BACKGROUND. APC8015 (sipuleucel-t) is a cellular prostate cancer vaccine containing

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1 67 Combination Immunotherapy With Prostatic Acid Phosphatase Pulsed Antigen-Presenting Cells (Provenge) Plus Bevacizumab in Patients With Serologic Progression of Prostate Cancer After Definitive Local Therapy Brian I. Rini, MD 1,2 Vivian Weinberg, PhD 2 Lawrence Fong, MD 2 Shauna Conry, BS 2 Robert M. Hershberg, MD 3 Eric J. Small, MD 2 1 Department of Solid Tumor Oncology and Urology, Cleveland Clinic Taussig Cancer Center, Cleveland, Ohio. 2 Department of Medicine, The University of California at San Francisco Comprehensive Cancer Center, San Francisco, California. 3 Dendreon Corporation, Seattle, Washington. Supported in part by Dendreon Corporation (Seattle, WA), the Cancer Therapy Evaluation Program (CTEP), the Prostate Cancer Foundation, and Grant P30CA82103 from the University of California at San Francisco Comprehensive Cancer Center. Dr. Rini s current address: Department of Solid Tumor Oncology and Urology, Cleveland Clinic Taussig Cancer Center, Cleveland, Ohio. Dr. Hershberg is currently Senior Vice President and Chief Medical Officer of Dendreon Corporation. As such, all his compensation and stock holdings are in the public domain, as per Securities and Exchange Commission guidelines. Dendreon provided APC8015 (Provenge) for the clinical trial herein, but the IND was held by the Cancer Therapy Evaluation Program (CTEP) at the National Cancer Institute, and data analysis was performed by Rini et al. Address for reprints: Brian I. Rini, MD, Department of Solid Tumor Oncology and Urology, Cleveland Clinic Taussig Cancer Center, 9500 Euclid Ave., Desk R35, Cleveland, OH 44195; Fax: (216) ; rinib2@ccf.org Received December 29, 2005; revision received February 1, 2006; accepted February 13, BACKGROUND. APC8015 (sipuleucel-t) is a cellular prostate cancer vaccine containing autologous antigen-presenting cells (APC) loaded with PA2024, a recombinant prostatic acid phosphatase/granulocyte-macrophage colony-stimulating factor fusion protein, as the immunogen. Bevacizumab is a recombinant antibody against vascular endothelial growth factor, a proangiogenic protein with inhibitory effects on APC. A clinical trial was conducted to determine the prostate-specific antigen (PSA) and immunomodulatory effects of this combination immunotherapy. METHODS. Patients with androgen-dependent prostate cancer who had received prior definitive therapy with nonmetastatic, recurrent disease as manifested by a rising PSA of between 0.4 ng/ml and 6.0 ng/ml were enrolled. APC8015 was given intravenously(i.v.) on Weeks 0, 2, and 4. Bevacizumab was given at a dose of 10 mg/kg i.v. on Weeks 0, 2, 4, and every 2 weeks thereafter until toxicity or disease progression. PSA changes were recorded and the PSA doubling time (PSADT) was calculated. Immune response versus PA2024 was measured at baseline and after treatment by T-cell proliferation and interferon- enzyme-linked immunospot (ELISPOT) assays. RESULTS. Twenty-two patients were treated. One patient achieved a 50% decrease in PSA. Nine patients exhibited some decrease in PSA from baseline, ranging from 6% to 72%, with the PSA of 3 patients decreasing at least 25%. The median pretreatment PSADT for the 20 evaluable patients was 6.9 months and the median posttreatment PSADT was 12.7 months (P.01). All patients demonstrated induction of an immune response against PA2024. CONCLUSIONS. The combination of APC8015 and bevacizumab induces an immune response and modulates PSA in patients with biochemically recurrent prostate cancer. Cancer 2006;107: American Cancer Society. KEYWORDS: APC8015, bevacizumab, prostate cancer, immunotherapy, sipuleucel-t. Patients with recurrent prostate cancer after definitive local therapy are often asymptomatic, with rising prostate-specific antigen (PSA) values as the sole manifestation of recurrence. Although androgen deprivation is a treatment option, the associated sequelae, including loss of libido, fatigue, anemia, and osteoporosis, make this therapy unacceptable to some patients. A window of opportunity exists in these patients with biochemically recurrent disease to investigate novel therapeutics before the initiation of hormone therapy American Cancer Society DOI /cncr Published online 30 May 2006 in Wiley InterScience (

