Comprehensive analysis of miRNA expression in T-cell subsets of rheumatoid arthritis patients reveals defined signatures of naive and memory Tregs.

Disturbed expression of microRNAs (miRNAs) in regulatory T cells (Tregs) leads to development of autoimmunity in experimental mouse models. However, the miRNA expression signature characterizing Tregs of autoimmune diseases, such as rheumatoid arthritis (RA) has not been determined yet. In this study, we have used a microarray approach to comprehensively analyze miRNA expression signatures of both naive Tregs (CD4+CD45RO-CD25++) and memory Tregs (CD4+CD45RO+CD25+++), as well as conventional naive (CD4+CD45RO-CD25-) and memory (CD4+CD45RO+CD25-) T cells (Tconvs) derived from peripheral blood of RA patients and matched healthy controls. Differential expression of selected miRNAs was validated by TaqMan-based quantitative reverse transcription-PCR. We found a positive correlation between increased expression of miR-451 in T cells of RA patients and disease activity score (DAS28), erythrocyte sedimentation rate levels and serum levels of interleukin-6. Moreover, we found characteristic, disease- and treatment-independent, global miRNA expression signatures defining naive Tregs, memory Tregs, naive Tconvs and memory Tconvs. The analysis allowed us to define miRNAs characteristic for a general naive phenotype (for example, miR-92a) and a general memory phenotype (for example, miR-21, miR-155). Importantly, the analysis allowed us to define miRNAs that are specifically expressed in both naive and memory Tregs, defining as such miRNA signature characterizing the Treg phenotype (that is, miR-146a, miR-3162, miR-1202, miR-1246 and miR-4281).

Regulation of pri-microRNA BIC transcription and processing in Burkitt lymphoma.

BIC is a primary microRNA (pri-miR-155) that can be processed to mature miR-155. In this study, we show the crucial involvement of protein kinase C (PKC) and nuclear factor-kappaB (NF-kappaB) in the regulation of BIC expression upon B-cell receptor triggering. Surprisingly, Northern blot analysis did not reveal any miR-155 expression upon induction of BIC expression in the Burkitt lymphoma-derived Ramos cell line, whereas other microRNAs were clearly detectable. Ectopic expression of BIC in Ramos and HEK293 cells resulted in miR-155 expression in HEK293, but not in Ramos cells, suggesting a specific block of BIC to miR-155 processing in Ramos. In line with the results obtained with Ramos, lack of miR-155 expression after induction of BIC expression was also observed in other Burkitt lymphoma cell lines, indicating a generic and specific blockade in the processing of BIC in Burkitt lymphoma. In contrast, induction of BIC expression in normal tonsillar B cells resulted in very high levels of miR-155 expression and induction of BIC expression in Hodgkin's lymphoma cell lines. It also resulted in elevated levels of miR-155. Our data provide evidence for two levels of regulation for mature miR-155 expression: one at the transcriptional level involving PKC and NF-kappaB, and one at the processing level. Burkitt lymphoma cells not only express low levels of BIC, but also prevent processing of BIC via an, as yet, unknown mechanism.

Corrigendum to "Hodgkin Lymphoma Cell Lines Are Characterized by a Specific miRNA Expression Profile." Neoplasia 2009, Feb;11(2):167-176.

[This corrects the article DOI: 10.1593/neo.08980.].

The role of microRNAs in normal hematopoiesis and hematopoietic malignancies.

Over the past few years, it has become evident that microRNAs (miRNAs) play an important regulatory role in various biological processes. Much effort has been put into the elucidation of their biogenesis, and this has led to the general concept that a number of key regulators are shared with the processing machinery of small interfering RNAs. Despite the recognition that several miRNAs play crucial roles in normal development and in diseases, little is known about their exact molecular function and the identity of their target genes. In this review, we report on the biological relevance of miRNAs for the differentiation of normal hematopoietic cells and on the contribution of deregulated miRNA expression in their malignant counterparts.

Bispecific antibody-mediated target cell-specific costimulation of resting T cells via CD5 and CD28.

