1195
22. Love LA, Leff RL, Fraser DD, et al. A new approach to the classification of idiopathic inflammatory myopathy: myositis-spccific autoantibodies define useful homogeneous patient groups. Medicine 1991; 70: 360-74. 23. Saiki RK, Gelfand DH, Stoffel S, et al. Primer-directed enzymatic amplification of DNA with a thermostable DNA polymerase. Science
1988; 239: 487-91. Greenberg SJ, Abbott MA. Detection of human T-cell lymphoma/leukemia viruses. In: Innis M, Gelfand D, Sninsky J, White T, eds.. PCR protocols: a guide to methods and applications. Orlando: Academic Press, 1990: 325-36.
24. Ehrlich GD,
25. Ehrlich
GD, Bryz-Gornia V, Maese J, Waldmann TA, Poiesz BJ, Greenberg SJ. Multiple sclerosis, retroviruses and PCR. Neurology
1991; 41: 335-43. 26. Mathews MB, Bernstein RM. Myositis autoantibody inhibits histidyltRNA synthetase: a model for autoimmunity. Nature 1983; 304: 177-79. 27. Salomon R, Littauer UZ. Enzymatic acylation of histidine to meningovirus RNA. Nature 1974; 249: 32-34. 28. Lindley IG, Stebbing N. Aminoacylation of encephalomyocarditis virus RNA. J Gen Virol 1977; 34: 177-82.
SHORT REPORT Response of refractory Hodgkin’s disease to monoclonal anti-CD30 immunotoxin
In
Hodgkin’s disease, Hodgkin and ReedSternberg cells consistently express the antigen CD30. We investigated the possible therapeutic role of an immunotoxin prepared by covalent linking of an anti-CD30 monoclonal antibody (Ber-H2) to saporin (SO6), a type-1 ribosome-inactivating protein. The immunotoxin (0·8 mg/kg in one or two doses) was given to four patients with advanced refractory Hodgkin’s disease. In three, there was rapid and substantial reduction in tumour mass (50% to >75%). Clinical responses were transient (6-10 weeks). In-vivo binding of the immunotoxin to tumour cells was shown by immunohistology in two patients.
Antibodies
immunotoxin developed
both parts in all patients.
to
of
the
dose of immunotoxin (see table) was infused for 4 h in 500 ml normal saline containing 5% human serum albumin, after intravenous premedication with 10 mg chlorpheniramine.** In-vivo targeting of Hodgkin and Reed-Sternberg cells by the Ber-H2 immunotoxin was assessed for two patients by incubation of acetone-fixed frozen sections of biopsy samples taken 18-24 h after treatment with rabbit antibody to mouse immunoglobulin followed by complexes of alkaline phosphatase and antibodies to that enzyme (APAAP).3 As negative controls, pretreatment biopsy samples were incubated with rabbit anti-mouse-immunoglobulin; positive controls were frozen sections incubated with Ber-H2. Serial frozen sections were stained for saporin with a rabbit antiserum and the peroxidase/antiperoxidase method. Pretreatment serum concentrations of soluble CD30 were measured by enzyme-linked immunosorbent assay (ELISA). Patient’s responses to the immunotoxin were assessed weekly by an ELISA. TREATMENT WITH BER-H2/S06 AND RESPONSE
*Measured by computed tomography (CT) scan and expressed diameters of all measurable lesions tMaxlmum tumour effect seen after first dose
as
product of two
In all four
The Ber-H2 monoclonal antibody is directed against an of the Hodgkin’s-disease-associated antigen CD30.1,2 Its binding to normal tissues is restricted in vitro and in vivo.3 Binding to Hodgkin and Reed-Sternberg cells has no antitumour effectsso for clinical use Ber-H2 will have to be coupled to cytotoxic agents. Immunotoxins made from antibodies to CD30 linked to ricin chain A are effective against Hodgkin cells in vitro4 and in transplanted nude mice.s We prepared an immunotoxin from Ber-H2 and saporin (S06), a potent single-chain ribosome-inactivating protein from Saponaria officinalis.6,7 We describe its clinical effects in four patients with advanced, refractory Hodgkin’s disease.
