[Prevention of fetomaternal rhesus-D allo-immunization. Perspectives]. / Perspectives.

At present, rhesus prophylaxis concerns RhD negative pregnant women, even though 30 to 40% of them are bearing a RhD negative child. Knowing the RhD fetal genotype could change this quite irrational practice of prophylaxis (exposing many more women than needed to blood derived products) without reducing its efficacy. RhD fetal genotype determined on amniotic fluid has an excellent sensitivity. Presence of silent D genes slightly impairs its specificity which remains acceptable. However women have to be informed of possible false positives. Fetal RhD genotyping on maternal blood is more complex. Sensitivity is good from 10 GW and excellent after 15 GW. In case of a first negative result, it is recommended to control fetal RhD on a second sample drawn a few weeks later. Another new perspective for rhesus prophylaxis is the attempt to substitute polyclonal IgG anti-D into human monoclonal IgG anti-D. The main difficulty is to elaborate monoclonal antibodies with a capacity to neutralize RhD positive red blood cells equivalent to those of polyclonal anti-D. A new generation of antibodies is in process and preliminary clinical results are suggesting a possible use of these monoclonal antibodies for future rhesus prophylaxis but long-term follow-up is required to draw further conclusions.

The rescue by phage display of human Fabs to gp120 HIV-1 glycoprotein using EBV transformed lymphocytes.

Human hybridomas secreting monoclonal antibodies in a stable manner are difficult to develop. The main difficulties are the restricted techniques for B-cell immortalization, the low number of sensitized B cells in peripheral blood, and the impossibility, for ethical reasons, to immunize humans with most antigens. Phage display has proved to be a powerful method for the generation of recombinant antibody fragments. This technology relies on the construction of recombinant Fab or scFv libraries and their display on phage M13. In order to rescue unstable B-cell clones secreting human antibodies we set up a method for the selection by phage display of human IgG fragments from Epstein-Barr virus (EBV)-transformed clones and applied it to the selection by phage display of Fabs directed against HIV-1 gp120, using a seropositive blood sample. The approach combines B-cell transformation by EBV of peripheral blood lymphocytes from a seropositive donor, preselection of specific IgG anti-gp120 producing clones, and the construction of a targeted human antibody library. In this library the percentage of heavy and light chain coding sequences expressed in Escherichia coli, amplified by a set of specific 5' primers for different antibody germ lines, was similar to that observed with the original untransformed B-cell sample. One round of panning was sufficient for the rescue of three Fabs specific for HIV-1 gp120 protein, which proves the efficiency of this technique.

Unusual N-glycosylation of a recombinant human erythropoietin expressed in a human lymphoblastoid cell line does not alter its biological properties.

Erythropoietin (Epo) is a 166 amino acids protein containing three N-glycosylation sites (Asn-24, Asn-38, and Asn-83) and 1 O- glycosylation site (Ser-126) and involved in the regulation of the level of red blood cells. Today, only one recombinant human Epo (rHuEpo), produced in CHO cell line, is extensively used in therapy to cure severe anemia. The structure of the glycan chains of this rHuEpo slightly differ of those of the urinary human Epo (uHuEpo), considered as the natural Epo molecule. In an attempt to produce a rHuEpo as close as possible to the uHuEpo, Epo gene was expressed in a human lymphoblastoid cell line, named RPMI 1788. In order to fully characterize the Epo-RPMI, structural characterizations of the protein skeleton as well as glycan chains were undergone. As expected, the amino acid sequence of the Epo-RPMI conformed to that of uHuEpo. Surprisingly, the structure of some N-glycan chains, as mainly determined by ESI-MS, revealed some unusual characteristics. Thus, 80% of N-glycans possess a bisecting GlcNAc residue, 25% bear a second fucose residue which is present, in a large part, in a sialyl Le(x)motif, and 13% contain more than three LacNAc repeats (up to five per molecule). Despite these unusual structural characteristics, the data concerning the in vitro and in vivo biological activities were not impaired when compared to Epo-CHO and uHuEpo.

