Antigen Priming with Enantiospecific Cationic Lipid Nanoparticles Induces Potent Antitumor CTL Responses through Novel Induction of a Type I IFN Response.

Certain types of cationic lipids have shown promise in cancer immunotherapy, but their mechanism of action is poorly understood. In this study, we describe the properties of an immunotherapeutic consisting of the pure cationic lipid enantiomer R-1,2-dioleoyl-3-trimethyl-ammonium-propane (R-DOTAP) formulated with modified viral or self-peptide Ags. R-DOTAP formulations with peptide Ags stimulate strong cross-presentation and potent CD8 T cell responses associated with a high frequency of polyfunctional CD8 T cells. In a human papillomavirus tumor model system, a single s.c. injection of tumor-bearing mice with R-DOTAP plus human papillomavirus Ags induces complete regression of large tumors associated with an influx of Ag-specific CD8 T cells and a reduction of the ratio of regulatory/Ag-specific CD8 T cells. R-DOTAP also synergizes with an anti-PD1 checkpoint inhibitor, resulting in a significant inhibition of B16 melanoma tumor growth. We found that R-DOTAP stimulates type I IFN production by dendritic cells in vivo and in vitro. s.c. injection of R-DOTAP results in an IFN-dependent increase in draining lymph node size and a concomitant increase in CD69 expression. Using knockout mice, we show that type I IFN is required for the induction of CD8 T cell activity following administration of R-DOTAP plus Ag. This response requires Myd88 but not TRIF or STING. We also show that R-DOTAP stimulates both TLR7 and 9. Collectively, these studies reveal that R-DOTAP stimulates endosomal TLRs, resulting in a Myd88-dependent production of type I IFN. When administered with Ag, this results in potent Ag-specific CD8 T cell responses and antitumor activity.

Recombinant Protein Vaccines Formulated with Enantio-Specific Cationic Lipid R-DOTAP Induce Protective Cellular and Antibody-Mediated Immune Responses in Mice.

Adjuvants are essential components of subunit vaccines added to enhance immune responses to antigens through immunomodulation. Very few adjuvants have been approved for human use by regulatory agencies due to safety concerns. Current subunit vaccine adjuvants approved for human use are very effective in promoting humoral immune responses but are less effective at promoting T-cell immunity. In this study, we evaluated a novel pure enantio-specific cationic lipid 1,2-dioleoyl-3-trimethylammonium-propane (R-DOTAP) as an immunomodulator for subunit vaccines capable of inducing both humoral- and cellular-mediated immunity. Using recombinant protein antigens derived from SARS-CoV2 spike or novel computationally optimized broadly reactive influenza antigen (COBRA) proteins, we demonstrated that R-DOTAP nanoparticles promoted strong cellular- and antibody-mediated immune responses in both monovalent and bivalent vaccines. R-DOTAP-based vaccines induced antigen-specific and polyfunctional CD8+ and CD4+ effector T cells and memory T cells, respectively. Antibody responses induced by R-DOTAP showed a balanced Th1/Th2 type immunity, neutralizing activity and protection of mice from challenge with live SARS-CoV2 or influenza viruses. R-DOTAP also facilitated significant dose sparing of the vaccine antigens. These studies demonstrate that R-DOTAP is an excellent immune stimulator for the production of next-generation subunit vaccines containing multiple recombinant proteins.

Monitoring EDTA process residuals in recombinant protein manufacturing using liquid chromatography.

We have developed a chromatographic method for the high sensitivity quantitation of EDTA process residuals in recombinant protein manufacturing validation studies. The reversed-phase HPLC method is based upon the detection of Cu(2+)/EDTA complexes at 254 nm, and has been qualified for use on intermediates from a purification process for a recombinant protein expressed in E. coli. Quantitation of EDTA in recombinant protein process intermediates is linear in the range of 0.2 to 64 microM with LOD/LOQ values below 2.0 microM. The assay is suitable for use in process backgrounds containing Tris, HEPES, MES, NaCl, hexanediol, NH(4)SO(4), and PEG. EDTA spike recovery values in all process samples tested were greater than 90% at the 4.0 microM concentration. System suitability parameters for the chromatographic method were developed based upon peak area and retention time precision, column efficiency and USP tailing. Peak area precision and intermediate precision values across the linear range of the assay exhibited C.V. values less than 15% at any concentration tested in all sample backgrounds. The assay robustness was tested by transfer of the assay to a second laboratory and analyst with use of multiple process intermediate lots, reagent/column lots, and HPLC systems.

