Monoclonal antibodies in the treatment of lung cancer

EJSO 32 (2006) 385–394

www.ejso.com

Review

Monoclonal antibodies in the treatment of lung cancer G. Egria,*, A. Taka´tsb a b

Department of Thoracic Surgery, Bajcsy-Zsilinszky Hospital, 89-91 Maglo´di Street, Budapest 1106, Hungary Department of Internal Medicine, Bajcsy-Zsilinszky Hospital, 89-91 Maglo´di Street, Budapest 1106, Hungary Accepted 18 January 2006 Available online 28 February 2006

Abstract Background: Lung cancer is an aggressive disease and its conventional therapy is far from success. There is a strong need for new, better approaches to improve survival, symptom control and quality of life. Methods: The authors searched the literature for indexed articles published over the past 30 years from Pubmed concentrating on all possibilities of monoclonal antibodies in the therapy of tumours and especially of lung cancer. Results: The search resulted in more then 200 published articles. Important major reports of the pre-clinical/clinical investigations of monoclonal antibodies in the therapy of tumours, with an emphasis on lung cancer were reviewed, screened and tracked for other relevant publications and the yielded data were summarized and systematized. Conclusion: It is concluded, that immunotherapy and the reviewed use of monoclonal antibodies in the therapy of tumours (including lung cancer) certainly carries a hope. However, studies of this topic are in a wide range of phases, from experiments to clinical trials, thereby their results are not comparable with each other. Based on the data available though the authors feel that active immunization with monoclonal antibodies as anti-idiotype vaccines, and antibody targeting with immunoconjugates (immunotoxins, radioimmunoconjugates and chemoimmunoconjugates) are the most promising methods. Radioimmunoguided surgery and immunoguided focal ablation are also valuable. Anti-growth factor monoclonal antibodies are the most evaluated agents so far. They certainly have an objective effect, though they are still not the ‘magic bullets’, waited for by many clinicians. The use of monoclonal antibodies against the escape mechanisms of tumours can be a good auxiliary method. There are too little data on the value of antibodies directly targeting tumour cells and on combined passive immunotherapy. Due to constant research, other modalities, such as prodrug activation, T cell activation, the use of intrabodies, T bodies, and conjugated antibody fragments might also prove to be valuable. q 2006 Elsevier Ltd. All rights reserved. Keywords: Lung cancer; Monoclonal antibodies; Anti-idiotype vaccines; Immunotoxins; (Radio)Immunoconjugates; Prodrug activation; T cell activation; Intrabodies; T bodies; Radioimmunoguided surgery; Immunoguided focal ablation

Introduction It seems that the conventional therapy of lung cancer (surgery, chemotherapy, radiotherapy) has reached a therapeutic plateau. The constant attempts to further improve their efficacy in survival, symptom control and quality of life have brought no real success yet. Moreover it is known that chemo-, and radiotherapy are neither specific, nor selective but are associated with significant * Corresponding author. 150 Ne´metvo¨lgyi Street, Budapest 1112, Hungary. Tel./fax: C36 13190962. E-mail address: [email protected] (G. Egri).

0748-7983/$ - see front matter q 2006 Elsevier Ltd. All rights reserved. doi:10.1016/j.ejso.2006.01.007

adverse events and toxicity, and surgery has its morbidity too. The search for new therapeutic strategies is understandable. The therapeutic application of antibodies against tumours is a longstanding desire. The target molecules of antitumour antibodies are the tumour associated antigens (TAAs), that are called lung cancer associated antigens (LCAAs) in the case of lung cancer. Data on the various known LCAAs and the numerous antibodies generated against them were collected and grouped by international workshops.1 Research, aiming to recognize still more LCAAs and antibodies against them has not stopped.

386

G. Egri, A. Taka´ts / EJSO 32 (2006) 385–394

The invention of hybridoma technology which allowed the production of monoclonal antibodies (mAbs) in large amounts was a cornerstone in research. Early investigations clarified some basic data and raised some problems on the use of mAbs in therapy of diseases. One issue that had been a fear earlier, the possible dangerous cross-reactivity of the mAbs with normal tissues, has luckily not been verified by trials. Another, still existing problem is associated with that most therapeutic antibodies are still produced by mouse cells. If we apply these antibodies in humans, however, T cells, and human anti-mouse antibodies (HAMAs) are generated against them.2,3 Attempts for the blocking of this human antimouse response resulted in changing the stems of the murine immunoglobulin molecules to human with genetic methods. The first chimeras contained about 30% mouse part, but further cutting the mouse component down to 5– 10%, ‘humanized’ chimeric antibodies were produced. It is agreed, that human monoclonals could solve finally the immunoreaction problem, however, because of technical difficulties, very few human antibodies have been tested yet.4 Parallel with the development of human antibodies by the way, there are also attempts for making advantage out of the murine part of the chimeric antibodies. Namely, the murine part is used for triggering host immune responses to destroy the whole antibodies, together with the bound tumour cells.5 A guideline of the American Medical Association for monoclonal antibody nomenclature proposes that all monoclonal antibody names should end with the suffix ‘mab’, indicating ‘monoclonal antibody’. The name of an antibody from a mouse source should add the letter ‘o’ to the suffix to become ‘omab’. For a chimeric antibody, the name should add the letters ‘xi’ to the suffix to become ‘ximab’ as in cetuximab. The name for a humanized antibody should add the letters ‘zu’ to the suffix to become ‘zumab’, as in trastuzumab, or bevicizumab.6 The application of mAbs in the therapy of lung cancer can be grouped as in Table 1.

Passive immunotherapy with monoclonal antibodies Monoclonal antibodies directly targeting tumour cells Mechanism In this approach the tumour cells are attacked directly by the monoclonals. After fixation to tumour cells, activation of the complement cascade (complement dependent citotoxicity), or the activation of certain effector cells happens (antibody dependent cell mediated citotoxicity), both of which results in tumour cell death.7 Some catalytic antibodies (‘abzymes’) follow, however, another pathway, they show direct citotoxicity, by the lysis of certain chemical bonds in the tumour cell membrane.8 Ways of research An interesting phenomenon of antigenic modulation, which involves internalisation, or sloughing of the antigens in response to binding by the antibodies can cause a decrease of the number of antibodies on the tumour cell surfaces to below the threshold required for citotoxicity. Thus, search is directed for non-modulating tumour associated antigens as targets for antibodies. Further research is also needed for the better knowledge of the abovementioned abzymes as well. Summarized opinion on the use of mAbs directly targeting tumour cells Although it seems to be the simplest and most obvious therapeutic mode of action, there are still more data available on the details of the technique than on clinical trials, concerning either pulmonary-, or other type of malignancies. Monoclonal antibodies targeting tumour growth- and proliferative factors Phase of research At the moment, this field of antitumour mAb therapy is the most widely investigated one, where research has

Table 1 The application of mAbs in the therapy of lung cancer Passive immunotherapy with mAbs