2 68 CANCER July 1, 2006 / Volume 107 / Number 1 Immune-based treatments may ideally be applied in these otherwise healthy patients with intact immune function and minimal disease burden. APC8015 (sipuleucel-t; Provenge ; Dendreon Corporation, Seattle, WA) is a cellular vaccine containing autologous antigen-presenting cells (APC) loaded with PA2024, a recombinant prostatic acid phosphatase (PAP)/granulocyte-macrophage colony-stimulating factor (GM-CSF) fusion protein, as the immunogen. Clinical trials of APC8015 in patients with androgen-independent prostate cancer have demonstrated safety and clinical activity consisting of PSA declines, an objective response, and prolongation of overall survival versus placebo. 1,2 In addition, induction of T-cell responses against PA2024 or native PAP have been demonstrated. Although further investigation is required to provide a causal link between these immune responses and the clinical benefit, the results provide a foundation on which to build immunotherapeutic approaches in prostate cancer. A greater understanding of the factors limiting dendritic cell (DC)-based immunotherapy such as APC8015 would allow for rational combinations to improve the antitumor immune response and ultimately enhance the clinical effect. In animal models, inadequate DC differentiation caused by tumor-derived factors, including vascular endothelial growth factor (VEGF), may contribute to defective DC function in tumor-bearing hosts. 3,4 In this model, infusion of VEGF resulted in decreased DC production. Furthermore, neutralizing anti-vegf antibody abrogated the inhibition of DC differentiation that was caused by tumor cell supernatant fluids. 5 Therefore, tumor-produced VEGF may contribute to the immunosuppressive tumor microenvironment and limit the generation of an antitumor immune response. In this same model, when tumor-bearing mice that had been immunized with peptide-pulsed DC were treated with an anti-vegf antibody, a more pronounced and longerlasting antitumor immune response was observed compared with mice treated with peptide-pulsed DC alone. These data suggest that the inhibition of VEGF may augment the antitumor effect of DC-based immunotherapy. Based on these data, it was hypothesized that inhibition of VEGF by bevacizumab could augment the immunostimulatory and antitumor effects of AP8015. Neutralization of VEGF could lead to augmentation of antitumor immune responses generated by APC8015, and therefore an enhanced clinical effect. Therefore, a Phase II trial was undertaken to determine the safety and PSA modulating effects of this combination therapy. MATERIALS AND METHODS Patients with histologically diagnosed adenocarcinoma of the prostate who had undergone prior definitive therapy for prostate cancer consisting of external beam radiotherapy, brachytherapy, or radical prostatectomy and who had experienced subsequent disease progression were eligible. Patients treated with adjuvant or salvage radiation therapy after radical prostatectomy were eligible provided the postprostatectomy PSA level was never 6.0 ng/ml. Progressive disease, defined as 2 climbing PSA values between 0.4 ng/ml and 6.0 ng/ml measured at least 2 weeks apart, was required. Patients with a local recurrence or evidence of metastases on bone scan or computed tomography (CT) scan were excluded. An Eastern Cooperative Oncology Group performance status of 0 or 1 was required. Adequate renal, hepatic, and bone marrow function (defined as blood urea nitrogen and serum creatinine 2.0 the upper limit of normal, total bilirubin and serum aspartate aminotransferase 2.0 the upper limit of normal, a white blood cell count 2500/ L, an absolute neutrophil count 1000/ L, and a platelet count 100,000/ L) were required. All patients provided signed, informed consent approved by the University of California San Francisco Committee on Human Research. Prior hormonal therapy of any type for the treatment of progressive disease was not permitted (up to 6 months of prior hormonal therapy used in an adjuvant or neoadjuvant setting was permitted, but the last day of effective androgen deprivation must have been at least 3 months before study entry). Prior chemotherapy, immunotherapy, or VEGF-targeted therapy for prostate cancer was not permitted. Patients with a history of deep venous thrombosis, bleeding disorder, or active autoimmune disease were excluded. Use of therapeutic oral or parenteral anticoagulants within 10 days preceding the first bevacizumab infusion was prohibited. Patients with clinically significant cardiac or pulmonary disease, uncontrolled infection, a history of surgery within the prior 4 weeks, central nervous system disease, active second malignancy, known human immunodeficiency virus (HIV) infection, or hepatitis, or who were receivin current systemic steroid therapy were excluded. Treatment Treatment was administered on an outpatient basis (Fig. 1). On Weeks 0, 2, and 4, APC8015 was prepared and administered as previously described. 1 Patients received the maximum manufacturable dose of APC8015, an estimated nucleated cells/m 2, with a minimum dose of DCs for all patients.