In vivo biotinylated recombinant antibodies: high efficiency of labelling and application to the cloning of active anti-human IgG1 Fab fragments.

In vivo biotinylation of antibody fragments with a gene fusion approach is a realistic alternative to conventional in vitro chemical labelling. We have previously reported the construction of a vector system suitable for the bacterial expression of the binding fragment of antibody (Fab) genetically linked to the C-terminal domain of Escherichia Coli biotin carboxy carrier protein (BCCP*). A minor fraction of the expressed hybrids was biotinylated in vivo and therefore able to interact with streptavidin. We now show that the large majority of bacterially-expressed Fab-BCCP* fusions are labelled with biotin when plasmid-encoded biotin holoenzyme synthetase (BirA) is co-expressed. The yield of biotinylated Fab is maximal when overexpression of BirA is driven by a second compatible plasmid. We took advantage of this property to develop a novel filter assay for the rapid identification of recombinant Fab reacting with immunoglobulin. Starting with total RNA of two newly established murine hybridoma cell lines producing anti-human IgG1 antibodies, we selected in a single experiment the bacterial clones that expressed in vivo biotinylated anti-IgG1 Fab. Sequence analysis of the isolated Fabs showed that they did not derive from a single B clone. In addition, we found that these recombinant Fabs labelled with biotin in vivo are useful for the specific detection of human IgG1 by a solid-phase immunoassay.

Human recombinant anti-Rh(D) monoclonal antibodies: improvement of biological properties by in vitro class-switch.

Therapeutic use of human monoclonal antibodies has so far been hampered by their poor availability. Recent developments in recombinant DNA technologies are expected to fill this gap; the variable and constant sequences of antibodies can be selected independently and then subsequently joined to express whole antibodies. We assessed here the potential of this methodology to obtain novel anti-D antibodies with improved biological characteristics. The sequences coding for heavy and light chains of two anti-Rh(D) monoclonal antibodies (one IgG1 and one IgG3) were isolated and co-expressed into murine myeloma cell P3X63.Ag8.653, either directly (parental antibodies) or after exchange of constant heavy chain sequences (class-switched antibodies). Parental antibodies produced either by transfectomas or by hybridomas behaved similarly in analysis of biochemical, binding and effector properties. Class-switched antibodies displayed altered functional properties over parental antibodies. Of particular interest, one of them showed improved phagocytosis potencies over both parental antibodies. Additionally, these results indicate that functional properties do not always simply reflect the addition of properties of constant and variable parts but that interactions between constant and variable regions may interfere.

Use of the DAF assay to assess the functional properties of polyclonal and monoclonal RhD antibodies.

The mechanism whereby passive Rh (D) immunoglobulins suppress the fetomaternal alloimmunization is still unclear. New in vitro tests are needed to better characterize the functional properties of polyclonal anti-Ds. The DAF assay was developed to monitor the antibody-dependent cell-mediated cytotoxicity (ADCC) and the phagocytosis of anti-Rh (D)-sensitized RBCs by effector cells. The principle of this test is based on the oxydization of the 2,7-diaminofluorene (DAF) by the pseudoperoxidase activity of free hemoglobin. The reaction is proportional to the hemoglobin concentration. This test was performed to determine and emphasize the efficacy of different polyclonal anti-D immunoglobulin preparations to mediate lysis and phagocytosis of sensitized RBCs by human peripheral mononuclear cells. The functional properties of different human RhD monoclonal antibodies were also analyzed and compared. The test was found to be convenient to perform and allowed the avoidance of radioactive labelling of RBCs for ADCC studies. It is mainly useful for the direct quantitation of phagocytosis.

Four epitopes on tumor necrosis factor-alpha defined by murine anti-tumor necrosis factor-alpha monoclonal antibodies.

