FORSCHUNG - TECHNOLOGIE 311 CHIMIA 45 (1991) Nr. 10 (Oktober)

Chimia 45 (1991) 311-316 active on quickly growing murine tumors, © Schweiz. Chemiker-Verband; ISSN 0009-4293 but has antitumoral activity on human tu- mors especially on colon carcinomas [29]. Clinical studies, however, had to be stopped due to high grade toxicity ofRodorubicin for endothelial cells [30][31]. Due to this expe- rience and facing the risk vs. benefit ratio we The Change in Research for the stopped the whole screening system. Another new approach in tumor research Therapy of Tumors was the clarification of the various mecha- nisms the tumor cell develops to become resistant to a tumor therapeutic drug. To these mechanisms belong the increase in H.-Harald Sedlacek*, Dieter Hoffmann, Jorg Czech, Cenek Kolar, Gerhard intracellular level of glutathion or increase Seemann, Dettef Giissow, and Klaus Bosslet in quantity and function of a Ca2+ ion de- pendent transmembraneous glycoprotein, which pumps toxic compounds out of the cell. Inhibition of these mechanisms, for Introduction gens, antiprogestins, aromatase inhibitors, instance by Ca2+ antagonists or lipophilic and LH-RH-antagonists) were likewise im- compounds, seems to restore sensitivity to Cytostatics, hormones, and hormone proved [15-17]. cytostatic drugs of the resistant tumor cell antagonists are families of drugs with proven However, in spite of the clinical success [4]. activity in a limited number of systemic, i.e. of cytostatics, hormones, and antihormones A further approach arose from the in- disseminated tumor diseases. The parenteral we are far behind our research aims in tumor creasing knowledge in the components, drug of each family has been detected dec- therapy. Today only 6-7% of tumors can be functions, and control mechanisms of growth ades ago. A considerable number of pri- cured by cytostatics. In about 40% of tumors of different nonnal cell types incl uding those mordial cytostatics were either detected by treatment with cytostatics or hormones or of the immune system. This knowledge an accidental specific clinical or preclinical hormone antagonists leads to a transient helped us to describe and to understand at observation [1-3] or on the basis of a broad tumor regression and/or to an increase in least in sections the misbehavior of cells, in vitro and in vivo tumor screening system survival time, which is limited by develop- especially of malignant cells, and the cor- [4]. The success of both approaches was ment of resistance of the tumor against any relation and interaction between tumor cells based on lucky chances. Some cytostatics further treatment. and the immune system. The hypothesis of and the hormones and hormone antagonists This development of resistance as well the immune surveillance of tumor growth were found on a more rational basis. With as the insensitivity of more than 50% of all restimulated the old research to find immu- increasing knowledge in the celIular syn- tumors to any kind of treatment is still the nological ways of treating tumor diseases thesis of the DNA and in the hormonal biggest provocation for academic as well as [32]. Antigen specific as well as nonspecific dependence of the growth of normal and industrial tumor research. approaches were tested preclinically as well tumor cells, biological structures (enzymes, as clinically. hormone receptors) could be identified the This challenge has been taken with var- The resu Its of more than three decades of blockage of which promised an inhibition of ious new approaches: intensive and broad evaluations of nonspe- cell proliferation. Once such targets were cific immunostimulators are less than mod- identified, substrate analogues to block the For instance the screening systems to erate. Indeed bovine mycobacteria (BCG) specific enzymes or of hormone derivatives detect new cytostatic compounds was locally applied into the urine bladderreduced to block the receptors were designed. When changed. The change consisted in the use of recurrence of [33], the im- recognizing the therapeutic potency as well slowly proliferating human tumors being munomodulator levamisole when applied in as the fai lure and toxicity of each parenteral clinically resistant to any kind of treatment combination with the 5-FU drug its structure was modified to find by instead of quickly proliferating murine tu- increased survival in colon carcinoma [34], trial and error derivatives with improved mors. The rational of this change was that a-Interferon proved to be curative for hairy activity. With the recognition of the structure compounds being active on slowly prolif- cell leucemia [35], and a-Interferon as well activity relationship the rational design of erating tumors might not be active on quickly as Interleukin-2 induced tumor regressions new and further improved compounds still proliferating tumors and thus might have in