3 APC8015 Bevacizumab in Prostate CA/Rini et al. 69 allergic reactions that were potentially life-threatening or any Grade 3 or 4 nonhematological toxicities, with the exception of Grade 3 hypertension that was well controlled with oral medication, Grade 3 nausea and vomiting that was controlled by antiemetics, and Grade 4 hematologic toxicities. FIGURE 1. On Weeks 0, 2, and 4, APC8015 was infused 48 hours after each leukapheresis procedure was completed. Bevacizumab (at a dose of 10 mg/kg intravenously) was administered on Weeks 0, 2, and 4 after APC8015 infusion and continued every 2 weeks thereafter until disease progression or unacceptable toxicity. PSA indicates prostate-specific antigen; RP, radical prostatectomy, XRT, radiation therapy, PD, progressive disease; DC, dendritic cells, APC, antigen-presenting cells. Patients received 3 total doses of APC8015. Patients were premedicated 30 minutes before APC8015 infusion with acetaminophen at a dose of 650 mg given orally and diphenhydramine at a dose of 50 mg given orally. Bevacizumab (at a dose of 10 mg/kg intravenously) was administered on Weeks 0, 2, and 4 after APC8015 infusion and continued every 2 weeks thereafter. The initial bevacizumab dose was administered over a minimum of 90 minutes. If no adverse reactions occurred, the second dose was administered over a minimum of 60 minutes. Again, if no adverse reactions occurred, the third and subsequent doses were administered over a minimum of 30 minutes. Treatment with bevacizumab was continued every 2 weeks until disease progression or unacceptable toxicity. PSA was assessed at baseline and every 4 weeks. Treatment continued until unacceptable toxicity or disease progression, defined as a doubling of the nadir or pretreatment PSA value (whichever was lowest) to at least 4 ng/ml maintained for at least 2 measurements that were at least 2 weeks apart, or the development of distant metastases. Patients underwent a repeat bone scan and CT scan of the abdomen and pelvis at the time of disease progression by PSA criteria or as clinically indicated. Toxicity Management The National Cancer Institute Common Terminology Criteria (version 3.0) were utilized to assess toxicity. Assessment of urinary protein by dipstick was performed before each dose of bevacizumab. If protein was found at a level of 2 on dipstick, a 24-hour urine collection was performed. For a 24-hour urine excretion of 2000 mg or 3 on dipstick, bevacizumab was held until protein was 1. If protein remained 1 for 6 weeks patients were removed from therapy. Patients were removed from therapy for Grade 2 Immune Monitoring Peripheral blood mononuclear cells (PBMCs) were isolated from each patient s leukapheresis at Week 0 (PCT-cell Processing Center, Mountain View, CA), frozen in media/serum containing dimethyl sulfoxide (DMSO), and stored in liquid nitrogen. Follow-up PBMC samples (Week 8 and/or Week 12) were isolated from whole blood at the University of California at San Francisco, frozen in media/serum containing DMSO, and stored in liquid nitrogen. All samples were shipped overnight on dry ice to Dendreon Corporation (Seattle, WA) for testing. All assay setup and PBMC preparation was performed under sterile conditions. Subject samples from all timepoints available were thawed and assayed on the same day. Samples were thawed and counted and viability was calculated using Trypan blue exclusion. T-Cell Proliferation Assay Proliferation assays were set up in triplicate in a 96- well U-bottom plate. PBMCs ( cells/well) were incubated with the indicated final concentration of PA2024. Control wells contained media only. Assays were incubated for 6 days, pulsed with 3 H-thymidine (Amersham, Piscataway, NJ) for the last 18 hours of incubation, and then harvested to filter mats using a Tomtec plate harvester. After the addition of scintillation cocktail (Perkin Elmer/Wallac, Norwalk, CT), assays were counted using a Wallac Beta Scintillation counter. Readout was reported in counts per minute (CPM). A stimulation index (SI) was calculated using the median CPM experimental/median CPM control ratio for each patient at each timepoint with an evaluable blood sample (Week 0/baseline, Week 8, and Week 12). Interferon-gamma ELISPOT Wells of Multiscreen-HA plates (Millipore, Bedford, MA) were coated overnight at 4 C with 100 L of antihuman interferon- (IFN- ) antibody (clone 1-DIK; Mabtech, Nacka, Sweden) at 15 g/ml in Dulbecco-phosphate-buffered saline (D-PBS) (Life Technologies, Baltimore, MD). After coating, plates were washed with phosphate buffered saline/tween (PBST; Sigma Chemical Company, St. Louis, MO) and blocked with 200 L D-PBS 10% horse serum (HS) for 2 hours at 37 C. Assays were set up in triplicate with 3

