@Copyright 1993 by Humana Press Inc. All rights of any nature whatsoever reserved.
0163-4992192/21113-23153.20
lmmunotoxins to Human Small-Cell Lung Cancer EDWARD J. WAWRZYNCZAK* AND ELAINE J. DERBYSHIRE Drug Targeting Laboratory, Institute of Cancer Research, Sutton, Surrey SM2 5NG, UK
ABSTRACT Ricin A chain ITs directed against a variety of the common cellsurface antigens associated with SCLC exerted selective toxic effects on SCLC cell lines. The potency of the cytotoxic effects matched or exceeded that previously reported for ricin A chain ITs directed against identical or similar antigens on other types of carcinoma, suggesting that SCLC may be uniquely sensitive to this type of IT.
IndexEntries-Ricin; immunotoxin; monoclonal antibody; small cell lung cancer; mucin. INTRODUCTION Small-cell lung carcinoma (SCLC) is a major clinical problem, both because of its frequency and the inability of conventional forms of treatment to improve patient survival significantly. The majority of SCLC *Author to whom all correspondence and reprint requests should be addressed. Cell Biophysics
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tumors are initially highly responsive to chemotherapy, and remissions are common. However, relapse with drug-resistant disease follows almost inevitabljfi and metastasis is widespread. New antitumor agents with the potential to act systemically and by novel mechanisms able to circumvent drug resistance would clearly be valuable. Ricin A chain immunotoxins (ITs) may constitute such a class of agents, because they can selectively intoxicate target cells by inactivating ribosomal protein synthesis and have been safely administered systemically to patients with metastatic cancer (1). SCLC possesses both epithelial and neuroendocrine characteristics, and might therefore be expected to express a variety of cell surface antigens that could prove suitable for IT therapy. Two International Workshops on SCLC Antigens, in which monoclonal antibodies (MAbs) were grouped by a mathematical clustering analysis on the basis of their reactivity with normal and tumor tissues, identified several cell-surface antigens commonly associated with SCLC (2,3). An indirect assay of IT cytotoxicity was used to examine the potential of all the MAbs submitted to the Second Workshop to form cytotoxic agents with ricin A chain. In addition, the cytotoxic properties of a panel of ITs made by directly linking ricin A chain to MAbs recognizing the major SCLCassociated antigens and the unclustered antimucin MAb BrE-3, which was predicted by the indirect screen to be toxic to SCLC, were examined in detail (4).
CYTOTOXIC PROPERTIES OF SCLC IMMUNOTOXINS The six SCLC MAbs used to make the ricin A chain ITs examined in this study, their Workshop cluster designations, and the identity or known molecular characteristics of their target antigens are summarized in Table 1.
Potency of IT Action The cytotoxic potency of the panel of ricin A chain ITs was measured using a conventional 48-h 3H-leucine incorporation assay in which three different SCLC cell lines--NCI-H69, SW2, and GLC-8mwere exposed continuously to IT or other agents in tissue culture. Figure 1 shows the range of IC50values determined against the three SCLC cell Cell Biophysics
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Table 1 Monoclonal Antibodies to Human Small-Cell Lung Cancer MAb
Workshop cluster
SEN36 MOC-31
1 2
SWA11 SWA20 MLuC1 BrE-3
w4 5A w6 Unclustered
Nature of antigen Neural cell adhesion molecule 35-kDa Epithelial glycoprotein 45-kDa Glycoprotein 40-, 100-, 180-kDa Sialoglycoproteins Y hapten High-molecular-weight mucin
ricin ricin A control ITs SEN36 IT
E
MOC-31 IT
t~
SWA11 IT
lSWA20 IT M L u C l IT BrE-3 IT I
I
I
!
|
I
"1210 -1110 -lo 10 -9 10 Concentration (M)
10 -+3 10
I
+ 10 -7
Fig. 1. Cytotoxic potency of SCLC ITs and other agents.
lines. None of the six SCLC ITs matched the potency of ricin. SEN36ricA had no selective activity compared with unconjugated ricin A chain and control isotype-matched ITs with irrelevant binding specificity. SWA20-ricA had moderate to weak activity. The other four SCLC ITs had strong to moderate activity. SWA11-ricA had the most consistently potent effects. MLuCl-ricA was highly active against two of the cell Cell Biophysics
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lines and about 100-fold less effective against the third. The differences in potency observed could not be simply ascribed to the variation in the level of target antigen, either between different antigens on the same cell line or the same antigen between different cell lines. None of the ITs in the panel had any selective cytotoxic activity against a human nonlung tumor cell line. The finding of weak activity for the SEN36 IT was in agreement with the predictions of the indirect IT assay in which SEN36 and seven other cluster I MAbs displayed only weak toxic effects in combination with the screening agent (5). The potent action of the IT made with MOC-31 is also typical of cluster 2 MAbs (5,6). BrE-3 was the single antimucin MAb in the Second Workshop displaying highly potent effects in the indirect assay (7).
