A novel monospecific tetravalent IgG1-(scFv)2 version shown enhanced neutralizing and Fc-mediated effector functions against SARS-CoV-2.

Neutralizing monoclonal antibodies (nMABs) have been proved to be effective therapeutics in treating coronavirus disease (COVID-19). To enhance the potency of nMAB 553-15, we generated a novel monospecific tetravalent IgG1-(scFv)2 version. This was achieved by covalently fusing two forms of 553-15-derived single chain variable fragments (scFv) to the C-terminus of the hIgG1 (human Immunoglobulin G1) Fc fragment. We found that the Fc-fused VL-linker-VH format achieved similar binding affinity and neutralizing behavior as 553-15. The tetravalent versions were constructed by fusing the scFv domains to the C-terminus of nMAB 553-15. As a result, the tetravalent version 55,315-VLVH exhibited significantly higher binding activity to target spike protein variants and enhanced neutralization against VOCs (variants of concern) pseudovirus compared to 553-15. We also measured the Fc effector responses of candidates using wild-type Spike-expressing CHOK1 cells. The 55,315-VLVH enhanced the function of ADCP (antibody-dependent cellular phagocytosis) but had similar IL-6 release levels compared to the bivalent 553-15. It seemed that the novel tetravalent version avoids the pro-inflammatory effect induced by macrophage activation. However, the 55,315-VLVH displayed slightly increased potency in ADCC (antibody-dependent cell-mediated cytotoxicity) and CDC (complement-dependent cytotoxicity), which might contribute to higher systemic inflammation. Further investigation is necessary to determine whether the tetravalent version is beneficial to balance efficiency and safety against COVID-19.

Development of the Novel Bifunctional Fusion Protein BR102 That Simultaneously Targets PD-L1 and TGF-ß for Anticancer Immunotherapy.

Immune checkpoint inhibitors (ICIs) are remarkable breakthroughs in treating various types of cancer, but many patients still do not derive long-term clinical benefits. Increasing evidence shows that TGF-ß can promote cancer progression and confer resistance to ICI therapies. Consequently, dual blocking of TGF-ß and immune checkpoint may provide an effective approach to enhance the effectiveness of ICI therapies. Here, we reported the development and preclinical characterization of a novel bifunctional anti-PD-L1/TGF-ß fusion protein, BR102. BR102 comprises an anti-PD-L1 antibody fused to the extracellular domain (ECD) of human TGF-ßRII. BR102 is capable of simultaneously binding to TGF-ß and PD-L1. Incorporating TGF-ßRII into BR102 does not alter the PD-L1 blocking activity of BR102. In vitro characterization further demonstrated that BR102 could disrupt TGF-ß-induced signaling. Moreover, BR102 significantly inhibits tumor growth in vivo and exerts a superior antitumor effect compared to anti-PD-L1. Administration of BR102 to cynomolgus monkeys is well-tolerated, with only minimal to moderate and reversing red cell changes noted. The data demonstrated the efficacy and safety of the novel anti-PD-L1/TGF-ß fusion protein and supported the further clinical development of BR102 for anticancer therapy.

Optimization strategies for expression of a novel bifunctional anti-PD-L1/TGFBR2-ECD fusion protein.

The novel anti-PD-L1/TGFBR2-ECD fusion protein (BR102) comprises an anti-PD-L1 antibody (HS636) which is fused at the C terminus of the heavy chain to a TGF-ß1 receptor Ⅱ ectodomain (TGFBR2-ECD), and which can sequester the PD-1/PD-L1 pathway and TGF-ß bioactivity in the immunosuppressive tumor microenvironment. In the expression of TGFBR2-ECD wild-type fused protein (BR102-WT), a 50 kDa clipped species was confirmed to be induced by proteolytic cleavage at a "QKS" site located in the N-terminus of the ectodomain, which resulted in the formation of IgG-like clipping. The matrix metalloproteinase-9 was determined to be associated with BR102-WT digestion. In addition, it was observed that the N-glycosylation modifications of the fusion protein were tightly involved in regulating proteolytic activity and the levels of cleavage could be significantly suppressed by MMP-inhibitors. To avoid proteolytic degradation, eliminating protease-sensitive amino acid motifs and introducing potential glycosylation were performed. Three sensitive motifs were mutated, and the levels of clipping were strongly restrained. The mutant candidates exhibited similar binding affinities to hPD-L1 and hTGF-ß1 as well as highly purified BR102-WT2. Furthermore, the mutants displayed more significant proteolytic resistance than that of BR102-WT2 in the lysate incubation reaction and the plasma stability test. Moreover, the bifunctional candidate Mu3 showed an additive antitumor effect in MC38/hPD-L1 bearing models as compared to that of with anti-PD-L1 antibody alone. In conclusion, in this study, the protease-sensitive features of BR102-WT were well characterized and efficient optimization was performed. The candidate BR102-Mutants exhibited advanced druggability in drug stability and displayed desirable antitumor activity.

