Expi293 Cells Research Articles

Highly selective split intein method for efficient separation and purification of recombinant therapeutic proteins from mammalian cell culture fluid

Biologics and vaccines have been successfully developed over the last few decades to treat many diseases. Each of these drugs must be highly purified for clinical use. Monoclonal antibodies (mAbs), the dominant therapeutic modality on the market, can be easily purified using the standard Protein A affinity platform. However, no generally applicable affinity platforms are available for the manufacture of other therapeutic proteins for clinical use. Thus, multicolumn chromatography processes for widely being used for product purification. These processes demand significant optimization to meet desired product quality attributes, where each step also decreases final yields. In this work, we demonstrate the novel self-removing iCapTagTM affinity tag, which provides a new platform for capturing, concentrating, and purifying recombinant proteins. Importantly, this system provides a tagless target protein, which is suitable for research and clinical use, where the only requirement for tag removal is a small change in buffer pH. No additional proteins, reagents or cofactors are required. We also present case studies demonstrating the use of iCapTagTM for highly efficient purification of untagged interferon alpha 2b, the ML39 single chain variable fragment (scFv), and the receptor binding domain (RBD) of SARS-CoV-2 spike protein. These proteins were expressed and secreted by Expi293 cells with the self-removing tag fused to their N-terminus. We were able to obtain highly pure (> 99%) tagless protein in a single purification step with high clearance of host cell DNA, tagged precursor, higher and lower molecular weight impurities. Based on these preliminary results, we propose the iCapTagTM as a universal capture platform for diverse classes of recombinant therapeutic proteins.

Suspension Culture Production and Purification of Adeno-Associated Virus by Iodixanol Density Gradient Centrifugation for In Vivo Applications.

This protocol describes recombinant adeno-associated virus (rAAV) production and purification by iodixanol density gradient centrifugation, a serotype-agnostic method of purifying AAV first described in 1999. rAAV vectors are widely used in gene therapy applications to deliver transgenes to various human cell types. In this work, the recombinant virus is produced by transfection of Expi293 cells in suspension culture with plasmids encoding the transgene, vector capsid, and adenoviral helper genes. Iodixanol density gradient centrifugation purifies full AAV particles based on particle density. Additionally, three steps are included in this now-ubiquitous methodology in order to increase total virus yield, decrease the risk of precipitation due to contaminating proteins, and further concentrate the final virus product, respectively: precipitation of viral particles from cell media using a solution of polyethylene glycol (PEG) and sodium chloride, the introduction of a second round of iodixanol density gradient centrifugation, and buffer exchange via a centrifugal filter. Using this method, it is possible to consistently achieve titers in the range of 1012 viral particles/mL of exceptional purity for in vivo use.

Fructose biphosphate aldolase: A new cassava allergen

BackgroundFood allergy has considerably increased in recent years and this situation has been aggravated mainly by the consumption of more processed and complex foods, since minor or potentially allergenic foods are not required to be labeled. Manihot esculenta (cassava) is a widely consumed food in South America, Africa, and Asia and can be used in the production of flour and starch, as well as several other products. This root can cause allergic reactions with symptoms ranging from mild to severe. MethodsThus, the aim of this study was the characterization of the immunogenic cassava proteins responsible for sensitizing patients allergic to it. Using a 2D-SDS-PAGE based proteomic approach, six proteins were identified, including Fructose Bisphosphate Aldolase (FBA). Recombinant FBA was produced in Expi293 cells and evaluated by immunoblotting with the serum of 10 individual study subjects. ResultsOur results showed six cassava IgE-reactive proteins. From those, recombinant fructose bisphosphate aldolase (FBA) showed a positivity of 80% among tested sera, proving to be a highly sensitizing protein. ConclusionThe recombinant FBA molecule obtained in this study can be important for in vivo diagnostic assays, by producing more accurate results, and for desensitization protocols, in which the use of the isolated molecule produces more precise results by avoiding secondary sensitization. Trial registrationAll patients signed a consent form approved by the internal ethics committee CAPPesq, Comissão de Ética para Análise de Projetos de Pesquisa do HC FMUSP (CAAE: 10420619.6.0000.0068).

