- Email: [email protected]
Specific Killing of Human and Mouse Tumor Cells by Immunotoxins
CAO. Drug Targeting
SPECIFIC KILLING OF HUMAN AND MOUSE TUMOR CELLS BY IMMUNOTOXINS P. CASELLAS,* H. E. BLYTHMAN,* J. P. BROWN,** O. GROS,* P. GROS,* K. E. HELLSTRÖM,** I. HELLSTRÖM,** F. K. JANSEN,* P. PONCELET* and H. VIDAL* ^Centre de Recherches CLIN MIDY, Montpellier, France **Fred Hutchinson Cancer Research Center, Seattle, Washington, U.S.A.
ABSTRACT Monoclonal antibodies (anti-Thy 1.2 and anti-human melanoma P97) were used to replace the binding moiety of ricin, the B-chain, in order to obtain hybrid molecules (immunotoxins) using as toxic moiety the purified A-chain of the toxin. A disulphide bridge was used to link both moieties. Both immunotoxins showed specific cytotoxicity for their respective target cells, WEHI-7 mouse lymphoma and Ml477 human melanoma. The specific activity was determined by the in vitro tests of protein synthesis inhibition and of inhibition of colony formation. The second test showed that both immunotoxins could specifically kill the last target cell without damaging the control cells, and a specific killing index could be calculated. KEYWORDS Immunotoxins, monoclonal antibodies, ricin, A-chain, Thy 1.2 antigen, tumor-associated antigen, immunotherapy, specific cytotoxicity, M1477 human melanoma cell line (P97). INTRODUCTION Numerous attempts to kill target cells using specific antibodies directed against cell surface antigens have been reported. Among the different approaches described, one of the most promising appears to be the use of specific antibodies as carriers of drugs or toxic proteins (1-3). This field has been considerably developed once lymphocyte hybridisation techniques made it possible to obtain extremely specific monoclonal antibodies. The toxic proteins most frequently used, diphtheria toxin and ricin, are extremely potent, because one molecule will suffice to kill a cell (4). Their molecular structure has been extensively described (5). The A-chain, responsible for the toxicity, is linked via one single disulphide bridge to the B-chain, which binds to cell membranes, facilitating the entry of A-chain into the cytoplasm (6). Highly specific results were obtained when the non-specific binding of the B-chain was circumvented by retaining the A-subunit, which is coupled, via a disulphide bridge, to a monoclonal antibody of a chosen specificity.
927
DRUG TARGETING
928
The validity of this approach has already been tested with an artificial model, (7) and the high potential of these hybrid molecules have now been further demonstrated using two natural models : anti-Thy 1.2 immunotoxins (using as targets WEHI-7 mouse leukemia cells) and monoclonal anti-melanoma antibodies, directed against human melanoma-associated antigen P97 (using as targets Ml477 human melanoma cells). MATERIAL AND METHODS Ricin, A-chain and immunotoxins were prepared as previously described (7). Briefly, ricin was extracted from Castor beans and purified by affinity chromatography on agarose. After cleavage of the disulphide bridge with 2ME, the A-chain was isolated on DEAE-Sepharose 6B and concentrated on CM-Sepharose 6B. Immunotoxins were prepared by coupling the A-chain to the antibodies via a disulphide linkage. Antibodies were reacted with 3-(2-pyridyl-dithio)-propionic acid activated by a limited quantity of carbodiimide. After dialysis, the modified antibodies were reacted with the