[Monoclonal anti-idiotype antibodies in immunotherapy of ovarian carcinoma (MAb ACA125) and breast carcinoma (MAb ACA14C5)]. / Monoklonale Anti-Idiotypen Antikörper zur Immuntherapie des Ovarialkarzinoms (MAk ACA125) und des Mammakarzinoms (MAk ACA14C5).

Anti-idiotypic antibodies, which imitate a tumor-associated antigen by their variable region, offer an elegant method for the induction of a specific immune response, when used as a surrogate antigen for immunization. We generated anti-idiotypic antibodies imitating 2 different tumor-associated antigens. I. CA125 for ovarian carcinomas and II. 14C5, a tumor-associated cell substrate adhesion molecule on breast cancer cells, whereas the first approach could be introduced in a first clinical trial and the second was evaluated in an immunocompetent animal model. For the induction of an immune response against CA125, 18 patients with advanced ovarian cancer (n = 6) or heavily pretreated recurrences (n = 12) were immunized with the anti-idiotypic antibody MAb ACA125. Patients were treated with 2 mg anti-idiotype antibody every two weeks for 4 injections i.m. and then monthly. 12 of 18 patients demonstrated an anti-anti-idiotypic (Ab3) response, which was to a lower extent also directed against CA125 and 9 of 18 patients developed a CA125 specific cellular immune response by their peripheral blood lymphocytes. Based on this data a follow-up clinical trial in advanced ovarian cancer patients with minimal residual disease in an adjuvant approach after primary therapy was started to evaluate the effect of the immune response on the progression free survival. For immunotherapy of breast cancer, we generated a murine monoclonal anti-idiotypic antibody (MAb ACA14C5), which imitates a cell substrate adhesion molecule on breast cancer cells. The anti-idiotype was introduced in an immunocompetent animal to prove his capability on induction of an immune and tumor response. The results showed a highly significant difference in the tumor growth of the ACA14C5 treated group in contrast to the controls starting the immunization on day 6 after tumor cell application with 10 of 12 animals being cured from their tumor burden. Prophylactic immunization against the invasion antigen of breast cancer by anti-idiotypic antibodies showed protection against increasing tumor burden. However, in the situation of established tumors only minor responses could be detected. Vaccination with anti-idiotypic antibodies comprises an effective method for induction of a specific immune response against non-immunogenic tumor-associated antigens and should be therefore considered in immunological approaches to tumor therapy, where the primary structure and sequence of the antigen, e.g. CA125, is up to now not available.

Immunological responses to the tumor-associated antigen CA125 in patients with advanced ovarian cancer induced by the murine monoclonal anti-idiotype vaccine ACA125.

We have generated an immunoglobulin G1 (IgG1) murine monoclonal anti-idiotype antibody (Ab2) designated ACA125, which mimics a specific epitope on the tumor-associated antigen CA125. This antigen is expressed by most of malignant ovarian tumors. Patients with CA125-positive tumors are immunologically tolerant to CA125. We used ACA125 as a surrogate for the tumor-associated antigen CA125 for vaccine therapy of 16 patients with advanced epithelial ovarian cancer or recurrences. Each of the patients received a minimum of 3 injections up to 19 injections of the complete anti-idiotype MAb ACA125 at a dosage of 2 mg per injection. Nine of 16 patients developed anti-anti-idiotypic (Ab3) responses to the ACA125. All 9 patients generated specific anti-CA125 antibody demonstrated by reactivity with purified CA125. Nine of 16 patients developed a CA125-specific cellular immune response by their peripheral blood lymphocytes (PBL) and 3 of 16 showed an increase in gamma-interferon concentrations accompanied by Ab3 responses. Toxicity was limited to abdominal pain in one case, which led to the withdrawal of further immunizations. The median progression free survival in those patients, who showed a specific immune response to the tumor-associated antigen CA125, was 11.0 +/- 5.6 months without any other therapy, in contrast to 8.0 +/- 4.2 months in the anti-anti-idiotype negative group. This is the first report of the induction of a specific active immunity to the tumor-associated antigen CA125 in patients with advanced ovarian cancer treated with an anti-idiotype antibody that "mimics" CA125. Patients showed the development of a specific humoral and cellular immune response to an otherwise nonimmunogenic tumor antigen. The immune responses in patients treated with this anti-idiotype vaccine, the low rate of side effects, and the improved time to progression after the induction of a specific immune response against the tumor-associated antigen CA125 justify follow-up clinical trials in advanced ovarian cancer patients with minimal residual disease in an adjuvant approach.

Production of a single-chain fragment of the murine anti-idiotypic antibody ACA125 as phage-displayed and soluble antibody by recombinant phage antibody technique.

