Inaba et al. (1993), J.
Exp. Med. 178:479-488; Cella et al. (1997), Nature
388:782-787).
[0040] III. Choice of Cytokines
[0041] The APCs of the present invention are genetically modified
to enhance expression of an immunostimulatory cytokine. Preferably,
the cytokine is one of the interleukins (e.g., IL-1.alpha.,
IL-1.beta., IL-2, IL-3, IL-4, IL-6, IL-7, IL-8, IL-9, IL-10, IL-12,
IL-18, IL-19, IL-20), the interferons (e.g., IFN-.alpha.,
IFN-.beta., IFN-.gamma.), tumor necrosis factor (TNF), transforming
growth factor-.beta. (TGF-.beta.), granulocyte colony stimulating
factors (G-CSF), macrophage colony stimulating factor (M-CSF),
granulocyte-macrophage colony stimulating factor (GM-CSF), the
Flt-3 ligand or the kit ligand. The amino acid sequences of these
cytokines are well known in the art. Thus, an amino acid sequence
of interleukin-4 may be found in, for example, Arai et al. (1989),
J. Immunol. 142(1):274-282; an amino acid sequence of interleukin-6
may be found in, for example, Yasukawa et al. (1987), EMBO J.
6(10):2939-2945; amino acid sequences of the p35 and p40 subunits
of interleukin-12 may be found in, for example, Wolf et al. (1991),
J. Immunol. 146(9):3074-3081; amino acid sequences of various
IFN-.alpha. subtypes may be found in, for example, Gren et al.
(1984), J. Interferon Res. 4(4):609-617, and Weismann et al.
(1982), Princess Takamatsu Symp. 12:1-22; an amino acid sequence of
TNF may be found in, for example, Pennica et al. (1984), Nature
312:724-729; an amino acid sequence of G-CSF may be found in, for
example, Hirano et al. (1986), Nature 324:73-76; and an amino acid
sequence of GM-CSF may be found in, for example, Cantrell et al.
(1985), Proc. Natl. Acad. Sci. (USA) 82(18):6250-6254. In order to
genetically modify APCs to enhance expression of one of these
cytokines, one of ordinary skill in the art may choose to use a
vector comprising a naturally occurring nucleic acid sequence which
encodes the cytokine (e.g., a genomic or cDNA sequence) or may,
utilizing the degeneracy of the genetic code, design and produce a
vector comprising a non-naturally occurring sequence which still
encodes a functional cytokine. In the case of heterodimeric
immunostimulatory cytokines (e.g., IL-12), the APCs of the
invention must be genetically modified to express both subunits of
the cytokine molecule.
[0042] The APCs of the invention may also be genetically modified
to express variants of these cytokines. For example, for those
cytokines having both pro-forms and mature forms (e.g., before and
after cleavage of a signal peptide, or before and after limited
proteolysis to yield an active fragment), the APCs of the invention
may be genetically modified to express either the pro- or mature
form. Other variants, such as fusion proteins between an active
fragment of a cytokine and a heterologous sequence (e.g., a
heterologous signal peptide), may also be employed. Species
variants may also be employed to the extent that they retain
activity in a human subject. Thus, for example, human APCs may be
genetically modified to express a murine, bovine, equine, ovine,
feline, canine, non-human primate or other mammalian variant of a
human cytokine if these species variants retain activity
substantially similar to their human homologues.
[0043] IV. Genetic Modification of APCs
[0044] The APCs of the invention may be genetically modified by any
standard technique known in the art to introduce nucleic acids
encoding the desired cytokines. For example, methods of genetic
modification of human dendritic cells by retroviral transduction
are described in Reeves et al. (1996), Cancer Res. 56:5672-5677,
and Specht et al. (1997), J. Exp. Med. 186:1213-1221. Similarly,
genetic modification of bone marrow cells by retroviral-mediated
transfer of the IL-4 gene is described in Chambers et al. (1992),
J. Immunol. 149(9):2899-2905. In addition, retroviral transduction
of human CD34.sup.+ progenitor cells, with subsequent cytokine
stimulation to promote differentiation and maturation into
dendritic cells, with or without centrifugation, has been described
in Henderson et al. (1996), Cancer Res. 56:3763-3770, and Reeves et
al. (1996), Cancer Res. 56:5672-5677. Use of a retroviral
supernatant, instead of co-cultivation with retroviral producer
cells (Specht et al. (1997)), has several advantages over other
strategies including: (1) there is no direct toxicity to the cells;
(2) stable gene expression is attained; (3) there is minimal
virus-specific CTL response unlike methods with adenoviral vectors
(see, e.g., Smith et al. (1996), J. Virol. 70:6733-6740); and (4)
there is more extensive clinical experience with the use of
retroviral supernatants.
