ACTIVITY OF A SINGLE-CHAIN IMMUNOTOXIN THAT SELECTIVELY KILLS LYMPHOMA AND OTHER B-LINEAGE CELLS EXPRESSING THE CD40 ANTIGEN

We have constructed anti-CD40 immunotoxins consisting of the single ch ain Fv (sFv) region of the anti-human CD40 mAb G28-5 fused to a trunca ted form of Pseudomonas exotoxin, PE40. CD40 is an integral membrane g lycoprotein found on the surface of B-lineage cells, including lymphom as and leukemias, as well as certain carcinomas. Two forms of the immu notoxin were constructed, one with the light chain variable (V-L) regi on of the sFv preceding the heavy chain variable region (V-H) [G28-5 s Fv(V-L-V-H)-PE40] and the second with the sFv in the opposite orientat ion [G28-5 sFv(V-H-V-L)-PE40]. Although both forms of G28-5 sFv-PE40 s pecifically bound to CD40 in ELISAs, the binding of g28-5 sFv(V-L-V-H) -PE40 was >10-fold higher. A number of malignant B- and T-cell lines w ere screened for CD40 expression and susceptibility to G28-5 sFv(V-L-V -H)-PE40. All of the B-lineage cells tested were CD40 positive and sen sitive to the anti-CD40 immunotoxin, with EC(50)s ranging from 2.5-70 ng/ml, whereas none of the T cell leukemias or lymphomas were antigen positive or were affected by the immunotoxins. Consistent with the ant igen-binding results, the V-L-V-H immunotoxin was >10-fold more cytoto xic than the V-H-V-L immunotoxin. The anti-CD40 single-chain immunotox in fusion protein G28-5 sFV(V-L-V-H)-PE40 represents a potent and spec ific cytotoxic agent for the elimination of normal and transformed B-l ineage cells expressing CD40.