4 70 CANCER July 1, 2006 / Volume 107 / Number PBMC cells/well and the indicated final concentration of PA2024. Negative control wells contained media alone. Assays were incubated at 37 C for hours. After 2 days cells and antigen were washed from the plate using PBST. The detection antibody, 100 L of biotinylated antihuman IFN- (Mabtech, clone 7-B6-1), was added to wells at 1 g/ml in PBST. Assays were incubated for 2.5 hours at room temperature. Plates were washed 6 times with PBST and 100 L of streptavidin alkaline phosphatase (Mabtech) diluted 1:1000 in PBST was added to assay wells. Assays were incubated for 1.5 hours and then washed 6 times with PBST. Then, 1-step BCIP/NBT solution (Pierce, Rockford, IL) was added at 100 L per well and incubated for 12 minutes to develop spots. To stop color development, wells were rinsed with sterile water. Plates were scanned and spots counted using an ImmunoSpot Analyzer and software (Cellular Technology, Cleveland, OH). Results are reported as spots per PBMC minus control spots for each timepoint. TABLE 1 Patient Characteristics (n 22) Characteristic No. of Patients Median age, y (range) 70 (55 79) Primary therapy XRT 3 (14%) RP 5 (23%) RP XRT 14 (63%) T classification at diagnosis RP and RP XRT patients (pathologic stage) pt2 9 (41%) pt3 10 (45%) XRT patients (clinical stage) ct1 2 (9%) ct2 1 (5%) Median pretreatment PSA (ng/ml) (range) 2.3 ( ) Median Gleason score (range) 7 (5 9) 5 3 (14%) 6 5 (23%) 7 10 (45%) 8 3 (14%) 9 1 (5%) Median pretreatment PSA doubling time (mo) (range) 6.9 ( ) XRT indicates external beam radiotherapy; RP, radical prostatectomy; PSA, prostate-specific antigen. Statistical Analysis The primary study endpoint for determining the sample size was the proportion of patients achieving a 50% PSA decline compared with baseline. The sample size for accrual utilized a 2-stage design. Treatment of 43 patients would be sufficient to determine if at least 30% of patients demonstrated PSA declines of 50% compared with a null hypothesis of 15% of patients. For the interim analysis, if 2 patients experienced a PSA decline of 50% among the first 20 patients treated, then accrual would stop. The probability of stopping accrual if the null hypothesis was true was 40% and 4% if the alternative was true. If accrual continued to 43, then the null hypothesis would be rejected if 11 responses were observed. The overall level of significance was.05 and power was Accrual was not stopped to perform the interim analysis for efficacy. For analysis of the data, the linear rate of increase of the PSA on the natural log (ln) scale over time before entry on protocol as well as the change in ln PSA calculated from the start of treatment were each estimated by the slope. The PSA doubling time both pretreatment and posttreatment were estimated using the relation of ln 2 divided by the slope. If a continuous decline in the rate of PSA with treatment was observed, this then resulted in a negative slope, and a doubling time could not be estimated. If the patient initially experienced a decline in PSA, then the posttreatment slope was calculated starting from the PSA nadir. Time to disease progression was defined as the time from the initiation of therapy to the first PSA meeting criteria for disease progression or the development of objective disease progression, whichever came first. The probability of remaining free of progressive disease and time to PSA nadir were estimated using the Kaplan Meier product limit method. The Wilcoxon matched pairs test was used to compare the distributions for the pre- and posttreatment doubling times. RESULTS Patient Characteristics Twenty-two patients were enrolled from October 2001 to November Further accrual was suspended based on the results of the interim analysis. Patients were typical of those with biochemical recurrence of prostate cancer (Table 1). The median age of patients was 70 years. Primary therapy included radiation (XRT) in 3 patients, radical prostatectomy (RP) in 5 patients, or RP followed by adjuvant XRT in 14 patients. Six patients (3 who received prior XRT, 1 who received RP, and 2 who received RP followed by XRT) had received neoadjuvant or adjuvant hormonal therapy. These patients received a median of 4.5 months of hormone therapy (range, 3-6 months) that was discontinued for a median of 49 months (range, months) before protocol treatment. The median maximum pretreatment PSA was 2.3 ng/ml (range, ng/ml). Fourteen patients (64%) had a Gleason score of 7.