Inhibition of IT Action by Excess MAb The ability of unconjugated antibody to inhibit the cytotoxic action of each of the SCLC ITs was measured by performing the 3Hleucine incorporation assay using the SW2 SCLC cell line in the presence of an excess, 1 x 10-7M, of the MAb relevant to each IT. Table 2 demonstrates that the ability to block IT action varied over a >500-fold range. This variability was not apparently related to the potency of the ITs judged from the ICs0values in the absence of competing MAb. In the most extreme case, the action of MLuCl-ricA could not be blocked even using protocols designed to minimize IT binding and internalization (8). In all cases of IT inhibition, the action of the blocking MAb was selective, since isotype-matched control MAbs had no effect on cytotoxic potency.
Kinetics of IT Action The kinetics of inhibition of protein synthesis by the SW2 cell line were determined using a short 3H-leucine pulse following exposure of the cells to the SCLC ITs or ricin for different periods of time. The rates at which the different ITs inhibited 3H-leucine incorporation varied widely (Fig. 2). SEN36-ricA, MOC-31-ricA, and BrE-3-ricA displayed relatively slow kinetics, reaching 50% inhibition after a time (t50)of about 20 h. SWAll-ricA had a ts0 of 6 h, a period that included a 4 h lag phase following exposure of the cells during which inhibition of protein synCell Biophysics
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lmmunotoxins
17 Table 2 Immunotoxin Inhibition by Excess Antibody IC50, M , a
1T
-MAb
SEN36-ricA MOC-31-ricA SWA11-ricA SWA20-ricA MLuCl-ricA BrE-3-ricA
2 6 2 4 7 2
x 10~
-X 10-8 x 10-9 x 10-1~ x 10-12 x 10-8
>1 1 2 2 >1
X 10-11
x x x x
Inhibition factor b
+MAb
10-11 10-11 10-12 10-11
->167 50 5 0 >500
aConcentration at which incorporation of 3H-leucine was inhibited by 50%.
100]
bRatio of IC50+ MAb/ICso- MAb.
~O'~o~101 ~ ~O
SEN36 IT MOC-31 IT BrE-3 IT
WAl I'~~T l --
3 i
i
i
i
0
8
16
24
Time
(h)
Fig. 2. Kinetics of cell intoxication by SCLC ITs and ricin. thesis could not be detected. SWA20-ricA acted rapidly with a ts0 of 2 h, but reached a plateau at about 85% inhibition consistent with the nonuniform expression of the cluster 5A antigen on SCLC cell lines (9). MLuC1 displayed exceptionally rapid kinetics of intoxication. The ts0 of the MLuC1 1T was only 0.5 h, equivalent to that of ricin toxin itself. A l t h o u g h no obvious relationship existed b e t w e e n the potencies of the SCLC ITs in the panel and their kinetics of action, the r a n k order of ITs in increasing rapidity of action, MOC-31/BrE-3 < SWA11 Cell Biophysics
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< SWA20 < MLuC1, correlated precisely with increasing difficulty in inhibiting their action by excess unconjugated MAb. Thus, the more rapid the action of the IT, the more difficult it was to block that action. This interesting correlation may imply that the rapidly acting ITs were able to bind to target cells with greater avidity than native MAb perhaps by means of dual recognition via the MAb and A chain components (8).
Clonogenic Efficiency of IT Action The fraction of clonogenic SW2 cells surviving a 48-h exposure to the SCLC ITs at a concentration of I x 10-8M in tissue culture was determined by means of a limiting dilution clonogenic assay scoring the development of spheroidal cell colonies after 14 d of culture (10). The four least effective ITs reduced the surviving clonogenic fraction to 3-15% of the ricin A chain control (Fig. 3). In the case of SWA20ricA, this relatively inefficient elimination of tumor cells was likely to have been the result of the presence of cells in the population expressing low levels of target antigen. This explanation may also be relevant to the other three ITs made with SEN36, MOC-31, and BrE-3. In addition, the slow kinetics of action of these three ITs would suggest that a proportion of tumor cells could escape destruction if cell division occurred before complete inactivation of protein synthesis. SWA11-ricA and MLuCl-ricA were highly effective ITs, reducing the surviving clonogenic fraction to only 0.02-0.03% of the ricin A chain control, a result reflecting both relatively homogeneous expression of target antigen and rapid kinetics of cell intoxication.
Potentiation of IT Activity SW2 cells were incubated in the presence of agents known to upregulate antigen expression, to potentiate the cytotoxic activity of ricin A chain ITs, or to be cytotoxic to SCLC, at various toxic and nontoxic concentrations either in the presence or absence of each of the SCLC ITs at the highest nontoxic concentration. In the 3H-leucine incorporation assay, the IC50for drug alone compared with drug in conjunction with IT was decreased by less than fourfold for all six ITs incubated with lysosomotropic agents--ammonium chloride, methylamine, and chloroquine with calcium antagonists--verapamil and perhexiline or with chemotherapeutic drugs--adriamycin, carboplatin, cisplaCell Biophysics
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SEN36 IT MOC-31 IT p, (1)
E t~ G)
SWA11 IT SWA20 IT
I i
!MLuC1 IT BrE-3 IT . . . . . . . .
