Specific Activation of the Prodrug Mitomycin Phosphate by a Bispecific Anti-CD30/Anti-Alkaline Phosphatase Monoclonal Antibody1

[CANCER RESEARCH 50. 6944-6948. November 1, 1990] Specific Activation of the Prodrug Mitomycin Phosphate by a Bispecific Anti-CD30/Anti-Alkaline Phosphatase Monoclonal Antibody1

Ugur Sahin, Frank Hartmann, Peter Senter, Christoph Pohl, Andreas EngerÃ,Volker Diehl, and Michael Pfreundschuh2

Laboratory of Tumor Biology, Medizinische Klinik I University of Cologne, D-5000 KÃ¶ln41, Federal Republic of Germany [U. S., F. H., C. P., A. E., V. D., M. P.], and Oncogen, Seattle, Washington 98121 [P. SJ

ABSTRACT occur as a result of the heterogeneity and instability of the malignant cell population within a given tumor (3). Finally, the The bispecific monoclonal antibody (Bi-MAb) HRS-3/AP-1 was de modification of antibodies with toxins or cytotoxic drugs by veloped by somatic hybridization of the 2 mouse hybridoma cell lines HRS-3 and AIM, which produce monoclonal antibodies with reactivity chemical means carries the risk of changing the binding prop against the Hodgkin's- and Reed-Sternberg cell-associated CD30 antigen erties of the antibody. and alkaline phosphatase, respectively. After an active incubation with An approach to address these problems has been described alkaline phosphatase, purified whole immunoglobulin molecules and in which antibodies are used to target enzymes that are capable F(ab')2 fragments of the Bi-MAb were equally effective in converting a of converting relatively nontoxic prodrugs into active anticancer relatively noncytotoxic prodrug, mitomycin phosphate (MOP), into ini- agents (6-8). It was postulated that the drugs thus formed at tomycin alcohol, which was 100 times more toxic to the Hodgkin's- and the surface of the antigen-positive tumor cells should be able to Reed-Sternberg cell line L540 (CD30+) than MOP. The cytotoxic activity act also on antigen-negative cells nearby (6, 8). of MOP was unaffected when the cells were pretreated with either the In order to circumvent the need to form MAb-enzyme con Bi-MAb or the enzyme alone. The Bi-MAb HRS-3/AP-1 did not bind to HPB-ALL cells (CD30") and was not able to activate MOP on these jugates chemically, we have developed a Bi-MAb that binds both AP and the CD30 antigen that is present on the surface cells. In cocultivation experiments with HPB-ALL and L540 cells, the of the Hodgkin's-derived cell line L540. Such Bi-MAbs may activation of MOP by the Bi-MAb HRS-3/AP-1 and alkaline phospha tase led to considerable cytotoxicity against the antigen-negative by have advantages over chemically formed conjugates (6-8) be stander cells. Thus, this immunotherapeutic approach might be effective cause of their uniform composition and low molecular weight. in tumors in which not all the tumor cells express the respective tumor We now demonstrate that our CD30/anti-AP Bi-MAb is useful for the specific activation of MOP on CD30+ Hodgkin's antigen. cells in vitro. Because the released drug can kill nearby antigen- negative cells, this immunotherapeutic approach might also be INTRODUCTION effective in tumors in which not all the cells carry the respective tumor-associated antigen. Since the establishment of the hybridoma technique for the production of MAbs3 with defined specificities by KÃ¶hlerand Milstein (1), many attempts have been made to use MAbs with MATERIALS AND METHODS selective reactivity to cell surface structures on tumor cells as carriers for cytotoxic drugs (2), toxins (3), and radioisotopes Hybridoma Cell Lines. The production of the parental hybridoma cell lines AP-1 (reactive against bovine alkaline phosphatase) and HRS- (4) in