Ion chromatographic quantification of cyanate in urea solutions: estimation of the efficiency of cyanate scavengers for use in recombinant protein manufacturing.

The chaotrope urea is commonly used during recombinant protein manufacturing as a denaturant/solublizing agent. The adventitious accumulation of cyanate in urea solutions during product manufacturing can cause unwanted carbamylation of proteins, leading to alterations in drug product structure, stability and function. We have developed an ion chromatographic method to quantify cyanate production in urea solutions, suitable for analysis of samples from manufacturing process buffers. We discuss assay development, system suitability criteria and limitations on assay applicability. The assay has a linear range from 2 to 250 microM, with LOQ/LOD values of 6 and 2 microM, respectively. Assay accuracy through spike/recovery testing were established and both precision and intermediate precision were estimated. We assessed the utility of the assay by testing a variety of biological buffers and potential cyanate scavengers, which could be used during protein purification processes, for their ability to control the level of cyanate in 8 M urea solutions buffered over the range of pH 5-10. Our results demonstrate pH dependence for prevention of cyanate accumulation by these buffers/scavengers and indicate useful buffers, pH ranges, and additives for controlling cyanate accumulation during recombinant protein manufacturing. The pertinence of these approaches in preventing protein carbamylation during manufacturing are discussed.

An enzyme-linked immunosorbent assay for host cell protein contaminants in recombinant PEGylated staphylokinase mutant SY161.

Staphylokinase, a bacterially-derived protein which functions as a plasminogen activator, has potential utility as a human therapeutic for thrombotic disorders. A recombinant version of this protein, SY161, contains 13 amino acid substitutions designed to decrease immunogenicity, and has been covalently modified by crosslinking a 5 kDa polyethyleneglycol (PEG) group to the N-terminal region to prolong the drug circulating half-life. The recombinant PEG-modified SY161 staphylokinase is currently in phase II clinical trials as a treatment for acute myocardial infarction. We have developed a sensitive product specific host cell protein (HCP) assay in the ELISA format to monitor in process host-derived contaminant clearance and final drug product purity. The assay is based upon use of goat polyclonal antibodies raised against E. coli host strain cell proteins from a null cell line, extracted by the same manufacturing process used to produce SY161. The identification and clearance of HCP contaminants was confirmed during drug product production using SDS-PAGE and Western blotting utilizing the same polyclonal HCP antibodies. The assay is specific for E. coli host cell strain proteins with a useful detection range from 1 to 100 ng/ml, and is not affected by product level. The level of residual HCPs in the clinical product produced by our manufacturing process was determined to be less than 1 ng/ml at a product concentration of 1 mg/ml.

Monitoring manufacturing process yields, purity and stability of structural variants of PEGylated staphylokinase mutant SY161 by quantitative reverse-phase chromatography.

Staphylokinase variant SY161 is a recombinant mutant of the Staphylococcus aureus polypeptide staphylokinase (Sak), and is currently in human clinical trials as a thrombolytic agent. The 15 kDa single chain SY161 protein is expressed as a soluble cytoplasmic product in E. coli with a single cysteine inserted near the N-terminus. The protein as extracted from E. coli is a mixture of both monomeric and intermolecularly disulfide crosslinked species. To improve protein purification yields SY161 is sulfitolyzed during the early stages of production, preventing disulfide formation. The protein is later modified during manufacturing to incorporate a single 5 kDa polyethylene glycol group on the single sulfhydryl sidechain. We have developed and qualified a reverse-phase chromatographic method to quantitate SY161 during product manufacturing. We discuss the use of the assay during manufacturing development to monitor fermentation yields, the SY161 PEGylation reaction, and as an in-process manufacturing control assay. The assay has been applied as a product purity and identity release assay and is suitable for use in assessing product structural integrity during stability testing. The assay has a linear range of quantitation for SY161 from at least 0.15 to 16 micro g, and is-in addition capable of detecting and quantitating protein de-PEGylation events and host cell-derived protein contaminants.