Blockade of dual immune checkpoint inhibitory signals with a CD47/PD-L1 bispecific antibody for cancer treatment.

Background: Even though PD-1/PD-L1 is an identified key "don't find me" signal to active adaptive immune system for cancer treatment, the overall response rate (ORR) for all cancer patients is still limited. Other effective therapeutic modalities to bridge the innate and adaptive immunity to improve ORR are urgently needed. Recently, CD47/SIRPα interaction is confirmed as a critical "don't eat me" signal to active innate immunity. However, the red blood cell (RBC) toxicity is the big concern for the development of CD47-based anti-cancer therapeutics. Methods: Here, we report the development of a CD47/PD-L1 bispecific antibody 6MW3211 to block both PD-1/PD-L1 and CD47/SIRPα signals, and studied the effects of 6MW3211 on anti-tumor immune functions in vitro and in vivo. The pharmacokinetic and toxicity profiles of 6MW3211 were evaluated in GLP non-human primate (NHP) studies. Results: The dual immune checkpoint inhibitory signaling blocker 6MW3211 shows high binding affinity to PD-L1 and low binding affinity to CD47. This inequivalent binding affinity design makes 6MW3211 preferentially bound to PD-L1 on tumor cells followed by disrupting the interaction of CD47/SIRPα. Complex structure determination and flow cytometry assay demonstrated that 6MW3211 has no binding to either human or rhesus monkey RBCs. 6MW3211 effectively blocked both PD-1/DP-L1 and CD47/SIRPα signaling and promoted macrophage phagocytosis of tumor cells. Potent therapeutic efficacies of 6MW3211 in three different mouse models were further observed. Moreover, 6MW3211 was demonstrated to have a fairly good safety profile in a GLP NHP study. In addition, multiplex fluorescent immunohistochemistry (mIHC) staining shows that PD-L1 and CD47 co-express on several different types of human tumor tissues. Conclusions: These results support the development of 6MW3211 for the treatment of PD-L1 and CD47 double positive cancers.

Remarkable photoluminescence of europium(ii)-doped phosphate cyan@red-emitting phosphors with highly dispersed luminescence centers.

Europium(ii)-doped phosphate cyan@red-emitting phosphors with highly dispersed luminescence centers were developed for the first time by using the deposition-precipitation method. Amazingly, when excited by near-ultraviolet light, the single phosphor generated warm white light with an adjustable correlated colour temperature (4000-2500 K) and high colour-rendering index (Ra ∼ 90).

The influence of YS-1 on the Dll4-Notch1 signaling pathway.

In this study, we investigated the role and molecular mechanism of p43 and YS-1 (recombinant human p43 protein) in Dll4-Notch1 signaling pathway. Active, small interfering RNA and recombinant plasmid targeting of p43 protein were used to infect human umbilical vein endothelial cells (HUVECs). Three-dimensional sprouting model, endothelial cell migration assay, and sprouting and tube formation assay were used to deduce the function of p43 and YS-1 in angiogenesis. Semi-quantitative reverse transcription-polymerase chain reaction and western blot analysis were performed to detect the efficiency of p43 in Dll4-Notch1 signaling in HUVECs. It was found that silencing and overexpression of p43 could upregulate Dll4-Notch and stimulate angiogenesis. p43 plays a complex role in angiogenesis. When the concentration is under 100 nM, it promotes angiogenesis; instead, when the concentration is over 100 nM, it inhibits angiogenesis. In this study, we found that the expression level of p43 was under 60 nM. However, recombinant human p43 protein, YS-1, inhibited endothelial cell sprouting, and 500 µg/ml of YS-1 attenuated the activation of Dll4-Notch1 signaling. These results suggested that YS-1 could directly inhibit angiogenesis through Dll4-Notch1 signal transduction pathway, while p43 plays a modulating role in this signaling pathway.

Characterization of a novel humanized anti-CD20 antibody with potent anti-tumor activity against non-Hodgkin's lymphoma.

BACKGROUND: Rituximab, a mouse Fab and human Fc chimeric antibody, has been widely used to treat Non-Hodgkin's lymphoma (NHL). However, only 48% of patients respond to the treatment and complete response rate is below 10%. Also, immunogenicity was reported in 17-20% patients receiving the treatment, making it unsuitable for long term diseases such as autoimmune disorders. It has been a hot research field to "humanize" rituximab toward improved efficacy and reduced immunogenicity. METHODS: In this study, an advanced antibody humanization technology was applied to the sequence of the anti-CD20 antibody 2B8, its sequence of which was based on the original murine monoclonal antibody of rituximab in Roche. The complementarity-determining regions (CDRs) of the humanized antibodies were further optimized through computer-aided molecular dock. RESULTS: Five novel humanized anti-CD20 antibodies 1-5(1635, 1534, 3637, 1634 and 1536) were generated and their immunogenicity was significantly decreased when compared to rituximab. The novel humanized anti-CD20 antibodies 1-5 retained the binding activity of their murine counterpart, as demonstrated by the fluorescence-activated cell-sorting analysis (FACS). When compared to rituximab, the humanized antibodies still have the similar properties on both complement-dependent cytotoxicity (CDC) and antibody-dependent cell-mediated cytotoxicity (ADCC). Furthermore, its anti-tumor efficacy in xenograft model is comparable to that of rituximab. CONCLUSION: The humanized anti-CD20 antibodies 1-5 have lower immunogenicity than rituximab. And at the same time, they still retain the anti-tumor effect both in vitro and vivo.

Topotecan inhibits cancer cell migration by down-regulation of chemokine CC motif receptor 7 and matrix metalloproteinases.

AIM: The aim of this study was to investigate the effect of topotecan (TPT) on cancer cell migration. METHODS: Growth inhibition of TPT was analyzed by MTT assay, and cancer cell migration was measured by transwell double chamber assay. To verify the effect of TPT on the chemokine receptors CXCR4 and CCR7, quantitative PCR, semi-quantitative PCR and Western blot analysis were performed. The secretion of MMP-2 and MMP-9 was detected by enzyme-linked immunosorbent assay (ELISA) and gelatin zymography. To evaluate possible contributions of CCR7 to MMP secretion, the overexpression vectors pcDNA3.1(+)-CCR7 and CCR7 siRNA were transiently transfected into MDA-MB-435 cells. RESULTS: TPT inhibited cancer cell migration in a dose-dependent manner. Additionally, TPT significantly decreased the expression of CCR7 in both MDA-MB-435 and MDA-MB-231 cells and moderately reduced the expression of CXCR4 in MDA-MB-435 cells. The secretion of MMPs (MMP-2, MMP-9) was also inhibited by TPT. Overexpression of CCR7 increased the secretion of MMP-2/9 and cancer cell migration, whereas knockdown of CCR7 reduced active MMP-2/9 production and migration of MDA-MB-435 cells. CONCLUSION: TPT inhibited cancer cell migration by down-regulation of CCR7 and MMPs (MMP-2 and MMP-9).