5 APC8015 Bevacizumab in Prostate CA/Rini et al. 71 TABLE 2 Pretreatment and Posttreatment PSA Characteristics for Evaluable Patients Characteristic Pretreatment Posttreatment FIGURE 2. the linear rate of increase of the prostate-specific antigen (PSA) on the natural log (ln) scale versus time for the patient who demonstrated a 50% decline in PSA. A rising PSA at the time of study entry (Week 0) continued to increase after initial therapy. A delayed subsequent decline was observed until the achievement of a PSA nadir at the cessation of therapy (Week 72). A rising PSA was noted on Week 108, 7 months after protocol therapy was discontinued. Up arrow indicates the initiation of protocol therapy; down arrow, end of protocol therapy. PSA Changes Twenty-one patients were evaluable for assessment of posttreatment PSA changes with a median follow-up of 11.4 months. One patient was not evaluable because of withdrawal of consent before repeat PSA measurement. This patient was included in the toxicity analysis. A PSA decline of 50% was observed in 1 patient (5%; 95% confidence interval, 0-24%). This patient had a Gleason 3 4 adenocarcinoma status after RP and adjuvant XRT. He had not received hormonal therapy at any time before protocol enrollment. Figure 2 depicts the ln PSA over time for this patient. An initial increase in PSA from a baseline value of 2.7 ng/ml to 5.6 ng/ml at Week 12 was followed by a subsequent decrease in PSA to a nadir value of 0.78 ng/ml at Week 72. This patient came off study with a PSA of 0.84 ng/ml due to toxicity (proteinuria) at Week 88 after initiation of protocol therapy. This patient remained off all therapy with a stable PSA until 7 months after the last bevacizumab infusion (Week 108), at which time a rising PSA was noted. Nine patients exhibited some decrease in PSA from baseline ranging from 6% to 72% with 3 patients decreasing at least 25% (Table 2). For these 9 patients, the median time to PSA nadir was 5.8 months (range, months). The median time to progression (PSA progression in all patients) was 11.2 months. For determination of pretreatment PSA doubling time, the 3 consecutive PSA values before the start of protocol therapy were used for all patients and were collected over a median of 4.2 months (range, months). The median estimated pretreatment PSADT Maximum pretreatment PSA (n 21) Median (range) 2.3 ng/ml ( ) PSA nadir (n 21) Median (range) 1.9 ng/ml ( ) Absolute PSA reduction 50% 1 (5%) 25% 3 (14%) Any reduction 9 (43%) PSA doubling time (n 20)* Median 6.9 mo 12.7 mo Mean 7.8 mo 13.0 mo Range ( ) ( ) PSA indicates prostate-specific antigen. * One patient with a decreasing PSA while on study was excluded. His pretreatment PSA doubling time was 4.8 months. for the 21 evaluable patients was 6.7 months, although there was a broad range ( months). The median posttreatment PSADT was 12.7 months (Wilcoxon matched pairs test, P.01). Six patients displayed at least a 200% increase over the pretreatment PSADT (range, %) and 4 patients displayed between a 100% and 200% increase in PSADT (range, %). Table 2 depicts the pretreatment and posttreatment PSA characteristics for all evaluable patients. Twelve patients developed progressive disease by PSA criteria, including 1 patient with only a single PSA value reaching the definition for progessive disease but who refused further PSA evaluation. Nine of these patients had a follow-up bone scan and CT scan, and none developed metastatic disease. Four patients were removed from study for toxicity (vide infra). Five patients remained on study at the time of last followup with treatment durations ranging from 18 months to 28 months. Toxicity The majority of side effects were Grade 1 or 2 including fatigue and infusion-related chills, myalgias, and fever (Table 3). A total of 6 Grade 3 toxicities were observed leading to the discontinuation of therapy in 4 patients (congestive heart failure, gastrointestinal hemorrhage, cerebrovascular ischemia, and proteinuria). There were no Grade 4 toxicities. Grade 3 congestive heart failure was observed in a 69-year-old man with a baseline history of hypertension, hypercholesterolemia, and asthma who developed dyspnea after 31 months of therapy. Echocardio-