Human-IgG-Neutralizing Monoclonal Antibodies Block the SARS-CoV-2 Infection.

Coronavirus disease 2019 (COVID-19) has become a worldwide threat to humans, and neutralizing antibodies have therapeutic potential. We have purified more than 1,000 memory B cells specific to SARS-CoV-2 S1 or its RBD (receptor binding domain) and obtain 729 paired heavy- and light-chain fragments. Among these, 178 antibodies test positive for antigen binding, and the majority of the top 17 binders with EC50 below 1 nM are RBD binders. Furthermore, we identify 11 neutralizing antibodies, eight of which show IC50 within 10 nM, and the best one, 414-1, with IC50 of 1.75 nM. Through epitope mapping, we find three main epitopes in RBD recognized by these antibodies, and epitope-B antibody 553-15 could substantially enhance the neutralizing abilities of most of the other antibodies. We also find that 515-5 could cross neutralize the SARS-CoV pseudovirus. Altogether, our study provides 11 potent human neutralizing antibodies for COVID-19 as therapeutic candidates.

A novel human lncRNA SANT1 cis-regulates the expression of SLC47A2 by altering SFPQ/E2F1/HDAC1 binding to the promoter region in renal cell carcinoma.

SLC47A2 encodes MATE 2-K in the kidney, which mediates the secretion of certain endogenous and exogenous compounds. SLC47A2 was dramatically repressed in patients with renal cell carcinoma (RCC), and a lower level of SLC47A2 might act as a negative prognostic marker, although the mechanism is not well understood. In this study, we aimed to investigate the mechanism via which SLC47A2 is downregulated in RCC. Based on the annotation information of the SLC47A2 locus available in the UCSC genome browser database, we identified a novel lncRNA, which is transcribed from the SLC47A2 locus and named it SANT1. Overexpression and knock-down assays were performed to investigate the effects of SANT1 on cis-regulation of SLC47A2. We verified the direct binding between SANT1 and SFPQ/E2F1/HDAC1 using the cross-linking and immunoprecipitation (CLIP) assay. Chromatin immunoprecipitation was performed to confirm the molecular mechanism via which SANT1 activates the transcription of the SLC47A2 coding region. We observed that SANT1 can cis-regulate its own genetic locus. In tumour-adjacent tissues, the SLC47A2 locus highly expresses SANT1, which can remove the regulatory SFPQ/E2F1/HDAC1 suppressor complex from the promoter region, thereby significantly increasing the levels of the H3K27ac modification and RNAPII binding. Owing to a low SANT1 level, the binding of this inhibitory complex in the promoter region is upregulated in RCC, which results in silencing of the SLC47A2 coding region. In conclusion, we identified a novel lncRNA and elucidated the mechanism via which it regulates SLC47A2 expression in RCC.

Inter-isoform Hetero-dimerization of Human UDP-Glucuronosyltransferases (UGTs) 1A1, 1A9, and 2B7 and Impacts on Glucuronidation Activity.

Human UDP-glucuronosyltransferases (UGTs) play a pivotal role in phase II metabolism by catalyzing the glucuronidation of endobiotics and xenobiotics. The catalytic activities of UGTs are highly impacted by both genetic polymorphisms and oligomerization. The present study aimed to assess the inter-isoform hetero-dimerization of UGT1A1, 1A9, and 2B7, including the wild type (1A1*1, 1A9*1, and 2B7*1) and the naturally occurring (1A1*1b, 1A9*2/*3/*5, and 2B7*71S/*2/*5) variants. The related enzymes were double expressed in Bac-to-Bac systems. The fluorescence resonance energy transfer (FRET) technique and co-immunoprecipitation (Co-IP) revealed stable hetero-dimerization of UGT1A1, 1A9, and 2B7 allozymes. Variable FRET efficiencies and donor-acceptor distances suggested that genetic polymorphisms resulted in altered affinities to the target protein. In addition, the metabolic activities of UGTs were differentially altered upon hetero-dimerization via double expression systems. Moreover, protein interactions also changed the regioselectivity of UGT1A9 for querectin glucuronidation. These findings provide in-depth understanding of human UGT dimerization as well as clues for complicated UGT dependent metabolism in humans.

Dimerization of human uridine diphosphate glucuronosyltransferase allozymes 1A1 and 1A9 alters their quercetin glucuronidation activities.

Uridine diphosphate glucuronosyltransferase 1A (UGT1A) is a major phase II drug-metabolism enzyme superfamily involved in the glucuronidation of endobiotics and xenobiotics in humans. Many polymorphisms in UGT1A genes are reported to inhibit or decrease UGT1A activity. In this study, two UGT1A1 allozymes, UGT1A1 wild-type and a splice mutant, as well as UGT1A9 wild-type and its three UGT1A9 allozymes, UGT1A9*2(C3Y), UGT1A9*3(M33T), and UGT1A9*5(D256N) were single- or double-expressed in a Bac-to-Bac expression system. Dimerization of UGT1A1 or UGT1A9 allozymes was observed via fluorescence resonance energy transfer (FRET) and co-immunoprecipitation analysis. SNPs of UGT1A altered the ability of protein-protein interaction, resulting in differential FRET efficiencies and donor-acceptor r distances. Dimerization changed the chemical regioselectivity, substrate-binding affinity, and enzymatic activity of UGT1A1 and UGT1A9 in glucuronidation of quercetin. These findings provide molecular insights into the consequences of homozygous and heterozygous UGT1A1 and UGT1A9 allozymes expression on quercetin glucuronidation.