[0045] The genetic modification of the APCs of the present
invention may be transient or stable. That is, the
cytokine-encoding nucleic acid sequences which are introduced into
the APCs may exist apart from the cells' genomic DNA and be
expressed only temporarily, or they may integrate into the cells'
genomes and continue to be expressed throughout the life of the
cells. For example, transient expression of IL-6 using adenovirus
vectors is described in Richards et al. (1995), Ann. NY Acad. Sci.
762:282-292. For transiently expressed sequences, it is preferred
that the expression of the immunostimulatory cytokines continue for
at least one and preferably several days, to allow adequate time
for the APCs to pick up antigen at the site of an infection or
tumor, to migrate to regional lymph nodes, and to activate cognate
T cells by presentation of antigen.
[0046] Preferably the genetic construct which is used to enhance
the expression of an immunostimulatory cytokine includes a
constitutive promoter which is operably joined to the sequences
encoding the cytokine to allow constitutive expression of the
cytokine by the genetically modified APCs. Alternatively, however,
inducible promoters may be used if the conditions for induction can
be met under physiological conditions with or without additional
treatment of the subject (e.g., administering an inducer).
[0047] In addition to the retroviral methods of genetically
modifying APCs described above and in the Examples below, many
other methods of producing appropriate vectors, genetically
modifying cells with those vectors, and identifying transformants
are well known in the art and are only briefly reviewed here (see,
for example, Sambrook et al. (1989) Molecular Cloning: A Laboratory
Manual, 2nd ed., Cold Spring Harbor Laboratory Press, Cold Spring
Harbor, N.Y.). A wide variety of vectors have been developed and
are commercially available which allow inducible (e.g., LacSwitch
expression vectors, Stratagene, La Jolla, Calif.) or constitutive
(e.g., pcDNA3 vectors, Invitrogen, Chatsworth, Calif.) expression
of nucleotide sequences under the regulation of an artificial
promoter element. Such promoter elements are often derived from CMV
or SV40 viral genes, although other strong promoter elements, which
are active in eukaryotic cells, can also be employed to induce
transcription. Typically, these vectors also contain an artificial
polyadenylation sequence and 3' UTR which can also be derived from
exogenous viral gene sequences or from other eukaryotic genes.
Furthermore, in some constructs, artificial, non-coding, introns
and exons may be included in the vector to enhance expression of
the cytokine sequence of interest. These expression systems are
commonly available from commercial sources and are typified by
vectors such as pcDNA3 and pZeoSV (Invitrogen, San Diego, Calif.).
Innumerable commercially-available as well as custom-designed
expression vectors are available from commercial sources to allow
expression of any desired transcript in more or less any desired
cell type, either constitutively or after exposure to an exogenous
stimulus (e.g., withdrawal of tetracycline or exposure to
IPTG).
[0048] Vectors may be introduced into the APCs by various methods
well known in the art including, but not limited to, calcium
phosphate transfection, strontium phosphate transfection, DEAE
dextran transfection, electroporation, lipofection (e.g., Dosper
Liposomal transfection reagent, Boehringer Mannheim, Germany),
microinjection, ballistic injection on micro-beads using a "gene
gun", or, for viral vectors, by infection with the recombinant
virus.
EXAMPLES
[0049] Retroviral vectors. The construction of retroviruses
DFG-mIL-12, TFG-mIL-12 and DFG-hCD80-neo have been described in
previous studies (Tahara et al. (1995); Zitvogel et al. (1996),
Eur. J. Immunol. 26:1335-1341). MFG-EGFP and MFG-Zeo were created
by subcloning the respective fragments obtained from pEGFP-N1
(Clontech, Palo Alto, Calif.) and pcDNA3.1/Zeo(-) (Invitrogen,
Carlsbad, Calif.) (Cormack et al. (1996), Gene 173:33-38).
