Radiol Oncol 2006; 40(2): 95-105.
review
Complement resistance impairs anti-tumour therapy
Thomas Konatschnig, Nicolas Geis, Stefan Schultz, Michael Kirschfink
Institute of Immunology, University of Heidelberg, Germany
Background. Various studies during the last two decades clearly indicate that resistance of human tumour cells to autologous complement is mainly based on the expression of membrane-bound complement regula-tory proteins (mCRP) like CD59, CD55 and CD46 with good evidence for a predominant role of CD59. Be-yond these in vitro findings the importance of this phenomenon for the patients` outcome now becomes evident from first clinical studies. Overcoming complement resistance of tumour cells is therefore considered a promising way to improve therapeutic options and prognosis in a variety of cancer diseases. In this short review two feasible approaches are discussed in more detail: (1) neutralisation of mCRP by monoclonal or recombinant antibodies and (2) gene silencing strategies to down-regulate mCRP by blocking the expression of these proteins on the RNA level using siRNA.
Conclusions. As mCRP are also present on all normal tissues like endothelial cells, parenchymatous organs (liver, kidney etc.) or blood cells, mCRP blocking strategies have to be targeted selectively to malignant cells sparing the surrounding healthy tissues from the deleterious complement attack. Despite first encouraging results, translation of mCRP inhibition to improve antibody-based immunotherapy into the clinic is still a great challenge.
Key words: neoplasms – drug therapy; immunology; complement inactivators
Introduction
The complement system is a cascade of serin proteases that plays an important role in the immune defense, linking innate and ac-quired immunity.1 Activation of comple-ment results in the release of highly potent proinflammatory molecules, the so-called anaphylatoxins, in the formation of the lytic membrane attack complex (MAC), C5b-9, as
Received 15 May 2006
Accepted 5 June 2006
Correspondence to: Michael Kirschfink, D.V.M. Ph.D., Im Neuenheimer Feld 305, 69120 Heidelberg. Tel: +49 6221 56 4076/4026; Fax: +49 6221 56 5586; Email: michael.kirschfink@urz.uni-heidelberg.de
well as in the opsonisation of pathogens and immune complexes for efficient phagocyto-sis. To protect themselves from unrestricted complement attack, all cells exposed to com-plement express various membrane comple-ment regulatory proteins (mCRP), such as membrane cofactor protein (MCP, CD46), decay accelerating factor (DAF, CD55) and CD59.2 In the last years, multiple studies ha-ve shown that complement resistance of tu-mour cells is a widespread phenomenon that is based on various mechanisms like se-cretion of soluble complement inhibitors or soluble forms of mCRP, respectively, into the microenvironment3-7, expression of sia-lic acid8 or complement cleaving proteases.9
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Also rebinding of secreted soluble comple-ment inhibitors to the tumour has been ob-served.10,11 The most important mechanism, however, is the overexpression of one ore more of the membrane-bound complement regulatory proteins CD46, CD55 and CD59.12,13 Although the influence of each mCRP varies between different tumour cell lines and has to be determined separately, there is strong evidence for an exceptional role of CD59, that blocks the assembly of the membrane attack complex (MAC) by in-terfering with the insertion of C9 thereby preventing the formation of the lytic pore. The functional importance of CD59 has been underlined by several approaches: whereas the mere number of mCRP only in part correlates with tumour cell resistance to complement-mediated lysis, transfection of CD59-negative tumour cells with CD59-cDNA increases their complement resistance considerably.14-16 Moreover, many studi-es have demonstrated that neutralisation of CD59 but also of other mCRP by using mo-noclonal antibodies significantly increases the susceptibility of cancer cells to comple-ment-mediated killing.13
Complement resistance as a prognostic factor?
There are only few clinical studies yet to underline the functional importance of complement resistance for tumour cell sur-vival and disease progression.
