Inhibition of bispecific monoclonal antibody (bsAb)-targeted cytolysis by human anti-mouse antibodies in ovarian carcinoma patients treated with bsAb-targeted activated T-lymphocytes

T lymphocytes of 8 patients with ovarian cancer were targeted to the tumor cells using F(ab')2 fragments of a bispecific monoclonal antibody (bsAb), specific for CD3 (a component of the T lymphocyte receptor for antigen) and for the folate receptor MOv18 (overexpressed by ovarian carcinoma cells) as part of a phase I/II study. Phase I (days 0 to 3) consisted of increasing intraperitoneal (i.p.) numbers (10(6)-10(9)) of bsAb-targeted T lymphocytes plus low-dose interleukin-2 (IL-2). Phase II (days 6 to 13, and 27 to 33) consisted of daily i.p. infusions of 10(9) targeted T lymphocytes, 2 mg soluble bsAb, and low-dose IL-2. Using enzyme-linked immunosorbent assays (ELISA), human anti-mouse antibodies (HAMA) were detected in all patients: in the serum from day 13 onwards and in the peritoneal fluid from day 20 onwards. A significant proportion of the HAMA appeared to be directed against the idiotypes of the bsAb specific for CD3 and MOv18, as suggested by (1) the clearly higher ELISA titers against OC/TR bsAb as compared to those against a monoclonal antibody (MAb) with unrelated specificity, and (2) failure to abrogate the capacity of peritoneal fluid containing HAMA to block the binding of OC/TR bsAb to MOv18+ or CD3+ cells by absorption of human anti-mouse IgG-framework antibodies in peritoneal fluid to immobilized mouse IgG. The OC/TR-targeted cytolysis of the MOv18+ ovarian carcinoma cell line Igrov-I by autologous T lymphocytes was inhibited by peritoneal fluid samples containing relatively high HAMA titers. Such inhibitory activity was never detected at the start of phase II, but coincided with the last series of i.p. infusions of targeted T lymphocytes in 2 patients.     

Evaluation of the diagnostic usefulness of CA125 immunoscintigraphy for ovarian carcinoma follow-up after treatment: contribution of this technique in Grenoble University Medical Center

Immunoscintigraphy using indium-111-labeled OC125 monoclonal antibody F(ab')2 fragments is a technic complementary of morphological imaging (i.e. ultrasonography and computed tomography). It allows early detection of recurrences of ovarian carcinomas. We performed immunoscintigraphy 30 times in 26 patients who previously underwent radical treatment for ovarian carcinoma, and were suspected to have a recurrence. Our purposes were appreciation of diagnostic accuracy of the method, and above all its impact on clinical decisions and evolution of the patients. There were, after reevaluation of the results, 18 true positives, 7 true negatives, 3 false negatives and 2 false positive cases (sensitivity 85.7%, specificity 77.8%). Bayesian analysis showed positive and negative predictive values of 86% and 87% when probability of recurrence a priori was 50%, and 80% and 58% when probability of recurrence a priori was 70%. The result of immunoscintigraphy contributed to clinical decisions in 24 cases out of 30, and led to a correct decision for the patient in 21 cases. Conversely, for the 6 cases in which the result has not been considered, to take this result into account would have been beneficial in 4 cases, but harmful in 2. Finally, survival tended to be longer when immunoscintigraphy was negative, which could be associated with a better prognosis. We conclude that OC125-immunoscintigraphy may be useful for ovarian carcinoma follow-up and may contribute to a better therapeutic strategy.     

Monitoring cancer antigen 125 in serum of ovarian cancer patients after administration of 131I-labeled F(ab')2 fragments of OC125 antibody

We evaluated the effect of repeated administration of OC125 F(ab')2 fragments on cancer antigen (CA) 125 determination in 210 serum samples from 30 patients. We found falsely high CA 125 concentrations in 142 (68%) samples, using a homologous CA 125 enzyme immunoassay (EIA) with OC125 antibodies. The Truquant OV2 method, which involves two other murine antibodies, and the IMx CA 125 method, which uses sheep antibodies as capture antibodies, resulted in only slightly increased (false-positive) values in some samples with exceptionally high CA 125 EIA values. We measured falsely low CA 125 values in 37 (18%) samples with the Truquant OV2 method. Interferences could be eliminated by removal of serum IgG. Our results suggest that interferences are to some extent caused by anti-idiotypic IgG induced by OC125 administration. Assays involving nonmurine anti-CA 125 antibodies as capture antibodies seem to be most suited for CA 125 determination after OC125 treatment, but in every case an apparent increase of CA 125 after OC125 infusion should be validated.     