order to deliver high tumoricidal doses to the malignant 3 (reactive against the Hodgkin's and Reed-Sternberg cell-associated target cells while minimizing side effects to nonmalignant cells. Despite impressive advances made in in vitro and in vivo models, CD30 antigen and only a small subpopulation of normal cells) have there remain considerable problems with these immunoconju- been described (9-11). Both antibodies are of the IgGl subclass. Development of the Hybrid-Hybridoma Cell Line HRS-3/AP-1. A gates. Due to the limited number of cytotoxic molecules that hypoxanthine-guanine phosphoribosyltransferase-deficient subclone of can be bound to an immunoglobulin without interfering with the hybridoma cell line HRS-3 was obtained by growing this hybridoma its antigen-binding capacity, sufficient concentrations of cyto in the presence of increasing concentrations of 8-azaguanine (GIBCO, toxic drugs in the tumor are achieved with great difficulty (5). Karlsruhe, Federal Republic of Germany) up to a final concentration Immunotoxins, such as MAb-ricin A chain are more potent, of 0.1 mM. AP-1 cells were treated immediately before fusion with 5 since it has been shown that a single toxin molecule can kill a mivi iodoacetamide in PBS for 5 min on ice according to the method tumor cell if the toxin reaches its specific site of action (3). This of Clark and Waldmann (12). Hypoxanthine-guanine phosphoribosyltransferase-deficient HRS-3 cells (5 x IO6) and iodoacetamide-pre- is, however, not possible if the antibody binds to an antigen treated AP-1 cells (1.5 x IO7)were mixed and fused using polyethylene that is not internalized or is degraded in lysosomes. Moreover, glycol as described (13). After fusion, cells were washed extensively, a prerequisite for the effectiveness of immunotoxins for treating resuspended at a concentration of IO6cells/ml in RPMI-1640 medium tumors is the expression of the immunotoxin-binding antigen containing 10% fetal calf serum and 1% hypoxanthine, aminopterin, on nearly all of the tumor cells, an event that is unlikely to and thymidine (GIBCO) and seeded into the wells of a 96-well micro- liter plate (Nunc, Wiesbaden, Federal Republic of Germany). Two Received 4/17/90; accepted 7/17/90. The costs of publication of this article were defrayed in part by the payment weeks after plating, supernatants from wells with growing clones were of page charges. This article must therefore be hereby marked advertisement in tested for the presence of Bi-MAbs using a modified form of the accordance with 18 U.S.C. Section 1734 solely to indicate this fact. previously described APAAP (14). In short, 5 M'of a suspension of the 1This study was supported by a grant from the Deutsche Forschungsgemein Hodgkin's-derived cell line L540 (10" cells/ml in RPMI 1640 + FCS) schaft. 2To whom requests for reprints should be addressed, at Medizinische Klinik were seeded into the wells of a 60-well Terasaki plate (Nunc) and fixed I University of Cologne, Josef-Stelzmann-Strasse 9, D-5000 KÃ¶ln41, Federal with 0.1% (v/v) glutaraldehyde in PBS. Five /jl of the supernatants Republic of Germany. 'The abbreviations used are: MAb, monoclonal antibody; AP, alkaline phos were incubated for 30 min. After washing, cells were incubated with phatase; APAAP, alkaline phosphatase/anti-alkaline phosphatase assay; Bi-MAb, AP (0.1 mg/ml) in PBS for 30 min. After repeated washing, the reaction bispecific monoclonal antibody; MOP, mitomycin phosphate; PBS, phosphate- was developed by incubation with 0.2 mg/ml naphthol-AS-Bl-phos- buffered saline; PCS, fetal calf serum; MMC, mitomycin C. phate and 1 mg/ml Fast Red TR salt (5-chloro-2-toluenediazonium 6944