An Efficient and Scalable Method for the Production of Immunogenic SARS-CoV-2 Virus-like Particles (VLP) from a Mammalian Suspension Cell Line.

The rapid evolution of new SARS-CoV-2 variants poses a continuing threat to human health. Vaccination has become the primary therapeutic intervention. The goal of the current work was the construction of immunogenic virus-like particles (VLPs). Here, we describe a human cell line for cost-efficient and scalable production of immunogenic SARS-CoV-2 VLPs. The modular design of the VLP-production platform facilitates rapid adaptation to new variants. Methods: The N, M-, and E-protein genes were integrated into the genome of Expi293 cells (ExpiVLP_MEN). Subsequently, this cell line was further modified for the constitutive expression of the SARS-CoV-2 spike protein. The resulting cell line (ExpiVLP_SMEN) released SARS-CoV-2 VLP upon exposure to doxycycline. ExpiVLP_SMEN cells were readily adapted for VLP production in a 5 L bioreactor. Purified VLPs were quantified by Western blot, ELISA, and nanoparticle tracking analysis and visualized by electron microscopy. Immunogenicity was tested in mice. Results: The generated VLPs contained all four structural proteins, are within the size range of authentic SARS-CoV-2 virus particles, and reacted strongly and specifically with immunoserum from naturally infected individuals. The VLPs were stable in suspension at 4 °C for at least 10 weeks. Mice immunized with VLPs developed neutralizing antibodies against lentiviruses pseudotyped with the SARS-CoV-2 spike protein. The flexibility of the VLP-production platform was demonstrated by the rapid switch of the spike protein to a new variant of concern (BA.1/Omicron). The present study describes an efficient, scalable, and adaptable production method of immunogenic SARS-CoV-2 VLPs with therapeutic potential.

Strategies for rapid production of crystallization quality coatomer WD40 domains

The vesicular secretion of soluble cargo proteins from the endoplasmic reticulum (ER) is accompanied by the export of ER-resident membrane proteins that are co-packaged in secretory vesicles. The cytosolic coatomer protein complex I (COPI) utilizes the N-terminal WD40 domains of α-COPI and β′-COPI subunits to bind these membrane protein “clients” for ER retrieval. These “αWD40” and “β'WD40” domains are structural homologs that demonstrate distinct selectivity for client proteins. However, elucidation of the atomic-level principles of coatomer-client interactions has been challenging due to the tendency of αWD40 domain to undergo aggregation during expression and purification. Here we describe a rapid recombinant production strategy from E. coli, which substantially enhances the quality of the purified αWD40 domain. The αWD40 purification and crystallization are completed within one day, which minimizes aggregation losses and yields a 1.9 Å resolution crystal structure. We demonstrate the versatility of this strategy by applying it to purify the β′WD40 domain, which yields crystal structures in the 1.2–1.3 Å resolution range. As an alternate recombinant production system, we develop a cost-effective strategy for αWD40 production in human Expi293 cells. Finally, we suggest a roadmap to simplify these protocols further, which is of significance for the production of WD40 mutants prone to rapid aggregation. The WD40 production strategies presented here are likely to have broad applications because the WD40 domain represents one of the largest families of biomolecular interaction modules in the eukaryotic proteome and is critical for trafficking of host as well as viral proteins such as the SARS-CoV-2 spike protein.

Pre-Clinical Development of a First in Class Half-Life Extended T Cell Engager Targeting ROR-1

