Clustering of missense mutations in the ataxia-telangiectasia gene in a sporadic T-cell leukaemia.

Ataxia-telangiectasia (A-T) is a recessive multi-system disorder caused by mutations in the ATM gene at 11q22-q23 (ref. 3). The risk of cancer, especially lymphoid neoplasias, is substantially elevated in A-T patients and has long been associated with chromosomal instability. By analysing tumour DNA from patients with sporadic T-cell prolymphocytic leukaemia (T-PLL), a rare clonal malignancy with similarities to a mature T-cell leukaemia seen in A-T, we demonstrate a high frequency of ATM mutations in T-PLL. In marked contrast to the ATM mutation pattern in A-T, the most frequent nucleotide changes in this leukaemia were missense mutations. These clustered in the region corresponding to the kinase domain, which is highly conserved in ATM-related proteins in mouse, yeast and Drosophila. The resulting amino-acid substitutions are predicted to interfere with ATP binding or substrate recognition. Two of seventeen mutated T-PLL samples had a previously reported A-T allele. In contrast, no mutations were detected in the p53 gene, suggesting that this tumour suppressor is not frequently altered in this leukaemia. Occasional missense mutations in ATM were also found in tumour DNA from patients with B-cell non-Hodgkin's lymphomas (B-NHL) and a B-NHL cell line. The evidence of a significant proportion of loss-of-function mutations and a complete absence of the normal copy of ATM in the majority of mutated tumours establishes somatic inactivation of this gene in the pathogenesis of sporadic T-PLL and suggests that ATM acts as a tumour suppressor. As constitutional DNA was not available, a putative hereditary predisposition to T-PLL will require further investigation.

Donor-derived human herpes virus 8-related Kaposi's sarcoma in renal allograft ureter.

We present a case of human herpes virus 8 (HHV8)-associated Kaposi sarcoma (KS) occurring in a renal allograft ureter from a male donor. The female patient presented with a rising creatinine due to ureteric obstruction, and subsequent histological examination of the excised tumor revealed a KS. The tumor tested positive for HHV8 antigen and, using in situ hybridization to identify X and Y chromosomes, we were able to demonstrate that the tumor was of male origin. In the absence of any other KS lesions, this suggested that the tumor arose due to reactivation of latent HHV8 in the donor tissue, permitted by the recipient's immunosuppression. The patient was managed by a gradual reduction in immunosuppression and there has been no subsequent recurrence of the tumor. KS in renal transplantation is discussed in detail including the possible utility of pre-transplant HHV8 screening.

Delivery of saporin to human B-cell lymphoma using bispecific antibody: targeting via CD22 but not CD19, CD37, or immunoglobulin results in efficient killing.

A panel of bispecific F(ab')2 antibodies (BsAb) have been constructed for delivering the ribosome-inactivating protein saporin to human B cell lymphoma. Each derivative was prepared with specificity for saporin and CD19, CD22, CD37, or immunoglobulin. In vitro studies measuring inhibition of [3H]leucine uptake by cultured Daudi and Raji cells demonstrated that, despite all BsAb capturing saporin on the cell surface, BsAb targeting through CD22 were far more cytotoxic than those functioning via CD19, CD37, or surface immunoglobulin. This exceptional activity of the CD22-specific BsAb appears to derive from its ability to deliver and accumulate saporin inside the target cells. Further studies showed that four CD22-specific BsAb all performed with equal potency and were able to increase saporin toxicity (50% inhibitory concentration) up to 1000-fold, from 2 x 10(-7) M to 2 x 10(-10) M. Pairs of anti-CD22 BsAb which recognized different nonblocking epitopes on the saporin molecule were able to bind saporin more avidly to the target cell and, as a consequence, increased cytotoxicity by at least an additional 10-fold, resulting in 50% inhibitory concentration for protein synthesis of 2 x 10(-11) M. These results suggest that selected combinations of BsAb which bind cooperatively to a toxin and the cell surface may provide an efficient way of delivering toxins to unwanted cells in patients.

Somatically mutated regions of immunoglobulin on human B-cell lymphomas code for peptides that bind to autologous major histocompatibility complex class I, providing a potential target for cytotoxic T cells.

