Br. J. Cancer
(1990), 62, Suppl. X,
Br. J. Cancer (1990), 62, Suppl.
X,
'."
1-5 5 1
©
Macmillan Press Ltd., 1990
Macmillan
Antibody transport and internalization into tumours S. Matzku', G. Moldenhauer2, H. Kalthoff3, Silvana Canevari4, Maria Colnaghi4, J. Schuhmacher' & H. Bihl5 'Institute of Radiology and Pathophysiology, German Cancer Research Center, D-6900 Heidelberg, 2Institute of Immunology and Genetics, German Cancer Research Center, 3Division of Immunology, University Hospital, D-3000 Hamburg, FRG, 4Division of Experimental Oncology, INT, I-20133 Milano, Italy and 5Division of Nuclear Medicine, University, D-6900 Heidelberg, FRG. Summary Internalization of monoclonal antibody (MAb) conjugates is an important feature of tumour targeting, both with respect to the therapeutic action of substances coupled to the antibody and to retention of radionuclides. Problems of analysing internalization in vitro and in vivo, of manipulating internalization, and of evaluating the involvement of normal tissues are illustrated by recent experimental data and are discussed in the light of published evidence.
The key elements of tumour targeting with antibody conjugates are the amount and rate of delivery into target tissue and possibly also into the target cell, as well as concurrent binding/retention in non-target tissues. They are determined by the biology of the antigen, the architecture of tumour tissue and by the pharmacodynamics of the conjugate. No strategy will be able to uniformly cope with all par'ameters of relevance in individual targeting situations. Since amounts of monoclonal antibody (MAb) conjugates reaching the tumour are generally too low (Jain, 1989), especially in the patient, measures for improving transport and delivery are needed to achieve sufficient therapeutic action. Protein engineering techniques will ultimately provide us with synthetic MAbs representing tailor-made combinations of function, molecule size and compatibility with the host's immune system. Yet, parameters determined by the nature of the tumour antigen, the architecture of tumour tissue and the laws of transport and degradation of macromolecules will not be outruled by species-related modifications of the carrier molecule. Being given this background of insufficiently solved problems, there remains a large field of 'conservative' concepts of improving tumour targeting with monoclonal antibodies. Materials and methods
Monoclonal antibodies, cell lines and xenografts MAbs mentioned in the text were purified from ascitic fluid by sequential chromatography on Protein A and Mono Q ion exchange columns (Pharmacia, Freiburg, FRG). Labelling with '25I or 13'I was carried out according to the standard IODO-Gen procedure. Labelling with "'In was performed according to Hnatowich et al. (1983), except that buffer exchange and separation of unreacted DTPA was achieved by centrifugation through columns of either Sepahdex G-25 or Bio-Gel P-30. Immunoreactivity of labelled MAbs was determined by the Lineweaver Burk method (Matzku et al., 1985). Cell lines were cultured in RPMI 1640 media supplemented with antibiotics, 4 mM glutamine and 10% fetal calf serum (GIBCO, Karlsruhe, FRG). Xenografts were raised by injecting 5-10 x 106 tissue cultured tumour cells into CD- 1 nu/nu mice (Charles River-WIGA, Sulzfeld, FRG).
Evaluation of MAb internalization by the pH 2.8 method The portion of labelled MAb that bound to the exterior of tissue cultured target cells was desorbed by treatment of MAb-loaded cells with an iso-osmolar buffer of pH 2.8
Correspondence: S. Matzku.
(0.05 M glycine/HCI, 0.1 M NaCI) for 20 min at 0°C as described previously (Matzku et al., 1986). Biodistribution of labelled MAbs in mice Biodistribution of MAbs labelled with either "'In-DTPA or I3'l was assessed in normal NMRI mice (Zentralinstitut fur Versuchstiere, Hannover, FRG). Groups of six animals were injected with 0.2 jig or 8.0 pg labelled MAb (no addition of unlabelled MAb) and dissected 1 h or 24 h later. Total liver and several other organs (not shown) were removed and counted on a GeLi gamma spectrometer. Intratumoral distribution of '25I-labelled MAb HD39 (F(ab')2) in B lymphoma BJAB xenografts was characterized autoradiographically as previously described (Matzku et al., 1989b). Results and discussion
Rationale of using several MAbs against a given tumour or antigen When dealing with a given targeting situation, which is largely defined by the biology of the target antigen and the composition of the target tissue, it seems logical to study as many MAbs as possible in order to hopefully find one that brings together a suitable complement of immunochemical and functional properties. First of all, cocktails of selected MAbs may be arranged which recognize different antigens on a heterogeneous tumour, thus directing MAb conjugates to all subpopulations of tumour cells (for reference see, for example, Epenetos et al., 1987; Stewart et al., 1989). Secondly, it may prove promising to study MAbs that are directed towards distinct epitopes of a selected antigen. These may exhibit functional diversity as has been convincingly demonstrated with various MAbs against the transferrin receptor (Hopkins & Trowbridge, 1983; Trowbridge et al., 1984; Lesley & Schulte, 1985). If, for example, internalizing antibodies are desired, it is obviously important to screen panels of MAbs in order to identify the most appropriate candidate(s). Evaluation of internalization by the pH 2.8 desorption method Most immune complexes are disrupted upon exposure to acid pH. When cells are treated with an iso-osmolar buffer of pH 2.8, externally exposed antibody will be desorbed while intracellular antibody and/or radioactivity will be left untouched. However, there is non-systematic evidence suggesting that pH 2.8 may not suffice to desorb 'very high' affinity antibodies. This reservation has to be kept in mind when experimental data are discussed. In our experience, differential internalization was regularly observed whenever more than one MAb was available for a
