U.S. patent application number 10/842185 was filed with the patent office on 2005-01-06 for mature type-1 polarized dendritic cells with enhanced il-12 production and methods of serum-free production and use.
Invention is credited to Kalinski, Pawel.
Application Number | 20050003533 10/842185 |
Document ID | / |
Family ID | 33555262 |
Filed Date | 2005-01-06 |
United States Patent
Application |
20050003533 |
Kind Code |
A1 |
Kalinski, Pawel |
January 6, 2005 |
Mature type-1 polarized dendritic cells with enhanced IL-12
production and methods of serum-free production and use
Abstract
The present invention discloses novel dendritic cell
maturation-inducing cytokine cocktails, and methods for inducting
type-1 polarized dendritic cells in serum-free conditions which
enhance the desirable properties of DC1s generated in
serum-supplemented cultures. The invention further discloses
methods and systems using IFN.gamma. and other ligands of the
IFN.gamma. receptor, in combination with IFN.alpha. (or other type
I interferons), poly I:C, and other IFN.alpha. (and IFN.beta.)
inducers to enhance the IL-12-producing properties of dendritic
cells. More specifically, the present invention discloses type-1
polarized dendritic cells that have a unique combination of a
fully-mature status and an elevated, instead of "exhausted",
ability to produce IL-12p70. allows for the generation of
fully-mature DC1s in serum-free AIM-V medium. The invention
discloses systems that use the foregoing products and methods to
facilitate the clinical application of DC1-based vaccines and the
identification of novel factors involved in the induction of Th1
and CTL responses by DC1.
Inventors: |
Kalinski, Pawel; (Allison
Park, PA) |
Correspondence
Address: |
Debra M. Parrish
Suite 200
615 Washington Road
Pittsburgh
PA
15228
US
|
Family ID: |
33555262 |
Appl. No.: |
10/842185 |
Filed: |
May 10, 2004 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
60468760 |
May 8, 2003 |
|
|
|
Current U.S.
Class: |
435/372 |
Current CPC
Class: |
C12N 5/064 20130101;
A61K 2039/5154 20130101; Y02A 50/30 20180101; A61K 2039/57
20130101; A61P 37/00 20180101; C12N 2501/23 20130101; Y02A 50/41
20180101; C12N 5/0634 20130101; C12N 2501/25 20130101; C12N 5/0639
20130101; C12N 2501/056 20130101 |
Class at
Publication: |
435/372 |
International
Class: |
C12N 005/08 |
Goverment Interests
[0002] Portions of the present invention were made with support of
the United States Government via a grant from the National Cancer
Institute under grant number 1R01CA82016. The U.S. Government may
therefore have certain rights in the invention.
Claims
What is claimed is:
1. A population of cells wherein a. said cells are
antigen-presenting cells; and b. said cells are activated or
matured by a combination of type I interferons, or analogs or
inducers of said type I interferons, and type II interferons or
analogs of said type II interferons.
2. The population of claim 1 wherein said cells are further
stimulated with maturation inducing agents.
3. The population of claim 2 wherein said maturation agent is one
or any combination from the group of inflammatory cytokines, LPS,
LPS derivatives, poly-I:C, heat shock proteins, derivatives of heat
shock proteins, endogenous mediators of antigen-presenting cell
activation, bacterial components, fungal components, and other
inducers of NF.kappa.b activation.
4. The population of claims 1 or 2 wherein said type I interferon
is IFN.alpha. or IFN.beta., or an analogue of IFN.alpha. or
IFN.beta., and said type II interferon is IFN.gamma. or an analogue
of IFN.gamma..
5. The population of claims 1 or 2 wherein said antigen-presenting
cells are dendritic cells.
6. The population of claim 5 wherein said dendritic cells are
alpha-type-1 polarized dendritic cells having fully mature status
wherein fully mature status is defined as a high expression of
dendritic cell markers CD83, CD86, and CCR7, a high ability to
migrate in response to CCR7 ligands), and an ability to produce
IL-12p70, in response to subsequent CD40L-dependent activation, at
levels above that produced by immature dendritic cells after
CD40L-dependent activation.
7. A method of producing a population of cells wherein
antigen-presenting cells are stimulated by exposure to a
combination of type I and type II interferons or their analogs.
8. The method of claim 7 wherein said antigen-presenting cells are
further stimulated with maturation inducing agents.
9. The method of claim 7 wherein said maturation agent is one or
any combination from the group of inflammatory cytokines LPS, LPS
derivatives, poly-I:C, heat shock proteins, derivatives of heat
shock proteins, endogenous mediators of antigen-presenting cell
activation, bacterial components, fungal components, and other
inducers of NF.kappa.b activation.
10. The method of claims 7 or 8 wherein said type I interferon is
IFN.alpha. or IFN.beta., or an analogue of IFN.alpha. or IFN.beta.,
and said type II interferon is IFN.gamma. or an analogue of
IFN.gamma..
11. The method of claims 7 and 8 wherein said antigen-presenting
cells are dendritic cells.
12. The method of claims 7 and 8 wherein said antigen-presenting
cells are .alpha.Type-1 polarized dendritic cells.
13. The method of claim 12 wherein .alpha.Type-1 polarized
dendritic cells are produced in a fetal calf serum (FCS)-free
medium.
14. A method of producing a population of .alpha.Type-1 polarized
dendritic cells comprising the steps of: a. isolating dendritic
cells or dendritic cell precursors from a donor; b. incubating said
dendritic cells with IFN.alpha. or IFN.beta. or functional
analogues of IFN.alpha. or IFN.beta., in the presence of IFN.gamma.
or IFN.gamma. functional analogues and dendritic cell maturation
factors.
15. The method of claim 14 further comprising the step of
presenting an antigen by said dendritic cells.
16. A method according to claim 15 wherein said presenting of an
antigen to said surface of said dendritic cells is achieved by one
of the group of: (a) contacting said dendritic cells with an
antigen or epitope differentially expressed on a cell; (b) pulsing
said dendritic cells with antigenic proteins; (c) loading said
dendritic cells with antigenic peptides; (d) transforming or
transducing said dendritic cells with nucleic acid molecules coding
for at least part of said antigen; (e) fusing said dendritic cells
with cells carrying specific antigens; (f) pulsing said dendritic
cells with apoptotic cells, apoptotic bodies, or cell lysates.
17. A method for stimulating or expanding T cells using a
population of alpha type 1 dendritic cells comprising at least the
following steps: a. isolating dendritic cells from a donor; b.
incubating said dendritic cells or dendritic cell precursors with
IFN.alpha. or IFN.beta. or functional analogues of IFN.alpha. or
IFN.beta., in the presence of IFN.gamma. or IFN.gamma. functional
analogues, and dendritic cell maturation factors; c. presenting a
peptide on the surface of said dendritic cells; and d. stimulating
a population of T cells with said population of dendritic
cells.