Induction of T-cell activation requires multiple signals provided by cell surface receptor interactions and/or cytokines. T-cell stimulation via the T-cell receptor/CD3 complex provides an important initial activation event which, when combined with the proper costimulatory signals, results in an activated effector T cell. In this report, we have investigated the effectiveness of epithelial glycoprotein-2- (EGP-2) positive tumor target cells to induce specific T-cell stimulation via CD3, CD5, and CD28 using various combinations of bispecific monoclonal antibodies (BsMab) directed against CD3, CD5, or CD28 on the one hand and the pancarcinoma-associated antigen EGP-2 on the other. Induction of T-cell activation was investigated by assessment of CD69 expression, induction of proliferation, and acquirement of cytolytic potential. EGP-2-specific induction of T-cell activation was observed using combinations of BsMab which simultaneous ligated CD3/CD5, CD3/CD28, or CD3/CD5/CD28 with EGP-2. Activation with CD3-, CD5-, or CD28-based BsMab alone did not result in significant induction of T-cell activation in the presence or absence of EGP-2-positive target cells. Simultaneous ligation via CD5/CD28 resulted in partial T-cell activation, including CD69 up-regulation and increased cytolytic activity. Stimulation via CD3 and CD5 or CD28 could be further increased by the addition of exogenously added recombinant Interleukin 2. In contrast, T-cell activation by simultaneous ligation of CD3/CD5/CD28 could not be further augmented by addition of exogenous interleukin 2, indicating that T-cell activation via the combination of CD3, CD5, and CD28 results in complete T-cell activation. Our results show that rapid and target cell-specific induction of T cells is possible using combinations of BsMab directed against different costimulatory molecules. Simultaneous costimulation via CD3/CD5/CD28 results in the most complete activation of T cells.

The epithelial glycoprotein 2 (EGP-2) promoter-driven epithelial-specific expression of EGP-2 in transgenic mice: a new model to study carcinoma-directed immunotherapy.

The human pancarcinoma-associated epithelial glycoprotein-2 (EGP-2), a M(r) 38,000 transmembrane antigen also known as 17-1A or Ep-CAM, is commonly used for targeted immunotherapy of carcinomas because it is strongly expressed by most carcinomas. EGP-2 is, however, also expressed in most normal epithelia. To evaluate anti-EGP-2-directed treatment-associated effects on tumors and on EGP-2-positive normal tissue, we generated EGP-2-expressing transgenic mice. A 55-kb DNA fragment consisting of the 14-kb genomic coding sequence of the human EGP-2 gene with approximately 10-kb-upstream and approximately 31-kb-downstream sequences was isolated and used to direct EGP-2 expression in an epithelium-specific manner. In the EGP-2 transgenic mice, EGP-2 appeared to be specifically expressed in all of those epithelial tissues that also express EGP-2 in humans, whereas all of the other tissues were negative. The specific in vivo localization of the i.v. administered anti-EGP-2 monoclonal antibody MOC31 was studied in EGP-2-positive and -negative tumors induced s.c. in this EGP-2 transgenic mouse model. Immunohistochemical analysis showed specific localization of MOC31 in the EGP-2-positive tumors but not in the EGP-2-negative tumors. No anti-EGP-2 monoclonal antibody localization was observed in any of the EGP-2-positive normal mouse tissues, which indicated a limited in vivo accessibility. In conclusion, an EGP-2 transgenic mouse model has been generated that expresses the EGP-2 antigen as in humans and, therefore, can serve as a model to evaluate the efficacy and safety of a variety of anti-EGP-2-based immunotherapeutic modalities in both tumors and normal tissue.

Purging of epithelial tumor cells from peripheral blood stem cells by means of the bispecific antibody BIS-1.

