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.

Glycan-Controlled Human PD-1 Variants Displaying Broad-Spectrum High Binding to PD-1 Ligands Potentiate T Cell.

Although therapeutic immunoglobulin G (IgG) antibodies that regulate the activity of immune checkpoints bring innovation to the field of immuno-oncology, they are still limited in their efficiency to infiltrate the tumor microenvironment due to their large molecular size (150 kDa) and the necessity of additional engineering works to ablate effector functions for antibodies targeting immune cells. To address these issues, the human PD-1 (hPD-1) ectodomain, a small protein moiety of 14-17 kDa, has been considered as a therapeutic agent. Here, we used bacterial display-based high-throughput directed evolution to successfully isolate glycan-controlled (aglycosylated or only single-N-linked glycosylated) human PD-1 variants exhibiting over 1000-fold increased hPD-L1 binding affinity compared to that of wild-type hPD-1. The resulting hPD-1 variants, aglycosylated JYQ12 and JYQ12-2 with a single-N-linked glycan chain, showed exceptionally high binding affinity to hPD-L1 and very high affinity to both hPD-L2 and mPD-L1. Moreover, the JYQ12-2 efficiently potentiated the proliferation of human T cells. hPD-1 variants with significantly improved binding affinities for hPD-1 ligands could be used as effective therapeutics or diagnostics that can be differentiated from large-sized IgG antibody-based molecules.

Selection and Characterization of YKL-40-Targeting Monoclonal Antibodies from Human Synthetic Fab Phage Display Libraries.

YKL-40, also known as chitinase-3-like 1 (CHI3L1), is a glycoprotein that is expressed and secreted by various cell types, including cancers and macrophages. Due to its implications for and upregulation in a variety of diseases, including inflammatory conditions, fibrotic disorders, and tumor growth, YKL-40 has been considered as a significant therapeutic biomarker. Here, we used a phage display to develop novel monoclonal antibodies (mAbs) targeting human YKL-40 (hYKL-40). Human synthetic antibody phage display libraries were panned against a recombinant hYKL-40 protein, yielding seven unique Fabs (Antigen-binding fragment), of which two Fabs (H1 and H2) were non-aggregating and thermally stable (75.5 °C and 76.5 °C, respectively) and had high apparent affinities (KD = 2.3 nM and 4.0 nM, respectively). Reformatting the Fabs into IgGs (Immunoglobulin Gs) increased their apparent affinities (notably, for H1 and H2, KD = 0.5 nM and 0.3 nM, respectively), presumably due to the effects of avidity, with little change to their non-aggregation property. The six anti-hYKL-40 IgGs were analyzed using a trans-well migration assay in vitro, revealing that three clones (H1, H2, and H4) were notably effective in reducing cell migration from both A549 and H460 lung cancer cell lines. The three clones were further analyzed in an in vivo animal test that assessed their anti-cancer activities, demonstrating that the tumor area and the number of tumor nodules were significantly reduced in the lung tissues treated with H1 (IgG). Given its high affinity and desirable properties, we expect that the H1 anti-hYKL-40 mAb will be a suitable candidate for developing anti-cancer therapeutics.

Effective clearance of rituximab-resistant tumor cells by breaking the mirror-symmetry of immunoglobulin G and simultaneous binding to CD55 and CD20.

Complement-dependent cytotoxicity (CDC), which eliminates aberrant target cells through the assembly and complex formation of serum complement molecules, is one of the major effector functions of anticancer therapeutic antibodies. In this study, we discovered that breaking the symmetry of natural immunoglobulin G (IgG) antibodies significantly increased the CDC activity of anti-CD20 antibodies. In addition, the expression of CD55 (a checkpoint inhibitor in the CDC cascade) was significantly increased in a rituximab-resistant cell line generated in-house, suggesting that CD55 overexpression might be a mechanism by which cancer cells acquire rituximab resistance. Based on these findings, we developed an asymmetric bispecific antibody (SBU-CD55 × CD20) that simultaneously targets both CD55 and CD20 to effectively eliminate rituximab-resistant cancer cells. In various cancer cell lines, including rituximab-resistant lymphoma cells, the SBU-CD55 × CD20 antibody showed significantly higher CDC activity than either anti-CD20 IgG antibody alone or a combination of anti-CD20 IgG antibody and anti-CD55 IgG antibody. Furthermore, the asymmetric bispecific antibody (SBU-CD55 × CD20) exhibited significantly higher CDC activity against rituximab-resistant cancer cells compared to other bispecific antibodies with symmetric features. These results demonstrate that enhancing CDC with an asymmetric CD55-binding bispecific antibody could be a new strategy for developing therapeutics to treat patients with relapsed or refractory cancers.

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.

Validation of Rapi-Fluor method for glycan profiling and application to commercial antibody drugs.

N-glycans influence the activity of antibody drugs such as antibody-dependent cell-mediated cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC). Thus, glycan profiling is considered a critical quality attribute (CQA) and requires routine and comprehensive monitoring. In this report, we validate the new glycan profiling method called Rapi-Fluor method, which reduced the sample preparation time and increased the FLR and MS intensities compared with conventional 2-AB method. Optimized glycan release, labeling, hydrophilic interaction liquid chromatography (HILIC) enrichment, and HILIC separation resulted in low variation and short preparation time. The method evaluated for human IgG standard varied from 100 µg/mL to 4000 µg/mL in 25 µL of water. The determination of coefficient (r2 > 0.9992), recovery (88.992% ~ 111.198%), limit of detection (LOD < 193.274 µg/mL), limit of quantification (LOQ < 585.679 µg/mL), and precision (Intra-day < 2.317%RSD and Inter-day < 4.287%RSD) were evaluated with four major glycans from antibody drugs. In addition, the method was used for glycan profiling of five different commercial antibodies. The method yielded precise results for IgG glycan analysis and demonstrated effective glycan profiling of commercial antibody drugs.

Optimal combination of beneficial mutations for improved ADCC effector function of aglycosylated antibodies.

The Fc region of IgG antibodies is crucial for binding to Fc receptors expressed on the surfaces of various immune leukocytes and eliciting therapeutic effector functions such as clearance of antibody-opsonized tumor cells. Despite abrogated Fc gamma receptor (FcγR) binding and therapeutic effector function in the absence of N-linked glycosylation at Asn297, the aglycosylated Fc region of IgG antibodies has bioprocessing advantages such as the absence of glycan heterogeneity and simple bacterial antibody production. Therefore, these antibodies have been comprehensively engineered as effector functional units for human therapy. In this work, we constructed a huge library of Fc variants with combinations of 25 beneficial mutations that were previously identified to improve binding of glycosylated or aglycosylated Fc regions to human FcγRs in previous studies. High-throughput screening of the resulting library led to the identification of an aglycosylated Fc variant that exhibited almost double the antibody-dependent cell-mediated cytotoxicity than wild-type glycosylated Fc. All mutations in this aglycosylated Fc variant were derived from previously identified beneficial mutations for engineered aglycosylated Fc variants as opposed to glycosylated variants, suggesting that significantly different sets of beneficial mutations are necessary to improve the effector function of aglycosylated Fc.