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Clinical Cancer Research Vol. 12, 869-877, February 2006
© 2006 American Association for Cancer Research

Cancer Therapy: Clinical

Mannan-MUC1–Pulsed Dendritic Cell Immunotherapy: A Phase I Trial in Patients with Adenocarcinoma

Bruce E. Loveland, Anne Zhao, Shane White, Hui Gan, Kate Hamilton, Pei-Xiang Xing, Geoffrey A. Pietersz, Vasso Apostolopoulos, Hilary Vaughan, Vaios Karanikas, Peter Kyriakou, Ian F.C. McKenzie and Paul L.R. Mitchell

Authors' Affiliation: Austin Research Institute, and Medical Oncology Unit, Austin Hospital, Heidelberg, Melbourne, Victoria, Australia

Requests for reprints: Bruce Loveland, The Austin Research Institute, Studley Road, Heidelberg, Victoria 3084, Australia. Phone: 61-3-9287-0666; Fax: 61-3-9287-0600; E-mail: b.loveland{at}ari.unimelb.edu.au.


   Abstract
Top
 Abstract
Materials and Methods
 Results
 Discussion
 References
 
Purpose: Tumor antigen-loaded dendritic cells show promise for cancer immunotherapy. This phase I study evaluated immunization with autologous dendritic cells pulsed with mannan-MUC1 fusion protein (MFP) to treat patients with advanced malignancy.

Experimental Design: Eligible patients had adenocarcinoma expressing MUC1, were of performance status 0 to 1, with no autoimmune disease. Patients underwent leukapheresis to generate dendritic cells by culture ex vivo with granulocyte macrophage colony-stimulating factor and interleukin 4 for 5 days. Dendritic cells were then pulsed overnight with MFP and harvested for reinjection. Patients underwent three cycles of leukapheresis and reinjection at monthly intervals. Patients with clinical benefit were able to continue with dendritic cell-MFP immunotherapy.

Results: Ten patients with a range of tumor types were enrolled, with median age of 60 years (range, 33-70 years); eight patients were of performance status 0 and two of performance status 1. Dendritic cell-MFP therapy led to strong T-cell IFN{gamma} Elispot responses to the vaccine and delayed-type hypersensitivity responses at injection sites in nine patients who completed treatments. Immune responses were sustained at 1 year in monitored patients. Antibody responses were seen in three patients only and were of low titer. Side effects were grade 1 only. Two patients with clearly progressive disease (ovarian and renal carcinoma) at entry were stable after initial therapy and went on to further leukapheresis and dendritic cell-MFP immunotherapy. These two patients have now each completed over 3 years of treatment.

Conclusions: Immunization produced T-cell responses in all patients with evidence of tumor stabilization in 2 of the 10 advanced cancer patients treated. These data support further clinical evaluation of this dendritic cell-MFP immunotherapy.

Dendritic cells as vectors for antigen delivery are a major focus of cancer immunotherapy. Early clinical studies have used diverse strategies, including the ex vivo loading of autologous monocyte-derived dendritic cells with antigens (synthetic peptides and recombinant proteins in melanoma, prostate, gastrointestinal, lung and breast cancer; refs. 1–6), with tumor cell lysates (in kidney and prostate cancer; refs. 7, 8), with DNA (9) or RNA (10), and with dendritic cell/tumor cell coinjection (11) or fusions (12). Patient benefit in these studies has been infrequent and inconsistent (reviewed in ref. 13), indicating the challenges to be surmounted if cellular therapy is to become clinically relevant.

We now report clinical and laboratory outcomes of a phase I clinical trial using monocyte-derived dendritic cells loaded with a mucin 1 (MUC1) VNTR recombinant protein conjugated to oxidized mannan [mannan-MUC1 fusion protein (MFP)]. The MUC1 antigen was chosen as it is highly and abnormally expressed with aberrant glycosylation by many epithelial tumors including breast, colon, kidney, lung, esophageal, stomach, and ovarian cancer. On normal tissues, low level expression of heavily glycosylated MUC1 is restricted to the apical cell surface facing the lumen of ducts and glands (14). Preclinical studies of the recombinant (not glycosylated) MUC1 antigen in mice indicated a particular advantage of conjugation to oxidized mannan, with enhanced stimulation of cellular immune responses (15). Unique features of mannan-conjugated antigen include targeting the complex to the mannose receptor of dendritic cells. Oxidized mannan rapidly traverses the endosome to deliver conjugated proteins (e.g., MUC1 VNTR) to the class I presentation pathway (16). However, in prior phase I clinical trials when MFP was injected (i.m., s.c., or i.p.) into >100 patients with advanced metastatic MUC1+ adenocarcinoma, while no toxicity was noted, there was a predominance of antibody-mediated, rather than cell-mediated, immunity and no clinical responses were observed (17–19).1 Mice and humans differ markedly in their immune responses to MUC1. We had shown that the naturally occurring human anti-Gal{alpha}(1,3)Gal antibodies in cancer patients cross-reacted with MUC1 peptides (20) and considered it likely that anti-Gal antibodies formed immune complexes with injected MFP, bound Fc receptors, and stimulated antibody production rather than cell-mediated immunity (21). This was clearly seen in Gal–/– mice (which in this regard resemble humans) where, in contrast to normal (Gal+/+) mice, the responses were predominantly antibody. The trend to antibody production in Gal–/– mice was reversed by the ex vivo loading of antigen-presenting cells with MFP (21). These studies led to the current clinical trial using autologous dendritic cells as vaccine carriers, aiming to maximize cellular immunity while minimizing the induction of antibody, which seemed to have no clinical benefit in the advanced disease setting (17–19).

This phase I study in patients with MUC1+ adenocarcinoma has shown that MFP loaded ex vivo into autologous monocyte-derived dendritic cells was without significant side effects, reliably led to T-cell immune responses, and was correlated with stabilization of disease in some patients.


