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Autologous Dendritic Cell Vaccines for Non–Small-Cell Lung Cancer

From the Division of Pulmonary and Critical Care Medicine, Department of Biostatistics, Department of Pathology, and Department of Microbiology and Immunology, University of Kentucky, Chandler Medical Center, Lexington Veteran's Administration Medical Center, Lexington, KY

Address reprint requests to Edward Hirschowitz, MD, Division of Pulmonary and Critical Care Medicine, University of Kentucky, Chandler Medical Center, 800 Rose St, Room MN 614, Lexington, KY 40536; e-mail: eahirs2{at}pop.uky.edu

	ABSTRACT

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION Authors' Disclosures of... REFERENCES

 
PURPOSE: Therapeutic outcomes of definitively treated non–small-cell lung cancer (NSCLC) are unacceptably poor. A wealth of preclinical information, and a modest amount of clinical information indicate that dendritic cell (DC) vaccines have therapeutic potential. Only a handful of NSCLC patients have been included in DC clinical trials. We delivered autologous DC vaccines to 16 individuals with stage IA to IIIB NSCLC treated with surgery, chemoradiation, or multimodality therapy. The objectives of the study were to evaluate tolerability and measure immunologic responses to DC vaccines in a heterogeneous group of NSCLC patients.

METHODS: DC vaccines were generated from CD14+ precursors, pulsed with apoptotic bodies of an allogeneic NSCLC cell line that overexpressed Her2/neu, CEA, WT1, Mage2, and survivin. DCs were partially matured with a factor that induced surface molecule expression but minimal cytokine production. Individuals were immunized intradermally two times, 1 month apart. Peripheral blood was drawn serially over 16 weeks, and immune responses were measured by interferon-gamma ELISPOT.

RESULTS: There were no unanticipated or serious adverse events. Immunologic responses followed three distinct patterns of reactivity: (1) five of 16 patients showed no clear immunologic response, (2) five of 16 patients showed a tumor-antigen independent response, and (3) six of 16 show an antigen specific response. Immunologic responses were independent of stage and prior therapy. Favorable and unfavorable clinical outcomes were independent of measured immunologic responses.

CONCLUSION: Vaccines were well tolerated and had biologic activity in a variety of NSCLC patients. Establishing an optimal approach will require comparative studies in well-defined NSCLC patient groups.

	INTRODUCTION

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Despite recent advances in treatment of non–small-cell lung cancer (NSCLC), clinical outcomes remain poor.^1,2 Recurrences in surgically resectable stage I to IIIA NSCLC patients treated with aggressive multimodality therapy are common.^1,2 Similarly, following chemotherapy and radiation for unresectable stage IIIA/B NSCLC, 5-year survival does not exceed 25%.¹ Tumor vaccines may have an adjuvant role in surgically resectable and unresectable NSCLC by consolidating responses to conventional therapy. Encouraging clinical results recently reported in NSCLC patients immunized with an autologous tumor cell vaccine expressing granulocyte macrophage colony-stimulating factor strongly support the rationale for additional investigation of immunotherapy in NSCLC.³

Dendritic cells (DCs) are potent antigen-presenting cells that have been under intensive investigation as components of tumor vaccines.^4-8 Numerous small clinical trials evaluating ex vivo antigen-loaded DCs in patients with a variety of solid and liquid tumors have been reported in the literature.^4-8 Comparative information is difficult to glean from this diverse group of studies that collectively includes fewer than 600 patients.⁸ There is not a standardized methodology for preparing vaccines and many questions remain about the optimal source or type of antigen and maturation state of DCs. Regardless, numerous DC vaccine trials have shown biologic activity suggesting additional investigation is warranted.^7,8 Literature also indicates that a percentage of individuals may derive therapeutic benefit, although, as expected from phase I/II trials, reports of clinical efficacy are anecdotal.^7,8

Notably, none of the clinical trials reported in the literature have evaluated DC vaccines exclusively in NSCLC, and collective experience in NSCLC is limited.^9-13 We initiated a DC vaccine clinical trial for NSCLC to determine feasibility, to gain information with which to build future studies, and work toward optimizing DC vaccines in NSCLC. This study was designed to evaluate immunologic responses to antigen-pulsed DC vaccines in a heterogeneous group of NSCLC patients treated surgically, medically, and with multimodality approaches. Data are analyzed in context of host factors that could influence vaccine efficacy and define appropriate application of this strategy.

