Expression and production of pigment epithelium-derived factor (PEDF) and PEDF receptor variants from mammalian and bacterial cells.

Human SERPINF1 gene codes for pigment epithelium-derived factor (PEDF), a secreted glycoprotein and member of the SERPIN superfamily. To obtain large amounts of recombinant PEDF proteins, we subcloned the coding sequence of human SERPINF1 mutated versions into the pCEP4 vector and generated stably transfected HEK.Ebna cells. The cells produced and secreted recombinant PEDF proteins into the culturing media. The recombinant PEDF proteins were purified by ion-exchange column chromatography and milligram amounts of highly purified protein were recovered. PEDF has affinity for PEDF-receptor (PEDF-R), a membrane-linked lipase encoded by the PNPLA2 gene. Recombinant PEDF-R truncated versions were obtained from Escherichia coli containing expression vectors with human PNPLA2 cDNAs with 3'end deletions and by induction with isopropyl ß-d-1-thiogalactopyranoside. The bacterially derived PEDF-R proteins in insoluble inclusion bodies were solubilized with urea and purified by cation-exchange column chromatography. C-terminally truncated PEDF-R versions containing the ligand binding region retained the ability to bind PEDF. The data demonstrate that mammalian-derived recombinant PEDF and bacterially derived recombinant PEDF-R can be produced and purified in large amounts for further use in structural and biological studies.

Affecting HEK293 Cell Growth and Production Performance by Modifying the Expression of Specific Genes.

The HEK293 cell line has earned its place as a producer of biotherapeutics. In addition to its ease of growth in serum-free suspension culture and its amenability to transfection, this cell line's most important attribute is its human origin, which makes it suitable to produce biologics intended for human use. At the present time, the growth and production properties of the HEK293 cell line are inferior to those of non-human cell lines, such as the Chinese hamster ovary (CHO) and the murine myeloma NSO cell lines. However, the modification of genes involved in cellular processes, such as cell proliferation, apoptosis, metabolism, glycosylation, secretion, and protein folding, in addition to bioprocess, media, and vector optimization, have greatly improved the performance of this cell line. This review provides a comprehensive summary of important achievements in HEK293 cell line engineering and on the global engineering approaches and functional genomic tools that have been employed to identify relevant genes for targeted engineering.

Knockout of the caspase 8-associated protein 2 gene improves recombinant protein expression in HEK293 cells through up-regulation of the cyclin-dependent kinase inhibitor 2A gene.

Cell lines used in bioproduction are routinely engineered to improve their production efficiency. Numerous strategies, such as random mutagenesis, RNA interference screens, and transcriptome analyses have been employed to identify effective engineering targets. A genome-wide small interfering RNA screen previously identified the CASP8AP2 gene as a potential engineering target for improved expression of recombinant protein in the HEK293 cell line. Here, we validate the CASP8AP2 gene as an engineering target in HEK293 cells by knocking it out using CRISPR/Cas9 genome editing and assessing the effect of its knockout on recombinant protein expression, cell growth, cell viability, and overall gene expression. HEK293 cells lacking CASP8AP2 showed a seven-fold increase in specific expression of recombinant luciferase and a 2.5-fold increase in specific expression of recombinant SEAP, without significantly affecting cell growth and viability. Transcriptome analysis revealed that the deregulation of the cell cycle, specifically the upregulation of the cyclin-dependent kinase inhibitor 2A (CDKN2A) gene, contributed to the improvement in recombinant protein expression in CASP8AP2 deficient cells. The results validate the CASP8AP2 gene is a viable engineering target for improved recombinant protein expression in the HEK293 cell line.

Improved protein expression in HEK293 cells by over-expressing miR-22 and knocking-out its target gene, HIPK1.

Stable cell lines can continuously produce a recombinant protein without the need to repeatedly engineer the genome. In a previous study HIPK1, Homeodomain-interacting Protein Kinase 1, was found to be a target of the microRNA miR-22 that, when repressed, improved expression of both an intracellular and a secreted protein. In this report, HEK293 cells stably over-expressing miR-22 were compared with HEK293 with knockout of HIPK1, executed by CRISPR/Cas9, for their ability to improve recombinant protein expression. In this model case of luciferase, over-expression of miR-22 improved overall activity 2.4-fold while the HIPK1 knockout improved overall activity 4.7-fold.

Potent Neutralization of Staphylococcal Enterotoxin B In Vivo by Antibodies that Block Binding to the T-Cell Receptor.