   Materials and Methods
Top
 Abstract
 Materials and Methods
Results
 Discussion
 References
 
Study design
The primary objective of the study was to assess toxicity with secondary objectives of assessing antitumor efficacy, immune responses, and procedure feasibility.

Eligible patients underwent leukapheresis at 4-weekly intervals on three occasions to obtain dendritic cell precursors. Autologous dendritic cells were cultured ex vivo and injected into patients at intradermal and s.c. sites. When it became evident that some patients were deriving clinical benefit, a protocol amendment allowed further dendritic cell-MFP immunotherapy.

Patients
Eligibility criteria were adenocarcinoma with no available curative therapy (i.e., either stage IV or with relapsed disease); no central nervous system involvement; no chemotherapy, radiotherapy, immunotherapy, or experimental treatment within the previous 4 weeks; life expectancy of >12 weeks; Eastern Cooperative Oncology Group performance status of 0 to 1; no other malignancy within 5 years except basal cell carcinoma of skin or noninvasive cervical cancer; no concurrent systemic corticosteroid therapy; adequate hematologic (Hb, >10 g/dL; WBC, >3 x 109/L; platelets, >109/L), renal (creatinine, <0.14 mmol/L), and hepatic function (bilirubin, <60 mmol/L), and no autoimmune disease apart from autoimmune thyroid disease. All patients were required to have strongly expressed MUC1 (VNTR) antigen on stored fixed tumor specimens. Tumor tissue was stained and scored for MUC1 expression on normal and cancer cells using the immunoperoxidase technique and four anti-MUC1-VNTR monoclonal antibodies (VA1, VA2, BC2, and 3E1.2, which recognize different epitopes, as reviewed in ref. 22). Patients gave written informed consent and the study was approved by the institutional Human Research Ethics Committee.

Investigations and monitoring
Baseline investigations were history and examination, blood tests for hematology, and clinical chemistry including electrolytes, renal and hepatic function, relevant tumor markers, autoantibody screen, and HLA typing. Hepatitis B and C and HIV serology were required to be negative for patients to undergo leukapheresis. Baseline imaging of disease sites was done within 3 weeks of study entry. Following each injection of cell product, patients were monitored for 3 hours. Patients were reviewed thrice at monthly intervals during the 12-week study period, including blood tests and repeat imaging of disease sites at 12 weeks. Patients who received ongoing therapy were reviewed with repeat imaging 3-monthly and monthly tumor markers.

Preparation of vaccine and test antigens
MFP vaccine was prepared as previously described (23, 24) The cDNA insert (pDF9.3; ref. 25) encoded 103 amino acids of the human MUC1 protein, including three copies of the VNTR motif (PDTRPAPGSTAPPAHGVTSA) flanked by natural variants of this sequence. Recombinant MFP, consisting of glutathione-S-transferase (26 kDa) and the VNTR-containing sequences (103 amino acids; 12 kDa), was produced in E. coli, solubilized by sonication, and purified by elution from glutathione-agarose columns (Sigma, St. Louis, MO). Mannan (Sigma) was oxidized with sodium periodate and conjugated to fusion protein to make MFP (23, 24). For in vitro assays of MUC1 vaccine–specific immune responses in delayed-type hypersensitivity (DTH), Elispot, and ELISA assays, an alternative source of recombinant MUC1 antigen (pVNTR), not containing glutathione-S-transferase, was prepared using the pTrcHisB vector (Invitrogen) and purified on a Nickel-NTA column (Qiagen, Melbourne, Australia). MFP and pVNTR were tested for sterility and for endotoxin content by the LAL assay to be <30 endotoxin units/250 µg protein.

Generation of dendritic cells in ex vivo cell culture, antigen loading, and injection
Patients underwent three leukaphereses at 4-weekly intervals to generate dendritic cells for three sets of injections within the 12-week trial period. Peripheral blood mononuclear cells (PBMC) were collected without priming over 2.5 to 3.5 hours using a Haemonetics MCS plus machine, were enriched for dendritic cells using standard monocyte adherence methods (reviewed in ref. 26), and cultured with recombinant human granulocyte macrophage colony-stimulating factor (500 units/mL; Schering-Plough Ltd., Sydney, Australia) and recombinant human interleukin 4 (500 units/mL; Peprotech, Inc., Rocky Hill, NJ) in AIM-V medium (Life Technologies, Gaithersburg, MD) and 2% v/v autologous serum. The MFP vaccine (10 µg/mL) was added to cell cultures on day 5 before recovering nonadherent dendritic cells on day 6, which were released for injection based on culture morphology (>50% dendritic cell-like) and Gram stain (negative). The cells were resuspended to 4 x 107/mL in BP saline for injection (Astra Pharmaceuticals, Sydney, Australia) with 2% v/v autologous serum, and injected into two intradermal sites (107 viable cells per site) with the remainder divided between two to four s.c. sites in the upper arms and/or legs. A sample was analyzed for phenotype by flow cytometry as described below.

Assessment of immune responses
PBMC and serum samples were cryopreserved at six time points: at each leukapheresis (weeks 1, 5, and 9) and 3 weeks after each injection (weeks 4, 8, and 12). Where possible, samples were again taken at 6 and 12 months. The intradermal injection sites were assessed at 48 hours for erythema and edema. In five patients (A07-A11), a protocol amendment allowed two skin biopsies of injection sites to be taken at week 10, with one formalin fixed for histology and the other cryopreserved and sectioned for immunohistochemistry (staining with anti-CD4, anti-CD8, and anti-CD86 monoclonal antibodies) to assess DTH. In five patients (A01-A05), skin tests at week 12 were used to assess general cellular immunity using MCI Multitest Kits (Pasteur Merieux, Lyon, France) for Tetanus, Diphtheria, Streptococcus (group C), Tuberculin, Candida (albicans), Trichophyton (mentagrophytes), and Proteus (mirabilis) antigens. In these patients, recombinant pVNTR (10 µg injected i.d.) was also used to assess DTH to the vaccine, recording the dimensions of erythema and edema for each antigen 24 hours after applying the test.

