Robust Antitumor Activity and Low Cytokine Production by Novel Humanized Anti-CD19 CAR T Cells.

Recent studies have described the remarkable clinical outcome of anti-CD19 chimeric antigen receptor (CAR) T cells in treating B-cell malignancies. However, over 50% of patients develop life-threatening toxicities associated with cytokine release syndrome which may limit its utilization in low-resource settings. To mitigate the toxicity, we designed a novel humanized anti-CD19 CAR T cells by humanizing the framework region of single-chain variable fragment (scFv) derived from a murine FMC63 mAb and combining it with CD8α transmembrane domain, 4-1BB costimulatory domain, and CD3ζ signaling domain (h1CAR19-8BBζ). Docking studies followed by molecular dynamics simulation revealed that the humanized anti-CD19 scFv (h1CAR19) establishes higher binding affinity and has a flexible molecular structure with CD19 antigen compared with murine scFv (mCAR19). Ex vivo studies with CAR T cells generated from healthy donors and patients with relapsed/refractory B-cell acute lymphoblastic leukemia (B-ALL) expressing either h1CAR19 or mCAR19 showed comparable antitumor activity and proliferation. More importantly, h1CAR19-8BBζ T cells produced lower levels of cytokines (IFNγ, TNFα) upon antigen encounter and reduced the induction of IL6 cytokine from monocytes than mCAR19-8BBζ T cells. There was a comparable proliferation of h1CAR19-8BBζ T cells and mCAR19-8BBζ T cells upon repeated antigen encounter. Finally, h1CAR19-8BBζ T cells efficiently eliminated NALM6 tumor cells in a preclinical model. In conclusion, the distinct structural modification in CAR design confers the novel humanized anti-CD19 CAR with a favorable balance of efficacy to toxicity providing a rationale to test this construct in a phase I trial.

Modulation of T-helper cytokines and inflammatory mediators by Atropa accuminata. Royle in adjuvant induced arthritic tissues.

ETHNOPHARMACOLOGICAL RELEVANCE: Atropa acuminata has been widely used in traditional medicine against arthritis and several associated inflammatory disorders. AIM OF THE STUDY: The present study was undertaken to investigate the anti-arthritic activities of ethanolic extract of Atropa accuminata (AAEE) and to explore the probable mechanism of action. MATERIALS AND METHODS: The anti-arthritic activity of AAEE was evaluated within a dose range of 125-500 mg/kg b.w. in adjuvant induced-arthritis in male wistar rats. An array of pro-inflammatory mediators (PGE2 NO, IL-1ß and LTB4) and T-cell-mediated cytokines (IL-2, TNF-a, IFN-c, IL-4, IL-10, IL-12, IL-17, IL-6) were assayed in arthritic paw tissue homogenate of the treated animals. In addition the effects on arthritic lesions, changes in body weight; haematological (Hb, ESR, WBC and RBC) and biochemical parameters (SOD, GSH, GR) and the serum markers (CRP, RF) were also observed. RESULTS: Significant anti-arthritic activity was observed for AAEE in the polyarthiritis test both in the developing and developed phase of the disease. This was associated with dose dependant suppression of pro-inflammatory mediators (PGE2, NO, IL-1ß and LTB4)., Th1-Th17 cytokines (IL-2, TNF-α, IFN-γ, IL-12, IL-17, IL-6) and upregulation of Th2 cytokines (IL-4 and IL-10). AAEE was also observed to protect rats against the primary and secondary arthritic lesions, body weight changes and haematological perturbations. In addition, inhibitory effects of AAEE on biochemical parameters and the serum markers further confirmed that it reduced signs on chronic inflammatory responses. CONCLUSION: The present investigation therefore suggested that AAEE is a potent anti-arthritic agent. The multipronged attack on the inflammatory mediators and T-helper cytokines and strong potency of AAEE may have relevance for inhibition of the chronic inflammatory responses in arthritis.

Effect of Ajuga bracteosa on systemic T-cell immunity in Balb/C mice: dual Th1/Th2 immunostimulatory effects.