epitope
The
study
patients
was
approved by the local ethics committee and all
informed
consent. No patient had received radiotherapy during the 3 months before the study. In all four patients, Hodgkin and Reed-Sternberg cells in pretreatment biopsy samples were strongly positive for CD30. Each
gave
chemotherapy
or
patients, the tumour mass decreased rapidly (5-7 days; figure). Patients 1 and 3 also had complete relief of systemic symptoms (table). Responses lasted 6-10 weeks; there was no growth at new sites but regrowth at all original sites except the skin of patient 4. There were no hypersensitivity reactions. All patients had fever (38-39°C) for 2-3 days after treatment, and malaise, anorexia, and fatigue were also common. Patient 1 had myalgia after the second dose, and patient 4 an increase in body weight (about 5 kg) and oedema of the lower legs. Serum aspartate and alanine aminotransferase activities (to 3-6 times normal) but returned to pretreatment values by days 7-10. Other transient abnormalities were a slight increase in liver alkaline phosphatase, rose
hypertriglyceridaemia, slight proteinuria (300-1000 mg/1), leucocytosis (> 1010/1; 70-80% neutrophils), and thrombocytopenia in patients 3 and 4 (5 x 1010/1 and 1.37 x l0"/l; lowest on day 6). *
A table
giving full details of the patients is available from
The Lancet.
1196
study3 unmodified Ber-H2 produced no clinical response.3 The rapidity of tumour regression is consistent with an imrnunotoxin-mediated effect. 10 No complete remission was obtained in these patients with advanced disease, but total clearance of tumour cells might be possible if the immunotoxin were given earlier or to patients with slight residual disease. The extent and duration of the clinical response are limited by the development of antibodies against both parts of the immunotoxin ; repeated doses cannot therefore be given. The use of chimeric (humanised) Ber-H2 linked to
immunologically distinct, type-1 ribosome-inactivating proteins could help to circumvent this problem. Our preliminary results suggest a role for the Ber-H2/ S06 immunotoxin in the treatment of minimum residual disease in certain types of Hodgkin’s disease and, possibly, of other CD30-expressing neoplasms, such as anaplastic
large-cell lymphomas.2 We thank Prof V. Kraft (Hannover, Germany) for the mouse antibody tests; Dr E. Weiss (Munich, Germany) for the mouse DNA contaminant test; and Mrs B. Bigema for technical assistance.
production
This study was supported by the Associazione Italiana Ricerca Cancro, CNR, Ministero dell’ Universita e Ricerca Scientifica, Regione EmiliaRomagna, and Pallotti’s Legacy for Cancer Research.
CT
lung
scans
of
patient
1 before
(A)
and 7
REFERENCES
days after (B)
Ber-H2/S06 treatment.
