| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
Advances in Brief |
Northwest Biotherapeutics, Inc., Seattle, Washington 98134 [P. A. L., L. A. J., R. A. B., M. L. S.], and Pacific Northwest Cancer Foundation, Seattle, Washington 98125 [G. P. M.]
 |
ABSTRACT |
|---|
 Top ABSTRACT IntroductionMaterials and MethodsResultsDiscussionREFERENCES |
|---|
|
Introduction |
|---|
|
TopABSTRACTIntroductionMaterials and MethodsResultsDiscussionREFERENCES |
|---|
|
Materials and Methods |
|---|
|
TopABSTRACTIntroductionMaterials and MethodsResultsDiscussionREFERENCES |
|---|
DC Generation and Infusion.
The generation of DCs from PBMCs has been described previously
(14)
. Briefly, PBMCs were isolated using Lymphoprep (Life
Technologies, Inc., Gaithersburg, MD) or Histopaque 1077 Ficoll (Sigma
Chemical Co., St. Louis, MO). After a 1-h, 37°C incubation step in
tissue culture flasks, adherent PBMCs were cultured with
granulocyte-macrophage colony-stimulating factor (500 units/ml) and
IL-4 (500 units/ml) for 7 days.
The treatment regimen has been detailed in previous work (12 , 13 , 16 , 17) . All patients had confirmed locally recurrent prostate cancer or hormone-refractory, advanced disease. Immediately prior to treatment, DCs were exogenously pulsed with peptide via incubation with 10 µg/ml PSM-P1 and PSM-P2 for 2 h. After washing, the DC suspension was infused i.v. over 30 min in a total volume of 10 ml of 0.9% saline. Patients received six infusions of peptide-pulsed autologous DCs at 6-week intervals. Patients were followed before, during, and after therapy with periodic PSA (Tandem-E PSA kit; Hybritech, Inc., San Diego, CA), chest X-rays, bone scans, and ProstaScint scans (18) . Clinical responses were determined by modified criteria of the National Prostatic Cancer Project (19) as well as PSA determinations.
Measurement of DTH.
DTH was measured prior to the first infusion and after the final
treatment with a Multitest CMI (Pasteur Merieux Connaught, Swiftwater,
PA). The recall antigens included in the test were: tetanus,
diphtheria, streptococcus, tuberculin, glycerin, Candida,
trychophyton, and proteus. Antigen (0.1 ml) was injected intradermally.
The skin test was read 48–72 h after administration. The widest
diameter of distinctly palpable induration was recorded in millimeters.
A 5-mm or greater induration was scored as a positive test.
Measurement of Nonspecific T-Cell Response.
Blood was drawn from patients prior to the first infusion and at time
points after every infusion. PBMCs were isolated at each time point and
stored in liquid nitrogen. PBMCs from all time points were evaluated in
the same assay for any one patient. To stimulate with anti-CD3, 100
µl of monoclonal antibody (Biosource International, Camarillo, CA) at
three concentrations (0.5, 0.25, and 0.1 µg/ml) was added in
triplicate in a 96-well, round-bottomed plate. After incubation
overnight at 4°C, antibody was removed, and 2 x 105 PBMCs were added in 0.2 ml of AIM V (Life
Technologies, Inc., Gaithersburg, MD). After a 48-h incubation,
supernatant was removed. A sandwich ELISA was used to determine the
amount of IFN-
in the supernatant. ELISA plates (Greiner, Palatine,
IL) were coated with 100 µl of capture antibody (Endogen, Inc.,
Woburn, MA) diluted in PBS at 1 µg/ml. After overnight incubation at
4°C, antibody was removed, and the plate was blocked with 100 µl of
PBS/BSA for 1 h at room temperature. Plates were then washed four
times. Sample or IFN- standard and detection antibody were added in
a volume of 100 µl, and the plate was incubated for 1.5 h at
room temperature. Plates were washed four times with PBS/0.05% Tween
20 using a Microplate Autowasher (Bio-Tek Instruments). Strepavidin
peroxidase (Zymed, South San Francisco, CA), diluted 1:10,000 in
PBS/Tween, was added in a volume of 100 µl and incubated for 30 min
at room temperature. Plates were washed four times, and then 100 µl
of OPD substrate (Zymed) was added to each well. The reaction was
incubated for 30 min at room temperature and then stopped with 100 µl
of 2N H2SO4.
The plates were read at 490 nm with a 630-nm reference using an EL 340
Microplate reader (Bio-Tek Instruments).