Elispot cytokine assay of PBMC T cells and test antigens. A standard Elispot assay of thawed PBMC was used (27). In brief, 96-well plates (MAIPS S45, Millipore, Sydney, Australia) were precoated with 50 µL of antihuman IFN{gamma} antibody (1D1K, Mabtech, Melbourne, Australia) and blocked with 10% v/v human AB serum. Each test well of triplicate groups contained 5 x 105 PBMC in 100 µL RPMI 1640 with 2.5% v/v human AB serum (Valley Biomedical, Winchester, VA) plus 25 µL of antigen to a final concentration of 20 µg/mL or medium (control). Where cell numbers allowed, CD4+ or CD8+ T cells were depleted from PBMC using Dynal magnetic beads (depletion >98% assessed by flow cytometry; data not shown) before the Elispot assay. Antigens were purified protein derivative (Tuberculin, Staten Serums Institute, Copenhagen, Denmark; positive control antigen) and recombinant pVNTR (specific test antigen). The plates were incubated for 18 hours at 37°C in 5% CO2, washed with PBS containing 0.1% v/v Tween 20, then with PBS, and incubated with biotin-conjugated antihuman IFN{gamma} antibody (7-B6-1, Mabtech) for 2 hours at room temperature before washing. Bound cytokine was visualized using 1 µg/mL streptavidin-alkaline phosphatase conjugate (Mabtech) incubated for 2 hours at room temperature, then incubated with the colorimetric AP detection kit according to the instructions of the manufacturer (Bio-Rad, Hercules, CA). Cytokine spots (spot-forming units) were counted with the AID Elispot Reader system (Autoimmun Diagnostika GmbH, Strassberg, Germany).

Serum antibody assays. Anti MUC1 VNTR antibodies in patient sera were measured by ELISA against pVNTR antigen as per the published method (19). A positive control serum (IFCM10_#2EC) was selected from a panel of patients previously immunized with MFP who had developed anti-VNTR antibodies. A negative control serum (ARI_#S2P) was selected, being from a normal individual with no detectable anti-VNTR antibody.

Flow cytometric cell analyses. Samples of the injected dendritic cell population and of leukapheresed PBMC, in aliquots of 3 x 105, were analyzed by flow cytometry (FacsCalibur II, Becton Dickinson, San Jose, CA) for phenotypic markers, in particular to assess dendritic cells content and maturity, using fluorochrome-conjugated monoclonal antibodies according to the recommendations of the manufacturer [anti-CD3.FITC, CD14.TC, CD40.FITC, CD80.TC, CD83.APC, CD86.PE, MHC-II(DR).APC (Caltag Laboratories, Burlingame, CA)], and anti-CD83.FITC (Becton Dickinson)] or using monoclonal antibody supernatants [W6/32 anti-MHC-I (ARI stocks), CMRF-44 (28), kindly provided by D. Hart, Master Medical Research Institute, Brisbane, Australia, and anti-CCR7, kindly provided by F. Sallusto, Institute for Research in Biomedicine, Bellinzona, Switzerland] with FITC-conjugated antimouse immunoglobulin. The mannose receptor was detected using FITC-conjugated mannan (29).


   Results
Top
 Abstract
 Materials and Methods
 Results
Discussion
 References
 
Patients. The characteristics of the 10 patients entered on study are detailed in Table 1. Median age was 60 years (range, 33-70 years) and performance status was 0 in eight patients and 1 in two patients. Tumor types (MUC1+) were renal carcinoma in three patients, breast carcinoma in two, ovarian/fallopian tube in two, and one patient each with non–small-cell lung, colon, and esophageal carcinoma. Eight patients had stage IV disease whereas two patients (A03 and A09) had recurrent, incurable stage III disease. All patients had received prior therapy in addition to surgery, which included chemotherapy in five patients, radiotherapy in four, IFN in two, and hormonal therapy in one patient.


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  		Table 1. Patient characteristics, immunologic responses, and antitumor efficacy

 
Treatment. Nine of 10 patients completed all three rounds of leukapheresis, cell culture, and immunization over the 12-week study period, with the third leukapheresis of #A01 abandoned due to poor vascular access. From these 29 leukaphereses, the mean PBMC yield was 2.8 ± 1.4 (SE) x 109 cells and after 6 days of culture with granulocyte macrophage colony-stimulating factor and interleukin 4, the mean viable cell recovery ("dendritic cells") was 8.8 ± 4.9 x 107 cells. However, within this broad range of cell recoveries, there were reproducible "patient-specific" features; i.e., some individuals had consistent low PBMC yields but high dendritic cells recoveries (e.g., #A11: 1.9 ± 0.4 x 109 PBMC; and 15.3 ± 3.4 x 107 dendritic cells, being 7.9% of the starting number) or the converse (e.g., #A09: 4.9 ± 0.6 x 109 PBMC; and 5.8 ± 1.3 x 107 dendritic cells, being 1.2% of the starting number). The content of dendritic cells in the harvested cultures varied considerably (estimated to be 60-85% based on flow cytometry as CD86+CD14–CD3– as detailed below) and the residual nondendritic cells (largely T cells by CD3 staining) were not expected to affect the immunogenicity of the injected cells. All recovered MFP-loaded cultured cells were injected into the patients: 10 million cells into each of two intradermal sites in the upper arm, with the remainder divided equally between two to four (for higher cell recoveries) s.c. sites.

