Structural basis of HLX10 PD-1 receptor recognition, a promising anti-PD-1 antibody clinical candidate for cancer immunotherapy.

Cancer immunotherapies, such as checkpoint blockade of programmed cell death protein-1 (PD-1), represents a breakthrough in cancer treatment, resulting in unprecedented results in terms of overall and progression-free survival. Discovery and development of novel anti PD-1 inhibitors remains a field of intense investigation, where novel monoclonal antibodies (mAbs) and novel antibody formats (e.g., novel isotype, bispecific mAb and low-molecular-weight compounds) are major source of future therapeutic candidates. HLX10, a fully humanized IgG4 monoclonal antibody against PD-1 receptor, increased functional activities of human T-cells and showed in vitro, and anti-tumor activity in several tumor models. The combined inhibition of PD-1/PDL-1 and angiogenesis pathways using anti-VEGF antibody may enhance a sustained suppression of cancer-related angiogenesis and tumor elimination. To elucidate HLX10's mode of action, we solved the structure of HLX10 in complex with PD-1 receptor. Detailed epitope analysis showed that HLX10 has a unique mode of recognition compared to the clinically approved PD1 antibodies Pembrolizumab and Nivolumab. Notably, HLX10's epitope was closer to Pembrolizumab's epitope than Nivolumab's epitope. However, HLX10 and Pembrolizumab showed an opposite heavy chain (HC) and light chain (LC) usage, which recognizes several overlapping amino acid residues on PD-1. We compared HLX10 to Nivolumab and Pembrolizumab and it showed similar or better bioactivity in vitro and in vivo, providing a rationale for clinical evaluation in cancer immunotherapy.

Distinguishing features of a novel humanized anti-EGFR monoclonal antibody based on cetuximab with superior antitumor efficacy.

BACKGROUND: Cetuximab, the first approved EGFR targeting therapeutic antibody, is currently used to treat colorectal cancer and head and neck cancer. While effective, cetuximab is associated with a higher rate of skin rash, infusion reactions, and gastrointestinal toxicity, which was suggested to be linked to the presence of heterogeneous glycan contents on the Fab of the SP2/0-produced cetuximab. OBJECTIVE AND METHODS: To improve efficacy and minimize toxicity of EGFR inhibition treatment, we re-engineered cetuximab by humanizing its Fab regions and minimizing its glycan contents to generate HLX07. RESULTS: HLX07 binds to EGFR with similar affinity as cetuximab and shows better bioactivity compared to cetuximab in vitro. In vivo studies demonstrated that HLX07 significantly inhibited the growth of A431, FaDu, NCI-H292, and WiDr tumor cells and synergized them with chemotherapeutics and immune simulator agents such as anti-PD-1. In cynomolgus monkeys, 13-week repeat-dose GLP toxicokinetic studies showed minimal-to-mild toxicities in the dose range of up to 60 mg/kg/wk. In the preliminary phase 1 dose-escalation study, HLX07 had showed lower incidence of skin rashes with grade >2 severities. CONCLUSION: HLX07 is currently under phase 1/2 clinical development. We believe HLX07 would potentially be an alternative for patients who have been suffering from cetuximab-mediated toxicity.

Downregulation of c-Myc determines sensitivity to 2-methoxyestradiol-induced apoptosis in human acute myeloid leukemia.