.01
I
.1
. . . . . . .
I
1
. . . . . . . .
l
. . . . . . . .
10
i
100
Surviving clonogenic fraction (% of control) Fig. 3. Efficiency of clonogenic tumor cell killing by SCLC ITs. tin, chlorambucil, (4-hydroperoxy)cyclo phosphamide, etoposide, methotrexate, and vincristine. In another experiment, no significant effect on the activity of SEN36-ricA was observed by treating SW2 cells with suramin or interferons 0~and 7, which can upregulate the expression of the neural cell adhesion molecule on neuroendocrine cells (11). In contrast with the other agents examined, the addition of the SCLC ITs to the carboxylic ionophore monensin decreased IC50 values by between 7- and 53-fold. The effect of monensin on IT activity was examined further by comparing the IC50values for the SCLC ITs in the presence of a fixed monensin concentration, 1 x 10-7M. In agreement with the predictions of the preliminary experiments, all six ITs in the panel were potentiated by monensin (Table 3). The effect of monensin was not entirely selective for the SCLC ITs. By comparison, the cytotoxic effects of ricin and ricin A chain were enhanced by 10- and 15fold, respectively. There was no obvious correlation between the enhancement factor in the presence of monensin and the potency, kinetics, or clonogenic efficiency of the SCLC ITs in the absence of potentiator. Cell Biophysics
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Wawrzynczak and Derbyshire Table 3 Immunotoxin Potentiation by Monensin IT
SEN36-ricA MOC-31-ricA SWA11-ricA SWA20-ricA MLuCl-ricA BrE-3-ricA
ICs~ M'a -Monensin +Monensin 1 1 3 1 2 3
x x x x x x
10-8 10-11 10-11 10-1~ 10-11 10-11
2 2 2 2 8 2
X 10-12 X 10-13
x 10-13 X 10-12 X 10-14
x 10-13
Enhancement factorb 5000 500 150 50 250 150
aConcentration at which incorporation of 3H-leucine was inhibited by 50%. bRatio of ICs0- monensin/ICs0 + monensin.
The 5000-fold enhancement in the activity of SEN36-ricA suggested that the failure of this IT to exert selective activity was owing to poor entry of A chain to the cytosol in the absence of ionophore. Detailed studies with SWA11 have revealed that although monensin had little effect on the rate of cluster w4 antigen internalization, the ionophore induced an earl~ transient delay in the delivery of internalized MAb to lysosomes. This correlated with an enhancement in the rate of protein synthesis inhibition by the SWA11 IT by twofold and elimination of the lag phase of cell intoxication implying a rapid promotion of A chain delivery to the cytosol (10).
CONCLUSION The ideal therapeutic IT would satisfy a number of criteria. First, activity criteria: It would intoxicate cells at low concentration, would do so rapidly, would kill a high proportion of cells within a tumor, and would be active against the majority of tumors. Second, selectivity criteria: The IT would be potently active only against cells bearing the target antigen, and would be toxic to tumor but not to normal tissues. On the basis of activity criteria alone, it was possible to exclude several ITs from consideration. SEN36-ricA bound to a high proportion of target cells, but was insufficiently active. SWA20-ricA had only moderate potency and recognized an antigen that was expressed too heterogeneously. Of the other four SCLC ITs, SWA11-ricAwas the one that CeUBiophysics
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came closest to fulfilling all the activity criteria (10). SWA11 ITs constructed with the aglycosyl A chain of abrin toxin or with chemically deglycosylated ricin A chain retained the high-potency and clonogenic efficiency of the standard ricin A chain IT and selectively inhibited the growth of established subcutaneous SCLC xenografts in a nude mouse model of systemic therapy (12,13). The prospects of systemic IT therapy in SCLC are further restricted by the relative lack of specificity of the SCLC Mabs. MOC-31 recognizes an antigen that is known to be present on a variety of carcinomas, but is also expressed by normal intestine and other normal tissues, although this has not precluded clinical trials with other immunoconjugates (6). Although the antigen recognized by SWA11 is present at high levels and with high frequency on SCLC, the biodistribution of the antigen is unlikely to prove favorable because cloning of a cluster w4 antigen cDNA and epitope mapping using synthetic peptides have revealed that SWA11 recognizes the polypeptide backbone of a phosphatidylinositoMinked glycoprotein, which is identical with or closely related to the leukocyte CD24 antigen (13,14; Weber et al., unpublished results). The Y hapten carbohydrate antigen recognized by MLuC1 is a ubiquitous epithelial antigen; only a proportion of patients could be anticipated to demonstrate tumor expression of the target antigen in the absence of substantial normal tissue expression (8). Antimucin ITs, such as BrE-3-ricA, may be more applicable to systemic therapy of SCLC because antimucin MAbs show a relatively restricted reactivity with normal tissues and some are reported to bind to the carcinoma-associated antigen with high selectivity (7). Indeed, SCLC may be especially suitable as the target for antimucin ITs,because patients who are in remission following chemotherapy will have a relatively small tumor burden and a low level of circulating mucin that can otherwise reduce the efficiency of targeting to solid tumors. In summary, ricin A chain ITs directed against a variety of the common cell-surface antigens associated with SCLC exerted selective toxic effects on SCLC cell lines. The potency of the cytotoxic effects matched or exceeded that previously reported for ricin A chain ITs directed against identical or similar antigens on other types of carcinoma, suggesting that SCLC may be uniquely sensitive to this type of IT (6-8). The substantial enhancement in the potency of all six SCLC ITs examined by monensin, but not by lysosomotropic amines suggests that lysosomal degradation was not the major factor limiting their potency and that CeUBiophysics
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Wawrzynczak and Derbyshire
the pathways of internalization of the different target antigens were grossly similar. SCLC cells, which are active in the synthesis and processing of polypeptide hormones for export, may be relatively rich in intracellular compartments associated with the trans-Gol~ from which translocation of the A chain to the cytosol occurs most efficiently. For this reason, the exploration and development of more selective antiSCLC ricin A chain ITs could prove rewarding.
