Development of anti-CD30 radioimmunoconstructs (RICs) for treatment of Hodgkin's lymphoma. Studies with cell lines and animal studies.

OBJECTIVES: Comparison of the binding affinity to a CD30-positive Hodgkin lymphoma (HL) cell line and biodistribution in HL bearing mice of new anti-CD30 radioimmunoconjugates (RICs) of varying structure and labelling nuclides. METHODS: The antibodies Ki-4 and 5F11 were radioiodinated by the chloramine T method or labelled with (111)In via p-NCS-Benzyl-DOTA. In addition, the Ki-4-dimer was investigated in the iodinated form. The RICs were analyzed for retained immunoreactivity by immunochromatography. In-vitro binding studies were performed on CD30-positive L540 cell lines. For in-vivo biodistribution studies, SCID mice bearing human HL xenografts were injected with the various radioimmunoconjugates. After 24 h, activities in the organs and tumour were measured for all 5 RICs. Tumour-free animals were studied in the same way with (131)I- Ki-4 24 h p. i. The three RICs with the highest tumour/background ratios 24 h p.i. ((131)I-Ki-4, (131)I-5F11, (111)In-bz-DOTA-Ki-4) were analysed further at 48 h and 72 h. RESULTS: All the RICs were successfully labelled with high specific activities (28-47 TBq/mmol) and sufficient radiochemical yields (>80%). Scatchard plot analysis proved high tumour affinity (KD = 20-220 nmol/l). In-vivo tumour accumulation in % of injected dose per g tissue (%ID/g) lay between 2.6 ((131)I-5F11) and 12.3 % ID/g ((131)I-Ki-4) with permanently high background in blood. Tumour/blood-ratios of all RICs were below one at all time points. CONCLUSIONS: In-vitro tumour cell affinities of all RICs were promising. However, in-vivo biokinetics tested in the mouse model did not meet expectations. This highlights the importance of developing and testing further new anti-CD30 conjugates.

Apoptosis in human ovarian tissue after conventional freezing or vitrification and xenotransplantation.

One of the new emerging techniques to preserve reproductive potential of cancer patients is cryopreservation of ovarian fragments prior to medical treatment and their retransplantation after healing. In order to investigate and compare apoptosis in human ovarian tissue after conventional ("slow") freezing and vitrification, we used a xenograft model in which conventionally frozen, vitrified and fresh non treated human ovarian tissue pieces were subcutaneously transplanted in SCID mice. The tissue samples were weekly, during four weeks, recovered from scarified SCID mice. The apoptosis was examined by immunohistochemical staining with the anti-caspase-3 antibody. There was a significant difference between the amount of apoptotic cells in cryopreserved ovarian tissue independent from mode of cooling compare to the control. The ovarian tissue after vitrification showed a significantly higher amount of apoptotic cells, than in slow frozen. The results obtained after comparative study of two different cryopreservation methods show that vitrification of human ovarian tissue could become a practice-relevant alternative to slow cryopreservation only after further improvement.

Portal hyperperfusion causes disturbance of microcirculation and increased rate of hepatocellular apoptosis: investigations in heterotopic rat liver transplantation with portal vein arterialization.

Clinical results of portal vein arterialization (PVA) in liver transplantation are controversial. One reason for this is the lack of a standardized flow regulation. Our experiments in rats compared PVA with blood-flow regulation to PVA with hyperperfusion in heterotopic auxiliary liver transplantation (HALT). In group I (n = 19), the graft's portal vein was completely arterialized via the right renal artery in-stent technique, using a 0.3-mm stent, leading to a physiological average portal blood flow. In group II (n = 19), a 0.5-mm stent was used. In group II, the average portal blood flow after reperfusion was significantly elevated (group II: 6.4 +/- 1.5; group I: 1.7 +/- 0.4 mL/min/g of liver weight; P < .001). The sinusoidal diameter after reperfusion was significantly greater in group II (9.8 +/- 0.5 microm) than in group I (5.5 +/- 0.2 microm; P < .001). Red blood cell velocity in the dilated sinusoids was significantly lower in group II (171 +/- 18 microm/s) than in group I (252 +/- 13 microm/s). Stasis of erythrocytes occurred; consequently, the functional sinusoidal density was significantly reduced in group II (38 +/- 7%) compared with group I (50 +/- 3%; P < .01). Two hours after reperfusion of the portal vein, the number of apoptotic hepatocytes was significantly higher in group II than in group I (I: 0 +/- 0 vs II: 7 +/- 9 M30-positive hepatocytes/10 high-power fields). The 6-week survival rate was 9 of 11 in both groups. In group II, 6 of 9 grafts showed massive hepatocellular necroses after 6 weeks, whereas in group I, only 1 of 9 presented a slight hepatocellular necrosis. Finally, our results demonstrate negative effects of portal hyperperfusion in transplanted livers, which are correctable by adequate flow regulation.

