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Novel Anti-CD30 Recombinant Immunotoxins Containing Disulfide-stabilized Fv Fragments

Laboratory of Molecular Biology, Center for Cancer Research, National Cancer Institute, NIH, Bethesda, Maryland 20892-4264

Purpose: To develop a novel targeting reagent to CD30 expressed on Hodgkin’sdisease and anaplastic large cell lymphoma, we made a panel of recombinant immunotoxins specific for CD30 using Fvs of newly produced anti-CD30 monoclonal antibodies (MAbs) and a M_r 38,000 truncated mutant of Pseudomonas exotoxin.

Experimental Design: A group of MAbs against CD30 was produced and characterized for their reactivity and epitopes. Recombinant immunotoxins were made using the Fv genes cloned from the hybridomas. Their cytotoxic activities were examined on various CD30-positive cell lines.

Results: Six MAbs were produced. All reacted with recombinant soluble CD30 and to a CD30-Fc fusion protein, and bound to native CD30 expressed on Hodgkin’s lymphoma-derived cell lines. The epitopes of the six MAbs were classified into two groups by a mutual competition assay for the binding to CD30 on cells. Sequencing the cDNAs revealed that all of the variable chains are unique except one valiable light that is shared by two MAbs. We made four disulfide stabilized Fv-based recombinant immunotoxins, in which the valiable heavy, which is genetically fused with truncated mutant of Pseudomonas exotoxin, forms a disulfide bond with the valiable light. The purified immunotoxins bound to recombinant soluble CD30 immobilized on a biosensor chip with K_ds of 4–400 nM. Fluorescence-activated cell sorter analysis confirmed their specific binding. In vitro cytotoxicity tests showed that the immunotoxins specifically kill a variety of CD30-positive lymphoma cell lines as well as CD30-transfected A431 cells. The IC₅₀ ranged from 0.3 to 100 ng/ml.

Conclusions: Four anti-CD30 disulfide stabilized Fv immunotoxins were successfully produced. Two of these showed good cytotoxic activity to various CD30-positive cell lines. These newly produced immunotoxins should be additionally evaluated for the treatment of CD30-positive lymphomas.

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