Increased tumor localization by monoclonal antibody A7 after F(ab')2 fragmentation in athymic nude mice bearing human pancreatic carcinomas

Much recent research has been directed toward the use of monoclonal antibodies (MoAbs) for the immunodetection of solid tumors. In pancreatic cancer, conventional immunoscintigraphy using intact MoAbs remains disappointing. In this study, 125I-labeled F(ab')2 fragments produced by pepsin digestion of MoAb A7 were injected intravenously into nude mice bearing human pancreatic cancer, HPC-YS, xenografts that have previously been shown to react specifically with MoAb A7. The tumor tissue/blood ratio of 125I-labeled F(ab')2 fragments of MoAb A7 increased with time and was much higher than those for normal tissues. Moreover, the tumor tissue/blood ratio of 125I-labeled F(ab')2 fragments was greater than that of intact MoAb A7, although the F(ab')2 accumulation was less than that of intact MoAb A7 in the tumor. These results suggest that F(ab')2 fragments of MoAb A7 may be suitable carriers of radionuclides for immunodetection of human pancreatic cancer.     

Distribution of radiolabeled monoclonal antibody MX35 F(ab')2 in tissue samples by storage phosphor screen image analysis: evaluation of antibody localization to micrometastatic disease in epithelial ovarian cancer

Our objective was to quantify the targeting of the monoclonal antibody (mAb) MX35 F(ab')2 to micrometastatic epithelial ovarian cancer. This mAb detects a Mr 95,000 glycoprotein with homogeneous distribution on 80% of ovarian tumor specimens. Six patients with minimal residual disease from an imaging trial were injected with 2 or 10 mg of 131I- and 125I-labeled mAb MX35 F(ab')2. Biopsied samples were removed at second-look laparotomy 1-5 days post-i.v. or -i.p. infusion of antibody. Serial cryostat sections were stained by indirect immunoperoxidase method for antigen distribution and exposed to storage phosphor screens for quantitative autoradiography. Coregistration of tumor histology, antigen expression, and radionuclide distribution demonstrated specific localization in micrometastatic tumor foci (50 micrometer to 1 mm) found within tissue stroma. The radiolabeled antibody uptake determined by well scintillation counts ranged between 5.2 and 223.5 x 10(-4) percentage of injected dose/g of tumor tissue for 131I. Specific localization of mAb in tumor was determined by tumor:normal tissue (fat) ratios ranging from 0.9:1 to 35.9:1 for 131I. The high resolution and linear response of the storage phosphor screen imager was used to estimate the radionuclide activity localized in each micrometastatic site. Quantitation of phosphor screen response revealed microCi/g values of 0.026-0.341 for normal tissue and 0.184-6.092 for tumor biopsies, evaluated 4 or 5 days post-antibody injection. The tumor:normal tissue (adjacent to tumor) ratios were between 1 and 4 times greater using the phosphor screen method than well counter measurements, but even larger variations of ratios up to 20:1 were observed between tumor cell foci and stromal cells within the same tissue section. This study has demonstrated that mAb MX35 F(ab')2 localizes to the micrometastatic ovarian carcinoma deposits within the peritoneal cavity. The dosimetry results suggest a therapeutic potential for this antibody in patients with minimal residual disease (<5 mm).     