Background: ROR1 is a cell surface receptor that is largely absent or expressed at low levels in healthy adult tissues but is expressed at high levels on a wide range of hematological malignancies as well as solid tumours, making it an ideal anti-cancer drug target. NVG-111 is a bispecific T cell engager (TCE) antibody targeting ROR1xCD3 using a tandem scFv format to redirect the cytotoxic activity of T cells to ROR1+ tumour cells. NVG-111 is currently showing efficacy with predictable and manageable toxicity in Phase I/II clinical development for the treatment of Chronic Lymphocytic Leukaemia (CLL) and Mantle Cell Lymphoma (MCL), with the scope to expand into other hematological malignancies and solid tumour indications. These clinical data provide the foundation for the clinical development of a half-life extended (HLE) TCE using the clinically derisked ROR1 and CD3 binders from NVG-111. Objective: Building on the safety and efficacy data emerging from the Phase I/II trial of NVG-111, develop a half-life extended ROR1xCD3 TCE for a more convenient once a week dosing regimen, without compromising the safety, efficacy and CMC attributes of NVG-111. Methods: Utilizing NVG-111 as a core for a ROR1xCD3 HLE-TCE, several formats were designed and evaluated. These incorporated different half-life extending technology solutions by increasing the molecule size and enabling FcRn-mediated recycling. NVG-111 was cloned in-format with an antibody Fc, human serum albumin (HSA), or an HSA-binding antibody into a suitable expression vector and the resultant clones expressed using Expi293 cells. Molecules were then purified using our proprietary downstream purification process. The potency of ROR1 targeting HLE-TCEs were evaluated in cytotoxicity and T cell activation co-culture assays using MCL derived JeKo-1 target cells and T cells from healthy donor PBMCs, with responses measured by flow cytometry. Biophysical properties were evaluated by determining the post-purification expression titer, aggregation profile, and stability at 37°C. Results: Reformatting NVG-111 into an IgG-like bispecific scFv-Fc heterodimer resulted in loss of cytotoxic potency, which may be due to a change in the geometry of binding or poorer target engagement. Fusing NVG-111 directly to an HSA-binding antibody resulted in complete loss of expression. A 3-fold reduction in expression titer was observed for NVG-111 fused directly with HSA, which was also accompanied with a >10-fold reduction in functional activity, loss of binding affinity and reduced stability compared with parental molecule. Fusing NVG-111 to Fc resulted in a molecule that maintained functional potency, with transient expression titer remaining >100mg/L, which is also aligned with the parental molecule. The lead Fc-fused HLE molecule was then further refined with appropriate Fc-silencing technologies, which displayed low levels of aggregation following purification, with a monomer content of >97% (Figure 1). Stability studies performed in formulation buffer and in human serum showed that the Fc-fused HLE molecule was at least as stable as parental NVG-111, retaining binding and no evidence of aggregation or fragmentation. The potency of HLE-TCE with respect to cytotoxicity was influenced by the silencing technologies (Figure 2), with technologies 1 and 2 retaining potency as measured by EC50 to within 2-fold to 5-fold of NVG-111. However, silencing technology 3 displayed a 10-fold lower potency than NVG-111 and was therefore not progressed. Finally, initial PK studies suggest that our selected ROR1 targeting HLE-TCE has a half-life that would support weekly dosing in humans. Conclusion: We have generated a first in class ROR1 targeting half-life extended TCE that maintains the functional and biophysical properties of the clinically promising NVG-111. Our systematic evaluation suggests that careful selection of HLE format is critical for the development of a molecule with good yield, biophysical characteristics and potency. The selected ROR1 HLE-TCE supports weekly administration whilst maintaining potency at sub-nanomolar concentrations, properties which differentiate it from other TCEs in development. IND enabling studies with our 1st in class ROR1 targeting HLE-TCE will conclude in 2023, supporting the initiation of a Phase I clinical trial in ROR1+ hematological malignancies. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Intranasal immunization with HRSV prefusion F protein and CpG adjuvant elicits robust protective effects in mice

Human respiratory syncytial virus (HRSV) is a leading cause of lower respiratory tract infections in elderly individuals and young children/infants and can cause bronchiolitis and even death. There is no licensed HRSV vaccine. An ideal vaccine should induce high titers of neutralizing antibodies and a Th1-biased immune response. In this study, we used EXPI293 cells to express the fusion (F) protein with a prefusion conformation (PrF) and compared the safety and efficacy of intranasal immunization with PrF in combination with two mucosal adjuvants (CpG ODN and liposomes) in mice. After two intranasal administrations, mice in the PrF + CpG group produced high titers of neutralizing antibodies (4961) and a Th1-biased immune response compared with the PrF + Lipo group. The lung viral load of mice in the PrF + CpG group was significantly reduced (3.5 log) compared with that in the adjuvant control group, and the survival rate was 100 %, while the survival rate of mice in the PrF + Lipo group was only 67 %. At the same time, this immunization strategy reduced the pathological damage to the lungs in mice. In conclusion, the combination of PrF and CpG adjuvant is immunogenic, elicits a Th1 type immune response, and completely protects mice from a lethal HRSV challenge. It is worthy of further evaluation as an HRSV vaccine in clinical trials.Clinical trial registration.This study was not related to human participation or experimentation.