The F(ab')2 fragment of the murine monoclonal anti-idiotypic antibody ACA125 mimicking the tumor-associated antigen CA125 is used as a vaccine for the induction of an anti-tumoral immunity in patients with ovarian carcinoma. We tried to generate a single-chain fragment (ScFv) composed of ACA125 heavy- and light-chain variable domains connected by a polypeptide linker as an alternative to the corresponding F(ab')2 fragment. Heavy- and light-chain genes of antibody-producing mouse hybridoma cell line were amplified separately and assembled into a ScFv gene with linker DNA by the polymerase chain reaction (PCR). The ScFv gene was ligated into the phagemid vector pCANTAB5E, which allows the production of both phage-displayed and soluble ScFv. Transformed Escherichia coli TG1 cells were infected with M13K07 helper phage to yield recombinant phage, which display ScFv fragments as a g3p fusion protein on the surface of the filamentous phage M13. Recombinant phages could be selected by binding to the idiotypic antibody OC125 after one round of panning and directly used to reinfect E. coli TG1 cells. The E. coli nonsuppressor strain HB2151 was infected with an antigen-positive phage clone, previously screened by enzyme-linked immunosorbent assay (ELISA), to express soluble ScFv fragments. Functional soluble ScFv binding to the idiotypic antibody OC125 F(ab')2 could be detected in the bacterial periplasm by Western blot and ELISA. The variable heavy- and light-chain genes of the ACA125 ScFv fragment were further sequenced and compared with known antibody sequences.

A monoclonal antiidiotypic antibody ACA 125 mimicking the tumor-associated antigen CA 125 for immunotherapy of ovarian cancer.

A new concept of oncological immunotherapy comprises the attempt to trigger the immune system of the host into a response against tumor cells. Antiidiotypic antibodies bearing the internal image of an antigen expressed on the surface of the tumor seem to be most suited for this purpose. We have generated a murine antiidiotypic antibody (ACA 125) functionally imitating the tumor-associated antigen CA 125, which can be detected in about 80% of ovarian carcinomas. The hybridoma cell was adapted to serum-free medium and antibody was produced in a hollow fiber cell culture system (Technomouse). ACA 125 (Ab2) shows high affinity for the paratope of Ab1 (affinity constant: 2.3 x 10(9) liters/mol) and binding of Ab2 to Ab1 is completely inhibited by the nominal antigen. Application of F(ab')2 fragments of ACA 125 to rats lead to an anti-CA 125 immunity by production of IgG and IgM antiantiidiotypic antibodies (Ab3) that bind to both ACA 125 and CA 125. Furthermore the induction of a non-MHC-restricted cell-mediated cytotoxicity for human ovarian adenocarcinoma cell line NIH-OVCAR3 (expressing CA 125 on its surface) could be proved; additionally complement-dependent cytotoxicity (CDC) as well as an antibody-dependent cellular cytotoxicity (ADCC) was observed. Thus, monoclonal antiidiotypic antibody ACA 125 fulfills recent criteria for an antibody, which might be successful in immunotherapy using the anti-idiotypic network approach.

Determination of digoxin in the blood of pregnant women, fetuses and neonates before and during anti-arrhythmic therapy, using four immunochemical methods.

Four immunochemical methods for digoxin assay were used to analyse control samples, 33 amniotic fluid samples, 57 samples from digitalis-treated, non-pregnant women, 90 pregnancy serum samples, and 72 samples of fetal or neonatal serum with or without digoxin therapy. One hundred and five samples were also submitted to ultrafiltration before analysis. Three methods (RIA, TDX, AMERLITE) showed practically the same precision, while the precision of the DELFIA was markedly inferior. In the analysis of serum samples from digoxin-treated, non-pregnant women, RIA and TDX gave practically the same values, whereas AMERLITE and DELFIA gave significantly higher values. Pregnancy serum and fetal serum contain "digoxin-like immunoreactive factors", and the qualitative and quantitative effects of these interfering factors are different for each of the four methods. The greatest sensitivity to "digoxin-like immunoreactive factors" is shown by TDX and DELFIA, while the lowest interference by "digoxin-like immunoreactive factors" is found in the analysis of ultrafiltered samples, using the TDX method. The composition of the "digoxin-like immunoreactive factors" in pregnancy serum and in fetal serum is altered by digoxin therapy, and these changes have different effects on the various analytical methods. The concentration of "digoxin-like immunoreactive factors" in the serum of fetuses receiving digoxin is markedly lower than that of healthy fetuses. For the reliable monitoring of digoxin therapy in the maternal and fetal circulation, the blood samples must be submitted to ultrafiltration before analysis.