Active immunotherapy with mAbs Antibody targeting with immunoconjugates

The use of mAbs against tumours in other, early phase applications

MAbs in surgery and in other ablative methods

mAbs directly targeting tumour cells mAbs targeting tumour growth- and proliferative factors mAbs targeting the escape mechanisms of tumour cells Combined passive immunotherapy with mAbs Anti-idiotype method Immunotoxins Radio-immunoconjugates Chemo-immunoconjugates Prodrug activation T cell activation Redirection of T cells with T bodies Intrabodies Antibody fragments Radioimmuno-guided surgery Immunoguided ablation techniques

G. Egri, A. Taka´ts / EJSO 32 (2006) 385–394

already advanced to the stage of Phase II/III clinical trials. Base of the method As growth factors, and their receptors certainly have a role in augmenting tumour cell proliferation, survival, and invasion, attempts to selectively block them seem obvious. The observed distinct overexpression of them in many malignancies confirms the concept of their use as targets in antitumour therapy. The most extensively evaluated growth factor receptor family are c-erb B-1 and c-erb B-2. The other names of c-erb B-1 are HER1 or epithelial growth factor receptor (EGFR). C-erb B-2 is called in other name HER2. EGFR and to a lesser extent HER2/neu is overexpressed in the majority of non-small cell cancer (NSCLC), principally in adenocarcinoma.9 The overexpression of these receptors is associated with an aggressive disease course and poorer prognosis. Strategies to inhibit this molecular switch in lung cancer are under active investigation.10,11 Interestingly, it seems that responses for EGFR inhibition are higher in Asians.12 EGFR of lung cancer cells can also be targeted by tyrosine kinase receptor inhibitors (gefitinib (ZD1839, Iressa) and erlotinib (Tarceva)), but these agents are not monoclonal antibodies, so are not the topic of this paper. Anti-c-erb B-1 (anti-EGFR-) monoclonal antibodies Cetuximab (C225, Erbitux) was initially found to have efficacy in the treatment of metastatic colorectal cancer, and had already been approved in this setting in 29 countries. Data are also accumulating about its value in lung cancer: † In a Phase II study cetuximab was combined with docetaxel in 47 chemotherapy refractory/resistant NSCLC patients. Twenty-eight percent partial response rate and 17% stable disease rate were observed. These rates are higher than the response rates usually seen with docetaxel alone.13 † In an ongoing Phase II study Cetuximab is under evaluation in EGFR-positive, chemotherapy refractory/recurrent NSCLC patients as a single agent. The interim results have been reported yet: 7% of 29 patients showed partial response and the stable disease rate was 17%.14 † The combination of cetuximab with cisplatinCvinorelbin, carboplatinCgemcitabine, or carboplatinCpaclitaxel in patients with previously untreated metastatic NSCLC gave also promising partial response rates, ranging from 22 to 32%, in Phase II studies.15–17 † It is suggested, that cetuximabCeither gefitinib, or erlotinib,18 or cetuximabCradiation (e.g. HART) instead of chemotherapyCradiation should also be tried.19,20 The most commonly reported grade three toxicities associated with cetuximab treatment were fatigue,

387

infections and acneiform rash. Papulopustular rash usually on the face and upper torso in a milder form, however, occurs in 70–80% of treated patients. Findings suggest that there is a relationship between the development of rash and response and/or survival, making rash a potential surrogate marker of activity.21,22 Rash can be easily treated with topical anti-inflammatory dermatologic agents.23 Panitumumab (ABX-EGF), a fully human IgG2 mAb, gave also good pre-clinical results, which encouraged clinicians to initiate clinical trials in colorectal- and in NSCLC patients.24 Anti-c-erb B-2 (anti-HER2) mAbs Trastuzumab (Herceptin) gained authority approval because of its significant benefit in metastatic breast cancer therapy, however, the results with this mAb in lung cancer therapy are disappointing. According to this, some investigators concluded that future efforts and resources should not be directed toward trastuzumab in pulmonologic oncology.25–28 Others, however, basing on the results of a study on Asian patients, do not share this opinion and still propose this mAb for those patients whose tumours overexpress HER2.29 Pertuzumab (Omnitarg) and 2C4, two other anti-HER2 mAbs are nevertheless still under evaluation. Their advantage is that unlike trastuzumab, neither of them is dependent on HER2 overexpression. Phase I/II clinical trials with pertuzumab in NSCLC patients after promising preclinical data are in progress,30 and 2C4 is in early clinical development too.31,32 Non-anti-c-erb monoclonals against other growth factors Bevacizumab (Avastin), recently gaining approval from FDA for colorectal cancer, targets vascular endothelial growth factor (VEGF). † In a Phase II combination trial, with bevacizumab and standard carboplatin/paclitaxel chemotherapy in 99 patients with previously untreated advanced metastatic NSCLC, it was shown that the addition of bevicizumab produces a significantly longer time to progression and greater response rates, than chemotherapy alone, with a moderate increase in survival too. The enhancement of survival was even emphasized in the subset of patients with non-squamous histologies (adenocarcinoma).33 † The good results were recently strengthened by another, similar combination-study in Phase III.34 † Another Phase I/II combination study of bevacizumab and non-antibody EGFR inhibitor erlotinib (Tarceva) in 40 advanced NSCLC patients also showed highly encouraging results in response/survival and in safety.35 † In a very interesting Phase II ongoing combination trial bevacizumab is administered in a neoadjuvant setting with paclitaxel plus carboplatin, in patients with stage IB-IIA NSCLC.36

388

G. Egri, A. Taka´ts / EJSO 32 (2006) 385–394

According to these trials, adverse events with bevacizumabCchemotherapy include hypertension, thrombosis, proteinuria, diarrhoea, rash, mucocutaneous haemorrhage, epistaxis, hematemesis and major haemoptysis, the latter appearing to be the main safety concern.37,38 We can also find reports on pre-clinical investigations of other growth-, or proliferative factor targets, such as intercellular adhesion molecule-1 (ICAM-1), cancer-associated fibroblasts, angioneogenic factors, etc.39–42

Combined passive immunotherapy with monoclonal antibodies Theory Using monoclonal antibodies in combination with other biological response modifiers (polytargeted therapy) aims that the antitumour effects supposedly add together, and that the limitations of the antibody therapy may be overcome. Examples

Summarized opinion on anti-growth factor monoclonals The fast growing body of research seems to show, that anti-growth factor monoclonals have an objective effect, though they are still not the ‘magic bullets’ against tumours/lung cancer. Nevertheless, the need for large prospective, randomized, placebo controlled, double-blind studies is warranted. Monoclonal antibodies targeting the escape mechanisms of tumour cells Theory and phase of research The theory of blocking the various escape mechanisms of the tumour cells in order to inhibit tumour growth is a logical one. Research of this methodology is still in preclinical phase. Examples † One tumour escape mechanism is the overexpression of CD95 ligand (Fas) by the tumour cells, which induces apoptosis of Fas-positive cytolytic T lymphocytes of the host. To block this mechanism, investigators used preincubation of T cells, in order to avoid their Fasmediated apoptosis, with Fas-blocking monoclonal antibodies.43 † Fas ligands themselves, together with seven other proteins, belong to the family of death receptors. These transmembrane proteins by detecting some specific extracellular death signals can rapidly trigger the destruction of the cells they are situated on by inducing apoptosis. Death receptors are expressed on cancer cells as well, but interestingly these cells somehow escape from this kind of apoptosis. An in vitro trial suggests that this escape mechanism could be broken by an agonist anti-fas monoclonal antibody (Jo2).44

Summarized opinion on targeting the escape mechanisms of tumour cells Much research is still needed. If further investigation will be positive, it might be an auxiliary way to other treatment modalities.