Peripheral blood stem cell (PBSC) support in breast cancer patients allows high-dose chemotherapy, but tumor cell contamination of the PBSCs is a potential source of relapse. Specific carcinoma cell killing can be obtained by retargeting activated T cells with bispecific antibody BIS-1, directed against epithelial glycoprotein-2 and CD3. To purge epithelial tumor cells from the PBSCs of breast cancer patients, activation of T cells in PBSCs and T-cell retargeting by BIS-1 was studied. PBSCs, obtained by leukapheresis after chemotherapy and recombinant human granulocyte colony-stimulating factor, were cultured in the presence of PBS, interleukin-2, OKT3, or interleukin-2/OKT3 for induction of T-cell activation. Subsequently, lysis of epithelial tumor cell lines by activated T cells of PBSCs in the presence or absence of BIS-1 was assessed with the 51Cr-release assay or immunocytochemical staining. The effect on PBSC hematopoietic colony formation (HCF) was evaluated by the granulocyte macrophage colony-stimulating units assay. Prior to activation, PBSCs from breast cancer patients contained higher levels of CD8+ T cells than peripheral blood from healthy volunteers (P < 0.05). The potential of PBSCs to sustain tumor cell lysis was increased after all prior activations and was further enhanced by BIS-1. Maximal BIS-1 effect was observed after OKT3 activation of PBSCs for 72 h (P < 0.0005), inducing a >3 log depletion of tumor cells. HCF was not affected by prior OKT3 activation and/or BIS-1. In conclusion, specific tumor cell lysis by PBSCs can be obtained in vitro by OKT3 activation and BIS-1 retargeting of T cells, without affecting HCF. At present, studies are evaluating this format for future clinical application.

Cancer immunotherapy: insights from transgenic animal models.

A wide range of strategies in cancer immunotherapy has been developed in the last decade, some of which are currently being used in clinical settings. The development of these immunotherapeutical strategies has been facilitated by the generation of relevant transgenic animal models. Since the different strategies in experimental immunotherapy of cancer each aim to activate different immune system components, a variety of transgenic animals have been generated either expressing tumor associated, HLA, oncogenic or immune effector cell molecule proteins. This review aims to discuss the existing transgenic mouse models generated to study and develop cancer immunotherapy strategies and the variable results obtained. The potential of the various transgenic animal models regarding the development of anti-cancer immunotherapeutical strategies is evaluated.

Increased production of antigen-specific B lymphocytes during in vitro immunization using carrier-specific T helper hybridomas.

An in vitro method to increase the production of hapten-specific antibody-forming B cells (AFC) using a carrier-specific T helper hybridoma and murine splenocytes is described. Naive splenocytes (6 x 10(6)/ml) are cultured in vitro in the presence of a hapten-carrier conjugate (DNP.OVA) and OVA-specific T helper hybridomas (0.5 x 10(6)/ml). After 4-5 days in vitro immunization (IVI), the maximum number of DNP-specific AFC were found using a spot-ELISA with twice the number of IgM positive cells as IgG positive AFC. The presence of antigen in the form of a hapten-carrier complex and the use of a carrier-specific Th hybridoma resulted in more hapten-specific AFC than when neither antigen nor Th hybridoma were present or when antigen alone or T help alone were used. Also when the hapten was conjugated to a carrier not recognised by the carrier-specific Th hybridoma there were considerably fewer (less than 50%) hapten specific AFC formed. When in vivo primed splenocytes (DNP) were boosted in vitro (IVB) under the same conditions as for IVI most hapten-specific AFC were found on day 4 and both anti-DNP IgM and IgG AFC were increased relative to IVI. Again most AFC were found when hapten was bound to the relevant carrier. In conclusion, carrier-specific T hybridomas can be used in an in vitro immunization procedure with naive or primed splenocytes to increase the frequency of anti-hapten AFC. This method offers an improvement over the current in vitro immunization procedures for the production of monoclonal antibodies.

Direct visualisation and quantification of cellular cytotoxicity using two colour flourescence.

A fluorescence method is described for the evaluation of cell death induced by cellular cytolytic activity. A green fluorescent membrane dye, D275, was used to label various target cell lines and propidium iodide (PI) uptake was used to assay cell death. Natural killer (NK), lymphokine activated killer (LAK) as well as targeted T cell mediated cellular cytotoxicity were quantified using the fluorescence method and compared to results obtained with the 51chromium (51Cr) release assay. A good correlation was found after an assay period of 4-8 h indicating that the fluorescence method is a reliable alternative to the 51Cr release assay.