Recently, Watson et al.17 showed that expression of CD59 goes along with a deterio-ration of the patients’ prognosis in colorectal carcinomas. Furthermore, the expression of CD59 correlated with local tumour progression and tissue dedifferentiation in prostate cancer.18 High levels of CD59 are associated with an earlier biochemical relapse measu-red by increasing PSA levels after radical prostatectomy. However, contradicting data
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for other tumours do not allow to generalise about the potential impact of mCRP expression levels on disease prognosis. In a study with breast cancer patients, the loss of CD59 expression could be found to go along with a
reduced over-all-survival.19
Also other mCRP and their association with the disease prognosis have been studi-ed. The overexpression of CD55 seems to predict a poorer prognosis in patients with colorectal cancer.20 The 7-year survival of patients with high expression levels of CD55 was remarkably lower than that of patients with low expression levels (24% vs. 50%). For breast tumours, Madjd et al.21 found that overexpression of CD46 correla-ted with worse histological staging and a higher risk of tumour recurrence. Intere-stingly, in certain malignancies the loss of CD55 or CD59 may also result in more aggressive tumour growth (bigger tumour si-ze, worse grading, higher rate of lymph no-de metastases) and a poorer prognosis.19,22 For gastric carcinomas a correlation bet-ween CD97(EGF) and CD55, respectively, and tumour invasion into the surrounding tissue is reported.23 High expression profiles of these two molecules go along with aggressive local tumour growth and a higher pathological and clinical staging.
All in all, overexpression of mCRP by cancer cells and its possible influence on patients’ mortality seems to be rather hete-rogenous and has to be examined separa-tely for each type of cancer.
Impact of complement resistance on immunotherapy
Complement resistance has gained signifi-cant importance with the introduction of anti-tumour immunotherapy. It not only in-fluences the course of disease but also the patients’ prognosis by impairing therapeu-tic options.
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The rapid progress in molecular biology and recombinant antibody technology du-ring the last two decades promoted immu-notherapy of malignant diseases. Since then, anti-tumour antibodies successfully made their way from the laboratories to the clinic and meanwhile present a well-esta-blished adjuvant therapy regimen for a va-riety of cancer diseases (Table 1).24
Classical murine monoclonal antibodies derived from hybridomas according to Köhler and Milstein25 could not succeed in clinical testing because of the risk of severe anaphylactic reactions and formation of ne-utralising human anti-mouse-antibodies (HAMA) with rapid loss of effector functi-ons.26 With the advent of recombinant technology, `designer’ antibodies became a
powerful tool in anti-cancer therapy. Be-yond the well-known classical antibody ef-fector functions such as antibody-depen-dent cellular cytotoxicity (ADCC) or com-plement-dependent cytotoxicity (CDC), there are additional effects on the target cells that rather depend on the epitope than on the antibody itself.27 These so-cal-led epitope-specific antibody effects can trigger apoptosis or can modulate the auto-and paracrine secretion of tumour cells, thus influencing the tumour’s microenvi-ronment.28 It is often difficult to determine which effect is most important for the anti-body’s anti-tumour response.