Treatment of ovarian cancer with photodynamic therapy and immunoconjugates in a murine ovarian cancer model

In photodynamic therapy (PDT), photosensitisers accumulate somewhat preferentially in malignant tissues; photoactivation with appropriate wavelength of light release toxic molecular species which lead to tumour tissue death. In order to target ovarian cancer with increased specificity, a chlorin-based photosensitiser (chlorin e6 monoethylendiamine monoamide) was conjugated to OC125, a monoclonal antibody recognising an antigen expressed in 80% of non-mucinous ovarian cancers. In previous work, this immunoconjugate (IC) was shown to be selectively phototoxic to cancer cells from ovarian cancer patients ex vivo and to localise preferentially in ovarian cancer tissue in vivo. In this study we report results from in vivo phototoxicology and photodynamic treatment studies using this IC in a murine model for ovarian cancer. A comparison of single vs multiple treatments was also made. For in vivo experimentation, Balb C nude mice were injected with 30 x 10(6) NIH:OVCAR 3 cancer cells to create an ascitic tumour model. Animals were then given intraperitoneal injections of the immunoconjugate (0.5 mg kg-1). Twenty-four hours later the intraperitoneal surfaces were exposed to 656 nm light from an argon-ion pumped-dye laser (50 mW, 656 nm), using a cylindrical diffusing tip fibre. The overall treatment was given either once or multiply. No animals died from treatment complications. Twenty-four hours following one and three PDT treatments, the percentage of viable tumour cells in the ascites of the treated animals analysed ex vivo was 34% and 5% of control for one and three treatments respectively. With respect to survival, all control mice (n = 18) died between 30 and 50 days. However, for those treated three times (n = 10), 40% were still alive after 50 days, and for those treated four times (n = 12) 58% were alive after 50 days. Evaluation with log-rank test revealed a significant survival with intraperitoneal PDT compared with controls (P = 0.0006). These preliminary results suggest that PDT with an OC125 immunoconjugate may be an effective therapy for the management of advanced ovarian cancer. Clinical application of this therapy needs to be further optimised and may require multiple treatments, similar to fractionated radiation therapy and cyclic chemotherapy, in order to control malignant disease with acceptable toxicity to normal tissue.     

Accurate measurement of intrinsic positive end-expiratory pressure: how to detect and correct for expiratory muscle activity

The basic amino acid L-lysine was administered to mice in an attempt to circumvent unwanted renal accumulation of 67Cu-labelled F(ab')2 fragments derived from the anti-NCAM IgG1, SEN7 and anti-CEA IgG1 monoclonal antibody (MAb)35. In control experiments, significant renal uptake of both 67Cu-labelled F(ab')2 fragments was observed, radiolabel being primarily localised to proximal tubules in the renal cortex. Following optimised L-lysine dosing protocols, renal uptake of 67Cu-MAb35 F(ab')2 was inhibited by up to 42%. Surprisingly, little inhibition (< 10%) of 67Cu-SEN7 F(ab')2 uptake was observed. Experiments to investigate this differential inhibition indicated that inhibition of MAb35 F(ab')2 uptake was relatively short-lived (approx. 6 hr), whilst no apparent differences were found in blood clearance rates between either 67Cu-F(ab')2 fragment. L-lysine administration caused a significant diuresis with high levels of intact 67Cu-labelled SEN7 and MAb35 F(ab')2 appearing in the urine, possibly due to blockade of renal uptake and lysine-induced increases in glomerular membrane permeability. Iso-electric focusing studies failed to identify any charge differences between the 67Cu-labelled F(ab')2 fragments, although a cathodal migration of all 67Cu-labelled samples, presumably due to the net positive charge conferred by addition of 67Cu2+ ions, was observed. Our results demonstrate that in addition to net charge, other unidentified characteristics may influence renal accumulation of radiometal-labelled F(ab')2 fragments and their inhibition by L-lysine.     

Bispecific monoclonal antibodies for intravenous treatment of carcinoma patients: immunobiologic aspects

Immunobiologic parameters measured during a phase I trial of intravenously (i.v.) administered bispecific monoclonal antibodies (BsmAb) in renal cell carcinoma (RCC) patients are described. The BsmAb used, BIS-1, is reactive with a pancarcinoma-associated 38 kDa transmembrane glycoprotein, EGP-2, as well with the CD3 complex. Patients received during a 2 h i.v. infusion F(ab')2 fragments of BIS-1 at doses of 1, 3, or 5 micrograms/kg body weight during concomitantly applied subcutaneous (s.c.) IL-2 treatment. Acute but transient BIS-1 F(ab')2-related toxicity was observed at the 3 and 5 micrograms/kg dose level, and the maximum tolerated dose (MTD) was set at 5 micrograms/kg. A dose-dependent binding of BIS-1 F(ab')2 to circulating T lymphocytes was found. The in vivo occupancy of CD3 molecules on T lymphocytes was highest at teh end of the infusion period and then rapidly decreased, as shown by flow cytometry. A much slower decrease of BIS-1 F(ab')2 binding was observed in vitro, suggesting migration of BIS-1 F(ab')2-loaded T lymphocytes from the circulation. A strong but transitory leukopenia was observed, in which LFA-1 alpha bright, CD3/CD8 double positive T cells left the circulation preferentially. This phenomenon was most likely induced by elevated TNF-alpha and IFN-gamma plasma levels, which were at a maximum shortly after the end of the infusion. Isolated peripheral blood mononuclear cells obtained from patients directly after treatment with BIS-1 F(ab')2 at the 3 and 5 micrograms/kg dose level showed increased EGP-2-directed antitumor activity.     

