An epidermal growth factor receptor-targeting immunotoxin based on IgG shows potent antitumor activity against head and neck cancer.

The epidermal growth factor receptor (EGFR) is an important target for cancer therapies. Many head and neck cancer (HNC) cells have been reported to overexpress EGFR; therefore, anti-EGFR therapies have been attempted in patients with HNC. However, its clinical efficacy is limited owing to the development of drug resistance. In this study, we developed an EGFR-targeting immunotoxin consisting of a clinically proven anti-EGFR IgG (cetuximab; CTX) and a toxin fragment (LR-LO10) derived from Pseudomonas exotoxin A (PE) using a novel site-specific conjugation technology (peptide-directed photo-crosslinking reaction), as an alternative option. The immunotoxin (CTX-LR-LO10) showed specific binding to EGFR and properties of a typical IgG, such as stability, interactions with receptors of immune cells, and pharmacokinetics, and inhibited protein synthesis via modification of elongation factor-2. Treatment of EGFR-positive HNC cells with the immunotoxin resulted in apoptotic cell death and the inhibition of cell migration and invasion. The efficacy of CTX-LR-LO10 was evaluated in xenograft mouse models, and the immunotoxin exhibited much stronger tumor suppression than CTX or LR-LO10. Transcriptome analyses revealed that the immunotoxins elicited immune responses and altered the expression of genes related to its mechanisms of action. These results support the notion that CTX-LR-LO10 may serve as a new therapeutic agent targeting EGFR-positive cancers.

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.

Engineered human FcγRIIa fusion: A novel strategy to extend serum half-life of therapeutic proteins.

The immunoglobulin G (IgG) molecule has a long circulating serum half-life (~3 weeks) through pH- dependent FcRn binding-mediated recycling. To hijack the intracellular trafficking and recycling mechanism of IgG as a way to extend serum persistence of non-antibody therapeutic proteins, we have evolved the ectodomain of a low-affinity human FcγRIIa for enhanced binding to the lower hinge and upper CH2 region of IgG, which is very far from the FcRn binding site (CH2-CH3 interface). High-throughput library screening enabled isolation of an FcγRIIa variant (2A45.1) with 32-fold increased binding affinity to human IgG1 Fc (equilibrium dissociation constant: 9.04 × 10-7 M for wild type FcγRIIa and 2.82 × 10-8 M for 2A45.1) and significantly improved affinity to mouse serum IgG compared to wild type human FcγRIIa. The in vivo pharmacokinetic profile of PD-L1 fused with engineered FcγRIIa (PD-L1-2A45.1) was compared with that of PD-L1 fused with wild type FcγRIIa (PD-L1-wild type FcγRIIa) and human PD-L1 in mice. PD-L1-2A45.1 showed 11.7- and 9.7-fold prolonged circulating half-life (t1/2 ) compared to PD-L1 when administered intravenously and intraperitoneally, respectively. In addition, the AUCinf of PD-L1-2A45.1 was two-fold higher compared to that of PD-L1-wild type FcγRIIa. These results demonstrate that engineered FcγRIIa fusion offers a novel and successful strategy for prolonging serum half-life of therapeutic proteins.

An Fc variant with two mutations confers prolonged serum half-life and enhanced effector functions on IgG antibodies.

The pH-selective interaction between the immunoglobulin G (IgG) fragment crystallizable region (Fc region) and the neonatal Fc receptor (FcRn) is critical for prolonging the circulating half-lives of IgG molecules through intracellular trafficking and recycling. By using directed evolution, we successfully identified Fc mutations that improve the pH-dependent binding of human FcRn and prolong the serum persistence of a model IgG antibody and an Fc-fusion protein. Strikingly, trastuzumab-PFc29 and aflibercept-PFc29, a model therapeutic IgG antibody and an Fc-fusion protein, respectively, when combined with our engineered Fc (Q311R/M428L), both exhibited significantly higher serum half-lives in human FcRn transgenic mice than their counterparts with wild-type Fc. Moreover, in a cynomolgus monkey model, trastuzumab-PFc29 displayed a superior pharmacokinetic profile to that of both trastuzumab-YTE and trastuzumab-LS, which contain the well-validated serum half-life extension Fcs YTE (M252Y/S254T/T256E) and LS (M428L/N434S), respectively. Furthermore, the introduction of two identified mutations of PFc29 (Q311R/M428L) into the model antibodies enhanced both complement-dependent cytotoxicity and antibody-dependent cell-mediated cytotoxicity activity, which are triggered by the association between IgG Fc and Fc binding ligands and are critical for clearing cancer cells. In addition, the effector functions could be turned off by combining the two mutations of PFc29 with effector function-silencing mutations, but the antibodies maintained their excellent pH-dependent human FcRn binding profile. We expect our Fc variants to be an excellent tool for enhancing the pharmacokinetic profiles and potencies of various therapeutic antibodies and Fc-fusion proteins.

Recent Achievements and Challenges in Prolonging the Serum Half-Lives of Therapeutic IgG Antibodies Through Fc Engineering.