Downloaded from cancerres.aacrjournals.org on September 30, 2021. © 1990 American Association for Cancer Research. PRODRUG ACTIVATION BY BISPECIFIC MONOCLONAL ANTIBODIES chloro-hemi-zinc chloride; Sigma, Munich, Federal Republic of Ger- addition of Nonidet P-40 (Sigma, Munich) to a final concentration of many) in Tris/HCl (pH 8.2) until a red reaction product developed. 2% (v/v). The hybrid hybridoma cell line producing the supernatant with the Cold Target Assay. To determine the effect of MOP activation on strongest reactivity in this modified APAAP was designated HRS-3/ antigen-negative cells, unlabeled L540 (IO4 cells/ml) and 5'Cr-labeled AP-1 and subcloned repeatedly by limiting dilution until a stable HPB-ALL cells (IO4 cells/ml) were pretreated as described previously, subclone was obtained. mixed at a ratio of 1:1, and incubated with MOP, MMC, or medium Purification and Characterization of the Bi-MAb HRS-3/AP-1. Anti as described above. bodies were produced in BALB/c mice by injection of 0.5-3 x IO6 hybrid hybridoma cells. An /V-hydroxysuccinimide-agarose column (Affi-Gel 15; Pharmacia, Freiburg, Federal Republic of Germany) was RESULTS coated with calf intestinal AP (Sigma) according to the manufacturer's Production of Bispecific Monoclonal Antibody. After fusion instructions. For immunoadsorption of HRS-3/AP-1, undiluted culture of 5 x IO6 HRS-3 cells with 1.5 x IO7 AP-1 cells, 28 clones supernatants or a 1:10 dilution of ascites in PBS was processed over grew out in the selective medium. The supernatants of 26 clones the AP/Affi-Gel 15 column and the bound fraction was diluted with a were positive in the screening assay using the modified APAAP 0.1 M sodium phosphate buffer gradient ranging from pH 7.2 to pH method (see "Materials and Methods") indicating the produc 3.0. The protein concentration of the eluate was monitored by deter tion of Bi-MAbs with reactivity against both CD30 and AP. mining the absorption at 280 nm. Aliquots of the eluate were tested for the presence of Bi-MAb as described above. Bi-MAb-containing sam The clone that produced the supernatant with the highest ples were analyzed by sodium dodecyl sulfate-polyacrylamide gel elec- reactivity was selected for repeated subcloning and established trophoresis under reducing conditions in 10% slab gels according to as Bi-MAb HRS-3/AP-1. Ascites containing the Bi-MAb was the method of Laemmli (15). Protein staining was performed using precipitated by ammonium sulfate and fractionated over an AP Coomassie brilliant blue (Pharmacia). column. Of the different antibody combinations produced by Preparation of F(ab')2 Fragments. F(ab')2 fragments of HRS-3/AP- the hybrid hybridoma cell line HRS-3/AP-1, MAbs with bi- 1 were prepared according to the method of Lamoyi (16). The purified valence (monospecific MAbs) and monovalence (i.e., bispecific Bi-MAbs were dialyzed against 0.1 M sodium acetate buffer (pH 7.0) at 4Â°Covernight. Immediately before digestion, the pH of the protein MAbs, the second valence being directed against the CD30 solution was adjusted to 4.2 by adding acetic acid, and incubated at antigen) for AP were bound to the column, while the antibodies 37Â°Cwitha 3:100 (w/w) pepsin:immunoglobulin ratio in 0.1 M acetate without reactivity to AP (i.e., monospecific anti-CD30 MAbs) buffer, pH 4.2. The reaction was stopped after 4 h by adding 0.5 N were found in the flowthrough. The elution curve of the AP- NaOH to a final pH of 8.0. The solution was then dialyzed against column showed 2 peaks, 1 at pH 5.3 and 1 at pH 3.4. The latter PBS. F(ab')2 fragments were purified on a protein A-Sepharose column is identical to the peak obtained with purified AP-1 (Fig. 1). that was equilibrated and washed with 1.5 M glycine; 3 M NaCl, pH The peak at pH 5.3 contained the Bi-MAb HRS-3/AP-1 as 8.9: followed by 0.1 M citrate, pH 3.0. F(ab')2 fragments were collected demonstrated by reactivity in the