Bivalent Fusion Research Articles

Structural insights into rapamycin-induced oligomerization of a FRB-FKBP fusion protein.

Inducible dimerization systems, such as rapamycin-induced dimerization of FK506 binding protein (FKBP) and FKBP-rapamycin binding (FRB) domain, are widely employed chemical biology tools to manipulate cellular functions. We previously advanced an inducible dimerization system into an inducible oligomerization system by developing a bivalent fusion protein, FRB-FKBP, which forms large oligomers upon rapamycin addition and can be used to manipulate cells. However, the oligomeric structure of FRB-FKBP remains unclear. Here, we report that FRB-FKBP forms a rotationally symmetric trimer in crystals, but a larger oligomer in solution, primarily tetramers and pentamers, which maintain similar inter-subunit contacts as in the crystal trimer. These findings expand the applications of the FRB-FKBP oligomerization system in diverse biological events.

Vaccine Based on Recombinant Fusion Protein Combining Hepatitis B Virus PreS with SARS-CoV-2 Wild-Type- and Omicron-Derived Receptor Binding Domain Strongly Induces Omicron-Neutralizing Antibodies in a Murine Model.

COVID-19, caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has become a recurrent endemic disease affecting the whole world. Since November 2021, Omicron and its subvariants have dominated in the spread of the disease. In order to prevent severe courses of disease, vaccines are needed to boost and maintain antibody levels capable of neutralizing Omicron. Recently, we produced and characterized a SARS-CoV-2 vaccine based on a recombinant fusion protein consisting of hepatitis B virus (HBV)-derived PreS and two SARS-CoV-2 wild-type RBDs. To develop a PreS-RBD vaccine which induces high levels of Omicron-specific neutralizing antibodies. We designed, produced, characterized and compared strain-specific (wild-type: W-PreS-W; Omicron: O-PreS-O), bivalent (mix of W-PreS-W and O-PreS-O) and chimeric (i.e., W-PreS-O) SARS-CoV-2 protein subunit vaccines. Immunogens were characterized in vitro using protein chemical methods, mass spectrometry, and circular dichroism in combination with thermal denaturation and immunological methods. In addition, BALB/c mice were immunized with aluminum-hydroxide-adsorbed proteins and aluminum hydroxide alone (i.e., placebo) to study the specific antibody and cytokine responses, safety and Omicron neutralization. Defined and pure immunogens could be produced in significant quantities as secreted and folded proteins in mammalian cells. The antibodies induced after vaccination with different doses of strain-specific, bivalent and chimeric PreS-RBD fusion proteins reacted with wild-type and Omicron RBD in a dose-dependent manner and resulted in a mixed Th1/Th2 immune response. Interestingly, the RBD-specific IgG levels induced with the different vaccines were comparable, but the W-PreS-O-induced virus neutralization titers against Omicron (median VNT50: 5000) were seven- and twofold higher than the W-PreS-W- and O-PreS-O-specific ones, respectively, and they were six-fold higher than those of the bivalent vaccine. Among the tested immunogens, the chimeric PreS-RBD subunit vaccine, W-PreS-O, induced the highest neutralizing antibody titers against Omicron. Thus, W-PreS-O seems to be a highly promising COVID-19 vaccine candidate for further preclinical and clinical evaluation.

Multivalent nanobody-based sandwich enzyme-linked immunosorbent assay for sensitive detection of porcine reproductive and respiratory syndrome virus

The pandemic of the porcine reproductive and respiratory syndrome virus (PRRSV) has caused huge economic losses and continues to threaten the swine industry worldwide. Nucleocapsid protein (N protein) is the primary antigen of PRRSV for development of sensitive diagnostic assays. Two high affinity nanobodies against N protein, Nb12 and Nb35, were selected and employed to develop a sandwich ELISA. Further we improved the ELISA method to obtain greater sensitivity, a trivalent nanobody (3 × Nb35) and a bivalent nanobody-HRP fusion protein (2 × Nb12-HRP) were expressed and used. This modified ELISA was found to have high sensitivity for detecting PRRSV, with a detection limit of 10 TCID50/ml (median tissue culture infectious dose), which was approximately 200-fold greater than the single-copy nanobody-based sandwich ELISA. The developed assay shows high specificity and can detect almost all circulating lineages of PRRSV-2 in China. This study provides suggestions for reforming nanobodies and for the further development of multivalent nanobody-based ELISAs for other various viruses.