Retroviral supernatant was generated by transfecting these proviral
constructs into BOSC23 or BING packaging cell lines (Tahara et al.
(1995)). CRE and CRIP cells producing DFG-hCD80-neo retroviruses
were created by the infection of these packaging cells with BING or
BOSC23-produced retroviruses, respectively with subsequent
selection with G418 (Geneticin; Life Technologies, Inc., Grand
Island, N.Y.). The titer of retroviral supernatants was calculated
from the G418-surviving colonies after the transduction into NIH3T3
cells.
[0050] Tumor cell lines and mouse strains. A variety of
transplantable murine tumor cell lines were injected into mice to
initiate tumors, which were then treated with the genetically
modified APCs of the invention. MCA205 methylcholanthrene-induced
fibrosarcoma was generously provided by S. A. Rosenberg (National
Cancer Institute, Bethesda, Md.). The B16F10 murine melanoma cell
line was kindly provided by E. Gorelik (University of Pittsburgh,
Pittsburgh, Pa.). The D122 highly metastatic variant of 3LL tumor
cells was kindly provided by L. Aisenbach (Weizmann Institute of
Science, Rehovot, Israel). YAC-1 was a generous gift by W. Chambers
(University of Pittsburgh, Pittsburgh, Pa.). These cell lines were
maintained in RPMI 1640 supplemented with 10% heat-inactivated
fetal bovine serum, 2 mM glutamine, 100 .mu.g/ml streptomycin, 100
IU/ml penicillin, and 5.times.10.sup.-5 M 2-ME (all from Life
Technologies, Inc., Grand Island, N.Y.), referred to henceforth as
complete medium (CM).
[0051] Female 6-8-week-old C57BL/6 (B6) mice were purchased from
Taconic Farms (Germantown, N.Y.) and used for all experiments at
the age of 8 to 10 weeks old.
[0052] Culture and genetic modification of APCs. BM-DC culture was
obtained using methods previously described (Mayordomo et al.
(1995); Zitvogel et al. (1996); Inaba et al. (1992), J. Exp. Med.
176:1693-1702). Briefly, murine bone marrow cells were harvested
from the femur and tibia of sacrificed mice. Contaminating
erythrocytes were lysed with 0.83M NH.sub.4Cl buffer and
lymphocytes were depleted with the cocktail of antibodies
(RA3-3A1/6.1, anti-B220; 2.43, anti-Lyt 2; GK1.5, anti-L3T4; all
from American Type Culture Collection, Rockville, Md.) and rabbit
complement (Accurate Chemical and Scientific Corp., Westbury, N.Y.)
on day 0. These cells were cultured overnight in CM to remove the
adherent macrophages, and then non-adherent cells were placed in
fresh CM containing rmGM-CSF (1000 U/ml) and rmIL-4 (1000 U/ml) (DC
media) on day 1. Cells were generally harvested on day 6. BM-DCs
were defined by morphology, phenotype and strong mixed lymphocyte
reaction-stimulating activity. Phenotypic analysis by flow
cytometry showed high expression of CD11b, CD11c, CD80, CD86, as
well as MHC class I and class II in the majority of the cultured
cells (60-95%).
[0053] For the retroviral transduction, 1.times.10.sup.6 BM cells
cultured in DC media for 24 h were aliquoted to 14-ml round bottom
tubes and suspended in 1 ml of the retroviral supernatant with 8
.mu.g/ml polybrene, 1000 U/ml rmGM-CSF and 1000 U/ml rmIL-4. These
cells were centrifuged at 2500.times.g at 30-32.degree. C. for 2 h
(Kotani et al. (1994), Hum. Gene Ther. 5:19-28; Bahnson et al.
