Dendritic Cell, the Immunotherapeutic Cell for Cancer

Dendritic cells play an important role in the development of effective cancer vaccines. These cells have the potential to present tumour-specific antigens and thereby induce an immune response. Various studies involving clinical trials have investigated the efficacy of administering antigen-loaded dendritic cells for cancer therapy. In order to design such experiments it is important to consider specific antigens, which initiate either a CD4+ or CDS+ response or both. The present review discusses the unique properties of dendritic cells as an immunotherapeutic cell for cancer.

Key words Dendritic cell – immunothcrapy – pcplidc – transfection – vaccine

The failure of conventional treatment for many forms of cancer has opened the doors for novel, experimental therapies. The first clinical trial with dendritic cell (DC) was started in the early 1990s though Steinman and Cohn discovered this cell in 1973′. The potency of the DC to present and sensitise the T cells has led to the development of effective vaccines. The first step in the induction of an effective anti-tumour therapy/vaccine is to identify a specific tumour antigen from an evergrowing list of tumour antigens. If a tumour antigen is presented to an antigen-presenting cell (APC), a more efficient and effective anti-tumour response is generated. Thus a critical target of vaccines would be APC, the most immunologically potent being the dendritic cell2,3.

DCs represent a heterogeneous cell population both phcnotypically and functionally, residing mostly in peripheral tissues where they represent 1-2 per cent of the total cell number4,5. DCs may be derived from either a pro-myeloid or a pro-lymphoid cell. In humans, two distinct subsets based on differential phenotype and function are, CD11c(-) plasmacytoid DCs (PDCs) and CD11c(+) myeloid DCs (MDCs)6. Myeloid DCs can be further subdivided into two fractions, one which is capable of differentiating into Langerhans cells (MDCl), and another, which is lackingthis ability (MDC2)7-9.

Morphologically, mature DCs are large cells with elongated and stellated processes. They express high levels of major histocompatibility complex (MHC) I and 11, CDIl a, b, c, CD40, CD54, CD58, CD80, CD83, CD86. The most typical markers at present arc MHC I, II and co-stimulatory markers such as CD80, CD86(10).

DCs are mainly localised in tissues and represent only a small proportion of less than 0.5 percent of peripheral blood leukocytes11. For therapeutic purposes, large numbers of DCs can be generated either from proliferating CD34+ bone-marrow precursor cells which differentiate under different cytokincs like IL-2. IL- 6, IL-7, IL-13, IL-15, hepatocyte growth factor (HGF), stem cell factor (SCF), granulocyte macrophage-colony stimulating factor (GM- CSF) or G-CSF, transforming growth factor-[beta] (TGF-[beta]) and tumour necrosis factor-7agr; (TNF-[alpha]) or from non- proliferating peripheral CD14+ cells (monocytes)12 Usually, CD34+ precursors are mobilized from bone marrow by G-CSF or GM-CSF and isolated by leucopheresis to obtain large number of peripheral cells for therapeutic purposes. The presence of FLT3-ligand (FLT3-L), SCF, and the differentiating growth factors GM-CSF and TNF-[alpha] leads to a sustained proliferation of CD34+ cells for long periods. These cells eem to be more efficient in the activation of tumourspecific cytotoxic T lymphocytes (CTLs) than CD14+ derived DCs13. Protocols for the generation of large number of monocyte-derived DCs exist since 1994 for both experimental and therapeutic purposes; different culture conditions have been reported14-16.

Briefly, the most common way of generation of DCs from CD14+ cells (monocytes) includes isolation of peripheral blood lymphocytes from buffy coats or from patients’ blood, by Ficoll-Hypaque densitycentrifugation17,18. The isolated adherent cells are cultured in RPMI 1640 with serum, GM-CSF and IL-4. Maturation of DCs has been achieved by using different cytokine cocktails19,20. Addition of cytokines like TNF[alpha] or IL-6 or IL-I[beta] or PgE2 and TNF- [alpha]21,23 or by culturing in monocyte conditioned medium elicits the required maturation signal.

Mature DCs are more potent in inducing Th1 and CTL responses in vitro. Immature DCs are not stable in vitro. They differentiate themselves to macrophages if the medium lacks GM-CSF and IL-424. After a week of culture, 25 to 50 per cent of the starting population differentiates into DCs which can be used for therapeutic purposes.