Eight murine anti-TNF alpha monoclonal antibodies (mAb) were produced after immunization of BALB/c mice with rhTNF alpha. Six of these mAbs were able to neutralize cytotoxic activity of TNF alpha against L929 cells. Two other mAbs had no neutralizing effect. Epitope mapping studies were performed by ELISA and by using a BIAcore system (Pharmacia). The described mAbs were allowed to define 4 different epitopes on TNF alpha. Three of them were involved in the binding of TNF alpha with its receptor (cytotoxic neutralization of TNF alpha). Another epitope was defined by non-neutralizing mAbs.

Bacterially expressed Fabs of monoclonal antibodies neutralizing tumour necrosis factor alpha in vitro retain full binding and biological activity.

Antibody fragments specific for the human tumour necrosis factor alpha (TNF alpha) have been cloned from lambda combinatorial expression libraries using total RNA obtained from three different hybridoma cell lines of therapeutic interest. The previously described bacteriophage lambda vectors, lambda HC2 and lambda LC1, were modified to create unique antibody cloning sites in the combinatorial construct and a novel tag peptide was inserted at the C-terminal end of the expressed Fd chain. Sequence analysis of the cloned Fabs indicated that two of them were derived from a single B cell. Expression in E. coli showed that the amount of recovered Fab in the bacterial culture medium was related to the sequences of the variable coding regions. Hybrid Fabs created by chain exchange of similar antibodies were as active as the originally paired Fabs in binding assays. The relative affinities and the capacities of the bacterially expressed Fabs to neutralize TNF alpha cytotoxicity in vitro were identical to those of the parental antibodies. The results demonstrate that, using an in vitro approach, it is possible to generate from existing hybridoma cell lines high affinity Fabs which retain antigen specificity. The cloning sites incorporated into the C-terminal parts of these Fabs will now permit their further modification to include additional functional characteristics not possible with the original hybridoma antibodies.

A plasma membrane protein is involved in cell contact-mediated regulation of tissue-specific genes in adult hepatocytes.

We have identified the liver-regulating protein (LRP), a cell surface protein involved in the maintenance of hepatocyte differentiation when cocultured with rat liver epithelial cells (RLEC). LRP was defined by immunoreactivity to a monoclonal antibody (mAb L8) prepared from RLEC. mAb L8 specifically detected two polypeptides of 85 and 73 kD in immunoprecipitation of both hepatocyte- and RLEC-iodinated plasma membranes. The involvement of these polypeptides, which are integral membrane proteins, in cell interaction-mediated regulation of hepatocytes was assessed by evaluating the perturbing effects of the antibody on cocultures with RLEC. Several parameters characteristic of differentiated hepatocytes were studied, such as liver-specific and house-keeping gene expression, cytoskeletal organization and deposition of extracellular matrix (ECM). An early cytoskeletal disturbance was evidenced and a marked alteration of hepatocyte functional capacity was observed in the presence of the antibody, together with a loss of ECM deposition. By contrast, cell-cell aggregation or cell adhesion to various extracellular matrix components were not affected. These findings suggest that LRP is distinct from an extracellular matrix receptor. The fact that early addition of mAb L8 during cell contact establishment was necessary to be effective may indicate that LRP is a novel plasma membrane protein that plays an early pivotal role in the coordinated metabolic changes which lead to the differentiated phenotype of mature hepatocytes.

A human anti-Jkb monoclonal antibody.

Peripheral blood mononuclear cells of an immunized patient were transformed with Epstein-Barr virus and then fused with P3X63Ag8 mouse myeloma cells by polyethylene glycol. After the cloning, a hybridoma cell line secreting specific anti-Jkb monoclonal antibody was isolated. The antibody was produced in supernatant form and tested for its use as a blood grouping reagent.

The conformation of the CD3 complex on T lymphocytes is modified by calcium ions.