kidney tumors and melanomas [35-36]. A belonging to each family tree of structures been missed in former screening programs breakthrough in tumor therapy, however, was possible. with murine tumors [4]. Indeed new cyto- has not been achieved till today. This approach took place with cytostat- static antibiotics active in vitro and in vivo The same is true for antigen specific ics like alkylating agents, antibiotics like on human tumors could be found [18-28] approaches irrespective, whether they are , mitomycins, with toxins in- but till now none of these compounds re- tumor cell vaccines or monoclonal antibodies activating enzymes (i.e. topoisomerase) or vealed a breakthrough in the treatment of [32][37] directed against tumor associated intracellular structures (i.e. tubulin), and tumor patients while with a part of these antigens. This overall frustrating balance anti metabolites (folic-acid antagonists and compounds new hitherto unknown toxic side led in several industrial institutions to the others) [5-14]. effects could be observed. consequence either to reduce or to stop cancer The antitumoral activity of hormones We were one of the first institutions, research. (progestins and androgens and derivatives) which made this experience using a battery The strongest motivation to engage or anti hormones (antiestrogens, antiandro- of slowly growing human tumors. Very early ourselves in tumor research was the in- we started a screening system for natural creasing knowledge in cell physiology, bi- compounds with antiproliferative activity. ology, and immunology of tumor cell growth, We had been successful so far that we de- enabled by the breakthrough in the devel- tected Rodorubicin (HLB 817) (7-L-rho- opment of new techniques in human tumor *Correspondence: PO Dr. H.-H. Sedlacek dosam inyI,2-desox y-L-fucos Y1-L-Cinerulo- Research Laboratories of Behringwerke xenotransplantation, in molecular biology P.O. Box 11 40 sy 1)-(1O-L-rhodosaminy 1)-f3-rhodomyci- resp. genetechnology, monoclonal antibody 0-3550 Marburg non. In vitro Rodorubicin (HLB 817) is not production, and antibody engineering. This FORSCHUNG - TECHNOLOGIE 312 CHIMIA45 (1991) Nr, 10 (Oklobcr) investment into tumor research revealed the Table I. Antitumoral Activity ofB 88 0308 [40] following main results: In vitro cytotoxicity IC50 = 0.001 g/ml Bifunctional Cytostatics 10 x stronger than cross-resistance no cross-resistance to The rational was the improvement of the anthracyclines (Doxorubicin) antitumoral activity of a compound by the topoisomerase inhibitors () synthetic combination of two structures with spindle toxins (Vinblastin) proven antitumoral activity [38], namely an DNA reacting compounds (, intercalating and an alky lating structure. One ) ofthecompounds synthesized, which carries the above mentioned structural and functional III vivo characteristics, is B 88 0308 [39] a com- murine tumors: pound, which demonstrates activity on tumor leukemia no cross-resistance to Doxorubicin cells being sensitive or being made resistant melanoma, ovarian carcinoma activity similar to Doxorubicin to standard cytostatic drugs [40]. On human colon carcinoma tumors transplanted into immunodeficient (nu/nu) mice, B 88 0308 revealed to be at Regression of human tumors least similar or stronger active than all standard anthracycl ines [40]. In all human human tumors >80% >50% <50% no effect ovarian carcinomas tested till now B 880308 induced either partial or even complete re- sponses and seems at least experimentally to Transplanted into kidneys of nude mice Number of tumors each from a different patient bronchial tumors B 88 0308 I I 5 be the most effective cytostatic compound on this tumor type (see Tahle 1) [49]. Bone Doxorubicin I 3 2 I 3 marrow toxicity seems to be the dose limiting colon tumors B 880308 Doxorubicin I 3 toxicity [40]. In cooperation with the NCI, ovarian tumors B 88 0308 2 I Bethesda, USA, further preclinical com- parative studies are ongoing now to evaluate Doxorubicin 2 in more detail the antitumoral activity and Transplanted subcutaneously into nude mice toxicity of this compound. bronchial tumors B 88 0308 I Doxorubicin 2 colon/Gl tumors B 88 0308 2 Doxorubicin 2 ovarian tumors B 88 0308 4 Doxorubicin 5

