Development and evaluation of a novel antibody-photon absorber conjugate reveals the possibility of photoimmunotherapy-induced vascular occlusion during treatment in vivo.

Photodynamic therapy (PDT) utilize a photosensitizing agent and light for cancer therapy. It exerts anti-cancer effect mainly by inducing vascular occlusion at the irradiated site. By controlling the irradiation area, PDT can be used in a tumor-specific manner. However, the non-specific cellular damage in the surrounding normal tissue is still a serious concern. Photoimmunotherapy (PIT) is a new type of targeted cancer therapy that uses an antibody-photon absorber conjugate (APC). The superiority of PIT to PDT is the improved target specificity, thereby reducing the damage to normal tissues. Here, we developed a novel APC targeting epithelial cell adhesion molecule (EpCAM) as well as a negative control APC that does not bind to the EpCAM antigen. Our in vitro analysis of APC cytotoxicity demonstrated that the EpCAM APC, but not the negative control, was cytotoxic to EpCAM expressing COLO 205 cells after photoirradiation, suggesting that the cytotoxicity is antigen-dependent. However, in our in vivo analysis using a mouse xenograft tumor model, decreased volume of the tumors was observed in all the mice treated with irradiation, regardless of whether they were treated with the EpCAM APC or the negative control. Detailed investigation of the mechanism of these in vivo reveal that both APCs induce vascular occlusion at the irradiation site. Furthermore, the level of vascular occlusion was correlated with the blood concentration of APC, not the tumor concentration. These results imply that, similar to PDT, PIT can also induce non-targeted vascular occlusion and further optimization is required before widespread clinical use.

Conformational effects of N-glycan core fucosylation of immunoglobulin G Fc region on its interaction with Fcγ receptor IIIa.

Antibody-dependent cellular cytotoxicity (ADCC) is promoted through interaction between the Fc region of immunoglobulin G1 (IgG1) and Fcγ receptor IIIa (FcγRIIIa), depending on N-glycosylation of these glycoproteins. In particular, core fucosylation of IgG1-Fc N-glycans negatively affects this interaction and thereby compromises ADCC activity. To address the mechanisms of this effect, we performed replica-exchange molecular dynamics simulations based on crystallographic analysis of a soluble form of FcγRIIIa (sFcγRIIIa) in complex with IgG1-Fc. Our simulation highlights increased conformational fluctuation of the N-glycan at Asn162 of sFcγRIIIa upon fucosylation of IgG1-Fc, consistent with crystallographic data giving no interpretable electron density for this N-glycan, except for the innermost part. The fucose residue disrupts optimum intermolecular carbohydrate-carbohydrate interactions, rendering this sFcγRIIIa glycan distal from the Fc glycan. Moreover, our simulation demonstrates that core fucosylation of IgG1-Fc affects conformational dynamics and rearrangements of surrounding amino acid residues, typified by Tyr296 of IgG1-Fc, which was more extensively involved in the interaction with sFcγRIIIa without Fc core fucosylation. Our findings offer a structural foundation for designing and developing therapeutic antibodies with improved ADCC activity.

Novel anticarcinoembryonic antigen antibody-drug conjugate has antitumor activity in the existence of soluble antigen.

Carcinoembryonic antigen (CEA) is a classic tumor-specific antigen that is overexpressed in several cancers, including gastric cancer. Although some anti-CEA antibodies have been tested, to the best of our knowledge, there are currently no clinically approved anti-CEA antibody therapies. Because of this, we have created the novel anti-CEA antibody, 15-1-32, which exhibits stronger binding to membrane-bound CEA on cancer cells than existing anti-CEA antibodies. 15-1-32 also shows poor affinity for soluble CEA; thus, the binding activity of 15-1-32 to membrane-bound CEA is not influenced by soluble CEA. In addition, we constructed a 15-1-32-monomethyl auristatin E conjugate (15-1-32-vcMMAE) to improve the therapeutic efficacy of 15-1-32. 15-1-32-vcMMAE showed enhanced antitumor activity against gastric cancer cell lines. Unlike with existing anti-CEA antibody therapies, antitumor activity of 15-1-32-vcMMAE was retained in the presence of high concentrations of soluble CEA.

Importance of the Side Chain at Position 296 of Antibody Fc in Interactions with FcγRIIIa and Other Fcγ Receptors.

