62, (1990), 62, Br. J. Cancer (1990),
Br. J. Cancer
Suppl. X, Suppl. X,
Q'I
85-88 85 88
Macmillan Press Ltd., 1990 1990
The role of humoral and cellular immunity in patients developing human anti-murine immunoglobulin antibody responses after radioimmunotherapy C. Kosmas', S. Man2, A.A. Epenetos' & N.S. Courtenay-Luck' 'ICRF Oncology Group, Department of Clinical Oncology and 2Department of Immunology, The Royal Postgraduate Medical School, Hammersmith Hospital, Du Cane Road, London W12 OHS, UK. Summary Epstein-Barr virus (EBV)-immortalised B cell lines were established from patients receiving multiple administrations (two or more) of radiolabelled murine monoclonal antibodies for the treatment of ovarian cancer. Cells secreting anti-id2 Ig, an immunoglobulin with binding specificities comparable to the administered murine monoclonal antibody, were isolated by using magnetic beads coated with tumour-associated antigen, incubated with the cells and concentrated with a magnetic particle concentrator. Cross-linking of the immunoglobulin receptors by the antigen-coated beads appears to stimulate proliferation, resulting in increased secretion of the human anti-tumour-associated antigen antibodies. The T cell responses were studied and it was found that monoclonal antibody therapy appears to lead to an increase in the population of T cells committed to proliferate in response to both specific antigen and non-specific mitogens. Multiple administrations of monoclonal antibody induce the generation of T cells which proliferate in vitro following stimulation with murine antibodies. The relevant (administered) monoclonal antibody induces higher proliferation rates than an idiotypically unrelated antibody of the same isotype, indicating the generation of idiotypically restricted T cell responses in these patients.
Within the last decade murine monoclonal antibodies have been employed in the therapy of neoplasia. As a consequence of their use a number of investigators have reported the development of anti-murine antibody responses (Schroff et al., 1985; Courtenay-Luck et al., 1986). Of these responses the most restricted are the anti-idiotypic. These responses are reported to develop mainly in patients receiving multiple administrations of murine monoclonal antibodies (Herlyn et al., 1986; Steinitz et al., 1988). In 1975 Jerne postulated the idiotypic network theory. This theory proposed that immunoregulation could result from the interaction of idiotype with anti-idiotype. Nearly all the reports on anti-idiotypic responses in patients receiving murine monoclonal antibody administrations have focused their attention on the humoral aspects of these responses. If Jerne's theory is correct then both B and T cells must interact in this idiotypic network. Having shown that the serum of patients receiving multiple administrations of murine monoclonal antibodies contains antibodies which reflect the internal image of the tumour-associated antigen (anti-id') in addition to human anti-anti-idiotypic antibodies (anti-id2) which have binding specificities similar to the administered murine monoclonal antibodies (Courtenay-Luck et al., 1988), we have immortalised peripheral blood B lymphocytes and from these positively selected anti-id2 antibody-secreting cells. In addition to the above study we have studied the in vitro T cell proliferation responses in an attempt to demonstrate the role of T cells in idiotypic immunoregulation.
Patients, materials and methods Patients Five patients with histological confirmation of ovarian cancer (stages 3 and 4) and malignant effusions were entered in the study protocol.
Antibody protocols For diagnostic (imaging) studies patients received intravenously 250 ltg murine monoclonal antibody labelled with 1-5 mCi (37-185 MBq) '23I. For therapy patients received
Correspondence: N.S. Courtenay-Luck.
between 5 and 15 mg monoclonal antibody labelled with injected either intraperitoneally or intrapleurally.
'`'I,
Monoclonal antibodies HMFG1 is a murine IgG, antibody shown to react with a large mucin-like molecule normally produced by the lactating breast but also expressed by the majority (>90%) of ovarian, breast and other carcinomas (Burchell et al., 1983). 11.4.1 is a murine IgG, antibody raised against murine major histocompatibility complex antigen H-2k (Oi et al., 1979). Immortalisation of peripheral blood B lymphocytes The lymphomononuclear fraction of the peripheral blood was isolated after density centrifugation of heparinised blood through lymphocyte separation medium (Flow, UK) at 2,000 r.p.m. for 30 min. The cells were then washed twice in RPMI medium (1,500 r.p.m.) for 10 and 5 min respectively and 107 cells were incubated with 1 ml of Epstein-Barr virus (EBV)-containing supernatant (B95.8 marmoset lymphoblastoid cell line) for 1 h at 37C. Subsequently the cells were washed twice and plated into 24-well tissue culture plates (Flow, UK) in RPMI containing 10% FCS and 2 fig ml' of phytohaemagglutinin (PHA; Wellcome, UK) at a concentration of 106 cells well-' in a final volume of 1 ml. Cultures were then left for 7 days at 37°C in which time T cell depletion occurred. After this time the lymphoblastoid cells were expanded into tissue culture flasks.
