Conditions of Tumor-Associated Antigens As a Proper Target for Therapeutic Antibodies Against Solid Cancers

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Review Article

Conditions of tumor-associated antigens as a proper target for therapeutic antibodies against solid cancers

Yoshikazu Kurosawa ______Institute for Comprehensive Medical Science, Fujita Health University, Toyoake, Aichi 470-1192, Japan

Abstract: Since the success of rituximab and trastuzumab for treatment of non-Hodgkin’s lymphoma and breast cancer, respectively, a huge therapeutic potential of monoclonal antibodies (mAbs) was realized and development of therapeutic mAbs has been widely tried against various cancers. However, the successful examples are still limited and therapeutic mAbs are not yet available for the majority of human cancers. We established a procedure for comprehensive identification of tumor-associated antigens (TAAs) through the extensive isolation of human mAbs that may become therapeutic. Thirty-twoTAAs have been identified and 555 mAbs that bound to one of the TAAs have been isolated to date. Now we are trying to select TAAs as proper targets for therapy and candidate mAbs as drugs from among them. The immunohistochemical analysis using many fresh lung cancer specimens suggested probabilities of proper targets, and moreover, presence of cancer-specific epitopes that could be distinguished from normal epitopes on the same molecules by mAbs. For Abs to efficiently kill the cancer cells they should have the ability to induce immunological cytotoxicity such as ADCC and/or CDC. They should also be able to inhibit the function mediated by the target Ags. For clinical point of view, the continuous presence of the target molecule on the cell surface until cell death might be essential for successful treatment. Therefore, it will be required for targets TAAs to play essential roles in tumorigenesis. Otherwise the cancer cells that do not express them could selectively survive during treatment and finally become dominant. It was also suggested that even the same molecules could play different roles in tumorigenesis quite often in different patients. Therefore when we develop therapeutic Abs, we should obtainwww.pubstemcell.com information about the conditions of patients including genetic background to whom the treatment will be effective. I will discuss how we can accomplish this purpose.

Introduction: antibodies (mAbs) and rushed into Since the success of rituximab and development of therapeutic Abs against trastuzumab for treatment of non- cancers [1,2]. In the commentary “Magic Hodgkin’s lymphoma and breast cancer, bullets hit the target” published in Nature, respectively, people realized a huge June issue of 2002, Trisha Gura described therapeutic potential of monoclonal the atmosphere of enthusiasm at that

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Journal of Stem Cells & Regenerative Medicine time and pointed out the problem as way of the use of the Ab such as a follows [3]. “The biggest issue to be delivery molecule to malignant cells [6]. In solved is cost. Although antibodies this review, however, I only focused on require much less investment in initial the TAAs that are preferentially research and development than overexpressed on the surface of conventional small-molecule drugs, they malignant cells and could be effective are hugely expensive to manufacture.” targets by IgG type of therapeutic mAbs. This comment presumed that many According to these criteria only EGFR groups would succeed in development of and HER2 are the antigens (Ags) against therapeutic mAbs against cancers in a which therapeutic mAbs have already near future. However, in the paper been approved by FDA to date and now “Colorectal cancer treatment: what’s are clinically available. While candidates next?” published in 2008, six years later, for target Ags should be abundantly Leonard Saltz described “although, present on the surface of cancer cells and initially, these new drugs appeared to accessible by Abs, they should not be offer enormous promise to radically highly expressed on normal cells change the landscape for patients with especially those that constitute vital metastatic colorectal cancer, the passage organs. Thus, anticancer mAbs can of time has begun to show us that the distinguish malignant cells from normal advances that have been made, while real, cells. It is also required that they are are more modest than we had expected homogeneously and consistently present or hoped” and he argued “more on the surface of cancer cells. In this sophisticated understanding of signal review I will summarize the strategies for transduction pathways, and of immune identification of TAAs, the kinds of TAAs surveillance and immunologically identified to date, classification of the mediated cytotoxicity, will help to reveal TAAs, conditions of proper target TAAs potential therapeutic options for and selection of effective mAbs as colorectal cancer” [4]. Thus, people therapeutic drugs. Since we have already started to think the necessity of a identified 29 TAAs and isolated 441 mAbs breakthrough for further revolutionary that bound to one of the TAAs to date success in this field. The simple strategy [7], we mainly focused on these TAAs composed of the following 4 steps: finding and mAbs in this review. Excellent of tumor-associated antigens (TAAs), reviews that treated the topics in more making mAbs against them, examining general way were given by other their antitumor activities in vitro and in researchers [8-10]. vivo and starting clinical tests, may not result in the www.pubstemcell.comsuccess in most cases. Strategies for identification of TAAs The success of rituximab, anti CD20 Completion of human genome mAb, and bevacizumab, anti VEGF mAb, project allowed us to comprehensively indicated that the targets for therapeutic examine all the proteins encoded on Abs are not necessarily TAAs that are human genome. In order to identify TAAs, located on the cell surface and profiling of mRNAs present in tumor vs preferentially expressed in malignant cells normal cells has been extensively [5]. In addition to the use of unmodified performed using cDNA or IgG, the success of Zevalin, anti CD20 oligonucleotide microarrays [11,12]. radiolabelled mAb, gave an alternative Although many candidate genes of TAAs Copyright © Journal of Stem cells and Regenerative medicine. All rights reserved JSRM/007010200003/Apr 01, 2011