Lymphoma-derived immunoglobulin idiotype (Id) is a well-characterized, tumor-specific antigen on B-cell malignancies. Immunotherapy using lymphoma immunoglobulin can lead to clinical responses mostly associated with anti-Id antibody. We cloned the Id from B-cell lymphomas, sequenced them, and used bioinformatics to select autologous MHC class I binding peptides from somatically mutated regions of the lymphoma Id. Peptides from patients who were HLA-A1, HLA-A2, HLA-A3, or HLA-A11 positive were analyzed in the T2 stabilization assay and a competitive peptide-binding assay. By both methods, approximately half of the peptides analyzed, regardless of HLA type, bound with intermediate or high affinity. Peptide binding affinity was similar to viral peptide sequences known to provide targets for cytotoxic T cells. Further investigation of lymphocyte responses to stimulation by autologous Id peptides versus Id peptides from other patients revealed that three of five patients in complete remission or with low volume, stable disease responded to self-peptides by IFN-gamma secretion greater than that seen with non-self peptides, whereas none of five patients with progressive disease responded to their own lymphoma Id. We have shown that mutated regions of lymphoma Id contain MHC class I binding peptides that are potential targets for cytotoxic T cells. Immunotherapy using the tumor-specific mutated regions from lymphoma Id avoids the need to break innate tolerance toward the germ-line protein sequences present on normal and malignant B cells.

Antitumor effects of ricin A chain immunotoxins prepared from intact antibodies and Fab' fragments on solid human Hodgkin's disease tumors in mice.

Three monoclonal antibodies which strongly bind to Hodgkin and Reed-Sternberg cells and two corresponding Fab' fragments were linked to deglycosylated ricin A chain (dg A) to evaluate their potential as immunotoxins for the treatment of Hodgkin's disease. Two of the antibodies, Ber-H2 and HRS-3, were shown to bind to the same epitope on the CD30 antigen, whereas the third antibody, IRac, bound to a different antigen. None of the antibodies significantly cross-reacted with normal human tissues as judged by indirect immunofluorescence and immunoperoxidase analyses on frozen sections from 28 normal tissues. All three antibodies formed potent and specific immunotoxins. They inhibited protein synthesis of the L540 Hodgkin's disease cell line in vitro by 50% at concentrations of 1 x 10(-11) M for IRac.dgA, 9 x 10(-11) M for HRS-3.dgA, and 2 x 10(-10) M for Ber-H2.dgA. HRS-3 Fab' and IRac Fab' immunotoxins were 7.8- and 60-fold less cytotoxic, respectively, than their intact counterparts in vitro. In vivo, a single i.v. injection of a dose of Ber-H2.dgA, HRS-3.dgA, or IRac.dgA corresponding to 40% of the LD50 induced lasting complete remissions in 38, 44, and 50%, respectively, of mice with solid s.c. L540 tumors of 60 to 80 mm3 size (0.5-cm diameter). At equivalent dosage (40% of the LD50), the HRS-3 Fab'.dgA and the IRac Fab'.dgA both induced lasting complete remissions in 25% of the mice, although the HRS-3 Fab'.dgA was significantly superior to IRac Fab'.dgA at retarding tumor growth in the remaining animals. The effectiveness of the immunotoxins depended on the size of the tumor at the time of injection, since IRac.dgA treatment induced complete remissions in 100% of mice with small tumors (10 to 20 mm3, approximately 0.3 cm in diameter) but only 13% of mice with larger tumors of 400 to 600 mm3 (approximately 1 cm in diameter). Tumors which regrew after IRac.dgA treatment mainly consisted of antigen-deficient mutants having reduced sensitivity to IRac.dgA but normal sensitivity to HRS-3.dgA. It is concluded that HRS-3.dgA, HRS-3 Fab'.dgA, and IRac.dgA are candidates for the treatment of Hodgkin's disease in humans.

Phase I immunotoxin trial in patients with B-cell lymphoma.

