Multivalent Anchoring and Oriented Display of Single-Domain Antibodies on Cellulose

Antibody engineering has allowed for the rapid generation of binding agents against virtually any antigen of interest, predominantly for therapeutic applications. Considerably less attention has been given to the development of diagnostic reagents and biosensors using engineered antibodies. Recently, we produced a novel pentavalent bispecific antibody (i.e., decabody) by pentamerizing two single-domain antibodies (sdAbs) through the verotoxin B subunit (VTB) and found both fusion partners to be functional. Using a similar approach, we have engineered a bispecific pentameric fusion protein consisting of five sdAbs and five cellulose-binding modules (CBMs) linked via VTB. To find an optimal design format, we constructed six bispecific pentamers consisting of three different CBMs, fused to the Staphylococcus aureus-specific human sdAb HVHP428, in both orientations. One bispecific pentamer, containing an N-terminal CBM9 and C-terminal HVHP428, was soluble, non-aggregating, and did not degrade upon storage at 4 °C for over six months. This molecule was dually functional as it bound to cellulose-based filters as well as S. aureus cells. When impregnated in cellulose filters, the bispecific pentamer recognized S. aureus cells in a flow-through detection assay. The ability of pentamerized CBMs to bind cellulose may form the basis of an immobilization platform for multivalent display of high-avidity binding reagents on cellulosic filters for sensing of pathogens, biomarkers and environmental pollutants

Pathogen Sensors

The development of sensors for detecting foodborne pathogens has been motivated by the need to produce safe foods and to provide better healthcare. However, in the more recent times, these needs have been expanded to encompass issues relating to biosecurity, detection of plant and soil pathogens, microbial communities, and the environment. The range of technologies that currently flood the sensor market encompass PCR and microarray-based methods, an assortment of optical sensors (including bioluminescence and fluorescence), in addition to biosensor-based approaches that include piezoelectric, potentiometric, amperometric, and conductometric sensors to name a few. More recently, nanosensors have come into limelight, as a more sensitive and portable alternative, with some commercial success. However, key issues affecting the sensor community is the lack of standardization of the testing protocols and portability, among other desirable elements, which include timeliness, cost-effectiveness, user-friendliness, sensitivity and specificity. [...

Multi-dimensional Glycan Microarrays with Glyco-macroligands

Glycan microarray has become a powerful high-throughput tool for examining binding interactions of carbohydrates with the carbohydrate binding biomolecules like proteins, enzymes, antibodies etc. It has shown great potential for biomedical research and applications, such as antibody detection and profiling, vaccine development, biomarker discovery, and drug screening. Most glycan microarrays were made with monovalent glycans immobilized directly onto the array surface via either covalent or non-covalent bond, which afford a multivalent glycans in two dimensional (2D) displaying. A variety of glyco-macroligands have been developed to mimic multivalent carbohydrate-protein interactions for studying carbohydrate-protein interactions and biomedical research and applications. Recently, a number of glyco-macroligands have been explored for glycan microarray fabrication, in particular to mimick the three dimensional (3D) multivalent display of cell surface carbohydrates. This review highlights these recent developments of glyco-macroligand-based microarrays, predominantly, novel glycan microarrays with glyco-macroligands like glycodendrimers, glycopolymers, glycoliposomes, neoglycoproteins, and glyconanoparticles with the effort in controlling the density and orientation of glycans on the array surface, which facilitate both their binding specificity and affinity and thus the high performance of glycan microarrays

Recombinant CBM-fusion technology : applications overview

Carbohydrate-binding modules (CBMs) are small components of several enzymes, which present an independent fold and function, and specific carbohydrate-binding activity. Their major function is to bind the enzyme to the substrate enhancing its catalytic activity, especially in the case of insoluble substrates. The immense diversity of CBMs, together with their unique properties, has long raised their attention for many biotechnological applications. Recombinant DNA technology has been used for cloning and characterizing new CBMs. In addition, it has been employed to improve the purity and availability of many CBMs, but mainly, to construct bi-functional CBM-fused proteins for specific applications. This review presents a comprehensive summary of the uses of CBMs recombinantly produced from heterologous organisms, or by the original host, along with the latest advances. Emphasis is given particularly to the applications of recombinant CBM-fusions in: (a) modification of fibers, (b) production, purification and immobilization of recombinant proteins, (c) functionalization of biomaterials and (d) development of microarrays and probes.Fundação para a Ciência e a Tecnologia (FCT), Portugal (grants SFRH/BDP/63831/ 2009 and SFRH/BPD/73850/2010, respectively). The authors thank the FCT GlycoCBMs Project REF. PTDC/AGR-FOR/3090/2012 — FCOMP-01- 0124-FEDER-027948, the FCT Strategic Project PEst-OE/EQB/LA0023/ 2013, and the Project “BioInd — Biotechnology and Bioengineering for improved Industrial and Agro-Food processes”, REF. NORTE-07-0124- FEDER-000028 Co-funded by the Programa Operacional Regional do Norte (ON.2 — O Novo Norte), QREN, FEDER