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Journal of Stem Cells & Regenerative Medicine have been identified, this strategy implied fluorescence dyes have been developed the potential problems due to [19]. After isolation of membrane discordance between mRNA levels and proteins from two different cells DIGE proteins levels [13]. If tumor-associated was applied for detection of differences in splice variants were discovered [14], protein expression [20]. furthermore, if their roles in While there had been suspicion tumorigenesis became evident as known whether authentic cancer-specific Ags in the case of CD44v6 and EGFRvIII were present and immunogenic, [15,16], they could be good TAAs therefore, recognized by the autologous recognized by therapeutic Abs. host, development of the method termed Proteomic analysis could directly SEREX (serological analysis of show which spots corresponded to TAAs recombinant cDNA expression libraries) on 2-dimensional (2D) gel. Development led to identification of many TAAs of mass spectrometry (MS) allowed us to [21,22]. Based on the success of SEREX, identify proteins corresponding to sera from patients with cancer are used respective spots even low in abundance. as the primary Ab to detect Since majority of the visible spots immunoreactive spots in 2D gel of detected by the ordinary 2D gel malignant cell-derived proteins, resulting technology were cytoplasmic proteins, in identification of various TAAs including however, further devices were required cancer/testis Ags [22]. for enrichment of membrane proteins. In these strategies, identification of Although membrane proteins could be TAAs was performed first, and at the selectively prepared by biotinylation of next step mAbs were prepared against cell surface proteins in viable cells them. When Ab technologies were followed by affinity enrichment using utilized for identification of TAAs at an streptavidin beads [17], this technology initial step, it became possible that the has not been successfully applied to experiments were designed in the identification of TAAs because of difficulty opposite direction [23]. MAbs against in quantitative preparation of all the TAAs were isolated first and the target proteins present on cell surface. A similar Ags would be identified at the next step. method was also reported. After The details are described in the next biotinylation of membrane proteins they section. were purified by avidin affinity chromatography. The isolated proteins Ab-directed identification of TAAs were applied to SDS-PAGE and visualized Given TAAs expressed on human by silver staining. Then, bands were cancer-derived cell lines are immunogenic excised and www.pubstemcell.comidentified by MS/MS analysis. in mice, mAbs against the TAAs could be Difference in abundance between cancer generated by using conventional cells and normal cells was examined by hybridoma technology. Since majority of HRP-avidin staining. Using this technology, the therapeutic Abs approved to date Hastie et al. [18] succeeded in were derived from mouse mAbs, we may identification of several TAAs. In order to expect that mAbs isolated by this method compare 2D gel images from different could be utilized as a source of samples, DIGE method where samples therapeutic Ab. According to this strategy, were differently labeled prior to however, living cells expressing many electrophoresis with spectrally resolvable molecules including TAAs were used as Copyright © Journal of Stem cells and Regenerative medicine. All rights reserved JSRM/007010200003/Apr 01, 2011