Intratumoral application of standardized mistletoe extracts down regulates tumor weight via decreased cell proliferation, increased apoptosis and necrosis in a murine model.

The cytotoxic in vitro activity of standardized mistletoe extracts (ME) was examined by established assays towards the human ductal breast carcinoma cell line BT474. A dose-dependent (optimum 25 mg/mL medium) and significantly (p < 0.05) enhanced cytotoxic activity towards the BT474 cells was demonstrated. In vivo experiments on the antitumor activity of ME-A and ME-M were performed in a BALB/c-mouse / BT474 ductal breast carcinoma model. ME-A and ME-M were intratumorally administered according to an application schedule which was found to be optimal concerning dosage and time of administration. Standardized intratumoral application of ME-A and ME-M induced a significantly (p < 0.05) decreased tumor weight in experimental mice. Histological investigations were performed comprising analysis of mitosis and proliferation rates (Ki67 expression), as well as necrosis and apoptosis induction (ssDNA detection). As compared to tumors of control mice with intratumoral phosphate-buffered saline (PBS) injections, tumors of the ME-A and ME-M treated groups showed a decreased cell proliferation rate, as well as an increased cell necrosis and apoptosis rate. Standardized mistletoe extracts, interfering with defined tumor cell functions, e.g., proliferation, necrosis and apoptosis, may have an impact on local cancer treatment.

Comparison of necrosis in human ovarian tissue after conventional slow freezing or vitrification and transplantation in ovariectomized SCID mice.

This paper examines and compares necrosis in human ovarian tissue after conventional slow freezing or vitrification and ensuing xenotranplantation. Slow cryoconserved or vitrified ovarian tissue samples and fresh controls from nine patients were subcutaneously transplanted into SCID mice. The tissue samples were explanted after 6 weeks and the necrotic areas were examined by staining with Lucifer yellow SV. The size of the necrotic areas in parallel cultivated ovarian tissue samples was compared, as was necrosis in cultivated prostate tumour spheroids where the emergence of necrosis and its pathophysiological correlation have been described. Examinations showed no significant rise in the proportion of necrotic areas after slow cryoconservation/transplantation and in the controls (transplanted fresh tissue, not transplanted fresh tissue, long-term culture). The proportion of necrotic areas in the tumour spheroids was significantly higher than in the ovarian tissue. Vitrification could, after these results, be presented as an alternative to conventional slow cryoconservation.

Ki-4(scFv)-ETA', a new recombinant anti-CD30 immunotoxin with highly specific cytotoxic activity against disseminated Hodgkin tumors in SCID mice.

The human lymphocyte activation marker CD30 is highly overexpressed on Hodgkin/Reed-Sternberg cells and represents an ideal target for selective immunotherapy. We used the murine anti-CD30 hybridoma Ki-4 to construct a new recombinant immunotoxin (rIT) for possible clinical use in patients with CD30(+) lymphoma. Hybridoma V genes were polymerase chain reaction-amplified, assembled, cloned, and expressed as a mini-library for display on filamentous phage. Functional Ki-4 scFv obtained by selection of binding phage on the CD30-expressing Hodgkin lymphoma cell line L540cy was inserted into the bacterial expression vector pBM1.1 and fused to a deletion mutant of Pseudomonas exotoxin A (ETA'). Periplasmically expressed Ki-4(scFv)-ETA' demonstrated specific activity against a variety of CD30(+) lymphoma cells as assessed by different in vitro assays. To evaluate in vivo antitumor activity, severe combined immunodeficient mice challenged with human lymphoma cell lines were treated with the immunotoxin. The blood distribution time t(1/2)alpha of Ki-4(scFv)-ETA' was 19 minutes, and its serum elimination time t(1/2)alpha was 193 minutes. A single intravenous injection of 40 microg rIT 1 day after tumor inoculation rendered 90% of the mice tumor free, extending the mean survival time to more than 200 days compared with 38.1 days in the phosphate-buffered saline control group (P <.001). This new rIT is a promising candidate for further clinical evaluation in patients with Hodgkin lymphoma or other CD30(+) malignancies. (Blood. 2000;95:3909-3914)

Recombinant anti-CD25 immunotoxin RFT5(SCFV)-ETA' demonstrates successful elimination of disseminated human Hodgkin lymphoma in SCID mice.