ACKNOWLEDGMENTS The work conducted at the Institute of Cancer Research was supported by the Cancer Research Campaign, London, UK, and, in part, by an EEC Concerted Action on Drug Targeting: Immunoconjugates for Cancer Therapy. We thank our many collaborators, especially R. Stahel, L. de Leij, and S. Menard for their substantial and willing cooperation.
REFERENCES 1. Wawrzynczak, E. J. (1991) Systemic imunotoxin therapy of cancer: advances and prospects. Br. J. Cancer 64, 624-630. 2. Souhami, R. L., Beverley, P. C. L., and Bobrow, L. (eds.) (1988) Proceedings of the first international workshop on small cell lung cancer antigens. Lung Cancer 4, 1-116. 3. Beverley, P. C. L., Bobrow, L. G., Gilks, W., and Souhami, R. L. (eds.) (1991) Second international workshop on small cell lung cancer antigens. Br. J. Cancer, Suppl. XW, 1-88. 4. Derbyshire, E. J. (1992) Immunotoxins to Human Small Cell Lung Cancer. Ph.D. Thesis, University of London. 5. Derbyshire, E. J. and Wawrzynczak, E. J. (1991) Monoclonal antibodies recognising the cluster 2 antigen associated with human small cell lung cancer mediate the toxic effects of ricin A chain in an indirect assay of immunotoxin cytotoxicity. Br. J. Cancer 63, Suppl. XIV, 74-77. 6. Derbyshire, E. J., de Leij, L., and Wawrzynczak, E. J. (1993) Refinement of an indirect immunotoxin assay of monoclonal antibodies recognising the human small cell lung cancer cluster 2 antigen. Br. J. Cancer 67, 1242-1247. Cell Biophysics
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7. Derbyshire, E. J. and Wawrzynczak, E. J. (1992) An anti-mucin immunotoxin BrE-3-ricin A chain is potently and selectively toxic to human small cell lung cancer. Int. J. Cancer 52, 624-630. 8. Derbyshire, E. J., Menard, S., and Wawrzynczak, E. J. (1992) Cytotoxic activity and MLuCl-ricin A chain recognising the Y hapten associated with human small cell lung carcinoma. Fund. Clin. Immunol., in press. 9. Derbyshire, E. J., Stahel, R. A., and Wawrzynczak, E. J. (1992) Cytotoxic properties of a ricin A chain immunotoxin recognising the cluster 5A antigen associated with human small cell lung cancer. Cancer Immunol. Immunother. 35, 417--420. 10. Derbyshire, E. J., Henry, R. V., Stahel, R. A., and Wawrzynczak, E. J. (1992) Potent cytotoxic action of the immunotoxin SWA11-ricin A chain against human small cell lung cancer cell lines. Br. J. Cancer 66, 444 451. 11. Derbyshire, E. J., Stahel, R. A., and Wawrzynczak, E. J. (1992) Potentiation of a weakly active ricin A chain immunotoxin recognising the neural cell adhesion molecule. Clin. Exp. Immunol. 89, 336-340. 12. Wawrzynczak, E. J., Zangemeister-Wittke, U., Waibel, R., and et al. (1992) Molecular and biological properties of an abrin A chain immunotoxin designed for therapy of human small cell lung cancer. Br. J. Cancer 66, 361-366. 13. Zangemeister-Wittke, U., Lehmann, H. -P., Waibel, R., and et al. (1992) Action of a CD24-specific deglycosylated ricin A chain immunotoxin in conventional and novel models of small cell lung cancer xenograft. Int. J. Cancer 53, 521-528. 14. Jackson, D., Waibel, R., and Weber, E., et al. (1992) CD24, a signal transducing molecule expressed on human B cells, is a major surface antigen on small-cell lung carcinomas. Cancer Res. 52, 1-7.