† Cytokines seem to be a reasonable choice to combine with monoclonals, as they have direct cytotoxic, as well as immunomodulative effects. Alpha-, beta-, gamma interferon (IFN) for instance increase the expression of certain tumour associated antigens,45 and gamma IFN, regulating the infiltration of antigen-specific cytolytic T lymphocytes into tumours, might give a general help in mAb therapy.46 However, the results with IFN in the case of small cell cancer (SCLC) were found a bit ambiguous.47 Interleukin-2 (IL-2) and tumour-necrosis factor (TNF) also enhance the cell-mediated citotoxicity,48,49 and as vasoactive agents, may help more antibody molecules to penetrate a tumour and so are subjects of further investigation too. † We have already mentioned the possibility of the combinations of anti-growth mAbs with other agents in ‘Monoclonal antibodies targeting tumour growth- and proliferative factors’. † Theoretically, other promising anti-growth agents, such as peptide antagonists, negative growth factors, G protein, etc. seem also feasible to be combined with monoclonal antibodies. Presumably due to biotechnological difficulties, there are no published data about the combination of mAbs with the abovementioned agents yet.

Phase of research, summarized opinion Although the idea is clear and logical, data are available still mostly from pre-clinical investigations. Active immunization (anti-idiotype method) with monoclonal antibodies Mechanism of the anti-idiotype method By immunising mice with human tumour cells (or only with tumour associated antigens), antitumour antibodies, the first antibodies (Ab1s) are gained. Ab1s recognize TAAs. Immunising other mice with these Ab1s, the production of anti-idiotype murine antibodies (Ab2s) are generated. Ab2s bind to Ab1s as a targets and so mimic the TAAs, i.e they can serve as surrogate antigens for vaccination of cancer patients. If we inject a vaccine made of these murine Ab2s into humans, the patients quickly start to produce anti-antiidiotype antibodies (Ab3s) against Ab2s. The produced

G. Egri, A. Taka´ts / EJSO 32 (2006) 385–394

Ab3s, having a binding portion just like the original Ab1s, attack not only the murine Ab2s, but through TAAs the cancer cells of the patients as well.50,51 Ways of technological refinements Since, the first reports, biotechnological attempts have been made for making mass-production of Ab2s easier, by using hybridomas instead of using mice to produce them.52

389

Benefits/precautions Toxins—if we compare one molecule of them—are more cytotoxic then chemotherapeutic agents, though toxin molecules are much larger. As the toxins are antigenic themselves, however, they also increase the immune response against the conjugate. Moreover, the large-scale production of immunotoxins is not easy, and producers also have to deal with serious safety issues.55–57 Clinical studies

Phase of research After the success of experiments with the anti-idiotype technique in animals with lung cancer,53 human clinical studies have been started. We have news about an ongoing Phase III study with BEC2 anti-idiotypic vaccine in SCLC patients after a first remission induced by chemotherapy.54 Benefits/precautions The advantages of the anti-idiotype method compared to passive antibody therapy include the easiness of maintaining high antibody titers with lower doses of antibodies, the lack of anti-antibody responses, the possibility of the generation of permanently activated B and T immune cells, and the lower costs of the therapy. The idiotype method is superior to the direct immunization of patients with the tumour associated antigens themselves as well, because the idiotype is more immunogenic so the efficacy is elevated. No specific precautions are known.

† The first report on the trial of an immunotoxin in connection with lung cancer came about the in vitro purging of bone marrow prior to autologous stem cell transplantation by 7-day continuous infusion, in refractory SCLC. The tested immunotoxin, containing antiNCAM (neural cell adhesion molecule) antibody and ricin, exhibited good specificity for SCLC cells without toxicity to the haematopoietic precursor cells. This Phase I trial also disconfirmed the fears from cardiac-, or neurological adverse events.58 † Other investigators published preliminary data of about another immunotoxin, with good effect against chemotherapy resistant lung cancer.59

Summarized opinion on the use of immunotoxins The method is very promising, but many more clinical trials are waited for, before one could propose its use. Radioimmunoconjugates

Summarized opinion on the anti-idiotype active immunisation method The smart method with many benefits gives strong hope for clinical results. More clinical trials are waited for. Antibody targeting with immunoconjugates (the use of monoclonals for delivering cytotoxic agents to tumour cells) After linking cytotoxic agents (toxins, isotopes, or drugs) chemically to monoclonal antibodies we can use these antibodies as vehicles for delivery of the agents to tumour cells. The creation of such immunoconjugates must be performed such a way that the immunoreactivity of the antibodies and the cytotoxicity of the conjugated agents are retained. Immunotoxins Immunotoxins are antibodies with potent toxins conjugated to them. Ricin from the castor bean is considered the prototype of toxins linked to monoclonals, but other plantand bacterial toxins (pokeweed antiviral protein, Pseudomonas-, diphtheria toxin, etc.), are under investigations too.

Radio-isotopes attached to monoclonal antibodies make radioimmune conjugates for radioimmunotherapy (RIT). Much work has been done to choose the best radionuclids for making radioimmune conjugates. Lutetium-177, yttrium-90 and iodine-131 isotope conjugated monoclonals are the most evaluated ones, in mouse models.60–62 Benefits/precautions General limitating factors of radioimmunotherapy can be total body irradiation caused by the prolonged circulation of the isotope-conjugated antibody; myelotoxicity which is typically the dose limiting factor; and human anti-mouse response if murine antibodies are used. Phase of research † Clinical data concerning the application of radioimmunoconjugates in lung cancer patients are reported only from one work-group: after good biodistribution results,63 Indium-111 labelled tumour necrosis treatment (TNT) antibodies were given systematically, or intratumorally to 97 patients with advanced NSCLC after the failure of prior radiotherapy, or chemotherapy. Thirty three percent objective response rate was observed.64

390

G. Egri, A. Taka´ts / EJSO 32 (2006) 385–394

† There are, however, some reports on ongoing refinements of radioimmunotherapy, suggesting, that the preload with unlabelled antibodies,65 or pre-treatment (pre-targeting) with optimal doses of various materials such as an interferon–antibody immunoconjugate, or the coadministration of IFN alpha66,67 can be advantageous. † The combination of radioimmunotherapy and radioimaging (to image with radio-antibodies in order to calculate dosimetry before therapy with other radioantibodies) is also an interesting strategy,68,69 and ofloregional administration of radioimmunoconjugates can also be a possibility of choice.70