factor receptors coded by protooncogenes In addition some tumors have truncated or and oncogenes. Since that time it is known mutated growth factor receptors. Such trun- that growth factors like epidermal growth cated receptors may lack the cell external Na+'o- Na+'O' I factor (EGF), transforming growth factor a part responsible for binding of the growth , 0=8=0 (TGFa), insulin like growth factor 1 (IGFI), factor. They possess, however, the cell inter- 0= 8 =0 oI o~o, 0 fibroblast growth factor (FGF), platelet de- nal part, which is constitutively activated. o~o rived growth factor (PDGF), and other fac- This constituted activation leads to a persist- 0=5=0 ? tors are mitogens, i.e. they stimulate normal ent growth stimulation, which can not be Na+.O' o=~=o Na+·O· cells as well as tumor cells to proliferate. In regulated by different concentrations of part these factors are produced by the tumor growth factors because the cell external part HoeJBay 946 cells themselves (autocrine stimulation), in of the receptor is lacking [41]. part they are produced by neighboring cells The aim of our research was to find (paracrine stimulation) irrespective, wheth- compounds able to inhibit TPK. At the be- OH 0 er they are tumor cells or normal cells. ginning of this work it was completely un- For being mitogenic the growth factors clear, whether TPK of different sources are bind to a specific receptor on the membrane different to such a degree that inhibitors can HO of the target cell. This binding is of a relative be found, which inhibit with preference high affinity (binding constants in the range growth of tumors [4]. Consequently, we of 109_1010 limo!). Binding of the growth screened for inhibitors with the use of the factor to its receptor leads to dimerization of TPK of the EGF receptor prepared from L868275 neighboring receptors and activation of the tumor cells [42]. According to our hopes we tyrosine phosphokinase (TPK), which is the found two different compounds, which in- cell internal partofthereceptor. The activated hibit the EGF receptor-associated TPK, block Growth Factor Inhibitors TPK activates via phosphorylation of serine the cell growth stimulating activity of growth kinases, which transfer the activation signal factors like EGF and bFGF, and suppress Quite different to cytostatic research is within the intracellular signal transduction. tumor proliferation in vitro and in vivo. To the project to find inhibitors for growth Nearly 70-80% of all tumors expose a con- our surprise both inhibitors seem not to factors. We started this project about 5 years siderable number of growth factor receptors effect at tumor therapeutic dosages the TPK ago based on the physiological data on cell (103-106 per cell) and consequently are in- of other origin as for instance the TPK of the growth control, on growth factors, and growth fluenced in their growth by growth factors. insulin receptor. FORSCHUNG - TECHNOLOGIE 313 CHI MIA 45 (1991) Nr. 10 (Oklobcr)

ficient specificity for tumor associated anti- Table 2. Enzyme Inhibition and Antiproliferative Activity of HoelBay 946 [43a] gens can be generated. After labeling, e.g. 99m Enzyme inhibition with Technetium (Tc ), such antibodies EGF receptor TPK lCso 6.0 J.Lg/ml are able to localize tumors of more than 0.5- PKA IC50 884.011g/ml I cm in diameter to such a degree that an immunoscintigraphic detection in the pa- In vitro anti proliferative activity tient is possible. Extensive clinical studies showed e.g. that in ca. 80-90% of patients activity on human tumors IC50 (lung, breast, ovar, prostate, (6 of 6 cell lines) recurrences or metastases of colon carcino- adrenal carcinoma, and sarcoma) mas can correctly be diagnosed by immuno- scintigraphy with the help of a Tc99m labeled 3 days incubation with drug > 1000 J.Lg/mI monoclonal antibody specific for CEA (BW 18 days incubation with drug 4-200 J.Lg/ml 431/26). In a number of patients this diag- nostic procedure revealed information, which In vivo antiproliferative activity on human tumors were decisive for therapy and could not be achieved with any other conventional diag- transplanted into kidney of acti ve on 10 of 15 tumors nude mice tumor regression between nostic procedure (for a review, see [37]). In (bronchial, gastrointestinal, 70 and 30% face of these advantages we are now pre- breast, kidney, and adrenal tumors) paring a drug license application for the antibody BW 431/26 in Europe. transplanted subcutaneously active on 2 of 2 tumors into nude mice tumor regression between The clinical in vivo diagnostic investi- (bronchial tumors) 40 and 30% gations made it possible to determine in the patient the distribution of murine antibodies, the localization rate in normal tissues and in Table 3. Emyme Inhibition and Antiproliferative Activity ofL 86 8275 143b] the tumor, and the organspecific metaboli- Enzyme inhibition zation.