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Ag for immunization [23]. Since it is not specifically bound to membrane proteins easy to efficiently isolate many mAbs with [27], and the methods, termed GFC and different characteristics against a single SITE, for systematic identification of Ags TAA, this method would be useful in recognized by many mAbs [28], I pointed identification of membrane proteins out two specific issues here. whose amounts are abundant and that are (1) The characteristics of clones isolated accessible by Abs. Candidates for by screening were very much diverged therapeutic mAbs would be isolated among the libraries and appeared to be afterwards since various capabilities largely influenced by the source of B cells should be required for mAbs to efficiently utilized in construction of Ab libraries. kill cancer cells.. We used two different libraries in this Using the phage-display Ab library we project: AIMS, composed of 1011 established a procedure for independent clones and the B cells were comprehensive identification of TAAs derived from 25 umbilical cord bloods, 20 through the extensive isolation of human tonsils, 2 peripheral bloods and 1 bone mAbs that may become therapeutic [7]. marrow; AOCK, whose size was the Our strategy was composed of four steps. same as that of AIMS but the B cell First we isolated a large number of mAbs source was spleens from 4 patients with that bind to the surface of cancer cells hepatocarcinomas. AIMS library gave us using a huge phage Ab library and many various mAbs with different kinds of cancer-derived cell lines. Then characteristics specific against probably using fresh tumor tissues we selected any kind of TAA but the repertoire of clones that gave significant staining of Abs formed in AOCK appeared to be malignant cells but were negative or very much less and biased. VH genes encoding weakly positive on the normal cells in the majority of the clones isolated from AIMS histological sections. At the third step the were germline-type without any mutation Ags recognized by the respective clones or with few. Although this result were isolated by immunoprecipitation (IP) indicated they were naive clones to Ags, and identified by MS analysis. Finally mAbs the Kd values between mAbs isolated and were converted to complete IgG1 and the the target TAAs were high enough, antitumor activity was examined. between 0.1 to 10 nM, in most cases. Although several papers reporting (2) When we started this project, we identification of TAAs by using similar presumed that many of the clones methods to ours were published [24-26], isolated might bind to Ags commonly the number of TAAs identified as well as present on both malignant and normal that of different kinds of mAbs bound cells, therefore, number of anti TAA each TAA werewww.pubstemcell.com much less than those by mAbs could not be many. Probably this our group. The quality of Ab library, the notion could have been commonly shared screening method, the definition of TAA among researchers, and in fact several for primary selection of clones, and the groups selected clones based on the method of identification of Ags restricted criteria of effective targets for recognized by respective mAbs should therapeutic Abs during the screening have given various effects on the results. process before identification of Ags [24- While we have already reported the 26]. Thus, they picked up only limited method, termed ICOS, for number of clones at the final stage for comprehensive isolation of mAbs identification of TAAs. For example, many Copyright © Journal of Stem cells and Regenerative medicine. All rights reserved JSRM/007010200003/Apr 01, 2011