Toxicity. The toxicity of leukapheresis and cell injections was mild. Two patients had anxiety and lower back pain related to leukapheresis. There was no clinical toxicity of the vaccine. One patient developed elevation of antithyroid antibodies up to a maximal titer of 1:400 with no evidence of clinical autoimmune disease, and another had anti–smooth muscle antibodies at 1:80.

Antitumor efficacy. In four patients (A02, A08, A09, and A10), disease remained stable during the 12-week study period (Table 1). This included two patients, with breast and colon carcinoma, respectively, who had stable or slowly progressing disease at study entry, which was not considered to have been altered by study therapy. The other two patients, with ovarian (A09) and renal carcinoma (A08), had documented progressive disease at study entry and their clinical courses are presented here in detail.

Patient A09, a 64-year-old woman, presented with stage III ovarian carcinoma 26 months before study entry, and underwent debulking surgery followed by six cycles of carboplatin and paclitaxel chemotherapy. Incurable recurrent disease, diagnosed by elevated serum CA125, occurred 20 months from presentation and she was treated with four cycles of carboplatin, with normalization of the CA125 level. Study therapy commenced 3 months following completion of chemotherapy, at which time serum CA125 remained normal at 14 (normal <35) with no evaluable disease on computed tomography scanning (as typical of many ovarian cancer cases). After 6 weeks on study, serum CA125 rose to 47 IU/mL (normal <35), then to 64 at 2 months from study entry, indicating definite progressive disease during the early stage of trial therapy. Following the second dendritic cell-MFP injection, CA125 stabilized and remained in the range of 57 to 70 for 4 months, after which the level again began to increase, increasing from 64 to 165 over 4 months (indicated as periods ii and iii in Fig. 1 using extrapolated trendlines). In view of the observed 4-month stabilization, then increasing CA125 levels, approval for a protocol amendment was obtained from the Institutional Human Research Ethics Committee to allow for further therapy. This resulted in a delay of 7 months before restarting intradermal dendritic cell-MFP immunotherapy, first at monthly intervals (three injections), then 3-monthly (using cryopreserved MFP-loaded dendritic cells). Serum CA125 on recommencing study therapy was 165, increasing to 191 during the next month. After the second of the additional injections at 11 months, the patient had largely stable disease for a period of 18 months, although with very slowly increasing CA125 levels which remained in the range of 182 to 258 IU/mL (Fig. 1, period iv). From ~27 months, there has been an inexorable increase in CA125 (most recently to 1,256 IU/mL, 34 months from recommencing therapy and 43 months since study entry), interspersed with brief intervals where dendritic cell-MFP injections are associated with a transient reduction in serum CA125 (e.g., at 31, 35, and 40 months; Fig. 1). Reversion to monthly injections at 35 months seemed to slow the CA125 increase for 5 months only. Through >3 years, there has been no evaluable disease on computed tomography scanning.


Figure 1
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  		Fig. 1. Serum CA125 levels in patient A09, monitored from pre-trial to 42 months, with the 23 MFP/dendritic cell injections (arrows) below the X axis. Injections were given as two triple injection series every month with a 6 month interval, then every 3 months to 33 months, then monthly. The dashed linear trendlines (Microsoft Excel), for periods of relatively systematic CA125 levels, indicate marked reductions in the rate of CA125 increase following the early injection series (sections ii and iv, marked by vertical lines) and acceleration from 28 months (section v).

 
The second patient, A08, a 69-year-old man, underwent right nephrectomy for a clear cell carcinoma of the kidney 22 months before study entry. The 7-cm tumor was adherent to the liver and extended into perinephric fat. The patient received adjuvant IFN for 6 months but stage IV disease with metastases to mediastinal nodes and liver was noted at 14 months from diagnosis. Computed tomography scanning at study entry showed progressive disease in mediastinal nodes (Fig. 2A, C, and E). The disease sites remained stable through the 12-week initial study period. After an interval of 8 months, during which the patient received no therapy and disease remained stable, the patient recommenced leukapheresis with three dendritic cell-MFP injections at monthly intervals followed by ongoing 3-monthly injections. Computed tomography scanning at 14 months showed the earlier disease sites to be stable or reduced in size (Fig. 2B, D, and F). The patient remained clinically well on dendritic cell-MFP therapy, with overall stable disease 36 months from commencing study therapy. At 20 months, a newly enlarged 2.0-cm aorto-pulmonary window lymph node was noted, which later increased to 3.5 cm in diameter but resolved by 36 months.


Figure 2
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  		Fig. 2. CT thorax scans of patient A08 at trial entry and after 14 months. Immediate pre-trial CT (A, B, and C) and 14 months later (corresponding D, E, and F), after receiving six dendritic cell/MFP injections at 1, 2, and 3 months (initial protocol), and 12, 13, and 14 months (extended treatment).

 
Dendritic cell yield and phenotype. In preliminary culture experiments to establish procedural details, the numbers and proportion of monocyte-derived dendritic cells (CD86+CD14–CD3–) increased markedly from day 4 to day 6 but little more to day 8 (data not shown). Because the cell yield was considered important, a 6-day culture period was used. Addition of the MFP antigen was on day 5, with rapid uptake into dendritic cells as monitored by surface and intracellular staining with anti-VNTR monoclonal antibodies after 0.5, 2, 6, and 24 hours (data not shown). Although no specific steps to mature the dendritic cells were undertaken, CD40, CD83, CD86, and CMRF44 expression on MFP-loaded dendritic cells tended to be greater than on untreated dendritic cells (Fig. 3) whereas other phenotypic markers did not change (data not shown).


Figure 3
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  		Fig. 3. Phenotypic changes to monocyte-derived dendritic cells following pulsing with MFP: representative results from two patients (A10 and A11). Dendritic cells were cultured for 5 days in vitro and then pulsed for an additional 18 hours with 10 µg/mL MFP (Figure 3) or without MFP (Figure 3). Harvested cells were stained with the panel of antibodies and flow cytometry data for CD40, CD83, CD86, and CMRF44 are shown here. Large mononuclear cells as determined by the wide and forward angle light scatter profile. Appropriate isotype controls (matching the isotype of the specific antibody) were included, with MFP (Figure 3) and no MFP ( - - - - ).