Fifteen patients with refractory B-cell lymphoma were treated in a Phase I dose escalation clinical trial with a highly potent immunotoxin consisting of the Fab' fragment of a monoclonal anti-CD22 antibody (RFB4) coupled to chemically deglycosylated ricin A chain. All patients had low, intermediate, or high grade non-Hodgkin's lymphoma. The immunotoxin was administered i.v. in two to six doses at 48-h intervals. The peak serum concentration and the t1/2 were not dose dependent among patients and averaged 1.3 micrograms/ml and 86 min, respectively. Three patients made antibody against A chain, and a fourth made antibody against both A chain and mouse immunoglobulin. Antibody responses were low (less than or equal to 85 micrograms/ml) in three patients and were not detected until 1 mo after treatment. The maximum tolerated dose of the immunotoxin was 75 mg/m2. Dose-related toxicities included vascular leak syndrome, fever, anorexia, and myalgia. Dose-limiting toxicities included pulmonary edema and/or effusion, expressive aphasia, and rhabdomyolysis (resulting in reversible kidney failure). There was no evidence of liver dysfunction. Partial responses were achieved in 38% of evaluable patients, and in those patients who had greater than 50% CD22+ tumor cells, 50% of the patients achieved a partial response. Clinical responses were not related to tumor grade and were generally transient, lasting between 1 and 4 mo.

Hodgkin's disease-derived cell lines--conflicting clues for the origin of Hodgkin's disease?

The origin of Hodgkin (H) and Reed-Sternberg (RS) cells remains a highly controversial issue. Studies on noncultured, freshly disaggregated biopsy material or of histological sections have not definitely established the cell lineage of HD cells, although evidence has been gathered that has allowed interpretation to favor one or the other hematopoietic cell type. In recent years a number of cell lines have been established from patients with Hodgkin's disease (HD) which are claimed to represent in vitro counterparts of H-RS cells. The phenotypic and functional properties of 7 HD-derived cell lines are reviewed here. The cell lines are not identical; they show many common features, but also a number of important differences in their phenotypes as well as in their in vitro behavior. The cell lines differ with some having characteristics of T cells, others of B cells, and yet others of the monocyte/macrophage cell lineage. This heterogeneity of H-RS established cell lines could be explained in a variety of ways: (a) the heterogeneity of HD itself might result from a disease process which leads to fusion of different cell types resulting in different phenotypic forms of H-RS cells depending on the cells involved; (b) the different cell types might reflect differences in the malignant cells present in the separate subtypes of HD; (c) the difficulty in successfully establishing H-RS cell lines might mean that unrepresentative cell types adapt to in vitro conditions. Rather than resolve the origin of H-RS cells, the established HD-derived cell lines have been consistent with the heterogeneity described in histochemical, immunohistological, and immunocytochemical studies.

Variable kappa gene rearrangement in lymphoproliferative disorders: an analysis of V kappa gene usage, VJ joining and somatic mutation.

We have studied a diverse group of lymphoproliferative disorders (acute lymphoblastic leukaemia (ALL), chronic lymphocytic leukaemia (CLL), non-Hodgkin lymphoma (NHL) and myeloma) to determine if V kappa gene use is random or disease-specific and whether somatic mutations (a late event in B-cell differentiation) can provide additional information on the type of B cell involved in the neoplastic clone. In this group of disorders V kappa gene selection is not random and some members of each V kappa family are preferentially rearranged. V kappa genes from the distal portion of the locus are seldom used, possibly because rearrangement of the proximal locus by deletion is more efficient than rearrangement of the distal locus by inversion. Although pseudogenes account for 46% of the V genes in the kappa locus none were ever rearranged, even in non-functional rearrangements of lambda-producing leukemias, suggesting the existence of a mechanism which down-regulates the rearrangement of pseudogenes. N regions were noted at the VJ junction in 20% of alleles (in six CLL, three NHL, two ALL and one myeloma) possibly the result of kappa-chain recombination during the early period of B-cell maturation in which TdT is expressed. Nucleotide addition or imprecise joining at the VJ junction, resulting in a shift in the reading frame, were the commonest causes of non-functional rearrangement. The occurrence of somatic mutation broadly correlated with the stage of B-cell maturation from which the different disorders are thought to arise. However, there was no strict association and somatic mutations were demonstrated in 'typical CLL' while V kappa genes were germline in some follicular lymphomas; these findings suggest either heterogeneity in the stage of B-cell maturation at which these disorders arise or some variability in the process of somatic mutation.

