1. Tab.2 Peptides derived from tumor and PCa-associated antigens used for loading of the DCs Introduction & Objectives Prostate cancer (PCa)  most common cancer diagnosis & 2 nd leading cause of cancer-related deaths 10 ‑ year cancer specific survival rates of 95-97% for radically prostatectomized patients, up to 1/3 disease recurrence accompanied by an increase of the serum marker prostate-specific antigen (PSA) in a median of 8 years after this relapse  metastatic disease  death within 2-5 years advanced, recurrent and metastatic PCa treated by androgen-deprivation convert to androgen-independent growth within a few years metastatic hormone-refractory prostate cancer (HRPC)  median survival of 16 months treatment options limited to few chemotherapies producing a survival benefit of 2-3 months  search for novel treatments for HRPC including immunotherapies based on dendritic cells (DCs)  DCs as professional antigen-presenting cells that induce, sustain & regulate T ‑ cell responses (Fig.1)  Phase I clinical trial: safety and feasibility of a vaccination with monocyte-derived DCs loaded with a cocktail consisting of HLA-A*0201-restricted peptides derived from the 5 different tumor-associated antigens (TAAs) PSA, PSMA, prostein, survivin & trp ‑ p8. Materials & Methods Patients: 8 patients with HRPC (Tab.1), positive for HLA-A*0201 allele, age 57-74 years (median 69) expected live span >3 months, Karnovsky index >60%, PSA 5-150ng/ml (increase of ≥20% within 2 months) anti-androgens withdrawn 4 weeks before the start, LHRH agonists continued Reagents, cytokines, peptides: all reagents (cytokines, peptides, buffers, media) were sterile, endotoxin-free and according to GMP guidelines X ‑ VIVO15 medium & DPBS (Cambrex Bio Science), IL1b, IL4, IL6, TNF ‑  (Cell Genix), GM ‑ CSF (Leukine Liquid Sargramostim, Berlex Laboratories), PGE2 (Minprostin E2, Pharmacia), human AB serum (CC Pro) HLA-A0201*-binding TAA-derived peptides (Tab.2) with a purity of ≥97% (Jerini Biotools) Immunomagnetic enrichment of monocytes and cultivation of DCs (Fig.2): 2 leukaphereses per patient (days -1 & 27) for 4 vacc., 1 st & 3 rd vacc. (days 8 & 36) with freshly cultivated and matured DCs (mDCs), 2 nd & 4 th vacc. (days 22 & 50) generated from cryopreserved immature DCs (iDCs) depletion of platelets by centrifugation, incubation with CliniMACS CD14 reagent (Miltenyi Biotec), immunomagnetic isolation of monocytes using CliniMACS device according to the manufacturer’s instructions samples for differential cell counting, flowcytometric analyses and sterility controls from unsorted cells & from sorted CD14-enriched and ‑ depleted fractions, viability assessed by trypan blue staining and cell counting differentiation to iDCs: 1.0 ‑ 1.2x10 9 monocytes in X ‑ VIVO15 + 100IU/ml penicillin & 10µg/ml streptomycin + 1% AB serum + 1000IU/ml GM ‑ CSF + 1000IU/ml IL ‑ 4 harvest of iDCs with cell scraperss 6 days after seeding seeding of 40% of the harvested iDCs for maturation with X ‑ VIVO15 + 1% AB serum, GM ‑ CSF, IL ‑ 4 IL ‑ 1b, IL ‑ 6 & TNF ‑  (each 1000IU/ml) + PGE2 (1µg/ml) remaining iDCs cryopreserved in plasma + 10% DMSO for vacc. 2 and 4 separate pulsing with 5 different peptides (final conc. 20µg/ml) on day 7 24h later harvest of peptide-loaded mDCs (cell scraper), 3 washing steps, resuspension in DPBS + 1% autologous inactivated plasma for vacc. Administration of peptide-pulsed DCs premedication: Paracetamol (analgetic), Fenistil & Cimetidine (histamine blockers) injection of each 1x10 7 mDCs intradermally (2ml) & intravenously (50ml) Flowcytometric analyses immunostaining with fluorescence-labeled antibodies incl. isotype controls purity of CD14 enriched population: CD14 / CD15 / CD45 DC differentiation and maturation: CD80 / CD83 / CD86 / HLA ‑ DR Clinical Monitoring at baseline & follow up visits on days 26 & 63 & at 3 months after the last vaccination medical history, physical examination, blood count, blood chemistry imaging studies: abdominal sonography, X ‑ ray of the chest, bone scan, magnetic resonance imaging or CT immune status (flowcytometric quantification of B- & T ‑ lymphocytes & natural killer cells; CD 3/4/8/19/16/56) bi-weekly measurement of serum levels of total PSA PSA response: - PSA decrease of  50%  partial