Microwell Cytokine Assay.
Our procedure was a modification of the semiquantitative assay
described recently by Romero et al. (Ref. 11
;
Fig. 1
). PBMCs from prostate cancer patients enrolled in the clinical trial
were used. IFN- secretion was measured using PBMCs collected before
the onset of therapy (pretreatment) and during or after the completion
of the regimen (posttreatment). They were added to 96-well plates at a
concentration of 1 x 106/ml in a
volume of 100 µl. They were cocultured with autologous DCs
exogenously pulsed for 2 h with 25 µg of peptide(s) consisting
of either: (a) PSM-P1 and PSM-P2, or (b) M1. An
E:T ratio of 10:1 was used. DMEM (Biofluids, Inc., Gaithersburg, MD)
supplemented with 10% human serum (Sigma Chemical Co., St. Louis, MO),
antibiotics, and gentamicin was used for all cultures. Cells were
cultured in a total volume of 200 µl. Three days after initial
stimulation, 300 IU/ml IL-2 were added to each 96-well culture.
Cultures were restimulated with peptide-pulsed DCs at day 10 of
culture. On day 11, 1 day after restimulation, 300 IU/ml IL-2 were
added to each microculture.
|
On day 17, all microcultures were gently resuspended. One hundred µl
of each effector T-cell suspension were added to 100 µl of T2 cells
(5 x 104/ml) that had previously
been exogenously pulsed with whichever peptide(s) (PSM-P1 and PSM-P2,
or M1) were used for the stimulations in vitro. The
remaining 100µl of each effector T-cell suspension was added to 100
µl of T2 cells (5 x 104/ml)
alone (unpulsed). Twenty-four h later, the supernatants were harvested
and measured for IFN- and IL-10 production in an ELISA using paired
antibodies from the manufacturer (Endogen, Inc., Woburn, MA). All
experimental assay points were in singlicate. Specific cytokine
secretion was determined by subtracting the amount of IFN- and IL-10
from a particular microwell in response to T2 alone from that secreted
in response to T2 plus peptide(s). Standards were performed in
duplicate.
Microwell Cytotoxicity Assay.
The procedure was identical to that of the microwell cytokine analysis,
with the exception that the T2 cells were 111In
labeled. Target cells (1 x 106)
were labeled with 111In (Nycomed Amersham,
Buckinghamshire, United Kingdom) in 0.5 ml of PBS or AIM-V (Lymphoprep;
Life Technologies, Inc.) for 10 min at 37°C, then washed three times
before the addition of T cells. Incubation of target and effector cells
was carried out for 4 h at 37°C. Supernatants were collected and
counted on a gamma counter. The percentage of specific lysis was
calculated as: (sample counts - spontaneous
counts/maximum counts - spontaneous counts) x 100%. Spontaneous release was determined from targets
incubated with AIM-V. Maximum release was determined from targets
incubated with 2% SDS. All experimental assay points were in
singlicate. Spontaneous and maximum releases were determined in
triplicate. Spontaneous counts did not exceed 30% of maximum
111In labeling. The percentage of specific
cytotoxicity was determined by subtracting the percentage of
cytotoxicity from a particular microwell in response to T2 alone from
the lytic activity in response to T2 plus peptide(s). Only the
percentage of specific cytotoxicity >10% was scored as an
antigen-specific microwell.
|
Results |
|---|
|
TopABSTRACTIntroductionMaterials and MethodsResultsDiscussionREFERENCES |
|---|
A cohort of patients was clinically evaluable (i.e., 46 for
DTH testing), and it is those patients who were assessed for this
report. The immune status of the patient was evaluated using two
different assays: (a) DTH; and (b) cytokine
secretion. The first assay was a skin test measuring DTH to six
different recall antigens: tetanus, diphtheria, streptococcus,
tuberculin, glycerin, Candida, trychophyton, and proteus.
Patients were tested prior to infusion and after completion of all six
infusions. As shown in Table 1
, patients that responded clinically to DC-based immunotherapy were more
likely to have a DTH response than those patients who did not.