	METHODS

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Human Subjects/Patient Characteristics
Individuals with histologically confirmed stage I to IIIB NSCLC who had completed definitive medical, surgical, or multimodality therapy, and had stable clinical disease at screening, were eligible for the study. Participants were approved under a protocol approved by the University of Kentucky's Medical institutional review board. Individuals entered the study anytime from 6 weeks to 3 years after definitive therapy (average, 8 months). The treatment group was heterogeneous with respect to stage, histology, treatment of primary disease, and risk of recurrence. Patient characteristics are summarized in Table 1.

Leukapheresis
Cobe Spectra (Gambro BCT, Lakewood, CO), used for all procedures, is a continuous flow machine that uses centrifugation to separate cells based on specific gravity. Plasma pump flow rates were adjusted to 1.0 mL/min, which is the appropriate interface to isolate mononuclear cells. Three total blood volumes were processed each procedure and took approximately 3 hours. All but one individual required placement of femoral double lumen hemodialysis catheters for access.

DC Preparation
CD14+ cells were isolated using a commercial magnetic bead separation protocol (Miltenyi Biotec, Auburn, CA). Ninety-five percent to 99% pure CD14+ cells were cultured in XVIVO-15 medium (10⁶ cell/mL; Biowhitaker, Walkersville, MD) supplemented with 5% heat-inactivated human AB serum (Biochemed, Winchester, VA), 2 mmol/L l-glutamine, 50 µg/mL penicillin/streptomycin, 10 µg/mL gentamycin, 100 µmol/L nonessential amino acids, 1 mmol/L sodium pyruvate, 50 µg/mL amphotericin-B, and 20 mmol/L HEPES buffer. Culture was supplemented with 20 ng/mL GMCSF and 20 ng/mL interleukin (IL) -4. Additional cytokines were added on days 2 and 5. Tumor antigens (described in Methods: Antigen Source and Preparation) were added at day 7. Four hours after antigen pulsing, Dendritic Cell/T Cell-derived Maturation Factor (DCTCMF described in this article), supplemented with 1,000u/mL interferon-gamma (IFN-), was added, and culture was continued for 18 hours.¹⁴ On day 8, 10⁸ 1650-pulsed DCs were resuspended in 3 mL of sterile saline for immediate injection.

Microbiologic Monitoring
Samples were taken from culture 24 hours before harvest for sterility testing at the University of Kentucky Clinical Microbiology Laboratory. Sterility was confirmed just before delivery. A sample of the final product was also submitted for microbiologic testing the day of delivery.

Antigen Source and Preparation
To generate a pluripotent vaccine, the adenocarcinoma cell line 1650 was used as a source of tumor-rejection antigens.¹⁵ 1650 overexpresses Her-2/neu, CEA, Mage 2, WT-1¹⁵ and survivin (unpublished data). The 1650 line does not express MAGE 1 or MAGE 3. The line was confirmed sterile by clinical laboratory analysis and Mycoplasma negative at regular intervals using polymerase chain reaction (Gen-Probe Inc, San Diego, CA). Just before DC pulsing, 1650 were apoptosed, then lethally irradiated. For apoptosis, 1650 (2 x 10⁶ cells/mL) were placed 6.5 cm from a 20 watt UV-b light source for 2 minutes. Cells were then irradiated with 10,000 rads using a Cesium-137 source. Treated 1650 were nonviable when monitored in extended culture. Apoptosis was confirmed by annexin-V staining. Apoptosed-irradiated 1650 were added to day 7 DC cultures at a DC:1650 ratio of 1:1.