Despite the great success of recombi-nant antibodies in cancer therapy, clinical oncologists and tumour immunologists are
Table 1. Anti-tumour antibodies in clinical use					
MAb name	Trade name	Target	Type	Approval date	Used to treat
Rituximab	Rituxan	CD20	IgG1, Chimeric	1997	Non-Hodgkin lymphoma
Trastuzumab	Herceptin	p185neu	IgG1, Humanised	1998	Breast cancer
Gemtuzumab-ozogamicin*	Mylotarg	CD33	IgG4, Humanised	2000	Acute myelogenous leukemia (AML)
Alemtuzumab	Campath	CD52	IgG1, Humanised	2001	Chronic lymphocytic leukemia (CLL)
In-111/Y-90-Ibritumomab-tiuxetan*	Zevalin	CD20	IgG1, Murine	2002	Non-Hodgkin lymphoma
Daclizumab	Zenapax	CD25	IgG1, Chimeric	2002	Acute and Chronic leukemia
I-131-Tositumomab*	Bexxar	CD20	IgG2, Murine	2003	Non-Hodgkin lymphoma
Bevacizumab	Avastin	VEGF	IgG1, Humanised	2004	Colorectal cancer
Cetuximab	Erbitux	EGFR	IgG1, Chimeric	2004	Colorectal cancer
* conjugated monoclonal antibodies
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confronted with limitations of this appro-ach. Similar to the well-known phenome-non of chemoresistance of tumours, i.e. the capacity of certain tumour cell clones to be-come refractory to cytostatic agents, there is also a phenomenon of resistance to anti-bodies.29 After repetitive treatment cycles, tumour cells get resistant against further antibody therapy. Several mechanisms may lead to antibody resistance, e.g down-regulation of the target epitope or dimini-shed effector functions. Various studies in-dicate that the up-regulation of mCRP, na-mely CD55 and CD59, is responsible for resistance against CD20 serotherapy with ri-tuximab.29-32 Blocking of these regulatory molecules can restore the tumour cells’ su-sceptibility to rituximab in vitro.30,31 The cytotoxic effects of the anti-her2/neu anti-body used in the therapy of metastased breast tumours could be augmented by blocking of mCRP in vitro.33
From these data mCRP appear as intere-sting target epitopes for new adjuvant the-rapeutic regimen.
Strategies for tackling complement resistance on human tumours
The significance of complement resistance of human tumours became obvious thro-ugh multiple experiments applying murine monoclonal    antibodies    that    blocked
mCRPs. 4,6,12,13,34,35
However, translation of these findings into the clinic is hampered by two major obstacles: (1) to find the most effective and secure way of mCRP neutralisiation and (2) restriction of the potentially dangerous intervention to cancer cells.
Different to the benchside situation, a therapeutic strategy must be tolerable for the patient. Blocking mCRP by murine mo-noclonal antibodies is not appropriate (for reasons as discribed above). Two promi-sing approaches have been developed for
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the future clinical application, which, ho-wever, still require comprehensive preclini-cal investigation.
Bispecific mCRP-blocking antibodies
For antibody-based immunotherapy the possibility to generate bispecific antibodies that can recognize two different epitopes by their two different antigen binding sites widens the scope and improves the chan-ces to generate truly tumour-specific »magic bullets«.36,37 Bispecific antibodies, which allow mCRP inhibition to be restric-ted to tumour cells in vitro have been pro-duced by various means.38-41 Harris et al.40 generated chimeric anti-CD59 x anti-CD19 and anti-CD59 x anti-CD38 antibodies by chemical linkage. B cell specific binding and lysis could be observed while sparing surrounding bystander cells. Although this work served as »proof of principle«, the chemical synthesis of bispecific antibodies is a cumbersome procedure and inappro-priate for clinical testing. Blok et al.41 obta-ined murine bispecific anti-CD55 x anti-G250 antibodies applying classical hybridoma or quadroma technology with good activity against renal cell carcinomas in vitro. Recently, a bispecific monoclonal anti-CD55 x anti-MHC class I antibody proved its efficacy on human colorectal and cervix carcinoma cell lines resulting in elevated C3-deposition and augmentation of com-plement-mediated cell lysis.39
For therapeutic approaches the use of humanised or at least chimeric antibodies is mandatory. These bispecific antibodies are nowadays constructed by recombinant
»antibody engineering«.42
However, despite all progress in the field of recombinant antibody technology it remains difficult to obtain continuously sufficient amounts of bispecific antibodies for in vivo testing in experimental animals or even clinical studies. The best established
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Figure 1. Blocking of CD59 augments tumour-directed complement activation: FACS-Scan for C3 (C3d) detection on human K562 erythroleukemic cell line after preincubation with polyclonal rabbit-anti-K562 or chimeric anti-CD59-miniantibody and pooled human serum as complement source. (A) Positive control with polyclonal rabbit-anti-K562 (green line), (B) Chimeric anti-CD59-scFv-Fc (green line). (Underlied curves each show two negative controls with heat inactivated serum or with irrelevant human IgG, respectively).
