Abstract
Background: Children and adolescents with primary multifocal, refractory or relapsed malignant
extracranial solid tumors still have a poor prognosis inspite of intensive standard
radio-/chemotherapy. Here complementary immunomodulatory treatment modalities may
prove beneficial as consolidation therapy following cytoreduction. Neuroblastoma,
Ewing tumor and soft tissue sarcoma cells have principally been shown to be susceptible
towards both cytotoxic and humoral effector mechanisms. Yet in vivo they are not capable of inducing an effective antitumor response which has been
attributed to low level MHC expression and lack of costimulatory surface molecules.
Professional antigen - presenting cells such as dendritic cells (DCs) in contrast
are capable of activating unprimed T cells and are therefore ideal tools for vaccine
generation.
Results: Here we demonstrate that DCs may be generated from CD34+ progenitor cells to clinical
scale in a three to four week cell culture process including an initial expansion
and subsequent differentiation and maturation steps. DCs derived from CD34+ progenitors
express the expected marker profile and are highly effective in stimulating allogeneic
T cell effectors. We also demonstrate that they effectively take up fluorescence-labelled
tumor cell lysate.
Discussion: Having established a cell culture process for clinical scale DC production utilizing
CD34+ progenitors as the cellular source we discuss the role of CD34+ derived DCs
in clinical vaccination protocols. The rationale for a phase I/II DC dose escalation
study for high risk pediatric patients with extracranial solid tumors assessing safety,
immunological and clinical efficacy of serial combined intranodal and subcutaneous
injections of tumor cell lysate-pulsed autologous DCs is delineated.
Zusammenfassung
Hintergrund: Kinder und Jugendliche mit primär metastasierten, therapierefraktären oder rezidivierten
malignen extrakranialen Tumoren haben trotz intensiver Strandard-Radio-/Chemotherapie
eine schlechte Prognose. Hier bieten sich ergänzend immuntherapeutische Behandlungsstrategien
an, die im Sinne einer immunologischen Konsolidierung nach zytoreduktiver Therapie
eingesetzt werden können. Sowohl Neuroblastome als auch Ewing-Tumoren und Weichteilsarkome
haben sich gegenüber zytotoxischen und humoralen immunologischen Effektormechanismen
als sensitiv erwiesen. Jedoch sind diese Tumoren nicht in der Lage, in vivo eine effektive anti-tumorspezifische Immunantwort zu induzieren. Diese reduzierte
Immunogenität wird auf die niedrige Expression von MHC- und kostimulatorischen Oberflächenmolekülen
zurückgeführt. Professionelle antigenpräsentierende Zellen wie dendritische Zellen
(DZ) sind dagegen in besonderer Weise befähigt, zytotoxische T-Zellen zu aktivieren,
und eignen sich daher in besonderem Maße zur Herstellung einer Tumorvakzine.
Ergebnisse: Wir zeigen, dass sich DZ in klinischem Maßstab aus CD34+-Vorläuferzellen innerhalb
von 3-4 Wochen in Zellkultur generieren lassen. Nach einer initialen Expansion der
Zellen wird durch Zugabe verschiedener Zytokine deren Differenzierung und schließlich
Ausreifung bewirkt. Die so gewonnenen DZ exprimieren die charakteristischen Marker
und kostimulatorischen Moleküle auf ihrer Oberfläche und weisen eine hohe T-Zell-stimulatorische
Kapazität auf. Auch hat sich zeigen lassen, dass aus CD34+-Progenitoren generierte
DZs fluoreszenzmarkierte Proteine bzw. Tumorzell-Lysat aufnehmen können.
Diskussion: Nach Etablierung eines Herstellungsprozesses von DZ wird der klinische Einsatz einer
von CD34+-Vorläuferzellen abgeleiteten dendritischen Zellvakzine diskutiert. Auf der
Grundlage der gewonnenen Ergebnisse wird die Rationale für die Entwicklung einer Phase-I/II-Studie
für pädiatrische Hochrisikopatienten mit malignen extrakranialen soliden Tumoren dargestellt.
Ziel dieser Dosisfindungsstudie ist, die Sicherheit sowie immunologische und klinische
Wirksamkeit einer seriellen Applikation von Tumorzell-Lysat-beladenen DZ zu untersuchen.
Key words
CD34+ derived DCs - vaccine - tumor lysate - solid tumors
Schlüsselwörter
DZ aus CD34+-Progenitoren - Vakzine - Tumorlysat - solide Tumoren
References
1
Matthay K K, Villablanca J G, Seeger R C, Stram D O, Harris R E, Ramsay N K, Swift P,
Shimada H, Black C T, Brodeur G M, Gerbing R B, Reynolds C P. Children's Cancer Group
.
Treatment of high-risk neuroblastoma with intensive chemotherapy, radiotherapy, autologous
bone marrow transplantation, and 13-cis-retinoic acid.
N Engl J Med.
1999;
341
1165-1173
2
Burdach S, Meyer-Bahlburg A, Laws H J, Haase R, van Kaik B, Metzner B, Wawer A, Finke R,
Gobel U, Haerting J, Pape H, Gadner H, Dunst J, Juergens H.
