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Rational immunotherapy with ribonuclease chimeras. An approach toward humanizing immunotoxins
S M Rybak1, H R Hoogenboom, D L Newton
1Surgical Neurology Branch, National Institute of Neurological Diseases and Stroke, National Institutes of Health, Bethesda, MD 20892.
Abstract:
Members of the pancreatic ribonuclease (RNase) family have diverse activities toward RNA that could cause them to function during host defense and physiological cell death pathways. This activity could be harnessed by coupling RNases to cell binding ligands for the purpose of engineering them into cell-type specific cytotoxins. Therefore, the cytotoxic potential of RNase was explored by linking bovine pancreatic ribonuclease A via a disulfide bond to human transferrin or antibodies to the transferrin receptor. The RNase hybrid proteins were cytotoxic to K562 human erythroleukemia cells in vitro with an IC50 around 10(-7) M, whereas > 10(-4) M of native RNase was required to inhibit protein synthesis. Cytotoxicity required both components of the conjugate since excess transferrin or ribonuclease inhibitors added to the medium protected the cells from the transferrin-RNase toxicity. Importantly, the RNase conjugates were found to have potent antitumor effects in vivo. Chimeric RNase fusion proteins were also developed. F(ab')2-like antibody-enzyme fusions were prepared by linking the gene for human RNase to a chimeric antitransferrin receptor heavy chain gene. The antibody enzyme fusion gene was introduced into a transfectoma that secreted the chimeric light chain of the same antibody, and cell lines were cloned that synthesized and secreted the antibody-enzyme fusion protein of the expected size at a concentration of 1-5 ng/mL. Culture supernatants from clones secreting the fusion protein caused inhibition of growth and protein synthesis toward K562 cells that express the human transferrin receptor but not toward a nonhuman derived cell line. Since human ribonucleases coupled to antibodies also exhibited receptor mediated toxicities, a new approach to selective cell killing is provided. This may allow the development of new therapeutics for cancer treatment that exhibit less systemic toxicity and, importantly, less immunogenicity than the currently employed ligand-toxin conjugates.
Insights
Researchers engineered ribonucleases (RNases) into targeted cancer toxins by linking them to antibodies. These RNase-antibody conjugates demonstrated potent antitumor effects in vitro and in vivo, offering a new therapeutic approach for cancer treatment.
Area of Science:
- Biochemistry
- Molecular Biology
- Immunology
Background:
- Pancreatic ribonucleases (RNases) possess RNA-degrading capabilities relevant to host defense and cell death.
- RNase activity can be exploited for targeted cell killing by conjugating them to cell-binding ligands.
Purpose of the Study:
- To explore the cytotoxic potential of RNase-based conjugates for targeted cancer therapy.
- To develop novel antibody-enzyme fusion proteins for selective cancer cell elimination.
Main Methods:
- Bovine pancreatic ribonuclease A was linked to human transferrin or anti-transferrin receptor antibodies via a disulfide bond.
- Chimeric RNase fusion proteins were engineered by linking human RNase genes to antitransferrin receptor antibody genes.
- Cytotoxicity was assessed in vitro against K562 cells and in vivo in antitumor models.
Main Results:
- RNase hybrid proteins exhibited potent cytotoxicity against K562 cells (IC50 ~10(-7) M), significantly more effective than native RNase.
- Cytotoxicity was dependent on both RNase and ligand components, as demonstrated by protection assays.
- RNase conjugates showed significant antitumor effects in vivo.
- Secreted antibody-enzyme fusion proteins inhibited K562 cell growth and protein synthesis in a receptor-mediated manner.
Conclusions:
- RNase-ligand conjugates and antibody-enzyme fusion proteins represent a promising strategy for targeted cancer cell killing.
- This approach offers potential for developing cancer therapeutics with reduced systemic toxicity and immunogenicity compared to current ligand-toxin conjugates.
- Receptor-mediated toxicity via engineered RNases provides a novel avenue for selective cell killing in cancer treatment.