EGF receptor TPK/CsO 10 J.Lg/ml According to these clinical investiga- PKA IC50 58 J.Lg/ml tions we know that the localization rate is (at its maximum) ca. 0.01 % of the applied anti- In vitro anti proliferative activity body per gram tumor (fora review, see [37]). activity on murine tumors IC50 Moreover, we know that the antibody spe- I h incubation with drug 63000 ng/ml cifically localized in the tumor is slower 10 days incubation with drug 50 ng/ml metabolized than the remaining 99.9% of cross-resistance no cross-resistance to Doxorubicin the administered antibody in blood or in activity on human tumors IC50 normal tissues (preferently liver and lung), T cclls (3 cell lines) 100 ng/ml which is metabolized within a period of ca. breast carcinoma (6 cell lines) 60-10 ng/ml lung carcinoma (6 cell lines) 9-80 ng/ml 10-14 d [45]. The antibodies binding to prostate carcinoma (3 cell lines) 500-600 ng/ml tumor antigens can be detected in tumor gastrointestinal tumors (I cell line) 60ng/ml tissues in considerable amounts even after 4 weeks [45]. In vivo activity on human tumors The rate of specific tumor targeting \IS. transplanted into kidney of nude mice active on 12 of 14 tumors background in normal tissues (a factor of 6- (bronchial, breast, ovarian, tumor regression between 70 and 20% 10 fold) is still at least by a factor of 10 too and colon tumors) low to allow successful therapy without damaging of normal tissues [37]. Conse- transplanted subcutaneously into nude mice active on 5 of 5 tumors tumor regression (bronchial, breast, eNS tumors) between 70 and 30% quently, in the majority of clinical studies for therapy of solid tumors with immunoto- xins, immunocytostatics, or radioimmuno- One compound is a polysulfated xylan Bethesda. To our knowledge Hoe/Bay 946 conjugates side effects have been observed (Hoe/Bay 946), which is currently in phase II as well as L 86 8275 are the first growth but altogether clinically significant tumor II clinical studies at the NCI, Bethesda, factor inhibitors, which inhibit tumor prolif- therapeutic effects have not been detected USA, to evaluate its maximal tolerated dose, eration in vivo in experimental systems and [46-53]. In leukemia and lymphoma the the dose limiting toxicity, and its antitumor- in an acceptable therapeutic dose range. It situation is more advantageous. In these al activity. Hoe/Bay 946 inhibits very se- remains to be seen, which therapeutic potency 'dispersed' tumors cells are directly acces- lectively the TPK and less the serine phos- these compounds will have in patients with sible for i.v. applied specific antibodies. phokinase (PKA). Hoe/Bay 946 is not active tumors of the , the lung, Consequently, the localization rate is much when applied perorally [43a] (see Table 2). and the breast (to name only the most impor- higher than in solid tumors. Thus, the chance The other compound is a derivative (L 86 tant frequent tumors being sensitive forTPK of an effective therapy with 'naked' antibo- 8275) of rohitukin, a natural flavon deriva- inhibitors). dies, with immunocytostatics, immunoto~- tive [43b] isolated out of dysoxylum binec- ins or antibody isotope conjugates is higher. tariferum. L 86 8275 inhibits the TPK as well Therapeutic activity of antibody conju- as the serine phosphokinase. Inhibition of Biphasic Tumor Therapy via Antibody gates in leukemia and lymphoma was ob- both types of enzymes may explain the very Targeting served in several clinical studies 1.54-57]. high antiproliferative effect of L 86 8275. This therapeutic activity of antibody conju- L 86 8275 is of broad antitumoral activity One of our most important project is the gates, however, competes with numerous irrespective, whether it is applied orally or evaluation of the therapeutic possibility of other therapeutic treatments, and it remains by parenteral routes [43b] (see Table 3). monoclonal antibodies. to be seen, which therapeutic treatment will L 86 8275 is now being evaluated inten- Data elaborated in various research be more effective. sively in various tumor pharmacological groups including ours coincidently show In contrast to leukemia and lymphoma assays at Behringwerke as well as at the NCI, that murine monoclonal antibodies with suf- most solid tumors are resistant to any kind of FORSCHUNG - TECHNOLOGIE 314 CHIMIA 45 (1991) Nr. 10 (Oktobcr)

jugate to generate the cytotoxic drug. The aspired result should be a local en- HlndUI Sail richment of the cytostatic drug at the tumor

pGIc site, sufficient to kill all tumor cells, irre- ~v I spective whether they have bound the anti- body enzyme conjugate or not. The ADEPT concept is presently being elaborated by different research groups. First results of clinical pilot studies are .,,,/ already available, which show on one hand ~H'"d'" Xbal that by ADEPT a regression of tumors resist- BamHI BamHI ant to conventional therapy is possible. On the other hand the results very clearly disclose CMV the problems. The most evident problem at enhancef the moment is the immunogenicity of the antibody enzyme conjugate [67]. Till now murine monoclonal antibodies pBRori were used to which xenogeneic enzymes (from bacteria or animals) were conjugated Fig. I. The intron/exon strucllIre of chemically. After application of such anti- the antihodyl f3-glucuronidase heavy body enzyme conjugates a humoral immune chain fusion gene and the plasmid I'ector pAB, which is lIsed for its response arises in the recipient, which inac- exprl'ssion tivates the antibody as well as the enzyme part of the antibody enzyme