Monoclonal antibody-mediated cytotoxicity against rat Beta cells detected in vitro does not cause Beta-cell destruction in vivo

Abstract

Two monoclonal Beta-cell surface antibodies M10H6 und K14D10 were obtained by fusion of spleen cells of Balb/c mice with the myeloma cell line P(3)0. The monoclonal antibody M10H6 was induced by immunization with rat insulinoma cells finally boostered with disintegrated rat islets, whereas the K14D10 was generated after immunization with porcine proinsulin. Both monoclonals belong to the IgG2A isotype and were screened with insulin-producing rat insulinoma cells by an indirect immunofluorescence test as well as by a cellular enzyme linked immunosorbent assay. In addition to the cell surface binding on living Beta cells the monoclonals react with islets on cryostat sections of rat pancreas. The anti-islet cytotoxic potential of these monoclonals was measured by 51Chromium-release in the presence of complement or Fc-receptor bearing leucocytes using 51Chromium-labelled rat islet cells as target. Both antibody secreting hybridomas were propagated in syngeneic mice resulting in high levels of islet cell surface antibodies in ascites and sera from the recipient. High anti-islet cytotoxicity was mediated by ascites fluid, but no mouse developed hyperglycaemia. Furthermore, the repeated injections of the monoclonals into rats did not exert a diabetogenic action and failed to reduce the pancreatic insulin content although the attraction of the K14D10 to the pancreatic islets in vivo could be demonstrated. We conclude that islet cell surface antibody-mediated Beta-cell lysis in vitro may not be relevant to Beta-cell destruction in vivo.

Quantitative Evaluation of a Monoclonal Antibody and its Fragment as Potential Markers for Pancreatic Beta Cell Mass

“…The low specificity for a number of small molecules obtained by the assay correlated well the lack of accumulation in the pancreas. In the present study, we have used this approach to assess the binding characteristics of a previously developed beta cell specific antibody K14D10 (Ziegler et al, 1992) and its Fab.…”

“…The in vitro predictions were borne out in in vivo studies, validating our notion that systematic in vitro screening can avoid wasteful in vivo experiments and radiosyntheses using compounds that have no chance of success. In the present study we have evaluated a recombinant Fab (rFab) of the beta-cell binding mAb K14D10 (Ziegler et al, 1992) using the previously-developed screening assay. The use of a standard method for evaluating potential imaging agents allows for a quantitative comparison of the antibodies to previously evaluated compounds.…”

Glutamate Decarboxylase (GAD) is not Detectable on the Surface of Rat Islet Cells Examined by Cytofluorometry and Complement-Dependent Antibody-Mediated Cytotoxicity of Monoclonal GAD Antibodies

“…injection. For fusion procedure, 3 x i0 splenocytes Schlosser, Witt, Besch and Ziegler (1992). Hybrid clones were selected by growing in RPMI 1640 supplemented with 20% heat-inactivated fetal calf serum and the selection intergradients io-mol/l hypoxanthine, 5 x 10-6 mol/I azaserine and 1.6x 10 mmol/l thymidine (Ziegler eta!.…”

“…The binding of the mc-GAD Ab was also examined on cryosections of human and rat pancreas and on viable rat islet cells by indirect immunofluorescence as described in detail previously (Ziegler et al 1992). To detect the surface binding of the monoclonals, islets were prepared by fractionated collagenase digestion of the pancreata from 10-14 day-old rats (Ziegler et al 1992;Schröder et al 1993). The islet cells were prepared by means of fractionated treatment of islets using 12mg/mi dispase in calcium-free Hank's balanced salt solution (HBSS) (Schröder eta!.…”

“…activity 9.25-18.5 GBq/mg chromium, Amersham, United Kingdom) in a total volume of 400 jil HBSS supplemented with 0.1 % bovine serum albumin (BSA). Then I x 10 washed 51Cr-labelled islet cells resuspended in 20 tl HBSS were mixed with 50 tl of the corresponding mc-GAD Ab or the monoclonal Beta-cell specific surface antibody KI4DIO (positive control) as well as non-inactivated WOK rat serum (negative control) and 30 Iii of rabbit complement (Ziegler et al 1992;Schroder et al 1993). The incubation mixture was held for I h at 37'C and 51Cr-release was measured in the supernatant as well as in the cell pellet by gamma counting.…”