response (PR) - PSA decrease of <50% or slower PSA increase  stable disease (SD) - PSA increase ≥ than before vaccination  progressive disease (PD) - duration: time of PSA response following the 1 st vacc. on day 8, or when highest PSA values were measured IFN ‑  ELISPOT assay frequency of peptide-reactive CD8 + T ‑ cells in the blood of vaccinated patients before & after treatment coating with mouse-anti-human IFN ‑  antibody (1 ‑ D1K) overnight at 4°C blocking with RPMI 1640 medium + 10% human serum addition of immunomagnetically isolated monocytes pulsed separately with TAA-derived peptides (100µg/ml) addition of 1x10 5 CD8 + immunomagnetically isolated T ‑ cells after 2h detection of captured cytokine by biotinylated anti-IFN ‑  antibody (7 ‑ B6 ‑ 1) for 2h incubation with avidin-biotin peroxidase complex (Vectastain Elite Kit) counting of spots using a stereomicroscope (40x magnification), subtracting mean background spot counts for control peptide derived from HIV-RT (not > 4 spots/1x10 5 cells in all experiments) from mean spot counts for each TAA-derived peptide Vaccination of hormone-refractory prostate cancer patients with peptide cocktail- loaded dendritic cells: results of a Phase I clinical trial A. Meye 1, S. Fuessel 1, M. Schmitz 2, S. Zastrow 1, C. Linné 1, K. Richter 1, B. Lobel 2, O.W. Hakenberg 1, K. Hoelig 3, E.P. Rieber 2, M.P. Wirth 1 1 Department of Urology, 2 Institute of Immunology, 3 Institute of Transfusion Medicine, Medical Faculty, Technical University Dresden, Germany peptide codepeptide sequencepositionreference PSA3 154-163VISNDVCAQV154-163 Correale et al. 1997 PSMA1 4-12LLHETDSAV4-12 Tjoa et al. 1996, Murphy et al. 1996 prostein 31-39CLAAGITYV31-39 Kiessling et al. 2004 survivin 95-104ELTLGEFLKL95-104 Schmitz et al. 2000, Andersen et al. 2001 trp ‑ p8 187-15 GLMKYIGEV187-195 Kiessling et al. 2003 Results Immunomagnetic enrichment of monocytes and cultivation of DCs leukaphereses: mean total numbers of 20.9x10 9 leukocytes (median 22.1x10 9 ) with mean percentages of CD14 + monocytes of 14.2% (median 13.1%) enriched monocytes: mean purity of 94.7% CD14 + /CD45 + cells (median 96.9%) DC differentiation (Fig.3): started with 1.0 ‑ 1.2x10 9 monocytes, recovery of iDCs after 6 days  mean 33.4% (median 30.0%) with a mean viability of 94.1% (median 95.8%), recovery of mDCs after 2 days of maturation  on the average 42% (median 41%) with a mean viability of 93.4% (median 96.8%) flowcytometric quantification (Fig.4) of the DC-markers HLA-DR, CD80, CD83 and CD86: freshly cultivated mDCs were on the average 66.6% CD80 + /CD86 + (median 72.4%) and 64.0% HLA-DR + /CD83 + (median 69.1%). Results of clinical monitoring characteristics and responses to therapy of the 8 patients: see Tab.1 & Fig.5 1 partial PSA response (patient #4): initial PSA increase followed by continuous decline of >50% after the 2 nd vaccination, stable PSA until day 105 (Fig.5a) 3 other patients (#8, #3 & #6) with stable disease (Fig.5b) for 4–17 weeks afterwards PSA increase in all 4 initial PSA responders continuous PSA progression in the remaining 4 patients (data not shown) vaccinations were well tolerated, no toxicity (hematological, hepatic, renal or neurological) or other side effects (allergic, autoimmune, fever, nausea or fatigue) except a temporary local skin reaction at the sites of intradermal injection (small edematous erythema of 4 ‑ 6 mm over 4 ‑ 6 h) no significant changes of metastatic load in any patient Results of immunological monitoring (IFN ‑  ELISPOT) evaluation for the presence of CD8 + T ‑ cells in blood samples reactive against the different peptides before and after treatment no pre-existing tumor peptide specific CD8 + T ‑ lymphocytes before vaccination frequency of CD8 + T ‑ cells with reactivity against the prostein, survivin and/or PSMA peptides increased to detectable levels in 4 of 8 patients (patients #3 - #6) after vaccination (Fig.6) no detectable reactivity against peptides derived from PSA or trp ‑ p8 3 of 4 ELISPOT responders showed also a PSA response (Tab.1), patient #8 (only short-term PSA stabilization over 4 weeks) was negative in ELISPOT http://urologie.uniklinikum-dresden.de/ susanne.fuessel@mailbox.tu-dresden.de Conclusions These data indicate that the application of