18. The method of claim 17 wherein the T cells are stimulated or
expanded in vivo through a vaccination with alpha type 1 dendritic
cells.
19. The method of claim 17 wherein the T cells are stimulated or
expanded ex vivo through adoptive immunotherapy with ex vivo
stimulated or expanded T cells.
20. The method according to claim 17 wherein said T cell is a CD4
cell or CD8 T cell.
21. A method for stimulating or expanding NK cells using a
population of alpha type 1 dendritic cells comprising at least the
following steps: a. isolating dendritic cells from a donor; b.
incubating said dendritic cells or dendritic cell precursors with
IFN.alpha. or IFN.alpha. functional analogues in the presence of
IFN.gamma. or IFN.gamma. functional analogues, and dendritic cell
maturation factors; c. stimulating a population of NK cells with
said population of dendritic cells.
22. The method of claim 21 wherein the NK cells are stimulated or
expanded in vivo through a vaccination with alpha type 1 dendritic
cells.
23. The method of claim 21 wherein the NK cells are stimulated or
expanded ex vivo through adoptive immunotherapy with ex vivo
stimulated or expanded NK cells.
24. A system using the population of cells of any of claims 1
through 6, for therapeutic, preventative or diagnostic purposes,
including the treatment of cancer, precancerous states,
precancerous lesions, viral infections, bacterial infections,
fungal infections, parasitic infections, allergies or autoimmune
diseases, including its use as vaccine adjuvant.
25. A system using the methods described in any of claims 7 through
19, for therapeutic, preventive, or diagnostic purposes, including
the treatment of cancer, precancerous states, precancerous lesions,
viral infections, bacterial infections, fungal infections,
parasitic infections, allergies or autoimmune diseases.
26. A system using the methods described in any of claims 7 through
19, as a tool to identify or develop additional therapeutic,
preventive, or diagnostic, tools or strategies, applicable for
cancer, precancerous states, precancerous lesions, viral
infections, bacterial infections, fungal infections, parasitic
infections, allergies or autoimmune diseases.
27. A system comprising IFN.alpha. or IFN.beta., or inducers or
functional analogues of IFN.alpha. or IFN.beta., in combination
with IFN.gamma., or IFN.gamma. functional analogues for the
preparation of alpha type 1 polarized dendritic cells according to
the methods of any of claims 7 through 19.
28. The system according to claim 27 further comprising dendritic
cell maturation factors.
29. The system according to claims 28 comprising IFN.alpha. or
IFN.beta., IFN.gamma., IL-1.beta., and TNF.alpha..
30. The system according to claim 29 further comprising
poly-I:C.
31. The system according to claims 28 comprising poly-I:C,
IFN.alpha., IL-1.beta., and TNF.alpha..
32. The system according to any of claims 27 through 31 further
comprising a diluent that is a serum supplemented medium.
33. The system according to any of claims 27 through 31 further
comprising a diluent that is serum-free medium.
34. The system according to any of claims 27 through 31, for the
preparation of alpha type 1 dendritic cells, according to any of
the claims 19 trough 21 further comprising a delivery route that is
one of the group of: intravenous, intratumoral, peritumoral,
intradermal, subcutaneous, intramuscular, intraperitoneal,
intracranial, intraventricular, intranodal, peri-nodal,
intralymphatic or topical.
Description
[0001] This application claims priority from U.S. application Ser.
No. 60/468,760, filed May 8, 2003, entitled "MATURE TYPE-1
POLARIZED DENDRITIC CELLS WITH ENHANCED IL-12 PRODUCTION AND
METHODS OF SERUM-FREE PRODUCTION AND USE".
BACKGROUND
[0003] 1. Field of the Invention
[0004] The invention relates to methods of generating mature
dendritic cells with enhanced IL-12 production and compositions and
systems for such dendritic cells.
[0005] 2. Discussion of the Background
[0006] Dendritic cells (DCs), the most potent antigen presenting
cells, are effective inducers of protective immunity against
infectious diseases and cancer (Banchereau & Steinman 1998).
The adjuvant function(s) of DCs has prompted intense interest in
the use of DCs as a vaccine component, particularly after the
advent of in vitro methods to generate large numbers of DCs from
monocytes (Peters et al 1993, Sallusto & Lanzavecchia 1994).
Over the past years, DC-based vaccines have been increasingly
applied in the clinical treatment of cancer patients (Steinman et
al., 2001; Parmiani et al., 2002). Following the initial success of
the multi-epitope melanoma trial (Nestle et al 1998; 30% objective
clinical responses), DCs have been used successfully to treat
patients with melanoma, lymphoma and renal cell carcinoma
(reviewed: Steinman et al., 2001; Parmiani et al., 2002). However,
the overall clinical response rates do not exceed the predictable
15% observed for alternate immunotherapies (idem), which is below
expectations, highlighting the need for improved design of DC-based
vaccines, including the selection of the most appropriate types of
DCs.
[0007] Although some of the early studies with DC-based vaccines
successfully used FCS-based protocols (Nestle et al., 1998), the
need to obtain the vaccine-applied DCs in possibly best-defined
conditions (and to overcome potential reproducibility and
regulatory issues) prompted the development of serum-free
approaches to grow DCs.
[0008] Extensive research of recent years convincingly demonstrated
that the effective induction of anti-tumor CTL responses requires
the participation of fully-mature DCs because immature DCs are
either ineffective, poorly immunogenic, or induce undesirable
IL-10-producing regulatory T cells (Jonuleit et al 2000, Dhodapkar
et al 2001). These considerations, in conjunction with the desire
to use the most strictly-defined and reproducible conditions of DC
generation for human use, established the dominant position of the
"complete cytokine cocktail" composed of the combination of
inflammatory cytokines IL-1.beta., TNF.alpha., IL-6, and PGE.sub.2
(Jonuleit et al. 1997), as the "gold standard" of DCs used in
cancer immunotherapy.
[0009] Fully-mature DCs induced by the combination of inflammatory
cytokines IL-1.beta., TNF.alpha., IL-6, and PGE.sub.2 (Jonuleit et
al. 1997) have been consistently observed as superior to immature
DCs in promoting a higher degree of specific T cell priming in
vitro and in vivo (Jonuleit et al., 2001, Schuler-Thurner et al.,
2000, Schuler-Thurner et al., 2002, Thurner et al., 1999, Dhodapkar
et al., 2001).
[0010] Unfortunately, the maturation stage of DCs obtained in the
currently-available protocols inversely correlates with their
ability to produce IL-12p70 (Kalinski et al., 1999, Langenkamp et
al., 2000), the cytokine with powerful anticancer Th1-and
CTL-inducing properties (Trinchieri, 1998b); (Shurin et al.,
1997).
[0011] Induction of Ag-specific CD8 T cells and Th 1-type CD4 T
cells depends on the ability of DCs to provide CD4 and CD8 T cell
precursors with high levels of co-stimulation and with
interleukin-12 (IL-12), the major DC-produced anti-tumor cytokine.