modified APAAP on L540 in the flow-through and undigested immunoglobulin was found in the cells, while the second peak contained MAbs with reactivity to acid eluate. AP only. Prodrugs and Cytotoxic Drugs. MMC was obtained from Medac The purity of the antibody preparations was then analyzed (Hamburg, Federal Republic of Germany). MOP was synthesized from on sodium dodecyl sulfate-polyacrylamide gel electrophoresis mitomycin A and 2-aminoethyldihydrogen phosphate as described pre (Fig. 2). The monospecific HRS-3 and AP-1 MAbs can be viously (8). Target Cells. The CD30+ Hodgkin's-derived cell line L540 (17) and distinguished by their different light chains, whereas their heavy the CD30~ cell line HPB-ALL, which is derived from a human acute chains cannot be distinguished electrophoretically. The peak at T-lymphocytic leukemia, were used for cytotoxic assays. All cell lines pH 5.3 contained 2 light chains that represented the light chains were cultured in RPMI 1640 supplemented with 10% fetal calf serum of each fusion partner in the Bi-MAb HRS-3/AP-1. (GIBCO). Trypan Blue Dye Exclusion Assay. Over a wide range of Trypan Blue Dye Exclusion Assay. Target cells (4 x 105/ml) were concentrations, MMC proved to be about 100-fold more toxic pretreated at 37Â°Cfor 30 min with the Bi-MAb HRS-3/AP-1 (10 ^g/ to the Hodgkin's-derived L540 cells than MOP (Fig. 3). When ml). After washing, cells were incubated with AP (0.1 mg/ml) for 30 the Hodgkin's cells were incubated with MOP after pretreat min. Untreated cells and cells pretreated with the Bi-MAb HRS-3/AP- ment with both the Bi-MAb HRS-3/AP-1 and AP, the cytotox 1 alone or AP alone were used as controls. One hundred n\ of the cell suspension (4 x 105/ml in RPMI 1640 + 10% FCS) were seeded into icity of MOP increased to the same level as MMC. In contrast, the wells of a 96-well microtiter plate, followed by incubation with pretreatment by either the monospecific antibody HRS-3, the various concentrations of MMC or MOP (ranging from 0.1 pM to 1 Bi-MAb HRS-3/AP-1, or AP alone did not alter the cytotoxic HIM)for 24 h at 37Â°C,5%CO2. Cells were harvested, mixed with trypan potency of MOP. blue solution, and counted. All experiments were performed in quad Similar to the Hodgkin's-derived cell line L540, the human ruplicate and repeated at least 3 times. acute T-lymphocytic leukemia cell line HPB-ALL proved to be Chromium Release Assay. The chromium release assay was per 100 times more sensitive to the cytotoxic activity of MMC formed according to the method of Sanderson (18) with the following compared with the prodrug MOP. However, in contrast to the modifications. Cells were labeled with MCr (100 MCi/10" cells for 1 h) CD30+ cell line L540, pretreatment of the CD30~ HPB-ALL and washed 3 times. Labeled cells were pretreated as described for the cells with Bi-MAb and AP did not increase the cytotoxic activity dye exclusion test. One hundred ^1of a suspension of labeled pretreated and control cells (IO4 cells/ml in RPMI 1640 + 10% FCS) were seeded of MOP (Fig. 3). into the wells of 96-well microtiter plate. MMC (10 nM), MOP (10 5lCr Release Assay. The drugs and prodrugs were tested at MM),or medium was added for 18 h at 37Â°Cat 5% CO2. The superna 10 ÃŸMbasedon the reproducible levels of significant cytotoxic tants of each well were harvested and assayed for radioactivity. The activity of both MMC and the released drug mitomycin alcohol percentage of cytotoxicity was calculated as: (the active derivative of MOP) and the minimal cytotoxicity of the prodrug MOP at this concentration (Figs. 3 and 4). The Counts experimental release â€”spontaneous release amount of 51Cr release for MMC was significantly higher (91% 100 x Maximal release - spontaneous release from L540, 82% from HPB-ALL) than for the prodrug MOP Maximal release of 5'Cr was defined as the release obtained by the (1% from each cell line; Fig. 4). Again, Bi-MAb HRS-3/AP-1 6945