 
Dendritic cell–like cells were largely CD3–CD86+ and contained minimal CD3+ and CD14+, high expression of MHC class I and class II, and high CD86, whereas CD83 expression was low to moderate and CMRF44 expression was moderate to strong. The mannose receptor, detected by the binding of FITC-conjugated mannan, was usually expressed on a majority of, but not all, dendritic cell–like cells (data not shown). There were no notably variant cultures generated from the 29 leukaphereses of these 10 patients.

Multitest CMI Kit and anti-VNTR DTH responses. Five patients were tested for skin DTH responses to seven common antigens using a Multitest CMI kit and to a local i.d. injection of the MUC1-VNTR vaccine antigen. All five had skin DTH reaction responses to tetanus and four had weak DTH responses to the low challenge dose of the VNTR peptide, with variable responses to the other antigens (data not shown). These results showed the participants to be immunocompetent and the DTH responses to MUC1-VNTR injection correlated with the in vitro T-cell assays.

Elispot assays of IFN{gamma} secretion. The ex vivo 18-hour Elispot assay did not require cell expansion as it detected memory effector cells (both CD4+ and CD8+ cytokine-producing). It is a sensitive method that has been used to monitor cellular immune responses in patients receiving immunotherapy (30, 31). Purified protein derivative was used as a positive control antigen to validate the stored PBMC samples, and to which all patients were expected to respond.

IFN{gamma}-secreting T cells were generated promptly in all 10 patients immunized with dendritic cells pulsed with oxidized mannan-MUC1, were specific for MUC1 pVNTR antigen (with no glutathione-S-transferase), and detected as early as 2 weeks after the first injection (Fig. 4A). The relative responses to the purified protein derivative and pVNTR antigens by a cell sample, rather than absolute values, were considered the most appropriate measure of immune response because of interassay variation. The patients developed specific VNTR IFN{gamma}-secreting T cells either 2 weeks after the first injection (A02, A04, and A11), 4 weeks after the first injection (A01, A03, A05, A07, A08, and A09), or 4 weeks after the second injection (A10). Follow-up tests after 6 to 12 months for A02 to A11 as available, and 2 to 3 years for A08 and A09 during extended treatments, showed a long-term sustained immune response to the vaccine antigen (Fig. 4A). In addition, when PBMC from patients A04 to A11 were depleted of CD4+ or CD8+ T cells and analyzed for VNTR-induced IFN{gamma} secretion, it was clear that both CD4+ and CD8+ T cells specific for MUC1 VNTR were present in all patents (A04 and A06 shown in Fig. 4B). Concurrent assays of intracellular IFN{gamma} production by flow cytometry showed that the responding cells were either CD4+ or CD8+ T cells and not other cells, excluding a significant natural killer cell response (data not shown).


Figure 4
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  		Fig. 4. Stimulation of serial PBMC specimens to assess vaccine immunogenicity, measured as T-cell secretion of IFN{gamma} in an Elispot assay. A, data from all patients, grouped according to magnitude of responses [spot-forming units (SFU)]. Patients received injections at weeks 2, 6, and 10, with specimens tested at various intervals after injections (some at 6 and 9 months). Patients A08 and A09 received extended treatments and tests to 28 and 38 months respectively, as shown. B, tests of PBMC depleted of CD4 or CD8 cells in 9- or 11-week samples from patients A04 and A07. Three stimulation conditions are shown: purified protein derivative antigen (positive control, Figure 4), VNTR recombinant protein antigen ({blacksquare}), and no antigen (negative control, {square}) using 5 x 105 thawed PBMC per well. w, week of blood sample; M, months after first injection.

 
Thus, all patients made vaccine-specific IFN{gamma} responses within the 12-week trial period after the three dendritic cell-MFP injections and these responses, in the nine patients who were further monitored, were shown to be sustained long-term.

Measurement of antibodies by ELISA assays. Low titer (1/40) immunoglobulin M (IgM) anti-VNTR serum antibodies were induced in 3 of 10 participants (A04, A08, and A10) and seroconverted from the second injection to low titer IgG in two of the patients (data not shown). A09 had unchanging low titre IgG and no IgM throughout the treatment period. There was no significant increase in antibody titers when assayed 6 to 12 months after the immunizations.

DTH responses. In previous phase I trials of MFP injection (see Karanikas et al. refs. 17, 19), we had assiduously measured DTH responses: most were weak and the biopsies showed few infiltrating cells. In this study, however, all but one patient developed DTH-type responses of 1 to 2 cm at intradermal injection sites 24 to 48 hours after the second or third injection of cell product. Patients A08 and A09, who then received ongoing dendritic cell-MFP therapy, continued to show DTH responses at injection sites. The exception was patient A01 who had no DTH after the second injection (although an in vitro cellular immune response; Fig. 4A) and had no further therapy due both to poor venous access precluding apheresis and to rapidly progressive disease. Typically, there was intense erythema up to 2 cm in diameter, with or without edema. These responses lasted 48 to 72 hours. Biopsy at 48 hours in 4 patients showed an infiltration of CD4+ T cells in substantial numbers whereas rarely could CD8+ cells be detected (data not shown).


   Discussion
Top
 Abstract
 Materials and Methods
 Results
 Discussion
References
 
Targeting dendritic cells to load a mannan-conjugated recombinant MUC1 VNTR (MFP) antigen for immunotherapy was well tolerated and was associated with prolonged essentially stable disease in 2 of 10 patients treated who had advanced ovarian and renal carcinoma, respectively. The patient with ovarian carcinoma was in second relapse at the time of study entry, indicating tumor escape from the effects of chemotherapy ceased 3 months earlier. The patient with renal cancer had cancer metastatic to liver and lymph nodes. These two patients, with progressive disease at study entry, continued on study therapy for over 3 years.