High-dose therapy for patients with primary multifocal and early relapsed Ewing's
tumors: results of two consecutive regimens assessing the role of total-body irradiation.
J Clin Oncol.
2003;
21
3072-3078
3
Koscielniak E, Harms D, Henze G, Jurgens H, Gadner H, Herbst M, Klingebiel T, Schmidt B F,
Morgan M, Knietig R, Treuner J.
Results of treatment for soft tissue sarcoma in childhood and adolescence: a final
report of the German Cooperative Soft Tissue Sarcoma Study CWS-86.
J Clin Oncol.
1999;
17
3706-3719
4
Koscielniak E, Handgretinger R, Dilloo D, Rosti G, Niethammer D, Treuner J, Klingebiel T.
Definition of the high risk patients: the EBMT-CWS experience and CWS proposal for
a therapeutic strategy for high risk sarcoma patients.
Bone Marrow Transplant.
2002;
30
33
5
Frohlich B, Ahrens S, Burdach S, Klingebiel T, Ladenstein R, Paulussen M, Zoubek A,
Jurgens H.
High-dosage chemotherapy in primary metastasized and relapsed Ewing’s sarcoma. (EI)CESS.
Klin Pädiatr.
1999;
211
284-290
6
Rosenberg S A.
A new era for cancer immunotherapy based on the genes that encode cancer antigens.
Immunity.
1999;
10
281-287
7
Soling A, Schurr P, Berthold F.
Expression and clinical relevance of NY-ESO-1, MAGE-1 and MAGE-3 in neuroblastoma.
Anticancer Res.
1999;
19
2205-2209
8
Delattre O, Zucman J, Melot T, Garau X S, Zucker J M, Lenoir G M, Ambros P F, Sheer D,
Turc-Carel C, Triche T J. et al .
The Ewing family of tumors - a subgroup of small-round-cell tumors defined by specific
chimeric transcripts.
N Engl J Med.
1994;
331
294-299
9
Galili N, Davis R J, Fredericks W J, Mukhopadhyay S, Rauscher F J, Emanuel B S, Rovera G,
Barr F G.
Fusion of a fork head domain gene to PAX3 in the solid tumour alveolar rhabdomyosarcoma.
Nat Genet.
1993;
5
230-235
10
Handgretinger R, Anderson K, Lang P, Dopfer R, Klingebiel T, Schrappe M, Reuland P,
Gillies S D, Reisfeld R A, Niethammer D.
A phase I study of human/mouse chimeric antiganglioside GD2 antibody ch14.18 in patients
with neuroblastoma.
Eur J Cancer.
1995;
2
261-267
11
Foreman N K, Rill D R, Coustan-Smith E, Douglass E C, Brenner M K.
Mechanisms of selective killing of neuroblastoma cells by natural killer cells and
lymphokine activated killer cells. Potential for residual disease eradication.
Br J Cancer.
1993;
67
933-938
12
Dilloo D, Laws H J, Hanenberg H, Korholz D, Nurnberger W, Burdach S E.
Induction of two distinct natural killer-cell populations, activated T cells and antineoplastic
cytokines, by interleukin-2 therapy in children with solid tumors.
Exp Hematol.
1994;
22
1081-1088
13
Mackall C, Berzofsky J, Helman L J.
Targeting tumor specific translocations in sarcomas in pediatric patients for immunotherapy.
Clin Orthop.
2000;
373
25-31
14
Banchereau J, Steinman R M.
Dendritic cells and the control of immunity.
Nature.
1998;
392
245-252
15
Ferlazzo G, Klein J, Paliard X, Wei W Z, Galy A.
Dendritic cells generated from CD34+ progenitor cells with flt3 ligand, c-kit ligand,
GM-CSF, IL-4, and TNF-alpha are functional antigen-presenting cells resembling mature
monocyte-derived dendritic cells.
J Immunother.
2000;
23
48-58
16
Pullarkat V, Lau R, Lee S M, Bender J G, Weber J S.
Large-scale monocyte enrichment coupled with a closed culture system for the generation
of human dendritic cells.
J Immunol Methods.
2002;
267
173-183
17
Sorg R V, Ozcan Z, Brefort T, Fischer J, Ackermann R, Muller M, Wernet P.
Clinical-scale generation of dendritic cells in a closed system.
J Immunother.
2003;
26
374-383
18
Nestle F O, Alijagic S, Gilliet M, Sun Y, Grabbe S, Dummer R, Burg G, Schadendorf D.
Vaccination of melanoma patients with peptide- or tumor lysate-pulsed dendritic cells.
Nat Med.
1998;
4
328-332
19
Mackensen A, Herbst B, Chen J L, Kohler G, Noppen C, Herr W, Spagnoli G C, Cerundolo V,
Lindemann A.
Phase I study in melanoma patients of a vaccine with peptide-pulsed dendritic cells
generated in vitro from CD34(+) hematopoietic progenitor cells.
Int J Cancer.