way to produce humanised bispecific anti-bodies takes advantage of expression vec-tors which contain the antibody genes. These vectors are commonly transfected into mammalian or insect cells that subsequ-ently secrete the recombinant antibody into the cell culture supernatant. However, this technology still suffers from difficulties in achieving stably transfected clones, varying and vanishing protein production yields, a highly inefficient heterodimerisation of the different antibody chains, and problems with the purification of the heterodimeric bispecific antibodies. Despite the fact that there are several strategies which may help to overcome these difficulties, construction and expression of recombinant bispecific
antibodies remains a »high risk challenge« far away from laboratory routine and with still unpredictable outcome.
We recently developed a chimeric mou-se/human anti-CD59 miniantibody (scFv-Fc) from a murine hydridoma (MEM43) that was able to trigger C3-deposition on human tumour cells via the Fc-mediated classical pathway although it failed to signi-ficantly augment complement-dependent killing (Figure 1). 43
Ziller et al.44 generated humanised anti-CD59 and anti-CD55 miniantibodies, that were able to trigger complement-mediated lysis on human lymphoma cell lines. Fur-thermore, the lytic effect of rituximab co-uld be augmented by these antibodies.
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Silencing of mCRP genes
Another approach for tackling complement resistance of human tumours is RNA inter-ference (RNAi). By using small interfering RNAs (siRNA) this technique offers great potential as a novel therapeutic strategy in tumour therapy but also in a wide field of other possible applications.
SiRNA technology, known since 2001, is based on short double-stranded RNA oligo-mers which cause highly specific and effici-ent silencing of target genes by posttrans-criptional gene knockdown (Figure 2).45 The antisense-strand of the siRNA molecu-le is complementary to the mRNA of the target protein.
SiRNAs induce the intracellular formation of a protein-complex, called „RNA-indu-ced silencing complex (RISC)« consisting of helicase and nuclease-acitivity among others. The RISC-complex induces the separation of the sense and antisense strand,
mediates the recognition of the target mRNA and catalyses the degradation of bo-und mRNA. The result is the specific inhibition of target-protein synthesis.
Although siRNA and its functionality in mammalian cells was detected just 5 years ago, plenty of studies demonstrating the therapeutic potential of siRNA have already been published. In vivo studies showed positive results applying siRNA for the the-rapy of neoplastic diseases46-48, the treat-ment of sepsis49 and the reduction of chole-sterol levels.50 Meanwhile the first clinical trial of siRNA therapy of the age-related ma-cula degeneration (AMD) has been started.
To better exploit complement for cancer cell eradication, we tried to reduce comple-ment-resistance of neoplastic cells by block-ing mCRP function using siRNA-techno-logy. SiRNAs targeting the mCRPs CD59, CD55 and CD46 were designed and tested concerning their downregulation efficiency in vitro. In this study siRNAs were either in-
I I I I I I I I I I I '......fill
J
siRNA: 21-nt dsRNA, 2-nt-overhangs
J !_J!! ' 'A J-1- vv ^   formation of the RNA-induced silencing complex („RISC-complex")
1
i i \ i i i i i i i
l i i i i i i i i t    target- m RNA recognition
i i i j......m 11 i j i i 11
-n   target-mRN A cleavage and further degradation
no target-protein synthesis
Figure 2. Schematic presentation of siRNA-induced silencing mechanism. Radiol Oncol 2006; 40(2): 95-105.
