Engineered Human Antibody with Improved Endothelin Receptor Type A Binding Affinity, Developability, and Serum Persistence Exhibits Excellent Antitumor Potency.

Endothelin receptor A (ETA), a class A G protein-coupled receptor (GPCR), is a promising tumor-associated antigen due to its close association with the progression and metastasis of many types of cancer, such as colorectal, breast, lung, ovarian, and prostate cancer. However, only small-molecule drugs have been developed as ETA antagonists with anticancer effects. In a previous study, we identified an antibody (AG8) with highly selective binding to human ETA through screening of a human naïve immune antibody library. Although both in vitro and in vivo experiments indicated that the identified AG8 had anticancer effects, there is a need for improvement in biochemical and physicochemical properties such as the ETA binding affinity, thermostability, and productivity. In this study, we engineered the framework regions of AG8 and isolated an anti-ETA antibody (MJF1) exhibiting significantly improved thermostability and ETA binding affinity. Subsequently, our previously isolated PFc29, an Fc variant with an enhanced pH-dependent human FcRn binding profile, was introduced to MJF1, and the resulting Fc-engineered anti-ETA antibody (MJF1-PFc29) inhibited the proliferation of tumor cells comparably to MJF1 and showed a 4.2-fold increased serum half-life in human FcRn transgenic mice. Moreover, MJF1-PFc29 elicited higher tumor growth inhibition in colorectal cancer xenograft mice compared to MJF1. Our results demonstrate that the engineered human anti-ETA antibody MJF1-PFc29 has great therapeutic potential and high antitumor potency against various types of cancers including colorectal cancer.

Antigen Design for Successful Isolation of Highly Challenging Therapeutic Anti-GPCR Antibodies.

G-protein-coupled receptors (GPCR) transmit extracellular signals into cells to regulate a variety of cellular functions and are closely related to the homeostasis of the human body and the progression of various types of diseases. Great attention has been paid to GPCRs as excellent drug targets, and there are many commercially available small-molecule chemical drugs against GPCRs. Despite this, the development of therapeutic anti-GPCR antibodies has been delayed and is challenging due to the difficulty in preparing active forms of GPCR antigens, resulting from their low cellular expression and complex structures. Here, we focus on anti-GPCR antibodies that have been approved or are subject to clinical trials and present various technologies to prepare active GPCR antigens that enable the isolation of therapeutic antibodies to proceed toward clinical validation.

A human antibody against human endothelin receptor type A that exhibits antitumor potency.

Endothelin receptor A (ETA), a class A G-protein-coupled receptor (GPCR), is involved in the progression and metastasis of colorectal, breast, lung, ovarian, and prostate cancer. We overexpressed and purified human endothelin receptor type A in Escherichia coli and reconstituted it with lipid and membrane scaffold proteins to prepare an ETA nanodisc as a functional antigen with a structure similar to that of native GPCR. By screening a human naive immune single-chain variable fragment phage library constructed in-house, we successfully isolated a human anti-ETA antibody (AG8) exhibiting high specificity for ETA in the ß-arrestin Tango assay and effective inhibitory activity against the ET-1-induced signaling cascade via ETA using either a CHO-K1 cell line stably expressing human ETA or HT-29 colorectal cancer cells, in which AG8 exhibited IC50 values of 56 and 51 nM, respectively. In addition, AG8 treatment repressed the transcription of inhibin ßA and reduced the ETA-induced phosphorylation of protein kinase B and extracellular regulated kinase. Furthermore, tumor growth was effectively inhibited by AG8 in a colorectal cancer mouse xenograft model. The human anti-ETA antibody isolated in this study could be used as a potential therapeutic for cancers, including colorectal cancer.

Structural consequences of aglycosylated IgG Fc variants evolved for FcγRI binding.

In contrast to the glycosylated IgG antibodies secreted by human plasma cells, the aglycosylated IgG antibodies produced by bacteria are unable to bind FcγRs expressed on the surface of immune effector cells and cannot trigger immune effector functions. To avoid glycan heterogeneity problems, elicit novel effector functions, and produce therapeutic antibodies with effector function using a simple bacterial expression system, FcγRI-specific Fc-engineered aglycosylated antibodies, Fc11 (E382V) and Fc (E382V/M428I), containing mutations in the CH3 region, were isolated in a previous study. To elucidate the relationship between FcγRI binding affinity and the structural dynamics of the upper CH2 region of Fc induced by the CH3 mutations, the conformational variation of Fc variants was observed by single-molecule Förster resonance energy transfer (FRET) analysis using alternating-laser excitation (ALEX). In sharp contrast to wild-type Fc, which exhibits a highly dynamic upper CH2 region, the mutations in the CH3 region significantly stabilized the upper CH2 region. The results indicate that conformational plasticity, as well as the openness of the upper CH2 region, is critical for FcγR binding and therapeutic effector functions of IgG antibodies.

Aglycosylated full-length IgG antibodies: steps toward next-generation immunotherapeutics.

Albeit the removal of Asn297 glycans of IgG perturbs the overall conformation and flexibility of the IgG CH2 domain, resulting in the loss of Fc-ligand interactions and therapeutically critical immune effector functions, aglycosylated full-length IgG antibodies are nearly identical to the glycosylated counterparts in terms of antigen binding, stability at physiological or low temperature conditions, pharmacokinetics, and biodistribution. To bypass the drawbacks of glycosylated antibodies that include glycan heterogeneity and requirement of high capital investment for biomanufacturing, aglycosylated antibodies have been developed and several are under clinical trials. Comprehensive cellular and bioprocess engineering has enabled to produce highly complex aglycosylated IgGs in a simple bacterial cultivation with comparable production level as that of mammalian cells. Moreover, extensive engineering of aglycosylated Fc has converted the aglycosylated IgG antibodies into a new class of effector functional human immunotherapeutics.