H 66

0.4 45

0.2 29

HRS-3/AP-1

1234567 M Fig. 2. Sodium dodccyl sulfate-polyacrylamide gel electrophoresis of MAb AP-1 (supernatant. Lane /), MAb HRS-3 (supernatant. Lane 2). purified Bi- MAb HRS-3/AP-1 [eluale peak at pH 5.3 (Fig. 1), Lanes 3 and 4; eluate peak at pH 3.4 (Fig. 1), Lanes 5 and 6], and MAb AP-1 [eluate peak at pH 3.4 (Fig. 1), lane 7]. The monospecific HRS-3 and AP-1 MAbs can be distinguished by their different light chains (Â¿).whereas their heavy chains (//) cannot be distinguished electrophorctically. The purified Bi-MAb HRS-3/AP-1 (Lanes 3 and 4) contains 2 light chains that represent the light chains of each fusion partner. M, marker proteins; numbers at righi, molecular weight in thousands.

DISCUSSION 0.2 Hybrid hybridoma cells produce the 2 parental light and AP-1 heavy chains, which are recombined by chance, so that as many

5.0 4.5 4.0 3.5 3.0 as 10 different immunoglobulin molecules are secreted into the pH supernatant (19). Purification of the Bi-MAb is mandatory in Fig. 1. Affinity chromatography of the Bi-MAb HRS-3/AP-1 (top) and of the order to separate the monospecific antibodies that can block MAb AP-1 (bottom) using an AP-conjugated column. The eluate contains 2 the functions of the Bi-MAbs. In the case of the Bi-MAb with peaks. The peak at pH 5.3 contains purified Bi-MAb HRS-3/AP-1. The peak at reactivity to the Hodgkin's-associated CD30 antigen and AP, pH 3.5 contains monospecific MAbs against AP and correlates with the peak of the MAb AP-1. purification was achieved using a column coated with AP. Since the monospecific antibodies with reactivity to AP have a higher alone was unable to increase the toxicity of the prodrug, whereas affinity to the antigen than do the Bi-MAbs, they can be eluted pretreatment with whole immunoglobulin molecules of the Bi- separately providing a highly purified preparation of Bi-MAb. Using the purified Bi-MAb HRS-3/AP-1 with reactivity to MAb and AP led to a cytotoxic activity against L540 that was the Hodgkin's-associated CD30 antigen and alkaline phospha- comparable with that of MMC (73%). Pretreatment of the CD30 antigen-negative HPB-ALL did not alter the toxic activ tase, we were able to demonstrate that the relatively nontoxic ity of the prodrug. The F(ab')2 fragments of the Bi-MAb HRS- prodrug MOP can be activated specifically on antigen-positive 3/AP-l were as effective as whole immunoglobulin molecules cells. The released drug mitomycin alcohol proved to be as toxic as the active compound MMC on a molar basis. We were in conferring prodrug activation after binding of AP (Fig. 4). Effect of Specific Prodrug Activation on Antigen-negative By able to demonstrate that the prodrug is specifically activated on the surface of the antigen-positive cell to which the enzyme stander Cells. To test whether the activation of the prodrug is bound via the Bi-MAb. The fact that F(ab'): fragments were MOP by the Bi-MAb and AP on CD30+ cells affects antigen- as effective as whole immunoglobulin molecules excludes the negative cells in the neighborhood of these cells, both unlabeled CD3CT L540 and MCr-labeled CD30 HPB-ALL cells were possibility that the binding of the BiMAb is mediated by Fc receptors. treated with Bi-MAb and AP and were mixed and incubated One advantage that antibody-enzyme complexes have over with the prodrug and the relevant controls (Fig. 5). Again, direct antibody-drug conjugates is that antigen-negative tumor MMC proved to be quite toxic to HPB-ALL. However, in cells in the neighborhood of antigen-positive cells can also be contrast to the results obtained when HPB-ALL cells were reached by the released drug. That this is indeed the case was incubated alone, the release of MCr from HPB-ALL cells in the demonstrated by the results of cocultivation experiments of the presence of L540 cells pretreated with Bi-MAb and AP in CD30+ L540 and the CD30" HPB-ALL cells: the released drug, creased significantly, even though the values of the release which is specifically generated on pretreated antigen-positive induced by the active compound MMC were not quite reached. Hodgkin's cells, can also gain access to antigen-negative cells Incubation of nonpretreated unlabeled L540 cells together with and exert cytotoxic activity. Thus, this immunotherapeutic Bi-MAb/AP-pretreated HPB-ALL cells did not increase the approach might be effective in vivo, where not all the tumor cytotoxicity of MOP (Fig. 5). cells carry the respective tumor antigen. It is expected that this 6946

100, L540 HPB-ALL

Fig. 3. Cytotoxicity of MMC and the prodrug MOP against the cells of the CD30* Hodgkin's-derived cell line L540 and the CD30" cell line HPB-ALL as determined by trypan blue exclusion. D, MMC and non-pretreated cells; O, MOP and non-pretreated cells; > +, MOP and cells pretreated with AP. A, MOP * and cells pretreated with Bi-MAb HRS-3/AP. *. MOP and cells pretreated with Bi-MAb HRS-3/AP and AP. Only pretreatment of the CD30* cell line L540 with both the Bi-MAb HRS-3/AP-1 and AP leads to an increase of the cytotoxicity of the prodrug against these cells.