6 72 CANCER July 1, 2006 / Volume 107 / Number 1 TABLE 3 Treatment-Related Adverse Events (n 22) Nonhematologic Toxicity* Grade 1 (%) Grade 2 (%) Grade 3 (%) Fatigue 16 (73) 2 (9) 0 Rigors, chills 13 (59) 1 (5) 0 Myalgia (muscle pain) 12 (55) 0 0 Fever 9 (41) 0 0 Hyperglycemia 8 (36) 1 (5) 0 Diarrhea 7 (32) 0 0 Epistaxis 7 (32) 0 0 Hoarseness 7 (32) 4 (18) 0 Nausea 6 (27) 0 0 Hypertension 6 (27) 1 (5) 1 (5) Headache 6 (27) 2 (9) 0 Proteinuria 5 (23) 1 (5) 1 (5) Hyponatremia 4 (18) 0 0 Rash 4 (18) 2 (9) 0 Gastrointestinal bleeding (5) Allergic reaction/hypersensitivity (5) Cardiac left ventricular function (5) CNS cerebrovascular ischemia (5) Dyspnea 0 3 (14) 0 Hematologic toxicity Anemia 5 (23) 0 0 Neutropenia 3 (14) 3 0 Leukopenia 0 1 (5) 0 CNS indicates central nervous system. * Toxicity was graded according to National Cancer Institute Common Toxicity Criteria (version 3.0). gram revealed a dilated left ventricle with an ejection fraction of 23%. No ischemia was noted on electrocardiogram and coronary catheterization revealed normal coronary arteries. Symptoms resolved and ejection fraction improved to 45% with supportive medication 2 months after discontinuation of protocol therapy. A Grade 3 gastrointestinal bleed was experienced by a 74-year-old man with a history of salvage radiation after prostatectomy and nonsteroidal antiinflammatory (NSAID) use for gout who presented with guaiac-positive stools requiring blood transfusion. Lower bowel endoscopy did not reveal an active bleeding source and the event was felt likely related to NSAID use, but a contribution of bevacizumab could not be entirely ruled out. Concurrently, the patient demonstrated PSA progression and was taken off study. A Grade 3 cerebrovascular ischemia was observed in a 60-year-old man with a history of atrial fibrillation who had undergone cardioversion to normal sinus rhythm 2 years prior. The patient then represented in atrial fibrillation after 10 months of therapy requiring repeat cardioversion. There was no evidence of ischemia or cardiac thrombus at that time, and the patient was placed on coumadin. The atrial fibrillation was believed to be unlikely related to protocol therapy. Three days after this episode, the patient presented with expressive aphasia and right arm weakness. There was no radiographic evidence of cerebrovascular ischemia or bleeding on CT scan and the symptoms resolved spontaneously. This event was believed to possibly be related to protocol therapy and the patient was removed from study. The patient who exhibited a 50% PSA decline developed proteinuria 22 months after the initiation of protocol therapy, which remained 1 for 6 weeks and therefore, per prespecified protocol criteria, was removed from therapy. Urinalysis performed 9 months after cessation of bevacizumab therapy demonstrated resolution of proteinuria. Two additional Grade 3 events were observed. A patient with baseline hypertension requiring 3 medicines developed Grade 3 hypertension after cycle 3 controlled with adjustment of existing antihypertension medications. Although blood pressure was controlled, the patient decided to withdraw consent at Cycle 5. Blood pressure returned to baseline status within 2 weeks after discontinuation of bevacizumab. Another patient demonstrated Grade 3 facial angioedema at cycle 58 of therapy, which was believed to most likely be related to food allergies or recently started lisinopril that resolved with prednisone and diphenhydramine. The patient received 2 subsequent doses of bevacizumab without incident before being taken off study for progressive disease. Immune Monitoring T-cell proliferation Nine patients had pre- and posttreatment blood samples evaluable for measurement of T-cell proliferation. Stimulation indices (SI) using a 50 g/ml concentration of PA2024 versus control are reported for baseline and highest posttreatment timepoint. The posttreatment timepoint utilized was dependent on availability of samples for analysis and was Week 8 in 6 patients and Week 12 in 3 patients. All 9 patients demonstrated increased T-cell proliferation versus antigen posttreatment compared with baseline. The median SI at Week 0 was 2.3 (range, ) and the median posttreatment SI was 25.9 (range, ). Due to the different follow-up observation times for a limited number of patients, estimating the correlation of T-cell proliferation and PSA modulation was precluded. Of note, however, the patient with the 50% PSA decline demonstrated the greatest increase in T-cell proliferation from an SI of 2.3 at baseline to at Week 8. Figure 3 depicts the paired baseline and Week 8 posttreatment SI for this patient using varying concentrations of PA2024. No T-cell proliferation data are available