OBJECTIVE: 2-Methoxyestradiol (2ME2) has been shown to induce apoptosis in leukemic cells, but its exact mechanism remains unclear. Because c-Myc plays a critical role in leukemogenesis, we evaluated whether 2ME2 acts on acute myeloid leukemia (AML) through modulation of c-Myc activity. MATERIALS AND METHODS: AML cell lines and primary AML leukemia were treated with 2ME2 and the relationship between 2ME2-induced apoptosis and changes in c-Myc activity was examined. RESULTS: 2ME2 induced mitochondrial apoptosis of human AML cells through increased reactive oxygen species. Further investigation showed that 2ME2 downregulated c-Myc expression in a time-dependent manner. Increased oxidative stress led to downregulation of c-Myc mRNA and protein, but did not affect the stability of c-Myc protein. To demonstrate the role of c-Myc in 2ME2-induced apoptosis, we ectopically expressed wild-type c-Myc in AML cells and found that ectopic expression of c-Myc abrogated the 2ME2-induced apoptosis. In addition, we showed that 2ME2 treatment inhibited phosphorylation of Akt and binding of nuclear factor-kappaB p65/p50 heterodimers to its DNA targets. As with results from cell lines studied, 2ME2 also induced cytotoxicity to primary AML cells and downregulated their c-Myc expression and induced apoptosis. CONCLUSION: Downregulation of c-Myc is critical for 2ME2-induced oxidative stress and apoptosis in AML cells. Our results might be extended to other types of cancers overexpressing c-Myc.

Downregulation of c-Myc is critical for valproic acid-induced growth arrest and myeloid differentiation of acute myeloid leukemia.

Valproic acid (VPA), an agent used for neurological disorders, has been shown to be a novel class of histone deacetylase inhibitor (HDACI), able to induce apoptosis and myeloid differentiation of acute myeloid leukemia (AML). In this study, we examined the underlying mechanisms in VPA-mediated activities in AML cells. VPA not only inhibited the growth of HL-60, U937 and NB4 cells by causing cell-cycle arrest at G(0)/G(1) phase and apoptosis, but also induced morphologic and phenotypic changes. VPA markedly increased p21WAF1, and downregulated c-Myc expression at transcriptional levels. Ectopic expression of wildtype c-Myc and T58A mutant significantly inhibited VPA-mediated growth inhibition. As with results from cell line studies, VPA also downregulated c-Myc levels, and induced apoptosis and myeloid differentiation of primary AML cells, leading to decreased colony-forming ability. Given the role of c-Myc in leukemogenesis, our study suggests that VPA might be a potential therapeutic agent for AML.

Conversion of human umbilical cord mesenchymal stem cells in Wharton's jelly to dopaminergic neurons in vitro: potential therapeutic application for Parkinsonism.

Human mesenchymal stem cells isolated from Wharton's jelly of the umbilical cord were induced to transform into dopaminergic neurons in vitro through stepwise culturing in neuron-conditioned medium, sonic hedgehog, and FGF8. The success rate was 12.7%, as characterized by positive staining for tyrosine hydroxylase (TH), the rate-limiting catecholaminergic synthesizing enzyme, and dopamine being released into the culture medium. Transplantation of such cells into the striatum of rats previously made Parkinsonian by unilateral striatal lesioning with the dopaminergic neurotoxin 6-hydroxydopamine partially corrected the lesion-induced amphetamine-evoked rotation. Viability of the transplanted cells at least 4 months after transplantation was identified by positive TH staining and migration of 1.4 mm both rostrally and caudally. These results suggest that human umbilical mesenchymal stem cells have the potential for treatment of Parkinson's disease.

Transformation of human umbilical mesenchymal cells into neurons in vitro.

Neuronal transplantation has provided a promising approach for treating neurodegenerative diseases. Recently, efforts have been directed at in vitro induction of various stem cells to transform into neurons. We report the first successful quantities in an in vitro attempt at directing the transformation into neurons of human umbilical mesenchymal cells, which are capable of rapid proliferation in vitro and are easily available. When cultured in neuronal conditioned medium, human umbilical mesenchymal cells started to express neuron-specific proteins such as NeuN and neurofilament (NF) on the 3rd day and exhibited retraction of the cell body, elaboration of processes, clustering of cells and expression of functional mRNA responsible for the synthesis of subunits of the kainate receptor and glutamate decarboxylase on the 6th day. Between the 9th and 12th days, the percentage of human umbilical mesenchymal cells expressing NF was as high as 87%, while functionality was demonstrated by glutamate invoking an inward current. At this stage, cells were differentiated into mature neurons in the post mitosis phase.