The study of the relationship between the granule size of immunoliposomes and the conjugate ratio of F(ab')2 of McAb HI30 (CD45)

The immunoliposomes were prepared by conjugating the F(ab')2 of monoclonal antibodies HI30(CD45) labelled by 125I with the liposomes labelled by 131I, and then were separated by a sephacryl S-300 column. The chromatography showed that the liposomes prepared by ultrasonic method were not homogeneous, and the liposomes of smaller granule size had a higher conjugate ratio. It was also found that the fragment F(ab')2 of McAb conjugated to liposomes could enlarge their granule size.     

Regulation of peroxisomal proteins and organelle proliferation by multiple carbon sources in the methylotrophic yeast, Candida boidinii

A methylotrophic yeast, Candida boidinii, was grown on various combinations of peroxisome-inducing carbon source(s) (PIC(s)), i.e. methanol, oleate and D-alanine, and the regulation of peroxisomal proteins (both matrix and membrane ones) and organelle proliferation were studied. This regulation was followed (1) at the protein or enzyme level by means of the peroxisomal enzyme activity and Western analysis; (2) at the mRNA level by Northern analysis; and (3) at the organelle level by direct observation of peroxisomes under a fluorescent microscope. Peroxisomal proliferation was followed in vivo by using a C. boidinii strain producing a green fluorescent protein having peroxisomal targeting signal 1. When multiple PICs were used for cell growth, C. boidinii induced specific peroxisomal proteins characteristic of all PIC(s) present in the medium, responding to all PIC(s) simultaneously. Thus, these PICs were considered to induce peroxisomal proliferation independently and not to repress peroxisomes induced by other PICs. Next, the sensitivity of the peroxisomal induction to glucose repression was studied. While the peroxisomal induction by methanol or oleate was completely repressed by glucose, the D-alanine-induced activities of D-amino acid oxidase and catalase, Pmp47, and the organelle proliferation were not. These results indicate that peroxisomal proliferation in yeasts is not necessarily sensitive to glucose repression. Lastly, this regulation was shown to occur at the mRNA level.     

Effects of intracerebroventricular administration of beta-funaltrexamine on DAMGO-stimulated [35S]GTP-gamma-S binding in rat brain sections

In a retrospective study, a total of 120 ovarian cancer patients were paired in terms of staging, grading, patent age, and operative and cytostatic therapy. Half of the patients underwent radioimmunoscintigraphy for diagnostic purposes. From the point of view of possible adjuvant immunological therapy, we investigated to what extent patients with treated ovarian cancer benefit from the application of OC 125 in terms of survival time. In our study we have been able to show that at favorable stages (FIGO II) the patient benefitted more from a radioimmunoscintigraphy than at prognostically unfavorable stages (FIGO IV). Even within FIGO stage III we have been able to show, thanks to the large number of cases, that patients with an NED situation benefitted significantly more form radioimmunoscintigraphy in terms of survival than those with residual tumors. Throughout FIGO stage III, patients with radioimmunoscintigraphy showed significantly superior 5-year survival rates (p < 0.05) than those without radioimmunoscintigraphy. These data would appear to justify prospective studies to establish to what extent ovarian cancer patients benefit from an adjuvant application of OC 125 in terms of the 5-years survival rate and the relapse-free interval.     

