Mitogenicity of anti-Thy-1 monoclonal antibodies attributable to an Fc-dependent mechanism

We analyzed the mechanism by which certain anti-Thy-1 monoclonal antibodies (mAb) activate T cells directly without additional stimuli. Using a panel of rat anti-Thy-1 antibodies which included more than 30 IgG2c mAb, we found that only the IgG2c isotype was able to induce a strong proliferative response in both resting T cells and a T cell lymphoma, suggesting that this form of T cell activation is isotype restricted and might be a consequence of a unique physico-chemical property of the IgG2c heavy chain. Results from surface distribution studies of Thy-1 molecules, following specific interactions with anti-Thy-1 antibodies of different isotypes, again showed that only IgG2c mAb formed Thy-1 aggregates of high valence on the surface of a T cell lymphoma, and such clustering always evoked a biological response. This led us to propose that IgG2c mAb have the inherent tendency to self-associate, probably through homophilic Fc-Fc contacts, and that this feature renders anti-Thy-1 mAb mitogenic. To prove this, we set up cross-inhibition studies with randomly selected mitogenic (IgG2c) and non-mitogenic (IgG2b) anti-Thy-1 mAb. The results clearly demonstrated that IgG2c antibodies enhance their own binding, analogous to the new form of antibody binding that was recently demonstrated between murine IgG3 mAb and a multivalent antigen. Confirmation of this was also provided by IgG2c-derived F(ab')₂ fragments, which were unable to cause proliferation. Furthermore, masking the Fc part of cell-bound IgG2c mAb with a monomeric and thus non-aggregating IgG-binding protein A-derived fragment cancelled their mitogenic ability. Finally, induction of T cell proliferation appeared to be independent of cross-linking via FcγR. The results support a model in which noncovalent intermolecular homophilic contacts of the Fc regions of the IgG2c isotype bring about effective aggregation of Thy-1 molecules, thereby stimulating the mitotic apparatus of the cell.     

抗人肿瘤相关核仁抗原单克隆抗体的建立

本研究以A549细胞系和人肺腺癌组织细胞核仁为抗原,建立了7株McAbs,并用ELISA技术对其反应性进行了初步分析。结果表明:各株McAb均能与人癌细胞核仁起反应,但各自的抗原却不尽相同。MA1、MA2、MA3和MA6株的抗原可能是HMNA类物质;MA4、MAS和ML1株的抗原可能是属于核仁的正常成份,但优势表达于癌细胞中。本组抗体的建立,对于研究肿瘤细胞核仁的分子组成和生物学功能,并进而利用HMNA为临床肿瘤病理服务可能有重要意义。     

Production of monoclonal antibodies for detection of antigens in both their native and denatured states

Different immunization procedures were tested to find methods to enhance the proportion of monoclonal antibodies reacting equally well with native and denatured proteins for use in food analysis. Antibodies to soybean trypsin inhibitor with the desired characteristics were obtained by immunization with the denatured protein. Antibodies to ovomucoid were obtained by long‐term immunization. The monoclonal antibodies to ovomucoid were of the IgM class.     

An antiglobulin microcytotoxicity assay to analyze non-complement fixing monoclonal antibodies to human histocompatibility antigens

An antiglobulin microcytotoxicity assay has been used to analyze non-complement fixing monoclonal antibodies to human histocompatibility antigens. The assay utilizes methodology similar to that of the widely used microcytotoxicity assay for HLA typing, requires low numbers of target cells and is suitable to test large numbers of samples. The sensitivity of the assay is influenced by the anti-mouse Ig antiserum used, by the sequence of addition of the various reagents and by the incubation time. The assay is suitable to screen supernatants of clones derived from hybridization experiments and to characterize the serological specificity of anti-HLA monoclonal antibodies.     

ICO-45 monoclonal antibodies to a new epitope of CD38 antigen

All-Union Oncologic Scientific Center, Academy of Medical Sciences of the USSR, Moscow. (Presented by Academician of the Academy of Medical Sciences of the USSR, N. N. Trapeznikov.) Translated from Byulleten' Éksperimental'noi Biologii i Meditsiny, Vol. 107, No. 6, pp. 729–731, June, 1989.     

The use of colloidal gold for screening monoclonal antibodies to cell surface antigens

An immunogold method in Terasaki plates is described which allows accurate and sensitive visualization of the binding of monoclonal antibodies to cell surface antigens and is suitable for large scale screening. Monolayers of fixed cells are prepared in the wells. The binding of monoclonal antibodies is detected by a protein A gold complex. The cell-bound gold can be visualized by either optical or transmission electron microscopy. The results obtained with various monoclonal antibodies are presented.     

Human anti-mouse antibody response to the injection of murine monoclonal antibodies against IL-6.