Previous work with DC transduced with IL-12 genes demonstrated that
high IL-12-producing DCs are effective inducers of tumor rejection
in experimental animals. However, use of IL-12 transduced DC in
humans creates substantial logistic problems. It also carries
potential risks associated with the administration of
genetically-manipulated material and the risks of direct IL-12
toxicity and of deregulating the immune system due to uncontrolled
IL-12 production.
[0012] Many have attempted to generate DC's using a variety of
methods. For example, U.S. Pat. Nos. 5,851,756, 5,994,126 and
5,475,483 (Steinman, Inaba and Schuler) disclose methods for
generating DCs from proliferating precursors and their maturation.
Further, U.S. Pat No. 5,866,115 discloses a method of developing
DCs from DC34+ blood progenitors and U.S. Pat Nos. 6,228,640 and
6,251,665 disclose a means of loading DCs developed from CD34+
progenitors with RNA or its expression products as a mean of
achieving the expression of tumor-related or other target-related
antigens. Similarly, U.S. Pat. No. 6,121,044 teaches a means of
developing DC in bulk monocytes-depleted PBMC cultures. These
patents focus on particular methods of generating immature
dendritic cells rather than the particular conditions of the
maturation of dendritic cells. More importantly, none of these
patents disclose or teach the generation of dendritic cells with
the unique properties described in the present invention.
Specifically, none of the patents disclose or teach the combination
of type I and type II interferons (such as IFN.alpha. and
IFN.gamma.), as a part of the cytokine cocktail used to produce
fully mature DCs with high IL-12 producing capacity.
[0013] Thus, despite the efforts of many, the desirable combination
of high immunostimulatory activity with a high capacity to produce
IL-12p70 could not be attained by all previous DC-based vaccines
which have employed either mature DCs exhibiting high
stimulatory/low IL-12-secreting functions or immature DCs that
display low stimulatory/high IL-12 secretion functions.
[0014] It is known in the art that the presence of IFN-.gamma.
during the either LPS-induced or IL-1.beta./TNF.alpha.- induced DC
maturation, results in the induction of stable type-1 polarized DCs
(DC1s) that produce up to 100-fold higher levels of IL-12p70 in
response to subsequent CD40L stimulation or the interaction with
CD40L-expressing CD4.sup.+ Th cells (Vieira et al., 2000, Mailliard
et al., 2002). Unfortunately, the original DC1-inducing cytokine
cocktail, composed of IL-1.beta., TNF.alpha., and IFN-.gamma.
(Vieira et al 2000), does not allow for the induction of DC1s in
serum-free media, which is desirable for clinical application.
[0015] DCs in the periphery can be exposed to a variety of
environmental "triggers" that result in DC "maturation" and
upregulation of factors critical to antigen-specific T-cell
activation, including IL-12 production. In some cases, these
signals are transmitted through Toll-like receptor ("TLRs") and
other cell-surface receptors expressed by DCs.
[0016] It is an object of the present invention to provide a means
of triggering DC maturation through innate signaling pathways to
enable DCs to express potent DC1-type function, regardless of the
presence of factors present in serum, enabling in vitro derivation
of DC1s for clinical applications.
[0017] It is an object of one preferred embodiment of the present
invention to add at least one from the group of IFN.alpha. or
IFN.beta. (type I interferons) or a type I interferon inducing
factor such as polyinosinic:polycytidylic acid (poly-I:C) to the
"classical" DC1-inducing cocktail
(TNF.alpha./IL-1.beta./IFN.gamma.) and to provide a means for
generating fully-mature DC1s in serum-free AIM-V medium.
[0018] It is further an object of the present invention to provide
an alpha-type-1 DC to induce up to 50-fold higher levels of
cancer-specific CTLs, and higher cytolytic activity of Th1 or NK
cells compared to the current "gold standard" DCs (matured by
IL-1.beta./TNF.alpha./IL-6/PGE2; Jonuleit et al., 1997).
SUMMARY
[0019] The present invention discloses novel dendritic cell ("DC")
maturation-inducing cytokine cocktails, and means for inducting
type-1 polarized dendritic cells ("DC1s") in serum-free conditions
which enhance the desirable properties of DC1s generated in
serum-supplemented cultures. The invention further discloses the
use of IFN.gamma. and other ligands of the IFN.gamma. receptor, in
combination with IFN.alpha. (or other type I interferons, such as
IFN.beta., known to bind to the same receptor), poly I:C, and other
IFN.alpha. (and IFN.beta.) inducers to enhance the IL-12-producing
properties of DCs. The invention also discloses the use of DC1s to
induce Ag-specific T cells against tumors, intracellular pathogens,
and atopic allergens for active and passive immunotherapy,
immunomonitoring and research purposes. More specifically, the
present invention discloses type-1 polarized DCs (DC1s) that have a
unique combination of a fully-mature status and an elevated,
instead of "exhausted", ability to produce IL-12p70. These
properties allow these DC1s to selectively induce high-intensity
Th1-, CTL-, and NK cell-mediated type-1 immune responses, including
those desirable in the treatment of cancer. Another preferred
embodiment of the present invention shows that the inclusion of
IFN.alpha. and/or poly-I:C to the "classical" DC1-inducing cocktail
(TNF.alpha./IL-1.beta./IFN.gamma.) allows for the generation of
fully-mature DC1s in serum- free AIM-V medium. In other preferred
embodiments, the present invention discloses serum-free protocols
of DC1 generation that facilitate the clinical application of
DC1-based therapies and the identification of novel factors
involved in the induction of Th1-, CTL-, and NK cell responses by
DC1.
DETAILED DESCRIPTION
[0020] To boost the immunogenic capacity of DCs and their ability
to induce high-intensity Th1 and CTL-mediated type-1 immune
responses, the present invention combines within one DC type a
fully-mature status and a high ability to produce high levels of
IL-12p70. In contrast to current methodologies in which the final
maturation of DCs induced by typical stimuli is associated with
reduced ability to produce IL-12 (Kalinski et al., 1999, Langenkamp
et al., 2000), the present invention provides for concomitant
exposure of immature DC to a maturation-inducing stimulus and to
IFN.gamma. which results in a strong enhancement of the subsequent
ability of mature DC to produce IL-12 and to induce Th1-dominated
responses (Vieira et al., 2000, Mailliard et al., 2002), and more
specifically the cancer-specific CTL responses.
[0021] Further, although current DC1-inducing protocols are
ineffective in serum-free conditions, the present invention
provides that IFN.alpha., a member of type I interferon family, and
poly-I:C, an IFN.alpha.-inducing factor, can both synergize with
the IFN-.gamma.-based type-1-polarizing cocktails, allowing for the
induction of fully-mature type-1 polarized DC in serum-free
conditions. The present invention provides for adding at least one
of the group of IFN.alpha. and poly-I:C to a cocktail of
TNF.alpha./IL-1.beta./IFN.gamma. in either a serum-free culture or
a serum-supplemented culture depending on the specifications of the
application.