Summarized opinion on radioimmunotherapy The method is promising, however, research to find the best agents ands cofactors must be continued. Chemoimmunoconjugates Linking sufficient numbers of chemotherapeutic drug molecules to monoclonal antibodies (gaining chemoimmunoconjugates) targets to reduce the systematic citotoxicity and improve the efficacy of known chemotherapeutics. The theory is based on the early observation that cellular internalization of a chemoimmunoconjugate decreases resistance to the drug attached to the antibody, possibly by decreasing efflux of the free drug.71 Phase of research There are mainly pre-clinical data available with the method yet. Many conjugated agents have been explored including doxorubicin, daunorubicin, methotrexate, chlorambucil, vinca alkaloids, paclitaxel, etc. Fab’ antibody fragments conjugated to doxorubicin-loaded liposomes (forming ‘immunoliposomes’) were also tested in vitro and in animal models.72 In spite of the good pre-clinical results, clinical experience is limited yet with drugimmunoconjugate preparations in lung cancer.73,74 Summarized opinion on the use of chemoimmunoconjugates This methodology is also very promising, but still a lot of research, in the form of clinical trials, is needed to decide on its value. The use of monoclonal antibodies against tumours in other, early phase applications The following very interesting applications are still far from clinical use (may be except prodrug activation), nevertheless it is worthwhile to know about them as they might play a role in the treatment of tumours such as lung cancer in the future. † During prodrug activation an enzyme, which is capable for activating a citotoxic prodrug is administered to the

patient first. The delivery and anchorage of the enzyme to the tumour cells is achieved by an antitumour monoclonal antibody, the enzyme is bound to. The next step is the administration of the citotoxic prodrug, which is activated only where the specific enzyme is present—at the tumour site. Many refinements have been suggested to the method since its first description,75–77 but unfortunately there is only one publication about its clinical trial, related to colorectal malignancies yet.78 † T cells can be activated against tumours, by the use of bifunctional antibodies. If we bind a tumour cell through its TAA to one arm of a bifunctional or bispecific monoclonal antibody, and a T cell through its CD2 or CD3 antigen to the other arm, we can achieve the elimination of the tumour cell by the T cell. The binding of the T cells through their CD2/CD3 antigen is crucial, as the binding of these antigens is the signal that activates these immune effector cells into a citotoxic state.79 There are many possible ways of refining the technique. Similar effect is hoped from the use of fusion proteins, made of superantigens-proteins with the capacity of activating a large proportion of T cells -and tumour specific monoclonal antibodies.80,81 Besides the genetic method for creating bispecific antibodies (fusion proteins), they can be made chemically (heteroconjugates), or by the use of qadromas, the combination of two mAb-secreting hybridomas, which directly secrete bifunctional monoclonals.82 Some investigators emphasize the importance of extracorporeal pre-activation of the T cells before applying the bispecifics,83,84 others constructed bispecific antibodies that bind to certain pairs of different tumour antigens that are coexpressed on the surface of the same tumour cells.85 † The use of T bodies for T cell redirection is a remarkable novel strategy. T-bodies are receptors, artificially expressed on cytolitic T lymphocytes, or on NK cells. These recombinant immunoreceptor molecules are usually constructed by transduction with viral vectors.86 They consist of an extra, and an intracellular domain. The extracellular domain is an antibody fragment (scFv) that is capable to bind to a tumour associated antigen (TAA), the intracellular domain is derived from a signalling receptor for cellular activation. When the antibody fragment part of the T body encounters its target TAA, the intracellular domain of the T-body activates the T cell. This activation is specific against the cancer cells expressing the TAA87,88 and is called the redirection of T cells. Refinements of the technique are in progress.89,90 † Intrabodies (intracellular antibodies) are antibody fragments expressed intracellularly. They have the ability to bind to various intracellular proteins91,92 and to knockout the function of various oncoproteins.93 This ‘intracellular immunization’ method is definitely more than attractive, though refinements are still needed, mainly in biotechnology.94,95 The possible use of

G. Egri, A. Taka´ts / EJSO 32 (2006) 385–394

genetically engineered bispecific intrabodies (intradiabodies) is an even newer concept. It is suggested that the therapeutic effect of bispecific antibodies can be significantly greater then of the corresponding monospecific intrabody agents.96 † The theoretic advantage of antibody fragments (Fabs) is that attaching different agents to them, we have still got reduced size of conjugates, having the advantage of penetrating tumours better. However, their real place has not been found yet. It seems they will most likely be used in conjugation with a large toxin subunit such as ricin A-chain. Besides this, the possibilities of the connection of more fragments of different antibodies together and the use of these heterofunctional antibodies should be investigated as well.

Monoclonal antibodies in radioimmuno-guided surgery and in immunoguided ablation Even though this review focuses on direct therapeutic application of mAbs in lung cancer, the technique of radioimmuno-guided surgery (RIGS, the use of radiolabelled mAbs for guiding surgery) should also be mentioned. The value of the method in the intraoperative determination of tumour-free margins and the evaluation of the lymphatic drainage stations by gamma-detection probes is evident. The integration of RIGS into clinical work needs, however, some further refinements.97,98 The same is true for the immuno-guided, locally ablative techniques, where the tumorous tissues are visualized by antibodies for a variety of methods to ablate them.99 Discussion It is well known, that treatment of patients with some malignancies, such as lung cancer is far from optimal and must be improved substantially. At present, a variety of new treatment modalities are emerging, and previously used approaches such as various forms of immunotherapy are reintroduced.100–102 The aim of the present paper was to review the literature on all aspects of monoclonal antibody therapy in lung cancer. We knew, that the investigation of therapeutic monoclonals for lung cancer has been preceded by the evaluation of them for other (e.g. colorectal-) malignancies. We intended to demonstrate, that the earlier doubting opinion about the use of antibodies as in vivo therapeutics in lung cancer patients should be forgotten for today. From the data of the reviewed literature one can make some basic observations. It can be assumed first, that antitumour monoclonals are more likely to be efficient on a limited tumour burden. It seems that in the therapy of solid tumours, such as non-small cell lung cancer they should be optimally used in an adjuvant setting, for eradicating dormant metastatic-, or residual cancer cells after surgery.