free thiol group of A-chain. Unreacted A-chain was eliminated by chromatography on Sephadex G200. Anti-Thy 1.2. Immunotoxins were prepared with monoclonal IgM antibodies purchased from Olac (England), anti-DNP-Immunotoxin using monoclonal IgG antibodies and anti-melanoma-Immunotoxins with monoclonal anti-P97. The antibody binding of the immunotoxin was analysed using a second fluorescent antibody with a fluorescent activated cell sorter (FACS IV Beckton-Dickinson). Enzymatic activity of the A-chain was assessed in a cell-free protein synthesis system in presence of (3-mercaptoethanol as well as in an in vitro target cell system. For this last test, cells at 4 x 10 cells per ml in 50 μΐ aliquots were seeded into wells of microtest culture plates and incubated with toxins or immunotoxin at various concentrations in a 100 μΐ final volume. The cells were incubated at 37°C for 20 hrs and again for 4 hrs after addition of 2μ Ci of 14C-leucine (25 Ci/mmole New England Nuclear). The cells were harvested from the wells with a multiple automated cell harvester. Incorporation of 14C-leucine into protein was measured in a Intertechnique scintillation counter. To increase the sensitivity of the measurement of Immunotoxin activity, another system was used, studying the inhibition of colony formation induced by IT treatment. For this test, target cells were cultured (WEHI-7) or plated (M1477) at various limiting dilutions to determine their colony formation or plating efficiency. Test cells were incubated for 24 hrs at various concentrations of toxins or immunotoxins. Cells were then washed and seeded into Petri dishes, on a feeder layer of 0,5 % agarose containing 1θ6 macrophages (for WEHI-7 cells) or seeded directly (without a feeder layer) in the case of adherent cells (M1477). After 10-15 days of incubation, the number of colonies (indicating cells surviving the treatment) were counted with an automatic colony counter (ARTEK 980 system). Colony formation or plating efficiency varied between 15-30 % for different experiments, but a linear relationship was observed between the number of cells seeded and the number of colonies recovered. RESULTS AND DISCUSSION Immunotoxins (IT). The coupling method allowed a linkage of active A-chain to antibody in the ratio of 3 to 1 for IgM anti-Thy 1.2 IT, and of 1.4 to 1 for the IgG anti-melanoma IT. The binding activity of the IT to its respective target cells was analyzed by immunofluorescence (FACS). The fluorescent intensity of the two immunotoxins compared to that of unmodified antibody showed a slight decrease of bin-
SPECIFIC KILLING BY IMMUNOTOXINS
929
ding in both cases, which may correspond either to a hindrance of the binding of the second Ab due to A-chain or to a loss of the binding capacity due to the conjugation (fig 1 a-b). This loss can be estimated as 10 % and 30 % for anti-Thy 1.2 IT and anti-melanoma IT respectively.
NUMBER OF CELLS
Anti-Thy 1-2 Immunotoxin IgM Anti-Thy 1-2
FLUORESCENCE INTENSITY
t- - - SCATTER
NUMBER OF CELLS
Anti Melanoma Immunotoxin
l/Nç [SCATTER
Melanoma IgG
FLUORESCENCE INTENSITY
Fig. 1. Binding capacity of anti-Thy 1.2 immunotoxin (a) and anti-melanoma immunotoxin (b) compared to their respective unmodified antibodies. Analysis was done anti-mouse IgG antibody. The analysis was with a FACS IV3 using a fluorescent obtained by 0.5 \ig/ml for anti-Thy 1.2 and performed at 50 7o of maximum binding3 1 \ig/ml for anti-melanoma antibodies.
