Antibody derivatives for mediating cellular cytotoxicity via the Fcy receptors

The ability of the three types of Fcγ receptor (FcγR) to mediate cellular cytotoxicity in vitro has been studied using a range of antibody derivatives. The derivatives used divide into two groups: mouse (Fab')/human (Fcγ) chimeric, and murine F(ab'γ)2 bispecific. These derivatives attach a Fab'γ arm to the target cell, and then mediate antibody-dependent cell-mediated cytotoxicity (ADCC) via an Fc-FcγR interaction (chimeric antibody), or redirected cellular cytotoxicity (RCC) through a Fab'-FcγR interaction, (bispecific antibodies). The two model target systems that have been used are chick red blood cells (CRBC) and the guinea-pig L_2C leukaemic lymphocytes. Human effector cells from venous blood enriched for monocytes (bearing FcγRI, II) and /or NK cells (bearing FcγRIII) were used for all cytotoxicity assays. The first part of the work describes the preparation and characterisation of a new anti-FcγRII (CDw32) monoclonal antibody (mAb), AT10. The mAb At10 gave an immunohistochemical staining profile that was intermediate between those given by mAb IV.3 and KB61 (previously identified CDw32 mAb). This mAb was shown to recognize a trigger molecule for lysis, on the surface of monocytes, both through RCC of CRBC and by the lysis of At10 hybridoma cells which attached via their surface mAb to monocytes. In the CRBC system all the FcγR are capable of mediating lysis in RCC using F(ab'γ)2 bispecific antibodies targeting individual FcγR. The degree of lysis was highly variable between donors. All the Fc-containing derivatives mediated efficient lysis which showed less donor variability than the bispecific antibodies. Using immunodepleted populations of cells it was shown that the Fc-containing derivatives can function through all three types of FcγR. In the presence of human Fcγ at serum concentrations the lysis mediated via the Fc-containing derivatives was blocked whereas the F(ab'γ)2 antibodies targeting FcγRI and II (but not FcγRIII) were not affected. Similar results were shown with the L2C tumour where all the FcγR mediated significant tumour lysis using the bispecific antibodies, through the level of lysis was lower than that given against CRBC. In general, the mouse/human chimeric antibodies gave a level of lysis similar to that given through the best bispecific antibody, anti-FcγRIII. Treatment of the effector cells with IFNγ gave an increase in lysis mediated through the anti-FcγRIII bispecific antibody and a representative Fc-containing derivative, whereas incubation with GM-CSF did not show any difference in cytotoxicity mediated by any FcγR. Finally, preliminary experiments were conducted which investigate how factors such as binding affinity and the protein kinase C (PKC) inhibitor, trifluoperazine, affect the levels of lysis. Construction of trimeric derivatives, possessing two (bispecific trimers) or three (trispecific) different specificities were compared with the F(ab'γ)2 bispecific antibodies in RCC. Bispecific trimers (anti-FcγR x (anti-target)_2) gave a modest increase in lysis, via all FcγR, but no increase in cytotoxicity was seen with the trispecific antibodies (anti-FcγRI x anti-FcγRII x anti-target). Experiments with trifluoperazine showed that NK cell lysis, mediated via the anti-FcγRIII bispecific antibody, uses a PKC-dependent mechanism. The work with monocytic cells is less conclusive, although consistent with a PKC-independent mechanism.