391

They can also be used for slowing down disease progression, alleviating symptoms and reducing complications, while preserving the quality of life in advanced NSCLC-, and also in SCLC patients. It also seems that the addition of monoclonal antibodies to chemotherapeutic regimens may improve overall response rates to chemotherapy, and influence positively short time survival.103 We can say that active immunisation with anti-idiotype antibodies is more feasible than with the poorly immunogenic LCAA-s, or passive immunisation. Antibody targeting with immunoconjugates (immunotoxins, radio- and chemoimmunoconjugates) is promising too. Radioimmunoguided surgery and immunoguided focal ablation are also valuable. New approaches, like prodrug activation, intrabodies, T cell activation, T cell redirection, Fab-s, all of which use mAb-s also open new possibilities. The bold, but logical issue of immunoprevention of cancer e.g. with monoclonals, applying to people with a high genetic or environmental risk of developing this disease, has also been raised.104 However, important issues of mAb therapy remain to be addressed. The biggest is the genetic instability and antigenic modulation of the malignant cells, which results in intrapatient and interpatient heterogeneity of the antigen pattern of tumour cells105–107 that makes the correct targeting with mAbs questionable or temporary. New techniques of recognising the actual antigenic expression (‘molecular signature’) of tumour cells in order to develop personalized treatments; or the use of rational combinations (‘cocktails’) of mAbs and other synergistic agents are needed as an answer to this problem.108,109 It is clear that much work still has to be done, nevertheless we believe that Dillman’s conclusion of 1989,110 that the current status of monoclonal antibody therapy in cancer is one of continued promise, is still correct. We hope that the expected help from the use of monoclonals in oncology and surgery will hopefully be not delayed for long.

References 1. Stahel RA, Gilks WR, Lehmann H-P, Schenker T. Third international workshop on lung tumor and differentiation antigens: overview of the results of central data analysis. Int J Cancer 1994;57(Suppl 8):6–26. 2. Courtenay-Luck NS, Epenetos AA, Moore R, Larche M, Pectasides D, Dhokia B, et al. Development of primary and secondary immune responses to mouse monoclonal antibodies used in the diagnosis and therapy of malignant neoplasms. Cancer Res 1986;46: 6489–93. 3. Kosmas C, Epenetos AA, Courtenay-Luck NS. Patients receiving murine monoclonal antibody therapy for malignancy develop T cells that proliferate in vitro in response to these antibodies as antigens. Br J Cancer 1991;64(3):494–500. 4. Yoshinari K, Kimura H, Arai K, Sugawara I, Noda K, Kihara M, et al. Generation of human monoclonal antibodies recognising membranous antigens of the lung adenocarcinoma cell line A549 using an AMEX immunohistostaining method. Br J Cancer 1996;74(3): 359–67.

392

G. Egri, A. Taka´ts / EJSO 32 (2006) 385–394

5. Clynes RA, Towers TL, Presta LG, Ravetch JV. Inhibitory Fc receptors modulate in vivo cytotoxicity against tumor targets. Nature Med 2000;6(4):443–6. 6. Van Laan S. Nomenclature for biological products: monoclonal antibodies. www.ama-assn.org/ama/pub/print/article/4781-4661.html [accessed 2002 Sep 3]. 7. Metzger H, Kinet JP. How antibodies work: focus on Fc receptors. FASEB J 1988;2:3–11. 8. Tramontano A, Janda KD, Lerner RA. Catalytic antibodies. Science 1986;234:1566–70. 9. Reissmann PT, Koga H, Figlin RA, Holmes EC, Slamon DJ. Amplification and overexpression of the cyclin D1 and epidermal growth factor receptor genes in non-small cell lung cancer. Lung Cancer Study Group. J Cancer Res Clin Oncol 1999;125:61–70. 10. Schiller JH. Epidermal growth factor receptor as a therapeutic target in lung cancer. Semin Resp Crit Care Med 2004;25(Suppl 1):11–16. 11. Nguyen DM, Schrump DS. Growth factor receptors as targets for lung cancer therapy. Semin Thor Cardiovasc Surg 2004;16(1):3–12. 12. Giaccone G. Epidermal growth factor receptor inhibitors in the treatment of non-small-cell lung cancer. J Clin Oncol 2005;23(14): 3235–42. 13. Kim ES, Mauer AM, Tran HT, Liu D, Gladish G, Dicke K, et al. A phase II study of cetuximab, an epidermal growth factor receptor (EGFR) blocking antibody, in combination with docetaxel in chemotherapy refractory/resistant patients with advanced non-small cell lung cancer: final report. Proc Am Soc Clin Oncol 2003;22:642a. 14. Lynch TJ, Lilenbaum R, Bonomi P, Ansari R, Govindan R, Janne PA, et al. A phase II trial of cetuximab as therapy for recurrent non-small cell lung cancer (NSCLC). Proc Am Soc Clin Oncol 2004;23:634a. 15. Rosell R, Daniel C, Ramlau R, Szczesna A, Constenla M, Mennecier B, et al. Randomized phase II study of cetuximab in combination with cisplatin (C) and vinorelbine (V) vs CV alone in the first-line treatment of patients (pts) with epidermal growth factor receptor (EGFR)-expressing advanced non-small-cell lung cancer (NSCLC). Proc Am Soc Clin Oncol 2004;23:618a. 16. Robert F, Blumenschein G, Dicke K, Tseng J, Saleh MN, Needle MN, et al. Phase Ib/IIa study of anti-epidermal growth factor receptor (EGFR) antibody, cetuximab, in combination with gemcitabine/carboplatin in patients with advanced non-small cell lung cancer (NSCLC). Proc Am Soc Clin Oncol 2003;22:643a. 17. Kelly K, Hanna N, Rosenberg A, Bunn PA, Needle MN. A multicentered phase I/II study of cetuximab in combination with paclitaxel and carboplatin in untreated patients with stage IV non-small cell lung cancer. Proc Am Soc Clin Oncol 2003;22:644a. 18. Huang S, Armstrong EA, Benavente S, Chinnaiyan P, Harari PM. Dual-agent molecular targeting of the epidermal growth factor receptor (EGFR): combining anti-EGFR antibody with tyrosine kinase inhibitor. Cancer Res 2004;64(15):5355–62. 19. Rogers SJ, Harrington KJ, Eccles SA, Nutting CM. Combination epidermal growth factor receptor inhibition and radical radiotherapy for NSCLC. Expert Rev Anticancer Ther 2004;4(4):569–83. 20. Raben D, Helfrich B, Chan DC, Ciardiello F, Zhao L, Franklin W, et al. The effects of cetuximab alone and in combination with radiation and/or chemotherapy in lung cancer. Clin Cancer Res 2005; 11(2 Pt 1):795–805. 21. Perez-Soler R. Can rash associated with HER1/EGFR inhibition be used as a marker of treatment outcome? Oncology (Willson Park) 2003;11(Suppl 12):23–8. 22. Perez-Soler R, Saltz L. Cutaneous adverse effects with HER1/EGFRtargeted agents: is there a silver lining? J Clin Oncol 2005;23(22): 5235–46. 23. Segaert S, Van Cutsem E. Clinical signs, pathophysiology and management of skin toxicity during therapy with epidermal growth factor receptor inhibitors. Ann Oncol 2005;12 [E-pub ahead of print]. 24. McIntyre JA, Martin L. Panitumumab: oncolytic anti-EGFR human monoclonal antibody. Drugs Future 2004;29(8):793–7.