(1995), J. Virol. Methods 54:131-143). After centrifugation, cells
were cultured in DC media. The transduction process was repeated on
day 3 and 4. Retroviral supernatant from the ecotropic producer
cells, BOSC23 and CRE, transduced murine BM-DCs more effectively
when compared with amphotropic viruses at comparable titers. The
retroviral supernatants from BOSC23 cells was used because they
produced the highest titer of virus (2-8.times.10.sup.6 cfu/ml). To
examine the transduction efficiency of murine BM-DCs, we generated
retroviral vectors with inserted human CD80 (B7.1) or EGFP genes as
transduction markers and determined the efficiency of transduction
by flow cytometry. Retrovirally modified DCs at high transduction
efficiency (22-75%) could express the transgenes for at least 12
days after the last transduction (on day 4) in culture.
Transduction efficiency was well correlated with the titer of
retroviral supernatants used. Two color immunofluorescence staining
showed that significant numbers of marker (hCD80)-positive cells
also expressed high levels of mCD80 as well as CD86, MHC class II
and DEC-205.
[0054] Transduction with the EGFP retroviral vector revealed that
murine BM-DCs can be retrovirally transduced at high efficiency.
Furthermore, BM-DCs were also transduced with a retroviral vector
modified to express both IL-12 genes (DFG-mIL-12). After completion
of the transduction procedures on day 4, the concentration of
mIL-12 p70 heterodimer in culture media was measured by ELISA. As
shown in FIG. 1A, the accumulation of heterodimeric IL-12 (p70) was
observed in cultures of DFG-mIL-12-transduced BM-DC, but not in
cultures of nontransduced or marker gene-transduced cells. On day
6, DCs were harvested, washed twice and transferred to a new plate.
At that time, IL-12-transduced DCs produced approximately 80
ng/10.sup.6 cells/48 h of heterodimeric IL-12 (FIG. 1B). The range
of IL-12 production from IL-12-transduced DCs was 8-80 ng/10.sup.6
cells/48 h, and was related to the titer of the retroviral
supernatant used in each experiment. IL-12 protein produced by the
genetically modified DCs was confirmed to be biologically active
and capable of stimulating IFN-.gamma. production from Con
A-treated splenocytes. To examine the effect of IL-12 transduction
on DC phenotype, various cell surface molecules were examined using
flow cytometry. IL-12-transduced DCs did not differ from non- or
Zeo-transduced DCs except that they expressed increased levels of
MHC class I and II molecules.
[0055] Culture and genetic modification of fibroblasts. For
comparison with the therapeutic effectiveness of the genetically
modified APCs of the invention, similarly modified fibroblasts were
prepared. In brief, primary culture of syngeneic fibroblasts were
obtained from the lungs of B6 mice. Small pieces of lung were
minced with scissors, and stirred in a triple enzyme solution of
collagenase IV, hyaluronidase V and deoxyribonuclease IV (Sigma, St
Louis, Mo.) for 3 h at room temperature. After rinsing twice with
HBSS, the cell suspension was cultured in CM to obtain the primary
culture of fibroblasts. IL-12-transduced fibroblasts were generated
by infection with the supernatant of CRIP-TFG-mIL-12-neo followed
by selection with G418.
[0056] Intratumoral injection of genetically modified APCs. To
produce animal models of cancer patients, mice (four or five
animals per group) were injected intradermally in the right flank
with 1.times.10.sup.5 cells of the MCA205, B16 and D122 tumor lines
on day 0. On day 7, when tumor sizes became 10-20 mm.sup.2,
10.sup.6 non-transduced or transduced BM-DCs were injected
intratumorally. IL-12-transduced syngeneic fibroblasts were used
for intratumoral injection after irradiation (5000 rad) as
described previously (Zitvogel et al. (1995)). When mice rejected
the established tumor, they were rechallenged with a larger number
of tumor cells (2.times.10.sup.5) on the opposite flank to evaluate
the induction of protective systemic immunity against tumor.