Moreover, there was an association between response to treatment and
the number of patients with three or more positive skin tests. Because
of the small number of complete responders and the number of patients
in each group overall, both Kruskal-Wallis analysis and P
test did not indicate that the differences between groups were
statistically significant. However, the tendency was for partial and
complete responders to have a greater percentage of patients with three
or more positive reactions, and this greater reactivity remained
constant throughout treatment.
|
production was measured 48 h
later by ELISA. As shown in Table 2
, production of low amounts of IFN- (<2000 pg/ml) correlated with
poor clinical response, whereas production of greater amounts (>300
pg/ml) indicated a probability of a positive clinical outcome. Although
two patients with progressive disease demonstrated high cytokine
secretion from preimmune PBMCs, it is important to point out that this
reactivity was not durable. That is, when using PBMCs from
posttreatment time points, only low IFN- secretion was observed.
This is in contrast with PBMCs from responders, whose high IFN-
secretion was maintained throughout treatment. In total, our cytokine
secretion data suggest that DC-based immunotherapy is more effective in
patients with a functional immune system.
|
We performed a semiquantitative analysis (Fig. 1)
for PSMA- and
M1-restricted T-cell reactivity by using a modified microwell ELISA
(Fig. 2)
and cytotoxicity assay (Fig. 3)
. Instead of a bulk method, usually using 24-well culture dishes, we
established singlicate microcultures of PBMCs in 96-well plates.
For patients in our clinical trial, both pre- and posttreatment PBMCs
were studied. In addition, both PSMA- and M1-restricted T-cell
reactivity was determined by conducting two stimulations in
vitro by testing against T2 cells with and without the stimulating
peptide in a split-well analysis.
|
|
. Twelve
of 24 microwells (50%) from pretreatment PBMCs showed specific IFN-
secretion >100 pg/ml. This reactivity was durable, because 50% of
microwells from posttreatment PBMCs showed specific IFN- secretion.
When IL-10 was analyzed, no microwells producing 
100 pg/ml were
observed.
In several patients achieving partial responses, the results of
microwell cytolytic assays again demonstrated the importance of
preexisting immunity (Fig. 3A)
. For Patient 122, who
achieved a 50% decrease in PSA with no new lesions as a result of
treatment (thereby being classified as a partial responder),
demonstrated the same strong lytic activity against the immunizing
peptides both before and after therapy (16 of 24 microwells; 67%).
PBMCs from a patient demonstrating progressive disease (no. 126), set
up at the same time, demonstrated minimal activity. In a smaller cohort
of partial responders, specific cytolytic activity began fairly low but
increased during treatment (Fig. 3B)
. Using PBMCs from
patient 94, cytotoxicity against the recall antigen M1 was low prior to
the first DC infusion (7 of 24 microwells; 29%) but was quite vibrant
after the sixth infusion (17 of 24 microwells; 71%). When performed
concurrently, PBMCs from patient 69, who did not respond to treatment,
showed minimal lytic activity.
|
Discussion |
|---|
|
TopABSTRACTIntroductionMaterials and MethodsResultsDiscussionREFERENCES |
|---|
Importantly, the data support the concept of prescreening patients to determine their immunocompetence before treating them with immune-based therapies. Determining which patients would potentially benefit from a given treatment is a crucial decision medical science faces on a daily basis. It is even more critical in the arena of advanced disease in which the subject has a limited life expectancy, and therefore may be facing their final opportunity for successful treatment. Lastly, for those whose cancer is of an earlier stage, monitoring a patient’s immune reactivity will be an important adjunct to imaging studies such as bone scans for the clinician to assess whether a patient is responding to immunotherapy.
It is noteworthy that the durable complete responder in our protocol demonstrated a Th1-type of immune response. This patient demonstrated specific cytokine secretion in when stimulated in vitro and subsequently tested against either the immunizing peptides or the recall antigen M1. The number of complete responders sampled is far too small to draw any firm conclusions. But it must be recognized that our results are interesting in light of the difficulty in producing any partial or complete responders in patients with hormone-refractory, metastatic prostate cancer.
Finally, in a finite cohort of partial responders, we demonstrated an association between clinical outcome and either cytokine secretion or lytic activity against the immunizing PSM-P1/P2 peptides or against the recall epitope M1. Pre-existing immunity seemed to be most strongly associated with clinical outcome for those microwell analyses that demonstrated antigen-specific cytokine secretion or cytotoxicity. But it must be mentioned that this observation, although not anecdotal, was not observed in a vast majority of patients. That is, we did not observe any repeatable, reliable correlation between peptide-specific immunoreactivity and clinical outcome. Therefore, it is highly questionable whether the time, effort, and cost of setting up microwell analyses of this sort are justified, and any observed correlation must be considered far from conclusive.