Up-regulation of endoglin on vascular endothelial cells in human solid tumors: implications for diagnosis and therapy.

We have characterized a murine IgM monoclonal antibody, TEC-11, that recognizes endoglin and may be suitable for targeting cytotoxic agents to human tumor vasculature. TEC-11 strongly stains endothelial cells in a broad range of solid human tumors while staining endothelial cells in the majority of normal, healthy adult tissues relatively weakly. Human umbilical vein endothelial cells (HUVECs) in sections of the umbilical vein react weakly with TEC-11, whereas proliferating HUVECs in tissue culture react strongly and uniformly. HUVEC cultures grown to confluence and then rested contain two subpopulations having high and low levels of endoglin expression. Flow cytometry revealed that a significant proportion of cells with high endoglin expression are cycling, having markedly increased levels of cellular protein, RNA, and DNA by comparison to low endoglin-expressing cells, which appear to be noncycling. Taken together, the increased binding of TEC-11 to tumor vasculature and to dividing as opposed to noncycling HUVECs in vitro suggests that endoglin is an endothelial cell proliferation-associated marker. An immunotoxin [TEC-11.deglycosylated ricin A chain (dgA)] composed of TEC-11 and dgA was 3000-fold more potent at inhibiting protein synthesis in proliferating HUVEC cultures than in confluent cultures. The confluent cells were no more sensitive to TEC-11.dgA than they were to an isotype-matched immunotoxin of irrelevant specificity. These findings suggest that TEC-11.dgA might have therapeutic value in the treatment of solid tumors in humans by selectively killing dividing endothelial cells which are prevalent in such tumors.

A new method of iodinating ovalbumin, a protein which lacks accessible tyrosine groups, by conjugation to a highly fluorescent coumarin active ester, CASE.

A simple and efficient method is described to overcome the difficulty in radiolabelling ovalbumin and is applicable to any protein. Coumarin-3-acetic acid-N-OH-succinimidyl ester (CASE) binds directly to ovalbumin in aqueous solution to form covalent bonds with free amino groups. The attached CASE residues can then be radiolabelled by the chloramine-T method. This offers an inexpensive alternative to the Bolton and Hunter reagent with the advantage that CASE is fluorescent. This property, apart from direct use as a fluorescent tag, allows one to monitor the conjugate before radiolabelling. Below a conjugation ratio of 9:1, CASE-ovalbumin retained full antigenic identity with ovalbumin.

The effect of a chimeric mouse-human CD7 antibody on human T, natural killer, and lymphokine-activated killer cell activity in vitro.

A chimeric CD7 antibody has been constructed with mouse variable and human constant regions and is currently being assessed in the prophylaxis of renal graft rejection. In this study we have investigated if this antibody or its murine parental form inhibits the function of a number of immune effector mechanisms involved in host defense against infection and/or malignancy. Most memory T cells and all natural killer cells express the CD7 antigen and could therefore be affected by CD7 antibody. Murine and chimeric CD7 antibodies significantly inhibit the alloproliferation of naive (65 +/- 4% and 66 +/- 8%, respectively) but not memory T cells (86 +/- 2% and 98 +/- 4%, respectively) in a primary mixed lymphocyte reaction relative to the negative control CD10 antibody (P less than 0.001). The memory T cell proliferative response to recall antigen is also largely unaffected by murine and chimeric CD7 antibodies relative to the negative control antibody (91 +/- 12% and 103 +/- 10%, respectively). The CD7 antigen is almost completely modulated from the surface of NK cells after incubation for 24 hr with either the murine or chimeric CD7, but not the CD10, negative control. The modulation of CD7 antigen by antibody, however, does not affect the cytotoxic function of either the NK or lymphokine-activated killer cells significantly. Preincubation with the chimeric antibody however, consistently showed a small inhibition relative to the negative control of 75-80% in NK assays and to 80-90% in LAK assays. These data suggest that both murine and chimeric CD7 antibodies may have a selective effect on alloproliferation but may largely spare a major component of the host's innate immunity as well as memory T cell proliferation to previously encountered antigens.