Lung nodules have almost completely disappeared and pericardial effusion (arrow) has regressed The immunotoxin bound to a smaller proportion of Hodgkin and Reed-Sternberg cells in vivo (75% patient 3, 50% patient 4) than in positive in-vitro controls (100%). The in-vivo binding was highly selective; no elements other than tumour cells were stained. In healthy controls8 and in patients 1 and 4 before treatment CD30 was undetectable in serum; patient 3 had 1.0 X 105 U/1 and patient 2 1.2 x 106 U/1. In patient 3, the immunotoxic activity circulating in the serum had fallen by 50% 36 h after infusion. In all patients antibodies against both the antibody and the toxin were present 2-4 weeks after treatment. All four patients had advanced, bulky disease which had become resistant to treatment. In three, the Ber-H2/S06 immunotoxin brought about rapid and substantial reduction of tumour masses at all disease sites. Although the best dose and schedule of administration of the immunotoxin are not yet clear, a single dose of 04 or 0-8 mg/kg induced an antitumour effect with only slight, transient hepatotoxic effects, as predicted from animal studies.9 The mechanism of the thrombocytopenia in patient 3 needs further study. The capillary leak syndrome, which has caused difficulties in other immunotoxin trials, 10 was not found; one patient gained weight because of oedema, but there was no decrease in serum albumin. The clinical responses and immunohistological findings in these patients show that the immunotoxin can reach tumour cells in various body sites. The specific binding may have been favoured by the low serum CD30 concentrations, restricted reactivity of Ber-H2 with normal tissues, and the high affinity of the antibody for CD30. An immunotoxin rather than an antibody effect seems likely to have brought about the reduction of tumour masses, since in a previous
Schwarting A, Gerdes J, Durkop H, Falini B, Pileri S, Stein H. Ber-H2: a new anti-Ki-1 (CD30) monoclonal antibody directed at a formolresistant epitope. Blood 1989; 74: 1678-89. 2. Stein H, Mason DY, Gerdes J, et al. The expression of the Hodgkin’s disease associated antigen Ki-1 in reactive and neoplastic lymphoid tissue: evidence that Reed-Stemberg and histiocytic malignancies are derived from activated lymphoid cells. Blood 1985; 66: 848-58. 3. Falini B, Flenghi L, Fedeli L, et al. In vivo targeting of Hodgkin and Reed-Stemberg cells of Hodgkin’s disease with monoclonal antibody Ber-H2 (CD30). Exp Hematol 1991; 19: 545. 4. Engert A, Burrows F, Jung W, et al. Evaluation of ricin A chaincontaining immunotoxms directed against the CD30 antigen as potential reagents for the treatment of Hodgkin’s disease. Cancer Res 1.
1990; 50: 84-88. 5.
Engert A, Martin G, Pfreundschuh M, et al. Anti-tumor effects of ricin A chain immunotoxins prepared from intact antibodies and Fab’ fragments on solid human Hodgkin’s disease tumors in mice. Cancer
6.
Stirpe F, Barbieri L. Ribosome-inactivating proteins up to date. FEBS
Res 1990; 50: 2929-35. Lett 1986; 195: 1-8. 7. Tazzari PL, Bolognesi A, Falini B, et al. Ber-HZ (anti-CD30)-saponin: a new tool for the treatment of Hodgkin’s disease and CD30+ lymphomas: in vitro evaluation. Br J Haematol (in press). 8. Pizzolo G, Vinante F, Chilosi M, et al. Serum levels of soluble CD30 molecule (Ki-1 antigen) in Hodgkin’s disease: relationship with disease activity and clinical stage. Br J Haematol 1990; 75: 282-84. 9. Stirpe F, Derenzini M, Barbieri L, et al. Hepatotoxicity of immunotoxins made with saporin, a ribosome-inactivating protein from Saponaria officinalis. Virchows Arch B Cell Pathol 1987; 53: 259-71 10. Vitetta ES, Stone M, Amlot P, et al. Phase I immunotoxin trial in patients with B-cell lymphomas. Cancer Res 1991; 51: 4052-58.
ADDRESSES: Institutes of
Haematology (Prof B. Falini, MD, L. Flenghi, MD, F. Aversa, MD, G. Barbabietola, MD, Prof M. F. Martelli, MD), and Radiology (P. Comeli, MD), University of Perugia; National Institute for Cancer Research, Genova, Italy (P. L. Tazzari, MD); Dako, Glostrup, Denmark (M. K. Broe, MD); Institute of Pathology, Free University of Berlin, Germany (Prof H. Stein, MD, H. Dürkop, MD); Department of Haematology, University of Verona (Prof G. Pizzolo, MD); and Department of Experimental Pathology (A. Bolognesi, PhD, Prof F. Stirpe, MD) and Institute of Haematology (E. Sabattini, MD, Prof S. Pileri, MD), University of Bologna, Italy. Correspondence to Prof B. Falini, Istituto di Ematologia, Policlinico, Monteluce, 06100 Perugia, Italy.
