CD BioGlyco has many years of experience in Carbohydrate-based Vaccine Development. We have successfully produced diverse types of antigens. 9-O-Acetyl-GD3 has been identified as a target for immune intervention due to its selective expression in malignant melanoma cells. This antigen is predominantly found on malignant melanoma cells in adult humans and offers a high likelihood of being recognized by antibodies due to its abundant presence. This antigen shows promise for immune-based therapies aimed at combating melanoma. Therefore, we produce it and conjugate it with carriers to help clients research the vaccine against malignant melanoma.

Fig.1 Structure of the ganglioside 9-O-acetyl-GD3. (Ritter, et al., 1995)

Tumor-Associated 9-O-Acetyl-GD3 Ganglioside Antigen Production Service at CD BioGlyco

• CD BioGlyco provides 9-O-acetyl-GD3 ganglioside synthesis by chemoenzymatic strategy by converting GD3 to 9-O-acetyl-GD3. We employ the conveniently accessible serine protease subtilisin to achieve the specific O-acetylation of GD3. In combination with enol esters, serine esterases, and proteases have been effectively utilized to accomplish regioselective acylations of carbohydrates that are challenging to achieve through chemical means. The use of subtilisin with DMF as a solvent has proven to be highly beneficial for acylating oligosaccharides.
• Initially, we investigate the enzymatic acetylation of Neu5Ac. It serves as the terminal saccharide unit in GD3 and is also a crucial recognition element on cell surfaces. The best results are achieved by carrying out the reactions at 37 °C in DMF with small quantities of buffer and triethylamine. These conditions have previously been observed to enhance the reaction rate of subtilisin-catalyzed reactions in organic solvents. This is also important for achieving good yields and increased reaction rates in the acetylation of Neu5Ac as well.
• We treat GD3 with similar reaction conditions. This ganglioside is also proving to be a substrate for subtilisin-catalyzed acetylation, yielding the desired 9-O-acetyl-GD3 along with smaller amounts of GD3 acetylated at non-terminal carbohydrate moieties. The choice of solvent system continues to exert a significant influence on the acetylation process. Insufficient triethylamine leads to slow reactions and low yields, while an excess of triethylamine results in polyacetylation of GD3, as it acetylates more than one primary hydroxyl group.

Fig.2 Enzymatic acetylation of GD3. (CD BioGlyco)

In order to enhance their immunogenicity, we employ a method of converting gangliosides into thymodependent antigens. This is achieved by incorporating purified gangliosides into heterologous very low-density lipoproteins (VLDL). Besides, we add recombinant interleukin 2 to further enhance the induction of an antibody response. Subsequently, we determine the antibody response by using ELISA and thin-layer chromatography (TLC) immunostaining. By this approach, we find that the immunogenicity of O-acetyl-GD3 in mice is enhanced.

Applications

Advantages

• By employing enol esters and serine esterases, we achieve regioselective acylation of carbohydrates. This is particularly useful for acylating challenging carbohydrate structures, which are difficult to achieve through traditional chemical means.
• The solvent system we chose, enhances the reaction rate of subtilisin-catalyzed reactions in organic solvents.
• Our chemoenzymatic strategy allows for specific and efficient O-acetylation of GD3 using the serine protease subtilisin, which offers several advantages over chemical methods.

Frequently Asked Question

• What are the functions of 9-O-acetyl-GD3 in organisms?

9-O-Acetyl GD3 ganglioside is an acetylated glycolipid that is found in the cellular membrane of a wide range of vertebrate cells. This ganglioside plays a vital role in the nervous system, including but not limited to cell adhesion to the extracellular matrix, regulation of neuronal migration, facilitation of neurite outgrowth in laboratory settings, and control of apoptosis. It is closely involved in the processes of nervous system development, differentiation, and regeneration. It is significantly upregulated in the regenerating nerve, as well as in the dorsal root ganglia and lumbar spinal cord.

CD BioGlyco, a renowned company specializing in glycobiology, offers a specialized Tumor-associated Ganglioside Antigen Production Service. We have a team of experienced researchers equipped with state-of-the-art technologies. We provide tailored solutions for the isolation, purification, and characterization of these antigens. If our proposition intrigues you, we invite you to contact us for additional information and to initiate discussions.

Reference

1. Ritter, G.; et al. Analysis of the antibody response to immunization with purified O-acetyl GD3 gangliosides in patients with malignant melanoma. International journal of cancer. 1995, 62(6): 668-672.