Quantifying Vitamin K-dependent Holoprotein Compaction caused by differential γ-carboxylation using HPSEC

This study uses high-pressure size exclusion chromatography (HPSEC) to quantify divalent metal ion (X2+)-induced compaction found in vitamin K dependent (VKD) proteins. Multiple X2+ binding sites formed by the presence of up to 12 -carboxyglutamic acid residues (Gla) are present in plasma-derived (pd-) and recombinant (r-) Factor IX (FIX). Analytical ultracentrifugation (AUC) was used to calibrate the Stokes radius (R) measured by HPSEC. A compaction of pd-FIX caused by the filling of Ca2+ and Mg2+ binding sites resulting in a 5-6% decrease in radius of hydration as observed by HPSEC. The filling of Ca2+ sites resulted greater compaction than for Mg2+ alone where this effect was additive or greater when both ions were present at physiologic levels. Less X2+ induced compaction was observed in r-FIX with lower Gla content populations which enabled the separation of biologically active from inactive r-FIX species by HPSEC. HPSEC was sensitive to R changes of ~0.01 nm that enabled the detection of FIX compaction that was likely cooperative in nature between lower avidity X2+ sites of the Gla domain and higher X2+ avidity sites of the EGF1-like domain

Engineering protein processing of the mammary gland to produce abundant hemophilia B therapy in milk

Both the low animal cell density of bioreactors and their ability to post-translationally process recombinant factor IX (rFIX) limit hemophilia B therapy to transgenic pigs to make rFIX in milk at about 3,000-fold higher output than provided by industrial bioreactors. However, this resulted in incomplete γ-carboxylation and propeptide cleavage where both processes are transmembrane mediated. We then bioengineered the co-expression of truncated, soluble human furin (rFurin) with pro-rFIX at a favorable enzyme to substrate ratio. This resulted in the complete conversion of pro-rFIX to rFIX while yielding a normal lactation. Importantly, these high levels of propeptide processing by soluble rFurin did not preempt γ-carboxylation in the ER and therefore was compartmentalized to the Trans-Golgi Network (TGN) and also to milk. The Golgi specific engineering demonstrated here segues the ER targeted enhancement of γ-carboxylation needed to biomanufacture coagulation proteins like rFIX using transgenic livestock

Engineering protein processing of the mammary gland to produce abundant hemophilia B therapy in milk

Both the low animal cell density of bioreactors and their ability to post-translationally process recombinant factor IX (rFIX) limit hemophilia B therapy to transgenic pigs to make rFIX in milk at about 3,000-fold higher output than provided by industrial bioreactors. However, this resulted in incomplete γ-carboxylation and propeptide cleavage where both processes are transmembrane mediated. We then bioengineered the co-expression of truncated, soluble human furin (rFurin) with pro-rFIX at a favorable enzyme to substrate ratio. This resulted in the complete conversion of pro-rFIX to rFIX while yielding a normal lactation. Importantly, these high levels of propeptide processing by soluble rFurin did not preempt γ-carboxylation in the ER and therefore was compartmentalized to the Trans-Golgi Network (TGN) and also to milk. The Golgi specific engineering demonstrated here segues the ER targeted enhancement of γ-carboxylation needed to biomanufacture coagulation proteins like rFIX using transgenic livestock

31st Annual Meeting and Associated Programs of the Society for Immunotherapy of Cancer (SITC 2016) : part two

Background The immunological escape of tumors represents one of the main ob- stacles to the treatment of malignancies. The blockade of PD-1 or CTLA-4 receptors represented a milestone in the history of immunotherapy. However, immune checkpoint inhibitors seem to be effective in specific cohorts of patients. It has been proposed that their efficacy relies on the presence of an immunological response. Thus, we hypothesized that disruption of the PD-L1/PD-1 axis would synergize with our oncolytic vaccine platform PeptiCRAd. Methods We used murine B16OVA in vivo tumor models and flow cytometry analysis to investigate the immunological background. Results First, we found that high-burden B16OVA tumors were refractory to combination immunotherapy. However, with a more aggressive schedule, tumors with a lower burden were more susceptible to the combination of PeptiCRAd and PD-L1 blockade. The therapy signifi- cantly increased the median survival of mice (Fig. 7). Interestingly, the reduced growth of contralaterally injected B16F10 cells sug- gested the presence of a long lasting immunological memory also against non-targeted antigens. Concerning the functional state of tumor infiltrating lymphocytes (TILs), we found that all the immune therapies would enhance the percentage of activated (PD-1pos TIM- 3neg) T lymphocytes and reduce the amount of exhausted (PD-1pos TIM-3pos) cells compared to placebo. As expected, we found that PeptiCRAd monotherapy could increase the number of antigen spe- cific CD8+ T cells compared to other treatments. However, only the combination with PD-L1 blockade could significantly increase the ra- tio between activated and exhausted pentamer positive cells (p= 0.0058), suggesting that by disrupting the PD-1/PD-L1 axis we could decrease the amount of dysfunctional antigen specific T cells. We ob- served that the anatomical location deeply influenced the state of CD4+ and CD8+ T lymphocytes. In fact, TIM-3 expression was in- creased by 2 fold on TILs compared to splenic and lymphoid T cells. In the CD8+ compartment, the expression of PD-1 on the surface seemed to be restricted to the tumor micro-environment, while CD4 + T cells had a high expression of PD-1 also in lymphoid organs. Interestingly, we found that the levels of PD-1 were significantly higher on CD8+ T cells than on CD4+ T cells into the tumor micro- environment (p < 0.0001). Conclusions In conclusion, we demonstrated that the efficacy of immune check- point inhibitors might be strongly enhanced by their combination with cancer vaccines. PeptiCRAd was able to increase the number of antigen-specific T cells and PD-L1 blockade prevented their exhaus- tion, resulting in long-lasting immunological memory and increased median survival

Pharmacokinetic Characterization of Procoagulation Proteins

The cessation of bleeding in mammals occurs due to a well-conserved sequence of protein activation known as the coagulation cascade. However, people who have a deficiency in one or more proteins in this cascade, whether due to genetics or blood loss, struggle to maintain hemostasis. In order to aid patients in the restoration of hemostasis, exogenous proteins are often administered in response to bleeding events. However, these proteins are limited and costly due to limited supply of donor blood available for the production of plasma-derived proteins and the high cost of mammalian cell bio-reactors required for the production of recombinant proteins. As an alternative to the two previously aforementioned methods, human recombinant coagulation proteins have also been produced in the mammary gland of mice, swine, and bovine. This technique offers high production of active coagulation protein at low scaled-up cost. The research in this dissertation details the preclinical trials and characterization of two such proteins, factor IX produced in swine (FIX) and fibrinogen produced in bovine (FI), as well as one traditional recombinant protein, factor XIII produced in Pichia pastoris (FXIII). FXIII and FI, in addition to thrombin, are the main components of fibrin sealant, which is typically used to seal a wound in the case of a catastrophic bleeding event. FIX is used in a completely different context: the treatment of hemophilia B, a disease where the body produces no active FIX. Pharmacokinetic analysis and characterization of FIX were performed both intravenously and buccally in mice and dogs. The analysis revealed that FIX produced in the mammary gland of swine exhibited enhanced endothelial binding while maintaining normal whole blood clotting times despite the reduced plasma retention times. FIX stored in the extravascular reservoir was shown to influence FIX retention times for samples infused over 24 hours after plasma levels had been depleted. FXIII produced in yeast was shown to be monomeric and containing an artificial activation peptide that enhanced the crosslinking of fibrinogen with a reduced activation time lag. Adviser: William H. Velande