Control of stroma-dependent hematopoiesis by basic fibroblast growth factor: stromal phenotypic plasticity and modified myelopoietic functions

The immunological agglutination reactions of physically absorbed F(ab')2 molecules onto anionic and cationic latex particles have been investigated by means of optical absorbance measurements. These measurements have been conducted under different conditions to determine the most influential factors. Surface F(ab')2 and BSA densities, particle concentration in the reaction medium and polyethylene glycol concentration are some of these factors. Sensitized cationic and anionic latexes differ considerably with respect to their colloidal stability and reactivity. As a general rule, the sensitized cationic latex has a relatively higher colloidal stability and hence, it provides reagents with a better optical response. Less than 0.025 microgram/ml of C-reactive protein has been detected using this particle enhanced optical immunoassay.     

15 years CA-125 antigen: usefulness and limits. Apropos of 3 clinical cases

Fifteen years ago, the first clinical assay of the serum tumour marker CA 125 was introduced as a commercial kit. This test is currently the most widely used tumour marker in gynaecologic oncology. Studies have shown that the CA 125 is a valuable tool in the follow-up management of ovarian cancer. As a teaching exercise we present the analysis of three clinical cases, in which CA 125 was determined. Two of these patients did not have an ovarian cancer. It appears that this tumour marker very often is not only used in the follow-up of ovarian cancer, but also as a screening test and as a diagnostic tool. This inappropriate use (due to the lack of specificity and therefore many false positive results) can be avoided by considering it's limits.     

Estimation of unit risk for coke oven emissions

We are investigating the hypothesis that biotin multimers can be used with streptavidin and monoclonal antibody conjugates in cancer pretargeting protocols to provide a method of increasing the amount of radioactivity bound on cancer cells in patients. As part of that investigation, a series of biotinylated Starburst dendrimers (BSBDs) have been prepared and evaluated in vitro and in vivo. In this study, a new biotinidase-stabilized, water-solubilizing biotinylation reagent was prepared and reacted with Starburst (PAMAM) dendrimers, generations 0, 1, 2, 3, and 4. The reaction conditions employed resulted in perbiotinylation of generation 0 (four biotin moieties conjugated), generation 1 (eight biotin moieties conjugated), generation 2 (16 biotin moieties conjugated), and generation 3 (32 biotin moieties conjugated). With generation 4, incomplete biotinylation was achieved resulting in the largest portion of that BSBD having 51 biotin moieties (of 64 possible) conjugated. The ability of each BSBD to cross-link streptavidin (SAv) was examined in an in vitro assay. In that assay, an assessment was made of the quantity of [125I]SAv bound with polystyrene-bound SAv after treatment with the synthesized BSBDs. All BSBDs cross-linked the polystyrene-bound SAv with [125I]SAv; however, the amount of [125I]SAv bound varied with the different BSBDs. Roughly 1 equiv of [125I]SAv was bound when Starburst dendrimers containing three or four biotin moieties (generation 0) were used. Two equivalents were bound with BSBD generation 1, and 4 equiv were bound with BSBDs generations 2, 3, and 4. To assess the distribution of BSBDs generations 0, 1, and 2 in mice (at 4 h postinjection), a method was developed for radioiodinating them using the NHS ester of p-[125I]iodobenzoate ([125I]PIB). It was found that the radioiodinated BSBDs had low blood concentrations (i.e., 0.13-0.20% ID/g) at the 4 h time point. In fact, most tissues examined had low concentrations of biotinylated dendrimers, except kidney and liver. Kidney had the highest concentration of [125I]-labeled BSBDs, and its concentration increased with increasing size and charge of dendrimer (e.g., 8-48% ID/g). On the basis of the increased radioactivity observed in the in vitro assay and the rapid clearance from blood in mice, additional in vivo studies with perbiotinylated Starburst dendrimer, generation 2, are planned.     