CeUBiophysics
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0163-4992192/21113-23153.20
lmmunotoxins to Human Small-Cell Lung Cancer EDWARD J. WAWRZYNCZAK* AND ELAINE J. DERBYSHIRE Drug Targeting Laboratory, Institute of Cancer Research, Sutton, Surrey SM2 5NG, UK
ABSTRACT Ricin A chain ITs directed against a variety of the common cellsurface antigens associated with SCLC exerted selective toxic effects on SCLC cell lines. The potency of the cytotoxic effects matched or exceeded that previously reported for ricin A chain ITs directed against identical or similar antigens on other types of carcinoma, suggesting that SCLC may be uniquely sensitive to this type of IT.
IndexEntries-Ricin; immunotoxin; monoclonal antibody; small cell lung cancer; mucin. INTRODUCTION Small-cell lung carcinoma (SCLC) is a major clinical problem, both because of its frequency and the inability of conventional forms of treatment to improve patient survival significantly. The majority of SCLC *Author to whom all correspondence and reprint requests should be addressed. Cell Biophysics
13
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14
Wawrzynczak and Derbyshire
tumors are initially highly responsive to chemotherapy, and remissions are common. However, relapse with drug-resistant disease follows almost inevitabljfi and metastasis is widespread. New antitumor agents with the potential to act systemically and by novel mechanisms able to circumvent drug resistance would clearly be valuable. Ricin A chain immunotoxins (ITs) may constitute such a class of agents, because they can selectively intoxicate target cells by inactivating ribosomal protein synthesis and have been safely administered systemically to patients with metastatic cancer (1). SCLC possesses both epithelial and neuroendocrine characteristics, and might therefore be expected to express a variety of cell surface antigens that could prove suitable for IT therapy. Two International Workshops on SCLC Antigens, in which monoclonal antibodies (MAbs) were grouped by a mathematical clustering analysis on the basis of their reactivity with normal and tumor tissues, identified several cell-surface antigens commonly associated with SCLC (2,3). An indirect assay of IT cytotoxicity was used to examine the potential of all the MAbs submitted to the Second Workshop to form cytotoxic agents with ricin A chain. In addition, the cytotoxic properties of a panel of ITs made by directly linking ricin A chain to MAbs recognizing the major SCLCassociated antigens and the unclustered antimucin MAb BrE-3, which was predicted by the indirect screen to be toxic to SCLC, were examined in detail (4).
CYTOTOXIC PROPERTIES OF SCLC IMMUNOTOXINS The six SCLC MAbs used to make the ricin A chain ITs examined in this study, their Workshop cluster designations, and the identity or known molecular characteristics of their target antigens are summarized in Table 1.
Potency of IT Action The cytotoxic potency of the panel of ricin A chain ITs was measured using a conventional 48-h 3H-leucine incorporation assay in which three different SCLC cell lines--NCI-H69, SW2, and GLC-8mwere exposed continuously to IT or other agents in tissue culture. Figure 1 shows the range of IC50values determined against the three SCLC cell Cell Biophysics
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Table 1 Monoclonal Antibodies to Human Small-Cell Lung Cancer MAb
Workshop cluster
SEN36 MOC-31
1 2
SWA11 SWA20 MLuC1 BrE-3
w4 5A w6 Unclustered
Nature of antigen Neural cell adhesion molecule 35-kDa Epithelial glycoprotein 45-kDa Glycoprotein 40-, 100-, 180-kDa Sialoglycoproteins Y hapten High-molecular-weight mucin
ricin ricin A control ITs SEN36 IT
E
MOC-31 IT
t~
SWA11 IT
lSWA20 IT M L u C l IT BrE-3 IT I
I
I
!
|
I
"1210 -1110 -lo 10 -9 10 Concentration (M)
10 -+3 10
I
+ 10 -7
Fig. 1. Cytotoxic potency of SCLC ITs and other agents.
lines. None of the six SCLC ITs matched the potency of ricin. SEN36ricA had no selective activity compared with unconjugated ricin A chain and control isotype-matched ITs with irrelevant binding specificity. SWA20-ricA had moderate to weak activity. The other four SCLC ITs had strong to moderate activity. SWA11-ricA had the most consistently potent effects. MLuCl-ricA was highly active against two of the cell Cell Biophysics
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lines and about 100-fold less effective against the third. The differences in potency observed could not be simply ascribed to the variation in the level of target antigen, either between different antigens on the same cell line or the same antigen between different cell lines. None of the ITs in the panel had any selective cytotoxic activity against a human nonlung tumor cell line. The finding of weak activity for the SEN36 IT was in agreement with the predictions of the indirect IT assay in which SEN36 and seven other cluster I MAbs displayed only weak toxic effects in combination with the screening agent (5). The potent action of the IT made with MOC-31 is also typical of cluster 2 MAbs (5,6). BrE-3 was the single antimucin MAb in the Second Workshop displaying highly potent effects in the indirect assay (7).