Anti-anti-idiotypic antibody assay Patients' pre- and post-therapy sera were screened against purified human milk fat globule (MFG) antigen, as were the EBV supernatants, for the presence of anti-id2 antibodies, using an enzyme linked immunosorbent assay (ELISA). Flatbottomed 96-well microtitre plates (Dynatech, UK) were coated overnight (18 h) at 4°C with 50 ng well-' of purified HMFG antigen (kindly provided by J. Taylor-Papadimitriou) in bicarbonate buffer, pH 9.6, by adding 50 tcl well-'. They were then washed once in PBS/0.05% Tween buffer, pH 7.4, and incubated with PBS/0.5% BSA solution to block nonspecific binding for 30 min at room temperature. Then they were washed in buffer (PBS/Tween) and serial dilutions (doubling) of 100 ftl supernatants of the cultures were applied and incubated at 37C for 2 h. After three washes in buffer for 4 min each, 100 IL of the species-specific sheep anti-
86
C. KOSMAS et al.
human immunoglobulin antiserum (Amersham International, UK) was added at 1:1,000 dilution in PBS/Tween (100pl well-') and incubated for 1 h at 37°C. Finally the plates were washed three times for 4 min each time in buffer and incubated at room temperature with 100 ftl well-' of 2,2'azino-di(3-ethyl-benzthiazoline sulphate) (Amersham International, UK) in the dark, and the absorbance determined in a Titertek Multiscan plate reader (Flow Irvine, Scotland) at 405 nm.
2-
E CD to ,s41
1-
0
ci
Selection of anti-anti-idiotypic B cells by antigen-coated magnetic beads Briefly, tosyl-activated Dynabeads M450 (Dynal, UK) were stored in 1 mM HCI and washed once in distilled water. The MFG antigen was dissolved in 0.2M borate buffer, pH 9.5, to a concentration of 150 fg ml-'. An equal volume of Dynabeads M-450 was added to the antigen solution (75pg:15mg antigen:bead ratio) and incubated for 24h at 22°C by slow end-over-end rotation. The beads were then collected using a magnetic particle concentrator and washed sequentially in 5 ml of 0.1 M PBS for 10 min, 5 ml of 1 M ethanolamine HCI, pH9.5, containing 0.1% Tween-20 for 2h adding the Tween-20 immediately before use to this buffer, then with 5 ml of 0.05 M Tris in 0.1 M NaCl, 0.1 % BSA, 0.01% merthiolate and 0.1% Tween-20, pH 7.5, for 12 h. The last washing step was repeated without Tween-20 for 2 h and the beads were then used in the cell separation experiments. A bead to cell ratio of 3:1 was used for positive selection. Using this procedure 75 LI of antigen-coated beads (3 x 107 beads) were mixed with 107 EBV-lymphoblastoid cells for 3-5 min at 4°C. Then isolated cells attached to beads were collected using the magnetic particle concentrator and plated out in 24-well tissue culture plates using 5 x 105 irradiated (3000 rad) peripheral blood mononuclear cells (PBMCs) as feeders per culture. Control EBV cells not producing anti-id2 antibodies were incubated with antigen-coated beads according to the above method, in order to detect non-specific binding by lymphocytes of irrelevant specificity. In addition, non-coated beads were incubated under identical conditions in order to determine any non-specific selection. T cell proliferation assays PBMCs were incubated at I05 cells well' in a 96-well flatbottomed microtitre plate, using increasing concentrations (10 fold) of monoclonal antibodies (0.001-1,000 Lg ml-') or PHA (1-100;Lgml-'). Control cultures contained cells in culture medium only. The cells were kept in culture for 72 h. After 56 h of the 72 h incubation time the cultures were pulsed with tritiated thymidine, 0.5 fCi well-' (Amersham International, UK) and harvested using an automatic cell harvester. The incorporated radioactivity was then determined by means of a P-scintillation counter. Results were expressed as c.p.m. culture-' using the mean value of the triplicate wells.
