Genetically Modified Hepatocytes Targeting Bilirubin and Ammonia Metabolism for the Construction of Bioartificial Liver System.

Acute liver failure (ALF) is a complex syndrome that impairs the liver's function to detoxify bilirubin, ammonia, and other toxic metabolites. Bioartificial liver (BAL) aims to help ALF patients to pass through the urgent period by temporarily undertaking the liver's detoxification functions and promoting the recovery of the injured liver. We genetically modified the hepatocellular cell line HepG2 by stably overexpressing genes encoding UGT1A1, OATP1B1, OTC, ARG1, and CPS1. The resulting SynHeps-II cell line, encapsulated by Cytopore microcarriers, dramatically reduced the serum levels of bilirubin and ammonia, as demonstrated both in vitro using patient plasma and in vivo using ALF animal models. More importantly, we have also completed the 3-dimensional (3D) culturing of cells to meet the demands for industrialized rapid and mass production, and subsequently assembled the plasma-cell contacting BAL (PCC-BAL) system to fulfill the requirements of preclinical experiments. Extracorporeal blood purification of ALF rabbits with SynHeps-II-embedded PCC-BAL saved more than 80% of the animals from rapid death. Mechanistically, SynHeps-II therapy ameliorated liver and brain inflammation caused by high levels of bilirubin and ammonia and promoted liver regeneration by modulating the nuclear factor κB (NF-κB) and signal transducer and activator of transcription 3 (STAT3) pathways. Also, SynHeps-II treatment reduced cerebral infiltration of neutrophils, reduced reactive oxygen species (ROS) levels, and mitigated hepatic encephalopathy. Taken together, SynHeps-II cell-based BAL was promising for the treatment of ALF patients and warrants clinical trials.

A novel LAG3 neutralizing antibody improves cancer immunotherapy by dual inhibition of MHC-II and FGL1 ligand binding.

LAG3 is an inhibitory immune checkpoint expressed on activated T and NK cells. Blocking the interaction of LAG3 with its ligands MHC-II and FGL1 renders T cells improved cytotoxicity to cancer cells. Current study generated a panel of LAG3 monoclonal antibodies (mAbs) through immunization of mice followed by phage display. Some of them bound to the D1-D2 domain of LAG3, which is known for the engagement of its ligands FGL1 and MHC-II. Three outperformers, M208, M226, and M234, showed stronger blocking activity than Relatlimab in the FGL1 binding. Furthermore, M234 showed dual inhibition of FGL1 (IC50 of 20.6 nM) and MHC-II binding (IC50 of 6.2 nM) to LAG3. In vitro functional tests showed that M234 significantly stimulated IFN-γ secretion from activated PBMC cells. In vivo studies in a mouse model of hepatocellular carcinoma xenografts demonstrated that combining M234 IgG with GPC3-targeted bispecific antibodies significantly improved efficacy. In addition, GPC3-targeted CAR-T cells secreting IL-21-M234 scFv fusion protein exhibited enhanced activity in inhibiting tumor growth and greatly increased the survival rate of mice. Taken together, M234 has potential in cancer immunotherapy and warrants further clinical trial.

Mesothelin CAR-T cells expressing tumor-targeted immunocytokine IL-12 yield durable efficacy and fewer side effects.

Chimeric antigen receptor (CAR)-modified T cell therapy has achieved remarkable efficacy in treating hematological malignancies, but it confronts many challenges in treating solid tumors, such as the immunosuppressive microenvironment of the solid tumors. These factors reduce the antitumor activity of CAR-T cells in clinical trials. Therefore, we used the immunocytokine interleukin-12 (IL-12) to enhance the efficacy of CAR-T cell therapy. In this study, we engineered CAR-IL12R54 T cells that targeted mesothelin (MSLN) and secreted a single-chain IL-12 fused to a scFv fragment R54 that recognized a different epitope on mesothelin. The evaluation of the anti-tumor activity of the CAR-IL12R54 T cells alone or in combination with anti-PD-1 antibody in vitro and in vivo was followed by the exploration of the functional mechanism by which the immunocytokine IL-12 enhanced the antitumor activity. CAR-IL12R54 T cells had potency to lyse mesothelin positive tumor cells in vitro. In vivo studies demonstrated that CAR-IL12R54 T cells were effective in controlling the growth of established tumors in a xenograft mouse model with fewer side effects than CAR-T cells that secreted naked IL-12. Furthermore, combination of PD-1 blockade antibody with CAR-IL12R54 T cells elicited durable anti-tumor responses. Mechanistic studies showed that IL12R54 enhanced Interferon-γ (IFN-γ) production and dampened the activity of regulatory T cells (Tregs). IL12R54 also upregulated CXCR6 expression in the T cells through the NF-κB pathway, which facilitated T cell infiltration and persistence in the tumor tissues. In summary, the studies provide a good therapeutic option for the clinical treatment of solid tumors.