The trial was intended to establish reliable and practical procedures to generate and use MFP-pulsed autologous monocyte-derived dendritic cells, to assess vaccine toxicity, and, by immunologic monitoring, to provide a measure of vaccine immunogenicity. The culture process minimized the use of components that are subject to batch variations and regulatory controls (e.g., minimal reagents to enrich monocytes or deplete other cells and no additional cytokines or chemicals to mature the dendritic cells) and was based on standard methods to generate dendritic cells from adherent monocytes with granulocyte macrophage colony-stimulating factor, interleukin 4, and autologous serum.

This immunotherapy produced cellular responses to the MUC1 immunogen in all patients treated, a result which had not previously been observed after direct injection of the MFP antigen (17–19), emphasizing the important adjuvant effect of the autologous dendritic cells to stimulate a much stronger immune response. Peripheral blood T cells from all 10 patients made IFN{gamma} in Elispot assays (frequency ~1/1,000) after only two or three dendritic cell-MFP injections at 4-week intervals. There was an extensive cellular infiltrate as a DTH response at the sites of boosting injections and little production of anti-MUC1 serum antibody. The maintenance of the DTH responses was consistent with the detection of IFN{gamma} Elispot results in vitro. We suspect that the rapid development of DTH responses, well established within 24 hours after the third or subsequent boosting injections, was a result of antigen-loaded dendritic cells being injected and no additional time was required for antigen to be taken up locally, processed, and presented (which usually takes several hours).

The low frequency of a serologic response was consistent with the ex vivo MFP vaccine uptake and internalization into dendritic cells with transient MUC1 detectable on the cell surface. Thus, it was likely that only low quantities of MUC1 protein were available to induce anti-vaccine serum antibodies whereas internalized protein was available for processing and presentation to stimulate T-cell responses.

Dendritic cells as antigen carriers are now receiving considerable attention, particularly in tumor immunotherapy where it has been difficult to obtain either appropriate immune responses or to produce tumor regression (13, 32). Apart from dendritic cells being the optimal antigen-presenting cell, there are additional advantages in targeting them ex vivo: removing them from an immunosuppressive cytokine milieu (e.g., interleukin 10) which does not encourage immune responses; ensuring the target antigen has ample opportunity to be taken up and processed by the dendritic cells; and, after injection intradermally, providing an opportunity for effector T-cell recruitment and activation to occur in draining lymph nodes distant from sites of metastatic disease. Although leukapheresis and cell culture in a GMP facility have logistical difficulties, there was no significant clinical toxicity and the quality of the immune responses generated, along with the clinical effects, justifies the additional procedures. In this phase I trial, each patient received all cells generated (though i.d. doses were standardized to 20 million cells), which did not allow assessment of cell dosage effects. A current follow-up phase II trial of this dendritic cell/MFP therapy is using standardized doses of 40 x 106 cells per treatment.

The T-cell responses to VNTR test antigen were striking, with all patients developing vaccine-specific IFN{gamma}-secreting T cells, both CD4+ and CD8+, which was not unexpected. The particular recombinant VNTR antigen contains 103 amino acids from MUC1 protein (three copies of the conserved 20-mer VNTR motif plus two flanking variants), providing considerable epitope diversity. Thus, both CD4 "helper" and CD8 "cytotoxic" responder cells were generated in the HLA-diverse patient group (Table 1), as proven by Elispot (IFN{gamma}) and Intracellular Cytokine assays of cell subsets. Two questions arise: why were the dendritic cell-MFP so effective in stimulating responses, and why was the response not eradicating tumors? Mannan-antigen targeted dendritic cells are likely to be potent because the mannose receptor enables rapid antigen uptake and its cross-linking initiates some of the progressive dendritic cell maturation processes (Fig. 3),2 which are likely to provide a direct immunogenic benefit after injection.

As to why clinical efficacy was less striking than T-cell responses, there are a number of potential factors whereby dendritic cell-MFP therapy was not delivered to the right patients or in an optimal fashion. First, it is increasingly being recognized that the mere detection of T-cell responses generated by an effective immmunization regimen, as used here, will not necessarily correlate with in vivo effector function (33) and we, like others, are seeking to identify relevant effector T-cell subpopulations and their specificities by careful stimulation and epitope mapping studies. Second, all patients had advanced disease and the physiologic effects of tumor burden may have been excessive (34). In the two patients with ongoing therapy, the 3-monthly interval between injections may have been too long to achieve a greater antitumor effect. The rate of CA125 change is an accepted surrogate marker of ovarian cancer progression (35), and initially in patient A09 there seemed to be a good correlation between treatment episodes and response; however, this deteriorated in the 3rd year of treatment even after reverting to monthly injections (Fig. 1). It is possible that effector T cells which are (newly) recruited by dendritic cell-MFP injection have only a limited period to destroy targeted cancer cells, and might not have the potential to renew, perhaps affected by the immunosuppressive environment of the tumor (36). Thus, protocols where the frequency of dendritic cell-based immunotherapy injections can be tailored to a patient with early stage (minimal residual) disease by observation of clinical response may have the best opportunity of a benefit. We note that in patient A09, recent dendritic cell-MFP injections were associated with a drop in CA125 levels 1 to 3 months later, which then rebounded, although the in vitro Elispot assay of unselected PBMC showed no meaningful change in the T-cell response to the vaccine antigen. Screening for the presence of MUC1+ was based on archival tissue specimens, rather than specimens of "current" tumor. Therefore, several of the trial participants may have developed MUC1-loss mutants.