We analysed human anti-mouse antibodies (HAMA) in 12 patients (six with multiple myeloma (MM) and six with metastatic renal cell carcinoma (MRCC) who were treated with B-E8, an IgG1 MoAb against IL-6. Efficiency of the treatment was evidenced by the drop in the serum levels of C-reactive protein (CRP), the in vivo production of which is under the control of IL-6. Three patients with MM and the six patients with MRCC became immunized to the injected MoAb. HAMA appeared between days 7 and 15 after the beginning of the treatment. The nine patients made IgG antibodies; four also made IgM. All immunized patients made anti-idiotype antibodies specific to B-E8. Two of them also developed HAMA directed to murine IgG1 isotype; in these two patients B-E8 MoAb cleared rapidly from the circulation with loss of treatment efficiency. In the patients who developed only anti-idiotype antibodies, serum levels of B-E8 remained unchanged and CRP production remained inhibited, indicating that treatment remained efficient in the presence of HAMA. Circulating B-E8 MoAbs were still able to bind to IL-6 and to inhibit IL-6-dependent proliferation despite the presence of anti-idiotypic HAMA. Therefore, in contrast to HAMA produced against MoAb directed against cellular targets, HAMA against anti-IL-6 MoAb idiotopes led neither to clearance nor to functional inactivation of the injected MoAb. This was further shown by resuming the B-E8 treatment with success in a patient who still had anti-idiotypic HAMA.     

Production of different antibodies after simultaneous immunization of animals with two antigens

We propose a method of simultaneous immunization with two different antigens for isolation of two types of antibodies from the same antiserum. Bacterial proteins (Bacillus stearothermophilus glyceraldehyde-3-phosphate dehydrogenase and Escherichia coli GroEL chaperonin) served as the antigens. Affinity purification of antibodies was carried out using two columns: with covalently immobilized glyceraldehyde-3-phosphate dehydrogenase or GroEL chaperonin. During stage I, the antiserum was applied onto the column with immobilized glyceraldehyde-3-phosphate dehydrogenase, after which antibodies to glyceraldehyde-3-phosphate dehydrogenase were eluted. During the next stage, the antiserum without antibodies to glyceraldehyde-3-phosphate dehydrogenase was passed through the column with immobilized GroEL and antibodies to chaperonin were isolated. Antibodies to glyceraldehyde-3-phosphate dehydrogenase and to GroEL had high titers and exhibited no cross-reaction. __________ Translated from Byulleten’ Eksperimental’noi Biologii i Meditsiny, Vol. 143, No. 6, pp. 718–720, June, 2007     

The value of immunohistological screening in the production of monoclonal antibodies

This paper describes an immunoperoxidase technique for labelling cryostat tissue sections which is routinely used in the authors' laboratories both in the initial screening of hybridoma culture supernatants, and also during the subsequent cloning and growth of antibody-secreting cell lines. The technique can readily be performed on 100 samples in less than 3 h and is free of non-specific background labelling. The staining pattern of a monoclonal antibody on a single tissue section allows semiquantitative assessment of its reactivity against a wide variety of tissue constituents and is thus inherently much more informative than conventional screening techniques (such as binding assays) which yield only a single numerical value for each test performed. In consequence it is often possible to identify the probable specificity of a new monoclonal antibody at the primary screening stage. A further important advantage of immunohistological screening is that it detects antigens on cells or other tissue structures which do not readily enter suspension and also antibodies against nuclear and cytoplasmic antigens.Examples of monoclonal antibodies analysed by immunohistological screening include antibodies against C3b receptor, HLA-DR, factor VIII-related antigen, human syncytiotrophoblast, dendritic reticulum cells and a proliferation-associated cell surface glyco-protein.     

Mimicking the humoral immune response in vitro results in antigen-specific isotype switching supported by specific autologous T helper cells: generation of human HIV-1-neutralizing IgG monoclonal antibodies from naive donors

Molecular and cellular requirements for antigen-specific isotype switch of human B cells have been investigated by mimicking signaling occurring in germinal centers. Peripheral blood mononuclear cells from healthy seronegative blood donors were first primary immunized in vitro, using a synthetic immunogen containing both a T and B cell epitope, which generated specific IgM-secreting B cells. We used the apex of the V3 loop of gp120 as B cell epitope linked to a promiscuous T helper epitope from tetanus toxin. In parallel, CD4⁺ T helper cell clones specific for the T epitope of the immunogen were established. In a secondary in vitro stimulation period, we co-cultured the antigen-specific T and B cells on CD32-transfected fibroblasts, together with an anti-CD40 monoclonal antibody. This resulted in isotype switching and human antigen-specific, IgG-secreting B cells were detected. This response was strictly dependent upon the presence of autologous T helper cells and the immunogen. Antigen-specific human B cells derived from this primary and secondary in vitro immunization were subsequently subjected to electrofield-induced somatic cell hybridization and hybridomas secreting human anti-V3 IgG monoclonal antibodies were isolated. One human antibody was further characterized and shown to be specific for the immunizing antigen with an affinity constant of 24 nM. This antibody also effectively neutralized different isolates of HIV-1, achieving a 50% neutralization at 0.46 μg/ml.