Inhibition of alloresponsive naive and memory T cells by CD7 and CD25 antibodies and by cyclosporine.

Human naive and memory T cells can be isolated from each other by their CD45RA and CD45RO expression, respectively. This enables the assessment of their differential sensitivities to immunosuppressive agents for the first time. We have investigated the ability of cyclosporine or CD7 and CD25 antibodies to selectively block alloantigen stimulated naive and memory T cells in vitro. CD7 antibodies blocked the proliferation of naive (P less than 0.025) but not memory T cells in a primary MLR. CD25 antibody inhibited both naive and memory subsets but a significantly greater effect was found on the memory T cells (P less than 0.005). The constitutive CD7 and CD25 antigen expression on resting naive or memory T cells was related to the inhibitory activities of these antibodies on both subsets. Accordingly, naive T cells expressed more CD7 antigen than memory cells while memory T cells displayed low levels of CD25 antigen that was absent from naive populations before activation. Cyclosporine, like CD25 antibody, inhibited both subsets in a primary MLR but had a greater effect on memory cells (P less than 0.02). Memory T cells, therefore, are more dependent than naive cells on IL-2 for proliferation. There was great individual variation in the ability of CsA to block the MLR. The simultaneous addition of CD25 or CD7 antibody together with CsA, however, enhanced the MLR inhibition as the effects of all three were additive. This suggested interference by these agents at different points during T cell activation. Thus, in CsA sensitive individuals, one-tenth of the optimal CsA concentration together with CD25 antibody maintained maximum immunosuppression in vitro. These results demonstrate the possibility of using CD7 and CD25 antibodies for selective inhibition of naive or memory T cells and also the possibility of augmenting the inhibition of and reducing the CsA concentration required for clinically effective immunosuppression.

Prolonged action of a chimeric interleukin-2 receptor (CD25) monoclonal antibody used in cadaveric renal transplantation.

A high affinity chimeric CD25 mAb (chRFT5: SDZ CHI 621) blocking interleukin-2 binding to the interleukin-2 receptor alpha-chain was evaluated in a phase I/II study in human renal cadaveric transplantation. The chRFT5 was well tolerated with no immediate adverse effects during 6 spaced infusions (from before transplantation to day 24) in 24 patients escalating from 2.5- to 25-mg dosages. The chRFT5 had a long terminal half-life with a mean of 13.1 days. There was good correlation between the detection of chRFT5 in the serum by radioimmunoassay, the coating and suppression of CD25 on T cells, and antibody activity in patient serum samples. The chRFT5 activity persisted in vivo for up to 120 days. No antibody response to the chRFT5 was detected in any of the patients, even though two patients who required treatment with antithymocyte globulin or OKT3 developed xenogeneic antiglobulin responses while chRFT5 was still present in vivo. There was a 33% incidence of rejection and the first rejection episode always occurred during chRFT5 therapy. Patients who did not reject during therapy did not reject during the first year following transplantation. Equal numbers of patients received dual and triple immunosuppressive therapy together with chRFT5. Posttransplant lymphoproliferative disorder developed in 2 patients, both on triple therapy, at 9 months after transplantation. The disorder did not develop in any patient receiving dual therapy, and no further cases have been observed to a minimum of 2 years' follow-up. No other viral, fungal, or bacterial infectious complications were prevalent in patients treated with chRFT5.

Immunohistology of Epstein-Barr virus-associated antigens in B cell disorders from immunocompromised individuals.

Proliferating B cell lesions developing in a series of immunosuppressed organ transplant recipients and patients with X-linked lymphoproliferative syndrome were examined for Epstein-Barr virus and cellular gene expression using immunocytochemistry and immunoblotting techniques. Results indicate that all the lesions examined from the patients in this series expressed Epstein-Barr virus gene products that were consistent with a latent, nonproductive type of infection. No lytic cycle antigens associated with productive viral infection were detected. This pattern is similar to the viral gene expression in normal B cells immortalized by Epstein-Barr virus in vitro. The demonstration in this study of Epstein-Barr virus viral gene expression in posttransplant and X-linked proliferative syndrome B cell disorders provides important new evidence for the primary role of Epstein-Barr virus in the development of these lesions. This is in contrast to the subsidiary role that the Epstein-Barr virus has in the etiology of Burkitt's lymphoma.