The barrier-to-autointegration protein is a host factor for HIV type 1 integration

In vivo, retroviral integration is mediated by a large nucleoprotein complex, termed the preintegration complex (PIC). PICs isolated from infected cells display in vitro integration activity. Here, we analyze the roles of different host cell factors in the structure and function of HIV type 1 (HIV-1) PICs. PICs purified by size exclusion after treatment with high salt lost their integration activity, and adding back an extract from uninfected cells restored this activity. In parallel, the native protein-DNA intasome structure detected at the ends of HIV-1 by Mu-mediated PCR footprinting was abolished by high salt and restored by the crude cell extract. Various purified proteins previously implicated in retroviral PIC function then were analyzed for their effects on the structure and function of salt-treated HIV-1 PICs. Whereas relatively low amounts (5-20 nM) of human barrier-to-autointegration factor (BAF) protein restored integration activity, substantially more (5-10 microM) human host factor HMG I(Y) was required. Similarly high levels (3-8 microM) of bovine RNase A, a DNA-binding protein used as a nonspecific control, also restored activity. Mu-mediated PCR footprinting revealed that of these three purified proteins, only BAF restored the native structure of the HIV-1 protein-DNA intasome. We suggest that BAF is a natural host cofactor for HIV-1 integration.     

Blockade of epidermal growth factor receptor function by bivalent and monovalent fragments of 225 anti-epidermal growth factor receptor monoclonal antibodies

We have previously described anti-epidermal growth factor (EGF) receptor monoclonal antibodies (MAbs) which can block binding of transforming growth factor alpha (TGF-alpha) and EGF to receptors and inhibit activation of receptor tyrosine kinase. Studies with these MAbs involving cell cultures and nude mouse xenografts demonstrated their capacity to inhibit the growth of a variety of tumor cell lines, which express EGF receptors and TGF-alpha and appear to depend upon receptor activation for cell proliferation. To explore the mechanism(s) by which anti-EGF receptor 225 MAb inhibits cell proliferation, we have compared the activity of native 225 MAb with the response to bivalent 225 F(ab')2 and monovalent 225 Fab' fragments. Both native 225 MAb and its fragments could inhibit the binding of 125I-EGF to EGF receptors. Scatchard analysis revealed that the Kd of 225 F(ab')2 is comparable to that of 225 MAb (1 nM), whereas the Kd of 225 Fab' is 5 nM. Both bivalent 225 MAb and 225 F(ab')2 and monovalent 225 Fab' were able to completely inhibit TGF-alpha-induced EGF receptor tyrosine kinase activation, as assayed by autophosphorylation of tyrosine residues of EGF receptors on MCF10A nonmalignant human mammary cells, MDA468 human breast adenocarcinoma cells, and A431 human vulvar squamous carcinoma cells. The bivalent forms of MAb could inhibit proliferation stimulated by endogenous (autocrine) TGF-alpha in cultures of these three cell lines. They also blocked growth stimulation by added exogenous TGF-alpha in cultures of MCF10A cells and the growth-inhibitory effect of exogenous TGF-alpha upon MDA468 and A431 cell cultures. Monovalent 225 Fab' had weaker inhibitory effects upon the proliferation of these cell lines. To determine whether the in vivo antiproliferative activity of anti-EGF receptor MAb can occur without the participation of the Fc portion of MAb, the capacities of 225 F(ab')2 and native 225 MAb to inhibit growth of s.c. A431 cell xenografts were compared. Equimolar amounts of either 225 MAb or 225 F(ab')2 were administered at intervals equivalent to the half-lives of the molecules, to attempt to maintain comparable plasma levels. Both 225 MAb and 225 F(ab')2 inhibited A431 cell xenograft growth in a dose-dependent manner, with a more sustained response in the case of the intact antibody.(ABSTRACT TRUNCATED AT 400 WORDS)     

Reduced expression of retinoblastoma gene product (pRB) and high expression of p53 are associated with poor prognosis in ovarian cancer

Paraffin sections (n = 168, 27 benign, 16 low malignant potential [LMP] and 125 malignant tumours) from epithelial ovarian tumours were evaluated immunohistochemically for expression of retinoblastoma gene product (pRB) and p53 protein, and the relationship among pRB, p53 and cyclin-dependent kinase inhibitor 2 (CDKN2) gene product p16INK4A (p16) was analysed, following our previous study of p16. Forty-one percent of the benign, 50% of the LMP and most (71%) of the malignant tumours showed high pRB expression. High expression of pRB (>50% pRB-positive cells) significantly correlated with non-mucinous histological subtypes. Reduced pRB expression, substage and residual disease were significant predictors for poor prognosis in stage I patients. All the benign and most of the LMP (81%) tumours were in either the p53-negative or low p53-positive category, but nearly half of the malignant tumours had high p53 expression. High p53 accumulation was found in non-mucinous, high grade and late stage tumours. For well-differentiated carcinomas, high p53 expression was a predictor of poor prognosis. However, even though high p53 expression was not associated with histological subtype, stage or the presence of residual disease, high p53 expression was not an independent predictor when all clinical parameters were combined. For all ovarian cancers, a close correlation was found between high p53 and high p16 expression. The relationship between the expression of pRB and p16 depended on tumour stage. In stage I tumours, high pRB was associated with low p16 reactivity. On the other hand, most advanced tumours showed both high pRB and high p16 reactivity.     

