This chapter contains sections titled: Introduction Production and characterization of H. polymorpha-derived hirudin Production and characterization of H. polymorpha-derived saratin
Platelets tether to collagen in both a von Willebrand factor (vWF)-dependent and a vWF-independent manner. We have recently characterized a recombinant protein, saratin, isolated from the saliva of the leech Hirudo medicinalis, expressed it in Hansenula polymorpha, and studied its effect on direct and indirect platelet-collagen interactions. Saratin dose dependently inhibited the binding of purified human vWF to human type I and III collagens (IC50 = 0.23 ± 0.004 and 0.81 ± 0.04 μg mL-1, respectively) and to calf skin collagen (IC50 = 0.44 ± 0.008 μg mL-1). Furthermore, saratin showed a similar inhibitory potency against the binding of human, rodent, and porcine plasma vWF to these collagens. In a flow chamber under conditions of elevated shear (2700 s-1), saratin dose dependently and potently inhibited platelet aggregate formation on a collagen-coated surface (IC50 = 0.96 ± 0.25 μg mL-1), but at reduced shear (1300 s-1) a rightward shift in the dose-response curve was noted (IC50 = 5.2 ± 1.4 μg mL-1). Surface plasmon resonance analysis revealed both high and low affinity binding sites for saratin on human collagen type III (Kd 5 × 10-8 M and 2 × 10-6 M, respectively). Although low concentrations of saratin, which inhibited platelet adhesion under increased shear (i.e., saturation of high-affinity binding sites), had no effect on vWF-independent collagen-induced platelet aggregation, high concentrations (i.e., saturation of low-affinity binding sites) were found to inhibit platelet aggregation. These data demonstrate that saratin is a potent inhibitor of vWF-dependent platelet adhesion to collagen and hence may have therapeutic potential as an antithrombotic agent.
<p>Supplemental Figure S2. Candidates with a majority of siRNAs inducing a > 4- fold shift in CPT EC50 (minimum of 7 siRNAs tested; left Qiagen siRNAs; right Ambion). Corresponding catalog numbers are listed in chart legends.</p>
<p>Supplemental Figure S2. Candidates with a majority of siRNAs inducing a > 4- fold shift in CPT EC50 (minimum of 7 siRNAs tested; left Qiagen siRNAs; right Ambion). Corresponding catalog numbers are listed in chart legends.</p>
<p>Supplemental Table S3. Summary of siRNA follow-up studies. siRNAs were evaluated in the context of a camptothecin dose response. The primary screen rank is listed for each gene along with the number of siRNAs yielding a 2-fold or greater sensitization. All genes were tested with at least 4 siRNAs from Qiagen (Q). Many genes were characterized with 3 additional siRNAs from Ambion (A). The percent of tested siRNAs yielding a 2-fold or greater sensitization is listed for each gene.</p>
<p>Supplemental Table S3. Summary of siRNA follow-up studies. siRNAs were evaluated in the context of a camptothecin dose response. The primary screen rank is listed for each gene along with the number of siRNAs yielding a 2-fold or greater sensitization. All genes were tested with at least 4 siRNAs from Qiagen (Q). Many genes were characterized with 3 additional siRNAs from Ambion (A). The percent of tested siRNAs yielding a 2-fold or greater sensitization is listed for each gene.</p>
Background Canonical α/β T-cell receptors (TCRs) bind to human leukocyte antigen (HLA) displaying antigenic peptides to elicit T cell−mediated cytotoxicity. TCR-engineered T-cell immunotherapies targeting cancer-specific peptide-HLA complexes (pHLA) are generating exciting clinical responses, but owing to HLA restriction they are only able to target a subset of antigen-positive patients. More recently, evidence has been published indicating that naturally occurring α/β TCRs can target cell surface proteins other than pHLA, which would address the challenges of HLA restriction. In this proof-of-concept study, we sought to identify and engineer so-called HLA-independent TCRs (HiTs) against the tumor-associated antigen mesothelin. Methods Using phage display, we identified a HiT that bound well to mesothelin, which when expressed in primary T cells, caused activation and cytotoxicity. We subsequently engineered this HiT to modulate the T-cell response to varying levels of mesothelin on the cell surface. Results The isolated HiT shows cytotoxic activity and demonstrates killing of both mesothelin-expressing cell lines and patient-derived xenograft models. Additionally, we demonstrated that HiT-transduced T cells do not require CD4 or CD8 co-receptors and, unlike a TCR fusion construct, are not inhibited by soluble mesothelin. Finally, we showed that HiT-transduced T cells are highly efficacious in vivo, completely eradicating xenografted human solid tumors. Conclusion HiTs can be isolated from fully human TCR–displaying phage libraries against cell surface-expressed antigens. HiTs are able to fully activate primary T cells both in vivo and in vitro. HiTs may enable the efficacy seen with pHLA-targeting TCRs in solid tumors to be translated to cell surface antigens.
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