Whereas dendritic cells used in a number of other studies induced good T-cell responses, the use here of oxidized mannan-coupled antigen induced high levels and sustained responses in all patients that have not been regularly reported in other studies (reviewed in refs. 37, 38). The oxidized mannan therefore offers an advantage, likely to result from targeting the mannose receptor and passage of the antigen into proteolytic pathways and class I and class II presentation to CD8 and CD4 cells, respectively. Both cell types are recruited following immunization. The roles of aldehydes and Schiff bases, created by oxidation of the mannan, also seem to be important to the immunogenicity of the MFP (29). Finally, the recombinant MUC1 antigen used, consisting of 103 amino acids, is a relatively complex antigen and clearly was processed into peptides recognized by T cells from all patients with a broad spectrum of HLA haplotypes (see Table 1). Therefore, the strategy to use MFP/autologous dendritic cells injections has been shown to be practical, safe, effective in generating strong cellular immunity, and in some patients has given indications of a therapeutic effect.


   Acknowledgments
 
We thank the following people for their valuable help with different aspects of the clinical trial: Belinda Bardsley, Sharon France, Vanessa Waddell, and Dr. John Bates (PRIMA BioMed Ltd.); Dr. Jayesh Desai and Wayne Saunders (Austin Hospital); Lesley Barber, Dr. Dominic Wall, and Kerrie Stokes (Centre for Blood Cell Therapies, Peter MacCallum Cancer Centre); Carla Osinski, Harry Aletras, Dr. Li Wenjun, and Violeta Bogdanoska (The Austin Research Institute); Dr. Carole Smith, Tamla Tait, and staff of the Apheresis Service (Austin Hospital); and Louise Keelan and Sharon Wilson (Department of Histopathology, Austin Hospital). Brendan Toohey prepared the pTRC-VNTR antigen. Thanks also to Dr. Pat Mottram for reviewing the manuscript.


   Footnotes
 
Grant support: The Austin Research Institute, AusIndustry START grant GRA02245; PRIMA BioMed Ltd., Kew, Victoria, Australia; National Health and Medical Research Council Senior Research Fellowship (B. Loveland); and National Health and Medical Research Council CJ Martin Research Fellowship, Australia (V. Apostolopoulos).

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 I.F.C. McKenzie, V. Karanikas, G.A. Pietersz, V. Apostolopoulos, and B.E. Loveland, unpublished data. Back

2 B.E. Loveland, A. Zhao, and V. Karanikas, manuscript in preparation. Back

Received 7/22/05; revised 11/13/05; accepted 11/16/05.


   References
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 Abstract
 Materials and Methods
 Results
 Discussion
 References
 