DRUG TARGETING
930
1
1
1
1
1
1
1
A)Target cells; WEHI-7 mouse leukemia cells (Thy 1-2positive) Anti-1-2 IT Ricin[
4
A-Chain
SPECIFICITY
1|Anti-DNP
FACTOR
B)Target cells i M T477 human melanoma cells (p97positive) Anti-melanoma IT Ricin [
10'12
10"11
4
10"10
A-Chain
Sp
EClFICITY FACTOR
10"9
10"8
10"7
JAnti-DNP
IT
10_6A-ChainIMI
Fig. 2. Specific in vitro activity of immunotoxins. Activity for each IT is expressed at 50 % of protein synthesis inhibition in a cellular system. Arrows indicate the specificity factor. Vertical bars indicate PI50 for ricin and A-chain. Dashed areas indicate PI50 for anti-DNP imrnunotoxin used as control. Anti-Thy 1.2 immunotoxin : WEHI-7 (Thy 1.2 positive) mouse lymphoma cells were used for testing the inhibition of protein synthesis in a cellular system. The ricin concentration producing 50 % inhibition (PI 50) was 2 x 10"H whereas for highly purified A-chain the PI50 = 6.10"7M. The PI 50 for anti-Thy 1.2 IT was 10" 10 while with an unrelated anti-DNP immunotoxin the PI 50 was similar to the one of A-chain alone. The specificity factor calculated between the PI 50 of A-chain and IT was about 6000 (figure 2a). The measure of the capacity of the cells to form colonies after IT treatment showed that IT was able to inhibit colony formation completely at 10"7M concentration, which is 100 times less than for A-chain (fig. 3). Those results suggest that there were no cells escaping immunotoxin treatment. With the highly sensitive method of colony formation inhibition, a clinical important parameter could be estimated, the specific killing index (the ratio between the concentration of IT which kills 100 % of tumor cells and the concentration which leaves intact 80 % or more normal cells). For anti-Thy 1.2 IT this index was of the order of 2. Anti-melanoma immunotoxin : the human melanoma cell line, Ml477 (bearing the P97 tumor-associated-antigen) (8) was used as the target for in vitro tests of inhibition of protein synthesis. While ricin showed a 3 x 1 0 ~ H M and free A-chain was 3 x 104 times less toxic, the PI 50 for IT was 5 x 10"10(figure 2b). A nonrelated IT (anti-DNP) behaved like free A-chain, although its PI 50 on DNP-labelled cells was of 10~9M. The specificity factor was of 2000 approximately. In the inhibition of colony formation, a more satisfactory activity could be demonstrated : 100 % of melanoma cells could be killed with an IT concentration comparable to the one needed by ricin itself = 2.10"9M. The specific killing index estimated was of 30. The unrelated anti-DNP IT showed no effect within the range of 2 x 103M concentrations.
931
SPECIFIC KILLING BY IMMUNOTOXINS SPECIFIC KILLING INDEX
104J c/> CD
ίιο3CD
α
- 1 0 0 % of control '- N Anti-Thy1.2 \ Immunotoxin
\
\
\
\
\
\
^A-Chain
\
2L
CO
-§10-1 \ 10" 8
10"
10" 6
A-Chain|
Fig. 3. Specific killing index for anti-Thy 1.2 IT. Inhibition by ricin, A-chain and anti-Thy 1.2 IT tested on WEHI-7 cells. index is indicated by the shaded area and arrow.
of colony formation The specific killing
CONCLUSIONS Antibody-toxin conjugates of high specific activity, have been reported, using antibodies directed either against tumor-associated antigens of colorectal carcinoma (10) or against an idiotype expressed by B-cell tumor (11). However, it is not possible from the published data, to calculate the specificity in the same way as we have done, since the PI 50 for a nonspecific conjugate was not indicated. In our study, immunotoxins were selectively toxic for the cells carrying the relevant antigen (differentiation or tumor-associated antigens), with a specificity factor of about 2000-6000. Another measure of specificity, the specific killing index, has been determined by means of the inhibition of colony formation test. This in vitro index corresponds to the in vivo therapeutic index and is the ratio between the IT concentration which kills the last tumor cell in vitro and the maximum A-chain concentration which does no harm to the same cells. The specific killing index obtained for anti-Thy 1.2 IT is of 2, and of 30 for antimelanoma IT. In the latter model, the activity of the IT, measured by the inhibition of colony formation test, is identical to the activity of ricin. This would indicate that the antibody could be as efficient as the B-chain of ricin to mediate the entry of A-chain into the cytoplasm. The high specificity of IT against their target cells, can be due to the low nonspecific activity, which depends on a highly purified A-chain, and to the use of monoclonal antibodies of high specificity. ACKNOWLEDGEMENT This study was supported in part by a grant from the Delegation Générale à la Recherche Scientifique et Technique, PARIS, FRANCE.
