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Novel anti-CD30 recombinant immunotoxins containing disulfide-stabilized Fv fragments
Satoshi Nagata1, Masanori Onda, Yoshito Numata
1Laboratory of Molecular Biology, Center for Cancer Research, National Cancer Institute, NIH, Bethesda, Maryland 20892-4264, USA.
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(d)s 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 IC(50) 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.
Insights
Researchers developed novel anti-CD30 immunotoxins for treating CD30-positive lymphomas. Two potent immunotoxins demonstrated significant cytotoxic activity against lymphoma cell lines, showing promise for cancer therapy.
Area of Science:
- Oncology
- Immunology
- Biotechnology
Background:
- CD30 is a key target antigen in Hodgkin's disease and anaplastic large cell lymphoma.
- Recombinant immunotoxins offer a targeted approach for cancer therapy.
Purpose of the Study:
- To create novel recombinant immunotoxins targeting CD30.
- To utilize Fv fragments from anti-CD30 monoclonal antibodies and a truncated Pseudomonas exotoxin.
Main Methods:
- Production and characterization of anti-CD30 monoclonal antibodies (MAbs).
- Construction of disulfide-stabilized Fv-based recombinant immunotoxins.
- Evaluation of binding affinity and in vitro cytotoxicity against CD30-positive cell lines.
Main Results:
- Six anti-CD30 MAbs were generated, binding to native and recombinant CD30.
- Four disulfide-stabilized Fv immunotoxins were successfully produced.
- Immunotoxins exhibited specific binding to CD30 and potent in vitro cytotoxicity (IC50: 0.3-100 ng/ml) against lymphoma cell lines.
Conclusions:
- Four novel anti-CD30 Fv immunotoxins were developed.
- Two immunotoxins demonstrated significant cytotoxic potential against CD30-positive cell lines.
- Further evaluation for treating CD30-positive lymphomas is warranted.