[0022] Although the current DC-based vaccines rely on either
immature DCs (with high ability to produce IL-12 but low
stimulatory capacity), or mature DCs (with high stimulatory
function, but reduced IL-12 production), the current invention
describes a method that provides a means of producing both of these
desirable features within a single DC1-based vaccine
preparation.
[0023] In addition, the DC1s of the present invention exhibit a
stable phenotype that is resistant to tumor-associated
immunosuppressive factors, including IL-10 and PGE.sub.2 (Kalinski
et al., 1998, Vieira et al., 2000). Moreover, DC1s of the present
invention can produce IL-12p70 upon the interaction with CD4.sup.+
T cells that are unable to produce IFN.gamma. or other IL-12
co-inducing factors (Vieira et al., 2000). These DC1s are able to
boost the clinical efficacy of cancer vaccines, despite the
presumed immunosuppressive environment of immunocompromised cancer
patients and their undesirable bias towards Th2-type immunity
(Tatsumi et al., 2002).
[0024] Previous work with DC transduced with IL-12 genes
demonstrated that high IL-12-producing DC are effective inducers of
tumor-specific Th1 cells and CTLs and of tumor rejection in
experimental animals (Zitvogel et al., 1996, Shimizu et al., 2001,
Tuting et al., ) (Chikamatsu et al., 1999, Tahara et al., 1994).
However, use of IL-12 transduced DC in humans suffers from
substantial logistic problems. It also carries potential risks
associated with the administration of genetically-manipulated
material as well as the risks of direct IL-12 toxicity and of
deregulating the immune system, due to uncontrolled IL-12
production.
[0025] The present invention provides a feasible way to generate
fully mature DC with high IL-12 producing capacity without any
genetic manipulation which overcomes the above obstacles, paving
the way to wide application of DC1-based immunotherapies. The
present invention's DC1-inducing cytokine cocktails are based on
the factors which are either FDA-approved drugs, or have been
approved by FDA for use as investigational drugs. Poly I: C have
been used as a biologic response modifier in cancer, as early as in
1976 in NCI, by the group of A. S. Levine (Robinson et al., 1976),
and subsequently in many other clinical trials, which demonstrated
its safety. Similar, IFN.alpha., IFN.gamma., IL-1.beta., and
TNF.alpha., are commonly-used biological agents, approved as drugs
or investigational drugs.
[0026] Because IL-12 production has been shown to be important for
the control of numerous intracellular pathogens, including
Leishmania, Listeria, Mycobacterial infections, and many viruses,
the DC1s of the present invention can be used to treat chronic
infections, including the infections with HIV, EBV, CMV, HCV, HBV,
mycobacteria (e.g. tuberculosis and lepromatous leprosy), or
parasites (e.g. Leishmaniasis). Further, the powerful Th1- and
CTL-inducing DC1s of the present invention may be used to revert
undesirable Th2 bias, and the B cell production of pathogenic
antibodies in atopic allergies (e.g. manifested as atopic
dermatitis or asthma) or autoimmune diseases, e.g. SLE, Graves
disease, IgA nephropathy, or autoimmune trombocytopenia. In
contrast to Th2 cells, Th1 cells and CTLs produced by the present
invention have a limited or no ability to support antibody
production, and can limit this process by killing the
antibody-producing B cells (Wierenga et al , Ju et al, Del Prete et
al 1991).
[0027] In addition to their therapeutic use as vaccine carriers,
the DC1s of the present invention will be a useful tool in the
development of additional novel therapies. The superior ability of
DC1s of the present invention to activate Ag-specific T cells in
vitro enables them to be used as immunomonitoring tools with
superior sensitivity in detecting low-intensity (or suppressed)
immune responses, facilitating the analysis of immune responses in
patients with cancer, HIV, and other diseases.
[0028] The serum-free protocols of DC1 induction of the present
invention can serve as a tool for defining the exact mechanism(s)
of the DC1-mediated induction of Th1 cells and CTLs. Although
IL-12's key role in the ability of DC to induce Th1 responses has
been demonstrated, it is likely that other factors may also be
important in this respect. The serum-free DC1 generation protocols
of the current invention enable the use of the powerful proteomic
approach to analyze the unique pattern of DC1 interaction with
other immune cells. This may lead to the identification of novel
Th1- and CTL-inducing factors, with potential additional
therapeutic applications.
[0029] The current data indicate the feasibility of generating
fully-mature DC in the absence of PGE2, the maturation-enhancing
factor with particularly-pronounced IL-12 antagonistic activity
(Kalinski et al., 1997, Kalinski et al., 1998, Kalinski et al.,
2001). The lack of the absolute requirement for PGE2 in the
induction of functional mature DC is in accord with the apparent
lack of immunosuppressive activity of COX-1 and COX-2-inhibitors,
used as non-steroid anti-inflammatory drugs. On the contrary, PGE2
has been shown to suppress the production of IL-12p70 in several
types of APC including DCs (van Der Pouw Kraan TC et al., 1995);
Kalinski et al., 1997), can directly suppresses Th1- cells (Betz
& Fox, 1991, Snijdewint et al., 1993), and may play a role in
tumor-associated immune dysfunction (reviewed in (Harris et al.,
2002).
[0030] Although other IFNs signaling through type I IFN receptors
are known to activate the similar signaling pathways and exert
similar biologic effects, the present invention allows testing
which of particular pathways induced by type I interferons
(including STAT-1, STAT-2, STAT-3, STAT-4 and NF.kappa.B) remain
critical for DC1 induction. Definition of the molecular mechanisms
of DC1 induction will pave the way for a pharmacological modulation
of DC, using appropriate small molecules. Further, the present
invention allows the identification of a wider panel of type-I
IFN-inducing agents able to promote DC1 induction, similar to
p-I:C.
[0031] DC-based vaccine targets enabled by the present invention
include the induction of type-1 immunity against HPV-related
antigens in cervical carcinoma patients. Further, the DCs of the
present invention provide a tool for understanding of the basic
principles of immuno-regulation and the treatment of infections
with pathogens resistant to standard forms of treatment, including
HIV, CMV, HBV, HCV, or tuberculosis.