While calcium ions are known to play a prominent role in signal transduction in the activation of T lymphocytes, its mechanism of action, target and function have not been elucidated. One crucial event in the calcium-dependent process is the activation of the CD3 complex, and this, too, is not understood. While studying the release of Ca2+ from intracellular stores by several monoclonal antibodies (mAb) against CD3, we found that one of them, mAb 141, was ineffective unless free Ca2+ was present in the external medium. By flow cytometric analysis of the binding of this mAb to Jurkat T cells and peripheral blood lymphocytes we showed that 141 does not recognize the CD3 complex when external Ca2+ is chelated by EGTA. The binding was restored by addition of Ca2+ but not Mg2+. Finally at least one subunit of the CD3 complex displayed a modified electrophoretic migration rate when immunoprecipitated by Leu-4 in the absence of external free Ca2+. These results suggest that the conformation of the CD3 complex depends on Ca2+, the epitope recognized by 141 being concealed at low Ca2+ concentration.

Distribution of radiolabelled monoclonal antibody Po66 after intravenous injection into nude mice bearing human lung cancer grafts.

Monoclonal antibody Po66, produced by immunization against a patient's lung squamous cell carcinoma was found suitable for the scintigraphic detection of human tumours. Surprisingly, the cellular antigen recognized by Po66 was abundant in the cytoplasm of tumour cells but could not be detected on the surface membrane. In the present work the biodistribution of radiolabelled Po66 and of an unrelated immunoglobulin were studied comparatively after intravenous injection into nude mice bearing lung squamous cell carcinoma grafts. Radioactivity distribution among mouse organs and tumour was analysed by gamma counting and autohistoradiography. After injection, radiolabelled Po66 decreased rapidly from the blood in tumour-bearing animals whereas, in controls, it remained at a level comparable to that of the unrelated immunoglobulin. The antibody seemed slowly trapped by the tumour and, 12 days after its injection, distribution ratios between tumour and mouse organs reached values of 20-30 as against 1 in animals injected with the non-specific immunoglobulin. Autohistoradiographic investigations in the tumour confirmed the slow diffusion rate of the antibody, which remained in the vascular spaces up to the 24th hour after injection and diffused afterwards throughout the clusters of tumor cells. Furthermore, radioactivity was detected in cells which, unexpectedly, seemed morphologically unaltered. These cells, the viability of which remains to be determined, were predominant in the central area of the tumours. The results presented constitute new evidence of the ability of an in vivo injected monoclonal antibody to reach a cytoplasmic target inside non-necrotic cells and suggest that the cells permeable to the antibody might be in defective nutritional conditions.

Characterization of the antigen identified by Po66. A monoclonal antibody raised against a lung squamous cell carcinoma.

The mouse monoclonal antibody (mAb) Po66 has been shown in previous work to be localized in nude mice xenografts of human lung tumours when injected intravenously [Dazord L et al. (1987) Cancer Immunol Immunother 24: 263-268] and to be suitable for the scintigraphic detection of lung cancers in patients [Dazord L, et al. (1987) in Klapdor (ed) New tumour markers and their monoclonal antibodies. Georg Thieme, Stuttgart, New York, pp 444-450]. The nature of the antigen recognized by Po66 has been investigated in the present work and comparisons are made with antigens recognized by other mAbs prepared in the laboratory. These mAbs were raised either against lung squamous cell carcinoma (mAbs Po43, Po60), or against a bronchio-alveolar carcinoma (mAbs BAM33, BAM45, BAM54 and BAM69). Radioiodinated purified Po66 did not compete for cell binding with any other mAb. All Po and BAM mAbs reacted with tumour cells both cultured in vitro and grown in vivo. They recognized cytoplasmic antigens as judged by immunofluorescence examination of fixed cells or by immunoperoxidase staining of cancer tissues, but could never be visualized by immunofluorescence on the surface membrane of culture cells. The mAbs of the BAM series reacted with vimentin as demonstrated by immunofluorescence staining, showing alterations in the aspect of the filaments under the effect of colchicine. Radiolabelled mAbs Po43, BAM33 and BAM45 bound to partially purified cytoplasmic cytoskeleton components. In contrast, Po66 was never seen associated with intermediary filaments. The sensitivity to enzyme digestion of the antigen associated with Po66 was studied in comparison with those associated with Po43, BAM33 and BAM45. All antigens were sensitive to protease digestion while only the Po66-identified antigen was sensitive to periodate, neuraminidase and alpha-fucosidase. Thus, mAb Po66 identified an antigen of 47 kDa (as determined before) present in the cytoplasm but not related to the cytoskeleton, not detected on the cell surface and glycoprotein in nature.