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Journal of Stem Cells & Regenerative Medicine groups that looked for TAAs used normal target recognized by therapeutic Abs, the tissue-derived cell lines as negative continuous presence on the cell surface controls. According to our experience, until cell death might be essential for when normal tissue-derived cells were successful treatment. For example in the established as cell lines they had to gain case of proteins classified into the ability of immortalization. Since the cancer/testis Ag, it does not seem to be genes involved in immortalization largely evident whether they are directly overlapped with those necessary for involved in tumorigenesis or they are tumorigenesis, they judged possible TAAs expressed in cancer cells by dysregulation as normally expressed genes. As long as of transcription without any specific role the membrane proteins that are in tumorigenesis. If the latter is the case, expressed on the cell surface of it would be possible that the cancer cells immortalized cell lines are not expressed that do not express them could on the normal tissues under the steady selectively survive during treatment and state conditions in vivo, they could be finally become dominant. Since proper targets by therapeutic Abs. We transcription is regulated mainly at selected the clones only based on the epigenetic level, such cells resistant to the immunohistochemical (IHC) analysis of treatment with therapeutic Ab might fresh cancer tissues. Then around one start to appear at some frequency. third of mAbs isolated were judged to Judging from their predicted function, give tumor-specific staining patterns [7]. majority of the TAAs identified according . to our Ab-directed strategy appeared to TAAs identified by various methods play some role in tumorigenesis. We have It is not so simple to define TAA identified 32 membrane proteins as TAAs commonly approved among researchers. and listed in Table 1. While many of them Since chimeric proteins such as Bcr-Abl could not be tumor-specific in the strict fusion protein generated by chromosome meaning because of expression in normal translocation resulting in chronic tissues, the characteristics of these myelogenous leukemia (CML) are not proteins commonly observed among present in any normal cells, they are them were: 1. they were abundantly genuinely cancer-specific. But this present on the cell surface of not only example appeared to be very rare in some cancer-derived established cell lines pathogenesis of cancers. If introduction of but also some fresh cancers; 2. The mAbs mutations into some protein caused against them gave tumor-specific staining oncogenicity, we could consider it a patterns in IHC analysis of some cancer cancer-specific molecule. However, it will tissues freshly resected, 3. not be practicallywww.pubstemcell.com easy to prepare mAbs Overexpression of these TAAs were that can distinguish the mutated protein observed not in specific cancers but in from normal one. From practical point of various kinds of carcinomas. We roughly view to developing therapeutic Abs, the estimated that the total number of TAAs important points would be how we that showed the characteristics indicated identify target TAAs on the surface of here and that could be potentially cancer cells that play essential roles in identified by our method might be 40 tumorigenesis and how we prepare mAbs kinds in total. that could inhibit their function. As a

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Classification of TAAs mAbs to these molecules based on various The 32 TAAs identified to date were strategies [35-41] classified into 15 groups in Table 1 According to the sequence data according to the predicted function. The obtained by human genome project, the largest group among them was cell total number of receptor tyrosine kinase adhesion molecules belonging to (RTKs) was estimated to be 58 [42]. immunoglobulin superfamily (IGSF). While Among them seven RTKs including EGFR it has been shown that they play roles in and HER2 have been identified to be cell-cell adhesion, they are also committed TAAs by our method. Since PTK7/CCK4 to some otherwww.pubstemcell.com phenomena. Although is a pseudokinase and the ligand has not CADM1 had been originally identified to been identified, it might be difficult to be tumor suppressor [33], we showed it examine the role of this molecule in could also function as oncogene in lung tumorigenesis [43]. Since it has been well adenocarcinoma [34]. While the roles of established that phosphorylation mediated these adhesion molecules in the by protein tyrosine kinases (PTK) plays a tumorigenesis have not yet been precisely crucial role in control of cell growth and revealed in most cases and they are that PTKs play an important role in almost expressed on various normal cells, several all types of cancer, not only EGFR and groups are trying to develop therapeutic HER2 but also IGF1R, HGFR and EphA2 Copyright © Journal of Stem cells and Regenerative medicine. All rights reserved JSRM/007010200003/Apr 01, 2011