Selection of tumour antigen for puhing of dendritic cells: Antigens are either tumour-specific or tumourassociated antigens (TAA). They are used for pulsing in the form of peptides, tumour extracts, apoptotic bodies25 or nucleic acids26,27. Single immuno- reactive peptide has been used to a cocktail of peptides or tumour lysates. Tumour lysates deliver an entire gamut of antigenic peptides resulting in a multivalent immune response28. The possible occurrence of antigen-loss cell variants within the same tumour may restrict the applicability of tumour-associated antigens25. The disadvantages of using DCs pulsed with synthetic TAAs include the uncertainty regarding longevity of antigen presentation, and restriction imposed by the patients’ haplotype29. The selection of the peptide used for vaccination depends on the type of tumour, the HLA type of the patient and successful induction of CTL-response in vitro or in vivo, etc30. The right choice would be the antigen, which would be efficiently processed and expressed on the cell surface of a DC and elicit the activation of CTLs, which in turn would lead to a significant antitumour response31. Analysis of tumour-associated antigens has determined immunodominant peptides for some HLA types. Several groups have shown that human DCs when pulsed with synthetic peptides in vitro, can elicit strong antigenspecific CTL response32,33. In some cancers like pancreatic carcinoma, allogeneic tumour cells have been used as a source of antigens34. Though more than 200 tumour-associated epitopes recognise T cells, most of them do not elicit a strong immune response35.

Pulsing of DCs with small peptides is the easiest method of delivering antigen to immune cells. The first clinical study involved injecting monocyte derived DCs pulsed with idiotype protein derived from B cell lymphoma36. Since then there have been many clinical studies using peptide pulsed DCs37. In the first study, 16 patients with melanoma were injected with monocytederived DCs pulsed with tumour lysatc of or a cocktail of melanoma antigens consisting of MART, tyrosinase and GP 100(38). Other methods using nucleic acids or genes arc also being tried out30.

Nucleic acids in the form of DNA or RNA have been increasingly used to transfect DCs. DNA vaccination has become an attractive strategy since it induces both cellular and humoral immunity but it has a limited potency to induce immune response38. Using adenovirus- MART and alpha-fetoprotein constructs, DCs were tranduced effectively and strong CTL response was reported39,40. Others have investigated the potential of RNA to deliver antigen to DCs41,42.

Capture, processing, and presentation of an antigen by DC: In general, immature DCs take up and process the antigen whereas mature DCs neither have the antigen capturing ability northc processing mechanism. Antigens are internalised, processed on either a MHC class I or II pathway and presented as on the surface as a peptidcMHC complex43,44. Antigenic peptides (about 8-10 amino acids) are normally loaded directly on to the MHC-I molecule on the cell surface whereas proteins from tumour lysatc are internalized and then presented with the MHC H-molecule on the cell surface43,44. Cross-presentation is achieved when tumour lysates arc used for pulsing or co-culturing. This implies that pulsing with tumour lysate activates both CD4+ and CD8+ T-cells by the conventional MHC- M and MHC-I pathways, respectively. Macropinocytosis is the method of choice for most of the soluble antigens or it could be a receptor mediated phenomenon using mannose receptors, Fc[gamma]RI and Fc[gamma]RII or by phagocytosis’1. Any of these methods results in efficient capture of the antigens.

Introducing genes into dendritic cells: Unfortunately, the advantages of peptidc vaccines are to some extent diminished by their inherent lack of immunogenicity. The immune system in most species has evolved through time to fight life threatening infectious agents, it should not be surprising that vaccines consisting of aseptic, endotoxinfree peptides are likely to be ignored and ineffective at inducing T cell immunity2345. The use of gene transfer techniques might prove to be more effective and specific methods to generate tumour-specific T-cells46. In order to enhance the tumour response, DCs are modified by introducing genes like cytokine genes interleukin-7, GM-CSF, interleukin-12, interferon-gamma and intcrferon-alpha or genes coding for tumour antigen by viral or non-viral methods47″49. Cytokine genes in most instances enhance tumour immunogenicity.

