Refinement of an indirect immunotoxin assay of monoclonal antibodies recognising the human small cell lung cancer cluster 2 antigen

Abstract

Monoclonal antibodies (Mabs) from the Second International Workshop on Small Cell Lung Cancer (SCLC) Antigens that recognise the cluster 2 SCLC-associated antigen mediated potent and selective cytotoxic effects in an indirect assay of immunotoxin cytotoxicity. In this assay, the NCI-H69 cell line was treated with each Mab at 4 degrees C, washed to remove unbound Mab, and then incubated at 37 degrees C in the presence of a fixed concentration, 1 x 10(-8) M, of the screening agent, sheep anti-mouse IgG-ricin A chain. The use of a fixed high concentration of screening agent led to a 300-fold overestimate of the potency of a cluster 2-directed immunotoxin, MOC-31-ricin A chain. In contrast, when the concentration of the screening agent was identical to the Mab concentration, a precise match to immunotoxin potency was obtained. MOC-31-ricin A chain selectivity inhibited the incorporation of [3H]leucine by the NCI-H69, SW2 and GLC-8 SCLC cell lines by 50% at a concentration between 3 x 10(-11) M and 3 x 10(-10) M, and by the NCI-H125 lung adenocarcinoma cell line at 7 x 10(-11) M, but exerted no selective toxic effects upon human lung and non-lung tumour cell lines lacking surface expression of the cluster 2 antigen.

Third international workshop on lung tumor and differentiation antigens: Overview of the results of the central data analysis

“…Sequence homologies of the partially purified antigen recognized by the cluster-2 antibody AUAl has proved that this antigen is identical to cluster 2 (Durbin et al, 1990). Cluster-2 antigen is internalized, making this antigen a potential target for rick-A-chain immunotoxins (Derbyshire et al, 1993). A small clinical trial has been performed with the antibody K1/4 alone, or conjugated to methotrexate, in patients with NSCLC.…”

Immunotoxin Screening System: A Rapid and Direct Approach to Obtain Functional Antibodies with Internalization Capacities

“…These techniques require purified ITs; therefore, they are inappropriate as a screening procedure to distinguish the hybridomas that secrete desired antibodies from those that do not produce them. More favorable approach is indirect IT assay wherein a toxin-labeled secondary antibody is utilized to identify a monoclonal antibody of interest [ 61 , 62 ]. While successful, since this procedure relies on the toxin-labeled secondary antibody, structural composition is not identical to the final form of IT or ADC where monoclonal (primary) antibody is directly linked to toxins or drugs.…”