A rapid and versatile method for harnessing scFv antibody fragments with various biological effector functions.

A versatile expression vector is described for the rapid construction and evaluation of bispecific scFvs and scFv-based fusion proteins. An important feature of this vector is the presence of two multiple cloning sites (MCS) separated by an in frame linker sequence. The first MCS was specifically designed to contain unique SfiI and NotI restriction enzyme sites that can be used for directional and in frame insertion of scFvs (or potentially any molecule) selected from established phage-display systems. Using this new vector, a functional bs-(scFv)(2) (2C11-MOC31) was constructed for retargeted T-cell cytotoxicity towards EGP2 positive tumor cells. The vector was also used for grafting of a number of promising biological effector principles onto scFv MOC31, including the prodrug converting enzyme cytosine deaminase, the anti-angiogenic factor angiostatin, and the thrombogenic molecule tissue factor. We aimed at producing biologically active fusion proteins by directing them through the endoplasmic reticulum-based protein folding machinery of eukaryotic cells (COS-7) using a kappa light chain leader, thereby taking advantage of the associated quality control mechanisms that allow only fully folded and processed fusion proteins to be secreted into the medium. Supernatants derived from fusion protein transfected COS-7 cells, which were transiently transfected at low transfection rates, were directly assayed for the biological and/or targeting activity of the excreted fusion proteins without any prior purification steps. This procedure might help to identify those fusion proteins that have favourable characteristics like stability and biological activity in the presence of serum and at low protein concentrations. Targeted delivery of all effector principles was subsequently assessed in an in vitro model system. The method we devised is both rapid and versatile and can be useful to construct and identify series of new chimeric proteins with enhanced therapeutic potential in human cancer therapy.

The use of bispecific antibodies in tumor cell and tumor vasculature directed immunotherapy.

To overcome dose limiting toxicities and to increase efficacy of immunotherapy of cancer, a number of strategies are under development for selectively redirecting effector cells/molecules towards tumor cells. Many of these strategies exploit the specificity of tumor associated antigen recognition by monoclonal antibodies. Using either hybridoma fusion, chemical derivatization or molecular biology technology, antibodies with dual specificity can be constructed. These so called biospecific antibodies (BsAbs) have been used to redirect the cytolytic activity of a variety of immune effector cells such as cytotoxic T lymphocytes, natural killer cells, neutrophils and monocytes/macrophages to tumor cells. Local administration of BsAbs, either alone or in combination with autologous effector cells, is highly effective in eradicating tumor cells. In contrast, systemic application of BsAb at present is only suitable for adjuvant treatment of minimal residual disease due to poor tumor cell accessibility. As an alternative, angiogenesis related determinants on tumor blood vessels can be exploited for the selective delivery of effector cells/molecules apart from being used to inhibit angiogenesis. Important advantages of this strategy is that the endothelial cell associated target epitope(s) are easy accessible. The dependence of tumor growth on the tumor's blood supply also renders tumor endothelial cells an attractive target for therapy. Although still in its infancy, attacking the tumor's blood supply for example by delivering coagulation factors or toxins, or by BsAb directed immunotherapies holds great promise for antineoplastic therapy.

CD3 directed bispecific antibodies induce increased lymphocyte-endothelial cell interactions in vitro.