Quantitative Approach to Supramolecular Assembly Engineering for Isolating and Activating Antigen-Specific T Cells

T cell immunotherapy is a novel therapeutic strategy that aims to leverage the antigen-specific nature of a T cell immune response to treat a variety of immunological conditions. Over the past twenty years, T cell immunotherapy has been applied to treat several types of cancer, autoimmune conditions, and chronic infections, culminating in the FDA approval of two highly effective chimeric antigen receptor (CAR) T cell therapies targeting hematological cancers in 2017. While the initial success of T cell immunotherapy has been encouraging, identifying appropriate antigenic targets and optimizing T cell activation to promote effective responses in vivo remain significant challenges. In this dissertation, we discuss the development and application of new molecular tools for identifying, isolating, and activating antigen-specific T cells, which are directly relevant to the current challenges facing T cell immunotherapy. One of the greatest obstacles to developing a successful T cell immunotherapy is the selection of appropriate antigenic targets. T cells naturally recognize antigen-derived peptides presented on polymorphic major histocompatibility complex (MHC) proteins, and different MHC alleles exhibit different peptide binding specificities. Therefore, peptides that promiscuously bind multiple MHC alleles representing a diverse population have significant potential in the development of broadly protective peptide-based therapeutics and vaccines. A number of high-throughput in silico strategies have been developed to predict peptide-MHC binding; however, the accuracy of these approaches is generally inadequate for the reliable prediction of class II peptide-MHC (MHCII) interactions. In contrast, most experimental systems designed to measure peptide-MHCII binding emphasize quantitative detail over throughput. In this dissertation, we develop and validate a high-throughput screening strategy to evaluate peptide binding to four common MHCII alleles. Using this strategy, which we have termed microsphere-assisted peptide screening (MAPs), we screened overlapping peptide libraries of antigenic viral proteins and identified 12 promiscuously MHCII-binding peptides. Subsequent structural analysis indicated that nearly half of these peptides overlapped with antibody neutralization sites on the respective viral protein. Together, these results indicate that the MAPS strategy can be used to rapidly identify promiscuously binding and immunodominant peptides that have therapeutic relevance. Another significant challenge limiting the successful application of T cell immunotherapy is expanding a clinically relevant number of therapeutically effective T cells. The effectiveness of a T cell response is largely determined by the spatial and stoichiometric organization of signals delivered to the T cell during T cell activation. One strategy for promoting an effective T cell response is to tune the presentation of stimulatory and costimulatory signals through artificial antigen presentation. However, existing technologies have a limited ability to control the spatial and stoichiometric organization of T cell ligands on 3D surfaces. In this dissertation, we introduce a novel strategy for presenting highly organized clusters of stimulatory and costimulatory ligands to T cells using protein-scaffold directed assembly. Using this approach, we systematically investigated how the global surface density, local valency, and stoichiometric ratio of T cell ligands on a 3D cellular (yeast) surface can be manipulated to tune T cell activation. After validating this approach, we further develop more complex scaffold-assembly schemes to enhance the controllability of isolating and activating antigen-specific T cells. We believe that MAPS and artificial antigen presentation using protein-scaffold directed assembly provide a robust toolset for identifying, isolating, and activating antigen-specific T cells for T cell immunotherapy.PHDChemical EngineeringUniversity of Michigan, Horace H. Rackham School of Graduate Studieshttps://deepblue.lib.umich.edu/bitstream/2027.42/147510/1/masonrsm_1.pd

Multivalent sialic acid binding proteins as novel therapeutics for influenza and parainfluenza infection