Preparation of DCTCMF
DCTCMF is a cytokine-enriched supernatant from autologous lymphocytes cultured for 48 hours in OKT-3 (anti-CD3 MoAb) coated flasks (10 µg/mL in coating buffer).¹⁴ Supernatant was centrifuged, filtered, supplemented with IFN- (1,000 units/mL) and stored at 4°C until use.

Immunization Protocol
The protocol, including route of administration, interval, and dose, was based on the best available information in the literature.⁸ A prime vaccine and a single boost were given one month apart intradermally in the thigh. The target dose was 10⁸ antigen-pulsed DCs in 3 mL volume. Average number of DCs injected in 16 prime injections was 9.1 x 10⁷, and 16 boost injections was 8.2 x 10⁷. Patients were monitored in the outpatient clinic for 2 hours following immunization for immediate unanticipated adverse events.

Clinical Evaluation
Follow-up by primary treating physicians included routine history and physical, chest x-ray, and/or computed tomography scans at regular intervals post-therapy or as directed by signs or symptoms of tumor recurrence.

Immunologic Assessment
Serial blood samples were drawn for immunologic testing (prevaccine, weeks 1, 4, 5, 8, 12, and 16 to complete the initial series; samples were also drawn at 6 and 12 months postvaccine). A portion of each sample was cryopreserved. Since each interval 50-mL sample yielded a limited and variable number of lymphocytes, a majority of the sample was amplified with phytohemaggluttinin (PHA; 5 µg/mL for 10 days) to obtain adequate numbers for analysis. PHA-stimulated lymphocytes, expanded 10- to 20-fold, were then cryopreserved. Before their use as responders in immunologic assays, cells were thawed and "rested" for 48 hours in PHA and IL-2–free medium yielding "PHA-rested blast cells." Duplicate assays were performed with non-PHA expanded lymphocytes from some subjects to confirm the accuracy and reproducibility of PHA-rested blast in this analysis.

IFN- ELISPOT assays were performed only when week-16 samples were collected and all samples could be run in parallel to avoid variability. ELISPOT was conducted as follows: flat-bottom Multiscreen-HA 96-well plates (Millipore, Bedford, MA) were precoated overnight at 4°C with antihuman IFN- ELISPOT antibody (1 µg/well in phosphate-buffered saline [PBS]; Mabtech, Mariemont, OH). The plates were then washed with PBS and blocked using complete RPMI-1640 for 1 hour.

Autologous tumor for testing immune responses was not available from our patients. As an alternative, 1650-antigen pulsed autologous DCs, prepared in an identical fashion to the vaccine, were used as targets for immune reactivity as previously described.^10,16 PHA-rested blasts used as responders were mixed with autologous 1650-antigen pulsed DCs at a lymphocyte-DC ratio of 10:1, and cocultured for 20 hours at 37°C. Controls included autologous DCs without antigen, prepared identically to 1650 antigen-pulsed DCs except for antigen pulsing, and T cells alone. PHA added to lymphocytes alone (10 µg/well) served as a control for lymphocyte proliferation. Each condition included six replicates.

Following 20-hour coculture, cell-containing medium was removed, plates were washed with 0.05% Tween-20 in PBS (Sigma, St Louis, MO), and incubated with antihuman IFN- biotinylated antibody (0.1 µg/well in 100 µL volume; Mabtech) at 37°C for 2 hours. Plates were again washed and incubated with APC solution (100 µL/well in PBS-0.1% Tween-20; Vecstatin kit, Vector labs, Burlingame, CA) at room temperature for 1 hour. During incubation, AEC solution (3-amino-9ethylcarbazole) was prepared by mixing 1-AEC tablet (Sigma) in 2.5 mL DMF (Sigma), diluted to 50 mL with acetate buffer (0.1N acetic acid, 0.1M sodium acetate, 25 µL 30% H₂O₂). The plates were washed free of APC solution, incubated with AEC solution (100 µL/well) for 4 minutes at room temperature in the dark, then the reaction stopped by rinsing plates with distilled water. Plates were dried overnight and analyzed using a Zeiss ELISPOT system.