[0032] Thus, the present invention's disclosure of .alpha.DC1s, as
powerful in inducers of CTL-, Th1- and NK cell activity, and of
CTL-, Th1-, and NK cell-mediated anti-tumor responses, indicate
several new therapeutic and preventive possibilities of the current
invention in cancer and pre-cancerous states as well as in chronic
infectious diseases, atopic allergies, and certain forms of
autoimmunity, where type-1 (CTL-, TH1, and NK cell-mediated)
immunity can also be beneficial. DC1, especially .alpha.DC1 induced
in the maturation conditions involving the combination of type I
and type II interferons (or their surrogates), can be used as
carriers of vaccines, or as the stimulating agents to activate and
expand immune cells ex-vivo, for their subsequent use in adoptive
immunotherapy. Moreover, the ability of DC1s to act as powerful
inducers of T cell responses in vitro, can also be a useful tool
for detecting the presence of pathogen-specific T cells in
circulation or in human tissues, even when T cells are difficult to
detect y standard methods, e.g. due to their suppression,
exhaustion or anergization. In addition, high potency of .alpha.DC1
in inducing CTL-, Th1- and NK cell activity makes them a useful
research tool for the identification of the genes and proteins
particularly important in activating the above types of immune
cells, facilitating the development of additional, potentially
novel targets of immune intervention, and potentially novel
factors, that can be used as immunomodulators, either in place of
.alpha.DC1, as self-standing therapeutic agents, or supplementing
other forms of (immuno)therapy.
[0033] Description of Preferred Embodiments
[0034] The following is a description of a preferred embodiment of
a method for generating DC1s according to the present
invention.
[0035] Many commercially available media can be used to generate
the DCs. By way of example, but not limitation, such media include:
IMDM with 10% FBS (both from Gibco, Grand Island, N.Y.), IMDM with
2% HS (Atlanta Biologicals, Atlanta, Ga.; additional, 1% and 10%
concentrations of human sera), and serum-free AIM-V medium
(Gibco/Invitrogen, Grand Island, N.Y.) or serum-free X-Vivo Medium
(Cambrex, East Rutherford, N.J.). Many cytokines, including, but
not limited to the following, can be used to obtain immature DCs,
induce their final maturation and polarization, and to generate
tumor-specific CTLs: rhu GM-CSF and IL-4 (both 1000 IU;
Schering-Plough (Kenilworth, N.J.); IFN-.alpha. (Intron A-
IFN-.alpha.-2b; Schering-Plough); IFN-.beta.; (Avonex; Biogen
Inc.,Cambridge, Mass.); IL-2 (Chiron Corp. ; Emeryville, Calif.);
rhuTNF-.alpha. (Strathmann Biotech Gmbh, Hannover, Germany);
rhuIL-1.beta. (Strathmann); rhuIFN-.gamma. (Strathmann); LPS (from
E.coli 011:B4; Sigma, St. Louis, Mo.); PGE2 (Sigma, St. Louis,
Mo.); rhuIL-7 (R&D Systems, Minneapolis, Minn.) poly-I:C
(Sigma, St. Louis, Mo.).
[0036] According to one embodiment of the present invention,
mononuclear cells obtained from the peripheral blood of healthy
donors or patients afflicted with a disease of interest, e.g.,
melanoma, are isolated by density gradient separation using a
variety of techniques including Lymphocyte Separation Medium
(Cellgro Mediatech, Herndon, Va.). To obtain immature (Sallusto
& Lanzavecchia, 1994), monocytes are isolated from peripheral
blood lymphocytes using a Percoll (Sigma) density separation
technique, followed by plastic adherence, as described (Kalinski et
al., 1997). Monocytes are cultured in well plates (Falcon, Becton
Dickinson Labware, Franklin Lakes, N.J.) in individual media
supplemented with rhu GM-CSF and IL-4 (both 1000 IU).
[0037] CD8.sup.+ T cells (96-98% purity) are isolated from PBMCs
using a variety of commonly known techniques including the
StemSep.TM. negative selection systems (StemCell Technologies Inc.,
Vancouver, BC, Canada). Phenotypic analysis is performed using
known methodologies including the WinMDI Version 2.8 Software
(Joseph Trotter, Scripps Research Institute, La Jolla, Calif.).
[0038] A comparison of the induction of DC maturation and
polarization according to the present invention and current methods
was performed to demonstrate the superiority of the present
invention. To conduct such a comparison, DC cultures (performed in
either serum-supplemented or serum-free conditions) were exposed to
different maturation regimens according to the following protocols:
(1) the current "gold standard" of clinically-used DC which
provides for DC matured by the "complete cytokine mix": IL-1.beta.,
TNF.alpha., IL-6, and PGE.sub.2 (control DC; Jonuleit et al.,
1997); (2) DC1-inducing protocols previously known in the art which
include (a) serum-supplemented cultures: maturation by IL-1.beta.,
TNF.alpha., and IFN.gamma.0 .alpha..nu..delta. (b)
Serum-supplemented cultures: maturation by LPS and IFN.gamma.; and
(3) DC1-inducing protocols of the present invention including (a)
serum-free culture (AIM-V medium): maturation by IL-1.beta.,
TNF.alpha., IFN.gamma. and IFN.alpha.; (b) serum-free culture
(AIM-V medium): maturation by IL-1.beta., TNF.alpha., IFN.gamma.,
IFN.alpha. and poly-I:C; (c) serum-free culture (AIM-V medium):
maturation by IL-1.beta., TNF.alpha., IFN.gamma., and poly-I:C; (d)
serum-supplemented culture: maturation by IL-1.beta., TNF.alpha.,
IFN.gamma. and IFN.alpha.; (e) serum-supplemented culture:
maturation by IL-1.beta., TNF.alpha., IFN.gamma., IFN.alpha. and
poly-I:C; and (f) serum-supplemented culture: maturation by
IL-1.beta., TNF.alpha., IFN.gamma., and poly-I:C.
[0039] A range of concentrations of each of the above factors can
be used (from 0.1 pg/mL to 10 mg/mL. One preferred embodiment,
depicted herein, uses the following concentrations of the above
factors: IL-1.beta. (25 ng/ml); TNF.alpha. (50 ng/ml), IFN.gamma.
(1000 U/ml); poly-I:C (20 .mu.g/ml); IFN.alpha. (3000 U/ml); LPS
(250 ng/ml). In an assay employing this preferred embodiment, the
DC cells produced by the disclosed protocols were harvested and
analyzed for the expression of maturation-associated surface
markers, the ability to produce IL-12p70, and to induce
melanoma-specific CTLs. To test and demonstrate the
IL12p70-producing capacity of DC, they were harvested, washed, and
plated in flat bottom well plates. To mimic the interaction with
CD40L expressing Th cells, CD40L-transfected J558 cells (University
of Birmingham, Birmingham, UK) were added (Cella et al., 1996).
Supernatants were collected and tested for the presence of IL-12p70
by ELISA.
[0040] Negatively-isolated CD8.sup.+ T cells from HLA-A2.sup.+
donors were sensitized by the individual populations (non-polarized
and polarized) of autologous DC pulsed with the HLA-A2-restricted
peptides MART-1 (27-35, AAGIGILTV), gp100 (209-217, ITDQVPFSV and
154-162, KTWGQYWQV), and tyrosinase (368-376, YMNGTMSQV). RhuIL-2
(50 U/ml) was added and the differentially-sensitized CD8.sup.+ T
cell cultures were expanded by an additional round of stimulation,
using peptide-pulsed autologous PBMC. The differentially-induced
CD8.sup.+ T cell lines were stimulated with peptide-pulsed
HLA-A2.sup.+ T2 cells to monitor the frequency of the
melanoma-specific CD8.sup.+ T cells by IFN-.gamma. ELISPOT.