Association of FcRn molecules to the Fc region of IgG in acidified endosomes and subsequent dissociation of the interaction in neutral pH serum enables IgG molecules to be recycled for prolonged serum persistence after internalization by endothelial cells, rather than being degraded in the serum and in the lysosomes inside the cells. Exploiting this intracellular trafficking and recycling mechanism, many researchers have engineered the Fc region to further extend the serum half-lives of therapeutic antibodies by optimizing the pH-dependent IgG Fc-FcRn interaction, and have generated various Fc variants exhibiting significantly improved circulating half-lives of therapeutic IgG antibodies. In order to estimate pharmacokinetic profiles of IgG Fc variants in human serum, not only a variety of in vitro techniques to determine the equilibrium binding constants and instantaneous rate constants for pH-dependent FcRn binding, but also diverse in vivo animal models including wild-type mouse, human FcRn transgenic mouse (Tg32 and Tg276), humanized mouse (Scarlet), or cynomolgus monkey have been harnessed. Currently, multiple IgG Fc variants that have been validated for their prolonged therapeutic potency in preclinical models have been successfully entered into human clinical trials for cancer, infectious diseases, and autoimmune diseases.

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.

Correction: A short PEG linker alters the in vivo pharmacokinetics of trastuzumab to yield high-contrast immuno-PET images.

Correction for 'A short PEG linker alters the in vivo pharmacokinetics of trastuzumab to yield high-contrast immuno-PET images' by Woonghee Lee et al., J. Mater. Chem. B, 2021, 9, 2993-2997, DOI: 10.1039/D0TB02911D.

A short PEG linker alters the in vivo pharmacokinetics of trastuzumab to yield high-contrast immuno-PET images.

The prolonged blood circulation of the radiolabeled antibody conjugates is problematic when using immuno-PET imaging due to the increased radiation exposure and longer hospitalization required until sufficient contrast develops. In contrast to the prevailing belief that PEGylation prolongs blood retention time, we observed that a PEGylated antibody with a short PEG8 linker cleared much faster from the blood while maintaining tumor uptake compared to its non-PEGylated counterpart. Breast tumors were clearly visualized with a very high tumor-to-background ratio as early as 24 h after injection in immuno-positron emission tomography (PET) imaging.

Discovery of Novel Pseudomonas putida Flavin-Binding Fluorescent Protein Variants with Significantly Improved Quantum Yield.

Oxygen-independent, flavin-binding fluorescent proteins (FbFPs) are emerging as alternatives to green fluorescent protein (GFP), which has limited applicability in studying anaerobic microorganisms, such as human gastrointestinal bacteria, which grow in oxygen-deficient environments. However, the utility of these FbFPs has been compromised because of their poor fluorescence emission. To overcome this limitation, we have employed a high-throughput library screening strategy and engineered an FbFP derived from Pseudomonas putida (SB2) for enhanced quantum yield. Of the resulting SB2 variants, KOFP-7 exhibited a significantly improved quantum yield (0.61) compared to other reported engineered FbFPs, which was even higher than that of enhanced GFP (EGFP, 0.60), with significantly enhanced tolerance against a strong reducing agent.

Effect of dietary calcium and 1,25-(OH)2D3 on the expression of calcium transport genes in calbindin-D9k and -D28k double knockout mice.

The phenotypes of calbindin-D9k (CaBP-9k) and -28k (CaBP-28k) single knockout (KO) mice are similar to wild-type (WT) mice due to the compensatory action of other calcium transport proteins. In this study, we generated CaBP-9k/CaBP-28k double knockout (DKO) mice in order to investigate the importance of CaBP-9k and CaBP-28k in active calcium processing. Under normal dietary conditions, DKO mice did not exhibit any changes in phenotype or the expression of active calcium transport genes as compared to WT or CaBP-28k KO mice. Under calcium-deficient dietary conditions, the phenotype and expression of calcium transport genes in CaBP-28k KO mice were similar to WT, whereas in DKO mice, serum calcium levels and bone length were decreased. The intestinal and renal expression of transient receptor potential vanilloid member 6 (TRPV6) mRNA was significantly decreased in DKO mice fed a calcium-deficient diet as compared to CaBP-28k KO or WT mice, and DKO mice died after 4 weeks on a calcium-deficient diet. Body weight, bone mineral density (BMD) and bone length were significantly reduced in all mice fed a calcium and 1,25-(OH)(2)D(3)-deficient diet, as compared to a normal diet, and none of the mice survived more than 4 weeks. These results indicate that deletion of CaBP-28k alone does not affect body calcium homeostasis, but that deletion of CaBP-9k and CaBP-28k has a significant effect on calcium processing under calcium-deficient conditions, confirming the importance of dietary calcium and 1,25-(OH)(2)D(3) during growth and development.

Engineered Arabidopsis Blue Light Receptor LOV Domain Variants with Improved Quantum Yield, Brightness, and Thermostability.