[Biochemical findings in gestosis patients with pulmonary complications]. / Biochemische Befunde bei Gestose-Patientinnen mit Lungenkomplikationen.

Lysosomal proteases from the granula of polymorphonuclear leucocytes play an important part in the development of the shock lung syndrome and its preliminary stages. If there is a local imbalance between the neutral proteinases (e.g. elastase) and its inhibitors, the interstitium consisting of collagenous fibres and glycosaminoglycanes is damaged. In 4 patients with gestosis and pulmonary complications it became evident that the plasma levels of the elastase-alpha 1-proteinase inhibitor complex correlate well with the clinical status and the x-ray findings. Significantly increased values are seen already prepartally; maximum values represent a severe interstitial oedema, and the drop of the values occurs parallel with the improvement of the clinical pattern. The increased consumption of inhibitors is expressed by the absence of the reactive increase of alpha 1-antitrypsin. By determining the elastase-alpha 1-PI complex it becomes possible to recognize the possible risk of development of an interstitial pulmonary oedema well in time, and the success of therapeutic measures is easily ascertained and controlled.

Comparison of a charcoal sorbent fiber with commercial charcoals for hemoperfusion.

A charcoal sorbent fiber (Enka, F.R.G.), was assessed for impurities, surface area, and adsorptive properties of its native charcoal, and compared with other uncoated activated charcoals. In vivo and in vitro hemocompatibility of the fiber were assessed as well as the adsorptive properties for endogenous toxins. The charcoal of the fiber had few impurities and a high surface area of 1,200 m2/g charcoal. For measuring the adsorptive speeds, 2 g of the uncoated charcoals were milled and screened to a particle size of 150-250 microns (Enka; 30-40 microns) and then mixed with the solutions of the individual solutes. The charcoal types of Enka, used in the charcoal sorbent fiber, and of Sutcliffe Speakman, used in the acrylic hydrogel coated charcoal, exhibited the highest adsorptive rates for bromthalein (middle molecular weight marker) and inulin (high molecular weight marker). No hematological differences among the various charcoals were found during the in vivo hemoperfusions. In the in vitro hemoperfusions with heparinized fresh blood, the fibers showed the lowest loss of leucocytes and thrombocytes. In the in vitro evaluation of the absorbents for hepatic support, the charcoal fiber and the petroleum pitch charcoal of Asahi had the best adsorptive properties for substances in the low molecular weight range.

Suppression of cell-mediated immunity in rats transplanted with kidneys from donors compatible at the major histocompatibility complex.

In order to study the immunological status of rats transplanted with H-1-compatible kidney allografts, LEW rats were grafted with F and (Fischer X Lewis)F1 (FLEWF1) kidneys. Most of the F kidneys were rejected within 55 days, only 4 of 24 surviving for more than 4 months. However, two-thirds of the FLEWF1 recipients survived for more than 4 months, the others having died within 64 days. During the first postoperative week, high levels of serum lymphocytotoxic antibodies were found in recipients of F kidneys, and thereafter there was little change. In this respect these rats did not differ from recipients of kidneys with major histocompatibility differences. However, recipients of FLEWF1 kidneys had low haemagglutinating and lymphocytotoxic antibody titres. No serum-blocking factor could be found in kidney of recipients by use of the microcytotoxicity assay (MCA) or inhibition of allorosette formation. Cellular immunity, which was studied by means of the graft-versus-host reaction (GVHR) and the microcytotoxicity assay, was detected in the first postoperative week. This immunity gradually declined, and after 6 weeks it was no longer detectable. The immunological status of the long-term surviving kidney recipients remained unchanged, even when they were provided with further antigenic challenge in the form of two successive donor strain skin grafts.

[Suppression of the cell-mediated immunity in minor histoincompatible rat kidney allograft recipients (author's transl)]. / Suppression der zellgebundenen Immunität bei schwach histoinkompatiblen Rattennierenallotransplantatempfängern.

To study the immunological status of recipients of major compatible and minor incompatible kidney allografts, we transplanted FiS and FLF1 kidneys into LEW rats. Most of the FiS kidneys were rejected within 55 days. Of 24 recipients, only 4 survived longer than 4 months. However, two-thirds of the FLF1 recipients survived longer than 4 months. The other third died with 64 days. During the first postoperation week a high level of lymphocytotoxin was detected in the serum of the FiS kidney recipients. Thereafter hardly any alternation of its titer was found, and no variation among the recipients of major histocompatible kidney allografts was shown. The FLF1 kidney recipients showed a low titer of antibody. The hemagglutinin titer showed the same trend as the lymphocytotoxin titer. A blocking serum factor could not be found in the serum of the kidney recipients with the microcytotoxity assay method or with the allorosette-formation inhibition test. Cellular immunity, which was studied with the GvH-reaction and microcytotoxity assay, was detected in the first postoperative week. However, this immunity was gradually supressed, and after 6 weeks was no longer to be found. This immunological status remained unchanged in the indefinitive surviving kidney-recipients in spite of antigen inoculation with two skin allografts of donor origin. This immunological status could be defined as "graft acceptance".

