Induction of ADCC by a folic acid-mAb conjugate prepared by tryptophan-selective reaction toward folate-receptor-positive cancer cells

Abstract

We developed conjugates between monoclonal antibody (mAb) and folic acid (FA) by using a tryptophan (Trp)-selective reaction, which yields relatively homogenous products compared to conventional methods. The obtained mAb-FA conjugates showed significant cellular cytotoxicity toward folate receptor-expressing cancer cells, demonstrating that the conjugates retained the Fc region's original function.

Fig. 1. Crystal structures of the Fc region of IgG1 with Trp residues shown in red. Each chain is shown in green and light green colors. Recognition residues of the three proteins are shown in different colors: magenta (FcγRIIIa, CD16a), cyan (C1q), blue (overlapped residues of FcγRIIIa and C1q), and orange (FcRn). PDB ID: 1E4K.

Fig. 2. The structures of the mAb–FA conjugates via Trp reside (A) and schematic illustration of ADCC induction by the mAb–FA conjugates which bind to the FR-α on a cancer cell via FA (B).

Fig. 3. Preparation of the mAb–FA conjugates.

Fig. 4. mAb–FA binds to the FR-α expressed on IGROV-1 cells. (A) IGROV-1 cells were incubated with anti-CD20–FA conjugates with different linker lengths (PEG, n = 4, 8, 12). After a washing step, FITC-labelled anti-IgG Fc polyclonal antibody was added to each sample, and the median of fluorescent intensity (FI) was measured using flow cytometry. (n = 3, mean ± SEM.) (B) IGROV-1 cells were seeded and incubated overnight. mAb, mAb–FA, or mAb–FA + excess FA were added. After washing, mAb–FA bound to the cells was detected using anti-IgG-Fc polyclonal IgG. Cells were also stained with Hoechst 33342. (C) IGROV-1 cells were treated with increasing concentrations of mAb–FA, and the antibodies bound to the cells were quantified. (n = 3, mean ± SEM.)

Fig. 5. mAb–FA induces ADCC against FR-α⁺ IGROV-1 cells. (A) IGROV-1 cells were treated with mAb (10 nM), mAb–FA (10 nM), or mAb–FA (10 nM) + FA (1 μM) and co-cultured with KHYG-1/CD16a-158V for 16 h. Cellular cytotoxicity was quantified by an LDH assay (n = 3, mean ± SEM). A statistically significant difference between “mAb–FA” and the others was observed at all effector/target ratios. (B) After co-culturing, culture supernatants were collected for detection of IFN-γ by ELISA (E/T = 4, n = 3, mean ± SEM). Statistical analyses were carried out using two-tailed Student's t-test or Welche's t-test depending on the result of F-test. **p < 0.01, ***p < 0.005.