Activity and biophysical properties related to clinical evaluation of a first-in-class EHL ROR1xCD3 T Cell Engager.

e14505 Background: NVG-111, a T cell engager (TCE) targeting ROR1 and CD3, is in clinical development for the treatment of Chronic Lymphocytic Leukemia (CLL) and Mantle Cell Leukemia (MCL). This 55KDa molecule consisting of humanised tandem scFvs is administered into patients as a continuous intravenous infusion. The intrinsically short half-life of NVG-111 has its advantages, such as rapid management of CRS by cessation of infusion. Early safety and efficacy clinical data supports the development of an extended half-life (EHL) ROR1xCD3 TCE for ease of administration and convenience for patients, building on the favorable activity and biophysical attributes of NVG-111. A next generation therapy possessing such an EHL has potential to improve patient experience by offering a different drug administration paradigm. Methods: Several formats were designed and evaluated, incorporating different EHL technology solutions by increasing the size and enabling FcRn-mediated recycling. EHL TCEs and non-EHL TCEs were cloned in-format with Fc, HSA, or an HSA binding moiety into a suitable expression vector. The resultant clones were expressed using Expi293 cells and the potency of TCEs were evaluated by co-culture assays using MCL derived JeKo-1 target cells and T cells from healthy donor PBMCs. Cytotoxic responses and T cell activation as determined by CD69 expression were measured by flow cytometry. T cell activation was also assessed by measuring CD3 signalling in a reporter gene assay. Biophysical properties were evaluated by determining the post-purification expression titer, the aggregation profile using analytical SEC, and the impact of stability challenge in formulation buffer and serum on target cell binding and activity. Results: Reformatting NVG-111 into an IgG-like heterodimeric bispecific scFv-Fc resulted in loss of potency, which may be due to a change in geometry of binding and/or poorer target engagement. Activity and expression titer were also deleteriously affected by directly fusing NVG-111 to human serum albumin (HSA), or to an HSA binding moiety. Fusing NVG-111 directly to Fc resulted in maintained potency in cytotoxicity and T cell activation assays, the expression titer of parental NVG-111, and exhibited very low levels of aggregation. Stability studies performed in formulation buffer and in human serum showed that NVG-111-Fc was at least as stable as parent. Furthermore, other studies with this molecular format indicate that the half-life in mice is comparable to a heterodimeric bispecific scFv-Fc. Conclusions: NVG-111 has been successfully engineered into an EHL format that increased the molecular size and has the potential to engage in FcRn-mediated recycling. This format maintains functional activity, is stable, and expresses well with a favorable aggregation profile. Further molecular refinements are being actively evaluated in readiness for IND enabling studies.

Biophysical Characterization of the Contribution of the Fab Region to the IgG-FcγRIIIa Interaction.

The cell-surface receptor FcγRIIIa is crucial to the efficacy of therapeutic antibodies as well as the immune response. The interaction of the Fc region of IgG molecules with FcγRIIIa has been characterized, but until recently, it was thought that the Fab regions were not involved in the interaction. To evaluate the influence of the Fab regions in a biophysical context, we carried out surface plasmon resonance analyses using recombinant FcγRIIIa ligands. A van't Hoff analysis revealed that compared to the interaction of the papain-digested Fc fragment with FcγRIIIa, the interaction of commercially available, full-length rituximab with FcγRIIIa had a more favorable binding enthalpy, a less favorable binding entropy, and a slower off rate. Similar results were obtained from analyses of IgG1 molecules and an IgG1-Fc fragment produced by Expi293 cells. For further validation, we also prepared a maltose-binding protein-linked IgG1-Fc fragment (MBP-Fc). The binding enthalpy of MBP-Fc was nearly equal to that of the IgG1-Fc fragment for the interaction with FcγRIIIa, indicating that such alternatives to the Fab domains as MBP do not positively contribute to the IgG-FcγRIIIa interactions. Our investigation strongly suggests that the Fab region directly interacts with FcγRIIIa, resulting in an increase in the binding enthalpy and a decrease in the dissociation rate, at the expense of favorable binding entropy.