Agonistic Bivalent Human scFvs-Fcγ Fusion Antibodies to OX40 Ectodomain Enhance T Cell Activities against Cancer.

(1) Background: Understanding how advanced cancers evade host innate and adaptive immune opponents has led to cancer immunotherapy. Among several immunotherapeutic strategies, the reversal of immunosuppression mediated by regulatory T cells in the tumor microenvironment (TME) using blockers of immune-checkpoint signaling in effector T cells is the most successful treatment measure. Furthermore, agonists of T cell costimulatory molecules (CD40, 4-1BB, OX40) play an additional anti-cancer role to that of checkpoint blocking in combined therapy and serve also as adjuvant/neoadjuvant/induction therapy to conventional cancer treatments, such as tumor resection and radio- and chemo- therapies. (2) Methods and Results: In this study, novel agonistic antibodies to the OX40/CD134 ectodomain (EcOX40), i.e., fully human bivalent single-chain variable fragments (HuscFvs) linked to IgG Fc (bivalent HuscFv-Fcγ fusion antibodies) were generated by using phage-display technology and genetic engineering. The HuscFvs in the fusion antibodies bound to the cysteine-rich domain-2 of the EcOX40, which is known to be involved in OX40-OX40L signaling for NF-κB activation in T cells. The fusion antibodies caused proliferation, and increased the survival and cytokine production of CD3-CD28-activated human T cells. They showed enhancement trends for other effector T cell activities like granzyme B production and lysis of ovarian cancer cells when added to the activated T cells. (3) Conclusions: The novel OX40 agonistic fusion antibodies should be further tested step-by-step toward their safe use as an adjunctive non-immunogenic cancer immunotherapeutic agent.

The Potent and Selective Antitumor Activity of CO-1 and Fully Humanized CO-1bi in Treatment of B Cell Non-Hodgkin's Lymphoma

Recent advancements have identified CD47 blockade as a promising approach for the treatment of B cell Non-Hodgkin's Lymphoma (B-NHL). CD47 blockade inhibits the CD47-SIRPα axis, which inhibits a “don't eat me” signal that facilitates the phagocytosis of tumor cells by macrophages [1,2]. While the effect of anti-CD47 monoclonal antibodies (mAbs) as monotherapy has been limited in B-NHL, combining CD47 mAbs with conventional treatment such as rituximab has demonstrated more encouraging outcomes. This combinatory approach may be attributed to the need for additional cytotoxic mechanisms to achieve clinically meaningful responses with CD47-mediated therapy in B-NHL [3]. Additionally, CD47 blockage has raised concerns about off-target effects and red blood cell depletion [4]. To address these challenges and improve treatment possibilities we developed a fully humanized CO-1bi based on the chimeric CO-1. The bifunctional CO-1 is an agonistic IgG4 anti-CD47 mAb designed to induce direct and rapid programmed cancer cell death (PCCD) and stimulate phagocytosis through blockage of CD47-SIRPα binding. CO-1bi is a humanized bivalent scFv-Fc fusion of CO-1 with superior safety (no red blood cell hemagglutination) while maintaining the functionality of CO-1. In this study, we evaluated the antitumor activity of CO-1 and CO-1bi on different B-NHL cell lines in vitro as well as in xenografts of B-NHL established in NOD- scid IL2Rγ null (NSG) mice. Both constructs demonstrated potent and fast cell death induction in lymphoma cell lines while sparing normal B cells. The robust antitumor activity demonstrated by CO-1 and CO-1bi as single agents in xenograft models of Burkitt's lymphoma underscores the significance of their additional agonistic PCCD capabilities compared to other anti-CD47 agents. The emergence of CO-1bi offers a unique strategy with dual functionality for the treatment of B-NHL and other malignancies without red blood cell depletion characterized by current anti-CD47 mAb in clinical trials. This innovative approach provides an improved and promising avenue for cancer treatment.

In silico design, production and immunization evaluation of a recombinant bivalent fusion protein candidate vaccine against E. coli O157:H7.