Since clinical phase-I/II trials in patients with resistant Hodgkin's lymphoma treated with the chemically linked anti-CD25 ricin-A-chain immunotoxin RFT5-SMPT-dgA indicate promising results for patients with minimal residual disease, we constructed a new immunotoxin by fusing the RFT5 single-chain variable fragment to a deletion mutant of Pseudomonas exotoxin A (ETA'). The recombinant protein was directed into the periplasmic space of E. coli by means of the pET-derived expression vector pBM1.1 and our newly developed expression/purification method. Biologically active RFT5(scFv)-ETA' was isolated by freezing/thawing and purified by immobilized metal-ion affinity and molecular-size-chromatography. RFT5(scFv)-ETA' was subsequently used for the treatment of disseminated human Hodgkin's lymphoma in a SCID-mouse model. The mean survival time (MST) of L540rec-challenged SCID mice was 38.1 days. A single i.v. injection of 40 microg recombinant immunotoxin (rIT) 1 day after tumor inoculation resulted in 100% tumor-free mice, extending the MST to more than 220 days (p < 0.0001). The blood-distribution time T(1/2)alpha was 39.65 min, the serum elimination time T(1/2)alpha, 756.6 min. All animals were assessed for soluble interleukin-2 receptor alpha, which is directly correlated to tumor burden. Soluble CD25 was not detectable in mice treated with the rIT. Our findings, concerning potent anti-tumor effects of a recombinant anti-CD25 immunotoxin against disseminated Hodgkin's lymphoma in SCID mice reported here demonstrate that RFT5(scFv)-ETA' might be suitable for further evaluation against Hodgkin's lymphoma in humans.

Antidisialoganglioside ricin A-chain immunotoxins show potent antitumor effects in vitro and in a disseminated human neuroblastoma severe combined immunodeficiency mouse model.

Several monoclonal antibodies (mAbs) were screened on different neuroblastoma cell lines to evaluate ricin A-chain immunotoxins for possible use against human neuroblastoma. Four mAbs were identified that exhibited high antitumor activity against neuroblastoma cell lines as measured in an indirect cytotoxicity assay. These mAbs, including 14G2a (antidisialoganglioside), ch14.18 (a humanized switch variant), BW704 (antidisialoganglioside), and chCE7 (anti-glycoprotein of M(r) 190,000), were subsequently linked via the bivalent linker N-succinimidyloxycarbonyl-alpha-methyl-alpha-(2-piridyldithio++ +)toluene to deglycosylated ricin A chain. The most potent immunotoxin, 14G2a.dgA, inhibited the protein synthesis of neuroblastoma cell lines IMR5 and NMB by 50% at concentrations of 6 x 10(-12) M. To test the antitumor efficacy of these immunotoxins in vivo, we developed a disseminated human neuroblastoma model in severe combined immunodeficiency mice. Treatment of tumor-bearing mice with 14G2a.dgA 12 days after tumor challenge resulted in a significant prolongation of survival as compared with phosphate-buffered saline-treated controls (16.8 versus 6.5 weeks). We conclude that ricin A-chain immunotoxins might be of potential use in the treatment of human neuroblastoma.

Preliminary report: growth of Hodgkin's lymphoma derived cells in immune compromised mice.

Until now there has been no satisfactory animal host for the in vivo growth of Hodgkin lymphoma cells. With the exception of one mutant subline (L540Cy) none of the other Hodgkin derived cell lines nor Hodgkin's disease (HD) derived lymphatic tissue could be propagated in suitable animal systems such as the T-cell deficient nude mouse. Recently, the severe combined immunodeficient (SCID-) mouse has been demonstrated as a possible recipient for human lymphatic tissue. In the present study, we have evaluated the SCID mouse as a possible in vivo model for Hodgkin's lymphoma. I) We demonstrate that seven permanent cell lines derived from patients with Hodgkin's disease grow progressively in SCID mice after subcutaneous and intraperitoneal inoculation. II) In addition, after intravenous injection, two of these lines (L540, L540Cy) show a disseminated growth pattern resembling the distribution of HD cells in man (involvement of lymph nodes, liver and bone marrow but not of spleen). The observed reproducible disseminated tumor growth establishes the SCID mouse as a new animal model for experimental treatment strategies in Hodgkin's lymphoma. III) We present preliminary results of the transplantation of primary material from 13 patients with Hodgkin's disease. Material from two patients induced human tumors in the SCID mice recipients, whereas material from two others led to the induction of mouse lymphomas. The human tumors showed three distinct histological patterns: 1) Lymphoproliferative disease (LPD); 2) anaplastic large cell lymphomas (ALCL); 3) Hodgkin like lesions (HLL). In vitro cell lines established from human SCID mouse tumors were of B-lymphoid origin, were EBV-positive and showed numerical and some structural chromosomal aberrations of varying degree.