peptide cocktail-loaded DCs is a safe and feasible approach which caused only few local side effects and induced transient clinical responses accompanied by the induction of peptide-reactive CD8 + T ‑ cells in 4 of 8 treated HRPC patients. The varying TAA-specific immunological responses as assessed by ELISPOT analyses comprising only 3 (prostein, survivin, PSMA) of the selected 5 TAAs highlight the importance of a reasoned choice of suitable, widespread, highly, and constantly expressed target antigens. In our opinion, this novel multi-target approach which is supposed to improve the efficacy of the induction of tumor-reactive CTLs in comparison to trials using single antigenic peptides represents a feasible and promising option of immunotherapy for HPRC patients and warrants further evaluation. Fig.5 Course of PSA changes before, during and after DC vaccination of 4 PSA responders a) partial responder (#4) with a PSA decrease within 7 weeks and further PSA stabilization for 5 weeks b) patients (#3, #6, #8) with stable PSA values or decelerated PSA increases (stable disease) Black arrows indicate the DC vaccinations. a) b) Fig.6 Detection of tumor peptide-reactive CD8+ T ‑ cells by IFN ‑  ELISPOT analysis Purified CD8 + T ‑ cells were coincubated with monocytes which were loaded separately with the different tumor peptides. The frequency of CD8 + T ‑ lymphocytes reactive against TAA-derived peptides 2 weeks (day 63) after treatment is demonstrated. Columns represent mean values of triplicate wells containing HIV-RT peptide-loaded monocytes subtracted from mean values of triplicate wells containing tumor peptide-pulsed monocytes. Asterisks indicate samples without detectable IFN ‑  -secretion. * * * * * * * * * * * * * References Andersen M.H. et al. Cancer Res 2001;61:869-872. Correale P. et al. J Natl Cancer Inst 1997;89:293-300. Kiessling A. et al. Prostate 2003;56:270-279. Kiessling A. et al. Br J Cancer 2004;90:1034-1040. Murphy G. et al. Prostate 1996;29:371-380. Schmitz M. Cancer Res 2000;60:4845-4849. Tjoa B. et al. Prostate 1996;28:65-69. Fig.4 Course of DC differentiation and maturation Flowcytometric analyses of expression changes of typical surface markers of monocytes (CD14) and DCs (CD80, CD83, CD86, HLA-DR) shown exemplarily for patient #3 (2 nd leukapheresis). CD86 iDC mono mDC CD80 iDC mono mDC CD86/CD80 : mono (0,39%) iDC (0,87 %) mDC (88,97%) HLA-DR iDC mono mDC CD83 iDC mono mDC HLA-DR / CD83 : mono (0,27%) iDC (1,45 %) mDC (85,72%) CD14 iDC mono mDC CD14/- : mono (90,11%) iDC (72,64 %) mDC (8,35%) monocytes  iDC  mDC monocyte marker CD14 DC differentiation marker HLA-DR / CD80 / CD83 / CD86 Tab.1 Patient’s characteristics: first treatment, disease stage, clinical and immunological responses abbreviations: CHT – chemotherapy, LN ‑ MS – lymph node metastases, LR – local recurrence, m – months, MS – metastases, NR – no response, OS ‑ MS – osseous metastases, PR – partial response, R – response, RAD – radiation, RPE – radical prostatectomy, SD – stable disease patientage first treatment (time before start of immunotherapy) current disease stage serum PSA at day –2 (in ng/ml) PSA-response & duration ELISPOT- response #157RAD ( ‑ 3 m)OS ‑ MS10.98NR # 270RPE ( ‑ 72 m) LR, LN ‑ MS, OS ‑ MS 7.94NR #367 no RPE or RAD, CHT ( ‑ 14 m) OS ‑ MS45.93 SD 5 weeks (days 26-64) R #470RAD ( ‑ 13 m)LN ‑ MS, OS ‑ MS70.15 PR 12 weeks (days 19- 105) R #565RPE ( ‑ 100 m)LR, OS ‑ MS14.16NRR #673RAD ( ‑ 80 m)OS ‑ MS68.68 SD 17 weeks (days 8-125) R #774RAD ( ‑ 22 m)no MS (cM0) 21.28 (day –8) NR #865no RPE or RADOS ‑ MS24.22 SD 4 weeks (days 8-36) NR monocytes iDCs mDCs (day 0)(day 6) (day 8) Fig.3 Course of DC differentiation and maturation Fig.2 Scheme of leukaphereses, immunomagnetic cell separations and DC vaccinations 1 st leukapheresis1 st CliniMACS 2 nd vaccination 1 st vaccination day -1 day 22 day 8day 0 2 nd leukapheresis2 nd CliniMACS 4 th vaccination 3 rd vaccination day 27 day 50 day 36day 28 Fig.1 Induction of cytotoxic T-cells by antigen-loaded DCs  organ-/tumor-specific over- expression of antigens (TAA)  presentation of TAA-derived peptides by MHC class I on DCs (green)  activation & expansion of cytotoxic T-cells (CTLs)  recognition of the same peptides on tumor cells  cytotoxic lysis of tumor cells