To develop an antibody (Ab) therapeutic against staphylococcal enterotoxin B (SEB), a potential incapacitating bioterrorism agent and a major cause of food poisoning, we developed a "class T" anti-SEB neutralizing Ab (GC132) targeting an epitope on SEB distinct from that of previously developed "class M" Abs. A systematic engineering approach was applied to affinity-mature Ab GC132 to yield an optimized therapeutic candidate (GC132a) with sub-nanomolar binding affinity. Mapping of the binding interface by hydrogen-deuterium exchange coupled to mass spectrometry revealed that the class T epitope on SEB overlapped with the T-cell receptor binding site, whereas other evidence suggested that the class M epitope overlapped with the binding site for the major histocompatibility complex. In the IgG format, GC132a showed ∼50-fold more potent toxin-neutralizing efficacy than the best class M Ab in vitro, and fully protected mice from lethal challenge in a toxic shock post-exposure model. We also engineered bispecific Abs (bsAbs) that bound tetravalently by utilizing two class M binding sites and two class T binding sites. The bsAbs displayed enhanced toxin neutralization efficacy compared with the respective monospecific Ab subunits as well as a mixture of the two, indicating that enhanced efficacy was due to heterotypic tetravalent binding to two non-overlapping epitopes on SEB. Together, these results suggest that class T anti-SEB Ab GC132a is an excellent candidate for clinical development and for bsAb engineering.

TBA225, a fusion toxoid vaccine for protection and broad neutralization of staphylococcal superantigens.

Superantigens (SAgs) play a major role in the pathogenesis of Staphylococcus aureus and are associated with several diseases, including food poisoning, bacterial arthritis, and toxic shock syndrome. Monoclonal antibodies to these SAgs, primarily TSST-1, SEB and SEA have been shown to provide protection in animal studies and to reduce clinical severity in bacteremic patients. Here we quantify the pre-existing antibodies against SAgs in many human plasma and IVIG samples and demonstrate that in a major portion of the population these antibody titers are suboptimal and IVIG therapy only incrementally elevates the anti-SAg titers. Our in vitro neutralization studies show that a combination of antibodies against SEA, SEB,and TSST-1 can provide broad neutralization of staphylococcal SAgs. We report a single fusion protein (TBA225) consisting of the toxoid versions of TSST-1, SEB and SEA and demonstrate its immunogenicity and protective efficacy in a mouse model of toxic shock. Antibodies raised against this fusion vaccine provide broad neutralization of purified SAgs and culture supernatants of multiple clinically relevant S. aureus strains. Our data strongly supports the use of this fusion protein as a component of an anti-virulence based multivalent toxoid vaccine against S. aureus disease.

Knocking out Ornithine Decarboxylase Antizyme 1 (OAZ1) Improves Recombinant Protein Expression in the HEK293 Cell Line.

Creating efficient cell lines is a priority for the biopharmaceutical industry, which produces biologicals for various uses. A recent approach to achieving this goal is the use of non-coding RNAs, microRNA (miRNA) and small interfering RNA (siRNA), to identify key genes that can potentially improve production or growth. The ornithine decarboxylase antizyme 1 (OAZ1) gene, a negative regulator of polyamine biosynthesis, was identified in a genome-wide siRNA screen as a potential engineering target, because its knock down by siRNA increased recombinant protein expression from human embryonic kidney 293 (HEK293) cells by two-fold. To investigate this further, the OAZ1 gene in HEK293 cells was knocked out using CRISPR genome editing. The OAZ1 knockout cell lines displayed up to four-fold higher expression of both stably and transiently expressed proteins, with comparable growth and metabolic activity to the parental cell line; and an approximately three-fold increase in intracellular polyamine content. The results indicate that genetic inactivation of OAZ1 in HEK293 cells is an effective strategy to improve recombinant protein expression in HEK293 cells.

Structurally designed attenuated subunit vaccines for S. aureus LukS-PV and LukF-PV confer protection in a mouse bacteremia model.

