A case study of a bispecific antibody manufacturability assessment and optimization during discovery stage and its implications.

The manufacturability assessment and optimization of bispecific antibodies (bsAbs) during the discovery stage are crucial for the success of the drug development process, impacting the speed and cost of advancing such therapeutics to the Investigational New Drug (IND) stage and ultimately to the market. The complexity of bsAbs creates challenges in employing effective evaluation methods to detect developability risks in early discovery stage, and poses difficulties in identifying the root causes and implementing subsequent engineering solutions. This study presents a case of engineering a bsAb that displayed a normal solution appearance during the discovery phase but underwent significant precipitation when subjected to agitation stress during 15 L Chemistry, Manufacturing, and Control (CMC) production Leveraging analytical tools, structural analysis, in silico prediction, and wet-lab validations, the key molecular origins responsible for the observed precipitation were identified and addressed. Sequence engineering to reduce protein surface hydrophobicity and enhance conformational stability proved effective in resolving agitation-induced aggregation. The refined bsAb sequences enabled successful mass production in CMC department. The findings of this case study contribute to the understanding of the fundamental mechanism of agitation-induced aggregation and offer a potential protein engineering procedure for addressing similar issues in bsAb. Furthermore, this case study emphasizes the significance of a close partnership between Discovery and CMC teams. Integrating CMC's rigorous evaluation methods with Discovery's engineering capability can facilitate a streamlined development process for bsAb molecules.

The complete mitochondrial genome of Semblis atrata (Trichoptera: Phryganeidae).

Semblis atrata is one of three Semblis species distributed in clean brooks and streams in northern Eurasia. Genomic DNA of an S. atrata sample was extracted and sequenced for assembly and annotation of its complete mitogenome. The complete mitochondrial genome of S. atrata was 14,909 bp in length and consisted of 13 protein-coding genes, 2 rRNA genes, and 22 tRNA genes. The S. atrata COX1 gene features a CGA start codon, and COX1, COX2, ND1, and ND5 exhibit incomplete stop codons that are presumed to be completed by the addition of 3' A residues to the mRNA. The nucleotide composition was highly AT biased, accounting for 77.71% of the whole mitogenome. Phylogenetic analysis placed Semblis as sister to Eubasilissa. The complete mitochondrial genome will be helpful for further studies on the population genetics of this species and phylogenetic analyses of Trichoptera.

Preclinical Characterization of GLS-010 (Zimberelimab), a Novel Fully Human Anti-PD-1 Therapeutic Monoclonal Antibody for Cancer.

BACKGROUND: Zimberelimab (GLS-010) is a novel fully human monoclonal immunoglobulin G4 (IgG4) against the programmed cell death-1 (PD-1) receptor. AIM: To evaluate the affinity, competitive blocking capability, T cell activation effect, cytotoxic effector functions by Fc, preliminary anti-tumor activity, and pharmacokinetics of GLS-010. METHODS: The affinity of GLS-010 to PD-1 and the ability of GLS-010 to block the PD-L1/2 to PD-1 interaction on the cell surface were measured. An allogeneic mixed lymphocyte reaction was conducted to evaluate the inhibitory effect of GLS-010 on Tregs and stimulatory effect on T cell proliferation and activation. Pharmacodynamics and pharmacokinetics were evaluated in tumor-bearing mice and cynomolgus monkeys, respectively. RESULTS: The equilibrium dissociation constant (KD) for the association between GLS-010 and PD-1 was 1.75×10-10 M. GLS-010 could effectively block the binding of PD-L1/2 to PD-1. GLS-010 showed statistically significant anti-tumor effects in the MC38 model in human PD-1 knock-in mice. The RO rate on in the low-, moderate-, and high-dose groups were 64.50%-48.53% in CD3+T, 58.87%-40.12% in CD8+T, and 66.26%-49.07% in CD4+T, respectively. With the increasing dose from 2 mg/kg to 18 mg/kg, the systemic exposure level of GLS-010 (AUC0-last) and C0 increased proportionally, while the proportion of AUC0-last was higher than the proportion of the increase in the dose. CONCLUSIONS: As a fully human anti-PD-1 monoclonal antibody, GLS-010 has a high affinity to PD-1 and shows potent anti-tumor effects in vivo and in vitro. The results support that GLS-010 could be investigated in clinical trials in tumor patients.

