After HPLC-SQ2 analysis suggested the reaction was completed, the reaction mixture was diluted with 2 mL water. anti-Rha antibody, antibody conjugates, chemoenzymatic synthesis, glycoengineering, immunotherapy Graphical Abstract A highly efficient chemoenzymatic synthesis of homogeneous antibody-rhamnose and antibody-Gal cluster conjugates is described. The synthesis was achieved through a modular antibody Fc-glycan remodeling that includes site-specific chemoenzymatic Fc-glycan functionalization and Tcf4 subsequent click conjugation of synthetic rhamnose- and Gal trisaccharide clusters. A comparative cell-based assay indicated that the antibody-rhamnose cluster conjugates showed potent complement-dependent targeted cancer cell killing by recruiting natural anti-rhamnose antibodies. Introduction Complement-dependent cytotoxicity (CDC) is one of the major mechanisms for antibody-mediated killing of target cells.[1] Nevertheless, many therapeutic antibodies are limited by their low potency in stimulating a strong complement-dependent cytotoxicity. One strategy to achieve complement-dependent targeted cell killing is to explore novel bi-functional molecules consisting of a target-binding motif and a specific antigenic structure to recruit naturally abundant antibodies, such as the anti-Gal and anti-rhamnose (anti-Rha) antibodies to the target cells.[2] The -Gal epitope, Gal1C3Gal1C4GlcNAc-R, is expressed abundantly on glycolipids and glycoproteins in non-human primates and New World monkeys, but it is not present on human cells. As a result, humans produce large quantities of anti-Gal antibodies in circulation: up to 1C2% of total serum IgG and 3C8% of total IgM natural antibodies are KRP-203 KRP-203 anti-Gal KRP-203 antibodies.[3] The interaction between Gal and anti-Gal antibodies are largely responsible for the rejection of the transplanted tissues following xenotransplantation, mainly due to activation of the complement systems.[4] On the other hand, anti-Rha antibodies are also abundant in human sera, which have been found even at higher concentrations than the anti-Gal antibodies in human serum samples.[2b, 5] Moreover, the majority of anti-Rha antibodies are of IgM type, while most of the anti-Gal antibodies are of IgG type.[2b, 6] It has been shown that the IgM type antibody is much more efficient than the monomeric IgG type antibody to initiate complement-dependent cytotoxicity, as the IgMs is inherently a pentamer that can efficiently bind to the multi-subunit C1q complement protein in a multivalent fashion to trigger the downstream effects, while the IgG antibody must form hexamer complex to engage an efficient multivalent interactions with the multi-subunit C1q complement protein.[7] To explore the potential of natural anti-Gal or anti-Rha antibodies for targeting cancer, bacterial and/or virus-infected cells, several groups have previously designed and synthesized bi-functional small molecules, by conjugating a target-binding ligand to an Gal oligosaccharide or a rhamnose moiety, to recruit natural anti-Gal or anti-Rha antibodies for complement-dependent cell killing.[2] For example, Kiessling and co-workers have reported the synthesis of Gal-integrin ligand conjugates for targeting KRP-203 cancer;[2a, 2b] Yi and co-workers have assembled bi-functional liposomes incorporating rhamnose and folic acid to recruit anti-Rha antibodies to target folate receptor-overexpressing tumor cells.[2e] Recently, Fukase and co-workers have reported the first synthesis of Gal oligosaccharide-antibody conjugates, and the cell-based assays have shown that the presentation of multiple copies of the Gal epitopes (multi-valency) was important for an efficient cancer cell killing.[8] While this study provides proof-of-concept data indicating the feasibility of using antibody-Gal conjugates for targeted cancer cell killing, the lysine-based antibody-Gal conjugation and the reduction of antibodies into monomeric antibodies for thiol-maleimide ligation both lead to mixtures of heterogeneous conjugates. We report in this paper a highly efficient chemoenzymatic synthesis and comparative study of structurally well-defined, KRP-203 homogeneous conjugates of an antibody with rhamnose and Gal oligosaccharide clusters, respectively. Trastuzumab, a therapeutic antibody targeting HER2 over-expressing cancer cells, was selected as a model antibody, and the carbohydrate antigens were introduced specifically at the Fc glycosylation site by a chemoenzymatic Fc glycan remodeling approach. The resulting glycoengineered antibodies carrying the rhamnose and Gal oligosaccharide clusters were designed to recruit natural.