22. Love LA, Leff RL, Fraser DD, et al. A new approach to the classification of idiopathic inflammatory myopathy: myositis-spccific autoantibodies define useful homogeneous patient groups. Medicine 1991; 70: 360-74. 23. Saiki RK, Gelfand DH, Stoffel S, et al. Primer-directed enzymatic amplification of DNA with a thermostable DNA polymerase. Science
1988; 239: 487-91. Greenberg SJ, Abbott MA. Detection of human T-cell lymphoma/leukemia viruses. In: Innis M, Gelfand D, Sninsky J, White T, eds.. PCR protocols: a guide to methods and applications. Orlando: Academic Press, 1990: 325-36.
24. Ehrlich GD,
25. Ehrlich
GD, Bryz-Gornia V, Maese J, Waldmann TA, Poiesz BJ, Greenberg SJ. Multiple sclerosis, retroviruses and PCR. Neurology
1991; 41: 335-43. 26. Mathews MB, Bernstein RM. Myositis autoantibody inhibits histidyltRNA synthetase: a model for autoimmunity. Nature 1983; 304: 177-79. 27. Salomon R, Littauer UZ. Enzymatic acylation of histidine to meningovirus RNA. Nature 1974; 249: 32-34. 28. Lindley IG, Stebbing N. Aminoacylation of encephalomyocarditis virus RNA. J Gen Virol 1977; 34: 177-82.
SHORT REPORT Response of refractory Hodgkin’s disease to monoclonal anti-CD30 immunotoxin
In
Hodgkin’s disease, Hodgkin and ReedSternberg cells consistently express the antigen CD30. We investigated the possible therapeutic role of an immunotoxin prepared by covalent linking of an anti-CD30 monoclonal antibody (Ber-H2) to saporin (SO6), a type-1 ribosome-inactivating protein. The immunotoxin (0·8 mg/kg in one or two doses) was given to four patients with advanced refractory Hodgkin’s disease. In three, there was rapid and substantial reduction in tumour mass (50% to >75%). Clinical responses were transient (6-10 weeks). In-vivo binding of the immunotoxin to tumour cells was shown by immunohistology in two patients.
Antibodies
immunotoxin developed
both parts in all patients.
to
of
the
dose of immunotoxin (see table) was infused for 4 h in 500 ml normal saline containing 5% human serum albumin, after intravenous premedication with 10 mg chlorpheniramine.** In-vivo targeting of Hodgkin and Reed-Sternberg cells by the Ber-H2 immunotoxin was assessed for two patients by incubation of acetone-fixed frozen sections of biopsy samples taken 18-24 h after treatment with rabbit antibody to mouse immunoglobulin followed by complexes of alkaline phosphatase and antibodies to that enzyme (APAAP).3 As negative controls, pretreatment biopsy samples were incubated with rabbit anti-mouse-immunoglobulin; positive controls were frozen sections incubated with Ber-H2. Serial frozen sections were stained for saporin with a rabbit antiserum and the peroxidase/antiperoxidase method. Pretreatment serum concentrations of soluble CD30 were measured by enzyme-linked immunosorbent assay (ELISA). Patient’s responses to the immunotoxin were assessed weekly by an ELISA. TREATMENT WITH BER-H2/S06 AND RESPONSE
*Measured by computed tomography (CT) scan and expressed diameters of all measurable lesions tMaxlmum tumour effect seen after first dose
as
product of two
In all four
The Ber-H2 monoclonal antibody is directed against an of the Hodgkin’s-disease-associated antigen CD30.1,2 Its binding to normal tissues is restricted in vitro and in vivo.3 Binding to Hodgkin and Reed-Sternberg cells has no antitumour effectsso for clinical use Ber-H2 will have to be coupled to cytotoxic agents. Immunotoxins made from antibodies to CD30 linked to ricin chain A are effective against Hodgkin cells in vitro4 and in transplanted nude mice.s We prepared an immunotoxin from Ber-H2 and saporin (S06), a potent single-chain ribosome-inactivating protein from Saponaria officinalis.6,7 We describe its clinical effects in four patients with advanced, refractory Hodgkin’s disease.