Complete regression of posttransplant lymphoproliferative disease using partially HLA-matched Epstein Barr virus-specific cytotoxic T cells.

BACKGROUND: Adoptive immunotherapy with autologous and donor-derived cytotoxic T lymphocytes (CTL) has recently been used to treat Epstein Barr virus (EBV)-positive posttransplant lymphoproliferative disease (PTLD). METHODS AND RESULTS: We report complete regression of EBV-positive PTLD in an 18-month-old small bowel and liver transplant recipient after one infusion of partially human leukocyte antigen (HLA)-matched EBV-specific CTL grown ex vivo from an EBV seropositive unrelated blood donor. No infusion-related toxicity or evidence of graft-versus-host disease was observed. The tumor showed signs of regression within 1 week and EBV load in peripheral blood dropped to undetectable levels. Limiting dilution analyses (LDA) detected no EBV-specific CTL precursor (CTLp) cells before the infusion, and high numbers of CTLp at 4 hr and 24 hr post-CTL infusion. There was a reversal of the CD4/8 ratio in peripheral blood and an increase in HLA-DR positive CD8 cells. The patient has been in complete remission for 24 months. CONCLUSION: If this success is repeated in more PTLD patients, then stored CTL could be used for antiviral and antitumor therapies in immunocompromised patients.

Independent clonal origin of T- and B-cell clones in a composite lymphoma.

We present a detailed immunohistological and genotypic analysis of an unusual case in which a peripheral T-cell lymphoma, with features of Lennert's and angioimmunoblastic lymphoma, occurred after treatment of a low grade plasmacytoid lymphoma. By analysis of immunoglobulin and T-cell receptor genes, we show that the two diseases had an independent clonal origin at the level of lymphoid commitment. However, by employing a novel polymerase chain reaction-based technique for analysis of B-cell clonality, we show the persistence of a residual minor clonal B-cell population in the subsequent T-cell lymphoma. Only 2 previous cases of composite lymphoma involving B- and T-cell clones have been demonstrated by molecular analysis. This study underlines the immunophenotypic and genotypic heterogeneity of peripheral T-cell lymphomas and illustrates an unusual disease course in which a T-cell lymphoma has arisen in the context of, and perhaps as a consequence of, a B-cell lymphoma.

T-cell receptor gene rearrangement in B-cell non-Hodgkin's lymphoma: correlation with methylation and expression.

Two of 19 B-cell non-Hodgkin's lymphoma (NHL) were found to have clonally rearranged T-cell receptor (TCR) beta chain genes with a germline TCR-gamma gene configuration. The inappropriate rearrangements had a similar structure to the TCR-beta rearrangements described in B-cell chronic lymphocytic leukemia. All cases analysed displayed a consistent germline transcription of TCR-beta and alpha chain genes which was independent of rearrangement. Consistent with this, all cases showed some degree of hypomethylation of the TCR-beta chain locus similar though distinguishable from the pattern seen in mature T cells, but quite different from that seen in normal mature B cells. Taken together, these data suggest that most NHL's arise from an immature B-lymphocyte precursor at a stage of differentiation where lineage commitment is incomplete.

A pilot study of treatment of active ulcerative colitis with natalizumab, a humanized monoclonal antibody to alpha-4 integrin.