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could be good targets for therapeutic Abs also well expressed on the various normal [44,45]. cell surfaces, it will be difficult to develop Tyrosine phosphorylation should be therapeutic Abs without adverse effects controlled by a balance of PTKs and on normal organs. It has been known that protein tyrosine phosphatases (PTP). Since v3 is highly expressed on mature an overexpression of PTKs has been osteoclasts, angiogenic endothelial cells shown to contribute to carcinogenesis, it and tumor cells, and development of seemed to be reasonable to argue that therapeutic Abs againstv3 was already PTPs may function as tumor suppressor started [50,51]. Both 31 and 64 were opposite to that mediated by PTKs [46]. major cellular receptors for laminin 5, However, PTP-LAR was identified to be their functions were different from each TAA while 22 different receptor-type other. Our IHC analysis suggested that PTPs have been identified in human 64 could be a proper target for genome [47]. Since PTPs and PTKs therapeutic Abs [52]. Trial for the interacted with each other not only development of therapeutic mAbs to functionally but also physically [48], there 64 was reported [53]. will be various ways to induce Tetraspanins are cell-surface proteins perturbation in the complexes by using that span the membrane four times and Abs. Therefore, it might be a good form a large family consisting of 32 candidate as the target for therapeutic Abs. members [54]. While some of them were So far there has been no report of identified as TAAs [55], our TAA developing therapeutic Abs to this protein. collection included CD9. Since Integrins are heterodimeric receptors tetraspanins are present on various consisting of and subunits. In mammals, normal cell surfaces, they will not be 18  and 8 subunits assemble 24 proper targets by therapeutic mAbs. From different receptors [49]. Integrins interact a different point of view, however, their with various extracellular matrix (ECM) function is a molecular organizer of multi- components such as laminin, collagen and protein complexes, located at the specific fibronectin and mediate cell-ECM adhesion. region termed tetraspanin-enriched Such interactions give the strong effects microdomains (TEMs), and it is involved in on cellular behavior through cellular signaling [56]. Since integrins, determination of structural features of a CD44 and tetraspanins are deeply tissue and transmission of signals from the involved in the signal pathway through surrounding microenvironment to forming a network of large complexes epithelial cells. Of the 24 integrins four with various signal molecules, it would be have been identifiedwww.pubstemcell.com as TAAs. Since mAbs possible to find a tumor-specific complex against 31 have been most frequently that could be distinguished by mAbs isolated by our method and it is [57,58]. Although CD44v6 has been well abundantly expressed at high frequency on known to be TAA, the phase I trial with the cancer cell surface, it seemed to be the immunoconjugate consisting of a toxin evident that it plays a crucial role in coupled to anti CD44v6 mAb recently tumorigenesis. This argument is supported failed because of serious skin toxicity [59]. by the experiments with siRNA. Cell Ecto-5’-nucleotidase (CD73) is a GPI- growth of various tumor cell lines could anchored cell-surface protein and be inhibited by siRNA of 3 and of 1 catalyzes the extracellular conversion of (unpublished results). However, since it is 5’-AMP to adenosine. While RNA Copyright © Journal of Stem cells and Regenerative medicine. All rights reserved JSRM/007010200003/Apr 01, 2011

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Journal of Stem Cells & Regenerative Medicine interference of CD73 suggested that it precisely performed [64]. In fact may have multiple functions independent development of therapeutic Abs to of the enzyme activity [60], we found that CDCP1 has already been started [66] anti CD73 mAb efficiently prevented cell Membrane cofactor protein growth (unpublished results). Although (MCP)/CD46, decay-accelerating protein this molecule is found on the surface of a (DAF)/CD55 and CD59 are well known variety of cell types, it might be a target by to be membrane-associated complement- therapeutic Abs if the cancer-specific inhibitory proteins that protect normal function could be revealed [61]. tissues from damage by autologous The transferrin receptor (TfR) has complement. All of these three molecules been one of the most hopeful targets by were frequently overexpressed on therapeutic mAbs [62,63]. The role of malignant cells and overexpression of transferrin (Tf) is to transport iron CD46 was especially distinct [67]. While through the blood and deliver it to cells the fact that the complement inhibitors through TfR. Therefore, TfR is expressed are overexpressed on tumor cells appears in normal tissues including liver, epidermis, to imply presence of autologous Abs intestinal epithelium and a part of blood against TAAs in patients with cancers, it cells in the bone marrow. But the would be possible that MCP might play an expression level is low in general. On the additional role in tumorigenesis. other hand, on the cell surface of many Expression of siRNA to MCP gave an tumors, extremely high expression was inhibitory effect on the growth of tumor observed. Moreover, some mAbs showed cells under complement-free conditions in strong activity of cell killing. In our case, vitro (unpublished results). for instance, the cells producing an anti Since extracellular matrix TfR mAb could not grow under ordinary metalloproteinase inducer culture conditions because of cell killing by (EMMPRIN)/CD147 containing two Ig the Ab secreted from the cells themselves superfamily domains induced production (unpublished results). Clinical trials using of MMP-1 and MMP-2 by surrounding several candidates for therapeutic mAbs fibroblasts, it has been argued that are ongoing. overexpression of this molecule gave Although involvement of CDCP1 in tumor cells the ability to grow under tumorignesis was recently discovered, it anchorage-independent conditions [68]. could be possible to develop therapeutic However, it is now clear that EMMPRIN is Abs to it in a near future. Since a precise a multifunctional protein and interacts review on CDCP1 was just published [64], with a wide range of binding partners [69]. I pointed out only the possibility of SiRNA experiment indicated that therapeutic Abswww.pubstemcell.com here. While this molecule EMMPRIN is indispensable for the growth plays a key role in the regulation of anoikis, of tumor cells [70]. Since the IHC analysis phosphorylation of conserved tyrosine gave tumor-specific staining patterns residues at the cytoplasmic domain of (unpublished results), EMMPRIN should be CDCP1 confers resistance of cancer cells a good target for therapeutic mAbs. to anoikis [65]. Moreover, since CDCP1 interacts with many key proteins including Lessons from success of trastuzumab integrins, tetraspanins and Src family and cetuximab kinases, analyses of antitumor activity It has been shown that the ability of mediated by anti CDCP1 mAbs could be Abs inducing immune responses initiated Copyright © Journal of Stem cells and Regenerative medicine. All rights reserved JSRM/007010200003/Apr 01, 2011