2
S. MATZKU et al.
given cell surface antigen, e.g. MAbs HD6 and HD39 (Dorken et al., 1986; Matzku et al., 1989a) against the CD22 differentiation antigen on B lymphoma cells, MAbs MOvl7/ 18/19 (Miotti et al., 1987) against the gp38 antigen on breast cancer and ovarian cancer cells, and a large variety of antiCEA MAbs. The CEA situation is illustrated in Figure 1 showing MAbs HEA19 (Moller et al., 1986) and CI-P83 (raised by H.K.); the former exhibits a very high, the latter only a very low level of internalization. Among the other CEA-detecting MAbs tested, no. 192 (Buchegger et al., 1988) (kindly provided by J-P. Mach, Lausanne) and HEA81 (Moller et al., 1986) showed high internalization, F023C5 (Mariani et al., 1984) (kindly provided by SORIN SpA, Saluggia) and BW431/26 (Bosslet et al., 1988) (kindly provided by Behringwerke, Marburg) showed weak internalization, while MAb no. 35 (Haskell et al., 1983) showed intermediate to low internalization depending on the target cells (i.e. MKN-45, 818-7). When these MAbs were tested in Western blots using cell extracts as well as 'purified CEA' (Dako, Santa Barbara, USA), MAbs CI-P83, BW431 /26, HEA19 and no. 35 were found to bind to material with Mr> 160 kD only, while MAbs F023C5, HEA81 and no. 192 additionally bound to cross-reacting material of Mr
(1990), 62, Suppl. X,
Br. J. Cancer (1990), 62, Suppl.
X,
'."
1-5 5 1
©
Macmillan Press Ltd., 1990
Macmillan
Antibody transport and internalization into tumours S. Matzku', G. Moldenhauer2, H. Kalthoff3, Silvana Canevari4, Maria Colnaghi4, J. Schuhmacher' & H. Bihl5 'Institute of Radiology and Pathophysiology, German Cancer Research Center, D-6900 Heidelberg, 2Institute of Immunology and Genetics, German Cancer Research Center, 3Division of Immunology, University Hospital, D-3000 Hamburg, FRG, 4Division of Experimental Oncology, INT, I-20133 Milano, Italy and 5Division of Nuclear Medicine, University, D-6900 Heidelberg, FRG. Summary Internalization of monoclonal antibody (MAb) conjugates is an important feature of tumour targeting, both with respect to the therapeutic action of substances coupled to the antibody and to retention of radionuclides. Problems of analysing internalization in vitro and in vivo, of manipulating internalization, and of evaluating the involvement of normal tissues are illustrated by recent experimental data and are discussed in the light of published evidence.
The key elements of tumour targeting with antibody conjugates are the amount and rate of delivery into target tissue and possibly also into the target cell, as well as concurrent binding/retention in non-target tissues. They are determined by the biology of the antigen, the architecture of tumour tissue and by the pharmacodynamics of the conjugate. No strategy will be able to uniformly cope with all par'ameters of relevance in individual targeting situations. Since amounts of monoclonal antibody (MAb) conjugates reaching the tumour are generally too low (Jain, 1989), especially in the patient, measures for improving transport and delivery are needed to achieve sufficient therapeutic action. Protein engineering techniques will ultimately provide us with synthetic MAbs representing tailor-made combinations of function, molecule size and compatibility with the host's immune system. Yet, parameters determined by the nature of the tumour antigen, the architecture of tumour tissue and the laws of transport and degradation of macromolecules will not be outruled by species-related modifications of the carrier molecule. Being given this background of insufficiently solved problems, there remains a large field of 'conservative' concepts of improving tumour targeting with monoclonal antibodies. Materials and methods
Monoclonal antibodies, cell lines and xenografts MAbs mentioned in the text were purified from ascitic fluid by sequential chromatography on Protein A and Mono Q ion exchange columns (Pharmacia, Freiburg, FRG). Labelling with '25I or 13'I was carried out according to the standard IODO-Gen procedure. Labelling with "'In was performed according to Hnatowich et al. (1983), except that buffer exchange and separation of unreacted DTPA was achieved by centrifugation through columns of either Sepahdex G-25 or Bio-Gel P-30. Immunoreactivity of labelled MAbs was determined by the Lineweaver Burk method (Matzku et al., 1985). Cell lines were cultured in RPMI 1640 media supplemented with antibiotics, 4 mM glutamine and 10% fetal calf serum (GIBCO, Karlsruhe, FRG). Xenografts were raised by injecting 5-10 x 106 tissue cultured tumour cells into CD- 1 nu/nu mice (Charles River-WIGA, Sulzfeld, FRG).
