Leonardo Flenghi

Response of refractory Hodgkin's disease to monoclonal anti-CD30 immunotoxin

In Hodgkins disease, Hodgkin and Reed-Sternberg 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 Hodgkins disease. In three, there was rapid and substantial reduction in tumour mass (50% to greater than 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 to both parts of the immunotoxin developed in all patients.

Expression of lymphoid-associated antigens on Hodgkin's and Reed-Sternberg cells of Hodgkin's disease. An immunocytochemical study on lymph node cytospins using monoclonal antibodies.

The aim of this study was to elucidate the origin of Hodgkins and Reed‐Sternberg cells. Lymph node cytospins and frozen sections from 20 cases of Hodgkins disease of different histological subtypes were immunostained by the immunoalkaline phosphatase technique using a panel of monoclonal antibodies. As expected, the Hodgkins and Reed‐Sternberg cells of all cases were positive for the CD30 (Ki‐1), CD 15 (hapten X) and CD25 (Tac) antigens. In eight cases, a variable percentage of typical Hodgkins and Reed‐Sternberg cells showed a clear‐cut cytoplasmic and/or surface positivity for the T‐cell‐associated antigens CD3, CD5, CD6 and CD4 (seven cases) or CD8 (one case), but consistently lacked B‐cell and macrophage‐associated markers. The best visualization of T‐cell antigens was obtained in cytocentrifuge preparations and in areas of lymph node frozen sections that had been infiltrated by clusters of Hodgkins and Reed‐Sternberg cells. In two cases of Hodgkins disease (nodular sclerosis, mixed cellularity) the neoplastic cells weakly expressed the B‐cell antigens CD19 and CD22, but not T‐cell or macrophage‐associated markers. In 10 cases, Hodgkins and Reed‐Sternberg cells were negative for all the lymphoid‐ and macrophage‐associated antigens. These results suggest a lymphoid (either T or B) rather than histiocytic origin for the Hodgkins and Reed‐Sternberg cells in a number of Hodgkins disease cases.

Ber-H2 (anti-CD30)-saporin immunotoxin : a new tool for the treatment of Hodgkin's disease and CD30+ lymphoma : in vitro evaluation

An immunotoxin containing an anti‐CD30 monoclonal antibody (Ber‐H2) and saporin, a ribosome‐inactivating protein type 1, is described. It specifically inhibits protein synthesis by Hodgkin derived target cell lines with a very high efficiency (IC50 ranging from 5 × 10–12 M to 5.10∼14 M, assaporin), while irrelevant immunotoxins do not. Present results suggest that this immunotoxin could be used for in vivo therapy as well as for ex vivo bone marrow purging in Hodgkins disease and CD304+ lymphomas.

Anti-CD30 (BER=H2) immunotoxins containing the type-1 ribosome-inactivating proteins momordin and PAP-S (pokeweed antiviral protein from seeds) display powerful antitumour activity against CD30+ tumour cells in vitro and in SCID mice.

The anti‐CD30 immunotoxin (IT) Ber‐H2/saporin is effective in patients with refractory Hodgkin’s disease. However, responses are short and partial, one of the main reasons being the inability to repeat IT doses because of formation of human antibodies against the murine antibody and/or the toxin. To overcome this problem, we constructed two new anti‐CD30 ITs by covalently linking the mouse monoclonal antibody Ber‐H2 to the type 1 ribosome‐inactivating proteins (RIPs) momordin (MOM) and pokeweed antiviral protein from seeds (PAP‐S), which do not cross‐react with each other or with saporin. Both ITs inhibited protein synthesis by Hodgkin’s disease and anaplastic large‐cell lymphoma (ALCL)‐derived CD30+ target cell lines with a very high efficiency (IC50 ranging from < 5 × 10−13 m to 2.75 × 10−11m, as RIP). In a SCID mouse model of xenografted CD30+ human ALCL, a 3 d treatment with non‐toxic doses of Ber‐H2/MOM (50% LD50), started 24 h after transplantation, prevented tumour development in about 40% of the animals and significantly delayed tumour growth rate in the others. Main toxicity signs in mice and rabbits were a dose‐related increase of serum transaminases (AST and ALT) and creatine phosphokinase (CPK). LD50 (as RIP) in Swiss mice was 7 mg/kg for Ber‐H2/MOM and 0.45 mg/kg for Ber‐H2/PAP‐S. Sequential administration of two anti‐CD30 ITs (Ber‐H2/MOM and Ber‐H2/saporin) was well tolerated and did not result in formation of antibodies cross‐reacting with the two plant toxins. The results presented in this paper suggest that, in the future, sequential administration of anti‐CD30 humanized antibodies linked to antigenically distinct type 1 RIPs (saporin, MOM, PAP‐S) should be feasible.