In nature, proteins with weak binding affinity often use a multivalency approach to enhance protein affinity via an avidity effect. Interested in this multivalency approach, we have isolated a carbohydrate binding module (CBM) that recognises sialic acid (known as a CBM40 domain) from both Vibrio cholerae (Vc) and Streptococcus pneumoniae (Sp) NanA sialidases, and generated multivalent polypeptides from them using molecular biology. Multivalent CBM40 constructs were designed either using a tandem repeat approach to produce trimeric or tetrameric forms that we call Vc3CBM and Vc4CBM, respectively, or through the addition of a trimerization domain derived from Pseudomonas aeruginosa pseudaminidase to produce three trimeric forms of proteins known as Vc-CBMTD (WT), Vc-CBMTD (Mutant) and Sp-CBMTD). Due to the position and flexibility of the linker between the trimerization domain and the CBM40 domain, site directed mutagenesis was employed to introduce a disulphide bond between the monomers at positions S164C and T83C of the CBM40 domain in order to promote a stable orientation of the binding site for easier access of sialic acids. Data from isothermal titration calorimetry (ITC) reveals that interaction of multivalent CBM40 proteins with α(2,3)-sialyllactose was mainly enthalpy driven with entropy contributing unfavorably to the interaction suggesting that these proteins establish a strong binding affinity to their ligand minimizing dissociation to produce stable multivalent molecules. However, using surface plasmon resonance (SPR), a mixed balance of entropy and enthalpy contributions was found with all constructs as determined by Van’t Hoff plots. This proved that binding does not occur through a simple protein-ligand interaction but through disruption of hydrophobic and/or ionic hydration that provide the driving force to the process. Interestingly, the valency of multiple-linked polypeptides also plays an important part in the protein stabilization. However, little is known about their detailed structure when in multivalent form, as attempts to crystallize the whole protein molecule of Vc-CBMTD (WT) failed due to linker and domain flexibility. Only the trimerization domain (TD) part from Pseudomonas aeruginosa pseudaminidase was successfully crystallized and structure was determined to 3.0 Å without its CBM40 domain attached. In this thesis, we have also reported on the potential anti-influenza and anti- parainfluenza properties of these proteins, which were found to block attachment and inhibit infection of several influenza A and parainfluenza virus strains in vitro. As widely mentioned in literature, terminal sialic acids on the cell surface of mammalian host tissue provide a target for various pathogenic organisms to bind. Levels of viral inhibition were greatest against A/Udorn/72 H3N2 virus for Vc4CBM and Vc3CBM constructs with the lowest EC50 of 0.59 µM and 0.94 µM respectively, however most of the multivalent proteins tested were also effective against A/WSN/33 H1N1 and A/PR8/34 H1N1 subtypes. For parainfluenza virus, all constructs containing V. cholerae sialidase CBM40 domain showed great effect in inhibiting virus infection during cell protection assay. The best EC50 values were 0.2 µM from Vc-CBMTD (WT) followed by 1.17 µM from Vc4CBM and 1.78 µM from Vc-CBMTD (Mutant) which was against hPIV2, hPIV3 and hPIV5 infections respectively. Only a construct from S. pneumoniae sialidase known as Sp-CBMTD showed negligible effect on cell protection. All constructs were further tested for cytotoxicity in mammalian cell culture as well as undergoing an inhibition study on viral replication proteins. For the in vivo study, we also demonstrated the effectiveness of Vc4CBM to protect cotton rats and mice from hPIV3 and Streptococcus pneumoniae infections, when given intranasally in advance or on the day of infection. Therefore, these novel multivalent proteins could be promising candidates as broad-spectrum inhibitors or as a prophylactic treatment for both influenza and parainfluenza associated diseases

Dextran-coated nanoparticles as immunosensing platforms: Consideration of polyaldehyde density, nanoparticle size and functionality

Magnetic nanoparticles (MNPs) can be used as antibody carriers in a wide range of immunosensing applications. The conjugation chemistry for preparing antibody-MNP bionanohybrids should assure the nanoparticle’s colloidal dispersity, directional conformation and high biofunctionality retention of attached antibodies. In this work, peroxidase (HRP) was selected as model target analyte, and stable antibody-MNP conjugates were prepared using polyaldehyde-dextrans as multivalent linkers, also to prevent nanoparticles agglomeration and steric shielding of non-specific proteins. Under the manipulation of the oxidation variables, MNP-conjugated antibody showed the highest Fab accessibility, of 1.32 μmol analyte per μmol antibody, corresponding to 139 μmol aldehyde per gram of nanocarrier (5 mM NaIO4, 4 h). Demonstrating anti-interference advantage up to 10% serum, colorimetric immunoassay gave a detection limit (LOD) of 300 ng mL− 1 , while electrochemical transduction led to a considerable (680 times) improvement, with a LOD of 0.44 ng mL− 1 . In addition, polyaldehydedextran showed priority over polycarboxylated-dextran as the multivalent antibody crosslinker for MNPs in terms of sensitivity and LOD value, while immunosensors constructed with carboxylated magnetic microbeads (HOOC-MBs) outperformed MNPs-based immunoplatforms. This work sheds light on the importance of surface chemistry (type and density of functional groups) and the dimension (nanosize vs micrometer) of magnetic carriers to conjugate antibodies with better directional orientation and improve the analytical performance of the resulting immunosensors