25. Hirsch FR, Langer CJ. The role of HER2/neu expression and trastuzumab in non-small cell lung cancer. Semin Oncol 2004; 31(Suppl 1):75–82. 26. Lara Jr PN, Laptalo L, Longmate J, Lau DHM, Gandour-Edwards R, Gumerlock PH, et al. Trastuzumab plus docetaxel in HER2/neupositive non-small-cell lung cancer: a California cancer consortium screening and phase II trial. Clin Lung Cancer 2004;5(4):231–6. 27. Clamon G, Herndon J, Kern J, Govindan R, Garst J, Watson D, et al. Cancer and leukemia group B: lack of trastuzumab activity in nonsmall cell lung carcinoma with overexpression of erb-B2: 39810: a phase II trial of cancer and leukemia group B. Cancer 2005;103(8): 1670–5. 28. Kalemkerian G. Trastuzumab in the treatment of advanced non-smallcell lung cancer: is there a role? Correspondence to the editor J Clin Oncol 2005;23(6):1325–6. 29. Langer CJ, Stephenson P, Thor A, Vangel M, Johnson DH. Eastern Cooperative Oncology Group Study 2598: trastuzumab in the treatment of advanced non-small-cell lung cancer: is there a role? Focus on Eastern Cooperative Oncology Group study 2598 J Clin Oncol 2004;22(7):1180–7. 30. Friess T, Scheuer W, Hasmann M. Combination treatment with erlotinib and pertuzumab against human tumor xenografts is superior to monotherapy. Clin Cancer Res 2005;11(14):5300–9. 31. Bianco AR. Targeting c-erbB2 and other receptors of the c-erbB family: rationale and clinical applications. J Chemother 2004; 16(Suppl 4):52–4. 32. Mass RD. The HER receptor family: a rich target for therapeutic development. Int J Radiat Oncol Biol Phys 2004;58(3):932–40. 33. Johnson DH, Fehrenbacher L, Novotny WF, Herbst RS, Nemunaitis JJ, Jablons DM, et al. Randomized phase II trial comparing bevacizumab plus carboplatin and paclitaxel with carboplatin and paclitaxel alone in previously untreated locally advanced or metastatic non-small-cell lung cancer. J Clin Oncol 2004;22(11):2184–91. 34. Tyagi P. Bevacizumab, when added to paclitaxel/carboplatin, prolongs survival in previously untreated patients with advanced non-small-cell lung cancer: preliminary results from the ECOG 4599 trial. Clin Lung Cancer 2005;6(5):276–8. 35. Herbst RS, Johnson DH, Mininberg E, Carbone DP, Henderson T, Kim ES, et al. Phase I/II trial evaluating the anti-vascular endothelial growth factor monoclonal antibody bevacizumab in combination with the HER-1/epidermal growth factor receptor tyrosine kinase inhibitor erlotinib for patients with recurrent non-small-cell lung cancer. J Clin Oncol 2005;23(11):2544–55. 36. Sandler AB, Johnson DH, Herbst RS. Anti-vascular endothelial growth factor monoclonals in non-small cell lung cancer. Clin Cancer Res 2004;10(12 Pt 2):4258s–462. 37. Kerr C. Bevacizumab and chemotherapy improves survival in NSCLC. Lancet Oncol 2005;6(5):266. 38. Herbst RS, Sandler AB. Non-small cell lung cancer and antiangiogenic therapy: What can be expected of bevacizumab? Oncologist 2004;9(Suppl 1):19–26. 39. Finzel AH, Reininger AJ, Bode PA, Wurzinger LJ. ICAM-1 supports adhesion of human small-cell lung carcinoma to endothelial cells. Clin Exp Metastasis 2004;21(3):185–9. 40. Raben D, Helfrich B. Angiogenesis inhibitors: a rational strategy for radiosensitization in the treatment of non-small-cell lung cancer. Clin Lung Cancer 2004;61(1):48–65. 41. Micke P, Ostman A. Tumour-stroma interaction: cancer-associated fibroblasts as novel targets in anti-cancer therapy? Lung Cancer 2004; 45(Suppl 2):S163–S75. 42. Dienst A, Grunow A, Unruh M, Rabausch B, Nor JE, Fries JW, et al. Specific occlusion of murine and human tumor vasculature by VCAM-1-targeted recombinant fusion proteins. J Natl Cancer Inst 2005;97(10):705–7.

G. Egri, A. Taka´ts / EJSO 32 (2006) 385–394 43. Mitani K, Nishioka Y, Yamabe K, Ogawa H, Miki T, Yanagawa H, et al. Soluble fas in malignant pleural effusion and its expression in lung cancer cells. Cancer Sci 2003;94(3):302–7. 44. French LE, Tschopp J. Protein-based therapeutic approaches targeting death receptors. Cell Death Differ 2003;10(1):117–23. 45. Hanibuchi M, Nishioka Y, Yanagawa H, Yano S, Parajuli P, Bando M, et al. Human interferon-gamma enhances expression of ganglioside GM2 on human lung cancer cells and their susceptibility for antiganglioside GM2 monoclonal antibody-dependent cellular cytotoxicity. Oncol Res 2000;12(4):173–9. 46. Wilcox RA, Flies DB, Wang H, Tamada K, Johnson AJ, Pease LR, et al. Impaired infiltration of tumor-specific cytolytic T cells in the absence of interferon-gamma despite their normal maturation in lymphoid organs during CD137 monoclonal antibody therapy. Cancer Res 2002;62(15):4413–8. 47. Sepherd FA. Alternatives to chemotherapy and radiotherapy in the treatment of small cell lung cancer. Semin Oncol 2001;28(2, Suppl 4): 30–7. 48. Kawase I, Komuta K, Hara H, Inoue T, Hosoe S, Ikeda T, et al. Combined therapy of mice bearing a lymphokine-activated killerresistant tumor with recombinant interleukin-2 and an antitumor monoclonal antibody capable of inducing antibody-dependent cellular cytotoxicity. Cancer Res 1988;48:1173–9. 49. Talmadge JE, Phillips H, Schneider M, Rowe T, Pennington R, Bowersox O, et al. Immunomodulatory properties of recombinant murine and human tumor necrosis factor. Cancer Res 1988;48(3): 544–50. 50. O’Connell MJ, Chen ZJ, Yang H, Yamada M, Massaro M, Mittelman A, et al. Active specific immunotherapy with anti-idiotypic antibodies in patients with solid tumors. Semin Surg Oncol 1989;5(6): 441–7. 51. Bhattacharya-Chatterjee M, Chatterjee SK, Foon KA. Anti-idiotype antibody vaccine therapy for cancer. Expert Opin Biol Therapy 2002; 2(8):869–81. 52. Saito H, Taniguchi M, Fukasawa T, Yamaguchi Y, Fujisawa T. Establishment of internal-image anti-idiotype monoclonal antibodies to a human antibody to lung cancer. Cancer Immunol Immunother 1997;44(2):83–7. 53. Lehmann HP, Zwicky C, Waibel R, Stahel RA. Tumor antigenspecific humoral immune response of animals to anti-idiotypic antibodies and comparative serological analysis of patients with small cell lung carcinoma. Int J Cancer 1992;50:86–92. 54. Chapman PB. Vaccinating against GD3 ganglioside using BEC2 antiidiotypic monoclonal antibody. Curr Opin Investig Drugs 2003;4(6): 710–5. 55. Kreitman RJ, Pastan I. Immunotoxins for targeted cancer therapy. Adv Drug Deliv Rev 1998;31(1–2):53–88. 56. Fan D, Yano S, Shinohara H, Solorzanol C, Van Arsdall M, Bucana CD, et al. Targeted therapy against human lung cancer in nude mice by high-affinity recombinant antimesothelin single-chain Fv immunotoxin. Mol Cancer Ther 2002;1(8):595–600. 57. Henry CJ, Buss MS, Hellstrom I, Hellstrom KE, Brewer WG, Bryan JN, et al. Clinical evaluation of BR96 sFv-PE40 immunotoxin therapy in canine models of spontaneously occurring invasive carcinoma. Clin Cancer Res 2005;11(2 Pt 1):751–5. 58. Lynch Jr TJ, Lambert JM, Coral F, Shefner J, Weu P, Blattler WA, et al. Immunotoxin therapy of small-cell lung cancer: a phase I study of N901-blocked ricin. J Clin Oncol 1997;15(2):723–34. 59. Zimmermann S, Wels W, Froesch BA, Gerstmayer B, Stahel RA, Zangemeister-Wittke U. A novel immunotoxin recognising the epithelial glycoprotein-2 has potent antitumoural activity on chemotherapy-resistant lung cancer. Cancer Immunol Immunother 1997;44(1):1–9. 60. Stein R, Govindan SV, Chen S, Reed L, Spiegelman H, Griffiths GL, et al. Radioimmunotherapy of a human lung cancer xenograft with

