Preclinical Characterization of a Novel Anti-Cancer PD-L1 Inhibitor RPH-120.

RPH-120 is a novel fully human anti-PD-L1 IgG1 monoclonal antibody with specifically designed Asn300Ala mutation in Fc fragment. Surface plasmon resonance assay showed that affinity of the RPH-120 to the dimeric form of human PD-L1-Fc fusion protein was much higher than affinity to the monomeric His-tagged PD-L1. Further binding studies demonstrated that RPH-120 is able to bind to human and monkey but not mouse PD-L1. Tissue cross-reactivity study showed good comparability of human and Cynomolgus monkeys tissue staining. Bioactivity was assessed using mixed lymphocyte reaction assay. This study revealed that RPH-120 was able to activate T cells preventing PD1/PD-L1 interaction. Antitumor efficacy was analyzed in HCC-827 lung cancer xenografts in humanized CD34+ mice at three dosage levels: 20, 80, and 200 mg/kg. RPH-120 demonstrated significant tumor growth inhibition, and this inhibition was comparable to that of atezolizumab. In a single dose toxicity, toxicokinetic and dose range finding study performed in Cynomolgus monkeys, RPH-120 was administered via intravenous (IV) bolus or 60-min IV infusion, followed by 8-weeks recovery period. An acceptable toxicokinetic profile was demonstrated and administration at doses of up to 200 mg/kg was well tolerated by all animals. In conclusion, RPH-120 revealed promising in vitro and in vivo activity and safety. RPH-120 is a potent anti-PD-L1 drug candidate for cancer immunotherapy.

Glycogen synthase kinase 3 phosphorylates RBL2/p130 during quiescence.

Phosphorylation of the retinoblastoma-related or pocket proteins RB1/pRb, RBL1/p107, and RBL2/p130 regulates cell cycle progression and exit. While all pocket proteins are phosphorylated by cyclin-dependent kinases (CDKs) during the G1/S-phase transition, p130 is also specifically phosphorylated in G0-arrested cells. We have previously identified several phosphorylated residues that match the consensus site for glycogen synthase kinase 3 (GSK3) in the G0 form of p130. Using small-molecule inhibitors of GSK3, site-specific mutants of p130, and phospho-specific antibodies, we demonstrate here that GSK3 phosphorylates p130 during G0. Phosphorylation of p130 by GSK3 contributes to the stability of p130 but does not affect its ability to interact with E2F4 or cyclins. Regulation of p130 by GSK3 provides a novel link between growth factor signaling and regulation of the cell cycle progression and exit.

Nucleocytoplasmic shuttling of p130/RBL2: novel regulatory mechanism.

The retinoblastoma-related pocket proteins pRb, p107, and p130 are implicated in the control of cell proliferation, differentiation, and transformation. The function of pocket proteins is in part mediated by their ability to inhibit specific E2F transcription factors. The transcriptional activity of E2Fs is controlled by alteration of their nucleocytoplasmic localization during the cell cycle. p130 was observed to shuttle between the nucleus and cytoplasm in a heterokaryon fusion assay, suggesting the presence of nuclear and cytoplasmic localization signals. Two independent nuclear localization signals (NLS) that could target reporter proteins to the nucleus in transient transfection and microinjection experiments were identified in the C terminus of p130. In addition to the C-terminal NLS, the intact pocket domain of p130 itself was sufficient for nuclear translocation. Moreover, an additional functional NLS was mapped within the unique Loop region of p130. An N-terminal domain that conferred cytoplasmic localization was identified. Removal of the entire N terminus did not affect the ability of p130 to interact with E2F and to induce growth arrest. A model suggesting that the activity of pRb family members can be regulated by intracellular trafficking of the proteins is proposed.

Western blot screening for monoclonal antibodies against human separase.

Separase is a cysteine protease that participates in separation of sister chromatids during mitosis. Human separase is a 230-kDa enzyme that is inhibited by binding to its protein inhibitor securin, specific phosphorylation, and subcellular localization. To further characterize human separase, we raised monoclonal antibodies specific against a C-terminal fragment of the protein. A critical step in monoclonal antibody production procedure is the primary screening of hybridoma supernatants. Here we report primary screening protocol utilizing Western blot analysis. The described screening protocol is carried out using fusion of a human separase fragment with two different purification tags, maltose-binding protein (MBP) and glutathione S-transferase (GST). Immunization by MBP-fusion was followed by primary screening with both MBP- and GST-separase fusions combined in the same preparation separated in SDS-PAGE. This highly sensitive screening approach reduced the number of positive signals by eliminating antibodies specific for the purification tag used in the immunization procedure. The described separase-specific antibodies were suitable for detection of endogenous separase in crude extracts, immunoprecipitation, and immunofluorescent cell staining experiments. The presented procedure is fast, reproducible and could be adopted as a primary screening scheme for a variety of protein antigens.

Processing, localization, and requirement of human separase for normal anaphase progression.

In all eukaryotes, anaphase is triggered by the activation of a protease called separase. Once activated, separase cleaves a subunit of cohesin, a complex that links replicated chromatids before anaphase. Separase and cohesin are conserved from yeasts to humans. Although the machinery for dissolving sister cohesion is conserved, the regulation of this process appears to be more complex in higher eukaryotes than in yeast. Here we report the cloning of full-length human separase cDNA and the characterization of the encoded protein. Human separase was observed at the poles of the mitotic spindle until anaphase, at which time its association with the mitotic spindle was abruptly lost. The dynamic pattern of localization of human separase during cell cycle progression differs from that of fungal separases. Human separase also appears to undergo an autocatalytic processing on anaphase entry. The processed forms of human separase were isolated and the identity of the cleavage sites was determined by N-terminal sequencing and site-directed mutagenesis. The processed catalytic domain was found to be stably associated with the processed N-terminal fragment. Finally, by depletion of endogenous separase with antisense oligonucleotides, we report direct evidence that separase is required for high-fidelity chromosome separation in human cells.