Viral methods use attenuated forms of virus50. Nonviral methods like nucleofection, electroporation, lipofection and gene-gun method have been tried to induce a better gene inoculation and to achieve a better T cell response30. Viral vectors can disturb the function of DCs as antigen presenting cells. They induce death, interfere with \antigen presentation, or affect maturation of DC30. The use ot viral vectors to present an antigen will lead into a phenomenon known as immunological dominance30. In such situations, other antigenic peptides like viral antigens mask or suppress the response to the tumour-specific antigen or else the CTLs may recognise and kill the DCs expressing both viral and specific tumour antigens. Therefore, a viral vector, which has a very limited or no viral protein expression will be the vector of choice. Regardless of these problems, viral vectors which are replicative defective like El, E3- deleted, replication-deficient recombinant adenoviruses, have been used extensively in immunotherapy. Adenoviral vectors seem to provide a most efficient transfection. For retroviral vectors, a lesser transfection efficiency has been reported since they only infect dividing cells30. There are reports on the use of other viral vectors like adeno-associated viruses, herpes simplex virus, influenza virus, fowl pox virus, lentivirus, avipoxvirus or vaccinia virus for transfection51’52. Recombinant adenoviruses provide a less efficient immune response. The immunogcnicity to adenoviruses lias been a major obstacle for its application for long-term genetic modifications. However, for immunotherapy, short-lived genetic transduction could suffice for T cell priming. Adenoviral transfection involves creating AdV-poly-L-lysine (PLL)-DNA complexes5334. Our studies showed gene transfer efficiency up to 80 per cent (unpublished observations).

Nucleofection of DCs provides a non-viral method of introducing genes. DCs were transfected by nucleofection using the electroporation system nuclcofector from Amaxa Biosystems GmbH (Cologne, Germany) under various conditions. This technique combines special electrical parameters and cell type specific solutions to deliver the DNA directly to the cell nucleus under mild conditions. An efficiency of 40 per cent expression was achieved using this technique55. For a short-term T cell priming use of this technique would be useful.

Clinical !rials: Results of phase I and 11 clinical studies proved that DCs could be pulsed with tumour lysate, and immune response against metastatic renal carcinoma could be achieved36. Fifteen patients were treated with a median of 3.9510^sup 6^ DCs administered and ultrasoundguided into a lymph node or into adjacent tissue. Seven patients remained with progressive disease, 7 showed stable disease (SD), and one patient displayed a partial response (PR). CD3+CD4+ and CD3+CD28+cclls as well as the proliferation rate of peripheral blood lymphocytes (PBL) increased significantly in the blood of patients during therapy^.

In vitro data and results of a clinical phase I/ll trial using DC tumour fusions in patients with progressive metastatic renal cell carcinoma’^ demonstrated an increase in cytotoxicity of peripheral blood lymphocytes against renal cell carcinoma cells during treatment. DC precursor cells were obtained from the peripheral blood mononucluar cells ufhcalthy donurs and were fused with either allogencic (8 patients) or autologous (4 patients) renal tumour cells. In total, 12 patients with progressive metastatic renal cell carcinoma were treated with an average of 2.810^sup 7^ tumour cells fused with 1.81O^sup 7^ DC each administered on days O, 28, and 56 intradermally. Fusion efficacy for the tumour cells used was 14.37.8 per cent. Cell viability was 59.86.8 per cent after fusion and irradiation. No adverse effects were observed and no difference in clinical outcome between the allogeneic and the autologous treatment was found. Eight patients remained in a progressive disease state and four in a stable disease state. The lack of adverse effects together with positive immunologie signs justifies further investigation of this novel therapeutic approach57.

The latest review of DC clinical trials is described on http:// www.mmri.mater.org.aU/pages/c I in ical_trial s/ ctu_table_v8.htm.

Future prospects’. The unique ability of DCs to induce and sustain primary immune responses makes them optimal candidates for vaccination protocols in cancer11-’0,5658-60. DCs definitely are the nature’s adjuvant for immune resistance and play a key role in immunity. Vaccine design extends beyond the identification of antigens. Vaccines with DC may harness the immunological mechanisms that lead to a strong and lasting immunity61. There is concern regarding development of unwanted immunity in cancer immunotherapy. Normal tissues expressing the tumour antigen may be harmed immunologically. Preventing immune escape, for example preventing immimosuppressive cytokines, and preventing recurrence of tumour lesions are important in successful immunotherapy62. With new methods of immunomonitoring and improved protocols of cancer immunotherapy, dendritic cell therapy most likely, will have an edge over the other modes of therapy.

References

1. Steinman RM. Cohn ZA. Identification of a novel cell type in peripheral Iymphoid organs of mice. I. Morphology, quanlilation. tissue distribution. J Exp Med 1973; 137 : 1142-62.

2. Shurin MR. Dendritic cells presenting tumor antigen. Cancer lmmunol Imniunolher 1996; -/J : 158-64.

3. Rcay PA. Dendritic cells: immunological features and utilisation for tumour immunotherapy. Expert Opin Ther Targets 2001; 5 491-506.

