Roles and Mechanisms of Choline Metabolism in Nonalcoholic Fatty Liver Disease and Cancers.

Choline participates in three major metabolic pathways: oxidation, phosphorylation, and acetylation. Through oxidation, choline is converted to betaine and contributes to methyl metabolism and epigenetic regulation. Through phosphorylation, choline participates in phospholipid metabolism, and serves as the precursor of phosphocholine, phosphatidylcholine, glycerophosphocholine, and other essential compounds, thereby modulating lipid metabolism and transport. Through acetylation, choline is transformed into acetylcholine in cholinergic neurons, playing a vital role in neurotransmission. Moreover, gut microbiota can metabolize choline into trimethylamine-N-oxide, and be involved in the pathogenesis of various diseases such as nonalcoholic fatty liver disease (NAFLD), cancer, cardiovascular disease, etc. Since choline metabolism is implicated in the development of NAFLD and diverse cancers, including liver cancer, it may serve as a therapeutic target for these diseases in the future. Currently, there are numerous therapeutic agents targeting choline metabolism to treat NAFLD and cancers, but most of them are ineffective and some even have adverse effects that lead to a series of complications. Therefore, further research and clinical validation are required to obtain safe and efficacious drugs. This review comprehensively summarizes the choline metabolic pathway and its regulatory mechanisms, elucidates the roles and mechanisms of choline metabolism in the aforementioned diseases, and provides a discussion of the current advances and immense potential of this field.

EGFRvâ ¢-targeted immunotoxin combined with temozolomide and bispecific antibody for the eradication of established glioblastoma.

EGFRvⅢ is an established target for immunotherapy of glioblastoma (GBM). Current study aims to explore the efficacy of EGFRvⅢ-targeted immunotoxin combined with temozolomide (TMZ) or T cell-engaged bispecific antibody for the treatment of GBM. We generated three rabbit monoclonal antibodies (R1, R2, and R6) that specifically bound to EGFRvⅢ, but not EGFR, with high affinity. Immunotoxins were made by fusing the scFv of these antibodies with engineered Pseudomonas exotoxin PE24. The in vitro cytotoxicity and specificity of the immunotoxins was rigorously validated by EGFRvⅢ and EGFR-expressed cell lines. The in vivo efficacy of immunotoxin monotherapy and in combination with TMZ or EGFRvⅢ-targeted bispecific antibody was evaluated in orthotopic and subcutaneous xenograft mouse models. EGFRvⅢ immunotoxins potently killed U87, U251 and GL261 cells that were forcefully expressing EGFRvⅢ, with IC50 values bellow 1.2 ng/ml. In a subcutaneous model, multiple intratumoral injections of immunotoxin at a dose of 2 mg/kg resulted in complete tumor regression in 3/5 of mice. In a C57BL/6 orthotopic glioblastoma model transplanted with GL261 cells that expressed a mouse version of EGFRvⅢ, two injections of 10 micrograms of immunotoxin in the lateral ventricles significantly improved the survival, with 2/5 mice being completely cured. Furthermore, in a subcutaneous xenograft model transplanted with EGFRvⅢ-expressed U87 cells, a single intratumoral injection of immuntoxin followed by i.v. injections of TMZ or EGFRvⅢ-targeted bispecific antibody achieved complete regression in mice. Taken together, EGFRvⅢ immunotoxin combined with TMZ or T cell-engaged bispecific antibody offers promise for curative treatment of GBM.

Strengthening of antitumor effects in breast cancer from a novel B7-H4- and CD3-targeting bispecific antibody by an oncolytic virus.