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by Ag/Ab complexes such as AADC and cell motility, invasiveness and metastasis CDC is very important for therapeutic through the stimulation of these signaling Abs to efficiently kill the cancer cells pathways, cetuximab eventually inhibits all [71,72]. In the case of TAAs identified by of these phenomena mediated by EGFR. our strategy, however, around a half of However, there are many ways for cancer the mAbs isolated showed AADC against cells to show resistance to EGFR-directed the tumor cells that expressed the therapeutics [75]. We isolated 12 kinds of abundant target TAAs [7]. It may be due anti EGFR mAbs as indicated in Table 1. to the following characteristics of the Using all of them plus cetuximab and TAAs identified by our method: they are panituzumab we systematically examined abundantly present on the tumor cell the antitumor activity such as inhibition of surface and easily accessible by Abs. While cell growth in vitro and in vivo against anti EpCAM mAb showed an extremely various cell lines. The results indicated the strong AADC activity in vitro [7], in the followings: all of them showed antitumor xenograft model using nude mice the activity with various intensities; the activity was largely dependent on the combinations of target cells and mAbs conditions of target tumor cells. When which showed either the strong activity or the Ab was injected into mice together less activity were very variable with cancer cells, it showed a strong (unpublished results). It suggested that antitumor activity. But if it was injected while EGFR mediated various kinds of after the cancer cells formed a certain activity, there could be differences in the volume of solid tumor, it showed only a activities which respective tumor cells weak antitumor activity (unpublished utilized in tumorigenesis. There could also results). Thus, the ability to induce AADC be differences in the activities which is one of the conditions necessary for respective mAbs could inhibit. It has been therapeutic Abs but not enough. shown that patients with a colorectal Crystallographic studies convincingly tumor bearing mutated K-ras did not revealed how EGFR is regulated by the benefit from cetuximab, where patients ligands [73]. Furthermore, structural with a tumor bearing wild-type K-ras did analysis of the complex between benefit from cetuximab [76]. It could be cetuximab and EGFR clearly indicated the possible that some other mAb than molecular mechanism of inhibition of the cetuximab might show antitumor activity ligand binding by cetuximab [74]. While it even against tumors with mutated K-ras. primarily inhibits the ligand binding, HER2 gave a good example of inhibits receptor dimerization and thus successful therapeutic mAbs. Trastuzumab blocks autophosphorylation of EGFR and binds to the juxtamebrane region of HER2 activation, www.pubstemcell.comit also induces receptor [77] and shows a strong antitumor activity internalization as occurs with binding of through blocking the various functions the natural ligand. Activation of EGFR mediated by HER2 in addition to ADCC leads to stimulation of various signaling [78]. Pertuzumab also binds to HER2 near pathways including p13K-AKT, MAPK, the center of domain II that contains a SRC and STAT by recruiting proteins to dimerization arm and inhibits dimerization specific phosphorylated tyrosine residues with HER1 or HER3 [79]. Thus, it has in the C terminal domain [75]. Since EGFR been expected that trastuzumab and overexpressed on the tumor cells plays pertuzumab will be effective in the essential roles in tumorigenesis such as treatment of different types of cancers Copyright © Journal of Stem cells and Regenerative medicine. All rights reserved JSRM/007010200003/Apr 01, 2011