4. Banchcrcau J, Steinman RM. Dendritic cells and the control of immunity. Nature 1998; 392 : 245-52.

5. Banchercau .1. Pac/csny S, Blanco P, Bennett L, Pascual V, l;ay J, et o/. Dendritic cells: controllers of the immune system and a new promise for immunotherapy. Ann N Y AcadSci 2003 ; 967: 180-7.

6. lto I. Amakawa R, Kaisho T, Hemmi H, Tajima K, lieh ira K, el al. Intcrfcron-alpha and intcrleukin-12 are induced differentially by ‘lull-like receptor 7 ligands in human blood dendritic cell subsets../ /Ur/) Med 2002; /PJ : 1507-12.

7. Dzionck ?, Fuchs ?, Schmidt P, Creme r S, Zysk M, Miltenyi S, ei al. BDCA-2, BDCA-3, und BDCA-4: three markers for distinct subsets of dendritic cells in human peripheral blood. JImmwwl 2000; / 6J : 6037-46.

8. Dzionck A, lnagaki Y, Okawa K, Nagalune J, Rock J, Sohma Y, et al. Plasmacyloid dendritic cells: from specific surface markers of specific cellular functions(l). I him !inmuno/2002: 63 : 1133-48.

9. lto T, lnaba M. lnaba K, Toki J, Sogo S. Iguchi T, Adachi Y, et al. ACDla+/CDl Ic+ subset of human blood dendritic cells is a direct precursor of Langcrhans cells. J Immunol 1999; /63 : 1409- 19.

10. Pong L, Engleman EG. Dendritic cells in cancer immunotherapy. Annu Rev Immunol 20OQ: 18: 245-73.

11. Marten A, Gretcn T, Ziske C, Rcnoth S. Schottker B, Buttgercil P, el al. Generation of activated and antigen-specific T cells with cyloxic activity after co-culture with dendritic cells. Cancer Immunol lmmunother 2002; 51 : 25-32.

12. Zhang W, Chcn Z, Li I, Kamencic H, juurlink B, Gordon JR, el al. Tumour necrosis factor-alpha (‘FNF-alpha) transgenecxpressing dendritic cells (DCs) undergo augmented cellular maturation and induce more robust T-cell activation and antitumour immunity than DCs generated in recombinant TNF-alpha. Immunology 2Q03: 108 : 177- 88.

13. Mortarini R, Anichini A, Di Nicola M, Siena S, Bregni M, Belli F, et al. Aulologous dendritic cells derived from CD34+ progenitors and from UiOIiOC)1IeS are not functionally equivalent antigen-presenting cells in the induction of melanA/Mart-l(27-35)- .spccific CTLs from peripheral blood lymphocytes of melanoma patients with low frequency of CTL precursors. Cancer Rex 1997; 57 : 5534-41.

14. Sallusto F, Lanzavccchia A. Efficient presentation of soluble antigen by cultured human dendritic cells is maintained by granulocyle/macrophagc colony-stimulating factor plus interleukin 4 and dovvnregulated by tumor necrosis factor alpha. JExp Med 1994; 179 : 1 109-18.

15. Zhou LJ, Tedder TF. CD 14+ blood monocytes can differentiate into functionally mature CD83-I- dendritic cells. I’roc NallAcad Sd USA 1996; 93: 2588-92.

16. Bender A, Sapp M. Schuler G, Sleinman RM, Bhardwai N. Improved methods for the generation of dendritic cells from nonprolifcrating progenitors in human b\ooa.J Immunol Methods 1996; 196: 121-35.

17. Romani N, Reidcr D. l leuer M, Ebner S. Kampgen E, Eibl B, et al. Generation of mature dendritic cells from human blood. An improved method with special regard to clinical applicability. J Immunol Methods 1996; 196: 137-51.

18. Romani N, Gruner S, Brang D, Kampgen E. Lenz A, Trockcnbacher B, et al. Proliferating dendritic cell progenitors in human blood. .1 Exp Med 1994; 180 : 83-93.

19. Lutz MB, Schnare M, Menges M, Rossner S, Rollinghoff M, Schuler G, et al. Differential functions of 1L-4 receptor types I and II for dendritic cell maturation and IL-12 production and their dependency on GM-CSK J Immunol 2002; 769 : 3574-80.

20. Berger TG. Feuerstein B, Strasscr E, Hirsch U, Schreiner D, Schulder G, et a/. Large-scale generation of mature monocytcderived dendritic cells for clinical application in cell factories. J Immunol Methods 2002; 268 : 131-40.