61.

62.

63.

64.

65.

66.

67.

68. 69.

70.

71.

72.

73.

74.

75.

76.

393

monoclonal antibody RS7: Evaluation of Lu-177 and comparison of its efficacy with that of Y-90 and residualizing I-131. J Nucl Med 2001;42(6):967–74. Zeng L, Ge D, Lin S, Ge X, Xhong G, Zhu J. Research on radioimmunotherapy of lung cancer in nude mice using lung cancer monoclonal antibody LC-1 combined with Y-90. Chin J Lung Cancer 2003;6(4):258–60. Wang B, Berger M, Masters G, Albona E, Yang Q, Sheedy J, et al. Radiotherapy of human xenograft NSCLC tumors in nude mice with a (90)Y-labeled anti-tissue factor antibody. Cancer Biother Radiopharm 2005;20(3):300–9. Khawli LA, Alauddin MM, Hu P, Epstein AL. Tumor targeting properties of Indium-111 labeled genetically engineered Fab’ and F(ab’)2 constructs of chimeric tumor necrosis treatment (chTNT)antibody. Cancer Biother Radiopharm 2003;18(6):931–40. Chen S, Yu L, Jiang C, Zhao Y, Sun D, Li S, et al. Pivotal study of iodine-131-labeled chimeric tumor necrosis treatment radioimmunotherapy in patients with advanced lung cancer. J Clin Oncol 2005; 23(7):1538–47. Watanabe N, Tanada S, Oriuchi N, Kim EE, Murata H, Sasaki Y. Tumor uptake of radioiodinated anti-human pulmonary surfactantassociated protein monoclonal antibody PE10 in nude mice bearing human pulmonary adenocarcinoma in combination with an unlabeled preload. Nucl Med Biol 2000;27(8):723–31. Pallela VR, Rao SP, Thakur ML. Interferon-alpha-2b immunocojugate for improving immunoscintigraphy and immunotherapy. J Nucl Med 2000;41(6):1108–13. Verhaar-Langereis MJ, Bongers V, De Klerk JMH, Van Dijk A, Blijham GH, Zonnenberg BA. Interferon-alpha induced changes in CEA expression in patients with CEA-producing tumours. Eur J Nucl Med 2000;27(2):209–13. Deelaloye AB. Radioimmunoimaging and radioimmunotherapy: will these be routine procedures? Semin Nucl Med 2000;30(3):186–94. Gruaz-Guyon A, Janevik-Ivanovska E, Raguin O, De LabriolleVaylet C, Barbet J. Radiolabeled bivalent haptens for tumor immunodetection and radioimmunotherapy. Quaterly J Nucl Med 2001;45(2):201–6. Paganelli G, Chinol M. Clinical applications of radiolabeled monoclonal antibodies for cancer diagnosis and therapy. Minerva Biotechnol 1998;10(4):146–61. Uadia P, Blair AH, Ghose T, Ferrone S. Uptake of methotrexate linked to polyclonal and monoclonal antimelanoma antibodies by a human melanoma cell line. J Natl Cancer Inst 1985;74:29–35. Sugano M, Egilmez NK, Yokota SJ, Chen FA, Harding J, Huang SK, et al. Antibody targeting of doxorubicin-loaded liposomes suppresses the growth and metastatic spread of established human lung tumor xenografts in severe combined immunodeficient mice. Cancer Res 2000;60(24):6942–9. Timms RM, Walker LE, Hirschowitz L, Reisfeld RA, Kline LE, Kroener JF. Human lung cancer KS1/4-methotrexate phase I trial. In: Dillman RO, Royston I, editors. Proceedings of the 2nd annual international conference on monoclonal antibody immunoconjugates for cancer, vol. 28. San Diego: University of California San Diego, Office of Continuing Medical Education; 1987 [abstract]. Saleh MN, Sugarman S, Murray J, Ostroff JB, Healey D, Jones D, et al. Phase I trial of the anti-Lewis Y drug immunoconjugate BR96doxorubicin in patients with Lewis Y-expressing epithelial tumors. J Clin Oncol 2000;18(11):2282–92. Wentworth P, Datta A, Blakey D, Boyle T, Partridge LJ, Blackburn GM. Toward antibody-directed ‘abzyme’ prodrug therapy, ADAPT: carbamate prodrug activation by a catalytic antibody and its in vitro application to human tumor cell killing. Proc Natl Acad Sci USA 1996;93(2):799–803. Kakinuma H, Fujii I, Nishi Y. Selective chemotherapeutic strategies using catalytic antibodies: a common pro-moiety for antibodydirected abzyme prodrug therapy. J Immunol Methods 2002;269(1-2): 269–81.