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[80]. Since there seem to be various suggested presence of a “cancer-specific pathways through which resistance to the epitope” that could be distinguished from established therapy is obtained [78], normal epitopes. different options for treatment of cancers Although cancers are very should be developed even against a single heterogeneous diseases, there should be target molecule. rules that govern the transformation of normal cells into malignant cancers. Search for proper targets and Hanahan and Weinberg [81] proposed six selection of candidates clones essential capabilities required for cancers. In order to search for target Ags and When normal cells newly obtained one of to select the candidates of therapeutic Abs the abilities during the process of to solid cancers we started the following tumorigenesis, for example self-sufficiency systematic studies. 1. IHC analysis using in growth signals, there should be many large number of fresh cancer tissues; 2. potential genes responsible for such an Examination of antitumor activities, in vitro activity which would be mutated or whose (ADCC, inhibitory effects on cell growth) expression might be up-regulated or and in vivo (xenograft using nude mice); 3. down-regulated. Furthermore, even one SiRNA of TAAs. Although the target gene could be involved in experiments have not been finished yet, tumorigenesis in various ways. For we could argue the followings. The IHC example, if we were able to precisely analyses were performed using 36 fresh analyze various cancer cells in which EGFR lung cancer specimens resected from was overexpressed, we would know the patients. Thee portions in lung, tumor roles of EGFR in tumorigenesis could be cells, bronchial epithelial cells and normal different among them. Therefore, when pulmonary alveolus cells including we develop therapeutic Abs, we should interalveolar septum were stained and also obtain information about the compared each other. The results were conditions of patients to whom the very informative since bronchial epithelial treatment with the Abs will be effective. cells appeared to represent the case This means that we should develop where cells are frequently turned over various options since the patients who can under healthy steady state conditions. In benefit from the treatment with a fact EGFR was expressed not only on the therapeutic Ab will be restricted. cancer cell surface but also on the surface of bronchial epithelial cells although at low Perspective level. Based on the results of IHC we In xenograft model using nude mice we concluded that HGFR, PTP-LAR, have already had many examples where EMMPRIN, TfR,www.pubstemcell.com CDCP1, integrin v3 mAbs showed strong antitumor activities. and integrin6 4 could be proper targets As I described examples of anti EGFR in addition to EGFR and HER2 [52]. One mAbs, the combinations of cancer cells more intriguing observation was obtained and mAbs that gave strong antitumor from IHC analysis. While we isolated activity were very heterogeneous. multiple clones against respective TAAs, Differences in response to the treatment they appeared to bind different epitopes may be mainly derived from the on the single TAA molecule from each differences in genetic background including other. As we already reported such the epigenetic level. Moreover, we examples using anti CADM1 mAbs [34], it observed many examples showing Copyright © Journal of Stem cells and Regenerative medicine. All rights reserved JSRM/007010200003/Apr 01, 2011

