Generation and Characterization of SORT1-Targeted Antibody-Drug Conjugate for the Treatment of SORT1-Positive Breast Tumor.

Antibody-drug conjugates (ADCs) have greatly improved the outcomes of advanced breast tumors. However, the treatment of breast tumors with existing ADCs is still hindered by many issues, such as tumor antigen heterogeneity and drug resistance. Therefore, ADCs against new targets would provide options for the treatment of these challenges. Sortilin-1 (SORT1) may be a promising target for ADC as it is upregulated in breast cancer. To evaluate the possibility of SORT1 as an ADC target, a humanized antibody_8D302 with high affinity against SORT1 was generated. Additionally, 8D302 was conjugated with MMAE and DXd to generate two ADCs_8D302-MMAE and 8D302-DXd, respectively. Both 8D302-MMAE and 8D302-DXd showed effective cytotoxicity against SORT1 positive breast tumor cell lines and induced bystander killing. Consequently, 8D302-MMAE showed relatively better anti-tumor activity than 8D302-DXd both in vitro and in vivo, but 8D302-DXd had superior safety profile and pharmacokinetics profile over 8D302-MMAE. Furthermore, SORT1 induced faster internalization and lysosomal trafficking of antibodies and had a higher turnover compared with HER2. Also, 8D302-DXd exhibited superior cell cytotoxicity and tumor suppression over trastuzumab-DXd, a HER2-targeted ADC. We hypothesize that the high turnover of SORT1 enables SORT1-targeted ADC to be a powerful agent for the treatment of SORT1-positive breast tumor.

Generation of a Novel SORT1×HER2 Bispecific Antibody-Drug Conjugate Targeting HER2-Low-Expression Tumor.

Human epidermal growth factor receptor 2 (HER2) is considered an ideal antibody-drug conjugate (ADC) target because the gene is overexpressed in many tumors compared to normal tissues. Multiple anti-HER2 ADCs conjugated with different toxic payloads bring benefits to patients with high HER2 expression. However, HER2-targeted ADC technology needs further optimization to improve its effect for the treatment of patients with low HER2 expression. We hypothesized that bispecific antibody-drug conjugate (bsADC) targeting HER2 and Sortilin-1 (SORT1) would overcome this limitation. SORT1 is a suitable target for pairing with HER2 to generate a bispecific antibody (BsAb) since the gene is co-expressed with HER2 in tumors and possesses rapid internalization. We developed a BsAb (bsSORT1×HER2) that exhibited strong binding and internalization activity on HER2-low-expression tumor cells and facilitated higher HER2 degradation. The bsSORT1×HER2 was further conjugated with DXd to generate a bsADC (bsSORT1×HER2-DXd) that showed strong cytotoxicity on HER2-low-expression tumor cells and antitumor efficacy in an MDA-MB-231 xenograft mice model. These results demonstrated that employment of a SORT1×HER2-targeted bsADC may be promising to improve the antitumor efficacy of HER2-targeted ADC for the treatment of tumors with low HER2 expression.

The retention of prion protein in the endoplasmic reticulum prevents N2A cells from proteasome inhibition-induced cytotoxicity.

Prion disease is a fatal neurodegenerative disease that may result from the conversion of normal cellular prion protein (PrPC) to the pathogenic scrapie PrP isoform (PrPSc), however, how proliferation of prion leads to neuronal apoptosis is still not clear. In this study, to explore the role of the endoplasmic reticulum (ER) in prion diseases, we engineered the KDEL ER-retention motif to the C-terminus of PrPC and studied its effect on N2A cell toxicity. The KDEL retention signal led to the accumulation of PrP in the ER, and KDEL signal could effectively deplete PrP from the cell surface and trap PrP in the ER/Cis-Golgi compartment. PrPC molecules were delayed in their transit along the early pathway of the secretory compartment, however, they did not aggregate, and were not resistant to Proteinase K (PK) or become detergent-insoluble. Moreover, we found that the ER was not the site where PrP became detergent-insoluble and acquired PK resistance. In addition, an MTT assay indicated cells expressing PrPC/N2A were sensitive to proteasome inhibition, but not N2A cells expressing PrPKDEL. Our findings suggest that the ER is not a compartment in which wild type PrPC is able to initiate aggregation, protease resistance or other scapie-like properties of PrP.

Generation and characterization of human B lymphocyte stimulator blocking monoclonal antibody.

The cytokine, B lymphocyte stimulator (Blys) is essential for activation and proliferation of B cells and is involved in the pathogenesis of B-cell mediated autoimmune diseases. Based on its essential activity, Blys may be a potential therapeutic target for human autoimmune diseases. In this article, we have described the development of a novel humanized anti-Blys antibody, NMB04, that binds with high affinity and specificity to both soluble and membrane bound Blys. This monoclonal antibody has the potential to block Blys binding to all its three receptors, TACI, BCMA and BR-3. Further in vivo studies revealed that NMB04 possessed more potent inhibitory activity against human Blys as compared to an existing antibody, Belimumab. Therefore, NMB04 may have potential as a therapeutic candidate targeting autoimmune diseases.

The mechanism of the G0/G1 cell cycle phase arrest induced by activation of PXR in human cells.

CONTEXT: Pregnane X receptor (PXR) is an important transcriptional regulator that plays important roles in the cell metabolism and cell growth by regulating the transcriptional of a sort of metabolizing enzymes. OBJECTIVE: To investigate whether rifampicin effected HepG2 cells growth and the inhibition was due to the G0/G1 phase arrest. METHODS: PXR-knockdown experiments using RNAi showed that the cell cycle phase arrest mediated by rifampicin based on activation of PXR. The results also indicated that cell phase arrest by rifampicin could protect cells form UVB-induced DNA damage. Retinoid X receptor alpha (RXRα) expression level in cells is another key factor for cell cycle phase arrest mediated by rifampicin. Both over expression and lacking expression of RXRα in cell reduced the cell arrest efficiency mediated by rifampicin. In the study, we found that rifampicin inhibited HepG2 cells growth and demonstrated that the inhibition is due to the G0/G1 phase arrest through flow cytometry analysis. CONCLUSION: The results showed that RXRα promote cell cycle phase transition rate of HepG2. Competitive bind of rifampicin-activated PXR with RXRα is one main reason to arrest cell cycle phase through inhibiting combination of RXRα with other partners. Rifampicin could promote cell growth rate when RXRα expressed more excessively than PXR in cells.