CS1003, a novel human and mouse cross-reactive PD-1 monoclonal antibody for cancer therapy.

The programmed cell death protein 1 (PD-1) is an immune-checkpoint that negatively regulates the immune system and a key mechanism that tumors utilize to escape from immune surveillance. PD-1 antibodies can block the interaction of PD-1 with its ligands (PD-L1 and PD-L2), restore T cells activation, and elicit antitumor activity. In this paper, we reported a novel PD-1 monoclonal antibody (mAb) CS1003, which is a humanized IgG4 PD-1 mAb generated by conventional hybridoma technology, and currently being developed in multiple clinical trials as monotherapy or in combination with other anticancer agents. We showed that CS1003 bound to recombinant human, cynomolgus monkey, and mouse PD-1 with EC50 values of 0.1757, 0.2459, and 0.3664 nM, respectively. CS1003 blocked PD-1 interaction with its ligands, dose-dependently enhanced T cell proliferation and secretion of cytokines (IL-2 and IFN-γ) to the levels comparable to the reference antibody pembrolizumab. Intraperitoneal administration of CS1003 (0.1, 0.5, 2.5 mg/kg, once every 3 days) dose-dependently suppressed the growth of MC38-hPD-L1 colon cancer in hPD-1 knock-in mice. Pharmacokinetics (PK) study revealed a linear PK profile within the dose range of 2-18 mg/kg following single intravenous administration in cynomolgus monkey. These data provide a comprehensive preclinical characterization of CS1003 that supports its clinical development for cancer immunotherapy.

Diversity and phylogeography of Northeast Asian brown frogs allied to Rana dybowskii (Anura, Ranidae).

We investigated the species diversity and phylogeography of the Northeast Asian brown frogs allied to Rana dybowskii (the R. dybowskii species complex: R. dybowskii, R. pirica, and R. uenoi) using four mitochondrial and three nuclear loci. Phylogenetic analyses confirmed the existence of three distinct species in this complex; using extensive molecular data, we confirm the validity of Rana uenoi recognized as a distinct species, and infer R. dybowskii and R. pirica to be sister species. Also, we included populations from previously unsampled regions in Northeast China, and identified them to be R. dybowskii. While many species in Northeast Asia diverged due to Pleistocene glaciation, divergence-dating analyses inferred older, Miocene speciation in the R. dybowskii species complex. Ancestral area reconstruction identified the orogenic movement of the Changbai Mountain Range and the opening of the Sea of Japan/East Sea being major events influencing allopatric speciation.

Phylogeny and biogeography of South Chinese brown frogs (Ranidae, Anura).

Few studies have explored the role of Cenozoic tectonic evolution in shaping the patterns and processes of extant animal distributions in and around East Asia. In this study, we selected South Chinese brown frogs as a model to examine the phylogenetic and biogeographical consequences of Miocene tectonic events within South China and its margins. We used mitochondrial and nuclear molecular data to reconstruct phylogenetic interrelationships among Chinese brown frogs using Bayesian and maximum likelihood analyses. The phylogeny results show that there are four main clades of Chinese brown frogs. Excepting the three commonly known Chinese brown frog species groups, R. maoershanensis forms an independent clade nearest to the R. japonica group. Phylogeny and P-distance analyses confirmed R. maoershanensis as a valid species. Among South Chinese brown frogs, there are four subclades associated with four geographical areas: (I) R. maoershanensis; (II) R. japonica; (III) R. chaochiaoensis; and (IV) other species of the R. longicrus species group. Divergence times, estimated using mitochondrial sequences, place the vicariance events among the four subclades in the middle to late Miocene epoch. Our results suggest that (1) South Chinese brown frogs originated due to a vicariance event separating them from the R. chensinensis species group at the time of the Geological movement (~18 million years ago, Ma) in southern Tibet and the Himalayan region; (2) the separation and speciation of R. maoershanensis from the R. japonica group occurred due to the dry climate at approximately 16 Ma; (3) South Chinese brown frogs migrated from South China to Japan at the time (~10.8 Ma) that the global sea-level fell and the East China Sea Shelf Basin was swamp facies, when a land gallery may have formed across the sea to connect the two areas; and (4) R. chaochiaoensis separated from other species of the R. longicrus species group during the uplift of the Tibetan Plateau at approximately 9.5 Ma.