BACKGROUND: Alpha-4 integrins facilitate leucocyte migration across vascular endothelium. AIM: To assess the safety and efficacy of natalizumab (Antegren), a humanized antibody to alpha-4 integrin, in patients with active ulcerative colitis. METHODS: Ten patients with active ulcerative colitis, defined by a Powell-Tuck activity score > 4, received a single 3 mg/kg natalizumab infusion. The primary end-point was the change in Powell-Tuck score at 2 weeks post-infusion. RESULTS: Significant decreases in the median Powell-Tuck score were observed at 2 and 4 weeks post-infusion (7.5 and 6, respectively) compared to the median baseline score (10). Five of 10 patients achieved a good clinical response at 2 weeks and one more patient by 4 weeks, defined by a Powell-Tuck score of < or = 5. Significant improvements in quality of life scores were found at week 4. Rescue medication was required by two (20%), three (30%) and eight (80%) patients by weeks 2, 4 and 8, respectively (median, 34 days; range, 8-43 days). One patient remained in remission at 12 weeks. The median C-reactive protein at 2 weeks (6 mg/L) was lower than that pre-treatment (16 mg/L). CONCLUSIONS: A single 3 mg/kg infusion of natalizumab was well tolerated by ulcerative colitis patients. The positive efficacy demonstrated in this study merits further investigation by randomized, placebo-controlled trials.

Experimental treatment of human Hodgkin's disease with ricin A-chain immunotoxins.

In the present paper we describe the evaluation of ricin A-chain immunotoxins for clinical application in Hodgkin's disease. The immunotoxins were constructed by chemically linking deglycosylated ricin-A to monoclonal antibodies (MoAb) recognising lymphocyte activation markers CD25, CD30, or IRac, which are expressed by Hodgkin's and Reed-Sternberg (H-RS) cells. The cytotoxic effects of the immunotoxins were investigated in vitro against L540Cy Hodgkin cells and in vivo against Hodgkin's tumors in nude mice and disseminated Hodgkin's tumors in SCID mice. MoAbs were evaluated for crossreactivity with normal human tissues and staining of sections from Hodgkin's disease tissue. Of 32 MoAbs, eight showed little crossreactivity with vital human organs and produced highly active immunotoxins. The most effective immunotoxin, RFT5 gamma l.dgA (CD25), inhibits the growth of H-RS cells at concentrations of 7 x 10(-12) M. RFT5 gamma l.dgA destroys about 60% of solid Hodgkin's tumors of 0.5 cm diameter in nude mice and induces complete remissions in 95% of SCID mice with disseminated Hodgkin's tumors when administered one day after tumor challenge. This immunotoxin binds to all H-RS cells in more than 90% of patients with Hodgkin's disease. Patients with refractory Hodgkin's disease are currently being treated in a phase-I/II clinical trial.

[New perspectives in oncology: is selective destruction of tumor cells with immunotoxins in Hodgkin's disease an additional therapeutic alternative?]. / Neue Perspektiven in der Onkologie: Bietet die gezielte Zerstörung von Tumorzellen durch Immuntoxine beim Morbus Hodgkin eine zusätzliche therapeutische Alternative?

In the present paper, the authors describe the production and testing of immunotoxins for clinical application in Hodgkin's disease. The immunotoxins were constructed by chemical coupling of deglycolysated ricin-A to monoclonal antibodies against antigens on Hodgkin's and Reed-Sternberg cells (CD25, CD30, IRac). The cytotoxic effect of the immunotoxins was investigated in vitro against Hodgkin's and Reed-Sternberg cells (H-RS) and in vivo against solid Hodgkin's tumors in nude mice and disseminated Hodgkin's tumors in SCID mice. Cross-reactivity with normal tissue and the staining behaviour observed in sections of Hodgkin's tissue of various subtypes proved important parameters for the assessment of clinical applicability. Of more than 30 evaluated MoAb's, eight immunotoxins were produced, of which six showed both, cytotoxic effects of considerable potency against Hodgkin's tumor cells and low cross-reactivity with vital human organs. The most effective immunotoxin, RFT5 gamma 1.dgA, (CD25) inhibits the growth of H-RS cells at concentrations of 7 pMol and destroys about 60% of solid Hodgkin's tumors of 0.5 cm in diameter in nude mice. This immunotoxin binds to virtually all tumor cells in more than 90% of patients with Hodgkin's disease. Sufficient quantities of RFT5 gamma 1.dgA were produced for the treatment of patients with refractory Hodgkin's disease. These patients are currently being treated in a phase I clinical trial.