conjugate. Facing this problem our work had the aim to treatment and, therefore, represent the major cule. Big molecules localize slower than reduce the immunogenicity of the antibody challenge for tumor therapy in future. small molecules. The size difference be- enzyme conjugate by humanization of the Till now the original idea to treat solid tween the antibody enzyme fusion molecule murine antibody and by the selection of a tumors with specific monoclonal antibodies and native antibodies should not be impor- suitable human enzyme. endowed with optimal effector functions tant as far as the diffusion rate is concerned. We selected the murine antibody BW 431 / (i.e. which are cytolytic via activation of the Thus, it is likely that the experiences in 26 [68], which is specific for an epitope on complement system and/or via activation of pharmacokinetic made with antibodies ap- CEA and which already proved to be suita- cytotoxic cells) has not met with the expect- ply also to the antibody enzyme conjugates. ble for the immunoscintigraphy of tumors, ed success. It may be speculated that this Accordingly, the conjugates will be metab- We succeeded in the humanization of this failure may be caused either by a strong olized relatively quickly in normal tissues antibody that means we transplanted the resistance of cells of solid tumors to humoral and blood, while they will remain in the antigen binding parts of the variable region or cellular lytic mechanisms or by a quali- tumor for a longer period of time. In case a from the murine antibody into a human tative or quantitative insufficiency of the will be applied after the antibody antibody framework by using recombinant lytic mechanisms in tumor patients. enzyme conjugate has been metabolized in DNA technologies [69]. The resulting hu- Another simple reason at least in a part of normal tissues, this prodrug will diffuse into manized anti CEA antibody BW hu 431/26 the clinical studies may be that dosages of the tumor very quickly due to its small showed the same specificity and avidity as the monoclonal antibodies have been applied, molecular weight and should preferentially the parenteral murine antibody. which are too low to achieve a therapeutic or even exclusively be cleaved there by the To the Fab fragments of the humanized effect. enzyme moiety ofthe antibody enzyme con- antibody the human enzyme j3-glucuroni- Therefore, it is likely that future strate- gies for the therapy of solid tumors with monoclonal antibodies will not be effective without exogenous cytotoxic molecules. Ideal Iy, these cytotoxic molecules should be interstitial space blood vessel active only on the tumor site but not on nonnal tissue. An approach, which may ful- ~ fill this requirement, is the biphasic immu- ').'). nospecific enzyme-mediated chemothera- py, in literature named ADEPT (antibody 0 ~ ~ dependent enzyme-mediated prodrug ther- apy) [58-60]. \ cd ADEPT consists of two components. In ~ a first phase an antibody enzyme conjugate ~ ~~ (whereby the selected enzyme should not be present extracellularly, e.g. in human blood) " is injected. In a second phase a hydrophilic ~ relatively untoxic prodrug of small molec- ular weight is applied. The latter should be ~ cleaved into a lipo- and cytophilic cytotoxic ~ drug by the enzyme linked to the antibody. After application to a tumor patient, the ADEPT antibody enzyme conjugate will localize the tumor according to its antigen specificity. The kinetic of localization is dependent on the diffusion rate and the size of the mole- Fig. 2. Principal reaction of antihody directed enzyme-mediated prodrug therapy (ADEPT) FORSCHUNG - TECHNOLOGIE 315 CHIMIA 45 (1991) Nr. 10 (Oklobcr) dase was successfully fused via an adequate should localize the tumor and should be The antitumoral activity is of considerable peptide linker again using recombinant DNA metabolized in normal tissue and blood as broadness. We will see, whether this pre- technology [70] (Fig. i). We now have a cell described earlier. After waiting an adequate clinical activity can also be found in the line, which is producing the human antibody time the isotope y90, complexed with the tumor patient. enzyme fusion product' BW hu431 /26 (Fab)- chelating agent, is injected. A part of the Two possibilities of a biphasic antibody j3-glucuronidase'. Both, the antibody spe- complexed y90 is bound to the tumor via the mediated tumortherapy with exogenous tox ic cificity and avidity as well as the enzymatic bispecific antibody and can carry out its molecules are presented. The first step of activity, are fully maintained in this anti- radiotoxic effect for the tumor cells. Non- these biphasic therapeutic approaches is the body enzyme fusion protein [71]. bound isotope complexes are eliminated via application of an untoxic antibody fusion In addition to the antibody enzyme fusion the kidney very quickly. Using