C Bindon

Cell lines have been established that secrete a matched set of human chimeric IgM, IgG1, IgG2, IgG3, IgG4, IgE, and IgA2 antibodies that are directed against the hapten 4-hydroxy-3-nitrophenacetyl. These chimeric antibodies secreted from mouse plasmacytoma cells behave exactly like their authentic human counterparts in SDS-PAGE analysis, binding to protein A and in a wide range of serological assays. The antibodies have been compared in their ability to bind human C1q as well as in their efficacy in mediating lysis of human erythrocytes in the presence of human complement. A major conclusion to emerge is that whereas IgG3 bound C1q better than did IgG1, the chimeric IgG1 was much more effective than all the other IgG subclasses in complement-dependent hemolysis. The IgG1 antibody was also the most effective in mediating antibody-dependent cell-mediated cytotoxicity using both human effector and human target cells. These results suggest that IgG1 might be the favoured IgG subclass for therapeutic applications.

Humanized antibodies are likely to have a major role in therapy and it is important to define their interaction with physiological effectors. By comparing a matched series of chimeric human mAbs we found that igG1 was most efficient in complement lysis, although IgG3 bound more C1q. To resolve this paradox we compared the ability of human IgG1, IgG2, IgG3, IgG4, and IgE and rat IgG2b to cause C1q binding, C1 binding and activation, C4 activation, C4b binding, and C3b binding. Rat IgG2b was included because this isotype has already successfully been used for therapy. Human IgG1 was less efficient than IgG3 and fixing C1q and C1 on the cell surface, but the number of C4 molecules bound per C1 was 10-fold greater for IgG1 than for IgG3. This difference, amplified through later stages of the complement cascade, can account for the superiority of IgG1 for cell lysis. The efficiency of IgG1 in fixing C4 was not due to a favored binding site on the antibody molecule, since virtually all of the bound C4b was attached to the cells. Rather, it appeared that the activation of C4 by C1s was greatly favored by IgG1 compared with IgG3. It should be possible to combine the optimal properties of IgG1 and IgG3 antibodies to produce an improved therapeutic reagent.

Lysis of human lymphocytes by autologous complement had been studied using a range of monoclonal antibodies against different antigens. Antigen specificity (and not antibody isotype) was the most important factor which influenced cell lysis and this could not be accounted for merely by differences in surface density between antigens. Three antigens with comparable surface density were studied in detail: CAMPATH-1 (lytic), major histocompatibility complex class I (lytic) and leukocyte common antigen (poorly lytic). C1q binding was roughly proportional to antibody binding and dependent on antibody isotype. However, the lytic antibodies were much better able to bind and activate whole C1 than the poorly lytic ones. This result would not have been predicted from traditional concepts of complement activation but can be interpreted in the light of models for C1 activation which involve Fc-Fc interactions, Fc-C1r2s2 interactions and a critical C1q stem-arm angle for C1 binding and activation.

The influence of interleukin 2(IL2) on the cytotoxic activity of lymphocytes from patients with melanoma against autologous and a variety of allogeneic melanoma cells was studied. IL2 was produced from blood lymphocytes cultured for 24 h with phytohaemagglutinin (PHA) and purified by membrane chromatography to exclude PHA. Lymphocytes from 13 patients with melanoma at various clinical stages were cultured fro 6 days with IL2 (2 U/ml) and then tested for cytotoxic activity against autologous melanoma cells, three allogeneic melanoma and three non-melanoma cells. Autologous cytotoxicity was generated by culture with IL2 alone and was not increased by culture with both IL2 and autologous tumour cells. Marked increases in cytotoxic activity were also generated against the allogeneic target cells and were maximal against the NK-insensitive Chang target cells. Similar degrees of cytotoxicity were induced by IL2 stimulation of lymphocytes from melanoma patients, patients with nonmelanoma carcinoma and normal subjects against the allogeneic target cells. Cold target inhibition studies were carried out against IL2 induced autologous cytotoxicity in five patients. In four of five studies the autologous target cells inhibited more than the allogeneic target cells. There was no significant difference between the inhibition produced by allogeneic melanoma cells and that produced by non-melanoma cells. Similarly, in studies against allogeneic target cells, there was no significant difference in the inhibition produced by allogeneic melanoma compared to non-melanoma target cells. This applied irrespective of whether effector cells were from melanoma or non-melanoma subjects. These results suggest that lymphocytes from patients with melanoma are primed against autologous antigens in vivo and that provision of a second signal, IL2, in vitro can induce cytotoxicity against the autologous tumour. The cytotoxicity generated against the allogeneic target cells did not appear to have specificity to melanoma. Several results, such as the pattern of cytotoxicity against the target cells and change in cell surface markers on the lymphocytes during culture, suggested that cytotoxicity was mediated by activated T cells rather than by nature killer cells. These findings appear to have important implications both in the understanding of tumor host relationships and for the use of IL2 in therapy.