A new immunoreagent for cell labeling. CD3 monoclonal antibody covalently coupled to fluorescent polymethacrylic nanoparticles.

The preparation and application of immunonanospheres are described. CD3 monoclonal antibodies were covalently coupled to fluorescent polymethacrylic nanoparticles by the glutaraldehyde reaction and the resultant conjugate purified by gel filtration on a Sepharose 4B column. Peripheral blood mononuclear cells labeled with this immunoreagent were observed by both fluorescence microscopy and scanning electron microscopy in order to evaluate the technique.

Scintigraphic detection in mice of inflammatory lesions and tumours by an indium-labelled monoclonal antibody directed against Mac-1 antigen.

The monoclonal antibody, 3A33, directed against Mac-1 antigen which is expressed essentially on macrophages and polymorphonuclear cells, was injected i.v. into mice, as part of an attempt to visualize inflammatory lesions and tumours by external scintigraphy. The monoclonal antibody, a rat IgG2a, was conjugated with a bifunctional chelating agent, diethylenetriaminepentaacetic acid at a 1:1 molecular ratio and complexed with 111-indium, a procedure which apparently did not alter its binding to peritoneal macrophages and provided relatively stable cell labelling. An unrelated rat IgG2a of unknown specificity radiolabelled in the same manner as 3A33 served as a control. The uptake of i.v. injected 3A33 by peritoneal macrophages was up to 50 times that of unrelated IgG2a. After i.v. inoculation, the antibody accumulated in the liver, spleen, lung, in foot-pad inflammatory reactions induced by injection of Freund's adjuvant and in experimentally grafted tumours. The 3A33: non-specific IgG2a uptake ratio in inflammatory lesions and tumours, however, was much lower than for peritoneal macrophages and was generally close to 2. This was sufficient to obtain scintigraphic images of inflammations and tumours. The images obtained after injection of 3A33 were clearly of better quality than those given by the non-specific immunoglobulin. They could be improved by subtraction of the vascular images obtained after injection of 99m-technetium serum albumin. The labelling of Mac-1-positive blood mononuclear cells by in vitro incubation with radioactive 3A33 was not intense enough to allow scintigraphic imaging after in vivo re-infusion but seemed more selective than the injection of whole antibody in detecting inflammatory reactions. These results seem interesting in view of the potential human application to the detection inflammatory lesions and the appreciation of tumour inflammatory components. Possible improvements in the technique are discussed.

[Anti-CD3 monoclonal antibodies. Characterization and function]. / Anticorps monoclonaux anti-CD3. Caractérisation et fonctions.

Four anti-T lymphocyte monoclonal antibodies were produced by the hybridoma technique. Percentages of miscellaneous labelled cell suspensions, as profiles of fluorescent histograms, showed a reactivity of these 4 reagents quite comparable to that observed with anti-CD3 monoclonal antibodies. Labelling summation studies, cell-sorting studies and blocking experiments gave similar results as gave other anti-CD3 reagents. Biochemical study and functional tests: modulation effects and mitogenic properties, argued for the belonging of these 4 antibodies to the CD3 cluster. The actual data concerning the CD3 molecule are briefly resumed to underline the interest of such antibodies in the understanding of T-lymphocyte activation mechanisms during the immune response.