Obtaining and characterizing rhodamine B immunoconjugates

BACKGROUND: Monoclonal antibody (mAb)–drug immunoconjugates are a promising option in cancer immunotherapy. However, developing effective techniques of their synthesis remains relevant, especially when a small molecule has no mAb binding sites.

AIM: The work aimed to synthesize immunoconjugates based on two modified rhodamine B molecules and four mAbs, and to examine their molecular and functional properties.

METHODS: mAbs were conjugated to Rod-SMCC via reduced disulfide groups of immunoglobulin G (IgG). Rod-piperazine was added to oxidized IgG oligosaccharides. The synthesis was assessed using spectrophotometry. The activity of the resulting conjugates was assessed using enzyme-linked immunosorbent assay. The molecular heterogeneity of the resulting conjugates was assessed using high-performance chromatography.

RESULTS: Rod-SMCC produced conjugates with all examined antibodies via thiol groups, with maximum saturation for each antibody. The maximum incorporation rate of approximately 10.96 ± 1.3 mol Rod per 1 mol IgG was observed for ICO 204, with approximately 5.19 ± 1.75 for trastuzumab. These Rod incorporation rates decreased after additional fractionation of the resulting immunoconjugates on a separating gel, amounting to 7.94 ± 1.8 for ICO 204 and 3.32 ± 1.2 for trastuzumab. The antigen-binding activity of ICO 204 immunoconjugates was comparable to that of native antibodies. The incorporation of Rod in immunoconjugates depended on the mAb fractionation technique. Acid elution during mAb fractionation on protein G decreased the incorporation of Rod in immunoconjugates. Rod-piperazine produced immunoconjugates with ICO 204 and trastuzumab via oxidized carbohydrate residues of antibodies. The maximum incorporation rate of rhodamine B in ICO 204 was 2.3 ± 0.3 mol per 1 mol IgG, with 0.2 ± 0.12 for trastuzumab.

CONCLUSION: Chemical synthesis was used to produce two derivatives of Rod, which initially had no IgG binding sites. These compounds were used to synthesize model immunoconjugates with high Rod incorporation rates while maintaining antigen-binding activity. This work demonstrated that immunoconjugates can be produced using pharmaceutical substances with carboxyl groups that are unsuitable for such synthesis. Furthermore, it was shown that immunoconjugates can be synthesized through carboxyl group modification of pharmaceutical substances using piperazine and SMCC.