DRUG TARGETING
932 REFERENCES
1. Mathé, G. Loc, T.B. and Bernard, J.C. (1958). C.r. hebd. Seanc. Acad. Sei., Paris, 246, 1626-1628. 2. Moolten, F.L., Zajdel, S. and Cooperband, S.R. (1976). Ann. N.Y. Acad. Sei. 277, 690-699. 3. Thorpe, P.E. and coll. (1978). Nature 271, 752-755. 4. Olsnes, S. and Pihl A. (1976). In P. Cuatrecasas (Ed) "Receptors and Recognition" Series B. "The Specificity and Action of Animal, Bacterial and Plant Toxins". Chapman and Hall (London), p 129-173. 5. Olsnes, S., Refnes, K., and Pihl, A. (1974). Nature., 249, 627-631. 6. Boquet, P., Silverman, M.S., Pappenheimer, A.M., Vernon, W.F., (1976). Proc. natn. Acad. Sei. USA, 73. 4449-4453. 7. Jansen, F.K. and coll. (1980). Imm. Lett. 2, 97-102. 8. Woodbury, R.G., J. Brown, M.Y., Yeh, I. Hellstrom, K.E. Hellstrom. (1980). Proc. natn. Acad. Sei. USA. 77, 2183-2187. 9. Pau B. and coll (1980). In H. Peeters (Ed) 28th colloquium Protides of the biological fluids, Pergamon Press, 497-500. 10. Gilliland, D.G. and coll. (1980). Proc. natn. Acad. Sei. USA. 77, 4539-4543. 11. Krolick, K.A., Villemze, C , Isakson, P., Uhr, J.W. & Vitetta, E.S. (1980). Proc. natn. Acad. Sei. USA. 77, 5419-5423.
SPECIFIC KILLING OF HUMAN AND MOUSE TUMOR CELLS BY IMMUNOTOXINS P. CASELLAS,* H. E. BLYTHMAN,* J. P. BROWN,** O. GROS,* P. GROS,* K. E. HELLSTRÖM,** I. HELLSTRÖM,** F. K. JANSEN,* P. PONCELET* and H. VIDAL* ^Centre de Recherches CLIN MIDY, Montpellier, France **Fred Hutchinson Cancer Research Center, Seattle, Washington, U.S.A.
ABSTRACT Monoclonal antibodies (anti-Thy 1.2 and anti-human melanoma P97) were used to replace the binding moiety of ricin, the B-chain, in order to obtain hybrid molecules (immunotoxins) using as toxic moiety the purified A-chain of the toxin. A disulphide bridge was used to link both moieties. Both immunotoxins showed specific cytotoxicity for their respective target cells, WEHI-7 mouse lymphoma and Ml477 human melanoma. The specific activity was determined by the in vitro tests of protein synthesis inhibition and of inhibition of colony formation. The second test showed that both immunotoxins could specifically kill the last target cell without damaging the control cells, and a specific killing index could be calculated. KEYWORDS Immunotoxins, monoclonal antibodies, ricin, A-chain, Thy 1.2 antigen, tumor-associated antigen, immunotherapy, specific cytotoxicity, M1477 human melanoma cell line (P97). INTRODUCTION Numerous attempts to kill target cells using specific antibodies directed against cell surface antigens have been reported. Among the different approaches described, one of the most promising appears to be the use of specific antibodies as carriers of drugs or toxic proteins (1-3). This field has been considerably developed once lymphocyte hybridisation techniques made it possible to obtain extremely specific monoclonal antibodies. The toxic proteins most frequently used, diphtheria toxin and ricin, are extremely potent, because one molecule will suffice to kill a cell (4). Their molecular structure has been extensively described (5). The A-chain, responsible for the toxicity, is linked via one single disulphide bridge to the B-chain, which binds to cell membranes, facilitating the entry of A-chain into the cytoplasm (6). Highly specific results were obtained when the non-specific binding of the B-chain was circumvented by retaining the A-subunit, which is coupled, via a disulphide bridge, to a monoclonal antibody of a chosen specificity.