2000;
86
385-392
20
Banchereau J, Palucka A K, Dhodapkar M, Burkeholder S, Taquet N, Rolland A, Taquet S,
Coquery S, Wittkowski K M, Bhardwaj N, Pineiro L, Steinman R, Fay J.
Immune and clinical responses in patients with metastatic melanoma to CD34(+) progenitor-derived
dendritic cell vaccine.
Cancer Res.
2001;
61
6451-6458
21
Hsu F J, Benike C, Fagnoni F, Liles T M, Czerwinski D, Taidi B, Engleman E G, Levy R.
Vaccination of patients with B-cell lymphoma using autologous antigen-pulsed dendritic
cells.
Nat Med.
1996;
2
52-58
22
Schott M, Feldkamp J, Lettmann M, Simon D, Scherbaum W A, Seissler J.
Dendritic cell immunotherapy in a neuroendocrine pancreas carcinoma.
Clin Endocrinol (Oxf).
2001;
55
271-277
23
Chang A E, Redman B G, Whitfield J R, Nickoloff B J, Braun T M, Lee P P, Geiger J D,
Mule J J.
A phase I trial of tumor lysate-pulsed dendritic cells in the treatment of advanced
cancer.
Clin Cancer Res.
2002;
8
1021-1032
24
Marten A, Flieger D, Renoth S, Weineck S, Albers P, Compes M, Schottker B, Ziske C,
Engelhart S, Hanfland P, Krizek L, Faber C, von Ruecker A, Muller S, Sauerbruch T,
Schmidt-Wolf I G.
Therapeutic vaccination against metastatic renal cell carcinoma by autologous dendritic
cells: preclinical results and outcome of a first clinical phase I/II trial.
Cancer Immunol Immunother.
2002;
51
637-644
25
Holtl L, Zelle-Rieser C, Gander H, Papesh C, Ramoner R, Bartsch G, Rogatsch H, Barsoum A L,
Coggin J H, Thurnher M.
Immunotherapy of metastatic renal cell carcinoma with tumor lysate-pulsed autologous
dendritic cells.
Clin Cancer Res.
2002;
8
3369-3376
26
Chakraborty N G, Sporn J R, Tortora A F, Kurtzman S H, Yamase H, Ergin M T, Mukherji B.
Immunization with a tumor-cell-lysate-loaded autologous-antigen-presenting-cell-based
vaccine in melanoma.
Cancer Immunol Immunother.
1998;
47
58-64
27
Geiger J, Hutchinson R, Hohenkirk L, McKenna E, Chang A, Mule J.
Treatment of solid tumours in children with tumour-lysate-pulsed dendritic cells.
Lancet.
2000;
356
1163-1165
28
Geiger J D, Hutchinson R J, Hohenkirk L F, McKenna E A, Yanik G A, Levine J E, Chang A E,
Braun T M, Mule J J.
Vaccination of pediatric solid tumor patients with tumor lysate-pulsed dendritic cells
can expand specific T cells and mediate tumor regression.
Cancer Res.
2001;
61
8513-8519
29
Di Nicola M, Carlo-Stella C, Anichini A, Mortarini R, Guidetti A, Tragni G, Gallino F,
Del Vecchio M, Ravagnani F, Morelli D, Chaplin P, Arndtz N, Sutter G, Drexler I, Parmiani G,
Cascinelli N, Gianni A M.
Clinical protocol. Immunization of patients with malignant melanoma with autologous
CD34(+) cell-derived dendritic cells transduced ex vivo with a recombinant replication-deficient
vaccinia vector encoding the human tyrosinase gene: a phase I trial.
Hum Gene Ther.
2003;
14
1347-1360
30
Banchereau J, Paczesny S, Blanco P, Bennett L, Pascual V, Fay J, Palucka A K.
Dendritic cells: controllers of the immune system and a new promise for immunotherapy.
Ann N Y Acad Sci.
2003;
987
180-187
31
Morse M A, Coleman R E, Akabani G, Niehaus N, Coleman D, Lyerly H K.
Migration of human dendritic cells after injection in patients with metastatic malignancies.
Cancer Res.
1999;
59
56-58
32
Timmerman J M, Czerwinski D K, Davis T A, Hsu F J, Benike C, Hao Z M, Taidi B, Rajapaksa R,
Caspar C B, Okada C Y, van Beckhoven A, Liles T M, Engleman E G, Levy R.
Idiotype-pulsed dendritic cell vaccination for B-cell lymphoma: clinical and immune
responses in 35 patients.
Blood.
2002;
99
1517-1526
33
Roskrow M A, Dilloo D, Suzuki N, Zhong W, Rooney C M, Brenner M K.
Autoimmune disease induced by dendritic cell immunization against leukemia.
Leuk Res.
1999;
23
549-557
34
Asavaroengchai W, Kotera Y, Mule J J.
Tumor lysate-pulsed dendritic cells can elicit an effective antitumor immune response
during early lymphoid recovery.
Proc Natl Acad Sci USA.
2002;
99
931-936
D. Dilloo
Clinic for Pediatric Oncology, Hematology and Immunology
University Clinic
Heinrich Heine University Düsseldorf
Germany