Statistical Methods
Significance was determined by analysis of variance. Correlation of events was assessed by linear regression analysis.

	RESULTS

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DC Characteristics
Final vaccine products were CD14- and a relative percent of the cell population expressed MHC-I (100%), MHC-II (80% to 90%), coexpressed CD80 and CD86 (60% to 75%), CD40 (50% to 80%), CD11c (85% to 90%), and the maturation marker CD83 (15% to 30%). Antigen-pulsed, DCTCMF treated DCs, however, appeared more immature with respect to cytokine secretion. Final DC products produced limited amounts (10⁶ DCs/mL/24 hours) of IL-12p40 (DCs alone: 28 ± 98 v DCs+DCTCMF: 338 ± 363; P > .1; n = 13), IL-10 (DCs alone: 13 ± 14 v DCs+DCTCMF: 87 ± 83; P > .1; n = 13) and to an even lesser extent IL-12p70 (DCs alone: 4 ± 10 versus DCs+DCTCMF: 12 ± 25; P > .1; n = 8). Additional experiments showed these cells were capable of producing significant amounts of each cytokine when stimulated with the standard laboratory maturation factor lipopdysaccharide/IFN- (IL-12p40: 1,585 ± 631, IL-10: 419 ± 184, and IL-12p70 175 ± 156; P < .01 all comparisons to DC alone and DC+DCTCMF).

Adverse Effects
A self-limited wheal and flare skin reaction appearing at the injection site 24 to 48 hours after immunization occurred in 10 of 16 subjects during at least one of the two immunizations. Two individuals noted a more profound reaction with the second vaccine. Maximal reaction was 6 cm. Three individuals noted minor fatigue for 1 to 2 days following the vaccine. Safety laboratories checked following immunization revealed no abnormalities in hematological parameters or serum chemistries.

Immunologic Responses to Vaccines
IFN- ELISPOT was performed at all available timepoints in all individuals (Fig 1). Blood samples did not yield adequate cell numbers for analysis at all timepoints in all individuals. Measurements were made of the number of IFN- producing lymphocytes (spots) in 24 hours culture with 1650-antigen pulsed DC targets (similar to vaccine product). Controls included lymphocytes plus DC without antigen and lymphocytes alone. Comparisons were made between each postvaccine measurement and the prevaccine response for each condition. The lowest measurable response in each condition was used as a baseline when prevaccine samples were not available. Comparisons were also made between conditions at individual timepoints (1650-pulsed DC targets v controls).

View larger version (35K): [in this window] [in a new window]   Fig 1. Interferon-gamma ELISPOT: Measurements of lymphocytes ("spots") from available timepoints responding to: (•)1650-pulsed autologous dendritic cells (DCs), () DCs alone, () lymphocytes alone. () indicates significance. Immune response patterns of 16 patients are grouped according to: (A) no significant change from baseline, (B) 1650-independent reactions, and (C) antigen-specific reactions.

Peak significant 1650-specific and 1650-independent reactions ranged from 25 to 331 spots (P .01 compared with cell-alone controls). Baselines (prevaccine or lowest available timepoint) ranged from 0 to 4 spots except in one individual (DC16) who had a significant and reproducible elevation of antigen specific reactions prevaccine (61 ± 23; P < .001) that also exceeded antigen-specific reactions at weeks 4, 8, and 12. Patterns of reactivity varied between individuals. Two of 16 manifested a significant immunologic reaction at a single timepoint. Four of 16 showed distinct reactions at two separate timepoints. One individual (DC10) showed a clearly boosted response temporally related to the second immunization (41 ± 3 spots week 1, v 99 ± 10 spots week 5; P = .0001 week 5 to week 1). One additional individual (DC2) showed a measurable and significant reaction at 1 week, and a much more profound reaction at 24 weeks (25 ± 7 spots week 1 v 62 ± 11 spots week 24; P < .0001 week 24 to week 1; and P < .0001 compared with controls).