927
DRUG TARGETING
928
The validity of this approach has already been tested with an artificial model, (7) and the high potential of these hybrid molecules have now been further demonstrated using two natural models : anti-Thy 1.2 immunotoxins (using as targets WEHI-7 mouse leukemia cells) and monoclonal anti-melanoma antibodies, directed against human melanoma-associated antigen P97 (using as targets Ml477 human melanoma cells). MATERIAL AND METHODS Ricin, A-chain and immunotoxins were prepared as previously described (7). Briefly, ricin was extracted from Castor beans and purified by affinity chromatography on agarose. After cleavage of the disulphide bridge with 2ME, the A-chain was isolated on DEAE-Sepharose 6B and concentrated on CM-Sepharose 6B. Immunotoxins were prepared by coupling the A-chain to the antibodies via a disulphide linkage. Antibodies were reacted with 3-(2-pyridyl-dithio)-propionic acid activated by a limited quantity of carbodiimide. After dialysis, the modified antibodies were reacted with the free thiol group of A-chain. Unreacted A-chain was eliminated by chromatography on Sephadex G200. Anti-Thy 1.2. Immunotoxins were prepared with monoclonal IgM antibodies purchased from Olac (England), anti-DNP-Immunotoxin using monoclonal IgG antibodies and anti-melanoma-Immunotoxins with monoclonal anti-P97. The antibody binding of the immunotoxin was analysed using a second fluorescent antibody with a fluorescent activated cell sorter (FACS IV Beckton-Dickinson). Enzymatic activity of the A-chain was assessed in a cell-free protein synthesis system in presence of (3-mercaptoethanol as well as in an in vitro target cell system. For this last test, cells at 4 x 10 cells per ml in 50 μΐ aliquots were seeded into wells of microtest culture plates and incubated with toxins or immunotoxin at various concentrations in a 100 μΐ final volume. The cells were incubated at 37°C for 20 hrs and again for 4 hrs after addition of 2μ Ci of 14C-leucine (25 Ci/mmole New England Nuclear). The cells were harvested from the wells with a multiple automated cell harvester. Incorporation of 14C-leucine into protein was measured in a Intertechnique scintillation counter. To increase the sensitivity of the measurement of Immunotoxin activity, another system was used, studying the inhibition of colony formation induced by IT treatment. For this test, target cells were cultured (WEHI-7) or plated (M1477) at various limiting dilutions to determine their colony formation or plating efficiency. Test cells were incubated for 24 hrs at various concentrations of toxins or immunotoxins. Cells were then washed and seeded into Petri dishes, on a feeder layer of 0,5 % agarose containing 1θ6 macrophages (for WEHI-7 cells) or seeded directly (without a feeder layer) in the case of adherent cells (M1477). After 10-15 days of incubation, the number of colonies (indicating cells surviving the treatment) were counted with an automatic colony counter (ARTEK 980 system). Colony formation or plating efficiency varied between 15-30 % for different experiments, but a linear relationship was observed between the number of cells seeded and the number of colonies recovered. RESULTS AND DISCUSSION Immunotoxins (IT). The coupling method allowed a linkage of active A-chain to antibody in the ratio of 3 to 1 for IgM anti-Thy 1.2 IT, and of 1.4 to 1 for the IgG anti-melanoma IT. The binding activity of the IT to its respective target cells was analyzed by immunofluorescence (FACS). The fluorescent intensity of the two immunotoxins compared to that of unmodified antibody showed a slight decrease of bin-
SPECIFIC KILLING BY IMMUNOTOXINS
929
ding in both cases, which may correspond either to a hindrance of the binding of the second Ab due to A-chain or to a loss of the binding capacity due to the conjugation (fig 1 a-b). This loss can be estimated as 10 % and 30 % for anti-Thy 1.2 IT and anti-melanoma IT respectively.