In silico techniques are highly suited for both the discovery of new and development of available vaccines. Escherichia coli O157: H7, a main cause of food poisoning can infect humans through the consumption of contaminated water or food. Vaccination is a choice strategy to combat the bacterium. In the present study, we designed, expressed and purified a chimeric protein comprising two antigens of Escherichia coli O157: H7, including intimin and flagellin proteins, as a vaccine candidate and evaluated its immunization ability in mice. Thein silicoresults showed that the proposed antigen has a high antigenicity and conformation to be used as a potent vaccine candidate. The protein was successfully expressed in E. coli expression system with a proper level of expression (0/8g/L). Immunization evaluation showed that the protein is able to evoke the mice's humoral immunity and can confer a protective immunity against E. coli O157:H7, so that 80% of the immunized animals were survived following the intraperitoneal injection of 100 LD50 of the live bacteria. Shedding analysis also showed the protectivity power of the protein. Bacterial excretion in control animals remained stable at about 108 CFU after 15days, while the excreted bacteria in the feces of immunized mice's decreased to about 102 after the same time. According to the results, the proposed protein is able to stimulate the immune responses of mice and protect them against E. coli O157:H7.

EGF‑IL2 bispecific and bivalent EGF fusion toxin efficacy against syngeneic head and neck cancer mouse models.

The epidermal growth factor receptor (EGFR) remains one of the best molecules for developing targeted therapy for multiple human malignancies, including head and neck squamous cell carcinoma (HNSCC). Small molecule inhibitors or antibodies targeting EGFR have been extensively developed in recent decades. Immunotoxin (IT)‑based therapy, which combines cell surface binding ligands or antibodies with a peptide toxin, represents another cancer treatment option. A total of 3 diphtheria toxin (DT)‑based fusion toxins that target human EGFR‑monovalent EGFR IT (mono‑EGF‑IT), bivalent EGFR IT (bi‑EGF‑IT), and a bispecific IT targeting both EGFR and interleukin‑2 receptor (bis‑EGF/IL2‑IT) were recently generated by the authors. Improved efficacy and reduced toxicity of bi‑EGF‑IT compared with mono‑EGF‑IT in immunocompromised HNSCC mouse models was reported. In the present study, bis‑EGF/IL2‑IT were generated using a unique DT‑resistant yeast expression system and evaluated the invitro and invivo efficacy and toxicity of the 3 EGF‑ITs in immunocompetent mice. The results demonstrated that while the three EGF‑ITs had different efficacies invitro and invivo against HNSCC, bi‑EGF‑IT and bis‑EGF/IL2‑IT had significantly improved invivo efficacy and remarkably less off‑target toxicity compared with mono‑EGF‑IT. In addition, bis‑EGF/IL2‑IT was superior to bi‑EGF‑IT in reducing tumor size and prolonging survival in the metastatic model. These data suggested that targeting either the tumor immune microenvironment or enhancing the binding affinity could improve the efficacy of IT‑based therapy. Bi‑EGF‑IT and bis‑EGF/IL2‑IT represent improved candidates for IT‑based therapy for future clinical development.

Bivalent HIV-1 fusion inhibitors based on peptidomimetics

Membrane fusion is a valid target for inhibition of HIV-1 replication. A 34-mer fragment peptide (C34), which is contained in the HIV-1 envelope protein gp41, has significant anti-HIV activity. Previously, a dimeric derivative of C34 linked by a disulfide bridge at its C-terminus was found to have more potent anti-HIV activity than the C34 peptide monomer. To date, several peptidomimetic small inhibitors have been reported, but most have lower potency than peptide derivatives related to C34. In the present study we applied this dimerization concept to these peptidomimetic small inhibitors and designed several bivalent peptidomimetic HIV-1 fusion inhibitors. The importance of the length of linkers crosslinking two peptidomimetic compounds was demonstrated and several potent bivalent inhibitors containing tethered peptidomimetics were produced.

Evaluating the Therapeutic Efficacy of Mono- and Bivalent Affibody-Based Fusion Proteins Targeting HER3 in a Pancreatic Cancer Xenograft Model.