Influence of geology and human activity on the genetic structure and demography of the Oriental fire-bellied toad (Bombina orientalis).

The Oriental fire-bellied toad (Bombina orientalis) is a commonly used study organism, but knowledge of its evolutionary history is incomplete. We analyze sequence data from four genetic markers (mtDNA genes encoding cytochrome c oxidase subunit I, cytochrome b, and 12S-16S rRNA; nuDNA gene encoding recombination activating gene 2) from 188 individuals across its range in Northeast Asia to elucidate phylogeographic patterns and to identify the historic events that shaped its evolutionary history. Although morphologically similar across its range, B. orientalis exhibits phylogeographic structure, which we infer was shaped by geologic, climatic, and anthropogenic events. Phylogenetic and divergence-dating analyses recover four genetically distinct groups of B. orientalis: Lineage 1-Shandong Province and Beijing (China); Lineage 2-Bukhan Mountain (Korea); Lineage 3-Russia, Northeast China, and northern South Korea; and Lineage 4-South Korea. Lineage 2 was previously unknown. Additionally, we discover an area of secondary contact on the Korean Peninsula, and infer a single dispersal event as the origin of the insular Jeju population. Skyline plots estimate different population histories for the four lineages: Lineages 1 and 2 experienced population decreases, Lineage 3 remained stable, while Lineage 4 experienced a sharp increase during the Holocene. The timing of the population expansion of Lineage 4 coincides with the advent of rice cultivation, which may have facilitated the increase in population size by providing additional breeding habitat.

The complete mitochondrial genome of the Bufo stejnegeri (Anura: Bufonidae).

The complete mitochondrial genome of Bufo stejnegeri was determined, which was 17 939 bp in length. It consists of 13 protein-coding genes, two rRNA genes, 22 tRNA genes, and one displacement loop (D-loop). The total length of D-loop region is 2533 bp, some tandem repeat units were found in this region. The phylogenetic trees of the 20 species from anura were reconstructed based on complete mtDNA sequences by Bayesian inference and maximum likelihood analyses. The result demonstrated that B. stejnegeri is the most closely related species with other Bufo species.

The complete mitochondrial genome of the Rana huanrensis (Anura: Ranidae).

We first determined complete mitochondrial genomes of R. huanrensis (Anura: Ranidae). The complete mtDNA sequence is 19 253 bp in length, including 13 protein-coding genes, 2 rRNA genes, 22 tRNA genes, and one displacement loop. The start/stop codons of protein-coding genes are similar to which of R. chensinensis. D-loop region of R. huanrensis is 3448 bp in size, contains many tandem repeat units. The phylogenetic trees of 18 species from Ranidae were reconstructed by BI and ML analyses. The result indicated that R. huanrensis is the most closely related species with other Rana species. The molecular data are expected to provide a useful tool for population genetics studies of this species and further phylogenetic analyses of Ranidae.

Germ-line-competent embryonic stem cells of the Chinese Kunming mouse strain with long-term self-renewal ability.