[0057] Suppression and/or rejection of established tumors. To
examine the antitumor effect of intratumoral injection with APCs
genetically modified to enhance expression of an immunostimulatory
cytokine, 10.sup.6 nontransduced or IL-12-transduced BM-DCs were
injected into the day 7 established tumors (MCA205, B16 and D122)
(tumor diameter; 3-5 mm). As shown in FIG. 2, IL-12-transduced DCs
significantly suppressed the growth of these established tumors,
resulting in eventual rejection in 2 of 5 mice injected with
MCA205. Neither non-nor Zeo-transduced DCs had any antitumor
effects. In these experiments, IL-12 production of genetically
modified DCs was 29 ng/10.sup.6 cells/48 h. In total, a single
treatment with IL-12-transduced DCs resulted in rejection of
established MCA205 tumors in 5 of 14 mice (36%). These tumor free
mice rejected a subsequent rechallenge with twice as many MCA205,
suggesting acquisition of immunological memory for the injected
tumor. As shown in FIG. 3, the antitumor effects of
IL-12-transduced DCs was correlated (r=-0.80, p<0.05) with IL-12
production on day 28 using Pearson's linear regression. In another
experiment, mice were initially injected i.d. with 1.times.10.sup.5
MCA205 cells and then, on day 7, HBSS (open circles), 10.sup.6
Zeo-transduced (triangles), IL-12-transduced BM-DCs (squares) or
IL-12-transduced syngeneic fibroblasts (solid circles) were
injected into established tumors. As shown in FIG. 4, the antitumor
effect of IL-12-transduced DCs was also compared with that of
IL-12-transduced fibroblasts. As previously reported (Zitvogel et
al. (1995)), IL-12-transduced fibroblasts suppressed the growth of
MCA205, whereas a single injection of them did not show any
rejection of tumors. However, IL-12-transduced DCs suppressed tumor
growth more efficiently when compared with IL-12-transduced
fibroblasts which expressed IL-12 at a similar, but slightly higher
level (IL-12 production of genetically modified DCs and fibroblasts
was 13 and 22 ng/10.sup.6 cells/48 h, respectively.). The
sequential treatment of intratumoral injection of IL-12-transduced
DCs every week was also examined. Briefly, mice were initially
injected i.d. with 1.times.10.sup.5 MCA205 cells. On days 7 and 14,
HBSS (open circles), 10.sup.6 IL-12-transduced BM-DCs (solid
circles), or IL-12-transduced syngeneic fibroblasts (triangles)
were injected into established tumors. As shown in FIG. 5, repeated
injection of IL-12-transduced DCs resulted in more significant and
prolonged regression (over 60 days) of tumor as compared with
single injection.
[0058] Systemic tumor-specific immune response. As mentioned above,
the local antitumor effects of IL-12-transduced DCs were more
profound than that observed with IL-12-transduced fibroblasts. To
determine whether intratumoral injection of IL-12-transduced DCs
could induce significant systemic immune responses specific for the
tumor, subcutaneous lymph nodes in the ipsilateral inguinal area of
the inoculated tumor (draining lymph nodes) as well as spleen were
harvested from tumor bearing mice 7 days after injection with DCs
(14 days after tumor inoculation). These lymphoid cells were
co-cultured with irradiated tumor cells (MCA205) in vitro, and
IFN-.gamma. and IL-4 production in the culture supernatant was
examined. Interestingly, injections with non- or Zeo-transduced DCs
enhanced tumor specific IFN-.gamma. production by lymphoid cells
harvested from draining lymph nodes and spleen when compared with
IL-12-transduced fibroblasts. Furthermore, intratumoral injection
of IL-12-transduced DCs resulted in greater enhancement of
IFN-.gamma. production in response to tumor re-stimulation by these
lymphoid cells. Interestingly, DC injection also enhanced IL-4
production to a lesser extent. IFN-.gamma. production was
specifically released following MCA205 stimulation, but not with
B16 or MCA207 tumors. These results suggest that intratumorally
injected DCs transduced with IL-12 traffic to the draining lymph
node and efficiently stimulate lymphocytes in situ to produce
IFN-.gamma.. To confirm this hypothesis, IL-12-transduced DCs were
stained with fluorescence dye (PKH-26) and injected intratumorally,
and the draining lymph and was examined 24 hours after injection. A
significant number of DCs were detected in the draining lymph node
24 h after injection.
[0059] In addition, CTL activity of splenocytes from treated mice
was also evaluated. Splenocytes were harvested and pooled from two
mice per group 7 days after intratumoral injection with BM-DC.