Bispecific antibody (BsMAb) BIS-1 has been developed to redirect the cytolytic activity of cytotoxic T lymphocytes (CTL) to epithelial glycoprotein-2 (EGP-2) expressing tumour cells. Intravenous administration of BIS-1 F(ab')2 to carcinoma patients in a phase I/II clinical trial, caused immunomodulation as demonstrated by a rapid lymphopenia prior to a rise in plasma tumour necrosis factor-alpha and interferon-gamma levels. Yet, no lymphocyte accumulation in the tumour tissue and no anti-tumour effect could be observed. These data suggest a BsMAb-induced lymphocyte adhesion to blood vessel walls and/or generalized redistribution of the lymphocytes into tissues. In this study, we describe the effects of BIS-1 F(ab')2 binding to peripheral blood mononuclear cells (PBMC) on their capacity to interact with resting endothelial cells in vitro. Resting and pre-activated PBMC exhibited a significant increase in adhesive interaction with endothelial cells when preincubated with BIS-1 F(ab')2, followed by an increase in transendothelial migration (tem). Binding of BIS-1 F(ab')2 to PBMC affected the expression of a number of adhesion molecules involved in lymphocyte adhesion/migration. Furthermore, PBMC preincubated with BIS-1 F(ab')2 induced the expression of endothelial cell adhesion molecules E-selectin, VCAM-1 and ICAM-1 during adhesion/tem. These phenomena were related to the CD3 recognizing antibody fragment of the BsMAb and dependent on lymphocyte-endothelial cell contact. Possibly, in patients, the BIS-1 F(ab')2 infusion induced lymphopenia is a result of generalized activation of endothelial cells, leading to the formation of a temporary sink for lymphocytes. This process may distract the lymphocytes from homing to the tumour cells, and hence prevent the occurrence of BIS-1 F(ab')2 - CTL-mediated tumour cell lysis.

Construction and characterization of a bispecific diabody for retargeting T cells to human carcinomas.

We describe the construction of a recombinant bispecific antibody fragment in the diabody format with specificity for both the well-established human pancarcinoma associated target antigen EGP2 (epithelial glycoprotein 2, also known as the CO17-1A antigen or KSA) and the CD3epsilon chain of human TCR/CD3 complex. The murine anti-EGP2 (MOC31) single chain variable fragment (scFv) and the humanized anti-CD3 (Ucht1v9) scFv were cast into a diabody format (designated Dia5v9) using a short 5 amino acid Gly-Ser linker between immunoglobulin heavy-chain and light-chain variable domains. Purification of the poly-histidine tagged Dia5v9 was achieved from extracts of protease deficient Escherichia coli by IMAC chromatography. The Dia5v9 diabody showed strong binding to both EGP2 and CD3 in transfected cells. The in vitro efficacy of Dia5v9 in mediating tumor cell lysis by interleukin-2 activated human T cells appeared to be similar to that of the hybrid-hybridoma-derived BsF(ab')2 Bis1 (anti-EGP2/anti-CD3) in a standard 4-hr 51Cr-release assay. This small and partially humanized recombinant bispecific antibody fragment may be valuable for T-cell-based immunotherapeutical treatment protocols, retargeting activated peripheral blood T lymphocytes to lyse various human carcinomas in vivo.

Analysis of in vitro lymphocyte adhesion and transendothelial migration by fluorescent-beads-based flow cytometric cell counting.

In this report, we describe a new and simple method for flow cytometric quantitation of lymphocyte numbers in lymphocyte-endothelial adhesion/ transendothelial migration assays. The method exploits fluorescent flow cytometer alignment beads as a counting reference. Known amounts of beads are added to samples with unknown lymphocyte numbers. Lymphocytes and a preset number of fluorescent beads are simultaneously analyzed. The total number of cells present in the sample can be subsequently calculated from the fixed ratio of added to analyzed fluorescent beads. Using this fluorescent-beads-based flow cytometric cell counting of lymphocyte numbers in adhesion/migration assays, labeling of cells and other time-consuming calibration procedures are not required and analysis time is short. Furthermore, we demonstrate that this cell counting method can be combined with concurrent single- or double-label fluorescence flow cytometric phenotyping of adherent and migrated lymphocytes. The method was applied to the in vitro study of the effects of lymphocyte activation status and binding of bispecific antibody (directed against CD3 x tumor cell-associated antigen) on lymphocyte adhesion and transendothelial migration.

The role of apoptosis in bispecific antibody-mediated T-cell cytotoxicity.