Antibody-dependent cellular cytotoxicity (ADCC) is an important effector function determining the clinical efficacy of therapeutic antibodies. Core fucose removal from N-glycans on the Fc portion of immunoglobulin G (IgG) improves the binding affinity for Fcγ receptor IIIa (FcγRIIIa) and dramatically enhances ADCC. Our previous structural analyses revealed that Tyr-296 of IgG1-Fc plays a critical role in the interaction with FcγRIIIa, particularly in the enhanced FcγRIIIa binding of nonfucosylated IgG1. However, the importance of the Tyr-296 residue in the antibody in the interaction with various Fcγ receptors has not yet been elucidated. To further clarify the biological importance of this residue, we established comprehensive Tyr-296 mutants as fucosylated and nonfucosylated anti-CD20 IgG1s rituximab variants and examined their binding to recombinant soluble human Fcγ receptors: shFcγRI, shFcγRIIa, shFcγRIIIa, and shFcγRIIIb. Some of the mutations affected the binding of antibody to not only shFcγRIIIa but also shFcγRIIa and shFcγRIIIb, suggesting that the Tyr-296 residue in the antibody was also involved in interactions with FcγRIIa and FcγRIIIb. For FcγRIIIa binding, almost all Tyr-296 variants showed lower binding affinities than the wild-type antibody, irrespective of their core fucosylation, particularly in Y296K and Y296P. Notably, only the Y296W mutant showed improved binding to FcγRIIIa. The 3.00 Å-resolution crystal structure of the nonfucosylated Y296W mutant in complex with shFcγRIIIa harboring two N-glycans revealed that the Tyr-to-Trp substitution increased the number of potential contact atoms in the complex, thus improving the binding of the antibody to shFcγRIIIa. The nonfucosylated Y296W mutant retained high ADCC activity, relative to the nonfucosylated wild-type IgG1, and showed greater binding affinity for FcγRIIa. Our data may improve our understanding of the biological importance of human IgG1-Fc Tyr-296 in interactions with various Fcγ receptors, and have applications in the modulation of the IgG1-Fc function of therapeutic antibodies.

Structural basis for improved efficacy of therapeutic antibodies on defucosylation of their Fc glycans.

Removal of the fucose residue from the N-glycans of the Fc portion of immunoglobulin G (IgG) results in a dramatic enhancement of antibody-dependent cellular cytotoxicity (ADCC) through improved affinity for Fcγ receptor IIIa (FcγRIIIa). Here, we present the 2.2-Šstructure of the complex formed between nonfucosylated IgG1-Fc and a soluble form of FcγRIIIa (sFcγRIIIa) with two N-glycosylation sites. The crystal structure shows that one of the two N-glycans of sFcγRIIIa mediates the interaction with nonfucosylated Fc, thereby stabilizing the complex. However, fucosylation of the Fc N-glycans inhibits this interaction, because of steric hindrance, and furthermore, negatively affects the dynamics of the receptor binding site. Our results offer a structural basis for improvement in ADCC of therapeutic antibodies by defucosylation.

Additional carbohydrate-binding modules enhance the insoluble substrate-hydrolytic activity of beta-1,3-glucanase from alkaliphilic Nocardiopsis sp. F96.

Beta-1,3-glucanase (BglF) from Nocardiopsis sp. F96 is composed of only a catalytic domain. To improve the enzymatic properties of BglF, we attempted to construct chimeric enzymes consisting of BglF and some carbohydrate-binding modules, such as the C-terminal additional domain (CAD) and the N-terminal additional domain (NAD) of beta-1,3-glucanase H from Bacillus circulans IAM1165 and the chitin-binding domain (ChBD) of chitinase from alkaliphilic Bacillus sp. J813. CAD-fused BglF (BglF-CAD), NAD-fused BglF (NAD-BglF), both NAD- and CAD-fused BglF (NAD-BglF-CAD) and ChBD-fused BglF (BglF-ChBD) were constructed and characterized. The addition of CAD caused increases in binding abilities and hydrolytic activities toward insoluble beta-1,3-glucans. As well as BglF-CAD, the binding ability and hydrolytic activity of BglF-ChBD toward pachyman were also increased. The hydrolytic activity of BglF-CAD at pH 9-10 was higher than that of BglF. The relative activities of BglF-CAD and BglF-ChBD at around 50-70 degrees C were higher than that of BglF.

Characterization of Nocardiopsis beta-1,3-glucanase with additional carbohydrate-binding domains.

beta-1,3-Glucanase F (BglF) from alkaliphilic Nocardiopsis sp. F96 is a single domain enzyme composed of only a catalytic domain. Chimeric BglFs with some carbohydrate-binding domains were constructed and characterized. By connecting the C-terminal additional domain of beta-1,3-glucanase H from Bacillus circulans IAM1165 and the chitin-binding domain of chitinase J from alkaliphilic Bacillus sp. J813, binding ability and hydrolyzing activity toward insoluble beta-1,3-glucans were both improved.