0-
CD%j
1Dv-sup. Figure 1 ELISA for anti-id2 antibody production of EBV-B cell supernatants of non-selected cells (EBV-sup.) and after selection with MFG antigen-coated magnetic beads (Bi, B2, B3 and B4 represent supernatants of the wells containing the selected B cells). _, 1:1. M, 1:2; E, 1:4. -..-
The selection of the lymphoblastoid cells from the patient with non-detectable anti-id2 serum levels did not show any anti-id2 production in the culture supernatants. Magnetic beads that were not coated with antigen did not show any non-specific selection of the patient's lymphoblastoid cells. The in vitro T cell responses were elevated and antibody concentration-dependent in three patients studied after the second therapy when compared with the pre-therapy responses (P
Br. J. Cancer
Suppl. X, Suppl. X,
Q'I
85-88 85 88
Macmillan Press Ltd., 1990 1990
The role of humoral and cellular immunity in patients developing human anti-murine immunoglobulin antibody responses after radioimmunotherapy C. Kosmas', S. Man2, A.A. Epenetos' & N.S. Courtenay-Luck' 'ICRF Oncology Group, Department of Clinical Oncology and 2Department of Immunology, The Royal Postgraduate Medical School, Hammersmith Hospital, Du Cane Road, London W12 OHS, UK. Summary Epstein-Barr virus (EBV)-immortalised B cell lines were established from patients receiving multiple administrations (two or more) of radiolabelled murine monoclonal antibodies for the treatment of ovarian cancer. Cells secreting anti-id2 Ig, an immunoglobulin with binding specificities comparable to the administered murine monoclonal antibody, were isolated by using magnetic beads coated with tumour-associated antigen, incubated with the cells and concentrated with a magnetic particle concentrator. Cross-linking of the immunoglobulin receptors by the antigen-coated beads appears to stimulate proliferation, resulting in increased secretion of the human anti-tumour-associated antigen antibodies. The T cell responses were studied and it was found that monoclonal antibody therapy appears to lead to an increase in the population of T cells committed to proliferate in response to both specific antigen and non-specific mitogens. Multiple administrations of monoclonal antibody induce the generation of T cells which proliferate in vitro following stimulation with murine antibodies. The relevant (administered) monoclonal antibody induces higher proliferation rates than an idiotypically unrelated antibody of the same isotype, indicating the generation of idiotypically restricted T cell responses in these patients.
Within the last decade murine monoclonal antibodies have been employed in the therapy of neoplasia. As a consequence of their use a number of investigators have reported the development of anti-murine antibody responses (Schroff et al., 1985; Courtenay-Luck et al., 1986). Of these responses the most restricted are the anti-idiotypic. These responses are reported to develop mainly in patients receiving multiple administrations of murine monoclonal antibodies (Herlyn et al., 1986; Steinitz et al., 1988). In 1975 Jerne postulated the idiotypic network theory. This theory proposed that immunoregulation could result from the interaction of idiotype with anti-idiotype. Nearly all the reports on anti-idiotypic responses in patients receiving murine monoclonal antibody administrations have focused their attention on the humoral aspects of these responses. If Jerne's theory is correct then both B and T cells must interact in this idiotypic network. Having shown that the serum of patients receiving multiple administrations of murine monoclonal antibodies contains antibodies which reflect the internal image of the tumour-associated antigen (anti-id') in addition to human anti-anti-idiotypic antibodies (anti-id2) which have binding specificities similar to the administered murine monoclonal antibodies (Courtenay-Luck et al., 1988), we have immortalised peripheral blood B lymphocytes and from these positively selected anti-id2 antibody-secreting cells. In addition to the above study we have studied the in vitro T cell proliferation responses in an attempt to demonstrate the role of T cells in idiotypic immunoregulation.
Patients, materials and methods Patients Five patients with histological confirmation of ovarian cancer (stages 3 and 4) and malignant effusions were entered in the study protocol.
Antibody protocols For diagnostic (imaging) studies patients received intravenously 250 ltg murine monoclonal antibody labelled with 1-5 mCi (37-185 MBq) '23I. For therapy patients received
Correspondence: N.S. Courtenay-Luck.
between 5 and 15 mg monoclonal antibody labelled with injected either intraperitoneally or intrapleurally.
'`'I,
Monoclonal antibodies HMFG1 is a murine IgG, antibody shown to react with a large mucin-like molecule normally produced by the lactating breast but also expressed by the majority (>90%) of ovarian, breast and other carcinomas (Burchell et al., 1983). 11.4.1 is a murine IgG, antibody raised against murine major histocompatibility complex antigen H-2k (Oi et al., 1979). Immortalisation of peripheral blood B lymphocytes The lymphomononuclear fraction of the peripheral blood was isolated after density centrifugation of heparinised blood through lymphocyte separation medium (Flow, UK) at 2,000 r.p.m. for 30 min. The cells were then washed twice in RPMI medium (1,500 r.p.m.) for 10 and 5 min respectively and 107 cells were incubated with 1 ml of Epstein-Barr virus (EBV)-containing supernatant (B95.8 marmoset lymphoblastoid cell line) for 1 h at 37C. Subsequently the cells were washed twice and plated into 24-well tissue culture plates (Flow, UK) in RPMI containing 10% FCS and 2 fig ml' of phytohaemagglutinin (PHA; Wellcome, UK) at a concentration of 106 cells well-' in a final volume of 1 ml. Cultures were then left for 7 days at 37°C in which time T cell depletion occurred. After this time the lymphoblastoid cells were expanded into tissue culture flasks.