Correlation of Immune Response With Clinical Criteria
Measurable immune responses were seen irrespective stage, histology, surgical resection, or prior chemotherapy, and/or radiation. There was no discernable correlation between immune responses and the wheal and flare skin reaction.

Clinical Outcomes
Clinical follow-up is available on all individuals for a minimum of twelve months from primary immunization. Data are presented in Table 2. Five individuals to date have documented disease recurrence or progression, three of whom died 4 to 9 months following detection and palliation of stage IV disease. One individual with unresectable stage IIIB NSCLC is being treated for local progression 19 months from chemoradiation and 16 months from initial vaccine. One individual (DC10) with stage IB NSCLC treated with multimodality therapy developed a solitary brain metastasis 2 months following initial vaccine, underwent local resection of the metastatic focus, and has no evidence of disease 15 months following resection of stage IV disease. A sixth individual (DC16) with surgically resectable stage IIIA NSCLC treated with multimodality therapy is currently receiving chemotherapy for a radiographically persistent nodule 21 months from completion of treatment and 12 months from the vaccine.

	DISCUSSION

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The data indicate biologically active autologous DC vaccines can be produced for a variety of NSCLC patients. Vaccines were well tolerated. Immunologic responses to vaccines were independent of stage, histology, or prior therapy. These responses did not appear to correlate with clinical events, though sample size and patient heterogeneity precluded meaningful statistical assessment of clinical outcomes. A summary of clinical outcomes includes five individuals with documented disease recurrence or progression. Three individuals have died from progressive disease. One individual with stage IB NSCLC and solitary brain metastasis survives 15 months following surgical resection of stage IV disease and 17 months postvaccine. Clinical outcomes that may indicate therapeutic efficacy include the above individual with resected solitary brain metastasis, and two individuals with unresectable stage III NSCLC who show no signs of disease progression at 35 and 23 months from chemoradiation, respectively. One individual with resected stage IIIB bronchoalveolar cell carcinoma also remains tumor-free 28 months post–surgical resection and 19 months postvaccine. Since these favorable clinical outcomes did not correlate with any specific immunologic response pattern, we are not sure whether our IFN- ELISPOT readout assay, that targets antigens used in the vaccine, is necessarily indicative of clinical benefit. Conversely, neither are we sure that lack of measurable reactions by IFN- ELISPOT indicates lack of therapeutic benefit.⁷

A number of variables related to both the host environment and the vaccine itself may be relevant to potential therapeutic efficacy.⁷ Effects of prior chemotherapy and radiation on immunologic responses are especially relevant since adjuvant chemotherapy, with or without radiation, is likely to become standard of care for all stages of resectable NSCLC and chemoradiation is routine for unresectable disease.^1,2 Chemoradiation, or radiation alone, either as definitive therapy or as part of a multimodality approach, had no apparent effect on vaccine production or immune reactivity. Neither did purported systemic tumor-related immunosuppression appear to limit vaccine production or response. Immunologic resistance of tumor to immune effector cells at the local level remains a potential limitation to vaccine efficacy. We are also unsure whether metastasis in typically immune privileged sites, specifically the brain, are accessible to immune effector cells.⁸