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Figure 3. Cell surface expression of mCRPs CD59, CD55 and CD46 on BT-474 breast carcinoma cells after trans-fection of the corresponding anti-mCRP siRNA individually or in combination, respectively.
dividually or combined transfected into Du145 prostate carcinoma cells or BT474 breast carcinoma cells, respectively. The inhibition of target protein expression was analysed both on protein level by FACS analysis and on mRNA level by RT-PCR. Downregulation of mCRP up to 80% could be achieved (Figure 3). Complement-resi-stance of CD55-, CD46- and/or CD59-defici-ent tumour cells, subsequently evaluated by cytotoxicity assays and by analysis of C3 deposition, clearly indicated that siRNA-indu-ced inhibition of mCRP expression sensiti-sed tumour cells to complement attack.51
Despite these encouraging findings and the outstanding potency and selectivity of siRNA, promising to improve targeted can-cer therapy, the systemic administration of aqueous siRNA, even chemically stabili-zed, is still limited by unspecific side ef-fects and a lack of activity in the target tis-sue due to limited blood stability on the one hand and poor intracellular uptake on
the other hand.46,52,53
The need for devices enabling systemic administration and targeted delivery to tu-
mour tissue and disseminated metastatic lesions is obvious.
Strategies based on viral vector delivery would be a possible approach but for safety reasons they are hitherto only of limited cli-nical use. A feasible approach, providing tissue selectivity and safe systemic delivery
is based on immunoliposome-technology.54
Liposomes are widely investigated for their properties as site-specific drug carriers allo-wing higher drug doses due to fewer syste-mic side effects.55,56 Liposomes are able to alter the pharmacokinetic profile of a drug, delivering the encapsulated agent preferen-tially to solid tumours, and acting as a slow-release depot for the drug in the dise-ased tissue.57 These attributes often result in a more favourable toxicity profile and an improved therapeutic window for the use of the agent.
Though conventional liposomes allow passive tumour site targeting to some de-gree, the idea of conjugation of cell-specific antibodies to liposomes (immunoliposo-mes) has been studied for selective drug
delivery.58-60
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Tumour-associated antigens can be utili-sed as appropriate target molecules. Mono-clonal antibodies against tumour-associa-ted antigens have been successfully adop-ted for targeting to various types of cancer
cells.61
Internalisation of immunoliposomes by receptor-mediated endocytosis into target-cells results in intracellular drug delivery.
A variety of cytotoxic drugs have been delivered to target cells in vitro by using im-munoliposomes; e.g. doxorubicin, vinorel-bine,   methotrexate62   and   daunomycin.63
Anti-HER2 immunoliposomal doxorubicin is awaiting Phase I clinical trials. Further-more, immunoliposomes have been emplo-yed to deliver oligodeoxyribonucleotides (ODN) designed to specifically inhibit gene expression by blocking translation, splicing or transcription process in vitro, thereby providing powerful therapeutic tools again-st viral diseases and cancer.64 Moreover, in vivo knockdown of gene expression with intravenous RNA interference (RNAi) us-ing a small hairpin RNA (shRNA) expression plasmid encapsulated in immunoliposo-mes has been shown.65
To conclude, immunoliposomes contai-ning siRNA combine specific antibody-me-diated tumour recognition with gene-speci-fic downregulation of target mRNAs.
Another promising approach of targeted siRNA delivery in vivo has been achieved by complexation of chemically unmodified siRNAs with polyethylenimine (PEI).66,67 Self-assembling nanoparticles constructed with polyethylenimine were adapted for siRNA. Target-specific delivery can be achi-eved by attaching peptide ligands (e.g. to bind to integrins) to the nanoparticle.
Furthermore, a protamine-antibody fusion protein for systemic, cell-type specific, antibody-mediated siRNA delivery was de-veloped recently.68 This approach takes advantage of the non-covalent nucleic acid-binding properties of protamine, which ori-Radiol Oncol 2006; 40(2): 95-105.
ginally nucleates DNA in sperm. In combi-nation with the site-specific delivery pro-perties of the antibody Fab fragment this fusion protein is a feasible device to admi-nister siRNA systemically.