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Journal of Stem Cells & Regenerative Medicine presence of a “cancer-specific epitope” 4) Saltz L. Colorectal cancer treatment: what’s recognized by mAbs. It is possible that next? (or: Is there life after EGFR and VEGF?) Gastrointest Cancer Res 2008;2:S20-2. such epitopes reflected presence of 5) Ferrara N, Hillan KJ, Gerber HP, Novotny “cancer-specific complexes”. In order to W. Discovery and development of bevacizumab, overcome the difficulty in clearing the final an anti-VEGF antibody for treating cancer. Nat clinical tests in which many patients Rev Drug Discov 2004;3:391-400. randomly divided to two groups, candidate 6) Bennett JM, Kaminski MS, Leonard JP, Vose JM, Zelenetz AD, Knox SJ, Horning S, Press Ab vs placebo, have to be treated, two OW, Radford JA, Kroll SM, Capizzi RL. things should be done before starting large Assessment of treatment-related scale clinical trials. The factors that myelodysplastic syndromes and acute myeloid determine the effectiveness of therapeutic leukemia in patients with non-Hodgkin Abs should be precisely examined. Based lymphoma treated with tositumomab and iodine I131 tositumomab. Blood 2005;105:4576- on the informed consent between patients 82. and clinical doctors, the patients that may 7) Kurosawa G, Akahori Y, Morita M, be curable would be searched and tested. Sumitomo M, Sato N, Muramatsu C, Eguchi K, This information allowed us to establish Matsuda K, Takasaki A, Tanaka M, Iba Y, the rationale for selection of patients. This Hamada-Tsutsumi S, Ukai Y, Shiraishi M, Suzuki K, Kurosawa M, Fujiyama S, Takahashi N, Kato can be performed only as a clinical R, Mizoguchi Y, Shamoto M, Tsuda H, Sugiura research termed “translational research” M, Hattori Y, Miyakawa S, Shiroki R, Hoshinaga by tight collaboration between basic K, Hayashi N, Sugioka A, Kurosawa Y. scientists and clinical doctors. Comprehensive screening for antigens overexpressed on carcinomas via isolation of Acknowledgments human mAbs that may be therapeutic. Proc Natl Acad Sci USA 2008;105:7287-92. 8) Carter P, Smith L, Ryan M. Identification Our work was supported in part by a and validation of cell surface antigens for grant-in-aid for the 21st Century Center of antibody targeting in oncology. Endocr Relat Excellence (COE) Program of Fujita Cancer 2004;11:659-87. Health University from the Ministry of 9) Reichert JM, Valge-Archer VE. Development trends for monoclonal antibody cancer Education, Culture, Sports, Science, and therapeutics. Nat Rev Drug Discov 2007;6:349- Technology and by a grant from the New 56. Energy and Industrial Technology 10) Ma WW, Adjei AA. Novel agents on the Development Organization (NEDO). horizon for cancer therapy. CA Cancer J Clin 2009;59:111-37. 11) Okabe H, Satoh S, Kato T, Kitahara O, References: Yanagawa R, Yamaoka Y, Tsunoda T, Furukawa Y, Nakamura Y. Genome-wide analysis of gene 1) Reff ME, Carner K, Chambers KS, Chinn PC, expression in human hepatocellular carcinomas Leonard JE,www.pubstemcell.com Raab R, Newman RA, Hanna N, using cDNA microarray: Identification of genes Anderson DR. Depletion of B cells in vivo by a involved in viral carcinogenesis and tumor chimeric mouse human monoclonal antibody to progression. Cancer Res 2001;61:2129-37. CD20. Blood 1994;83:435-45. 12) Hippo Y, Taniguchi H, Tsutsumi S, Machida 2) Carter P, Presta L, Gorman CM, Ridgway N, Chong JM, Fukayama M, Kodama T, JBB, Henner D, Wong WLT, Rowland AM, Aburatani H. Global gene expression analysis of Kotts C, Carver ME, Shepard HM. gastric cancer by oligonucleotide microarrays. HER2 Humanization of anti-p185 antibody for Cancer Res 2002;62:233-40. human cancer therapy. Proc Natl Acad Sci USA 13) Chen G, Gharib TG, Huang CC, Taylor 1992;89:4285-9. JMG, Misek DE, Kardia SLR, Giordano TJ, 3) Gura T. Magic bullets hit the target. Nature Iannettoni MD, Orringer MB, Hanash SM, Beer 2002;417:584-6. DG. Discordant protein and mRNA expression

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