epitope
The
study
patients
was
approved by the local ethics committee and all
informed
consent. No patient had received radiotherapy during the 3 months before the study. In all four patients, Hodgkin and Reed-Sternberg cells in pretreatment biopsy samples were strongly positive for CD30. Each
gave
chemotherapy
or
patients, the tumour mass decreased rapidly (5-7 days; figure). Patients 1 and 3 also had complete relief of systemic symptoms (table). Responses lasted 6-10 weeks; there was no growth at new sites but regrowth at all original sites except the skin of patient 4. There were no hypersensitivity reactions. All patients had fever (38-39°C) for 2-3 days after treatment, and malaise, anorexia, and fatigue were also common. Patient 1 had myalgia after the second dose, and patient 4 an increase in body weight (about 5 kg) and oedema of the lower legs. Serum aspartate and alanine aminotransferase activities (to 3-6 times normal) but returned to pretreatment values by days 7-10. Other transient abnormalities were a slight increase in liver alkaline phosphatase, rose
hypertriglyceridaemia, slight proteinuria (300-1000 mg/1), leucocytosis (> 1010/1; 70-80% neutrophils), and thrombocytopenia in patients 3 and 4 (5 x 1010/1 and 1.37 x l0"/l; lowest on day 6). *
A table
giving full details of the patients is available from
The Lancet.
1196
study3 unmodified Ber-H2 produced no clinical response.3 The rapidity of tumour regression is consistent with an imrnunotoxin-mediated effect. 10 No complete remission was obtained in these patients with advanced disease, but total clearance of tumour cells might be possible if the immunotoxin were given earlier or to patients with slight residual disease. The extent and duration of the clinical response are limited by the development of antibodies against both parts of the immunotoxin ; repeated doses cannot therefore be given. The use of chimeric (humanised) Ber-H2 linked to
immunologically distinct, type-1 ribosome-inactivating proteins could help to circumvent this problem. Our preliminary results suggest a role for the Ber-H2/ S06 immunotoxin in the treatment of minimum residual disease in certain types of Hodgkin’s disease and, possibly, of other CD30-expressing neoplasms, such as anaplastic
large-cell lymphomas.2 We thank Prof V. Kraft (Hannover, Germany) for the mouse antibody tests; Dr E. Weiss (Munich, Germany) for the mouse DNA contaminant test; and Mrs B. Bigema for technical assistance.
production
This study was supported by the Associazione Italiana Ricerca Cancro, CNR, Ministero dell’ Universita e Ricerca Scientifica, Regione EmiliaRomagna, and Pallotti’s Legacy for Cancer Research.
CT
lung
scans
of
patient
1 before
(A)
and 7
REFERENCES
days after (B)
Ber-H2/S06 treatment.