Cytolytic activity of the differentially-sensitized CTL cultures
was determined by performing standard .sup.51Cr-release assays with
results calculated and reported in the percent of target lysis at
individual effector-to-target ratios as described(Friberg et al.,
1996). Concentrations of IL-12p70 in cell supernatants were
determined by specific ELISAs, performed with matched antibody
pairs, standards, and reagents.
[0041] The presence of IFN.gamma. during DC maturation induced by
IL-1.beta., and TNF.alpha., or induced by LPS, results in the
development of stable type-1-polarized DC (DC1), characterized by
high ability to produce IL-12p70 upon subsequent stimulation (FIG.
1), and by mature phenotype (FIG. 2). Although the expression of
surface maturation-associated markers on DC1 is similar to
IL-1.beta./TNF.alpha./IL-6/PGE.sub.2-matured control DCs (cDCs),
their IL-12-producing capacity is at least 1-log higher. Both DC1s
and cDCs uniformly expressed CCR7, the predictive marker of their
ability to migrate to the lymph nodes, indicating their utility in
vaccines.
[0042] Although it has been previously demonstrated that DC1s show
superior ability to induce Th1 cytokine profiles in naive CD4.sup.+
T cells (Vieira et al., 2000, Mailliard et al., 2002), the present
invention discloses a method that uses DC1s as superior inducers of
CTL-responses against melanoma-related antigens in healthy donors
and melanoma patients. A single round of short-term stimulation of
CD8.sup.+ T cells, results in the induction of strongly elevated
numbers of IFN.gamma.-producing MART-1-specific CTLs in the blood
of healthy donors (FIG. 3). The most pronounced differences were
observed in the case of long-lived CTL responses, when the
differential sensitization with DC1s as opposed to cDC was followed
by the subsequent expansion of the differentially-induced CTL lines
(on autologous PBMCs), to mimick the situation when vaccine-induced
CTLs will need to be sustained by endogenous APCs in
cancer-affected patients (FIG. 3). A preferred embodiment of the
present invention using the blood of two melanoma patients,
provides for DC1 that induce superior expansion of
melanoma-specific CTLs, against MART-1, tyrosinase and gp100 (FIG.
4). After a single round of differential IVS, a 4- to 8-fold higher
frequency of DC1-induced peptide-specific CTL is observed, the
advantage of using DC1 is more evident after secondary
restimulation of the differentially-sensitized cultures with
peptide-pulsed autologous PBMC, reflecting the enhanced persistence
of the DC1-induced CTL responses according to the present
invention. The overall magnitude of such responses is over 50-fold
higher than in control cultures (FIG. 4). Induction of long-lived
CTL responses against multiple MAAs indicates that DC1s of the
present invention are highly suitable to induce meaningful
anti-tumor responses in vivo, which will have beneficial
therapeutic effects in patients with cancer.
[0043] According to the methods of the present invention,
IFN.alpha. and p-I:C synergize with an IFN.gamma.-based polarizing
cocktail to generate DC1 in serum-free conditions. The combination
of IFN.gamma. with IL-1.beta. and TNF.alpha. allows the development
of mature DC1 in the fetal calf serum (FCS)-supplemented media, but
not in the presence of human serum, nor in serum-free AIM-V medium
(FIG. 5). In contrast, the addition of IFN.gamma. to the widely
used "complete cytokine mix"(IL-1.beta./TNF.alpha./IL-6/PGE.sub.2;
Jonuleit et al., 1997) is ineffective in the induction of high
IL-12producing DC1 in serum-free conditions (FIG. 5).
[0044] Although neither IFN.alpha. nor poly-IC alone (nor in
combination with IL-1.beta. and TNF.alpha.) promote the induction
of DC1 (Vieira et al., 2000, current data not shown), pursuant to
methods of the present invention, the addition of each IFN.alpha.
and to a lesser extent poly I:C to the cocktail of IFN.gamma.,
IL-1.beta., and TNF.alpha., provides for a serum-independent
development of DC1 with a strongly enhanced ability to produce
IL-12p70 after subsequent stimulation (FIG. 6). In a most preferred
embodiment, both IFN.alpha. and poly-IC are present. Further, the
presence of IFN.alpha. and poly-I:C during DC maturation
individually or in combination, enhance the already high
IL-12-producing capacity of the DC1 obtained in the presence of
FCS.
[0045] Serum-free DC1s obtained in the presence of all five factors
(IFN.gamma., IL-1.beta., TNF.alpha., IFN.alpha., and poly-I:C) show
a similar fully-mature surface phenotype as control DC (matured by
the "complete cytokine mix": IL-1.beta., TNF.alpha., IL-6, and
PGE.sub.2), or DC1 induced by LPS and IFN.gamma. in
FCS-supplemented medium, showing similar expression levels of such
maturation-associated markers as CD83, CD86 and CCR7 (FIG. 7). The
fully-mature DC phenotype exist when both IFN.alpha. and poly-IC
are present (FIG. 7).
[0046] Alpha type-1 DC (.alpha.DC1) of the present invention have a
superior ability to induce melanoma-specific CTL responses. To
analyze their CTL-inducing activity, individual populations of DC1
(generated under serum-free, or serum-supplemented conditions), or
serum-free control IL-1.beta./TNF.alpha./IL-6/PGE.sub.2-matured
cDC, were pulsed with melanoma-associated antigenic peptides, and
used to sensitize autologous CD8.sup.+ T cells from HLA-A2.sup.+
melanoma patients. The long-term CD8.sup.+ T cell lines obtained by
further expansion with autologous PBMCs were harvested and used as
responder cells against T2 cells pulsed with individual peptides,
or their combination. As shown in FIG. 8, each of the DC1s obtained
in each of the protocols tested induced significantly higher
numbers of melanoma specific (MART-1-, gp100-, and
tyrosinase-specific) CD8.sup.+ T cells, when compared to the DCs
matured in the presence of
IL-1.beta./TNF.alpha./IL-6/TNF.alpha./IL-6 and PGE.sub.2 ("control
DC": Jonuleit et al, 1997). In addition, to being superior to
control DCs in the induction high numbers of melanoma-specific
CD8.sup.+ T cells, alpha-type-1 DC also proved superior in the
induction of high cytotoxic activity against target cells pulsed
with MAA peptides (FIG. 9).
[0047] In addition to being superior inducers of the cytolytic
activity of tumor-specific CD8+ T cells (CTLs), .alpha.DC1 also
proved superior in their ability to induce similar, cytolytic
functions in CD4+ Th cells, and in isolated NK cells, allowing them
to efficiently kill transformed cells (FIG. 10).