such a sys- protein. After the antibody fusion protein protein we now need the second component, tem we hope to get access to a successful has localized and is preserved at the tumor a suitable prodrug. The synthesis of this biphasic radioimmunotherapy of tumors. site but has already been metabolized in prodrug is in process in cooperation with The application of bispecific antibodies normal tissue and blood, the second step of different partners. As a first example we in biphasic treatments is also not restricted the treatment is performed, which consists synthesized etoposid 4'-fJ-glucuronide [72]. to tumor therapy. Similar as with ADEPT of the application of an exogenous cytotoxic Our hope for the near future is to have both the specific elimination of cells of the im- compound. components in sufficient amounts in our mune system, of virus infected cells or of In case the antibody fusion protein is an hands, so that we can perform the essential parasites or bacteria or the targeting of fi- antibody enzyme conjugate, the cytotoxic tumor pharmacological investigations. brinolysis [82] may be possible. According compound is an untoxic prodrug, which is to the chosen indication the target cell spe- cleaved into a cytotoxic drug at the tumor Etoposid 4'-6-g1ucoronide cificity of the bispecific antibody and the site by the enzyme linked to the antibody. In kind of the cytotoxic molecule has to be case the antibody fusion protein is a bispe- selected. cific antibody, the cytotoxic compound is As an alternative to therapy, bispecific e.g. an isotope (j3-emitter) complexed with a antibody molecules may be used for the in chelating agent. Only those isotope com- vitro and in vivo diagnosis of various dis- plexes, which bind to the tumor via the bis- eases. A speciality with an enormous poten- pecific antibody, can be radiotoxic. Isotope tial would be the use ofbispecific antibodies complexes, not bound by the antibody, will to increase the immunogenicity of vaccines be excreted very quickly via the kidney. [83]. Such bispecific antibodies should link Both approaches of the biphasic tumor the immunogen with MHC class II antigens. therapy represent a combination of target cell localization of the antibody part (first phase) and (second phase) of an antibody Summary directed cytotoxic activity of a synthetic exogenous compound. Three research projects for tumor thera- As the limits of the tumor therapeutic It can be foreseen that the field of appli- py are presented, which are based on the activity of antibodies as well as of synthetic cation of ADEPT is not restricted to tumor experience gained so far in tumor physiolo- cytotoxic compounds are more or less well- therapy (Fig. 2). Important therapeutic in- gy and therapy. The conception of and re- known, the combination of both may lead to dications may additionally be the elimina- search in these projects have been possible a new generation of drugs, which can not tion of cells of the immune system, of virus by the increasing knowledge in the growth only be of importance in the elimination of infected cells (e.g. in chronic HBV or HIV behavior and control of normal as well as tumor cells but also of immune cells, virus infection) or even of bacteria or parasites tumor cells and in the role of protooncogenes infected cells, blood clots, bacteriae, or par- being resistant to conventional chemother- and oncogenes, moreover, in the insight we asites. apy or antibiotics. Moreover, the ADEPT have achieved in the components of the system may also be suitable for local fibri- immune system, and in the interaction with The authors thank Mrs. Manuela Rogala for the nolysis of blood clots [73]. each other and tumor cells, and in the tech- skilled typing of the manuscript. In such additional therapeutic indica- niques of generating monoclonal antibodies tions different from tumor therapy the anti- and modifying them by antibody engineer- body specificity and the prodrug/drug mg. [I] A. Gilman, F.S. Philips, Science 1946. 103. 409. component in the ADEPT system has to be A bifunctional cytostatic (B 88 0308) has [2] A. Gilmann, Am. J. Surg. 1963, 105, 574. exchanged or adapted accordingly. been synthesized consisting in an intercalat- [3] S. Farber, L.K. Diamond, R.D. Mercer, R.F. A further possibility for a biphasic therapy ing and a sugar moiety with an Sylvester, Jr., J.A. Wolff, Nell' Engl. J. Med. with the use of exogenous cytotoxic mole- epoxy site chain. Its preclinical antitumoral 1948,238,787. 14] H.H. Sedlacek, D. Hoffmann, G. Schulz,J. Czech, cules is the application ofbispecific antibody activity is superior to conventional anthra- B. Greifenberg, G. Dickneite, H.