Antibody Light Chains: Key to Increased Monoclonal Antibody Yields in Expi293 Cells?

When constructing isogenic recombinant IgM–IgG pairs, we discovered that μ heavy chains strongly prefer partnering with λ light chains for optimal IgM expression in transiently cotransfected Expi293 cells. When μ chains were paired with κ light chains, IgM yields were low but increased by logs—up to 20,000 X—by using λ chains instead. Switching light chains did not alter epitope specificity. For dimeric IgA2, optimal expression involved pairing with λ chains, whereas light-chain preference varied for other immunoglobulin classes. In summary, recombinant IgM production can be drastically increased by using λ chains, an important finding in the use of IgM for mucosal immunoprophylaxis.

Biophysical Fitness Landscape of the SARS-CoV-2 Delta Variant Receptor Binding Domain

Among the five known SARS-CoV-2 variants of concern, Delta is the most virulent leading to severe symptoms and increased mortality among infected people. Our study seeks to examine how the biophysical parameters of the Delta variant correlate to the clinical observations. Receptor binding domain (RBD) is the first point of contact with the human host cells and is the immunodominant form of the spike protein. Delta variant RBD contains two novel mutations L452R and T478K. We examined the effect of single as well as the double mutations on RBD expression in human Expi293 cells, RBD stability using urea and thermal denaturation, and RBD binding to angiotensin converting enzyme 2 (ACE2) receptor and to neutralizing antibodies using isothermal titration calorimetry. Delta variant RBD showed significantly higher expression compared to the wild-type RBD, and the increased expression is due to L452R mutation. Despite their non-conservative nature, none of the mutations significantly affected RBD structure and stability. All mutants showed similar binding affinity to ACE2 and to Class 1 antibodies (CC12.1 and LY-CoV016) as that of the wild-type. Delta double mutant L452R/T478K showed no binding to Class 2 antibodies (P2B-2F6 and LY-CoV555) and a hundred-fold weaker binding to a Class 3 antibody (REGN10987), and the decreased antibody binding is determined by the L452R mutation. These results indicate that the immune escape from neutralizing antibodies, rather than increased receptor binding, is the main biophysical parameter that determined the fitness landscape of the Delta variant RBD.

Transient Gene Expression in Human Expi293 Cells.

Transient gene expression (TGE) is an important tool for generating recombinant proteins in a short period of time. The human cell line HEK293 is widely used for this purpose since it can grow in suspension to a high cell density in serum-free media. In addition, this cell line is amenable to several transfection methods and produces recombinant proteins in satisfactory quantities for functional and structural analysis. This chapter describes the methodology for TGE using the Expi293 system, which provides higher expression levels than other HEK293-based systems.

Recombinant Protein Expression and Purification of N, S1, and RBD of SARS-CoV-2 from Mammalian Cells and Their Potential Applications.

The coronavirus disease 2019 (COVID-19) pandemic has reached an unprecedented level. There is a strong demand for diagnostic and serological supplies worldwide, making it necessary for countries to establish their own technologies to produce high-quality biomolecules. The two main viral antigens used for the diagnostics for severe acute respiratory syndrome coronavirus (SARS-CoV-2) are the structural proteins spike (S) protein and nucleocapsid (N) protein. The spike protein of SARS-CoV-2 is cleaved into S1 and S2, in which the S1 subunit has the receptor-binding domain (RBD), which induces the production of neutralizing antibodies, whereas nucleocapsid is an ideal target for viral antigen-based detection. In this study, we designed plasmids, pcDNA3.1/S1 and pcDNA3.1/N, and optimized their expression of the recombinant S1 and N proteins from SARS-CoV-2 in a mammalian system. The RBD was used as a control. The antigens were successfully purified from Expi293 cells, with high yields of the S1, N, and RBD proteins. The immunogenic abilities of these proteins were demonstrated in a mouse model. Further, enzyme-linked immunosorbent assays with human serum samples showed that the SARS-CoV-2 antigens are a suitable alternative for serological assays to identify patients infected with COVID-19.