21. Nestle FO, Banchereau J, Hart D. Dendritic cells: On the move from bench to bedside. Nature Med2QO; 7:761-5.

22. Nestle FO. Dendritic cell vaccination for cancer therapy. Oncogens 2000; 19 : 6673-9.

23. Cella M, Sallusto F, Lanzavccchia ?. Origin, maturation and antigen presenting function of dendritic cells. Curr Opin Immunol 1997; 9: 10-6.

24. Palucka KA. Taquet N, Sanchez-Chapuis F. Gluckman JC. Dendritic cells as the terminal stage of monocyte differentiation. J Immunol 1998; /60 : 4587-95.

25. Gregoire M, Ligcza-Poisson C, jugc-Morineau N, Spisck R. Anti- cancer therapy using dendritic cells and apoptotic tumour cells: pre- clinical data in human mesothelioma and acute myeloid leukaemia. Vaccine 2003; 21 : 791-4.

26. Pawelec G, Engel ?, Adibzadeh M. Prerequisites for the immunotherapy of cancer. Cancer Immunol lmmunolher 1999; Jg: 214-7.

27. Gunzcr M, J an ich S, Varga G, G\rabbc S. Dendritic cells and tumor immunity. Semin Immunol 2001 ; /3 : 291-302.

28. Thumann P, Moc I, Uumrich J, Berger TG, Schultz ES, Schulder G. Antigen loading ofdendritic cells with whole tumor cell preparations. JImmunol Methods 2003; 277 : 1-16.

29. Stingl G, Bergstresscr PR. Dendritic cells: a major story unfolds. Immunol Today 1995; 16 : 330-3.

30. Reinhard G, Marten A, Kiske SM, Feil F, Bicbcr T, Schmidt- Wolf IG. Generation of dendritic cell-based vaccines for cancer therapy. Br J Cancer 2002; 86 : 1529-33.

31. Armstrong AC, Eaton D, Ewing JC. Science, medicine, and the future: Cellular immunotherapy for cancer. /J/WJ 2001; 323 : 1289- 93.

32. Murphy GP, Tjoa BA, Simmons SJ, Ragde II, Rogers M, Elgamal A, et ?/. Phase Il prostate cancer vaccine trial: report of a study involving 37 patients with disease recurrence following primary treatment. Prostate 1999; 39 : 54-9.

33. Murphy GP, Tjoa BA, Ragde II, Kenny G, Boynton A. Phase I clinical trial: T-cell therapy for prostate cancer using autologous dendritic cells pulsed with HLA-A0201-specific peptidcs from prostate-specific membrane antigen. Prostate 1996; 29: 371-80.

34. Stift A, Fricdl J, Dubsky P, Bachleitner-Hofmann T, Bcnkoc T, Brostjan C. In vivo induction of dendritic cell-mediated cytotoxicity against allogeneic pancreatic carcinoma cells. Int.I Oncol 2003; 22 : 651-6.

35. Renkvist N, Castelli C, Robbins PF, Parmiani G. A listing of human tumor antigens recognized by T cells. Cancer Immunol Immunother20Ql;50: 3-15.

36. Onaitis M, KaInJy M ?, Pruitt S, Ilcr I)S. Dendritic cull gene therapy. ,SVg Oncol Clin N Am 2002; 11 : 645-60.

37. Berger TG, Schnitz ES. Dendritic cell-based immunotherapy. Cui-r Top Micmbiol Immunot 2003; 276 : 163-97.

38. You Z, I-Iuang X, Hester J, Toh UC. Chen SY. Targeting dendritic cells to enhance DNA vaccine potency. Cancer /tes 2001; 67 : 3704-11.

39. Butterfield LH, Jilani SM, Chakraborty NCi. Bui LA, Ribas A, Disette VB. Generation of melanoma-specific cytotoxic T lymphocytes by dendritic cells transduced with a MART-1 adenovirus. Jlmmunol 1998; 76/ : 5607-13.

40. Butterfield LH. Koh A. Meng W, Vollmer CM, Ribas A, Disette VB. Generation of human T-cell responses to an HLA-A2. 1 – restricted peplide cpitope derived from aplha-fetoprotein. Cancer Res 1999; 59 : 3134-42.

41. Nair SK, Morse M, Boc/.kowski D, Gumming RL, Vasovic L, Gilboa E. Induction of tumor-specific cytoloxic T lymphocytes in cancer patients by autologous tumor RNA-transfectecl dendritic cells. BA Gibola; Surg 2002; 235 : 540-9.