Lung nodules have almost completely disappeared and pericardial effusion (arrow) has regressed The immunotoxin bound to a smaller proportion of Hodgkin and Reed-Sternberg cells in vivo (75% patient 3, 50% patient 4) than in positive in-vitro controls (100%). The in-vivo binding was highly selective; no elements other than tumour cells were stained. In healthy controls8 and in patients 1 and 4 before treatment CD30 was undetectable in serum; patient 3 had 1.0 X 105 U/1 and patient 2 1.2 x 106 U/1. In patient 3, the immunotoxic activity circulating in the serum had fallen by 50% 36 h after infusion. In all patients antibodies against both the antibody and the toxin were present 2-4 weeks after treatment. All four patients had advanced, bulky disease which had become resistant to treatment. In three, the Ber-H2/S06 immunotoxin brought about rapid and substantial reduction of tumour masses at all disease sites. Although the best dose and schedule of administration of the immunotoxin are not yet clear, a single dose of 04 or 0-8 mg/kg induced an antitumour effect with only slight, transient hepatotoxic effects, as predicted from animal studies.9 The mechanism of the thrombocytopenia in patient 3 needs further study. The capillary leak syndrome, which has caused difficulties in other immunotoxin trials, 10 was not found; one patient gained weight because of oedema, but there was no decrease in serum albumin. The clinical responses and immunohistological findings in these patients show that the immunotoxin can reach tumour cells in various body sites. The specific binding may have been favoured by the low serum CD30 concentrations, restricted reactivity of Ber-H2 with normal tissues, and the high affinity of the antibody for CD30. An immunotoxin rather than an antibody effect seems likely to have brought about the reduction of tumour masses, since in a previous
Schwarting A, Gerdes J, Durkop H, Falini B, Pileri S, Stein H. Ber-H2: a new anti-Ki-1 (CD30) monoclonal antibody directed at a formolresistant epitope. Blood 1989; 74: 1678-89. 2. Stein H, Mason DY, Gerdes J, et al. The expression of the Hodgkin’s disease associated antigen Ki-1 in reactive and neoplastic lymphoid tissue: evidence that Reed-Stemberg and histiocytic malignancies are derived from activated lymphoid cells. Blood 1985; 66: 848-58. 3. Falini B, Flenghi L, Fedeli L, et al. In vivo targeting of Hodgkin and Reed-Stemberg cells of Hodgkin’s disease with monoclonal antibody Ber-H2 (CD30). Exp Hematol 1991; 19: 545. 4. Engert A, Burrows F, Jung W, et al. Evaluation of ricin A chaincontaining immunotoxms directed against the CD30 antigen as potential reagents for the treatment of Hodgkin’s disease. Cancer Res 1.
1990; 50: 84-88. 5.
Engert A, Martin G, Pfreundschuh M, et al. Anti-tumor effects of ricin A chain immunotoxins prepared from intact antibodies and Fab’ fragments on solid human Hodgkin’s disease tumors in mice. Cancer
6.
Stirpe F, Barbieri L. Ribosome-inactivating proteins up to date. FEBS
Res 1990; 50: 2929-35. Lett 1986; 195: 1-8. 7. Tazzari PL, Bolognesi A, Falini B, et al. Ber-HZ (anti-CD30)-saponin: a new tool for the treatment of Hodgkin’s disease and CD30+ lymphomas: in vitro evaluation. Br J Haematol (in press). 8. Pizzolo G, Vinante F, Chilosi M, et al. Serum levels of soluble CD30 molecule (Ki-1 antigen) in Hodgkin’s disease: relationship with disease activity and clinical stage. Br J Haematol 1990; 75: 282-84. 9. Stirpe F, Derenzini M, Barbieri L, et al. Hepatotoxicity of immunotoxins made with saporin, a ribosome-inactivating protein from Saponaria officinalis. Virchows Arch B Cell Pathol 1987; 53: 259-71 10. Vitetta ES, Stone M, Amlot P, et al. Phase I immunotoxin trial in patients with B-cell lymphomas. Cancer Res 1991; 51: 4052-58.
ADDRESSES: Institutes of
Haematology (Prof B. Falini, MD, L. Flenghi, MD, F. Aversa, MD, G. Barbabietola, MD, Prof M. F. Martelli, MD), and Radiology (P. Comeli, MD), University of Perugia; National Institute for Cancer Research, Genova, Italy (P. L. Tazzari, MD); Dako, Glostrup, Denmark (M. K. Broe, MD); Institute of Pathology, Free University of Berlin, Germany (Prof H. Stein, MD, H. Dürkop, MD); Department of Haematology, University of Verona (Prof G. Pizzolo, MD); and Department of Experimental Pathology (A. Bolognesi, PhD, Prof F. Stirpe, MD) and Institute of Haematology (E. Sabattini, MD, Prof S. Pileri, MD), University of Bologna, Italy. Correspondence to Prof B. Falini, Istituto di Ematologia, Policlinico, Monteluce, 06100 Perugia, Italy.