Immunogenicity and Protective Efficacy of a Highly Thermotolerant, Trimeric SARS-CoV-2 Receptor Binding Domain Derivative.

The receptor binding domain (RBD) of SARS-CoV-2 is the primary target of neutralizing antibodies. We designed a trimeric, highly thermotolerant glycan engineered RBD by fusion to a heterologous, poorly immunogenic disulfide linked trimerization domain derived from cartilage matrix protein. The protein expressed at a yield of ∼80–100 mg/L in transiently transfected Expi293 cells, as well as CHO and HEK293 stable cell lines and formed homogeneous disulfide-linked trimers. When lyophilized, these possessed remarkable functional stability to transient thermal stress of up to 100 °C and were stable to long-term storage of over 4 weeks at 37 °C unlike an alternative RBD-trimer with a different trimerization domain. Two intramuscular immunizations with a human-compatible SWE adjuvanted formulation elicited antibodies with pseudoviral neutralizing titers in guinea pigs and mice that were 25–250 fold higher than corresponding values in human convalescent sera. Against the beta (B.1.351) variant of concern (VOC), pseudoviral neutralization titers for RBD trimer were ∼3-fold lower than against wildtype B.1 virus. RBD was also displayed on a designed ferritin-like Msdps2 nanoparticle. This showed decreased yield and immunogenicity relative to trimeric RBD. Replicative virus neutralization assays using mouse sera demonstrated that antibodies induced by the trimers neutralized all four VOC to date, namely B.1.1.7, B.1.351, P.1, and B.1.617.2 without significant differences. Trimeric RBD immunized hamsters were protected from viral challenge. The excellent immunogenicity, thermotolerance, and high yield of these immunogens suggest that they are a promising modality to combat COVID-19, including all SARS-CoV-2 VOC to date.

Proteomic identification and validation of novel interactions of the putative tumor suppressor PRELP with membrane proteins including IGFI-R and p75NTR

Proline and arginine-rich end leucine-rich repeat protein (PRELP) is a member of the small leucine-rich repeat proteoglycans (SLRPs) family. Levels of PRELP mRNA are downregulated in many types of cancer, and PRELP has been reported to have suppressive effects on tumor cell growth, although the molecular mechanism has yet to be fully elucidated. Given that other SLRPs regulate signaling pathways through interactions with various membrane proteins, we reasoned that PRELP likely interacts with membrane proteins to maintain cellular homeostasis. To identify membrane proteins that interact with PRELP, we carried out coimmunoprecipitation coupled with mass spectrometry (CoIP-MS). We prepared membrane fractions from Expi293 cells transfected to overexpress FLAG-tagged PRELP or control cells and analyzed samples precipitated with anti-FLAG antibody by mass spectrometry. Comparison of membrane proteins in each sample identified several that seem to interact with PRELP; among them, we noted two growth factor receptors, insulin-like growth factor I receptor (IGFI-R) and low-affinity nerve growth factor receptor (p75NTR), interactions with which might help to explain PRELP’s links to cancer. We demonstrated that PRELP directly binds to extracellular domains of these two growth factor receptors with low micromolar affinities by surface plasmon resonance analysis using recombinant proteins. Furthermore, cell-based analysis using recombinant PRELP protein showed that PRELP suppressed cell growth and affected cell morphology of A549 lung carcinoma cells, also at micromolar concentration. These results suggest that PRELP regulates cellular functions through interactions with IGFI-R and p75NTR and provide a broader set of candidate partners for further exploration.