  1. Thurner B, Haendle I, Roder C, et al. Vaccination with mage-3A1 peptide-pulsed mature, monocyte-derived dendritic cells expands specific cytotoxic T cells and induces regression of some metastases in advanced stage IV melanoma. J Exp Med 1999;190:1669–78.[Abstract/Free Full Text]
  2. Lodge PA, Jones LA, Bader RA, et al. Dendritic cell-based immunotherapy of prostate cancer: immune monitoring of a phase II clinical trial. Cancer Res 2000;60:829–33.[Abstract/Free Full Text]
  3. Panelli MC, Wunderlich J, Jeffries J, et al. Phase 1 study in patients with metastatic melanoma of immunization with dendritic cells presenting epitopes derived from the melanoma-associated antigens MART-1 and gp100. J Immunother 2000;23:487–98.
  4. Hersey P, Menzies SW, Halliday GM, et al. Phase I/II study of treatment with dendritic cell vaccines in patients with disseminated melanoma. Cancer Immunol Immunother 2004;53:125–34.[CrossRef][Medline]
  5. Chen Q, Jackson H, Parente P, et al. Immunodominant CD4+ responses identified in a patient vaccinated with full-length NY-ESO-1 formulated with ISCOMATRIX adjuvant. Proc Natl Acad Sci U S A 2004;101:9363–8.[Abstract/Free Full Text]
  6. Maraskovsky E, Sjolander S, Drane DP, et al. NY-ESO-1 protein formulated in ISCOMATRIX adjuvant is a potent anticancer vaccine inducing both humoral and CD8+ T-cell-mediated immunity and protection against NY-ESO-1+ tumors. Clin Cancer Res 2004;10:2879–90.[Abstract/Free Full Text]
  7. Nestle FO, Alijagic S, Gilliet M, et al. Vaccination of melanoma patients with peptide- or tumor lysate-pulsed dendritic cells. Nat Med 1998;4:328–32.[CrossRef][Medline]
  8. Rieser C, Ramoner R, Holtl L, et al. Mature dendritic cells induce T-helper type-1-dominant immune responses in patients with metastatic renal cell carcinoma. Urol Int 1999;63:151–9.[CrossRef][Medline]
  9. Rosenberg SA, Zhai Y, Yang JC, et al. Immunizing patients with metastatic melanoma using recombinant adenoviruses encoding MART-1 or gp100 melanoma antigens. J Natl Cancer Inst 1998;90:1894–900.[Abstract/Free Full Text]
  10. Morse MA, Nair SK, Mosca PJ, et al. Immunotherapy with autologous, human dendritic cells transfected with carcinoembryonic antigen mRNA. Cancer Invest 2003;21:341–9.[CrossRef][Medline]
  11. O'Rourke MG, Johnson M, Lanagan C, et al. Durable complete clinical responses in a phase I/II trial using an autologous melanoma cell/dendritic cell vaccine. Cancer Immunol Immunother 2003;52:387–95.[Medline]
  12. Homma S, Matai K, Irie M, et al. Immunotherapy using fusions of autologous dendritic cells and tumor cells showed effective clinical response in a patient with advanced gastric carcinoma. J Gastroenterol 2003;38:989–94.[Medline]
  13. Mocellin S. Cancer vaccines: the challenge of developing an ideal tumor killing system. Front Biosci 2005;10:2285–305.[Medline]
  14. Hollingsworth MA, Swanson BJ. Mucins in cancer: protection and control of the cell surface. Nat Rev Cancer 2004;4:45–60.[CrossRef][Medline]
  15. Apostolopoulos V, Pietersz GA, Loveland BE, et al. Oxidative/reductive conjugation of mannan to antigen selects for T1 or T2 immune responses. Proc Natl Acad Sci U S A 1995;92:10128–32.[Abstract/Free Full Text]
  16. Apostolopoulos V, Pietersz GA, Gordon S, et al. Aldehyde-mannan antigen complexes target the MHC class I antigen-presentation pathway. Eur J Immunol 2000;30:1714–23.[CrossRef][Medline]
  17. Karanikas V, Hwang LA, Pearson J, et al. Antibody and T cell responses of patients with adenocarcinoma immunized with mannan-MUC1 fusion protein. J Clin Invest 1997;100:2783–92.[Medline]
  18. Karanikas V, Lodding J, Maino VC, McKenzie IF. Flow cytometric measurement of intracellular cytokines detects immune responses in MUC1 immunotherapy. Clin Cancer Res 2000;6:829–37.[Abstract/Free Full Text]
  19. Karanikas V, Thynne G, Mitchell P, et al. Mannan Mucin-1 Peptide Immunization: Influence of Cyclophosphamide and the Route of Injection. J Immunother 2001;24:172–83.[CrossRef][Medline]
  20. Sandrin MS, Vaughan HA, Xing PX, McKenzie IF. Natural human anti-Gal{alpha}(1,3)Gal antibodies react with human mucin peptides. Glycoconj J 1997;14:97–105.[CrossRef][Medline]
  21. Apostolopoulos V, Osinski C, McKenzie IF. MUC1 cross-reactive Gal{alpha}(1,3)Gal antibodies in humans switch immune responses from cellular to humoral. Nat Med 1998;4:315–20.[CrossRef][Medline]
  22. Xing PX, Apostolopoulos V, Pietersz G, McKenzie IF. Anti-mucin monoclonal antibodies. Front Biosci 2001;6:D1284–95.[Medline]
  23. Apostolopoulos V, Xing P, Trapani J, McKenzie I. Production of anti-breast cancer monoclonal antibodies using a glutathione-S-transferase-MUC1 bacterial fusion protein. Br J Cancer 1993;67:713–20.[Medline]
  24. Apostolopoulos V, Pietersz G, McKenzie I. Cell-mediated immune responses to MUC1 fusion protein coupled to mannan. Vaccine 1996;14:930–8.[CrossRef][Medline]
  25. Siddiqui J, Abe M, Hayes D, et al. Isolation and sequencing of a cDNA coding for the human DF3 breast carcinoma-associated antigen. Proc Natl Acad Sci U S A 1988;85:2320–3.[Abstract/Free Full Text]
  26. Banchereau J, Briere F, Caux C, et al. Immunobiology of dendritic cells. Annu Rev Immunol 2000;18:767–811.[CrossRef][Medline]
  27. Clay TM, Hobeika AC, Mosca PJ, et al. Assays for monitoring cellular immune responses to active immunotherapy of cancer. Clin Cancer Res 2001;7:1127–35.[Abstract/Free Full Text]
  28. Hock BD, Starling GC, Daniel PB, Hart DN. Characterization of CMRF-44, a novel monoclonal antibody to an activation antigen expressed by the allostimulatory cells within peripheral blood, including dendritic cells. Immunology 1994;83:573–81.[Medline]
  29. Apostolopoulos V, Barnes N, Pietersz GA, McKenzie IF. Ex vivo targeting of the macrophage mannose receptor generates anti-tumor CTL responses. Vaccine 2000;18:3174–84.[CrossRef][Medline]
  30. Schmittel A, Keilholz U, Thiel E, Scheibenbogen C. Quantification of tumor-specific T lymphocytes with the ELISPOT assay. J Immunother 2000;23:289–95.
  31. Speiser DE, Pittet MJ, Guillaume P, et al. Ex vivo analysis of human antigen-specific CD8+ T-cell responses: quality assessment of fluorescent HLA-A2 multimer and interferon-{gamma} ELISPOT assays for patient immune monitoring. J Immunother 2004;27:298–308.
  32. Schuler G, Schuler-Thurner B, Steinman RM. The use of dendritic cells in cancer immunotherapy. Curr Opin Immunol 2003;15:138–47.[CrossRef][Medline]
  33. Rosenberg SA, Sherry RM, Morton KE, et al. Tumor progression can occur despite the induction of very high levels of self/tumor antigen-specific CD8+ T cells in patients with Melanoma. J Immunol 2005;175:6169–76.[Abstract/Free Full Text]
  34. Dunn GP, Old LJ, Schreiber RD. The three Es of cancer immunoediting. Annu Rev Immunol 2004;22:329–60.[CrossRef][Medline]
  35. Rustin GJ, Bast RC, Jr., Kelloff GJ, et al. Use of CA-125 in clinical trial evaluation of new therapeutic drugs for ovarian cancer. Clin Cancer Res 2004;10:3919–26.[Free Full Text]
  36. Mukherjee P, Ginardi AR, Madsen CS, et al. MUC1-specific CTLs are non-functional within a pancreatic tumor microenvironment. Glycoconj J 2001;18:931–42.[CrossRef][Medline]
  37. Ridgway D. The first 1000 dendritic cell vaccinees. Cancer Invest 2003;21:873–86.[CrossRef][Medline]
  38. Cranmer LD, Trevor KT, Hersh EM. Clinical applications of dendritic cell vaccination in the treatment of cancer. Cancer Immunol Immunother 2004;53:275–306.[CrossRef][Medline]



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