The paucity of immunological data concerning PSMA could be attributable to several reasons. The antigen may be poorly immunogenic. It is not inconsequential to note that there is not a single report in the literature of T-cell reactivity against PSMA-derived peptides. Alternatively, the assays used may not have been sensitive enough to detect PSMA-specific responses. T-cell receptor-ligand interactions are relatively weak compared with antigen-antibody interactions. In addition, there is great variation in the sensitivity of immune monitoring methods, depending upon the approach used (11) , and if the precursor T-cell frequency was below 1/105, it would likely be below the threshold of detection of the methods we used. Perhaps PSMA-specific cells are sequestered at tumor sites and not found in the peripheral blood. Finally, the mechanism responsible for the clinical response may have not been addressed. For example, DCs have been reported recently to stimulate natural killer cells (20) : natural killer activity would not have been detected by any of the assays used in our evaluation.
There has been a great deal of work concerning immune monitoring (reviewed in Ref. 10 ).3 Five methods commonly used to detect B- or T-cell responses in PBMCs are: (a) antibody titer determinations; (b) cytotoxicity; (c) cytokine release (via ELISA, ELISPOT, or intracellular staining); (d) molecular analysis of T-cell receptor usage; and (e) direct quantification of antigen-specific cells using tetramers (11) . A complimentary tactic is to look for defects in a patient’s immune cells (21, 22, 23) , or prostate tumor escape mechanisms (24 , 25) , as reasons for a subject to fail treatment. There is currently no consensus on which methods to use or how a monitoring scheme should be designed. This is appropriate, considering that the field of cancer vaccines directed against defined antigenic targets is in its infancy. One fundamental, inherent problem with true immunological monitoring is that a sufficient number of clinical responses must be achieved before statistical correlations between in vitro and in vivo parameters are declared; for experimental immunotherapy of advanced cancers, this is not trivial. For tumor types other than melanoma, there exists another dilemma: the glaring lack of basic knowledge concerning human tumor-associated antigens. It is a problem that must be addressed if monitoring and the immune-based therapies they are based upon are to fulfill their early promise.
|
FOOTNOTES |
|---|
1 To whom requests for reprints should be
addressed, at Northwest Biotherapeutics, Inc., 2203 Airport Way South,
Suite 200, Seattle, WA 98134. Phone: (425) 608-3052; Fax:
(425) 608-3004; E-mail: mls{at}nwbio.com 
2 The abbreviations used are: PSMA,
prostate-specific membrane antigen; PSA, prostate-specific antigen;
HLA, human leukocyte antigen; IL, interleukin; DC, dendritic cell;
PBMC, peripheral blood mononuclear cell; DTH, delayed-type
hypersensitivity; Th1, T helper cell type 1.
3 G. Shankar and M. L. Salgaller. Immune
monitoring of cancer patients undergoing experimental immunotherapy.
Curr. Opin. Mol. Ther., in press.
Received 10/ 7/99. Accepted 1/ 3/00.
|
REFERENCES |
|---|
|
TopABSTRACTIntroductionMaterials and MethodsResultsDiscussionREFERENCES |
|---|
and lipopolysaccharide. Cancer Immunol. Immunother., 45: 250-256, 1998.[Medline]
J. J., Rosenberg S. A., Bennick J. R. Identification of human cancers deficient in antigen processing. J. Exp. Med., 177: 265-272, 1993.This article has been cited by other articles:
|
|
 |
|
  A. Mukhopadhaya, J. Mendecki, X. Dong, L. Liu, S. Kalnicki, M. Garg, A. Alfieri, and C. Guha Localized Hyperthermia Combined with Intratumoral Dendritic Cells Induces Systemic Antitumor Immunity Cancer Res., August 15, 2007; 67(16): 7798 - 7806. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 C. P. Tarassoff, P. M. Arlen, and J. L. Gulley Therapeutic vaccines for prostate cancer. Oncologist, May 1, 2006; 11(5): 451 - 462. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 B. E. Loveland, A. Zhao, S. White, H. Gan, K. Hamilton, P.-X. Xing, G. A. Pietersz, V. Apostolopoulos, H. Vaughan, V. Karanikas, et al. Mannan-MUC1-Pulsed Dendritic Cell Immunotherapy: A Phase I Trial in Patients with Adenocarcinoma Clin. Cancer Res., February 1, 2006; 12(3): 869 - 877. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