Adiponectin and related C1q/TNF-related proteins bind selectively to anionic phospholipids and sphingolipids

Adiponectin (Acrp30) is an adipokine associated with protection from cardiovascular disease, insulin resistance, and inflammation. Although its effects are conventionally attributed to binding Adipor1/2 and T-cadherin, its abundance in circulation, role in ceramide metabolism, and homology to C1q suggest an overlooked role as a lipid-binding protein, possibly generalizable to other C1q/TNF-related proteins (CTRPs) and C1q family members. To investigate this, adiponectin, representative family members, and variants were expressed in Expi293 cells and tested for binding to lipids in liposomes using density centrifugation. Binding to physiological lipids were also analyzed using gradient ultracentrifugation, liquid chromatography-mass spectrometry, and shotgun lipidomics. Interestingly, adiponectin selectively bound several anionic phospholipids and sphingolipids, including phosphatidylserine, ceramide-1-phosphate, glucosylceramide, and sulfatide, via the C1q domain in an oligomerization-dependent fashion. Binding to lipids was observed in liposomes, low-density lipoproteins, cell membranes, and plasma. Other CTRPs and C1q family members (Cbln1, CTRP1, CTRP5, and CTRP13) also bound similar lipids. These findings suggest that adiponectin and CTRPs function not only as hormones, but also as lipid opsonins, as may other C1q family proteins.

Impact of the expression system on the immune responses to self-assembling protein nanoparticles (SAPNs) displaying HIV-1 V1V2 loop

The V1V2 loop of the Env protein is a major target for HIV-1 vaccine development because in multiple studies antibodies to this region correlated with protection. Although SAPNs expressed in E. coli elicited anti-V1V2 antibodies, the Env protein is heavily glycosylated. In this study the technology has been adapted for expression in mammalian cells. SAPNs containing a V1V2 loop from a B-subtype transmitter/founder virus were expressed in E. coli, ExpiCHO, and Expi293 cells. Independent of the expression host, particles were well-formed. All SAPNs raised high titers of V1V2-specific antibodies, however, SAPNE.coli induced a mainly anti-V1 response, while SAPNExpiCHO and SAPNExpi293 induced a predominantly anti-V2 response. In an ADCP assay, sera from animals immunized with the SAPNExpiCHO or SAPNExpi293 induced a significant increase in phagocytic activity. This novel way of producing SAPNs displaying glycosylated epitopes could increase the antibody titer, functional activity, and shift the immune response towards the desired pathway.

Novel synthetic immune modulators for the activation of tumor antigen-specific T cells.

e15203 Background: Although checkpoint inhibitor immunotherapy and adoptive T-cell therapy revolutionized cancer treatments, such approaches suffer either from lack of target specificity for checkpoint inhibitors or inability to target intracellular tumor-related antigens from CAR-T therapy. Here, we report the development of novel Tavo Immune Modulator (TIM) biologics molecules which can specifically recognize tumor antigen-specific T cells through an engineered pMHC complex with peptides derived from intracellular tumor-related antigens. These molecules can selectively activate such T cells through engineered T cell co-stimulatory modulators for enhanced tumor cell killing. Methods: NY-ESO-1 and MAGE-A10 TIM molecules were constructed as fusions of HLA-A*02:01 MHC complexed with either NY-ESO-1 (157-165) or MAGE-A10 (254-262) epitope peptides at the N-termini and various T cell costimulatory modulators at the C-termini of IgG heavy and light chains. TIM molecules were expressed in Expi293 cells and purified by Protein A affinity chromatography. Specific binding of TIM with cancer specific T cells was evaluated by immunostaining. The activation and proliferation of tumor specific CD8+ T cells were confirmed in T cell activation and recall assays. Results: Both NY-ESO-1 and MAGE-A10 specific TIM molecules were generated which recognized corresponding tumor specific T cells. NY-ESO-1 TIM engineered with IL2 could activate NY-ESO-1 specific CD8+ T cell exclusively. Engineering additional T cell costimulatory factors along with IL2 on NY-ESO-1 TIM molecule could further boost T cell proliferation and activation in T cell recall assays. Besides NY-ESO-1, combinations of T cell costimulatory factors with MAGE-A10 TIM molecules enhanced specific T cell activation. Additional in vitro and in vivo studies are ongoing to demonstrate efficacy of such novel TIM molecules in eliminating different types of NY-ESO-1 and MAGE-A10 which are over-expressed on tumor cells. Conclusions: This study demonstrates the utility of NY-ESO-1 and MAGE-A10 TIM molecules in the selective recognition and activation of tumor antigen-specific T cells. Such novel biologics molecules may provide target specificity in tumor treatment, and potential targeting of intracellular tumor-related antigens presented as peptides in MHC complexes on cell surfaces. Selective activation of tumor-specific T cells may provide a unique method for the treatment of various solid tumors and warrants further investigation.