Mesothelin-targeted CAR-T therapy combined with irinotecan for the treatment of solid cancer.

BACKGROUND: Chimeric antigen receptor (CAR) T cell therapy has shown promising results in treating blood cancers, but it has limited efficacy against solid tumors that express mesothelin (MSLN). One of the reasons is the immunosuppressive tumor microenvironment, which consists of physical barriers, multiple mechanisms of immune evasion, and various biochemical factors that favor tumor growth and survival. These factors reduce the antitumor activity of MSLN-targeted CAR T cells in clinical trials. Therefore, new therapeutic strategies are needed to enhance the effectiveness of MSLN-targeted CAR T cell therapy. METHODS: To investigate whether the antitumor efficacy of anti-MSLN CAR-T cells depends on the epitopes they recognize, we generated MSLN-targeted CAR T cells that bind to different regions of MSLN (Region I, II, III and Full length). We then evaluated the antitumor activity of MSLN-targeted CAR T cells alone or in combination with the chemotherapeutic drug irinotecan or an anti-PD-1 antibody in vitro and in vivo. RESULTS: We found that MSLN-targeted CAR T cells effectively killed MSLN-positive cancer cells (H9, H226 and Panc-1), but not MSLN-negative cells (A431) in vitro. In a mouse model of H9 tumor xenografts, all CAR T cells showed similar tumor suppression, but an MSLN-targeted scFv with Region I epitope, R47, performed slightly better. Combining irinotecan with CAR_R47 T cells enhanced tumor control synergistically in both H9 xenograft mice and patient-derived xenograft mice. CONCLUSIONS: Our results suggest that irinotecan can enhance the antitumor activity of MSLN-targeted CAR T cells, and offer a promising combination therapy strategy for MSLN-positive solid tumors.

Molecular cloning and expression patterns of a sex-biased transcriptional factor Foxl2 in the giant freshwater prawn (Macrobrachium rosenbergii).

BACKGROUD: Macrobrachium rosenbergii is an economically important species that is widely cultivated in some Asian nations. Foxl2 is a transcriptional regulator of ovarian differentiation and development. The aim of this study was to study the bioinformatics features and expression patterns of M. rosenbergii Foxl2 (MrFoxl2). METHODS: In this study, all experimental animals were mature M. rosenbergii (9-12 cm) individuals. The foxl2 gene was identified and characterized in the genome of M. rosenbergii using molecular cloning, bioinformatic analysis, in situ hybridization, and quantitative analysis. RESULTS: The identified cDNA encoded a putative 489-amino-acid MrFoxl2 protein. Bioinformatics analysis revealed a low identity of MrFoxl2 to other crustacean orthologues. The closest phylogenetic relationship was to Foxl2 of Eriocheir sinensis. The result of in situ hybridization demonstrated that transcripts of MrFoxl2 in M. rosenbergii were identified in spermatocytes, oocytes, and secretory epithelial cells of the vas deferens. The result of q-PCR suggested that a high expression of MrFoxl2 was identified in the testis, vas deferens, and ovaries. During ovarian development, MrFoxl2 expression was the highest in stage I. CONCLUSION: Our findings suggest that MrFoxl2 may play a role in gonadal development in both female and male M. rosenbergii.

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.

Combination Therapy of Hepatocellular Carcinoma by GPC3-Targeted Bispecific Antibody and Irinotecan is Potent in Suppressing Tumor Growth in Mice.

Hepatocellular carcinoma (HCC) is a world leading cause of cancer-related mortality, and currently no curative treatment for advanced HCC is available. Glypican-3 (GPC3) is an attractive target for HCC immunotherapy. This study explored the efficacy of six GPC3-targeted bispecific antibodies, alone or in combination with chemotherapeutic drug Irinotecan, for the treatment of HCC. The bispecific antibodies were constructed using three different structures, knob-into-hole (KH), scFv-scFv-hFc, and scFv-hFc-scFv, where CD3-targeting mAb OKT3 (scFv) was paired with two representative GPC3 mAbs hYP7 (scFv) and HN3 (VH only) that target different epitopes. The In vitro cell killing assay revealed that all bispecific antibodies efficiently killed GPC3 positive cancer cells, with hYP7-KH, hYP7-OKT3-hFc, and HN3-KH being most potent. In vivo xenograft mouse studies demonstrated that all bispecific antibodies suppressed tumor growth similarly, with hYP7-OKT3-hFc performing slightly better. Combination of hYP7-OKT3-hFc with Irinotecan dramatically improved the efficacy and arrested tumor growth of HepG2, Hep3B, and G1 in xenograft mice. Our results demonstrated that the cell surface proximal bispecific antibody hYP7-OKT3-hFc was superior in terms of potency and the GPC3-targeted bispecific antibody combined with Irinotecan was much potent to control HCC growth.

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.

Combined DLL3-targeted bispecific antibody with PD-1 inhibition is efficient to suppress small cell lung cancer growth.

BACKGROUND: Small cell lung cancer (SCLC) accounts for 15% of lung cancers, and the primary treatment of this malignancy is chemotherapy and radiotherapy. Delta-like 3 (DLL3) is an attractive target for SCLC immunotherapy since its expression is highly restricted to SCLC with a neglectable appearance on normal adult tissues. In the current study, we aimed to explore the efficacy of DLL3-targeted SCLC immunotherapy via the engagement of T cell. METHODS: As a proof of concept, we constructed DLL3-targeted bispecific antibody and chimeric antigen receptor (CAR)-modified T cells. In vitro and in vivo tumor-suppression activity of these treatments alone or in combination with a Program Death-1 (PD-1) inhibitory antibody was evaluated. RESULTS: In vitro studies showed that both DLL3 bispecific antibody and CAR-T efficiently killed DLL3-positive cancer cells, including the native SCLC cell lines H446, H196, H82, and the artificial A431 cells that were forcefully overexpressing DLL3. In vivo studies in xenograft mouse models demonstrated that both bispecific antibody and CAR-T suppressed the tumor growth, and combination therapy with PD-1 inhibitory antibody dramatically improved the efficacy of the DLL3 bispecific antibody, but not the CAR-T cells. CONCLUSIONS: Our results demonstrated that DLL3-targeted bispecific antibody plus PD-1 inhibition was effective in controlling SCLC growth.

Effects of different dietary energy and protein levels and sex on growth performance, carcass characteristics and meat quality of F1 Angus × Chinese Xiangxi yellow cattle.

BACKGROUND: The experiment evaluated the effect of nutrition levels and sex on the growth performance, carcass characteristics and meat quality of F1 Angus × Chinese Xiangxi yellow cattle. METHODS: During the background period of 184 d,23 steers and 24 heifers were fed the same ration,then put into a 2 × 2 × 2 factorial arrangement under two levels of - dietary energy (TDN: 70/80% DM), protein (CP: 11.9/14.3% DM) and sex (S: male/female) during the finishing phase of 146 d. The treatments were - (1) high energy/low protein (HELP), (2) high energy/high protein (HEHP), (3) low energy/low protein (LELP) and (4) low energy/high protein (LEHP). Each treatment used 6 steers and 6 heifers, except for HELP- 5 steers and 6 heifers. RESULTS: Growth rate and final carcass weight were unaffected by dietary energy and protein levels or by sex. Compared with the LE diet group, the HE group had significantly lower dry matter intake (DMI, 6.76 vs. 7.48 kg DM/d), greater chest girth increments (46.1 vs. 36.8 cm), higher carcass fat (19.9 vs.16.3%) and intramuscular fat content (29.9 vs. 22.8% DM). The HE group also had improved yields of top and medium top grade commercial meat cuts (39.9 vs.36.5%). The dressing percentage was higher for the HP group than the LP group (53.4 vs. 54.9%). Steers had a greater length increment (9.0 vs. 8.3 cm), but lower carcass fat content (16.8 vs. 19.4%) than heifers. The meat quality traits (shear force value, drip loss, cooking loss and water holding capacity) were not affected by treatments or sex, averaging 3.14 kg, 2.5, 31.5 and 52.9%, respectively. The nutritive profiles (both fatty and amino acid composition) were not influenced by the energy or protein levels or by sex. CONCLUSIONS: The dietary energy and protein levels and sex significantly influenced the carcass characteristics and chemical composition of meat but not thegrowth performance, meat quality traits and nutritive profiles.