394

G. Egri, A. Taka´ts / EJSO 32 (2006) 385–394

77. Bagshawe KD, Sharma SK, Begent RHJ. Antibody-directed enzyme prodrug therapy (ADEPT) for cancer. Expert Opin Biol Ther 2004; 4(11):1777–89. 78. Napier MP, Sharma SK, Springer CJ, Bagshawe KD, Green AJ, Martin J, et al. Antibody-directed enzyme prodrug therapy: efficacy and mechanism of action in colorectal carcinoma. Clin Cancer Res 2000;6(3):765–72. 79. Baeuerle PA, Kufer P, Lutterbuse R. Bispecific antibodies for polyclonal T-cell engagement. Curr Opin Mol Ther 2003;5(4):413–9. 80. Forsberg G, Ohlsson L, Brodin T, Bjork P, Lando PA, Shaw D, et al. Therapy of human non-small-cell lung carcinoma using antibody targeting of a modified superantigen. Br J Cancer 2001;85(1):129–36. 81. Rosendahl A, Hansson J, Sundstedt A, Kalland T, Dohlsten M. Immune response during tumor therapy with antibody–superantigen fusion proteins. Int J Cancer 1996;68(1):109–13. 82. Wong JF, Colvin RB. Bi-specific monoclonal antibodies: selective binding and complement fixation to cells that express two different surface antigens. J Immunol 1987;139:1369–74. 83. Kroesen BJ, Nieken J, Sleijfer DT, Molema G, deVries EG, Groen HJ, et al. Approaches to lung cancer treatment using the CD3!EGP-2directed bispecific monoclonal antibody BIS-1. Cancer Immunol Immunother 1997;45(3-4):203–6. 84. Schweizer C, Strauss G, Lindner M, Marme A, Deo YM, Moldenhauer G. Efficient carcinoma cell killing by activated polymorphonuclear neutrophils targeted with an Ep-CAMCD64 (HEA125197) bispecific antibody. Cancer Immunol Immunother 2002;51(11–12):621–9. 85. Shi T, Hsieh-Ma ST, Reeder J, Ring DB. Murine bispecific antibody 1A10 directed to human transferring receptor and a 42-kd tumorassociated glycoprotein. Clin Immunol Immunopathol 1996;78(2): 188–95. 86. Gyobu H, Tsuji T, Suzuki Y, Ohkuri T, Chamoto K, Kuroki M, et al. Generation and targeting of human tumor-specific Tc1 and Th1 cells transduced with a lentivirus containing a chimeric immunoglobulin T-cell receptor. Cancer Res 2004;64(4):1490–5. 87. Eshhar Z. Tumor-specific T-bodies: towards clinical application. Cancer Immunol Immunother 1997;45(3–4):131–6. 88. Abken H, Hombach A, Heuser C. Immune response manipulation: recombinant immunoreceptors endow T-cells with predefined specificity. Curr Pharm Des 2003;9(24):1992–2001. 89. Alvarez-Vallina L, Russell SJ. Efficient discrimination between different densities of target antigen by tetracycline-regulatable T bodies. Hum Gene Ther 1999;10(4):559–63. 90. Pinthus JH, Waks T, Kaufman-Francis K, Schindler DG, Harmelin A, Kanety H, et al. Immuno-gene therapy of established prostate tumours using chimeric receptor-redirected human lymphocytes. Cancer Res 2003;63(10):2470–6. 91. Marasco WA. Intrabodies: turning the humoral immune system outside in for intracellular immunization. Gene Ther 1997;4(1): 11–15. 92. Lobato MN, Rabbitts TH. Intracellular antibodies and challenges facing their use as therapeutic agents. Trends Mol Med 2003;9(9): 390–6.

93. Arafat W, Gomez-Navarro J, Xiang J, Siegal GP, Alvarez RD, Curiel DT. Antineoplastic effect of anti-erbB-2 intrabody is not correlated with scFv affinity for its target. Cancer Gene Ther 2000; 7(9):1250–6. 94. Hyland S, Beerli RR, Barbas III CF, Hynes NE, Wels W. Generating and functional characterization of intracellular antibodies interacting with the kinase domain of human EGF receptor. Oncogene 2003; 22(10):1557–67. 95. Donini M, Morea V, Desiderio A, Pashkoulov D, Villani ME, Tramontano A, et al. Engineering stable cytoplasmic intrabodies with designed specificity. J Mol Biol 2003;330(2):323–32. 96. Jendreyko N, Popkov M, Beerli RR, Chung J, McGavern DB, Rader C, et al. Intrabodies, bispecific, tetravalent antibodies for the simultaneous functional knockout of two cell surface receptors. J Biol Chem 2003;278(48):47812–9. 97. Bakalakos EA, Burak Jr WE. The radioimmunoguided surgery (RIGS) system as a diagnostic tool. Surg Oncol Clin North Am 1999; 8(1):129–44. 98. Mansi L, Di Lieto E, Rambaldi PF, Bergaminelli C, Fallanca F, Vicidomini G, et al. Preliminary experiences in radioimmunoguided surgery for primary lung neoplasms. Minerva Chir 1998;3(5):369–72. 99. Goldberg SN, Grassi CJ, Cardella JF, Charboneau JW, Dodd 3rd GD, Dupuy DE, et al. Image-guided tumor ablation: standardization of terminology and reporting criteria. J Vasc Interv Radiol 2005;16(6): 765–78. 100. Parish CR. Cancer immunotherapy: the past, the present and the future. Immunol Cell Biol 2003;81(2):106–13. 101. Ross JS, Gray K, Gray GS, Worland PJ, Rolfe M. Anticancer antibodies. Am J Clin Pathol 2003;119(4):472–85. 102. Gatto B. Monoclonal antibodies in cancer therapy. Curr Med Chem— Anticancer Agents 2004;4(5):411–4. 103. Mitchel MS. Combinations of anticancer drugs and immunotherapy. Cancer Immunol Immunother 2003;52(11):686–92. 104. Forni G, Lollini PG, Musiani P, Colombo MP. Immunoprevention of cancer: is the time ripe? Cancer Res 2000;60:2571–5. 105. Konemann S, Schuck A, Malath J, Rupek T, Horn K, Baumann M, et al. Cell heterogeneity and subpopulations in solid tumors characterized by simultaneous immunophenotyping and DNA content analysis. Cytometry 2000;41(3):172–7. 106. Riker A, Cormier J, Panelli M, Kammula U, Wang E, Abati A, et al. Immune selection after antigen-specific immunotherapy of melanoma. Surgery (St Louis) 1990;126:112–20. 107. Fishel R. Genomic instability, mutation, and the development of cancer: is there a role for p53? J Natl Cancer Inst 1996;88:1608–9. 108. Livingston PO, Hood C, Krug LM, Warren N, Kris MG, Brezicka T, et al. Selection of GM2, fucosyl GM1, globo H and polysialic acid as targets on small cell lung cancers for antibody mediated immunotherapy. Cancer Immunol Immunother 2005;31 [E-pub ahead of print]. 109. Krug JM. Vaccine therapy for small cell lung cancer. Semin Oncol 2004;31(1 Suppl 1):112–6. 110. Dillman RO. Monoclonal antibodies for treating cancer. Ann Int Med 1989;111:592–603.