Human epidermal growth factor receptor 3 (HER3) has been increasingly scrutinized as a potential drug target since the elucidation of its role in mediating tumor growth and acquired therapy resistance. Affibody molecules are so-called scaffold proteins with favorable biophysical properties, such as a small size for improved tissue penetration and extravasation, thermal and chemical stability, and a high tolerance to modifications. Additionally, affibody molecules are efficiently produced in prokaryotic hosts or by chemical peptide synthesis. We have previously evaluated the biodistribution profiles of five mono- and bivalent anti-HER3 affibody molecules (designated as 3) fused to an albumin-binding domain (designated as A), 3A, 33A, 3A3, A33, and A3, that inhibit ligand-dependent phosphorylation. In the present study, we examined the therapeutic efficacy of the three most promising variants, 3A, 33A, and 3A3, in a direct comparison with the HER3-targeting monoclonal antibody seribantumab (MM-121) in a preclinical BxPC-3 pancreatic cancer model. Xenografted mice were treated with either an affibody construct or MM-121 and the tumor growth was compared to a vehicle group. Receptor occupancy was estimated by positron emission tomography/computed tomography (PET/CT) imaging using a HER3-targeting affibody imaging agent [68Ga]Ga-(HE)3-Z08698-NODAGA. The affibody molecules could inhibit ligand-dependent phosphorylation and cell proliferation in vitro and demonstrated tumor growth inhibition in vivo comparable to that of MM-121. PET/CT imaging showed full receptor occupancy for all tested drug candidates. Treatment with 3A and 3A3 affibody constructs was more efficient than with 33A and similar to the anti-HER3 antibody seribantumab, showing that the molecular design of affibody-based therapeutics targeting HER3 in terms of the relative position of functional domains and valency has an impact on therapeutic effect.

Evaluating single-domain antibodies as carriers for targeted vaccine delivery to the small intestinal epithelium

Targeting a vaccine to the mucosal surface has recently been recognized as a promising approach to efficiently induce mucosal immune responses against enteric pathogens. However, poor uptake and inefficient transport of orally delivered subunit vaccines across the intestinal epithelium combined with weak immune responses still present important bottlenecks for mucosal vaccination. A possible strategy suggested to surmount these hurdles is to target the selected antigen to transcytotic receptors, such as aminopeptidase N (APN) present on enterocytes and antigen-presenting cells (APCs). Therefore, we aimed to identify potent and selective VHHs against porcine aminopeptidase N (pAPN), that were fused to the fragment crystallizable (Fc) domain of the murine IgG2a, resulting in dimeric VHH-MG fusions. Out of a library of 30 VHH-MG fusion candidates, two fusions displaying the best binding on pAPN-expressing cells were selected and showed in vivo internalization across the porcine gut epithelium. One of these fusions triggered systemic and intestinal IgA responses upon oral administration. Our results demonstrate the potential of bivalent VHH-MG fusions as delivery vehicles for vaccine antigens. VHH-mediated targeting of antigens to APN to generate protective immunity at the mucosal surface remains to be further validated.

A new bivalent fluorescent fusion protein for differential Cu(II) and Zn(II) ion detection in aqueous solution

Simple and easy to engineer metal-sensing molecules that are capable of differentiating metal ions and producing metal-specific signals are highly desirable. Metal ions affect the thermal stability of proteins by increasing or decreasing their resistance to unfolding. This work illustrates a new strategy for designing bivalent fluorescent fusion proteins capable of differentiating metal ions in solution through their distinct effects on a protein’s thermal stability. A new dual purpose metal sensor was developed consisting of biotin protein ligase (BirA) from B. pseudomallei (Bp) fused to green fluorescent protein (GFP). When coupled with differential scanning fluorimetry of GFP-tagged proteins (DSF-GTP) for signal-transduction detection, Bp BirA-GFP yields distinct protein unfolding signatures with Zn(II) and Cu(II) ions in aqueous solutions. The limit of detection of the system is ∼1 μM for both metal species. The system can be used in a variety of high-throughput assay formats including for the screening of metal-binding proteins and chelators. Bp BirA-GFP has also the additional benefit of being useful in Cu(II) ion field-testing applications through simple visual observation of a temperature-dependent loss of fluorescence. Bp BirA-GFP is the first example of a protein-based dual purpose Cu(II) and Zn(II) ion sensor compatible with two different yet complementary signal-transduction detection systems.

Abstract 222: Novel protein drug conjugates targeting ROR1 through the development and exploitation of a drug discovery platform based on small, engineered VNAR domains