Kunming (KM) mice are the most widely used strain in China. However, authentic embryonic stem cells (ESCs) from KM mice have never been available, and this hampers the genetic manipulation of this valuable mice strain. In this study, we show that KM ESCs can be efficiently derived and maintained in chemically defined N2B27 medium with the presence of two small molecules PD0325901 and CHIR99021 (2i medium). These KM ESCs exhibit all features of ESCs, including long-term self-renewal ability, expression of key molecular markers (Oct4, Nanog, and Sox2), the ability to form teratomas, and the capacity to incorporate into the developing embryo and then transmit through the germ line.

Conversion of rat embryonic stem cells into neural precursors in chemical-defined medium.

Rat embryonic stem (ES) cells hold great interest for the research of neurodevelopment and neurodegenerative diseases. However, neural conversion of rat ES cells in vitro has proven to be a challenge owing to the proliferation arrest and apoptosis. Here we report that rat ES cells can commit efficiently to a neural fate in the presence of CHIR99021 and Y-27632 (CY medium). In addition, CHIR99021 is crucial for maintaining the metabolic activity of differentiated rat ES cells, while Y-27632 facilitates the neural differentiation of rat ES cells by inhibiting bone morphogenetic protein expression. The chemical-defined CY medium also provides a platform for exploring the mechanism of neural commitment and optimizing the production efficiency of neural precursor from rat ES cells.

Enormous influence of TNIK knockdown on intracellular signals and cell survival.

Basic cellular activities and coordinated cell actions are governed by intracellular signals, among which the Wnt signaling cascade plays an important role in tissue polarity and cell adhesion or movement through the activation of c-Jun N-terminal kinase (JNK) pathway. As one of the central transcriptional factors, Traf2- and Nck-interacting kinase (TNIK) mediates the transactivation of Wnt target genes and promotes the activity of c-Jun N-terminus kinase (JNK)2 when overexpressed. To further understand the function of TNIK, changes in intracellular signals were detected in colon cancer cell lines using a knockdown strategy. In this study, we found that the short-hairpin RNA-mediated knockdown of TNIK decreased the expressions of CD44, c-MYC and cyclin D1, which was consistent with the results of a TCF-4 reporter assay. Our data showed, for the first time, that the activation of both JNK1 and JNK2 by TNFα could be blocked through TNIK knockdown, which dampened the AP1 luciferase activity accordingly. In addition, adenovirus mediated the downregulation of TNIK-triggered intrinsic apoptosis in SW480 cells by activating caspase-9 and PARP-1. We conclude that TNIK is essential for the activation of both the canonical Wnt pathway and the JNK pathway, and serves as a pro-survival factor.

[The comparative study on maturation of metacarpal bone in puberty children during their growth spurt period between urban and rural areas].

OBJECTIVES: To study maturation of the metacarpal bone in puberty children during their growth spurt period and its difference between urban and rural areas. METHODS: Totally, 560 pupils/students were selected from primary and secondary schools in urban and rural areas each, with 35 children in each gender and age group, ranging 12 - 15 years of age for boys and 10 - 13 for girls. An X-ray film of left hand-wrist site was taken for each of them. Length and width of the metacarpal bone were measured and the metacarpal index was calculated. RESULTS: Increment of length of the metacarpal bone was great in puberty children both in urban and rural areas, (6.26 - 9.31) mm in boys and (5.28 - 9.12) mm in girls. Mean length of the metacarpal bone was longer in children of urban areas than that of rural ones, regardless of their age and gender. There was significant difference in mean length of the metacarpal bone between boys aged 14 - 15 years and girls aged 12. Mean width of the metacarpal bone in most children was wider in rural areas than that in urban ones. Mean metacarpal index in children was higher in urban areas than that in rural ones, with very statistical significance, except for girls of 13 year age group. The peak age of metacarpal maturation was 1 year earlier in urban areas than in rural ones. CONCLUSIONS: Maturation of the metacarpal bone was rapid during puberty growth spurt period, with relatively significant difference in urban and rural ares.