Antibody-Cell Conjugation: Enhance Targeted Cancer Immunotherapy

Background

Targeted immunotherapy refers to the precise killing of tumors through the specific recognition of cancer cells. For example, the introduction of immune cells into malignant tissue and the eradication of malignant tumor cells by antibodies is known as antibody-dependent cytotoxicity (ADCC). The antibody here refers to the participant who mediates the action of cellular ADCC: the Fab domain of the immunoglobulin G (IgG) antibody binds to antigens on the surface of cancer cells, while the Fc domain binds to Fc receptors on the surface of immune effector cells.

Fig.1 Schematic diagram of the site-selective Tyr reaction.¹

In this study, a chemical enzymatic reaction was developed to specifically functionalize a single tyrosine. Tyrosinase catalyzes the oxidation of tyrosine to o-quinone, which is then subjected to a one-pot reaction with vinyl ether for [3+2] photoaddition. This reaction mounts a fluorescent molecule or bioorthogonal group on the C-terminal Y-tag Y296 of an IgG or engineered nanobody. Tyr-specific reactions are used to construct monofunctionalized antibody-drug conjugates (ADCs) and antibody/nanobody-conjugated effector cells, such as natural killer cells or macrophages.

Research Results

Reaction development (left) and expansion of peptide substrates (right).
Fig.2 Reaction development (left) and expansion of peptide substrates (right).1
Site-selective Tyr reaction of IgGs.
Fig.3 Site-selective Tyr reaction of IgGs.1	Trastuzumab (HER2+ specific) was used for the validation experiments. First, the N297 glycans are removed using PNGase F enzyme pretreatment to expose Y296 in the Fc domain. Then, deglycosylated Tras were incubated with VEN3 (100 equivalent) tyrosinase (0.4 equivalent) under 456 nm blue light irradiation for 8 hours at 4 degrees Celsius. The photoaddition reaction is then confirmed by the covalent bond of the fluorochrome. The SDS-PAGE results showed that the heavy chain of Tras was successfully labeled by the DBCO-TAMRA light chain. The specific targeting ability was demonstrated by flow cytometry and confocal experiments.
Monovalent Tras-Vc-MMAE ADC.
Fig.4 Monovalent Tras-Vc-MMAE ADC.1	Then, through this conjugation strategy, MMAE was ligated and made into an ADC drug for cell viability verification. The results showed significant differences.
Antibody-cell conjugation for immunotherapy.
Fig.5 Antibody-cell conjugation for immunotherapy.1	NK-92 cells are a cytotoxic but non-targeting interleukin-2 (IL2)-dependent natural killer cell line. In this study, nbHER2-BPA was covalently conjugated with NK-92 cells. The cytotoxicity of nbHER2-NK-92 was significantly higher than that of unbound NK-92 cells (Fig.5 D). This enhanced ADCC effect is HER2-dependent because HER2-negative MDA-MB-231 cells are not killed by nbHER2-NK-92 cells. In addition, the same response was applied to trastuzumab. As shown in Fig.5 E-F, the site-selective functionalization of Tras-BPA/AF488 coupled to THP1 cells. The cytotoxicity of TrasNK-92 to SK-OV-3 cells was also significantly higher than that of non-Tras-conjugated NK-92 cells (Fig.5 G).

Conclusion

In summary, nanobodies or antibodies promote the contact between immune effector cells and target cancer cells through site-selective tyrosine reaction, enhancing the killing effect of cancer cells.

Reference

1. Chen Hongfei.; et al. "Site-selective tyrosine reaction for antibody-cell conjugation and targeted immunotherapy." Advanced Science. (2023).