Inhibition of IT Action by Excess MAb The ability of unconjugated antibody to inhibit the cytotoxic action of each of the SCLC ITs was measured by performing the 3Hleucine incorporation assay using the SW2 SCLC cell line in the presence of an excess, 1 x 10-7M, of the MAb relevant to each IT. Table 2 demonstrates that the ability to block IT action varied over a >500-fold range. This variability was not apparently related to the potency of the ITs judged from the ICs0values in the absence of competing MAb. In the most extreme case, the action of MLuCl-ricA could not be blocked even using protocols designed to minimize IT binding and internalization (8). In all cases of IT inhibition, the action of the blocking MAb was selective, since isotype-matched control MAbs had no effect on cytotoxic potency.
Kinetics of IT Action The kinetics of inhibition of protein synthesis by the SW2 cell line were determined using a short 3H-leucine pulse following exposure of the cells to the SCLC ITs or ricin for different periods of time. The rates at which the different ITs inhibited 3H-leucine incorporation varied widely (Fig. 2). SEN36-ricA, MOC-31-ricA, and BrE-3-ricA displayed relatively slow kinetics, reaching 50% inhibition after a time (t50)of about 20 h. SWAll-ricA had a ts0 of 6 h, a period that included a 4 h lag phase following exposure of the cells during which inhibition of protein synCell Biophysics
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lmmunotoxins
17 Table 2 Immunotoxin Inhibition by Excess Antibody IC50, M , a
1T
-MAb
SEN36-ricA MOC-31-ricA SWA11-ricA SWA20-ricA MLuCl-ricA BrE-3-ricA
2 6 2 4 7 2
x 10~
-X 10-8 x 10-9 x 10-1~ x 10-12 x 10-8
>1 1 2 2 >1
X 10-11
x x x x
Inhibition factor b
+MAb
10-11 10-11 10-12 10-11
->167 50 5 0 >500
aConcentration at which incorporation of 3H-leucine was inhibited by 50%.
100]
bRatio of IC50+ MAb/ICso- MAb.
~O'~o~101 ~ ~O
SEN36 IT MOC-31 IT BrE-3 IT
WAl I'~~T l --
3 i
i
i
i
0
8
16
24
Time
(h)
Fig. 2. Kinetics of cell intoxication by SCLC ITs and ricin. thesis could not be detected. SWA20-ricA acted rapidly with a ts0 of 2 h, but reached a plateau at about 85% inhibition consistent with the nonuniform expression of the cluster 5A antigen on SCLC cell lines (9). MLuC1 displayed exceptionally rapid kinetics of intoxication. The ts0 of the MLuC1 1T was only 0.5 h, equivalent to that of ricin toxin itself. A l t h o u g h no obvious relationship existed b e t w e e n the potencies of the SCLC ITs in the panel and their kinetics of action, the r a n k order of ITs in increasing rapidity of action, MOC-31/BrE-3 < SWA11 Cell Biophysics
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< SWA20 < MLuC1, correlated precisely with increasing difficulty in inhibiting their action by excess unconjugated MAb. Thus, the more rapid the action of the IT, the more difficult it was to block that action. This interesting correlation may imply that the rapidly acting ITs were able to bind to target cells with greater avidity than native MAb perhaps by means of dual recognition via the MAb and A chain components (8).
Clonogenic Efficiency of IT Action The fraction of clonogenic SW2 cells surviving a 48-h exposure to the SCLC ITs at a concentration of I x 10-8M in tissue culture was determined by means of a limiting dilution clonogenic assay scoring the development of spheroidal cell colonies after 14 d of culture (10). The four least effective ITs reduced the surviving clonogenic fraction to 3-15% of the ricin A chain control (Fig. 3). In the case of SWA20ricA, this relatively inefficient elimination of tumor cells was likely to have been the result of the presence of cells in the population expressing low levels of target antigen. This explanation may also be relevant to the other three ITs made with SEN36, MOC-31, and BrE-3. In addition, the slow kinetics of action of these three ITs would suggest that a proportion of tumor cells could escape destruction if cell division occurred before complete inactivation of protein synthesis. SWA11-ricA and MLuCl-ricA were highly effective ITs, reducing the surviving clonogenic fraction to only 0.02-0.03% of the ricin A chain control, a result reflecting both relatively homogeneous expression of target antigen and rapid kinetics of cell intoxication.
Potentiation of IT Activity SW2 cells were incubated in the presence of agents known to upregulate antigen expression, to potentiate the cytotoxic activity of ricin A chain ITs, or to be cytotoxic to SCLC, at various toxic and nontoxic concentrations either in the presence or absence of each of the SCLC ITs at the highest nontoxic concentration. In the 3H-leucine incorporation assay, the IC50for drug alone compared with drug in conjunction with IT was decreased by less than fourfold for all six ITs incubated with lysosomotropic agents--ammonium chloride, methylamine, and chloroquine with calcium antagonists--verapamil and perhexiline or with chemotherapeutic drugs--adriamycin, carboplatin, cisplaCell Biophysics
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lmmunotoxins
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SEN36 IT MOC-31 IT p, (1)
E t~ G)
SWA11 IT SWA20 IT
I i
!MLuC1 IT BrE-3 IT . . . . . . . .