[0048] Of particular importance for their ability to function in
vivo, as carriers of anticancer vaccines, .alpha.DC1 can
effectively migrate in response to CCR7 ligands (FIG. 11A), known
to be produced in the T cell areas of the lymph nodes and to be
responsible for the local accumulation of immune cells.
Importantly, exclusively .alpha.DC1, but not standard mature DCs
nor immature DCs could produce IL-12p70 in response to CD40L
stimulation, following their CCR7-ligand-induced migration (FIG.
11B).
[0049] The current description of .alpha.DC1, as powerful in
inducers of CTL-, Th1- and NK cell activity, and of CTL-, Th1-, and
NK cell-mediated antitumor responses, indicate several new
therapeutic and preventive possibilities of the current invention
in cancer and precancerous states as well as in chronic infectious
diseases, atopic allergies, and certain forms of autoimmunity,
where type-1 (CTL-, Th1, and NK cell-mediated) immunity can be
beneficial. DC1, especially .alpha.DC1 induced in the maturation
conditions involving the combination of type I and type II
interferons (or their surrogates), can be used as carriers of
vaccines, or as the stimulating agents to activate and expand
immune cells ex-vivo, for their subsequent use in adoptive
immunotherapy. Moreover, the ability of DC1 to act as powerful
inducers of T cell responses in vitro, can also be a useful tool
for the detecting the presence of pathogen-specific T cells in
circulation or in human tissues, even in cases when T cells are
difficult to detect y standard methods, e.g. due to their
suppression, exhaustion or anergization. In addition, high potency
of .alpha.DC1 in inducing CTL-, Th1- and NK cell activity makes
them a useful research tool for the identification of the genes and
proteins particularly important in activating the above types of
immune cells, facilitating the development of additional,
potentially novel targets of immune intervention , and potentially
novel factors, that can be used as immunomodulators, either in
place of .alpha.DC1, as a self-standing therapeutic agents, or
supplementing other forms of (immuno)therapy.
BRIEF DESCRIPTION OF THE FIGURES
[0050] FIG. 1. DC1 of the present invention show strongly
upregulated ability to produce IL-12p70. DC1 or cDC induced in 48
hour-long maturation cultures, were washed, counted and stimulated
with CD40L-transfected J558 cells. 24 hour supernatants were
analyzed for IL-12p70 contents with IL-12p70 ELISA. DC1 were
obtained in FCS-supplemented medium and matured under the influence
of IL-1.beta./TNF.alpha. and IFN.gamma.. Control DC, obtained in
serum-free medium (AIM-V) were matured by
IL-1.beta./TNF.alpha./IL-6/PGE.sub.2. (See Materials and Methods
for the concentrations of the individual DC1-maturation-inducing
factors, PGE2 was used at 10.sup.-6M, IL-6 was used at 1000
units/mL). The data obtained from 9 different donors is shown as
the ratios of the levels of IL-12p70 produced by DC1 of the present
invention compared to the levels of IL-12p70 production by control
DC.
[0051] FIG. 2. (A). DC1 of the present invention and control DC
express similar levels of the maturation-associated markers: CD83
and CD86. (B). DC1 uniformly express CCR7. 48 hour-long maturation
cultures: DC1 were obtained in FCS-supplemented medium and matured
under the influence of IL-1.beta./TNF.alpha. and IFN.gamma.. cDC
were grown in serum-free conditions and matured by
IL-1/TNF.alpha./IL-6/PGE2.
[0052] FIG. 3. Polarized DC1 induce stronger responses of
MART-1-specific CD8.sup.+ CTLs. CD8.sup.+ T cells from a
HLA-A2.sup.+ healthy donor were primed with MART-MHC class
I-restricted peptide presented either by DC1 (obtained the standard
protocols in the presence of serum) or standard cDC. At day 14 and
day 28, the expanded CTL cultures were tested for specific
responses against MART-1. Left: 1 round of IVS, using DC1 or cDC
matured by IL-1/TNF/IL-6/PGE2. Right: 1 round of IVS with DC1 as
compared to cDC, followed by second round of stimulation with
peptide-pulsed PBMC, to demonstrate the persistence of the
DC-induced specific CTLs. In both cases, the ELISPOT assay was
performed using the peptide-pulsed T2 cells. Non-peptide-coated T2
cells served as nonspecific control. The levels of nonspecific
responses (to peptide-unpulsed T2 cells) were subtracted. Similar
data was obtained using the blood from two additional healthy
donors.
[0053] FIG. 4. DC1 generated from melanoma patients show superior
ability to induce CD8.sup.+ T cell responses against multiple
melanoma-specific epitopes. DC1 (induced by LPS/IFN.gamma.) or
IL-1.beta./TNF.alpha./IL-6/P- GE.sub.2-matured cDC, generated from
an HLA-A2.sup.+ melanoma patient, were pulsed with a mix of the 4
melanoma-associated antigenic peptides, washed, counted and
co-cultured with negatively-isolated CD8.sup.+ T cells and
(irradiated) CD40L-tansfected J558 cells as a surrogate source of
CD40L-mediated helper signals. The differentially-sensitized
CD8.sup.+ T cells, were further expanded by restimulation (d14)
with irradiated autologous PBMCs pulsed with the same mix of
peptides. At day 28, CTL lines were harvested, washed, and used as
responder cells against T2 cells pulsed with individual peptides
(nonpulsed T2 cells served as nonspecific control, which was
subtracted).
[0054] FIG. 5. "Traditional" type-1-polarizing protocols do not
support the induction of DC1 in serum-free medium. DCs grown in
media supplemented with FCS (10%), HS (2%)-, or in serum free
medium (AIM-V) were matured in different "traditional protocols"
either in the absence or in the presence of the DC1-polarizing
factor; IFN.gamma.. Following 48 hour-long maturation, DC were
washed, counted and stimulated with CD40L-transfected J558 cells.
24 hour supernatants were analyzed for IL-12p70 contents with
IL-12p70 ELISA. The data are shown as a summary of data obtained
from 7-9 different donors. In each experiment, the levels of
IL-12p70 produced by DC1 were compared to the levels of IL-12
production by control DC (DC matured in
IL-1.alpha./TNF.alpha./IL-6/PGE.s- ub.2). In case of the human
serum, its additional concentrations (1% and 10%; using several
different batches of serum from different suppliers, yielded
similar negative results (data not shown).
[0055] FIG. 6. Novel Serum Free Protocols of DC1 generation. The
addition of IFN.alpha. (and to a lesser extent of poly I:C) to DC
maturation-inducing cytokines (IL-1.beta./TNF.alpha./IFN.gamma.)
allows the induction of DC1 in serum free culture conditions. In
all cases, 24 h-matured DCs were harvested, washed and stimulated
with CD40L to induce IL-12 production. Please, note that the
optimal expression of all of the maturation-associated maturation
markers required the participation of all 5 components of the
"alpha-type-1"-polarizing cocktail. The data are from a
representative experiment of five.