Abstract Whilst antibody drug conjugates (ADCs) have been successfully used to target highly potent cytotoxic agents to tumours, there is a continued emphasis on developing technologies for improving the therapeutic effectiveness of this class of agents. The vast majority of ADCs that have entered the clinic have been based on large intact immunoglobulins, however, there is significant interest in exploiting small protein domains as the payload delivery vehicle. These offer a number of potential benefits over full length antibodies including increased tumour penetration, amenability to protein engineering and site-specific conjugation, and improved tolerability. To this end, we have developed a therapeutics platform based on shark Variable New Antigen Receptor (VNAR) proteins. Shark VNAR domains are the smallest naturally occurring antigen binding domain (~11 kDa), with a binding mechanism distinct from traditional antibodies. Here we report the application of this platform to the development of homogenous VNAR-drug conjugates targeting the onco-embryonic receptor tyrosine kinase ROR1. ROR1 expression has been observed across a broad range of solid tumours and haematological malignancies, and is reported to correlate with poor clinical outcome for a number of cancer indications, but is largely absent from normal adult tissue. As such, the expression pattern of ROR1, coupled with its functional role in tumourogenesis and disease progression, make it an attractive PDC target. Using a combination of direct immunisation and synthetic VNAR library screening, we have generated high affinity binders to the extracellular domain of ROR1 which show species cross-reactivity, do not bind the closely related family member ROR2, and can target distinct non-overlapping regions of the protein. By exploiting the ability of VNAR domains to be flexibly reformatted, the lead sequences have been configured as a series of bivalent Fc fusions, VNAR multimers and novel bi-paratopic binders, and constructs identified that show further enhanced binding to ROR1 and which can induce internalisation and lysosomal trafficking in ROR1 expressing cancer cell-lines. Subsequent attachment of highly potent DNA-damaging cytotoxic payloads yielded homogenous VNAR-drug conjugates which demonstrated extremely potent (sub-nanomolar) in vitro cytotoxicity against selected cancer cell-lines in a receptor mediated fashion. Studies to assess the in vivo efficacy of these conjugates are in progress. These ROR1 VNAR constructs have also been successfully fused to sdAbs and scFv directed to other cell-surface protein targets, demonstrating the power of the platform and opening the way for novel bi-specific approaches for targeting both solid and haematological tumours. Citation Format: Graham Cotton, Jennifer Thom, Paul Trumper, Stacey Bell, Andrei Kamenski, Mark Wappett, Caroline Barelle, Marina Kovaleva, John Steven, Andy Porter, Estelle McLean, Chiara Saladino, Aidan McCann, Aaron Cranston, Tim Harrison. Novel protein drug conjugates targeting ROR1 through the development and exploitation of a drug discovery platform based on small, engineered VNAR domains [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 222.

A bivalent fusion vaccine composed of recombinant Apx proteins shows strong protection against Actinobacillus pleuroneumoniae serovar 1 and 2 in a mouse model.

Actinobacillus pleuropneumonia (APP) causes porcine pleuropneumoniae, resulting in severe economic losses in the swine industry. Since there are diverse serotypes of APP, it is necessary for vaccines to induce cross-protection. In this report, we developed a bivalent fusion vaccine, the L vaccine composed of ApxIA and ApxIIA fragments. According to the experimental results of the L vaccine, recombinant protein specific-IgG antibody level increased significantly as well as Apx toxin specific-IgG antibody, suggesting toxin-neutralizing effect. Also, the production of both IgG1 and IgG2a indicates this fusion vaccine induces Th1 and Th2 immune reactions. In addition, lymphocytes were proliferated and immune related-cytokines of TNF-α, IL-12, IFN-γ and IL-5 were detected in the serum after the vaccination. The L vaccine showed a perfect cross-protection against APP serovar 1 and 2 that each secrete different Apx exotoxins. These findings reveal that the fusion L vaccine induces specific humoral and cellular immunity, leading to a perfect cross-protection against A. pleuropneumoniae infections in a murine model.

Immune efficacy of single, bivalent fusion and combined DNA vaccines based on oprL and oprF genes of Pseudomonas aeruginosa

Immune efficacy of single, bivalent fusion and combined DNA vaccines based on oprL and oprF genes of Pseudomonas aeruginosa

Needle-free delivery of DNA: Targeting of hemagglutinin to MHC class II molecules protects rhesus macaques against H1N1 influenza

Conventional influenza vaccines are hampered by slow and limited production capabilities, whereas DNA vaccines can be rapidly produced for global coverage in the event of an emerging pandemic. However, a drawback of DNA vaccines is their generally low immunogenicity in non-human primates and humans. We have previously demonstrated that targeting of influenza hemagglutinin to human HLA class II molecules can increase antibody responses in larger animals such as ferrets and pigs. Here, we extend these observations by immunizing non-human primates (rhesus macaques) with a DNA vaccine encoding a bivalent fusion protein that targets influenza virus hemagglutinin (HA) to Mamu class II molecules. Such immunization induced neutralizing antibodies and antigen-specific T cells. The DNA was delivered by pain- and needle-free jet injections intradermally. No adverse effects were observed. Most importantly, the immunized rhesus macaques were protected against a challenge with influenza virus.