Despite remarkable contribution of green fluorescent protein and its variants for better understanding of various biological functions, its application for anaerobic microorganisms has been limited because molecular oxygen is essential for chromophore formation. To overcome the limitation, we engineered a plant-derived light, oxygen, or voltage (LOV) domain containing flavin mononucleotide for enhanced spectral properties. The resulting LOV variants exhibited improved fluorescence intensity (20 and 70% higher for SH3 and 70% for BR1, respectively) compared to iLOV, an LOV variant isolated in a previous study, and the quantum yields of the LOV variants (0.40 for SH3 and 0.45 for BR1) were also improved relative to that of iLOV (Q = 0.37). In addition to fluorescence intensity, the identified mutations of SH3 enabled an improved thermostability of the protein. The engineered LOV variants with enhanced spectral properties could provide a valuable tool for fluorescent molecular probes under anaerobic conditions.

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.

The beneficial effect of the sap of Acer mono in an animal with low-calcium diet-induced osteoporosis-like symptoms.

The sap of Acer mono has been called 'bone-benefit-water' in Korea because of its mineral and sugar content. In particular, the calcium concentration of the sap of A. mono is 37.5 times higher than commercial spring water. In the current study, we examined whether A. mono sap could improve or prevent osteoporosis-like symptoms in a mouse model. Male mice (3 weeks old) were fed a low-calcium diet supplemented with 25, 50 or 100 % A. mono sap, commercial spring water or a high calcium-containing solution as a beverage for 7 weeks. There were no differences in weekly weight gain and food intake among all the groups. Mice that were given a low-calcium diet supplemented with commercial spring water developed osteoporosis-like symptoms. To assess the effect of sap on osteoporosis-like symptoms, we examined serum calcium concentration, and femur density and length, and carried out a histological examination. Serum calcium levels were significantly lower in mice that received a low-calcium diet supplemented with commercial spring water (the negative control group), and in the 25 % sap group compared to mice fed a normal diet, but were normal in the 50 and 100 % sap and high-calcium solution groups. Femur density and length were significantly reduced in the negative control and 25 % sap groups. These results indicate that a 50 % sap solution can mitigate osteoporosis-like symptoms induced by a low-calcium diet. We also examined the regulation of expression of calcium-processing genes in the duodenum and kidney. Duodenal TRPV6 and renal calbindin-D9k were up-regulated dose-dependently by sap, and the levels of these factors were higher than those attained in the spring water-treated control. The results demonstrate that the sap of A. mono ameliorates the low bone density induced by a low-calcium diet, most likely by increasing calcium ion absorption.

Determination of the endothelin-1 recognition sites of endothelin receptor type A by the directed-degeneration method.

G-protein coupled receptors (GPCRs) play indispensable physiological roles in cell proliferation, differentiation, and migration; therefore, identifying the mechanisms by which ligands bind to GPCRs is crucial for developing GPCR-targeting pharmaceutics and for understanding critical biological functions. Although some structural information is available regarding the interactions between GPCRs and their small molecule ligands, knowledge of how GPCRs interact with their corresponding macromolecule ligands, such as peptides and proteins, remains elusive. In this study, we have developed a novel strategy to investigate the precise ligand recognition mechanisms involved in the interaction of endothelin receptor type A (ETA) with its ligand, endothelin-1 (ET-1); we call this method "directed degeneration" method. Through flow cytometric screening of a randomized ETA library, statistical analysis of the identified sequences, and biochemical studies, the ligand interaction map was successfully obtained.

Dietary calcium and 1,25-dihydroxyvitamin D3 regulate transcription of calcium transporter genes in calbindin-D9k knockout mice.

The effect(s) of oral calcium and vitamin D(3) were examined on the expression of duodenal and renal active calcium transport genes, i.e., calbindin-D9k (CaBP-9k) and calbindin-D28k (CaBP-28k), transient receptor potential cation channels (TRPV5 and TRPV6), Na(+)/Ca(2+) exchanger 1 (NCX1) and plasma membrane calcium ATPase 1b (PMCA1b), in CaBP-9k KO mice. Wild-type (WT) and KO mice were provided with calcium and vitamin D(3)-deficient diets for 10 weeks. The deficient diet significantly decreased body weights compared with the normal diet groups. The serum calcium concentration of the WT mice was decreased by the deficient diet but was unchanged in the KO mice. The deficient diet significantly increased duodenal transcription of CaBP-9k and TRPV6 in the WT mice, but no alteration was observed in the KO mice. In the kidney, the deficient diet significantly increased renal transcripts of CaBP-9k, TRPV6, PMCA1b, CaBP-28k and TRPV5 in the WT mice but did not alter calcium-relating genes in the KO mice. Two potential mediators of calcium-processing genes, vitamin D receptor (VDR) and parathyroid hormone receptor (PTHR), have been suggested to be useful for elucidating these differential regulations in the calcium-related genes of the KO mice. Expression of VDR was not significantly affected by diet or the KO mutation. Renal PTHR mRNA levels were reduced by the diet, and reduced expression was also seen in the KO mice given the normal diet. Taken together, these results suggest that the active calcium transporting genes in KO mice may have resistance to the deficiency diet of calcium and vitamin D(3).