NUMBER OF CELLS
Anti-Thy 1-2 Immunotoxin IgM Anti-Thy 1-2
FLUORESCENCE INTENSITY
t- - - SCATTER
NUMBER OF CELLS
Anti Melanoma Immunotoxin
l/Nç [SCATTER
Melanoma IgG
FLUORESCENCE INTENSITY
Fig. 1. Binding capacity of anti-Thy 1.2 immunotoxin (a) and anti-melanoma immunotoxin (b) compared to their respective unmodified antibodies. Analysis was done anti-mouse IgG antibody. The analysis was with a FACS IV3 using a fluorescent obtained by 0.5 \ig/ml for anti-Thy 1.2 and performed at 50 7o of maximum binding3 1 \ig/ml for anti-melanoma antibodies.
DRUG TARGETING
930
1
1
1
1
1
1
1
A)Target cells; WEHI-7 mouse leukemia cells (Thy 1-2positive) Anti-1-2 IT Ricin[
4
A-Chain
SPECIFICITY
1|Anti-DNP
FACTOR
B)Target cells i M T477 human melanoma cells (p97positive) Anti-melanoma IT Ricin [
10'12
10"11
4
10"10
A-Chain
Sp
EClFICITY FACTOR
10"9
10"8
10"7
JAnti-DNP
IT
10_6A-ChainIMI
Fig. 2. Specific in vitro activity of immunotoxins. Activity for each IT is expressed at 50 % of protein synthesis inhibition in a cellular system. Arrows indicate the specificity factor. Vertical bars indicate PI50 for ricin and A-chain. Dashed areas indicate PI50 for anti-DNP imrnunotoxin used as control. Anti-Thy 1.2 immunotoxin : WEHI-7 (Thy 1.2 positive) mouse lymphoma cells were used for testing the inhibition of protein synthesis in a cellular system. The ricin concentration producing 50 % inhibition (PI 50) was 2 x 10"H whereas for highly purified A-chain the PI50 = 6.10"7M. The PI 50 for anti-Thy 1.2 IT was 10" 10 while with an unrelated anti-DNP immunotoxin the PI 50 was similar to the one of A-chain alone. The specificity factor calculated between the PI 50 of A-chain and IT was about 6000 (figure 2a). The measure of the capacity of the cells to form colonies after IT treatment showed that IT was able to inhibit colony formation completely at 10"7M concentration, which is 100 times less than for A-chain (fig. 3). Those results suggest that there were no cells escaping immunotoxin treatment. With the highly sensitive method of colony formation inhibition, a clinical important parameter could be estimated, the specific killing index (the ratio between the concentration of IT which kills 100 % of tumor cells and the concentration which leaves intact 80 % or more normal cells). For anti-Thy 1.2 IT this index was of the order of 2. Anti-melanoma immunotoxin : the human melanoma cell line, Ml477 (bearing the P97 tumor-associated-antigen) (8) was used as the target for in vitro tests of inhibition of protein synthesis. While ricin showed a 3 x 1 0 ~ H M and free A-chain was 3 x 104 times less toxic, the PI 50 for IT was 5 x 10"10(figure 2b). A nonrelated IT (anti-DNP) behaved like free A-chain, although its PI 50 on DNP-labelled cells was of 10~9M. The specificity factor was of 2000 approximately. In the inhibition of colony formation, a more satisfactory activity could be demonstrated : 100 % of melanoma cells could be killed with an IT concentration comparable to the one needed by ricin itself = 2.10"9M. The specific killing index estimated was of 30. The unrelated anti-DNP IT showed no effect within the range of 2 x 103M concentrations.