7 APC8015 Bevacizumab in Prostate CA/Rini et al. 73 FIGURE 3. Paired baseline and posttreatment T-cell proliferation stimulation indices for the patient who demonstrated a 50% decline in prostate-specific antigen (PSA) using varying concentrations of PA2024. from this patient at the time of PSA nadir or subsequently to evaluate the duration of T-cell response. IFN- ELISPOT Six patients had pre- and posttreatment blood samples evaluable for measurement of IFN- production via ELISPOT. All 6 patients demonstrated increased IFN- producing T-cells versus PA2024 posttreatment compared with baseline. The median number of spots (minus control spots) for PBMCs at a PA2024 concentration of 50 mg/ L was 1 (range, 0-3) at baseline and the median number of spots posttreatment was 87 (range, ). DISCUSSION APC8015 treatment in combination with bevacizumab in patients with biochemically recurrent prostate cancer patients resulted in PSA modulation as demonstrated by PSA declines and alterations in PSA doubling time. Furthermore, immunomodulation was demonstrated by induction of an immune response against PA2024. Despite these encouraging observations, the current study must be interpreted as hypothesis-generating due to several limitations. This small, single-arm study without a concurrent control arm of either APC8015 or bevacizumab monotherapy did not allow estimation of the potential additive or synergistic clinical or immune effects of this combination. Nevertheless, a study of APC8015 monotherapy in this group of patients appeared to have less PSA-modulating effects, with no PSA declines 50% and no significant difference between pre- and posttreatment PSADT. 6 In addition, a study of bevacizumab monotherapy in patients with hormone-refractory prostate cancer failed to demonstrate objective responses or PSA declines 50%. 7 Direct comparison among these trials is not possible, however, and no immune parameters were measured in the monotherapy trials. Furthermore, prospective investigation in a randomized setting is needed for a more precise estimation of the potential additive or synergistic immunomodulatory or clinical benefit of this combination therapy. The serious cardiac and bleeding toxicities potentially related to therapy require close monitoring with further investigation of this combination. Although each agent has demonstrated acceptable safety as monotherapy in large populations of patients, the combination of therapy and/or patient population may result in increased or unique toxicity. Induction of an immune response versus PA2024 was demonstrated in the current study. Although the clinical relevance of this effect in prostate cancer is unknown, the patient with the greatest PSA effect from therapy also demonstrated the greatest induction of an immune response. In addition, the magnitude of the PA2024-specific T-cell immune response noted in this study is consistent with that seen in other studies with APC ,2 The immunologic assays performed in the current study were limited to a small number of patients, precluding statistical correlation of induced immune response and PSA modulation. No functional testing of induced immune cells (e.g., cytotoxic effect against prostate cancer cells) was undertaken to investigate the relevance of the immune cells generated. Furthermore, as noted earlier, the putative mechanism of effect of APC8015 and potential enhancement from VEGF blockade is through the DC component. No direct assay of DC type, quantity, or function was undertaken in this study. Availability of adequate numbers of cells for assays was limited, and therefore assays to measure T-cell activity