In this report we describe the role of apoptosis in the process of tumour cell killing by bispecific monoclonal antibody (BsMAb)-redirected cytolytic T cells. The BsMAb used, BIS-1, has dual specificity for the CD3 complex on T cells and the pancarcinoma-associated 38 kDa transmembrane antigen EGP-2. BIS-1 allows activated T cells to specifically recognise and kill EGP-2-positive but not EGP-2-negative target cells. An assay was developed to quantify apoptosis in cells by separation of 3H-thymidine-labelled low-molecular, i.e. fragmented, from high-molecular, i.e. non-fragmented DNA. The presence of low molecular weight DNA was measured both within the target cells and in the cell-free supernatant. After exposure to BIS-1-redirected, -activated T cells, apoptosis was observed in EGP-2-positive target cells but not in EGP-2-negative target cells. Also no DNA fragmentation proved to be induced in the activated effector cells during assay. The degree of EGP-2-positive target DNA fragmentation depended on the concentration of BsMAb, the E/T ratio and the incubation time. Using a low E/T ratio (1/1), DNA fragmentation in and 51Cr release from target cells showed similar characteristics and kinetics. At higher E/T ratio (20/1), the 51Cr release from the target cells increased to a greater extent than the percentage fragmented target cell DNA. Inhibitors of DNA fragmentation added to the cytotoxicity assay inhibited not only DNA fragmentation, but also the release of chromium-51 from the target cells, suggesting that apoptosis and cell lysis are closely related in BsMAb-mediated cell killing.

Induction of apoptosis through B-cell receptor cross-linking occurs via de novo generated C16-ceramide and involves mitochondria.

B-cells, triggered via their surface B-cell receptor (BcR), start an apoptotic program known as activation-induced cell death (AICD), and it is widely believed that this phenomenon plays a role in the restriction and focusing of the immune response. Although both ceramide and caspases have been proposed to be involved in AICD, the contribution of either and the exact molecular events through which AICD commences are still unknown. Here we show that in Ramos B-cells, BcR-triggered cell death is associated with an early rise of C16 ceramide that derives from activation of the de novo pathway, as demonstrated using a specific inhibitor of ceramide synthase, fumonisin B1 (FB1), and using pulse labeling with the metabolic sphingolipid precursor, palmitate. There was no evidence for activation of sphingomyelinases or hydrolysis of sphingomyelin. Importantly, FB1 inhibited several specific apoptotic hallmarks such as poly(A)DP-ribose polymerase cleavage and DNA fragmentation. Electron microscopy revealed morphological evidence of mitochondrial damage, suggesting the involvement of mitochondria in BcR-triggered apoptosis, and this was inhibited by FB1. Moreover, a loss of mitochondrial membrane potential was observed in Ramos cells after BcR cross-linking, which was inhibited by the addition of FB1. Interestingly, benzyloxycarbonyl-Val-Ala-dl-Asp, a broad spectrum caspase inhibitor did not inhibit BcR-induced mitochondrial membrane permeability transition but did block DNA fragmentation. These results suggest a crucial role for de novo generated C16 ceramide in the execution of AICD, and they further suggest an ordered and more specific sequence of biochemical events in which de novo generated C16 ceramide is involved in mitochondrial damage resulting in a downstream activation of caspases and apoptosis.

FAS activation induces dephosphorylation of SR proteins; dependence on the de novo generation of ceramide and activation of protein phosphatase 1.