931
SPECIFIC KILLING BY IMMUNOTOXINS SPECIFIC KILLING INDEX
104J c/> CD
ίιο3CD
α
- 1 0 0 % of control '- N Anti-Thy1.2 \ Immunotoxin
\
\
\
\
\
\
^A-Chain
\
2L
CO
-§10-1 \ 10" 8
10"
10" 6
A-Chain|
Fig. 3. Specific killing index for anti-Thy 1.2 IT. Inhibition by ricin, A-chain and anti-Thy 1.2 IT tested on WEHI-7 cells. index is indicated by the shaded area and arrow.
of colony formation The specific killing
CONCLUSIONS Antibody-toxin conjugates of high specific activity, have been reported, using antibodies directed either against tumor-associated antigens of colorectal carcinoma (10) or against an idiotype expressed by B-cell tumor (11). However, it is not possible from the published data, to calculate the specificity in the same way as we have done, since the PI 50 for a nonspecific conjugate was not indicated. In our study, immunotoxins were selectively toxic for the cells carrying the relevant antigen (differentiation or tumor-associated antigens), with a specificity factor of about 2000-6000. Another measure of specificity, the specific killing index, has been determined by means of the inhibition of colony formation test. This in vitro index corresponds to the in vivo therapeutic index and is the ratio between the IT concentration which kills the last tumor cell in vitro and the maximum A-chain concentration which does no harm to the same cells. The specific killing index obtained for anti-Thy 1.2 IT is of 2, and of 30 for antimelanoma IT. In the latter model, the activity of the IT, measured by the inhibition of colony formation test, is identical to the activity of ricin. This would indicate that the antibody could be as efficient as the B-chain of ricin to mediate the entry of A-chain into the cytoplasm. The high specificity of IT against their target cells, can be due to the low nonspecific activity, which depends on a highly purified A-chain, and to the use of monoclonal antibodies of high specificity. ACKNOWLEDGEMENT This study was supported in part by a grant from the Delegation Générale à la Recherche Scientifique et Technique, PARIS, FRANCE.
DRUG TARGETING
932 REFERENCES
1. Mathé, G. Loc, T.B. and Bernard, J.C. (1958). C.r. hebd. Seanc. Acad. Sei., Paris, 246, 1626-1628. 2. Moolten, F.L., Zajdel, S. and Cooperband, S.R. (1976). Ann. N.Y. Acad. Sei. 277, 690-699. 3. Thorpe, P.E. and coll. (1978). Nature 271, 752-755. 4. Olsnes, S. and Pihl A. (1976). In P. Cuatrecasas (Ed) "Receptors and Recognition" Series B. "The Specificity and Action of Animal, Bacterial and Plant Toxins". Chapman and Hall (London), p 129-173. 5. Olsnes, S., Refnes, K., and Pihl, A. (1974). Nature., 249, 627-631. 6. Boquet, P., Silverman, M.S., Pappenheimer, A.M., Vernon, W.F., (1976). Proc. natn. Acad. Sei. USA, 73. 4449-4453. 7. Jansen, F.K. and coll. (1980). Imm. Lett. 2, 97-102. 8. Woodbury, R.G., J. Brown, M.Y., Yeh, I. Hellstrom, K.E. Hellstrom. (1980). Proc. natn. Acad. Sei. USA. 77, 2183-2187. 9. Pau B. and coll (1980). In H. Peeters (Ed) 28th colloquium Protides of the biological fluids, Pergamon Press, 497-500. 10. Gilliland, D.G. and coll. (1980). Proc. natn. Acad. Sei. USA. 77, 4539-4543. 11. Krolick, K.A., Villemze, C , Isakson, P., Uhr, J.W. & Vitetta, E.S. (1980). Proc. natn. Acad. Sei. USA. 77, 5419-5423.