Evaluation of MAb internalization by the pH 2.8 method The portion of labelled MAb that bound to the exterior of tissue cultured target cells was desorbed by treatment of MAb-loaded cells with an iso-osmolar buffer of pH 2.8
Correspondence: S. Matzku.
(0.05 M glycine/HCI, 0.1 M NaCI) for 20 min at 0°C as described previously (Matzku et al., 1986). Biodistribution of labelled MAbs in mice Biodistribution of MAbs labelled with either "'In-DTPA or I3'l was assessed in normal NMRI mice (Zentralinstitut fur Versuchstiere, Hannover, FRG). Groups of six animals were injected with 0.2 jig or 8.0 pg labelled MAb (no addition of unlabelled MAb) and dissected 1 h or 24 h later. Total liver and several other organs (not shown) were removed and counted on a GeLi gamma spectrometer. Intratumoral distribution of '25I-labelled MAb HD39 (F(ab')2) in B lymphoma BJAB xenografts was characterized autoradiographically as previously described (Matzku et al., 1989b). Results and discussion
Rationale of using several MAbs against a given tumour or antigen When dealing with a given targeting situation, which is largely defined by the biology of the target antigen and the composition of the target tissue, it seems logical to study as many MAbs as possible in order to hopefully find one that brings together a suitable complement of immunochemical and functional properties. First of all, cocktails of selected MAbs may be arranged which recognize different antigens on a heterogeneous tumour, thus directing MAb conjugates to all subpopulations of tumour cells (for reference see, for example, Epenetos et al., 1987; Stewart et al., 1989). Secondly, it may prove promising to study MAbs that are directed towards distinct epitopes of a selected antigen. These may exhibit functional diversity as has been convincingly demonstrated with various MAbs against the transferrin receptor (Hopkins & Trowbridge, 1983; Trowbridge et al., 1984; Lesley & Schulte, 1985). If, for example, internalizing antibodies are desired, it is obviously important to screen panels of MAbs in order to identify the most appropriate candidate(s). Evaluation of internalization by the pH 2.8 desorption method Most immune complexes are disrupted upon exposure to acid pH. When cells are treated with an iso-osmolar buffer of pH 2.8, externally exposed antibody will be desorbed while intracellular antibody and/or radioactivity will be left untouched. However, there is non-systematic evidence suggesting that pH 2.8 may not suffice to desorb 'very high' affinity antibodies. This reservation has to be kept in mind when experimental data are discussed. In our experience, differential internalization was regularly observed whenever more than one MAb was available for a
2
S. MATZKU et al.
given cell surface antigen, e.g. MAbs HD6 and HD39 (Dorken et al., 1986; Matzku et al., 1989a) against the CD22 differentiation antigen on B lymphoma cells, MAbs MOvl7/ 18/19 (Miotti et al., 1987) against the gp38 antigen on breast cancer and ovarian cancer cells, and a large variety of antiCEA MAbs. The CEA situation is illustrated in Figure 1 showing MAbs HEA19 (Moller et al., 1986) and CI-P83 (raised by H.K.); the former exhibits a very high, the latter only a very low level of internalization. Among the other CEA-detecting MAbs tested, no. 192 (Buchegger et al., 1988) (kindly provided by J-P. Mach, Lausanne) and HEA81 (Moller et al., 1986) showed high internalization, F023C5 (Mariani et al., 1984) (kindly provided by SORIN SpA, Saluggia) and BW431/26 (Bosslet et al., 1988) (kindly provided by Behringwerke, Marburg) showed weak internalization, while MAb no. 35 (Haskell et al., 1983) showed intermediate to low internalization depending on the target cells (i.e. MKN-45, 818-7). When these MAbs were tested in Western blots using cell extracts as well as 'purified CEA' (Dako, Santa Barbara, USA), MAbs CI-P83, BW431 /26, HEA19 and no. 35 were found to bind to material with Mr> 160 kD only, while MAbs F023C5, HEA81 and no. 192 additionally bound to cross-reacting material of Mr