Ajuga bracteosa (AB) has been widely used in folk medicine in Asian countries against gout, hepatitis, pneumonia, rheumatism, and various neuro inflammatory disorders. The aim of this study was to investigate the possible immunoregulatory effects of the ethanolic extract of Ajuga bracteosa (ABEE) on systemic Th1/Th2 immunity in SRBC immunized Balb/C mice. Animals were orally administered with graded doses of ABEE from 6.25 mg/kg to 100 mg/kg. Post sub-cutaneous immunization with SRBCs and circulating antibody titers, DTH responses and splenocyte proliferation was monitored as markers of Th2 and Th1 responses. Cyclophosphamide and levamisole were used as controls. Lymphocyte immunophenotying (CD4/CD8 cell counts) and intracellular Th1/Th2 cytokine concentrations were determined using flow cytometry. Treatment with ABEE demonstrated significant biphasic immunostimulation of effector T-helper immunity. ABEE at 50 mg/kg dose resulted in maximal increase in antibody titers, DTH responses and CD4+/CD8+ T-cell percentages indicating maximal activation and proliferation of T and B lymphocytes at this dose. ABEE, at the same dose, also showed maximal up regulation of LPS and CON A stimulated splenocyte proliferation and also maximal up-regulation of both Th1 (IL-2, IFN-γ) and Th2 (IL-4) cytokines which suggest its mixed Th1/Th2 immunostimulatory activity. Comparatively at higher doses (100 mg/kg), significant down regulation of all these effector T-helper (Th) immune responses was observed. The study therefore suggests mixed biphasic immunostimulatory Th1/Th2 activity of ABEE that could support its immunoadjuvant potential.

Atropa acuminata Royle Ex Lindl. blunts production of pro-inflammatory mediators eicosanoids., leukotrienes, cytokines in vitro and in vivo models of acute inflammatory responses.

ETHNOPHARMACOLOGICAL RELEVANCE: Atropa acuminata Royle Ex Lindl. has been widely used in folk medicine for several inflammatory disorders such as arthritis, asthma, conjunctivitis, encephalitis, pancreatitis, peritonitis, acute infections and neuroinflammatory disorders. AIM OF THE STUDY: Our aim was to evaluate Atropa acuminata for its anti-inflammatory properties and to delineate its possible mechanism of action on the modulation of the inflammatory mediators. MATERIALS AND METHODS: We investigated the inhibitory action of ethanolic extract of Atropa acuminata (AAEE) on production of NO, TNF-α and IL-1ß in lipopolysaccharide (LPS)-stimulated RAW264.7 cells and also assayed it for COX 1/2 and 5-LOX inhibitory activities. Next AAEE was tested in acute inflammatory animal models., carragenean induced rat paw edema, carragenean induce pleurisy in rats and vascular permeability in mice and the effects on NO, PGE2 and LTB4 production in the pleural fluid and paw exudates were evaluated. In addition the effects on leukocyte migration and exudation and vascular permeability were also observed. RESULTS: Our findings summarized novel anti-inflammatory mechanisms for Atropa acuminata based on dual in vitro cyclooxygenase 1/2/ and 5-Lipoxygenase inhibitory activities and also significant downregulation of nitric oxide (NO) and pro-inflammatory cytokin (TNF-α and Il-1 ß) release in LPS-stimulated RAW 246.7 macrophage cell line. In acute inflammatory models in vivo (carragenean induced edema, carragenean induced pleurisy in rats and vascular permeability in mice), AAEE exhibited an extensive diverse mechanism for anti-inflammatory properties. This was indicated on the basis of dose dependent suppression of multi targeted inflammatory mediators., NO, TNF-α and IL-1ß, eicosanoids., PGE2 and leukotrienes., LTB4 along with significantly decreased leucocyte migration, exudation and decreased vascular permeability. These effects were more potent and prolonged than traditional NSAIDS, thereby indicating fewer side effects. AAEE was found to be safe for long term administration, as confirmed by the results of acute toxicity studies and MTT assay. The complex mode of action of the herbs was attributed possibly due to the high polyphenolic, flavanol and flavonoid content present in the extracts as observed by means of quantitative screening for phytochemicals. CONCLUSION: Our study provides scientific evidence to support the traditional anti-inflammatory uses of Atropa acuminata and is probably due to inhibitory effects on multiple inflammatory mediators which indicates a promising potential for the development of a strong anti-inflammatory agent from this plant.