-P. Kraemer, molecules [74]. Hereby one specificity of cyclines. No cross-resistance to any known 'Contributions to Oncology', Karger Verlag, the bispecific antibody is binding to the cytostatic compound could be detected. Miinchen, 1989, Vol. 34. target cell, e.g. tumor cell, the second spe- Special activity could be observed in human [5] J.E. McCormick, R.S. McElhinney, Ellr. 1. cificity is catching the exogenous cytotoxic ovarian carcinoma, and it remains to be seen, Cancel' 1990, 3,207. [61 P. Hilgard, Cancer Trealmelll Rev. 1987, /4, 187. molecule [75-79]. Currently, we are en- whether this compound will clinically fulfill [7] D. Newell, A. Gescher, St. Harland, D. Ross, Ch. gaged in construction of such bispecific the expectations, which arose out of the Rutty,CancerChemother. Pharmaeol.1987, 19, antibody molecules by recombinant DNA preclinical studies. 91. technology [80][81]. As cytotoxic molecule Two growth factor inhibitors could be [8] C. Wasternack, B. Hause, Die Pharma:ie 1987, 2,73. we selected a complex formed by a chelating found (Hoe/Bay 946, a poly sulfated xylan, [9] P.J. O'Dwyer,S.A. King, D.F. Hoth, B. Leyland- agent and y90, an emitter of {3-radiation. In and L 86 8275, a flavone derivative origi- Jones, Cancel' Res. 1987,47,3911. the complex with the chelating agent y90 is nating from rohitukin), which both block the [10] J.H. Beijnen, H. Lingeman, 1-l.A. van Munster, quickly secreted via the kidney and looses growth factor receptor associated tyrosine W.J.M. Underberg,./. Pharmae. Biomed. Anal. its affinity for bones and its toxicity for bone phosphokinase to such a degree and specif- 1986,4,275. [11] R.B. Weiss,G.Sarosy, K.Glagell-Carr.M. Russo, marrow. icity that in vitro as well as in vivo a signif- B.Leyland-Jones, Cancel' Chemolher. Pharma- After application the bispecific antibody icant antitumoral activity could be observed. col. 1986, 18, 185. FORSCHUNG - TECHNO LOGIE 316 CHIMIA 45(1991) Nr. 10 (Oktobcr)

[12] C.J. Allegra, J.e. Drake, J. Joliven, B.A. Chab- D.G. Haller, J.A. Laurie, PJ. Goodman, 1.S. body Immunoconjugates, San Diego, 1991. ner, Pmc. Natl. Acad. Sci. U.S.A. 1985, 82,4881. Ungerleider, W.A. Emerson, D.e. Tormey, J.H. [56] D.M. Goldenberg, 6th Int. Conf. on Monoclonal [ 13] A.R. Beard, M.G.B. Drew, P. Hilgard, B.D. Glick, M.H. Veeder, J.A. Mailliard,New Engl.J. Antibody Immunoconjugates, San Diego, \991. Hudson, J. Mann, S. Neidle, L.F.T. Wong, Anti- Med. 1990, 352. [57] W.A. Blattler, 6th Int. Conf. on Monoclonal Cancer Drug Design 1987, 2, 247. [35] J.F. Smyth, Cancer Treatment Rev. 1988, /5, Antibody Immunoconjugates, San Diego, 199 I. [14] J. Haiduc, C. Silvestru, Coord. Chem. ReI'. 1990, supp!. A 3. [58] G.W.Philpott, R.I. Bower, Ch.W. Parker, Surgery 99,253. [36] J Atzpodien, H. Kirchner, KUn. Wochenschr. 1973, 74, 51. [15] J.G.M.Klijn, F.H.deJong,G.H. Bakker, St.W.J. 1990,68, I. [59] G.W. Philpott, R.I. Bower, K.L. Parker, W.T. Lamberts, C.J. Rodenburg, J. Alexieva-Figusch, [37] H.H. Sedlacek, G. Schulz, A. Steinstraesser, L. Shearer, Ch.W. Parker, Cancer Res. 1974,34, Cancer Res. 1989,49,2851. Kuhlmann, A. Schwarz, L. Seidel, G. Seemann, 2159. [16] HJ. Harmsen, Jr., AJ. Porsius, Eur. J. Cancer H.-P. Kraemer, K. Bosslet, 'Contributions to [60] G.W. Philpott, E.H. Grass, Ch.W. Parker, Can- CUn. Oncol. 1988,24, 1099. Oncology. Karger Verlag, Miinchen, 1988, p. 32. cer Res. 1979,39,2084. [17] A. Howell, OJ. Dodwell, H.Anderson,Bailliere's [38] U. Stache, H.H. Sedlacek, D. Hoffmann, H.-P. [61] P.L. Carl, 'Development of target-oriented anti- Clin. Endocr. Metah. 1990,4,67. Kraemer, J. Cancer Res. Clin. Onco!.1990, / /6, cancer drugs.' Raven Press ••, 1983. [18] J.M. Cassady, W .M. Baird, e. Chang,J. Nat. Prod. supp!. part I, 439. [62] K.D. Bagshawe, Br. J. Cancer 1987, 56, 531. 1990,53,23. [39] P. Hermentin, E. Raab, e. Kolar, M. Gerken, D. [63] K.D. Bagshawe, Br. J. Cancer 1989,60,275. [19] WJ. Slichenmyer, D.O. Von Hoff, .I. Clin. Hoffmann, H.-P. Kraemer, U. Stache, Eur. Pat- [64] P.O. Senter, GJ. Schreiber, D.L. Hirschberg, Pharmacol. 1990,30,770. entanmeldung. VerOffentlichungsnr.: 0345598. S.A. Ashe, K.E. Hellstrom, 1. Hellstrom, Cancer [20] B. Winograd, M.W. Lobbezoo, H.M. Pinedo, Anmeldenr.: 89109689.3 1989. Res. 1989,49,5789. J.H. Beijnen, F.E. Durr, 6th NCI-EORTC sym- [40] D. Hoffmann, M. Gerken, P. Hermentin, H.H. [65] D.E. Kerr, P.D. Senter, W.V. Burnett, D.L. Hir- posium on new drugs in cancer therapy, Amster- Sedlacek, 82nd Annual Meetingofthe AACR for schberg, 1. Hellstrom, K.E. Hellstrom, Cancer dam, 1989. cancer research, Houston, 1991. Immunol. Inlllllfllotiler. 1990,3/,202. [21] J.M. Brown,E.M. Zeman, K.A. Bidermann, M.J. [41] A. Ullrich, 1. Schlessinger, Cell 1990, 61,203. [66] P.D. Senter, FASEB J. 1990,4, 188. Lemmon, M. Tracy, 6th NCI-EORTC symposi- [42] 1. Czech, H.H. Sedlacek, Cancer Cilemother. [67] K.D. Bagshawe, 6th Int. Conf. on Monoclonal um on new drugs in cancer therapy. Amsterdam, Pharmacol. 1989,24, supp!. 2, 72. Antibody Immunoconjugates, San Diego, 1991. 