Conclusion
Complement resistance is a widespread and nowadays well examined mechanism that enables tumour cells to withstand au-tologous immune attack. A magnitude of in vitro and several in vivo studies support the notion that blocking of mCRP is a feasible approach for tackling cancer cells. By me-ans of modern recombinant technologies humanised bispecific anti-mCRP-anti-tu-mour antibodies and siRNA based immu-noliposomes for mCRP gene silencing are promising strategies that could allow transferring experimental complement research to clinical application. Encouraging results from in vitro and animal studies have to be reproduced and then could widen the sco-pe of clinical anti-tumour therapy.
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Radiol Oncol 2006; 40(2): 95-105.
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Slovenian abstracts
Radiol Oncol 2006; 40(2): 95-105.
Odpornost na komplement ovira onkološko zdravljenje
Konatschnig T, Geis N, Scultz S, Kirschfink M
Izhodišča. Različne in vitro raziskave, ki so bile narejene v zadnjih dveh desetletjih, jasno kažejo, da je odpornost človeških tumorskih celic na avtologni komplement pogojena z na membrano vezanimi regulatornimi proteini komplementa (mCRP). Takšna proteina sta CD55 in CD46, najpomembnejšo vlogo pa ima CD59. Ta imunska dogajanja zelo vplivajo na potek bolezni, kar potrjujejo novejše klinične raziskave. Odpraviti odpornost na komplement obeta izboljšanje zdravljenja bolnikov z različnim rakom, s tem pa tudi izboljšanje napovedi izhoda bolezni. V pričujočem kratkem preglednem članku podrobneje predstavljamo: (1) nevtrali-zacijo proteinov mCRP z monoklonskimi ali rekombinantnimi protitelesi in (2) strategijo »utišanja« genov za proteine mCRP z delovanjem na nivoju RNA ob uporabi siRNA. Zaključki. Ker so proteini mCRP prisotni v vseh normalnih tkivih endotelnih celic paren-himskih organov (jetra, ledvica, itd...) in v krvnih celicah, je zelo pomembno, da je blokiranje delovanja proteinov mCRP selektivno in da tako ne prizadene zdravega tkiva. Čeprav so prvi rezultati ohrabrujoči, je vplivanje na delovanje proteinov MCRP, da bi izboljšali imunoterapijo, še vedno velik izziv v klinični praksi.
Radiol Oncol 2006; 40(2): 107-13.
Katepsini cisteinske skupine in njihovi inhibitorji pri raku glave in
vratu: pregled raziskovalnega dela na Onkološkem inštitutu Ljubljana
in Kliniki za otorinolaringologijo Kliničnega centra Ljubljana
Strojan P
Za odločitev o vrsti in intenzivnosti terapije, potrebne za uspešno ozdravitev raka, kot tudi za napoved izida bolezni je potrebna natančna ocena agresivnosti bolezni. Hipoteza, ki predpostavlja napovedni in prognostični pomen posameznih katepsinov in njihovih inhibitorjev, temelji na vpletenosti enih in drugih v obcelične proteolitične procese. Ti so sestavni del večine aktivnosti, povezanih z življenjem normalne celice, kot tudi procesov, povezanih z razgradnjo zunajceličnega matriksa med procesom invazije in zasevanja tu-morskih celic. Vlogo katepsinov in njihovih inhibitorjev pri raku lahko razčlenimo na naslednje skupine: markerji za presejanje; markerji za napoved prisotnosti zasevkov v področnih bezgavkah; markerji za napoved odgovora na zdravljenje in ponovitev bolezni; prognostični markerji. Čeprav je raziskav s področja katepsinov in njihovih endogenih inhibitorjev pri raku glave in vratu malo, rezultati opravičujejo nadaljna preučevanja. V pričujočem pregledu smo predstavili naše izkušnje in rezultate iz desetletnega obdobja klinično usmerjenega razsikovalnega dela in podali mnenje o njihovi napovedni in prognostični vlogi za potrebe vsakodnevne klinične prakse.
Radiol Oncol 2006; 40(2): 133-8.