.01
I
.1
. . . . . . .
I
1
. . . . . . . .
l
. . . . . . . .
10
i
100
Surviving clonogenic fraction (% of control) Fig. 3. Efficiency of clonogenic tumor cell killing by SCLC ITs. tin, chlorambucil, (4-hydroperoxy)cyclo phosphamide, etoposide, methotrexate, and vincristine. In another experiment, no significant effect on the activity of SEN36-ricA was observed by treating SW2 cells with suramin or interferons 0~and 7, which can upregulate the expression of the neural cell adhesion molecule on neuroendocrine cells (11). In contrast with the other agents examined, the addition of the SCLC ITs to the carboxylic ionophore monensin decreased IC50 values by between 7- and 53-fold. The effect of monensin on IT activity was examined further by comparing the IC50values for the SCLC ITs in the presence of a fixed monensin concentration, 1 x 10-7M. In agreement with the predictions of the preliminary experiments, all six ITs in the panel were potentiated by monensin (Table 3). The effect of monensin was not entirely selective for the SCLC ITs. By comparison, the cytotoxic effects of ricin and ricin A chain were enhanced by 10- and 15fold, respectively. There was no obvious correlation between the enhancement factor in the presence of monensin and the potency, kinetics, or clonogenic efficiency of the SCLC ITs in the absence of potentiator. Cell Biophysics
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Wawrzynczak and Derbyshire Table 3 Immunotoxin Potentiation by Monensin IT
SEN36-ricA MOC-31-ricA SWA11-ricA SWA20-ricA MLuCl-ricA BrE-3-ricA
ICs~ M'a -Monensin +Monensin 1 1 3 1 2 3
x x x x x x
10-8 10-11 10-11 10-1~ 10-11 10-11
2 2 2 2 8 2
X 10-12 X 10-13
x 10-13 X 10-12 X 10-14
x 10-13
Enhancement factorb 5000 500 150 50 250 150
aConcentration at which incorporation of 3H-leucine was inhibited by 50%. bRatio of ICs0- monensin/ICs0 + monensin.
The 5000-fold enhancement in the activity of SEN36-ricA suggested that the failure of this IT to exert selective activity was owing to poor entry of A chain to the cytosol in the absence of ionophore. Detailed studies with SWA11 have revealed that although monensin had little effect on the rate of cluster w4 antigen internalization, the ionophore induced an earl~ transient delay in the delivery of internalized MAb to lysosomes. This correlated with an enhancement in the rate of protein synthesis inhibition by the SWA11 IT by twofold and elimination of the lag phase of cell intoxication implying a rapid promotion of A chain delivery to the cytosol (10).
CONCLUSION The ideal therapeutic IT would satisfy a number of criteria. First, activity criteria: It would intoxicate cells at low concentration, would do so rapidly, would kill a high proportion of cells within a tumor, and would be active against the majority of tumors. Second, selectivity criteria: The IT would be potently active only against cells bearing the target antigen, and would be toxic to tumor but not to normal tissues. On the basis of activity criteria alone, it was possible to exclude several ITs from consideration. SEN36-ricA bound to a high proportion of target cells, but was insufficiently active. SWA20-ricA had only moderate potency and recognized an antigen that was expressed too heterogeneously. Of the other four SCLC ITs, SWA11-ricAwas the one that CeUBiophysics
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came closest to fulfilling all the activity criteria (10). SWA11 ITs constructed with the aglycosyl A chain of abrin toxin or with chemically deglycosylated ricin A chain retained the high-potency and clonogenic efficiency of the standard ricin A chain IT and selectively inhibited the growth of established subcutaneous SCLC xenografts in a nude mouse model of systemic therapy (12,13). The prospects of systemic IT therapy in SCLC are further restricted by the relative lack of specificity of the SCLC Mabs. MOC-31 recognizes an antigen that is known to be present on a variety of carcinomas, but is also expressed by normal intestine and other normal tissues, although this has not precluded clinical trials with other immunoconjugates (6). Although the antigen recognized by SWA11 is present at high levels and with high frequency on SCLC, the biodistribution of the antigen is unlikely to prove favorable because cloning of a cluster w4 antigen cDNA and epitope mapping using synthetic peptides have revealed that SWA11 recognizes the polypeptide backbone of a phosphatidylinositoMinked glycoprotein, which is identical with or closely related to the leukocyte CD24 antigen (13,14; Weber et al., unpublished results). The Y hapten carbohydrate antigen recognized by MLuC1 is a ubiquitous epithelial antigen; only a proportion of patients could be anticipated to demonstrate tumor expression of the target antigen in the absence of substantial normal tissue expression (8). Antimucin ITs, such as BrE-3-ricA, may be more applicable to systemic therapy of SCLC because antimucin MAbs show a relatively restricted reactivity with normal tissues and some are reported to bind to the carcinoma-associated antigen with high selectivity (7). Indeed, SCLC may be especially suitable as the target for antimucin ITs,because patients who are in remission following chemotherapy will have a relatively small tumor burden and a low level of circulating mucin that can otherwise reduce the efficiency of targeting to solid tumors. In summary, ricin A chain ITs directed against a variety of the common cell-surface antigens associated with SCLC exerted selective toxic effects on SCLC cell lines. The potency of the cytotoxic effects matched or exceeded that previously reported for ricin A chain ITs directed against identical or similar antigens on other types of carcinoma, suggesting that SCLC may be uniquely sensitive to this type of IT (6-8). The substantial enhancement in the potency of all six SCLC ITs examined by monensin, but not by lysosomotropic amines suggests that lysosomal degradation was not the major factor limiting their potency and that CeUBiophysics
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Wawrzynczak and Derbyshire
the pathways of internalization of the different target antigens were grossly similar. SCLC cells, which are active in the synthesis and processing of polypeptide hormones for export, may be relatively rich in intracellular compartments associated with the trans-Gol~ from which translocation of the A chain to the cytosol occurs most efficiently. For this reason, the exploration and development of more selective antiSCLC ricin A chain ITs could prove rewarding.