Background: For programmed cell death protein 1/programmed death-ligand 1 (PD-L1)-positive triple-negative breast cancer (TNBC), the addition of immune checkpoint inhibitors (ICIs) to chemotherapy has been shown to increase the objective response rate (ORR) by only 13.4-16.3%. Thus, examining converting tumor immunogenicity and targeting novel pathways may be needed to improve response to immunotherapy. B7-H4 is an emerging target for breast cancer immunotherapy. However, the antitumor effect of B7-H4 is unstable during cell generation and further research is necessary to strengthen the antitumor efficacy of B7-H4. Methods: To explore the potential of B7-H4-targeted immunotherapy of breast cancer, current study generated four monoclonal antibodies (mAbs) against B7-H4 through immunization of mice followed by phage display technology. T cell-engaged bispecific antibodies and immunotoxins were created based on these mAbs. To evaluate the anti-tumor activity of B7-H4-targeting bispecific antibody and immunotoxin, the anti-tumor efficacy test in vitro and in mice models in vivo were performed. Moreover, to boost the efficacy, oncolytic virus herpes simplex virus type 2 (HSV-2) was combined with the bispecific antibody and tested in mice models. Results: Rigorous in vitro cytotoxicity assay showed that both B7-H4 bispecific antibodies and immunotoxins were toxic to B7-H4 positive cells, with the former being more potent. However, in vivo studies showed that immunotoxin was slightly more effective in suppressing tumor growth. Further, bispecific antibody combined with oncolytic virus HSV-2 revealed a synergistic effect in suppressing tumor growth without causing obvious adverse effects. Conclusions: Collectively, our findings uncover a novel strategy of combining oncolytic virus HSV-2 with bispecific antibody reinforce antitumor immune response, which warranted further translational investigation.

Degradation study of rutin as template from magnetic composite molecularly imprinted polymer supernatant samples by liquid chromatography-mass spectrometry.

Compound structure change of the template molecule from rutin samples might affect the selectivity and adsorption of molecularly imprinted polymers based on magnetic halloysite nanotubes (MHNTs@MIPs). In the present study, not only MHNTs@MIPs were successfully characterized by TEM, SEM, EDS, FT-IR, TGA and VSM, but related compounds and potential degradation factors of template molecule rutin were also investigated in the polymerization and elution process of MHNTs@MIPs by high-performance liquid chromatography together with photodiode array and tandem mass spectrometry (HPLC-PDA-MS/MS) in negative ion mode. Nine flavonol components were detected in rutin active pharmaceutical ingredient samples, such as rutin, isoquercetrin, kaempferol-3-O-rutoside, isorhamnetin-3-O-rutoside, quercetin, kaempferol, isorhamnetin and two unknown triglycosides. Under acidic, basic, oxidative and solvent conditions, two common degradants with negative ESI-MS ions at m/z 317 and 349 were found. Meanwhile, protocatechuic acid and methyl ester were also observed as two characteristic oxidative degradants. Compared to above degradation results, rutin-dimethylsulfoxide adduct and oxidative degradant were observed in the synthesis supernatant, which demonstrated that appropriate solvents and strict control of the oxygen level were critical in the synthesis process. Therefore, degradation studies provide a solid foundation for the optimization of MHNT@MIP synthesis with rutin as a template, which can be potentially applied to other templates for further material performance investigation.

Highly Potent Immunotoxins Targeting the Membrane-distal N-lobe of GPC3 for Immunotherapy of Hepatocellular Carcinoma.