Choice of antigens is also relevant to therapeutic efficacy and potentially to immunologic responses to vaccines. We used allogeneic tumor to produce a multivalent vaccine that allowed us to immunize individuals who did not have autologous tumor available for vaccine construction. Moreover, use of allogeneic tumor simplified the clinical protocol, and antigen-standardized vaccines facilitated comparison of immunologic reactions across a heterogeneous patient group. Consequently, our choice of antigens, that includes CEA, HER2/neu, WT-1, survivin, and Mage-2 may not have been optimal for all individuals. Notably, characterization of tumors for relevant antigens was not possible for a majority of subjects, nor practical in context of our budget and initial scientific goals. Retrospective analysis of specific antigens expressed by patient tumors is being considered. For practical reasons we have made only a best guess as to the targets presented by an individual's tumor. Since a single vaccine, even a multivalent vaccine, may not have a corresponding target, we paid special attention to wheal and flare skin reactions, as this might suggest the vaccine contained recall antigens. No clear correlation, however, could be made between the wheal and flare reaction and immunologic responses or purported therapeutic efficacy. Since antigen delivered with the DC vaccines also included foreign MHC derived from 1650, a wheal and flare skin reaction or immunologic response measured by IFN- ELISPOT based solely on alloreactivity is possible. 1650-independent responses noted in five of sixteen individuals might also indicate reactivity to antigens ingested by DCs during culture, such as serum supplements. Future protocols will therefore incorporate only autologous serum obtained from leukapheresis. Ultimately we cannot be certain that 1650-specific responses were to 1650-derived tumor protein(s), to other nontumor antigens including foreign MHC, or to both. Importantly, we questioned whether host immune response to foreign MHC or other nontumor antigens could be beneficial as adjuvants for corecognition of tumor proteins.

Lastly, there is no standardized methodology for vaccine preparation.^7,8 The DC vaccines used in this trial appeared to be mature by cell surface phenotype, yet incompletely activated with respect to cytokine secretion. However, using a standard maturation factor for in vitro studies, (LPS and IFN-), the DCs were capable of producing significant amounts of IL-12p70, a principal Th1 inducing cytokine indicative of final DC maturation.^19-23 By not fully maturing DCs before delivery we did not commit DC vaccines to a dominant IL-10 or IL-12p70 producing phenotype.^22-24 It is possible that DCs producing greater amounts of IL-10 could induce tolerance rather than immune stimulation.^22-25 Regardless, this vaccine induced measurable immunologic responses in a number of individuals, suggesting this DC preparation does have biologic activity. We therefore hypothesize that following injection the partially mature DCs interact with host inflammatory and resident cells to complete their maturation in vivo and subsequently induce the immunologic responses noted in this study.^26-28 Questions remain whether fully mature DCs, expressing high levels of IL-12, would be more biologically active or conversely whether DCs less mature than those used in this study might be optimal.

In summary, the current study shows it is possible to make biologically active DC vaccines for variety of NSCLC patients. Future studies might incorporate antigen characterization and evaluate effects of DC maturation on immunologic responses.^8,29 Alternative dosing, immunization schedules and routes of administration can be considered.^8,29 Comparative trials will help define an optimal approach and greater number of patients in controlled treatment groups will better define therapeutic efficacy.^8,29 Vaccines may ultimately find a permanent role as adjuvants that consolidate responses to definitive medical or multimodality therapy for all stages of NSCLC.

	Authors' Disclosures of Potential Conflicts of Interest

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION Authors' Disclosures of... REFERENCES

 
The authors indicated no potential conflicts of interest.

	Acknowledgment

 
We thank Joanne Wroblewski for her help in making vaccines, and Jennie Batsel and Jennie Bowden of the UK Leukapheresis Center for their outstanding care of the research patients.

	NOTES

 
These studies were supported by the Cancer Treatment Research Foundation grant # G-01-009, Kentucky Lung Cancer Grants Association, and the Veteran's Administration.

Authors' disclosures of potential conflicts of interest are found at the end of this article.

	REFERENCES

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION Authors' Disclosures of... REFERENCES

 
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Submitted January 14, 2004; accepted April 17, 2004.

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