Novel HPV-E7 immune modulators to activate HPV-specific T cells to eliminate cervical cancer.

e18027 Background: Human papilloma virus (HPV) infection can lead to several types of cancers in both men and women. HPV+ tumor cells constitutively express the HPV-E7 antigen which can act as an oncogene to promote tumor growth and malignant transformation. Here, we report the application of novel Tavo Immune Modulator (TIM) biologics molecules which are consisted of a pMHC complex with an epitope peptide derived from HPV-E7 and co-stimulatory modulators of T cell activity. The HPV-E7 TIM molecules can specifically recognize and activate HPV-E7-specific T cells for the elimination of HPV affected cells. Methods: HPV-E7 TIM molecules were engineered as fusion molecules with HLA-A*02:01 MHC complexed with an HPV-E7 (11-20) epitope peptide at the N-termini, and various T cell costimulatory modulators at the C-termini of IgG heavy and light chains. TIM molecules were expressed in Expi293 cells and purified by Protein A affinity chromatography. Specific binding of TIM with HPV-E7 specific T cells was confirmed by immunostaining and flow cytometry. The activation and expansion of antigen specific CD8+ T cells were elucidated in T cell activation and recall assays. Results: HPV-E7 TIM molecules with various T cell co-stimulator molecules were engineered to specifically recognize HPV-specific T cells. Activation of T cells was antigen-specific and depended on the presence of an engineered T cell modulatory component on the TIM framework. The effects of various costimulatory molecules in different combinations on T cell activation were explored and an optimal combination was identified which facilitated high potency antigen-specific T cell activation. Such molecular combinations could facilitate T cell expansion and activation in T cell recall assays. Efficacy of HPV-E7 TIM molecules by inhibiting tumor growth in a syngeneic tumor model is ongoing. Conclusions: This study demonstrates that HPV-E7 TIM molecules selectively recognize and activate HPV-specific CD8+ T cells in the presence of a combination of two T cell costimulatory factors. Such novel biologics provide distinctive approaches in the treatment of HPV-related cancers and warrant further investigation. Additional in vitro and in vivo studies are ongoing to demonstrate the utility in eliminating HPV-infected tumor cells. Full data will be presented at the meeting.

Computer-designed active human butyrylcholinesterase double mutant with a new catalytic triad

A computer-designed mutant of human butyrylcholinesterase (BChE), N322E/E325G, with a novel catalytic triad was made. The catalytic triad of the wild-type enzyme (S198·H438·E325) was replaced by S198·H438·N322E in silico. Molecular dynamics for 1.5 μs and Markov state model analysis showed that the new catalytic triad should be operative in the mutant enzyme, suggesting functionality. QM/MM modeling performed for the reaction of wild-type BChE and double mutant with echothiophate showed high reactivity of the mutant towards the organophosphate. A truncated monomeric (L530 stop) double mutant was expressed in Expi293 cells. Non-purified transfected cell culture medium was analyzed. Polyacrylamide gel electrophoresis under native conditions followed by activity staining with BTC as the substrate provided evidence that the monomeric BChE mutant was active. Inhibition of the double mutant by echothiophate followed by polyacrylamide gel electrophoresis and activity staining showed that this enzyme slowly self-reactivated. However, because Expi293 cells secrete an endogenous BChE tetramer and several organophosphate-reacting enzymes, catalytic parameters and self-reactivation constants after phosphorylation of the new mutant were not determined in the crude cell culture medium. The study shows that the computer-designed double mutant (N322E/E325G) with a new catalytic triad (S198·H438·N322E) is a suitable template for design of novel active human BChE mutants that display an organophosphate hydrolase activity.