Glypican-3 (GPC3) has become a compelling target for immunotherapy of hepatocellular carcinoma, including antibody-drug conjugate (ADC), and ADC-like immunotoxin. To investigate the impact of epitopes on the potency of ADCs, current study generated a large panel of chicken monoclonal antibodies (mAbs) that targeted 12 different and over-lapping epitopes on GPC3. These mAbs demonstrated a very high affinity with Kd values in the range of 10-9-10-14 M, and the highest affinity (Kd value of 0.0214 pM) was 40-fold higher than the previously generated high-affinity mAb YP7 (Kd value of 0.876 nM). Additionally, these mAbs exhibited excellent thermostability with Tm values in the range of 45-82 °C. As a proof-of-concept study for ADC, we made immunotoxins (scFv fused with PE24, the 24-kDa cytotoxic domain of Pseudomonas exotoxin A) based on these mAbs, and we found that immunotoxins targeting the N-lobe of GPC3 were overall much more potent than those targeting the C-lobe and other locations. One representative N-lobe-targeting immunotoxin J80A-PE24 demonstrated 3 to 13-fold more potency than the hitherto best immunotoxin HN3-PE24 that was previously developed. J80A-PE24 could suppress tumor growth much greater than HN3-PE24 in a xenograft mouse model. Combination of J80A-PE24 with an angiogenesis inhibitor FGF401 showed additive effect, which dramatically shrank tumor growth. Our work demonstrated that, due to high affinity, excellent thermostability and potency, chicken mAbs targeting the N-lobe of GPC3 are appealing candidates to develop potent ADCs for immunotherapy of liver cancer.

Generation of TIM3 inhibitory single-domain antibodies to boost the antitumor activity of chimeric antigen receptor T cells.

Targeting inhibitory immune checkpoint molecules has significantly altered cancer treatment regimens. T cell immunoglobulin and mucin domain 3 (TIM3) is one of the major inhibitory immune checkpoints expressed on T cells. Blocking the engagement of TIM3 and its inhibitory ligand galectin-9 may potentiate the effects of immunotherapy or overcome the adaptive resistance to the therapeutic blockade of programmed cell death protein 1, cytotoxic T-lymphocyte-associated protein 4, B- and T-lymphocyte attenuator and lymphocyte-activation gene 3, amongst others, as each of these immune checkpoints harbors unique properties that set it apart from the rest. Heavy chain variable fragment (VH)-derived single-domain antibodies (sdAbs) represent a class of expanding drug candidates. These sdAbs have unique advantages, including their minimal size in the antibody class, ease of expression, broad scope for modular structure design and re-engineering, and excellent tumor penetration. In the present study, two sdAbs, TIM3-R23 and TIM3-R53, were generated by immunizing rabbits with the recombinant extracellular domain of TIM3 and applying phage display technology. These sdAbs were easily expressed in mammalian cells. The purified sdAbs were able to bind to both recombinant and cell surface TIM3, and blocked it from binding to the ligand galectin-9. In vivo studies demonstrated that TIM3-R53 was able to potentiate the antitumor activity of chimeric antigen receptor T cells that targeted mesothelin. In conclusion, the results of the present study suggested that TIM3-R53 may be a novel and attractive immune checkpoint inhibitor against TIM3, which is worthy of further investigation.

Angptl7 promotes insulin resistance and type 2 diabetes mellitus by multiple mechanisms including SOCS3-mediated IRS1 degradation.

Angptl7 is a secreted and circulating cytokine that belongs to Angiopoietin-like family. The current knowledge about the function of Angptl7 is still limited, and its biological role is only marginally known, such as in the promotion of angiogenesis and inflammation. Here, we demonstrated that Angptl7 promotes insulin resistance and type 2 diabetes mellitus (T2DM). We found that the circulating Angptl7 levels in T2DM patient and mouse models were significantly elevated. Artificial overexpression of Angptl7 in hepatic cells inhibited glucose uptake and impaired insulin signaling pathway. Furthermore, in vivo overexpression of Angptl7 in experimental healthy mice also caused insulin resistance-like characteristics. Mechanistic studies revealed that Angptl7 can upregulate SOCS3 expression, leading to the IRS1 degradation in proteasome. Furthermore, over-expressed Angptl7 inhibited the phosphorylation of Akt and promoted the phosphorylation of ERK1/2, which was known to be associated with insulin resistance. Taken together, our study provided strong evidence that Angptl7 promotes insulin resistance and T2DM by multiple mechanisms, which made Angptl7 a new potential therapeutic target for treatment of insulin resistance and T2DM.