42. Sullengcr BA, Gibola E. Emerging clinical applications of RNA. Nature 2002; 418: 252-8.

43. Sleinman RM. The dendritic cell system and its role in immunogenicity. Annu Rev lmmunol 1991 ; 9 : 271 -96.

44. Rcid DC. Dendritic cells and immunotherapy for malignant disease. BrJHaemalol200\; 112 : 874-87.

45. Celis E. Getting peptidc vaccines to work; just a matter of quality control? J Clin Invest 2002; 110 : 1765-8.

46. Terando A, Chang AE. Applications of gene transfer to cellular immunotherapy. Surg Oncol CUn N Am 2002; 11 : 621-43.

47. Engleman EG. Dendritic cell-based cancer immunotherapy. Semin Oncol 2003; 30: 23-9.

48. Schmidt-Wolf GD, Schmidt- WoIfIGl I. Non-viral and hybrid vectors in human gene therapy: an update. Trends MoI Med 2003; 9: 67- 72.

49. Pecher G. DNA-based tumor vaccines. Onkologie 2002; 25 : 528- 32.

50. Humrich J, Jennc L. Viral vectors for dendritic cell-based immunotherapy. Curr Top Microbiol lmmunol 2003; 270 : 241-59.

51. Rosenberg SA. Progress in human tumour immunology and immunotherapy Nature 2001; 411 : 380-4.

52. Jenne L, Schuler G, Steinkasserer A. Viral vectors for dendritic cell-based immunotherapy. Trends lmmunol 2001 22 : 102-7.

53. Cristiano RJ. Smith LC, Kay MA, Brinkley BR, Woo SL. Hepatic gene therapy; efficient gene delivery and expression in primary hepalocytcs utili/.ing a conjugated adenovirus-DNA complex. ProcNatlAcadSci USA 1993; 90 : 11548-52.

54. Mulders P, Pang S, Dannull J, Kaboo R, Hinkcl A. Michel K, el al. I lighly efficient and consistent gene transfer into dendritic cells utilizing a combination of ultraviolet-irradiated adcnovirus and polv(I,-lysine) conjugates. Cancer Res 1998; 58 : 956-61.

55. Srinivas N. jucrgen Neurnan, Koch N, Schmidt-Wolf IGI-I. Class II associated invariant chain – C2GnT epitope against pancreatic carcinoma. MoI Ther 2003: 7 : S270.

56. Marten ?. Flieger D, Renoth S. Weineck S, Albers P, Compes M. Therapeutic vaccination against mctastatic renal cell carcinoma by autologous dendritic cells: preclinical results and outcome of a first clinical phase I/II trial. Cancer lmmunol lmmunother 2002: Sl : 637-44.

57. Marten A. Renoth S. Heinicke T, Albers P, Pauli A, Mcy U. Allogeneic dendritic cells fused with tumor cells: preclinical results and outcome of a clinical phase I/II trial in patients metaslatic renal cell carcinoma. Hum Gene Ther 2003; 14 : 483-94.

58. Lemoli RM, Curti A, Fogli M, Fcrri I. Baccarani M. The therapeutic role of dendritic cells in cancer immunolherapy. Ilaemalologica 2002; 87 : 62-6.

59. Satthaporn S, Bremin O. Dendritic cells (II): Role and therapeutic implications in cancer../R CollSnrg Edinh 2001:46: 159- 67.

60. Marten A, Ziske C, Schottker B, Renoth S. Wcineck S, Buttgereit P. Increase in the inimunosliimilatory effect of dendritic cells by pulsing with serum derived from pancreatic and colorcctal cancer patients, hit J (Oloreckil Dis 2000; 15 : 197- 205.

61. Armstrong AC. Katon D, Ewing JC. Cellular vaccine therapy for cancer. Expert Rev !”ciccincs 2002; / : 303-16.

62. Cohcn IC P. DNA-hascd vaccines for the treatment of cancer – an experimental mode, ‘/’rends MoI Med 200 1 ; 7: 175-9.

S. Nagaraj, C. Ziske & I.G.H. Schmidt-Wolf

Department of Internal Medicine I, Rheinische Friedrich-Wilhelms- Universitat, Bonn, Germany

Received February 19.2003

Reprint requests : Prof. Dr I.G.11. Schmidt-Wolf, Medizinische Universitasklinik und Poliklinik l Sigmund Freud Str. 25. 53 105 Bonn, Germany

e-mail: picasso@uni-bonn.de

Copyright Indian Council of Medical Research Apr 2004