ACKNOWLEDGMENTS The work conducted at the Institute of Cancer Research was supported by the Cancer Research Campaign, London, UK, and, in part, by an EEC Concerted Action on Drug Targeting: Immunoconjugates for Cancer Therapy. We thank our many collaborators, especially R. Stahel, L. de Leij, and S. Menard for their substantial and willing cooperation.
REFERENCES 1. Wawrzynczak, E. J. (1991) Systemic imunotoxin therapy of cancer: advances and prospects. Br. J. Cancer 64, 624-630. 2. Souhami, R. L., Beverley, P. C. L., and Bobrow, L. (eds.) (1988) Proceedings of the first international workshop on small cell lung cancer antigens. Lung Cancer 4, 1-116. 3. Beverley, P. C. L., Bobrow, L. G., Gilks, W., and Souhami, R. L. (eds.) (1991) Second international workshop on small cell lung cancer antigens. Br. J. Cancer, Suppl. XW, 1-88. 4. Derbyshire, E. J. (1992) Immunotoxins to Human Small Cell Lung Cancer. Ph.D. Thesis, University of London. 5. Derbyshire, E. J. and Wawrzynczak, E. J. (1991) Monoclonal antibodies recognising the cluster 2 antigen associated with human small cell lung cancer mediate the toxic effects of ricin A chain in an indirect assay of immunotoxin cytotoxicity. Br. J. Cancer 63, Suppl. XIV, 74-77. 6. Derbyshire, E. J., de Leij, L., and Wawrzynczak, E. J. (1993) Refinement of an indirect immunotoxin assay of monoclonal antibodies recognising the human small cell lung cancer cluster 2 antigen. Br. J. Cancer 67, 1242-1247. Cell Biophysics
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7. Derbyshire, E. J. and Wawrzynczak, E. J. (1992) An anti-mucin immunotoxin BrE-3-ricin A chain is potently and selectively toxic to human small cell lung cancer. Int. J. Cancer 52, 624-630. 8. Derbyshire, E. J., Menard, S., and Wawrzynczak, E. J. (1992) Cytotoxic activity and MLuCl-ricin A chain recognising the Y hapten associated with human small cell lung carcinoma. Fund. Clin. Immunol., in press. 9. Derbyshire, E. J., Stahel, R. A., and Wawrzynczak, E. J. (1992) Cytotoxic properties of a ricin A chain immunotoxin recognising the cluster 5A antigen associated with human small cell lung cancer. Cancer Immunol. Immunother. 35, 417--420. 10. Derbyshire, E. J., Henry, R. V., Stahel, R. A., and Wawrzynczak, E. J. (1992) Potent cytotoxic action of the immunotoxin SWA11-ricin A chain against human small cell lung cancer cell lines. Br. J. Cancer 66, 444 451. 11. Derbyshire, E. J., Stahel, R. A., and Wawrzynczak, E. J. (1992) Potentiation of a weakly active ricin A chain immunotoxin recognising the neural cell adhesion molecule. Clin. Exp. Immunol. 89, 336-340. 12. Wawrzynczak, E. J., Zangemeister-Wittke, U., Waibel, R., and et al. (1992) Molecular and biological properties of an abrin A chain immunotoxin designed for therapy of human small cell lung cancer. Br. J. Cancer 66, 361-366. 13. Zangemeister-Wittke, U., Lehmann, H. -P., Waibel, R., and et al. (1992) Action of a CD24-specific deglycosylated ricin A chain immunotoxin in conventional and novel models of small cell lung cancer xenograft. Int. J. Cancer 53, 521-528. 14. Jackson, D., Waibel, R., and Weber, E., et al. (1992) CD24, a signal transducing molecule expressed on human B cells, is a major surface antigen on small-cell lung carcinomas. Cancer Res. 52, 1-7.
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