Neutralization of Clostridium difficile toxin B with VHH-Fc fusions targeting the delivery and CROPs domains.

An increasing number of antibody-based therapies are being considered for controlling bacterial infections, including Clostridium difficile by targeting toxins A and B. In an effort to develop novel C. difficile immunotherapeutics, we previously isolated several single-domain antibodies (VHHs) capable of toxin A neutralization through recognition of the extreme C-terminal combined repetitive oligopeptides (CROPs) domain, but failed at identifying neutralizing VHHs that bound a similar region on toxin B. Here we report the isolation of a panel of 29 VHHs targeting at least seven unique epitopes on a toxin B immunogen composed of a portion of the central delivery domain and the entire CROPs domain. Despite monovalent affinities as high as KD = 70 pM, none of the VHHs tested were capable of toxin B neutralization; however, modest toxin B inhibition was observed with VHH-VHH dimers and to a much greater extent with VHH-Fc fusions, reaching the neutralizing potency of the recently approved anti-toxin B monoclonal antibody bezlotoxumab in in vitro assays. Epitope binning revealed that several VHH-Fcs bound toxin B at sites distinct from the region recognized by bezlotoxumab, while other VHH-Fcs partially competed with bezlotoxumab for toxin binding. Therefore, the VHHs described here are effective at toxin B neutralization when formatted as bivalent VHH-Fc fusions by targeting toxin B at regions both similar and distinct from the bezlotoxumab binding site.

Immune efficacy of DNA vaccines based on oprL and oprF genes of Pseudomonas aeruginosa in chickens

Pseudomonas aeruginosa (P. aeruginosa) is a zoonotic pathogen that can infect a variety of animals, including poultry. However, as there is no commercial vaccine available it is imperative that new and effective vaccines are developed. In this study, 2 monovalent DNA vaccines (pOPRL and pOPRF), one divalent combination DNA vaccine (pOPRL+pOPRF) and one fusion DNA vaccine (pOPRLF) were constructed based on the oprL and oprF genes of P. aeruginosa. These vaccines were administered to chickens, an outer membrane protein vaccine (OMP vaccine) and inactivated vaccine used as positive controls. The serum antibody, interferon-γ (IFN-γ), interleukin-2 (IL-2) and interleukin-4 (IL-4) concentrations were determined and lymphocyte proliferation assays were performed. After challenging with virulent P. aeruginosa, protective efficacy was evaluated. Following vaccination, serum antibodies, stimulation index (SI) values, concentrations of IL-2 and IFN-γ in chickens vaccinated with the bivalent combination DNA vaccine and fusion DNA vaccine were found to be significantly higher than in those chickens vaccinated with the 2 monovalent DNA vaccines. Moreover, the immune indexes in the bivalent combination DNA vaccine group were higher than those in the fusion DNA vaccine group. However, the concentrations of IL-4 in the 4 DNA vaccine groups were of no significant difference. The protective efficacy rate provided by pOPRL, pOPRF, pOPRLF, pOPRL+pOPRF, inactivated vaccine and OMP vaccine were 53.3%, 40%, 66.7%, 80%, 93.3%, and 80%, respectively. The results indicate that DNA vaccines constructed with the oprL and oprF genes of P. aeruginosa, particularly the divalent combination DNA vaccine, represent better potential vaccines. This study has laid a foundation for the design and application of future DNA vaccines of P. aeruginosa.

A novel bivalent fusion vaccine induces broad immunoprotection against Staphylococcus aureus infection in different murine models

With more and more drug-resistant Staphylococcus aureus strains emerging in hospitals, there is an urgent need to develop an effective vaccine to combat S. aureus infection. In this study, we constructed a novel bivalent fusion vaccine, SpA-DKKAA-FnBPA37-507 (SF), based on the D domain of staphylococcal protein A (SpA) and the A domain of fibronectin-binding protein A (FnBPA). Immunisation with SF induced a more ideal protective effect compared with the single components alone in a sepsis model. It also showed broad immunoprotection against seven FnBPA isotypes. Vaccination with SF induced strong antibodies responses and Th1/Th17 polarized cellular responses. Further we demonstrated the protective effect of antibodies by the opsonophagocytic assay (OPA) and passive immunisation. Moreover, vaccination with SF showed protective efficacy in a murine pneumonia model and skin abscess model. These results suggest that SF can be regarded as a promising vaccine candidate for the prevention of S. aureus infections.