Immunocytochemical evaluation of the percentage of proliferating cells in pathological bone marrow and peripheral blood samples with the Ki‐67 and anti‐bromo‐deoxyuridine monoclonal antibodies

The monoclonal antibody Ki‐67, directed against a nuclear antigen expressed by dividing cells in all the phases of cell cycle except G0 and early G1, was used in combination with an anti‐BrdU monoclonal antibody, reacting selectively with cells in S‐phase, for assessing the percentage of proliferating cells in bone marrow and peripheral blood samples from patients with lymphoma, leukaemia and multiple myeloma. Immunocytochemical labelling of proliferating cells was performed on marrow frozen sections and/or cytospins using an immunoalkaline phosphatase (APAAP) technique that made it possible to obtain proliferative index measurements in a few hours in contrast to the 3–7 d needed with tritiated thymidine. In the 54 marrow lymphoma cases studied a highly significant correlation was observed between the proportion of Ki‐67 (+) cells and the separation into low‐ and high‐grade malignant lymphomas according to the Kiel classification. In patients with multiple myeloma at the first diagnosis, the percentage of Ki‐67 (+) cells was low (6–10%). In contrast, a high percentage of Ki‐67 (+) cells (40–50%) was observed in a young adult with multiple myeloma, in a patient who first presented at the clinical observation with an extradural mass and in three patients who developed extramedullary masses several years after the initial diagnosis of myeloma. In acute lymphoblastic leukaemias of common type the mean value of Ki‐67 labelling was 31.3%. Because of their simplicity and rapidity, immunocytochemical techniques may be expected to replace autoradiography and flow cytometry for the detection of proliferating cells in haematological samples.

Randomized trial comparing R-CHOP versus high-dose sequential chemotherapy in high-risk patients with diffuse large B-cell lymphomas

Purpose The benefit of high-dose chemotherapy with autologous stem-cell transplantation (ASCT) as first-line treatment in patients with diffuse large B-cell lymphomas is still a matter of debate. To address this point, we designed a randomized phase III trial to compare rituximab plus cyclophosphamide, doxorubicin, vincristine, and prednisone (R-CHOP)-14 (eight cycles) with rituximab plus high-dose sequential chemotherapy (R-HDS) with ASCT. Patients and Methods From June 2005 to June 2011, 246 high-risk patients with a high-intermediate (56%) or high (44%) International Prognostic Index score were randomly assigned to the R-CHOP or R-HDS arm, and 235 were analyzed by intent to treat. The primary efficacy end point of the study was 3-year event-free survival, and results were analyzed on an intent-to-treat basis. Results Clinical response (complete response, 78% v 76%; partial response, 5% v 9%) and failures (no response, 15% v 11%; and early treatment-related mortality, 2% v 3%) were similar after R-CHOP versus R-HDS, respectively. After a median follow-up of 5 years, the 3-year event-free survival was 62% versus 65% ( P = .83). At 3 years, compared with the R-CHOP arm, the R-HDS arm had better disease-free survival (79% v 91%, respectively; P = .034), but this subsequently vanished because of late-occurring treatment-related deaths. No difference was detected in terms of progression-free survival (65% v 75%, respectively; P = .12), or overall survival (74% v 77%, respectively; P = .64). Significantly higher hematologic toxicity ( P < .001) and more infectious complications ( P < .001) were observed in the R-HDS arm. Conclusion In this study, front-line intensive R-HDS chemotherapy with ASCT did not improve the outcome of high-risk patients with diffuse large B-cell lymphomas.

LF61: a new monoclonal antibody directed against a trimeric molecule (150 kDa, 125 kDa, 105 kDa) associated with hairy cell leukaemia.