DRUG CONCENTRATION DRUG CONCENTRATION approach will lead to some normal cell destruction, but hope MAb-enzyme complex-bearing cell. Therefore, most of the fully less than that which would occur through systemic drug toxic effects to normal cells should be confined to cells located administration, since a large portion of the active drug will be in the neighborhood of tumor cells. released at the target site. The observation that the cytotoxic Our results extend previous observations on the enhancement activity is weaker on antigen-negative bystander cells than on of antitumor activity of phosphorylated prodrugs such as MOP antigen-positive cells suggests that a considerable proportion and etoposide phosphate on colorectal and lung cancers in vitro of the active compounds are directly internalized into the Bi- and in vivo (6, 8). In these previous studies, immunoconjugates were produced by covalenti}1 binding alkaline phosphatase to an antitumor antibody via a thioether bridge. Immunoconju 100 gates formed in this manner have several theoretical and prac , r., L540 F â€¢ tical disadvantages when compared with Bi-MAbs; the process 80 of the chemical coupling of a MAb alters its affinity, and the stability of the conjugate is often not satisfactory. In contrast, the use of Bi-MAbs [whole immunoglobulin molecules and S 'o F(ab')2 fragments] provides a stable product with consistent E binding properties and uniform composition. Particularly i F(ab')2 fragments may prove to be valuable for therapeutic S) 40 SÂ« applications in vivo because of their shorter half-time in plasma, better tumor penetration, and the lack of unspecific binding via 20

r-i r-B I 1 I 1 100

MOP Bi-MAb Bi-MAb Bi-MAb +AP MMC

+MOP 80

100 HPB-ALL 80

60 a Q. en 40

HPB-ALL'/Bi-MAb/AP HPB-ALL'/Bi-MAb/AP HPB-ALL'/Bi-MAb/AP 20 L540/-/- L540/Bi-MAb/AP L540/Bi-MAb/AP MOP MOP MMC Fig. 5. Effect of specific activation of MOP on CD30-positive Hodgkin's- 0 derived L540 cells pretreated with Bi-MAb HRS-3/AP-I [whole molecules or MOP Bi-MAb Bi-MAb Bi-MAb. AP MMC F(ab')2 fragments] plus AP on CD30-negative bystander HPB-ALL cells. There + MOP + MOP is a significantly increased cytotoxicity of MOP against antigen-negative HPB- Fig. 4. Specific release of "Cr from the CD30 antigen-positive cell line L540 ALL cells if these cells are coincubated with pretreated antigen-positive Hodg (top) and the CD30 antigen-negative HPB-ALL (bottom) after pretreatment with kin's-derived L540 cells (middle columns) as compared with cocultivation with MOP, Bi-MAb HRS-3/AP-1, Bi-MAb plus MOP, Bi-MAb plus AP plus MOP, non-pretreated L540 cells (left columns) even though the values of the release and MMC. Specific activation of MOP occurs only after pretreatment of the induced by the active compound MMC (right columns) are not quite reached. antigen-positive L540 cells with both the Bi-MAb and the enzyme. F(ab')2 Columns indicate specific MCr release Â±SE from HPB-ALL cells (HPB-ALL*) fragments of the Bi-MAb HRS-3/AP-1 are as effective as whole-immunoglobulin as determined in 3 independent experiments performed in quadruplicate. IgG, molecules in inducing prodrug activation. IgG, whole immunoglobulin molecules whole immunoglobulin molecules of Bi-MAb HRS-3/AP-I; F(ab). F(ab'); frag of Bi-MAb HRS-3/AP-1; F(ab), F(ab')2 fragments of HRS-3/AP-I. ments of HRS-3/AP-I. 6947