against the vaccine immunogen were employed because ultimately it is cytotoxic T-cells that are believed to exert the antitumor effect of immunotherapy. Last, the posttreatment immune assays were performed at limited timepoints. The timing of induction of an immune response from this therapy is unknown, and collection of blood at additional timepoints could have allowed for further characterization of the immune response. Such insight could also assist in the design of future clinical trials of this approach in determining the need for booster APC8015 treatments in the case of an insufficient or waning immune response. Therefore, although the observation of an induced immune response against the immunogen is encouraging, further investigation in a larger number of patients is needed to more definitively characterize the immune response generated by this combination. In addition,

8 74 CANCER July 1, 2006 / Volume 107 / Number 1 defining the precise correlation between immune and clinical endpoints remains a significant challenge in trials of novel immunotherapies. Absolute PSA declines are delayed and often limited after immunotherapy, further compounding interpretation of the clinical activity of a given approach in a single-arm study. 1 Indeed, the most dramatic PSA effect observed in the current study occurred only after a significant PSA rise. Therefore, absolute PSA changes may be of limited value and alternative measures of PSA modulation are more frequently being employed. The relevance of PSADT modulation in this disease state is not yet established to our knowledge, and placebo-controlled randomized studies in this setting lend caution to overinterpretation of differences in pretreatment and posttreatment PSADT. 8 The use of a limited number of PSA values and a short duration of collection limits the precision of PSADT estimation. Additional investigation to validate intermediate endpoints (e.g., immune parameters or PSA modulation) is needed for immunotherapy trials in this population of patients. Despite these limitations, the preclinical data suggesting that VEGF blockade may enhance immune function and the PSA modulating and immune effects observed in this study warrant further investigation. A randomized trial of APC8015 with or without bevacizumab in patients with hormon-refractory prostate cancer is being considered to further investigate the clinical and immunologic effects achieved with the addition of VEGF blockade to APC8015. REFERENCES 1. Small EJ, Fratesi P, Reese DM, et al. Immunotherapy of hormone-refractory prostate cancer with antigen-loaded dendritic cells. J Clin Oncol. 2000;18: Burch PA, Croghan GA, Gastineau DA, et al. Immunotherapy (APC8015, Provenge) targeting prostatic acid phosphatase can induce durable remission of metastatic androgen-independent prostate cancer: a Phase 2 trial. Prostate. 2004;60: Gabrilovich D, Ishida T, Oyama T, et al. Vascular endothelial growth factor inhibits the development of dendritic cells and dramatically affects the differentiation of multiple hematopoietic lineages in vivo. Blood. 1998;92: Gabrilovich DI, Chen HL, Girgis KR, et al. Production of vascular endothelial growth factor by human tumors inhibits the functional maturation of dendritic cells. Nat Med. 1996;2: Gabrilovich DI, Ishida T, Nadaf S, Ohm JE, Carbone DP. Antibodies to vascular endothelial growth factor enhance the efficacy of cancer immunotherapy by improving endogenous dendritic cell function. Clin Cancer Res. 1999;5: Beinart G, Rini BI, Weinberg V, Small EJ. Antigen-presenting cells 8015 (Provenge) in patients with androgen-dependent, biochemically relapsed prostate cancer. Clin Prostate Cancer. 2005;4: Reese DM, Frohlich M, Bok R, Corry M, Novotny W, Small EJ. A phase II trial of humanized monoclonal anti-vascular endothelial growth factor antibody (rhumab VEGF) in hormone refractory prostate cancer (HRPC). Programs and Proceedings of the American Society of Clinical Oncology, Abstract Smith MR, Manola J, Kaufman DS, et al. Rosiglitazone versus placebo for men with prostate carcinoma and a rising serum prostate-specific antigen level after radical prostatectomy and/or radiation therapy. Cancer. 2004;101: