BCA101 Is a Tumor-Targeted Bifunctional Fusion Antibody That Simultaneously Inhibits EGFR and TGFß Signaling to Durably Suppress Tumor Growth.

The EGFR and TGFß signaling pathways are important mediators of tumorigenesis, and cross-talk between them contributes to cancer progression and drug resistance. Therapies capable of simultaneously targeting EGFR and TGFß could help improve patient outcomes across various cancer types. Here, we developed BCA101, an anti-EGFR IgG1 mAb linked to an extracellular domain of human TGFßRII. The TGFß "trap" fused to the light chain in BCA101 did not sterically interfere with its ability to bind EGFR, inhibit cell proliferation, or mediate antibody-dependent cellular cytotoxicity. Functional neutralization of TGFß by BCA101 was demonstrated by several in vitro assays. BCA101 increased production of proinflammatory cytokines and key markers associated with T-cell and natural killer-cell activation, while suppressing VEGF secretion. In addition, BCA101 inhibited differentiation of naïve CD4+ T cells to inducible regulatory T cells (iTreg) more strongly than the anti-EGFR antibody cetuximab. BCA101 localized to tumor tissues in xenograft mouse models with comparable kinetics to cetuximab, both having better tumor tissue retention over TGFß "trap." TGFß in tumors was neutralized by approximately 90% in animals dosed with 10 mg/kg of BCA101 compared with 54% in animals dosed with equimolar TGFßRII-Fc. In patient-derived xenograft mouse models of head and neck squamous cell carcinoma, BCA101 showed durable response after dose cessation. The combination of BCA101 and anti-PD1 antibody improved tumor inhibition in both B16-hEGFR-expressing syngeneic mouse models and in humanized HuNOG-EXL mice bearing human PC-3 xenografts. Together, these results support the clinical development of BCA101 as a monotherapy and in combination with immune checkpoint therapy. SIGNIFICANCE: The bifunctional mAb fusion design of BCA101 targets it to the tumor microenvironment where it inhibits EGFR and neutralizes TGFß to induce immune activation and to suppress tumor growth.

Correlation of abrin-mediated inhibition of protein synthesis and apoptosis.

The plant toxin, abrin, a type-II ribosome inactivating protein, is extremely lethal, the human fatal dose being ~1 µg/kg body weight. Abrin has been classified as an agent for bioterrorism, which is of concern. Conversely, the high toxic property of abrin has been employed in generating immunotoxins, whereas its toxin moiety is conjugated to cell surface marker-specific antibodies for cell-targeted killing. Different cell types exhibit variable levels of sensitivity to abrin toxicity; therefore, adequate knowledge of the molecular mechanism that governs the activity of the protein would be a safeguard. To gain insights into this, two cell lines requiring strikingly different concentrations of abrin for inactivating ribosomes were studied. Employing conjugates of the wild-type and active site mutant of abrin A chain with the ricin B chain, it was found that abrin-induced apoptosis was dependent on inhibition of protein synthesis (PSI) leading to ER-stress in Ovcar-3 cells, but not in KB cells. Abrin was also observed to cause direct DNA damage in KB cells, while in Ovcar-3 cells abrin-induced DNA damage was found to be dependent on caspases. Overall, the study demonstrates that the correlation of abrin-mediated PSI and apoptosis is cell-specific and abrin can induce more than one pathway to cause cell death. © 2018 IUBMB Life, 71(3):357-363, 2019.

A chimeric protein of abrin and Abrus precatorius agglutinin that neutralizes abrin mediated lethality in mice.

Abrin, a type II ribosome inactivating protein from the Abrus precatorius plant, is extremely toxic. It has been shown to be 75 times more potent than its infamous sister toxin, ricin and their potential use in bio-warfare is a cause of major concern. Although several vaccine candidates are under clinical trials for ricin, none are available against abrin. The present study proposes a chimeric protein, comprising of 1-123 amino acids taken from the A chain of abrin and 124-175 amino acids from Abrus precatorius agglutinin A chain, as a vaccine candidate against abrin intoxication. The design was based on the inclusion of the immunogenic region of the full length protein and the minimal essential folding domains required for inducing neutralizing antibody response. The chimera also contains the epitope for the only two neutralizing antibodies; D6F10 and A7C4, reported against abrin till now. Active immunization with the chimera protected all the mice challenged with 45 X LD50 of abrin. Also, passive transfer of antibodies raised against the chimera rescued all mice challenged with 50 X LD50 of toxin. Hence the chimeric protein appears to be a promising vaccine candidate against abrin induced lethality.

Sequestration of the abrin A chain to the nucleus by BASP1 increases the resistance of cells to abrin toxicity.

Abrin, a type II ribosome-inactivating protein, comprises A and B subunits wherein the A subunit harbours toxin activity and the B subunit has a galactose-specific lectin activity. The entry of the protein inside the cell is through the binding of the B chain to cell surface glycoproteins followed by receptor-mediated endocytosis and retrograde transport. A previous study from our laboratory showed that different cell lines exhibited differences of as great as ~200-fold in abrin toxicity, prompting the present study to compare the trafficking of the toxin within cells. Observations made in this regard revealed that the abrin A chain, after being released into the cytosol, is sequestered into the nucleus through interaction with a cellular protein of ~25 kDa, BASP1 (brain acid-soluble protein 1). The nuclear localization of the A chain is seen predominantly in cells that are less sensitive to abrin toxicity and dependent on the levels of BASP1 in cells. The sequestration by BASP1 renders cells increasingly resistant to the inhibition of protein synthesis by abrin and the nucleus act as a sink to overcome cellular stress induced by the toxin.

Diabetogenic effects of Parthinium hysterophorous induced allergic rhinitis.

Diabetes mellitus (DM) is known to be associated with the cytokines secreted by Th1 cells, while allergic rhinitis (AR) is mainly regulated by Th2 cytokines. According to Th1/Th2 paradigm, there is an inverse relationship between Th1 and Th2 cytokines and resultantly, both the aforesaid diseases are also inversely correlated. On the other hand, numbers of clinical reports suggest every possible correlation between DM and AR including positive, negative and neutral. However, till to date, no experimental report available, suggesting the changes in glucose homeostasis of animal model(s) of allergic rhinitis, if any. Therefore, in the present study we have observed the changes in glucose homeostasis of the animals bearing AR induced by Parthinium hysterophorous (PH). The condition of AR was induced by intranasal instillations of the acetone extract of PH. At the end of experimentation, various parameters for AR and DM were evaluated. A significant increase was observed in total leukocytes in nasal fluid, serum glucose, thyroxine, dyslipidemia and activity of α-amylase, pancreatic lipid peroxidation, serum and pancreatic nitrite with a concomitant reduction in serum calcium, triiodothyronine, hepatic glycogen and activity of phosphoglucomutase. However, serum insulin, TSH, pancreatic calcium and hepatic glucokinase increased non-significantly. Immune cells infiltration and increased intra alveolar space were observed in lungs tissue, while alterations were also observed in pancreas of AR treated animals. The induction of AR led to the diabetogenic changes to rats via exerting multifaceted metabolic defects in the biochemical machinery regulating glucose homeostasis.

DPP-IV inhibitory potential of naringin: an in silico, in vitro and in vivo study.

The incretin based therapies are an emerging class of antidiabetic drugs, with two categories: one is glucagone like peptide-1 (GLP-1) agonists and the other one is dipeptidyl peptidase (CD26; DPP-IV) inhibitors. However, in the DPP-IV inhibitors category only few compounds are commercially available and also have some undesirable effects. Therefore, in the present work we tried to explore a naturally occurring compound naringin for its potential DPP-IV inhibition and antidiabetic potential. It is noteworthy that this compound is abundantly present in orange peel and thus may provide cost effective treatment for diabetes, especially type 2 diabetes mellitus. In the present study, we have conducted virtual docking study and observed tight binding of naringin, as shown by higher negative values of H bond lengths, while in vitro DPP-IV inhibition assay has also shown better inhibition by naringin. In vivo study, in response to 10 days administration of 40 mg/kg of naringin twice daily to Wistar albino rats, inhibited the serum levels of DPP-IV activity, random glucose concentration with concomitant increase in insulin levels. All the comparisons were made with the standard commercially available drug sitagliptin.

Coexpression of 16.8 kDa antigen of Mycobacterium avium paratuberculosis and murine gamma interferon in a bicistronic vector and studies on its potential as DNA vaccine.

Paratuberculosis or Johne's disease is a chronic gastric disease of ruminants. For this disease there is no effective treatment or preventive measure available. 16.8 kDa protein is an immunogenic protein of Mycobacterium avium paratuberculosis and can be an ideal candidate for developing a DNA vaccine construct. In present study a bicistronic DNA vaccine construct pIR16.8/IFN was developed using eukaryotic vector pIRES 6.1. Two genes MPT (expressing 16.8 kDa protein) and murine IFNgamma were cloned, expressed and immunoreactivity was studied in murine model. Immunoreactivity was also compared with monocistronic construct pIR16.8 expressing 16.8 kDa protein. Both pIR16.8 and pIR16.8/IFN showed eukaryotic expression of respective proteins in BHK21 cells. The expressed proteins also showed immunoreactivity when reacted with hyperimmune sera raised against recombinant 16.8 kDa protein in western blot assay and immunofluorence assay. Both constructs were used as DNA vaccine in murine model and immunogenecity was studied by DTH, lymphocyte proliferation assay and NO determination. DTH reaction was significantly high in pIR16.8/IFN than pIR16.8 group, similarly lymphocyte proliferation and NO release was higher in pIR16.8/IFN group than pIR16.8 group. This indicated T cell epitopic nature of 16.8 kDa protein. The study also showed that co-expression of IFNgamma with mycobacterial gene can enhance immunogenecity of DNA vaccine and can be used as immunoadjuvant.