Endosomal trafficking regulates receptor-mediated transcytosis of antibodies across the blood brain barrier

Current methods for examining antibody trafficking are either non-quantitative such as immunocytochemistry or require antibody labeling with tracers. We have developed a multiplexed quantitative method for antibody ‘tracking’ in endosomal compartments of brain endothelial cells. Rat brain endothelial cells were co-incubated with blood-brain barrier (BBB)-crossing FC5, monovalent FC5Fc or bivalent FC5Fc fusion antibodies and control antibodies. Endosomes were separated using sucrose-density gradient ultracentrifugation and analyzed using multiplexed mass spectrometry to simultaneously quantify endosomal markers, receptor-mediated transcytosis (RMT) receptors and the co-incubated antibodies in each fraction. The quantitation showed that markers of early endosomes were enriched in high-density fractions (HDF), whereas markers of late endosomes and lysosomes were enriched in low-density fractions (LDF). RMT receptors, including transferrin receptor, showed a profile similar to that of early endosome markers. The in vitro BBB transcytosis rates of antibodies were directly proportional to their partition into early endosome fractions of brain endothelial cells. Addition of the Fc domain resulted in facilitated antibody ‘redistribution’ from LDF into HDF and additionally into multivesicular bodies (MVB). Sorting of various FC5 antibody formats away from late endosomes and lysosomes and into early endosomes and a subset of MVB results in increased antibody transcytosis at the abluminal side of the BBB.

Abstract 3631: Delivery of a CD20 transferrin receptor VNAR bispecific antibody to the brain for CNS lymphoma

Abstract Penetration of the blood brain barrier (BBB) remains a significant hurdle in the development of biologic therapeutics for CNS-related cancers. Antibody-based molecules typically do not cross the BBB in amounts required for therapeutic efficacy. Receptor-mediated transporters in the luminal membrane of brain capillary endothelium, which normally transport circulating endogenous peptides and proteins, offer a non-invasive approach for the delivery of therapeutic molecules to the brain.Using a combination of in vitro and in vivo phage display technology, we isolated a panel of cross species (rodent, primate and human) binders to the transferrin receptor 1 (TfR1) from synthetic libraries of shark variable antigen receptors (VNARs). Initially, the anti-TfR1 VNARs were fused to an immunoglobulin Fc backbone for testing in mice. At therapeutic (2 mg/kg) doses delivered by tail vein injection, high levels of the VNAR-Fc antibodies were found in the brain after 18 hours, which exceeded 5% of the plasma levels. Brain fractionation studies showed that the VNAR-Fc antibodies to TfR1 translocated to the brain parenchyma and were not retained in the capillaries. The lead anti-TfR1 VNAR was then used to target aberrant B cells in the brain for conditions such as cerebral lymphoma where existing CD20 therapies to not penetrate the CNS at therapeutic doses. A family of anti-CD20/TfR1 bispecific molecules were produced by linking the small VNAR to either the N- and C- terminus of heavy and light chains to create both monovalent and bivalent fusions . We used rituxan as the CD20 agent. The bispecifics were assessed for binding to the two cognate antigens and then tested for brain penetration after tail vein injection. At least 4 different formats gave excellent brain penetration 18 hours after dosing at 3.75mg/kg. Unmodified rituxan had a brain plasma ratio of 0.25 in line with published data while the best anti-TfR1-rituximab bispecific formats were between 2 to 5%. The bispecific formats also retain antibody-dependent cell-mediated cytotoxicity and will be further tested for brain penetration in non-human primates and in rodent models of CNS lymphoma. If these results successfully translate from rodents to larger animals, the peripheral delivery of a brain penetrant CD20/TfR1 bispecific antibody would provide a safer and more effective alternative to CD20 antibody delivery by the intrathecal route and could have broad utility in B cell driven brain cancer. Citation Format: Frank S. Walsh, Krzysztof Wicher, Jaroslaw Szary, Pawel Stocki, Michael Demydchuk, Lynn Rutkowski. Delivery of a CD20 transferrin receptor VNAR bispecific antibody to the brain for CNS lymphoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3631. doi:10.1158/1538-7445.AM2017-3631