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 & 2nd 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 & MethodsPatients:• 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60%, PSA 5-150ng/ml (increase of ≥20% within 2 months)• anti-androgens withdrawn 4 weeks before the start, LHRH agonists continuedReagents, cytokines, peptides:• all reagents (cytokines, peptides, buffers, media) were sterile,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., 1st & 3rd vacc. (days 8 & 36) with freshly cultivated and

matured DCs (mDCs), 2nd & 4th 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: 1.0‑1.2x109 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 1x107 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‑DRClinical 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 1st vacc. on day 8, or when highest PSA values were measuredIFN‑ 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 1x105 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/1x105 cells in all experiments) from mean spot counts for each TAA-derived peptide

Vaccination of hormone-refractory prostate cancer patients with peptide cocktail-Vaccination of hormone-refractory prostate cancer patients with peptide cocktail-loaded dendritic cells: results of a Phase I clinical trialloaded dendritic cells: results of a Phase I clinical trial

A. MeyeA. Meye11, S. Fuessel, S. Fuessel11, M. Schmitz, M. Schmitz22, S. Zastrow, S. Zastrow11, C. Linné, C. Linné11, K. Richter, K. Richter11, B. Lobel, B. Lobel22, O.W. Hakenberg, O.W. Hakenberg11, K. Hoelig, K. Hoelig33, E.P. Rieber, E.P. Rieber22, M.P. Wirth, M.P. Wirth11

11 Department of Urology, Department of Urology, 22 Institute of Immunology, Institute of Immunology, 33 Institute of Transfusion Medicine, Medical Faculty, Technical University Dresden, Germany Institute of Transfusion Medicine, Medical Faculty, Technical University Dresden, Germany

peptide code peptide sequence position reference

PSA3 154-163 VISNDVCAQV 154-163 Correale et al. 1997

PSMA1 4-12 LLHETDSAV 4-12 Tjoa et al. 1996, Murphy et al. 1996

prostein 31-39 CLAAGITYV 31-39 Kiessling et al. 2004

survivin 95-104 ELTLGEFLKL 95-104 Schmitz et al. 2000, Andersen et al. 2001

trp‑p8 187-15 GLMKYIGEV 187-195 Kiessling et al. 2003

ResultsImmunomagnetic enrichment of monocytes and cultivation of DCs

• leukaphereses: mean total numbers of 20.9x109 leukocytes (median 22.1x109)

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 1.0‑1.2x109 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 2nd 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

ConclusionsThese 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 respondersa) partial responder (#4) with a PSA decrease within 7 weeks and further PSA stabilization for 5 weeksb) patients (#3, #6, #8) with stable PSA values or decelerated PSA increases (stable disease)Black arrows indicate the DC vaccinations.

a)

b)

0

20

40

60

80

100

-80 -60 -40 -20 0 20 40 60 80 100 120 140

day

PS

A (

ng

/ml)

# 6: 17 weeks

# 3: 5 weeks

# 8: 4 weeks

0

20

40

60

80

100

-80 -60 -40 -20 0 20 40 60 80 100 120 140

day

PS

A (

ng

/ml)

# 4: 12 weeks

Fig.6 Detection of tumor peptide-reactive CD8+ T‑cells by IFN‑ ELISPOT analysisPurified 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.

0

20

40

60

80

100

120

140

160

180

200

# 3 # 4 # 5 # 6patient

sp

ots

/ 5

x 1

05 CD

8+

T c

ells

PSA

PSMA

trp-p8

prostein

survivin

* * * * * * * * * * * * *

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 (2nd leukapheresis).

CD86CD86

iDCmono

mDC

CD80CD80

iDCmono

mDC

CD86/CD80: mono (0,39%)

iDC (0,87 %)

mDC (88,97%)

HLA-DRHLA-DR

iDCmono

mDC

CD83CD83

iDCmono mDC

HLA-DR / CD83: mono (0,27%)

iDC (1,45 %)

mDC (85,72%)

CD14CD14

iDCmono

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

patient age first treatment(time before start

of immunotherapy)

current disease stage

serum PSAat day –2(in ng/ml)

PSA-response

& duration

ELISPOT-

response

#1 57 RAD (‑3 m) OS‑MS 10.98 NR NR

# 2 70 RPE (‑72 m) LR, LN‑MS, OS‑MS

7.94 NR NR

#3 67 no RPE or RAD,CHT (‑14 m)

OS‑MS 45.93SD

5 weeks (days 26-64)R

#4 70 RAD (‑13 m) LN‑MS, OS‑MS 70.15PR

12 weeks (days 19-105)

R

#5 65 RPE (‑100 m) LR, OS‑MS 14.16 NR R

#6 73 RAD (‑80 m) OS‑MS 68.68SD

17 weeks (days 8-125)R

#7 74 RAD (‑22 m) no MS (cM0) 21.28(day –8)

NR NR

#8 65 no RPE or RAD OS‑MS 24.22SD

4 weeks (days 8-36)NR

monocytes iDCs mDCs (day 0) (day 6) (day 8)

Fig.3 Course of DC differentiation and maturationFig.2 Scheme of leukaphereses, immunomagnetic cell separations and DC vaccinations

1st leukapheresis 1st CliniMACS

2nd vaccination

1st vaccination

day -1

day 22

day 8day 0

2nd leukapheresis 2nd CliniMACS

4th vaccination

3rd 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