[Active enhancement of canine liver allograft by pretreatment with polyspecific spleen alloantigen (author's transl)]. / Aktives Enhancement von Hundeleberallotransplantaten. Vorbehandlung mit polyspezifischem semisolublen Milzalloantigen.

Because it is not possible to use donor specific antigens for the induction of immunological enhancement in cadaveric organtransplantation, attempts were made to use polyspecific antigens in the enhancement of orthotopic canine liver allotransplants. Of 34 mongrel recipients, 17 controls survived for 6.9 +/- 1.5 days. Six recipients (group 3) were given 750 mg/kg polyspecific, semisoluble antigen prepared from 20 spleens (PSEA 20) together with 10 mg/kg prednisolone on days 15, 8 and 1 before transplantation. The mean survival time of this group was 10.1 +/- 2.0 days (P less than 0.01 compared with the control group). Six other recipients (group 4) were treated similiarly, except that the antigen had been prepared from a pool of 70 spleens (PSEA 70). Survival was variable here: 3 survived for more than 3 weeks and the other 3 died on days 1, 7 and 8 post-operatively, with signs of accelerated rejection. Donors and recipients were not identical for LD determinants, but one donor recipient pair with near identity showed a higher degree of enhancement. The recipients displaying accelerated rejection had markedly higher lymphocytotoxic and haemagglutinating antibodies. Animals surviving for longer periods had low antibody titres. In addition, all recipients progressive rejection were found to show inhibition of leucocyte migration. After three antigen doses rosette-forming lymphocytes were present in increased numbers in peripheral blood, and remained unchanged thereafter.

Treatment of extrahepatic occlusive jaundice with activated charcoal hemoperfusion in dogs.

To find the feasibility of treatment for congenital bile duct atresia, we studied the usefullness of extracorporeal hemoperfusion over activated charcoal in canine obstructive jaundice. One, three and five weeks after ligation and disection of common bile duct in 5 dogs we performed the hemoperfusion over activated charcoal extracorporeally (group 3). In this animals we examined hematological and blood coagulation studies, serum electrolyte levels, kidney function tests and liver chemistries. As control in 5 animals we carried out after sham operation the perfusion without common bile duct ligation (group 2) and in 5 animals only common bile duct ligation without perfusion (group 1). In the liver chemistries we found 2 weeks after 2nd and 3rd perfusion (5 and 7 weeks after bile duct ligation) lower levels of serum bilirubin, GOT, GPT and SDH in treated group than in non-treated jaundiced animals. It suggest the effectiveness of hemoperfusion with activated charcoal in the treatment of occlusive jaundice. There were no alteration in the hematological studies, serum electrolyte levels and kidney function tests. PT and PTT was prolonged in the jaundiced animals there were no significant differences with and without hemoperfusion.

Active enhancement of rat kidney allografts. Effect of pretreatment with prednisolone and donor-specific antigen.

(Lewis x Brown Norway) F1 hybrid rat kidney allografts were transplanted to bilaterally nephrectomized Lewis recipients pretreated in various ways. The mean survival time of untreated controls was 16.1 +/- 1.7 days. All rats pretreated with 1.67 g/kg of semi-soluble Brown Norway spleen extract and 5 mg/kg of prednisolone on days 15, 8, and 1 before transplantation survived indefinitely. Pretreatment with semi-soluble or soluble extract alone prolonged survival modestly (36.5 +/- 13.6 and 30.8 +/- 5.6 days, respectively), but the former induced indefinite survival in two of eight animals. Prednisolone on its own failed to bring about prolongation of survival and the combined use of soluble extract and prednisolone did not reveal a synergistic effect. Cytotoxic antibody titres in animals showing indefinite survival were very low, and there was no correlation between antibody titres and prolonged survival. It is assumed that the pretreatment with semi-soluble extract and prednisolone inhibited the formation of cytotoxic antibodies as well as cell-mediated immunity, and encouraged the formation of enhancing antibodies. To study the cellular and humoral reactivity of five prolonged survived kidney recipients, 1st and 2nd donor-specific skin grafts were carried out. The humoral and cell-mediated responses were somewhat delayed in these recipients but otherwise normal except for the absence of the second-set phenomenon.