[0056] FIG. 7. Fully-mature status of ".alpha.lpha type-1" DC
(.alpha.DC1) obtained in serum-free media. Expression of the
maturation-associated markers (CD83, CD86, and CCR7) on cDC
(IL-1.beta./TNF.alpha./IL-6/PGE.sub- .2-matured DC), DC1 induced in
serum-supported protocol, and serum-free DC1 (induced by
IL-1.beta./TNF.alpha./p-I:C/IFN.alpha., and IFN.gamma.,) data from
a representative of three experiments, that all gave similar
results. Please, note that the optimal expression of all of the
maturation-associated maturation markers required the participation
of all 5 components of the "alpha-type-1"-polarizing cocktail.
Similar data were obtained in at least three additional
experiments.
[0057] FIG. 8. Alpha type-1 DC (.alpha.DC1), generated from
melanoma patients in serum-free conditions (an in
serum-supplemented media) show superior ability to induce CD8.sup.+
T cell responses against multiple melanoma-specific epitopes, when
compared to the currently-applied DCs. Individual populations of
DC1 (generated under serum-free, or serum-supplemented conditions),
or serum-free control IL-1.beta./TNF.alpha./IL-6/PGE.sub.2-matured
cDC, were pulsed with melanoma-associated antigenic peptides, and
used to sensitize autologous CD8.sup.+ T cells from HLA-A2.sup.+
melanoma patient. In brief, DCs were pulsed with a mix of the 4
melanoma-associated antigenic petides [MART-1 (27-35), gp100
(209-217 & 154-162), tyrosinase (368-376)], washed, counted and
co-cultured with negatively-isolated CD8.sup.+ T cells and
(irradiated) CD40L-tansfected J558 cells as a surrogate source of
CD40L-mediated helper signals. The resulting
differentially-sensitized CD8.sup.+ T cell lines were further
expanded by restimulation (d14) with irradiated autologous PBMCs
pulsed with the same mix of peptides, and at day 28, they were
harvested, washed, and used as responder cells against T2 cells
pulsed with individual peptides The levels of nonspecific
background (obtained with peptide-unpulsed T2 cells) were
subtracted. (nonpulsed T2 cells served as nonspecific control,
which was subtracted). A. Analysis of the responses to individual
MAA peptides; top: Comparison of the CTL-inducing cultures
utilizing individual serum-free and serum-supplemented protocols of
DC generation; bottom: back-to-back comparison of CTL-inducing
efficacy of cDC and alpha type-1 DC (.alpha.DC1) generated in
serum-free conditions B. .alpha.DC1 are highly-efficient
cross-presenting cells. Apoptotic melanoma cells (FEM-X) were fed
to immature DCs or .alpha.DC1 generated from the blood of healthy
HLA-A2+ donor and used to prime autologous CD8+ T cells (see
directly above). At day 28, the numbers of IFN.gamma.-producing
CD8+ T cells recognizing the original melanoma cell line (FEM-X),
or T2 cells pulsed with a defined melanoma associated epitope (MART
27-35). The levels of nonspecific IFN.gamma.-producing cells
(obtained respectively with non-HLA-A2-expressing melanoma cell
line, or with T2 cells pulsed with melanoma-nonrelevant peptide)
were subtracted.
[0058] FIG. 9. Alpha type-1 DC (.alpha.DC1) show superior ability
to induce cytotoxic T cell (CTL) responses against multiple
melanoma-specific epitopes, when compared to the currently-applied
DCs. Alpha type-1 DC (.alpha.DC1), generated under serum-free
conditions, or IL-1/TNF/IL-6/PGE.sub.2-matured cDC, were pulsed
with melanoma-associated antigenic peptides [MART-1 (27-35), gp100
(209-217 & 154-162), tyrosinase (368-376)], and used to
sensitize autologous CD8.sup.+ T cells from HLA-A2.sup.+ melanoma
patient (see the legend to FIG. 8 for the experimental details). A.
The cumulative responses of differentially sensitized T cells (at
day 28 post IVS) to all of the peptides used for in vitro
sensitization, in 3 patients with stage II-stage IV melanoma
(IFN.gamma. ELISPOT). The levels of nonspecific backgrounds were
subtracted. B. To analyze their cytotoxic (CTL) activity, the
differentially-induced CD8.sup.+ T cells were harvested at day 28,
washed, and used as effector cells against .sup.51Cr-labelled T2
cells pulsed with all MAA antigenic peptides as target cells (Top).
Bottom: The levels of nonspecific killing (obtained with irrelevant
peptide-unpulsed T2 cells) were low and similar in all cases.
[0059] FIG. 10. Mature .alpha.DC1 generated in serum-free cultures
are superior inducers of cytotoxic activity of CD4+ T cells and NK
cells against tumor cells. A. Standard DC
(IL-1.beta./TNF.alpha./IL-6/PGE.sub.2- -mature and .alpha.DC1
induced in serum free cultures by IL-1.beta., TNF.alpha., p-I:C,
IFN.alpha., and IFN.gamma., were loaded with superantigen (SEB) and
used to prime freshly-isolated CD4+ T cells. After two week
expansion of CD4+ T cell cultures, the cytotoxic potential of
differentially-primed CD4+ T cells was tested in 4 hour or 16 hour
chromium release assay, using SEB-coated melanoma cells as targets.
Data from a representative experiment of 3. B. NK cell activating
potential of differentially-matured DCs. C. Cytotoxic activity of
NK cells against chromium-labeled K562 target cells, either without
prestimulation or prestimulated overnight by (left) serum free
generated .alpha.DC1
(IL-1.beta./TNF.alpha./IFN.alpha./p-I:C/IFN.gamma.-induced DC1) and
standard DCs (IL-1.beta./TNF.alpha./IL-6/PGE.sub.2-matured DC) or
(right) traditional DC1 (generated in FCS-supplemented cultures and
matured with LPS and IFN.gamma.) or standard
(IL-1.beta./TNF.alpha./IL-6/PGE.sub.2-mat- ured) DC generated in
the presence of FCS. 4 hour-long chromium release assay at 10:1 T:T
ratio. Data from one of two experiments that both showed similar
results.
[0060] FIG. 11. Mature .alpha.DC1 generated from melanoma patients
in serum-free cultures are highly-responsive to 6C-kine and
maintain high IL-12p70 producing capacity following migration. A.
6C-kine-induced migration of differentially-matured DCs. B.
IL-1.beta./TNF.alpha./IFN.alp- ha./p-I:C/IFN.gamma.-induced
.alpha.DC1 (but not standard DCs nor iDC) retain their ability to
produce high levels of IL-12p70 in response to CD40-ligation,
following the migration in 6C-kine gradients. The inset: The
corresponding relative numbers of DCs migrating in response to
6C-kine.
* * * * *