Summary The newly produced monoclonal antibody (mAb) LF61 detects a molecule restricted to hairy cell leukaemia (HCL) among B‐cell non‐Hodgkins lymphomas. In particutar, the percentage of LF61+HCL cells in different cases ranges from 10% to 100%. In normal lympho‐haemopoietic tissues LF61 reacts with only about 2% of T‐cells, mostly of the CD8 subset in the peripheral blood. extrafollicular areas of the tonsil, red pulp of the spleen and thymic medulla. Expression of the LF61 molecule is observed following stimulation of peripheral blood lymphocytes with phyto‐haemagglutinin (PHA) or pokeweed mitogen (PWM), suggesting that it represents an activation antigen. Due to its restricted reactivity with a small subset of normal CD8 + T‐cells, LF61 in combination with a CD22 mAb is highly suitable for monitoring residual disease in interferon or deoxycoformicin‐treated HCL patients. Polyacrylamide gel gradient electrophoresis shows that LF61 precipitates a 150 kDa. 125 kDa, 105 kDa trimeric molecule from the surface of HCL cells. Immunohistological and immunobiochemical results show that this molecule is the same as the one recognized by the still unclustered anti‐HCL mAb B‐ly7.

Selection of a panel of monoclonal antibodies for monitoring residual disease in peripheral blood and bone marrow of interferon‐treated hairy cell leukaemia patients

Summary A panel of monoclonal antibodies (mAbs) directed against B‐cell and hairy cell leukaemia (HCL)‐associated antigens was used to identify residual hairy cells in the peripheral blood and/or bone marrow samples from 20 patients with HCL. following treatment with interferon‐alpha (IFN‐alpha) or interferon‐beta (IFN‐beta). In all cases, hairy cells retained their characteristic phenotype, e.g. positivity for CD22, CD11c, CD25, CD32, and the HCL‐associated trimeric protein (t‐GP) recognized by the mAbs HML‐1, B‐ly7, LF61 and Ber‐Act8. The most specific marker for identifying a small percentage of hairy cells in peripheral blood cytospins. was t‐GP. In alkaline phosphatase/anti alkaline phosphatase (APAAP) stained preparations, t‐GP+ hairy cells (provided with large cytoplasm and hairy surface) could be usually distinguished from t‐GP+ normal lymphocytes (small‐sized cells with smooth surface). In doubtful cases the percentage of residual hairy cells could exactly be estimated by double immunofluorescence staining for CD22 (B‐cell marker) and t‐GP. The rationale of the test is based on the finding that the small percentage (about 1%) of t‐GP + lymphocytes circulating in the peripheral blood of normal individuals are T‐cells of the CD8 subset and not B‐cells. The best markers for identifying residual hairy cells in routine bone marrow biopsies were CD45RA (mAb 4KB5) and CD20 (mAb L26). Immunohistological labelling was superior to morphological examination in picking up scattered hairy cells in bone marrow biopsies showing either severe hypoplasia or exuberant hyperplasia of normal haemopoietic series.

Expression of the intestinal T-lymphocyte associated molecule HML-1: analysis of 75 non-Hodgkin's lymphomas and description of the first HML-1 positive T-lymphoblastic tumour.

The expression of the gut intra‐epithelial T‐cell associated molecule HML‐1, a trimeric protein of 150, 125, 105 kD, was studied in 75 T‐cell lymphomas of different subtypes: 20 T‐lymphoblastic lymphomas/leukaemias; 50 nodal peripheral T‐cell lymphomas; and five intestinal T‐cell lymphomas. Our results confirm: (i) the usefulness of the HML‐1 monoclonal antibody as an immunohistochemical marker for intestinal T‐cell lymphomas; and (ii) the lack of reactivity of HML‐1 with nodal peripheral T‐cell lymphomas. Moreover, expression of the HML‐1 molecule was found for the first time in a case of T‐lymphoblastic lymphoma/leukaemia. The patient presented with a mediastinal mass which consisted of HML‐1 + neoplastic cells displaying a phenotypic profile consistent with early thymocytes. Genes coding for the α, β, γ, and δ chains of the T‐cell receptor were in a germline configuration. The neoplastic cells could have been derived from the small subset of HML‐1 + thymocytes detectable in the cortex of normal human thymus.

Vaccine Therapy of B Cell Malignancies: Different Strategies for a Novel Approach

This review deals with the theoretical principles and experimental results of immunotherapy for B cell malignancies, namely for non-Hodgkin lymphomas (NHLs) and multiple myeloma. Its focus is the use of vaccines in clinical practice with particular emphasis on the most recent developments and therapeutic opportunities arising from combination therapies. Previous studies will be reviewed and the present status of vaccine technology summarized.