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A New Combination: Anti Glypican-3 scFv and Diphtheria Toxin with the Best Flexible Linker
Hamid Hashemi Yeganeh1,2, Mohammad Heiat1, Seyed Moayed Alavian1
1Baqiyatallah Research Center for Gastroenterology and Liver Diseases, Baqiyatallah University of Medical Science, Tehran, Iran.
Abstract:
Along with all cancer treatments, including chemotherapy, radiotherapy, and surgery, targeting therapy is a new treatment manner. Immunotoxins are new recombinant structures that kill cancer cells by targeting specific antigens. Immunotoxins are composed of two parts: toxin moiety, which disrupts protein synthesis process, and antigen binding moiety that bind to antigens on the surface of cancer cells. Glypican 3 (GPC3) is an oncofetal antigen on the surface of Hepatocellular carcinoma (HCC) cells. In this study, truncated Diphtheria toxin (DT389) was fused to humanized scFv YP7 by one, two and three repeats of GGGGS linkers (DT389-(GGGGS)1-3YP7). In-silico and experimental investigation were performed to find out how many repeats of linker between toxin and scFv moieties are sufficient. Results of in-silico investigations revealed that the difference in the number of linkers does not have a significant effect on the main structures of the immunotoxin; however, the three-dimensional structure of two repeats of linker had a more appropriate structure compared to others with one and three linker replications. In addition, with enhancing the number of linkers, the probability of protein solubility has increased. Generally, the bioinformatics results of DT389-(GGGGS)2-YP7 structure showed that expression and folding is suitable; and YP7 scFv has appropriate orientation to bind GPC3. The experimental investigations indicated that the fusion protein was expressed as near to 50% soluble. Due to the high binding affinity of YP7 scFv and the proven potency of diphtheria in inhibiting protein synthesis, the proposed DT389-(GGGGS)2-YP7 immunotoxin is expected to function well in inhibiting HCC.
Insights
This study developed a novel immunotoxin for hepatocellular carcinoma (HCC) treatment. The optimal linker length was determined to enhance protein solubility and binding for targeted cancer therapy.
Area of Science:
- Oncology
- Biotechnology
- Molecular Biology
Background:
- Targeted therapy represents a novel approach in cancer treatment, complementing traditional methods like chemotherapy and radiotherapy.
- Immunotoxins are engineered proteins combining a toxin payload with an antigen-binding moiety for specific cancer cell targeting.
- Hepatocellular carcinoma (HCC) expresses the oncofetal antigen Glypican 3 (GPC3) on its surface, making it a potential target for immunotoxin therapy.
Purpose of the Study:
- To investigate the optimal linker length for fusing truncated Diphtheria toxin (DT389) to the humanized scFv YP7 antibody fragment.
- To evaluate the in-silico and experimental characteristics of immunotoxins with varying linker lengths (one, two, or three GGGGS repeats).
- To assess the potential of the optimized immunotoxin, DT389-(GGGGS)2-YP7, for inhibiting HCC growth.
Main Methods:
- In-silico structural analysis of immunotoxin constructs with different linker lengths (DT389-(GGGGS)1-3-YP7).
- Computational assessment of protein structure, solubility, and antigen-binding orientation.
- Experimental expression and solubility analysis of the fusion proteins.
Main Results:
- In-silico analysis indicated that linker length minimally impacted overall structure but suggested two repeats (DT389-(GGGGS)2-YP7) offered a more favorable three-dimensional conformation.
- Increased linker length correlated with enhanced protein solubility, with experimental expression yielding approximately 50% soluble protein.
- Bioinformatic evaluation of DT389-(GGGGS)2-YP7 confirmed suitable expression, folding, and YP7 scFv orientation for GPC3 binding.
Conclusions:
- The DT389-(GGGGS)2-YP7 immunotoxin exhibits promising characteristics for HCC targeted therapy.
- The fusion protein's design facilitates effective binding to GPC3 on HCC cells.
- The combination of Diphtheria toxin's protein synthesis inhibition and YP7 scFv's binding affinity suggests significant therapeutic potential against HCC.
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