Extended Biocatalytic Halogenation Cascades Involving a Single-Polypeptide Regeneration System for Diffusible FADH2.

Flavin-dependent halogenases have attracted increasing interest for aryl halogenation at unactivated C-H positions because they are characterised by high regioselectivity, while requiring only FADH2 , halide salts, and O2 . Their use in combined crosslinked enzyme aggregates (combiCLEAs) together with an NADH-dependent flavin reductase and an NADH-regeneration system for the preparative halogenation of tryptophan and indole derivatives has been previously described. However, multiple cultivations and protein purification steps are necessary for their production. We present a bifunctional regeneration enzyme for two-step catalytic flavin regeneration using phosphite as an inexpensive sacrificial substrate. This fusion protein proved amenable to co-expression with various flavin-dependent Trp-halogenases and enables carrier-free immobilisation as combiCLEAs from a single cultivation for protein production and the preparative synthesis of halotryptophan. The scalability of this system was demonstrated by fed-batch fermentation in bench-top bioreactors on a 2.5 L scale. Furthermore, the inclusion of a 6-halotryptophan-specific dioxygenase into the co-expression strain further converts the halogenation product to the kynurenine derivative. This reaction cascade enables the one-pot synthesis of l-4-Cl-kynurenine and its brominated analogue on a preparative scale.

Optimized Heterologous Expression and Efficient Purification of a New TRAIL-Based Antitumor Fusion Protein SRH-DR5-B with Dual VEGFR2 and DR5 Receptor Specificity.

In the last two decades, bifunctional proteins have been created by genetic and protein engineering methods to increase therapeutic effects in various diseases, including cancer. Unlike conventional small molecule or monotargeted drugs, bifunctional proteins have increased biological activity while maintaining low systemic toxicity. The recombinant anti-cancer cytokine TRAIL has shown a limited therapeutic effect in clinical trials. To enhance the efficacy of TRAIL, we designed the HRH-DR5-B fusion protein based on the DR5-selective mutant variant of TRAIL fused to the anti-angiogenic synthetic peptide HRHTKQRHTALH. Initially low expression of HRH-DR5-B was enhanced by the substitution of E. coli-optimized codons with AT-rich codons in the DNA sequence encoding the first 7 amino acid residues of the HRH peptide. However, the HRH-DR5-B degraded during purification to form two adjacent protein bands on the SDS-PAGE gel. The replacement of His by Ser at position P2 immediately after the initiator Met dramatically minimized degradation, allowing more than 20 mg of protein to be obtained from 200 mL of cell culture. The resulting SRH-DR5-B fusion bound the VEGFR2 and DR5 receptors with high affinity and showed increased cytotoxic activity in 3D multicellular tumor spheroids. SRH-DR5-B can be considered as a promising candidate for therapeutic applications.

Novel method for effectively amplifying human peripheral blood T cells in vitro.

The use of chimeric antigen receptor-modified T cells (CAR T cells) is an effective therapy for advanced cancer, especially hematological malignancies, and this method has attracted widespread attention in the last several years. The type, number and vitality of the effector cells clearly play important roles in this approach. In this study, to expand the possibility of curing cancer through adoptive cell therapy (ACT), we developed a novel method for effectively obtaining abundant T cells in vitro. The fusion proteins of three cytokines, SA-hIL-2, SA-hIL-7 and SA-hIL-21, were anchored onto biotin magnetic beads to increase the number of cytokines on the surface of the magnetic beads, which increased the local concentration of cytokines and thus promoted the binding of cytokines to T cells. Next, we examined the effects of these modified magnetic beads on the proliferation rate of T cells and CD19 CAR T cells. In this study, we report the expression and purification of the active bifunctional fusion proteins SA-hIL-2, SA-hIL-7 and SA-hIL-21, which were bound to biotin magnetic beads to develop a platform that was employed to increase the local concentration of cytokines. When the cells had been cultured for 14 days, the proliferation rate of the CD3+ T cells in the group that received cytokine-coupled biotin magnetic beads (Beads-SA-CK) was higher than that of the cells in the groups that received soluble cytokines (Soluble-SA-CK) and that of the cells in the standard group (Standard-CK). We speculate that this difference may be the result of the increased expression of Bcl-2 and the increased phosphorylation of Stat5. Moreover, our results preliminarily indicate that compared with the other two treatments, Soluble-SA-CK and Standard-CK, adding cytokine-coupled biotin magnetic beads more effectively increases the proliferation rate of CD19 CAR-T cells. As expected, the CD19 CAR-T cells stimulated by Beads-SA-CK had a stronger anticancer effect than the cells stimulated by the other two treatments. An effective method of preparing abundant T cells in vitro was developed, and it may provide a novel strategy for ACT.

Bifunctional Fusion Proteins Derived from Tumstatin and 4-1BBL for Targeted Cancer Therapy.

The therapeutic utilities of antiangiogenesis and immunotherapy have been proven in clinics, and cancer patients have benefited from respective therapy. Given that the combination of both therapeutic strategies may further improve the effectiveness, a recombinant human 4-1BBL/tumstatin fusion protein (rh4TFP) library was constructed in the present study to target both angiogenesis and T lymphocyte activation, in which the fragments of an endogenous angiogenesis inhibitor tumstatin and a T lymphocyte costimulatory 4-1BBL are coupled with different linkers. After comparison of different combinations, rh4TFP-2 was found to show a promise on potential antiangiogenic immunotherapy. On one hand, rh4TFP-2 inhibited proliferation and migration of human umbilical vein endothelial cells, exhibiting the antiangiogenic activity similar to tumstatin. On the other hand, rh4TFP-2 led to significant increase of T lymphocyte activation for the release of IL-2 and IFN-γ, showing the T lymphocyte activation by 4-1BBL. Moreover, administration of rh4TFP-2 suppressed tumor growth and prolonged survival in a B16F10 melanoma-bearing mouse model. Taken together, the present study provides a new approach of using bifunctional fusion proteins to target both angiogenesis and T lymphocyte activation for cancer therapy.