Anti-diabetic effects of CTB-APSL fusion protein in type 2 diabetic mice.

To determine whether cholera toxin B subunit and active peptide from shark liver (CTB-APSL) fusion protein plays a role in treatment of type 2 diabetic mice, the CTB-APSL gene was cloned and expressed in silkworm (Bombyx mori) baculovirus expression vector system (BEVS), then the fusion protein was orally administrated at a dose of 100 mg/kg for five weeks in diabetic mice. The results demonstrated that the oral administration of CTB-APSL fusion protein can effectively reduce the levels of both fasting blood glucose (FBG) and glycosylated hemoglobin (GHb), promote insulin secretion and improve insulin resistance, significantly improve lipid metabolism, reduce triglycerides (TG), total cholesterol (TC) and low density lipoprotein (LDL) levels and increase high density lipoprotein (HDL) levels, as well as effectively improve the inflammatory response of type 2 diabetic mice through the reduction of the levels of inflammatory cytokines tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6). Histopathology shows that the fusion protein can significantly repair damaged pancreatic tissue in type 2 diabetic mice, significantly improve hepatic steatosis and hepatic cell cloudy swelling, reduce the content of lipid droplets in type 2 diabetic mice, effectively inhibit renal interstitial inflammatory cells invasion and improve renal tubular epithelial cell nucleus pyknosis, thus providing an experimental basis for the development of a new type of oral therapy for type 2 diabetes.

ORF4-protein deficient PCV2 mutants enhance virus-induced apoptosis and show differential expression of mRNAs in vitro.

Porcine circovirus type 2 (PCV2) is the essential infectious agent of PCV associated disease (PCVAD). During previous in vitro studies, 11 RNAs and four viral proteins have been detected in PCV2-infected cells. Open reading frame (ORF) 4 is 180bp in length and has been identified at the transcription and the translation level. It overlaps completely with ORF3, which has a role in virus-induced apoptosis. In this study, start codon mutations (M1-PCV2) or in-frame termination mutations (M2-PCV2) were utilized to construct two ORF4-protein deficient viruses aiming to investigate its role in viral infection. The abilities of M1-PCV2 and M2-PCV2 to replicate, transcribe, express viral proteins, and to cause cellular apoptosis were evaluated. Viral DNA replication curves supported that the ORF4 protein is not essential for viral replication, but inhibits viral replication in the early stage of infection. Comparison of the expression level of ORF3 mRNA among wild-type and ORF4-deficient viruses in infected PK-15 cell demonstrated enhanced ORF3 transcription of both ORF4 mutants suggesting that the ORF4 protein may play an important role by restricting ORF3 transcription thereby preventing virus-induced apoptosis. This is further confirmed by the significantly higher caspase 3 and 8 activities in M1-PCV2 and M2-PCV2 compared to wild-type PCV2. Furthermore, the role of ORF4 in cell apoptosis and a possible interaction with the ORF1 associated Rep protein could perhaps explain the rapid viral growth in the early stage of infection and the higher expression level of ORF1 mRNA in ORF4 protein deficient PCV2 mutants.

Identification and characterization of two novel transcription units of porcine circovirus 2.

Porcine circovirus type 2 (PCV2) infection is associated with porcine circovirus-associated diseases in pigs, which is a serious threat to the swine industry worldwide. To date, only three open reading frames (ORFs) within the PCV2 genome have been reported: ORF1 codes for two replicase proteins (Rep and Rep'), ORF2 for the structural protein (Cap), and ORF3 for a protein implicated in cellular apoptosis. In this study, based on transcription analysis of ORF3 mRNA, a potential ORF4 mRNA was detected and characterized by real-time RT-PCR and rapid amplification of cDNA ends analysis. The results indicate that the ORF4 gene is expressed at the level of transcription in the PCV2-infected cells. In addition, a novel ORF3 associated (ORF3') mRNA was identified during virus replication in PK15 cells. Moreover, a 3' poly(A) addition signal sequence (AUUAAA, nt 258-263) was found 10-30 nucleotides upstream of the cleavage site in the novel ORF4 mRNA in the complementary-strand of the PCV2 genome. Furthermore, alternate trans-splicing was identified in the ORF3' mRNA between orientation diverse transcripts with typical GT-AG donor/acceptor junctions. Similar strategies as in this work can be applied to examine the transcription of other potential ORFs in PCV in the future.