Previous efforts towards S. aureus vaccine development have largely focused on cell surface antigens to induce opsonophagocytic killing aimed at providing sterile immunity, a concept successfully applied to other Gram-positive pathogens such as Streptococcus pneumoniae. However, these approaches have largely failed, possibly in part due to the remarkable diversity of the staphylococcal virulence factors such as secreted immunosuppressive and tissue destructive toxins. S. aureus produces several pore-forming toxins including the single subunit alpha hemolysin as well as bicomponent leukotoxins such as Panton-Valentine leukocidin (PVL), gamma hemolysins (Hlg), and LukED. Here we report the generation of highly attenuated mutants of PVL subunits LukS-PV and LukF-PV that were rationally designed, based on an octameric structural model of the toxin, to be deficient in oligomerization. The attenuated subunit vaccines were highly immunogenic and showed significant protection in a mouse model of S. aureus USA300 sepsis. Protection against sepsis was also demonstrated by passive transfer of rabbit immunoglobulin raised against LukS-PV. Antibodies to LukS-PV inhibited the homologous oligomerization of LukS-PV with LukF-PV as well heterologous oligomerization with HlgB. Importantly, immune sera from mice vaccinated with the LukS mutant not only inhibited the PMN lytic activity produced by the PVL-positive USA300 but also blocked PMN lysis induced by supernatants of PVL-negative strains suggesting a broad protective activity towards other bicomponent toxins. These findings strongly support the novel concept of an anti-virulence, toxin-based vaccine intended for prevention of clinical S. aureus invasive disease, rather than achieving sterile immunity. Such a multivalent vaccine may include attenuated leukotoxins, alpha hemolysin, and superantigens.

Novel structurally designed vaccine for S. aureus α-hemolysin: protection against bacteremia and pneumonia.

Staphylococcus aureus (S. aureus) is a human pathogen associated with skin and soft tissue infections (SSTI) and life threatening sepsis and pneumonia. Efforts to develop effective vaccines against S. aureus have been largely unsuccessful, in part due to the variety of virulence factors produced by this organism. S. aureus alpha-hemolysin (Hla) is a pore-forming toxin expressed by most S. aureus strains and reported to play a key role in the pathogenesis of SSTI and pneumonia. Here we report a novel recombinant subunit vaccine candidate for Hla, rationally designed based on the heptameric crystal structure. This vaccine candidate, denoted AT-62aa, was tested in pneumonia and bacteremia infection models using S. aureus strain Newman and the pandemic strain USA300 (LAC). Significant protection from lethal bacteremia/sepsis and pneumonia was observed upon vaccination with AT-62aa along with a Glucopyranosyl Lipid Adjuvant-Stable Emulsion (GLA-SE) that is currently in clinical trials. Passive transfer of rabbit immunoglobulin against AT-62aa (AT62-IgG) protected mice against intraperitoneal and intranasal challenge with USA300 and produced significant reduction in bacterial burden in blood, spleen, kidney, and lungs. Our Hla-based vaccine is the first to be reported to reduce bacterial dissemination and to provide protection in a sepsis model of S. aureus infection. AT62-IgG and sera from vaccinated mice effectively neutralized the toxin in vitro and AT62-IgG inhibited the formation of Hla heptamers, suggesting antibody-mediated neutralization as the primary mechanism of action. This remarkable efficacy makes this Hla-based vaccine a prime candidate for inclusion in future multivalent S. aureus vaccine. Furthermore, identification of protective epitopes within AT-62aa could lead to novel immunotherapy for S. aureus infection.

Synthetic human monoclonal antibodies toward staphylococcal enterotoxin B (SEB) protective against toxic shock syndrome.

Staphylococcal enterotoxin B (SEB) is a potent toxin that can cause toxic shock syndrome and act as a lethal and incapacitating agent when used as a bioweapon. There are currently no vaccines or immunotherapeutics available against this toxin. Using phage display technology, human antigen-binding fragments (Fabs) were selected against SEB, and proteins were produced in Escherichia coli cells and characterized for their binding affinity and their toxin neutralizing activity in vitro and in vivo. Highly protective Fabs were converted into full-length IgGs and produced in mammalian cells. Additionally, the production of anti-SEB antibodies was explored in the Nicotiana benthamiana plant expression system. Affinity maturation was performed to produce optimized lead anti-SEB antibody candidates with subnanomolar affinities. IgGs produced in N. benthamiana showed characteristics comparable with those of counterparts produced in mammalian cells. IgGs were tested for their therapeutic efficacy in the mouse toxic shock model using different challenge doses of SEB and a treatment with 200 µg of IgGs 1 h after SEB challenge. The lead candidates displayed full protection from lethal challenge over a wide range of SEB challenge doses. Furthermore, mice that were treated with anti-SEB IgG had significantly lower IFNγ and IL-2 levels in serum compared with mock-treated mice. In summary, these anti-SEB monoclonal antibodies represent excellent therapeutic candidates for further preclinical and clinical development.