Development of Antibody-recruiting Strategy to Enhance the Immune Responses

概要

Antibody-recruiting molecules (ARMs) are bifunctional molecules capable of recruiting endogenous antibodies that are naturally found in human blood stream to induce immune responses against disease-relevant proteins, cells, or organisms, ARMs typically consist of two covalently-linked units: target-binding terminus (TBT), possessing the ability to recognize disease-associated protein target, and antibody-binding terminus (ABT), binding to endogenous antibodies. Simultaneous interaction of TBT to cell surface receptors and ABT to antibodies allows the formation of a ternary complex of target cells, ARMs and antibodies, subsequently eliciting antibody-dependent immune responses to promote cell destruction by various mechanisms.

In this report, the author investigated the antibody-recruiting strategy using the interaction between carbohydrate antigens and their corresponding antibodies to develop a potential novel cancer immunotherapy with high selectivity and potency. The development of antibody-recruiting molecules (ARM) using human serum albumin (HSA) as protein scaffold were described in this research, as well as the synthesis of α-gal epitope. The synthesis of HSA-based ARM with α-gal epitope as ABT and sialyl glycan as TBT was successfully achieved. However, the binding affinity between the synthesized HSA conjugate and lymphoma cells was inadequate to induce immune responses. These results attributed to the weak non-covalent trans interaction between Siglec-2 and sialyl glycan and the fierce competition with cis interaction.

An alternative strategy has been developed to attach ABT onto the membrane of target cells. Metabolic labeling, an effective and facile method to label cell membrane, has been employed in this research to covalently bind α-gal, B antigen, and rhamnose onto the surface of Raji cells. The display of α-gal, B antigen, and rhamnose on the surface of Raji cells has successfully recruit their natural antibodies to the target cells to induce cytotoxicity through Complement- Dependent Cytotoxicity (CDC). Based on CDC assay result, rhamnose induced an extremely high cytotoxicity, compared to α-gal and B antigen. In addition, the position of azide group expression in glycocalyx was also investigated and revealed to influence the CDC activity. Azide introduction on non-reducing terminus using Ac4ManNAz showed higher CDC activity compared to reducing terminus using Ac4GalNAz.

Metabolic glycan labeling, however, can take place in both healthy and cancerous cells. Therefore, to achieve cancer cell selective antibody recruitment, the caging of carbohydrate antigen was investigated. Rha-DBCO was successfully modified with the photocaging group, 2-nitrobenzyl group, to give caged-rha-DBCO. This photocaging-antigen strategy is expected to prevent the trapping of natural antibody in blood stream. Moreover, it can also be used to markedly improved targeting efficiency of immune responses by the combination application with cancer-targeted metabolic labeling.