These cells (2.times.10.sup.6) were restimulated in vitro with
2.times.10.sup.5 irradiated (5000 rad) MCA205 in the presence of 25
IU/ml of rhIL-2. Restimulated cells were tested 5 days later in a
standard .sup.51Cr-release assay. CTL activities by splenocytes
from the groups treated with HBSS, Zeo-transduced and
IL-12-transduced BM-DCs, and IL-12-transduced fibroblasts were
tested against MCA205 (FIG. 6A). Zeo-transduced DCs were more
effective in inducing CTL activity than IL-12-fibroblasts.
IL-12-transduced DCs induced significantly higher CTL activity than
that observed using any other strategy. CTL activity by splenocytes
from mice injected with IL-12-transduced BM-DCs was tested against
MCA205, YAC-1 and syngeneic fibroblasts (FIG. 6B). This activity
appeared to be specific for the MCA205 tumor and could be blocked
40-50% with anti-CD8 antibody and 24-35% with anti-H-2K.sup.b
antibody.
[0060] To further confirm the induction of systemic immunity, the
growth of a contralateral nontreated tumor was examined. Mice were
injected i.d. with 1.times.10.sup.5 MCA205 and B16 tumor cells in
both flanks, and IL-12-transduced DCs were injected in the tumor in
the right flank on day 7, when tumor area reached 13-20 mm.sup.2.
Tumor growth in both flanks was monitored. FIG. 7 shows that
intratumoral injection with IL-12-transduced DCs significantly
suppressed the growth not only of the injected tumor (FIG. 7A) but
also of the contralateral, non-injected tumor (FIG. 7B). In these
experiments, no IL-12 was detected in murine sera 2 days after
injection with IL-12-transduced DCs.
[0061] Flow cytometry. For phenotypic analysis of BM-DCs, PE- or
FITC-conjugated monoclonal antibodies against murine cell surface
molecules (CD11b, CD11c, CD80, CD86, Gr-1, H-2K.sup.b, I-A.sup.b
and appropriate isotype controls, all from PharMingen, San Diego,
Calif.) were used. DEC-205 was detected by staining with NLDC-145
antibody (Serotec Ltd., Oxford, UK). The transduction marker hCD80
was stained with FITC-conjugated anti-hCD80 antibody (PharMingen,
San Diego, Calif.), which does not cross-react with mouse CD80.
[0062] In vitro cytokine release assays. Lymphoid cells were
obtained from the draining (inguinal) lymph node and spleen
harvested from each of two mice which had received intratumoral
injection with BM-DCs 7 days earlier. These cells
(2.times.10.sup.6)were co-cultured in 24-well plates with
2.times.10.sup.5 irradiated (5000 rad) MCA205 in the presence of 25
IU/ml rhIL-2 (Chiron, Emeryville, Calif.) for 36 h as described
previously (Zitvogel et al. (1996)). The supernatants were
collected and assessed in an ELISA for mIFN-.gamma. and mIL-4
expression (PharMingen, San Diego, Calif.). The lower limit of
sensitivity for each assay was 18 and 36 pg/ml, respectively.
[0063] Cytotoxic T lymphocyte assays. Splenocytes were harvested
and pooled from two mice per group, 7 days after intratumoral
injection with BM-DCs. These cells (2.times.10.sup.6) were
restimulated in vitro with 2.times.10.sup.5 irradiated (5000 rad)
MCA205 in the presence of 25 IU/ml of rhIL-2. Five days later,
re-stimulated cells were used as effectors for the standard 4
h-.sup.51Cr release assay against MCA205, YAC-1 and syngeneic
fibroblasts. In brief, 10.sup.6 of each target cell were labeled
with 100 .mu.Ci of Na.sub.2.sup.51CrO.sub.4 for 1 h. After washing
twice, these effector and target cells were plated at an
appropriate E/T ratio in 96-well round bottom plates. The
supernatant (100 .mu.l) was collected after 4 h incubation and the
radioactivity was counted with a .gamma.-counter. The percentage of
specific lysis was calculated as the following formula: % specific
lysis=100.times.(experime- ntal release-spontaneous
release)/(maximal release-spontaneous release).
[0064] Statistical analysis. Statistical analysis was performed
using the unpaired two-tailed Student's t-test. Pearson's linear
regression was applied to examine the correlation. Differences were
considered significant when the p value was less than 0.05.
* * * * *