Anti-anti-idiotypic antibody assay Patients' pre- and post-therapy sera were screened against purified human milk fat globule (MFG) antigen, as were the EBV supernatants, for the presence of anti-id2 antibodies, using an enzyme linked immunosorbent assay (ELISA). Flatbottomed 96-well microtitre plates (Dynatech, UK) were coated overnight (18 h) at 4°C with 50 ng well-' of purified HMFG antigen (kindly provided by J. Taylor-Papadimitriou) in bicarbonate buffer, pH 9.6, by adding 50 tcl well-'. They were then washed once in PBS/0.05% Tween buffer, pH 7.4, and incubated with PBS/0.5% BSA solution to block nonspecific binding for 30 min at room temperature. Then they were washed in buffer (PBS/Tween) and serial dilutions (doubling) of 100 ftl supernatants of the cultures were applied and incubated at 37C for 2 h. After three washes in buffer for 4 min each, 100 IL of the species-specific sheep anti-
86
C. KOSMAS et al.
human immunoglobulin antiserum (Amersham International, UK) was added at 1:1,000 dilution in PBS/Tween (100pl well-') and incubated for 1 h at 37°C. Finally the plates were washed three times for 4 min each time in buffer and incubated at room temperature with 100 ftl well-' of 2,2'azino-di(3-ethyl-benzthiazoline sulphate) (Amersham International, UK) in the dark, and the absorbance determined in a Titertek Multiscan plate reader (Flow Irvine, Scotland) at 405 nm.
2-
E CD to ,s41
1-
0
ci
Selection of anti-anti-idiotypic B cells by antigen-coated magnetic beads Briefly, tosyl-activated Dynabeads M450 (Dynal, UK) were stored in 1 mM HCI and washed once in distilled water. The MFG antigen was dissolved in 0.2M borate buffer, pH 9.5, to a concentration of 150 fg ml-'. An equal volume of Dynabeads M-450 was added to the antigen solution (75pg:15mg antigen:bead ratio) and incubated for 24h at 22°C by slow end-over-end rotation. The beads were then collected using a magnetic particle concentrator and washed sequentially in 5 ml of 0.1 M PBS for 10 min, 5 ml of 1 M ethanolamine HCI, pH9.5, containing 0.1% Tween-20 for 2h adding the Tween-20 immediately before use to this buffer, then with 5 ml of 0.05 M Tris in 0.1 M NaCl, 0.1 % BSA, 0.01% merthiolate and 0.1% Tween-20, pH 7.5, for 12 h. The last washing step was repeated without Tween-20 for 2 h and the beads were then used in the cell separation experiments. A bead to cell ratio of 3:1 was used for positive selection. Using this procedure 75 LI of antigen-coated beads (3 x 107 beads) were mixed with 107 EBV-lymphoblastoid cells for 3-5 min at 4°C. Then isolated cells attached to beads were collected using the magnetic particle concentrator and plated out in 24-well tissue culture plates using 5 x 105 irradiated (3000 rad) peripheral blood mononuclear cells (PBMCs) as feeders per culture. Control EBV cells not producing anti-id2 antibodies were incubated with antigen-coated beads according to the above method, in order to detect non-specific binding by lymphocytes of irrelevant specificity. In addition, non-coated beads were incubated under identical conditions in order to determine any non-specific selection. T cell proliferation assays PBMCs were incubated at I05 cells well' in a 96-well flatbottomed microtitre plate, using increasing concentrations (10 fold) of monoclonal antibodies (0.001-1,000 Lg ml-') or PHA (1-100;Lgml-'). Control cultures contained cells in culture medium only. The cells were kept in culture for 72 h. After 56 h of the 72 h incubation time the cultures were pulsed with tritiated thymidine, 0.5 fCi well-' (Amersham International, UK) and harvested using an automatic cell harvester. The incorporated radioactivity was then determined by means of a P-scintillation counter. Results were expressed as c.p.m. culture-' using the mean value of the triplicate wells.
0-
CD%j
1Dv-sup. Figure 1 ELISA for anti-id2 antibody production of EBV-B cell supernatants of non-selected cells (EBV-sup.) and after selection with MFG antigen-coated magnetic beads (Bi, B2, B3 and B4 represent supernatants of the wells containing the selected B cells). _, 1:1. M, 1:2; E, 1:4. -..-
The selection of the lymphoblastoid cells from the patient with non-detectable anti-id2 serum levels did not show any anti-id2 production in the culture supernatants. Magnetic beads that were not coated with antigen did not show any non-specific selection of the patient's lymphoblastoid cells. The in vitro T cell responses were elevated and antibody concentration-dependent in three patients studied after the second therapy when compared with the pre-therapy responses (P