Nonmitogenic anti-CD3F(ab')2 fragments inhibit lethal murine graft-versus-host disease induced across the major histocompatibility barrier

We have investigated the in vivo administration of nonmitogenic anti-CD3F(ab')2 fragments for the prevention of lethal graft-vs-host disease (GVHD) in irradiated recipients of fully allogeneic bone marrow cells plus splenocyte (BMS) inocula. Recipients of anti-CD3F(ab')2 fragments administered for 1 mo post-bone marrow transplantation (BMT) had 100% survival without clinical or histopathological evidence of GVHD. Controls given saline injections succumbed by 39 days post-BMT. Similar results were obtained in groups of recipient mice given BMS in which T cells were depleted by in vitro anti-Thy-1.2 plus C' treatment. Further studies were undertaken to define mechanistic differences in the two approaches. Using Ly-5 congenic sources of donor bone marrow and spleen, we determined that anti-CD3F(ab')2 fragments induced TCR modulation and T cell depletion. Mature splenic-derived CD4+ cells were depleted to a greater extent than CD8+ cells. Early post-BMT, recipients receiving injections with control saline had the highest number of CD4+ and CD8+ cells (which may cause GVHD) followed by recipients of anti-CD3F(ab')2 fragments, with the fewest CD8+ cells observed in the anti-Thy-1.2 + C' treated group. CD3+CD4-CD8- cells (which may suppress GVHD generation) were present in higher numbers early post-BMT in recipients given anti-CD3F(ab')2 fragments as compared to recipients given anti-Thy-1.2 + C'-treated BMS. In long term survivors, a mononuclear T cell containing infiltrate without evidence of destruction was observed in sites of GVHD (lung and liver), consistent with a "Quilty" effect, which was not observed in either of the other two groups. Although survivors were tolerant of donor skin grafts and rejected third party grafts, recipients given anti-CD3F(ab')2 fragments but not anti-Thy-1.2 + C'-treated BMS had vigorous anti-host proliferative responses. These results demonstrate that although in vitro anti-Thy-1.2 + C' treatment of BMS (which is highly depletionary) and in vivo administration of anti-CD3F(ab')2 fragments (which is modulatory and less depletionary) are both effective strategies for GVHD, the cellular events involved in achieving GVHD prevention are indeed different.     

Modulation of associated ovarian carcinoma antigens by 5 cytokines used as single agents or in combination

Optimization of intraperitoneal radioimmunotherapy of ovarian cancer depends on increasing the antigenic expression of tumor cells. For this purpose, we studied the effect of 5 cytokines (IFN-alpha, IFN-beta, IFN-gamma, TNF-alpha and TGF-beta), used as single agents or in combination, on 4 ovarian cancer cell lines which present different antigenic profiles with the monoclonal antibodies (MAbs) tested (OC125, OVTL-3, MOv 18 and MOv 19). Analyses were performed by flow cytometry and the Scatchard technique in order to study antigenic modulation. The effect on proliferation was determined by cell counting. Expression of O3 antigen, recognized by the OVTL3 MAb, was increased up to 2.5 times after IFNs and TNF-alpha (used as single agent) on the 2 lines presenting low basal expression (SHIN-3 and IGROVI). The expression of CA125 antigen and the antigens recognized by MOv 18 and MOv 19 MAbs was not increased by any of the cytokines tested. The combination IFN-gamma+TNF-alpha was synergistic on cytotoxicity and enhanced O3 expression, providing 10 times as many sites per cell on the SHIN-3 line. For 3 other associations (IFN-alpha+IFN-gamma, IFN-beta+IFN-gamma and IFN-alpha+TNF-alpha), there was an additive effect on O3 expression and on cell cytotoxicity.