1989. [43] a) J. Czech, D. Hoffmann, G. Dickneite, C.E. [68] K. Bosslet, A. Steinstraesser, A. Schwarz, H.P. [22] R.L. Tuttle, V.S. Lucas, N.J. Clendeninn, J.D. Myers, H.H. Sedlacek,.I. Cancer Res. CUn. On- Harthus, G. Lueben, L. Kuhlmann, H.H. Sed- Purvis, 6th NCI-EORTC symposium on new col. 1990, / /6, supp!., part 1,474; b) 1. Czech, D. lacek, Eur . .I. Nuc/. Med. 1988, /4,523. drugs in cancer therapy, Amsterdam, 1989. Hoffmann, Ch.E. Myers, 1.D. Horak, E.A. Saus- [69] D. Guessow, G. Seemann, Meth. En:ymol. (ac- [23] e. Sessa, M. Zucchetti, Y. Willems, M. D'incalci, ville, H.H. Sedlacek, 4th Int. Congress on Hor- cepted for publication, 1991). F. Cavalli, 6th NCI-EORTC symposium on new mones and Cancer, Amsterdam, 1991. [70] G. Seemann, P. Lorenz, 1. Czech, H.H. Sedlacek, drugs in cancer therapy, Amsterdam, 1989. [44] R.G. Naik, B. Lal, R.H. Rupp, H.H. Sedlacek, G. M. Schuermann, K. Bosslet, in preparation. [24] 1.P. Armand, G. Recando, A. Gouyette, M. Bele- Dickneite, J. Czech, Patentanmeldung Nr.: [71] K. Bosslet, J. Czech, H.H. Sedlacek, G. See- rahdek, M. Bonnay. G. Chabot. C. L'Homme. 89119710.5,1989. mann, 8th Int. Hammersmith Meeting, Greece. 6th NCI-EORTC symposium on new drugs in [45] R.HJ. Begent, 6th Int. Conf. on Monoclonal 1991. cancer therapy, Amsterdam, ]989. Antibody Immunoconjugates for Cancer, San [72] C. Kolar, J. Czech, K. Bosslet, G. Seemann, H.H. [25] A.M. Casazza, J.E. Schurig, S. Forenza, B.H. Diego, 1991. Sedlacek, Patentanmeldung Nr. P 39 35 016.9 Long, W.C. Rose, J.J. Catino, H. Kamei, Y. [46] T. Takahashi, T. Yamaguchi, K. Kitamura, H. 1989. Nishiyama, W.T. Bradner, R. Trail,J. Matson, A. Suzuyama, M. Honda, T. Yokota, H. Kotanagi, [73] Ch. Bode, G. R. Matsueda, K. Y. Hui, E. Haber, Crosswell, T.W. Doyle, 6th NCI-EORTC sym- M. Takahashi, Y. Hashimoto, Cancer 1988, 6/, Science 1985, 229, 765. posium on new drugs in cancer therapy, Amster- 881. [74] O. Nolan, R. O'Kennedy,Biocilim. Biophys.Acra dam. 1989. [47] P. von Wussow, L. Spitler, B. Block, H. Deicher, 1990, /040, I. [261 G.B. Grindey, L.W. Hertel, V. Heinemann, W. Eur.1. Cancer Clin. Oneal. 1988,24, supp\. 2, [75] U. Sahin, F. Hartmann, P. Senter, Ch. Pohl, A. Plunkett, P.J. Houghton, J.A. Houghton, 6th NCI- 69. Engert, V. Diehl, M. Pfreundschuh, Cancer Res. EORTC symposium on new drugs in cancer [48] B.J. Gould, MJ. Borowitz, E.S. Groves, P.W. 1990, 50, 6944 . therapy, Amsterdam, 1989. Carter, D. Anthony, L.M. Weiner, A.E. Frankel, [76] 1.L. Phelps, D.E. Beidler, R.A. Jue, B.W. Unger, [27] R.L. Schilsk'y, M. Ratain, J. Grayhack, K. J. Natl. Cancer /nst. 1989,8/,775. MJ. Johnson,.I. Immunol. 1990, /45, 1200. Breedlove, S. Lucas. R. Tuttle, 6th NCI-EORTC [49] L.M. Weiner,J. O'Dwyer,J. Kitson. R.L. Comis, [77] W. Smith, V.A. Gore, D.R. Brandon. D.N. Lynch, symposium on new drugs in cancer therapy, A.E. Frankel, R.J. Bauer, M.S. Konrad, E.S. S.A. Cranstone, J.R.F. Corvalan, Cancer Immll- Amsterdam, 1989. Groves, Cancer Res. 1989,49,4062. nol. Immllnother. 1990.3/, 157 . 1281 PJ. Hoghton, J.A. Houghton, G.B. Grindey, 6th [50] V.S. Byers, R. Rodvien, K. Grant, L.G. Durrant, [78] S. Honda, Y. Ichimor, S. Iwasa, Cytotechnology NCI-EORTC symposium on new drugs ineancer K.H. Hudson, R.W. Baldwin, PJ. Scannon, 1990,4,59. therapy, Amsterdam, 1989. Cancer Res. 1989,49,6153. [79] J.-M. Le Doussal, A. Gruaz-Guyon, M. Martin, [29] H.P. Kraemer, H.G. Berscheid, H. Ronneberger, [51] St.M. Larson, A. Raubitschek, 1.e. Reynolds, E. Gautherot, M. Delaage, 1. Barbet, Cancer Res. H. Zilg, H.H. Sedlacek, 5th NCI-EORTC sym- R.D. Neumann, K.-E. Hellstrom, I. Hellstrom, D. 1990,50,3445. posium on new drugs in cancer therapy, Amster- Colcher, J. Schlom, E. Glatstein,J.A. Carrasquil- [80] K. Bosslel, P. Hernlentin, L. Kuhlmann, A. dam, 1986. 10, Nllc/. Med. Bioi. 1989, /6, 153. Steinstraesser, G. Seemann, H.H. Sedlacek, [30] J. Verweij, M.E.L. van der Burg, W.LJ. von [52] J.B. Alexander, M.B. McEwan, 6th Int. Conf. on Cancer Treatment Re\'. 1990, 17,355. Putten, H.P. Kraemer, E. Weidmann, G. Stoler, Monoclonal Antibody Immunoconjugates for [81] K. Bosslet, A. Steinstraesser, P. Hemlentin, L. Proceedings 4th European conference on clinical Cancer, San Diego, 1991. Kuhlmann, A. Bruynck, M. Magerstaedt, G. oncology and cancer nursing, Madrid, 1987. [53] D.G. Haller, 6th Int. Conf. on Monoclonal Anti- Seemann, A. Schwarz, H.H. Sedlacek, submitted [31] G. Falkson, H.e. Falskon, A.S. Alberts, Eur. 1. body Immunoconiugates for Cancer, San Diego, to Br. J. Cancer Cancer 1990, 26,71. 1991. [82] M.S. Runge, Ch. Bode, Ch.E. Savard, G.R. [32] H.H. Sedlacek, 'Beitrage zur Onkologie', Karger [54] B. Parker, 6th Int. Conf. on Monoclonal Anti- Matsueda, E. Haber, Bioconjugate Chem. 1990, Verlag, Miinchen, 1987, p. 25. body Immunoconiugates for Cancer, San Diego, 1,274. 1331 D. Newling, Cancer 1990, 61,497. 1991. [83] D.P. Snider, A. Kaubiseh, D.M. Segal, J. Exp. [34] Ch.G. Moertel, T.R. Fleming, J.S. Macdonald, [55] e.R. Divgi, 6th Int. Conf. on Monoclonal Anti- Med.1990, /7/, 1957.