The search for potential targets for ceramide action led to the identification of ceramide-activated protein phosphatases (CAPP). To date, two serine/threonine protein phosphatases, protein phosphatase 2A (PP2A) and protein phosphatase 1 (PP1), have been demonstrated to function as ceramide-activated protein phosphatases. In this study, we show that treatment with either anti-FAS IgM (CH-11) (150 ng/ml) or exogenous d-(e)-C(6-)ceramide (20 microm) induces the dephosphorylation of the PP1 substrates, serine/arginine-rich (SR) proteins, in Jurkat acute leukemia T-cells. The serine/threonine protein phosphatase inhibitor, calyculin A, but not the PP2A-specific inhibitor, okadaic acid, inhibited both FAS- and ceramide-induced dephosphorylation of SR proteins. Anti-FAS IgM treatment of Jurkat cells led to a significant increase in levels of endogenous ceramide beginning at 2 h with a maximal increase of 10-fold after 7 h. A 2-h pretreatment of Jurkat cells with fumonisin B(1) (100 microm), a specific inhibitor of CoA-dependent ceramide synthase, blocked 80% of the ceramide generated and completely inhibited the dephosphorylation of SR proteins in response to anti-FAS IgM. Moreover, pretreatment of Jurkat cells with myriocin, a specific inhibitor of serine-palmitoyl transferase (the first step in de novo synthesis of ceramide), also blocked FAS-induced SR protein dephosphorylation, thus demonstrating a role for de novo ceramide. These results were further supported using A549 lung adenocarcinoma cells treated with d-(e)-C(6-)ceramide. Dephosphorylation of SR proteins was inhibited by fumonisin B(1) and by overexpression of glucosylceramide synthase; again implicating endogenous ceramide generated de novo in regulating the dephosphorylation of SR proteins in response to FAS activation. These results establish a specific intracellular pathway involving both de novo ceramide generation and activation of PP1 to mediate the effects of FAS activation on SR proteins.

Reduction of EGP-2-positive pulmonary metastases by bispecific-antibody-redirected T cells in an immunocompetent rat model.

Effectiveness of bispecific-monoclonal-antibody (BsMAb)-mediated cellular anti-tumour activity was evaluated in vitro and in vivo in relation to the additional need for T-cell activation in a new immunocompetent rat tumour model. L37 tumour cells, derived from a squamous-cell carcinoma of the lung of Wag/Rij rats, were transfected with the cDNA coding for the human 38-kDa transmembrane pan-carcinoma-associated antigen EGP-2. Intravenous inoculation of EGP-2-positive L37 cells resulted in a rapid outgrowth of EGP-2-positive tumour nodules in the lungs. A BsMAb BIS-19, recognizing EGP-2 on the transfected tumour cells and the T-cell receptor of the rat, was made and allowed specific lysis of EGP-2-transfected L37 tumour cells by activated rat T lymphocytes in vitro. In vivo T-cell activation, assessed by up-regulation of IL-2-receptor expression, could be induced by daily injection of rat rIL-2. Intravenous treatment of tumour-bearing EGP-2-positive L37 tumour with BIS-19 together with rat rIL-2 resulted in almost complete disappearance of established tumour. In contrast, animals treated with BIS-19 alone, IL-2 alone or a combination of anti-EGP-2, anti-TcR and IL-2 showed much less or no tumour reduction. These results show effectiveness of systemic treatment with BsMAbs to induce anti-tumour activity in established tumours. Immune activation prior to or during treatment with BsMAbs, as achieved with IL-2, appears to be a prerequisite for successful treatment.

Immunomodulatory effects of intravenous BIS-1 F(ab')2 administration in renal cell cancer patients.

We report the immunomodulatory effects of an intravenous treatment with F(ab')2 fragments of the bispecific monoclonal antibody BIS-1 during subcutaneous recombinant interleukin 2 (rIL-2) therapy of renal cell cancer (RCC) patients. BIS-1 is directed against both the CD3 antigen on T cells and the EGP-2 molecule on carcinoma cells and some normal epithelia. The amount of BIS-1 F(ab')2 bound to peripheral blood lymphocytes (PBLs) increased dose-dependently. This occupation degree was highest at the end of the 2 h infusion and rapidly decreased subsequently. During the first hour of BIS-1 F(ab')2 infusion the number of PBLs decreased slowly. This was followed by an increase in serum tumour necrosis factor alpha (TNF-alpha) concentrations and a rapid decrease in the numbers of peripheral blood lymphocytes, monocytes and eosinophils. In our view, the most likely explanation for the observed decrease in occupation degree of BIS-1 F(ab')2 and the rise in TNF-alpha levels is based on the assumption that BIS-1-carrying T cells leave the circulation. The CD3 antigens on these extravasated T cells become cross-linked by EGP-2 antigens, inducing TNF-alpha secretion. This results in an enhanced decrease in the numbers of PBLs, monocytes and eosinophils. These preliminary results suggest that BIS-1 F(ab')2 treatment during IL-2 therapy may induce local T-cell activation.