6. Senter. P. D., Saulnier. M. G.. Schreiber. G. J., Hirschberg, D. L., Brown, Fc receptors. In addition, when using antibodies against the AP J. P., HellstrÃ¶m, I., and HellstrÃ¶m. K. E. Anti-tumor effects of antibody- type of the tumor-bearing host, the Bi-MAb may be able to alkaline phosphatase conjugates in combination with etoposide phosphate. localize endogenous phosphatases present in the body, thereby Proc. Nati. Acad. Sci. USA, 85: 4842-4846, 1988. 7. Bagshawe, K. D., Springer, C. J., Searle, F.. Antoniw, P.. Sharma, S. K., reducing prodrug hydrolysis at nontarget sites. Melton. R. G., and Sherwood, R. F. A cytotoxic agent can be generated In summary, our results obtained with Bi-MAbs against a selectively at cancer sites. Br. J. Cancer. 58: 700-703, 1988. Hodgkin's-associated antigen and AP may serve as a model for 8. Senter. P. D.. Schreiber, G. J.. Hirschbcrg, D. L., Ashe. S. A.. HellstrÃ¶m, K. E., and HellstrÃ¶m. 1. Enhancement of the in vitro and in vivo antitumor many tumors for which MAbs with restricted reactivity to activities of phosphorylated mitomycin and etoposide derivatives by mono tumor cells are available. The AP-mediated activation of cyto- clonal antibody-alkaline phosphatase conjugates. Cancer Res., 49: 5789- toxic prodrugs on tumor cells is not restricted to phosphates of 5792, 1988. 9. Steinmetz, T.. and Pfreundschuh. M. Production of monoclonal antibodies etoposide or mitomycin since other hydroxyl-containing cyto- against glucose oxidase, alkaline phosphatase, and horseradish peroxidase. toxic drugs may be amenable to phosphorylation, thus opening J. Immunol. Methods, 101: 252-259. 1987. the possibility of polychemoimmunotherapy. Because of its 10. Engert, A., Burrows, F.. Jung, W., Ta/./ari, P. L., Stein, H., Pfreundschuh. restricted reactivity to Hodgkin's and Reed-Sternberg cells, the M., Diehl, V., and Thorpe, P. Evaluation of ricin A chain-containing immunotoxins directed against the CD30 antigen as potential reagents for the malignant cells of certain lymphomas and only a small subpop- treatment of Hodgkin's disease. Cancer Res.. 50: 82-88. 1990. ulation of normal cells (13), HRS-3 might be a good candidate 11. Schwarting, R.. and Stein. H. Cluster report: CD30. In: W. Knapp, B. DÃ¶rken,W. R. Gilks. E. P. Rieber. R. E. Schmidt. H. Stein, and A. E. G. for immunodiagnostics and immunotherapy in patients. Indeed, von dem Borne (eds.). Leucocyte Typing IV. White Cell Differentiation we recently demonstrated that HRS-3 binds to the malignant Antigens, pp. 419-422. Oxford. United Kingdom: Oxford University Press, Hodgkin's and Reed-Sternberg cells of patients with Hodgkin's 1989. 12. Clark. M. R., and Waldmann, H. T-cell killing of target cells induced by disease in vivo (20, 21). Studies using less immunogenic ("hu hybrid antibodies: comparison of two bispecific monoclonal antibodies. J. manized") Bi-MAbs with reactivity against human AP will be Nati. Cancer Inst.. 79: 1393-1402, 1987. 13. Pfreundschuh, M., Mommcrtz, E., Meissner, M., Feller, A. C., Hassa, R., the subject of future investigations to show whether this ap Krueger, G. R. F.. and Diehl. V. Hodgkin and Reed-Sternberg associated proach opens new avenues in the treatment of human cancer. antibodies react with activated cells of lymphoid and monocytoid origin. Anticancer Res., 8: 217-225, 1988. 14. Pfreundschuh, M., Lehmann, M.. Steinmetz, T.. Kirchner. H. H., and Dichl, V. Monoclonal glucose-oxidase-antiglucose-oxidase (GAG) immunosand- ACKNOWLEDGMENTS wich assay for the detection of monoclonal antibodies on routine hematolog- ical smears. Blut. 56: 125-130. 1988. We thank Gisela SchÃ¶nfor excellent technical assistance. 15. Laemmli, U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature (Lond.). 227: 680-685. 1979. 16. Lamoyi, E. 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Downloaded from cancerres.aacrjournals.org on September 30, 2021. © 1990 American Association for Cancer Research. Specific Activation of the Prodrug Mitomycin Phosphate by a Bispecific Anti-CD30/Anti-Alkaline Phosphatase Monoclonal Antibody

Ugur Sahin, Frank Hartmann, Peter Senter, et al.

Cancer Res 1990;50:6944-6948.

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