F. Chagnon, S. Tanguay, O. L. Ozdal, M. Guan, Z. Z. Ozen, J.-S. Ripeau, M. Chevrette, M. M. Elhilali, and L. A. Thompson-Snipes Potentiation of a Dendritic Cell Vaccine for Murine Renal Cell Carcinoma by CpG Oligonucleotides Clin. Cancer Res., February 1, 2005; 11(3): 1302 - 1311. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 S. Watanabe, H. Kagamu, H. Yoshizawa, N. Fujita, H. Tanaka, J. Tanaka, and F. Gejyo The Duration of Signaling through CD40 Directs Biological Ability of Dendritic Cells to Induce Antitumor Immunity J. Immunol., December 1, 2003; 171(11): 5828 - 5836. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
T. Satoh, T. Saika, S. Ebara, N. Kusaka, T. L. Timme, G. Yang, J. Wang, V. Mouraviev, G. Cao, E. M. A. Fattah, et al. Macrophages Transduced with an Adenoviral Vector Expressing Interleukin 12 Suppress Tumor Growth and Metastasis in a Preclinical Metastatic Prostate Cancer Model Cancer Res., November 15, 2003; 63(22): 7853 - 7860. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 K. Tschoep, T. C. Manning, H. Harlin, C. George, M. Johnson, and T. F. Gajewski Disparate functions of immature and mature human myeloid dendritic cells: implications for dendritic cell-based vaccines J. Leukoc. Biol., July 1, 2003; 74(1): 69 - 80. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. Gilliet, M. Kleinhans, E. Lantelme, D. Schadendorf, G. Burg, and F. O. Nestle Intranodal injection of semimature monocyte-derived dendritic cells induces T helper type 1 responses to protein neoantigen Blood, July 1, 2003; 102(1): 36 - 42. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 L. Samady, E. Costigliola, L. MacCormac, Y. McGrath, S. Cleverley, C. E. Lilley, J. Smith, D. S. Latchman, B. Chain, and R. S. Coffin Deletion of the Virion Host Shutoff Protein (vhs) from Herpes Simplex Virus (HSV) Relieves the Viral Block to Dendritic Cell Activation: Potential of vhs- HSV Vectors for Dendritic Cell-Mediated Immunotherapy J. Virol., March 15, 2003; 77(6): 3768 - 3776. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. Lu and E. Celis Recognition of Prostate Tumor Cells by Cytotoxic T Lymphocytes Specific for Prostate-specific Membrane Antigen Cancer Res., October 15, 2002; 62(20): 5807 - 5812. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 G. Parmiani, C. Castelli, P. Dalerba, R. Mortarini, L. Rivoltini, F. M. Marincola, and A. Anichini Cancer Immunotherapy With Peptide-Based Vaccines: What Have We Achieved? Where Are We Going? J Natl Cancer Inst, June 5, 2002; 94(11): 805 - 818. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. Ross, S. D. Spencer, I. Holcomb, C. Tan, J. Hongo, B. Devaux, L. Rangell, G. A. Keller, P. Schow, R. M. Steeves, et al. Prostate Stem Cell Antigen as Therapy Target: Tissue Expression and in Vivo Efficacy of an Immunoconjugate Cancer Res., May 1, 2002; 62(9): 2546 - 2553. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 C. Sanchez-Torres, G. S. Garcia-Romo, M. A. Cornejo-Cortes, A. Rivas-Carvalho, and G. Sanchez-Schmitz CD16+ and CD16- human blood monocyte subsets differentiate in vitro to dendritic cells with different abilities to stimulate CD4+ T cells Int. Immunol., December 1, 2001; 13(12): 1571 - 1581. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
N. Okada, T. Saito, Y. Masunaga, Y. Tsukada, S. Nakagawa, H. Mizuguchi, K. Mori, Y. Okada, T. Fujita, T. Hayakawa, et al. Efficient Antigen Gene Transduction Using Arg-Gly-Asp Fiber-Mutant Adenovirus Vectors Can Potentiate Antitumor Vaccine Efficacy and Maturation of Murine Dendritic Cells Cancer Res., November 1, 2001; 61(21): 7913 - 7919. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
H. Zheng, P. Palese, and A. García-Sastre Antitumor Properties of Influenza Virus Vectors Cancer Res., December 1, 2000; 60(24): 6972 - 6976. [Abstract] [Full Text]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
| Cancer Research | Clinical Cancer Research |
| Cancer Epidemiology Biomarkers & Prevention | Molecular Cancer Therapeutics |
| Molecular Cancer Research | Cancer Prevention Research |
| Cancer Prevention Journals Portal | Cancer Reviews Online |
| Annual Meeting Education Book | Meeting Abstracts Online |
