Linker Conjugation Research Articles

Design and Synthesis of BODIPY-Cored Near IR-emitting Lipophilic and Water-Soluble Dendritic Platforms

Capitalizing on our experience in constructing branched and dendritic monomers with functionalizable aromatic moiety at the focal point and various terminal groups, we prepared lipophilic and water-soluble dendrimers with BODIPY fluorophore in the core, emitting in the green and near IR spectral regions. The dendrimers, incorporating aliphatic tails, exhibited excellent fluorescence properties in apolar organic solvents, while those with the triethylenglycol-based tails were highly fluorescent in polar organic solvents as well. The water solubility induced by the terminal hexaethyleneglycol tails enables fluorescence of the dyes also in aqueous media. The phenolic hydroxyls in the molecules enable attachment of linkers for prospective conjugation of additional units.

Synthesis of Group B Streptococcus type III polysaccharide fragments for evaluation of their interactions with monoclonal antibodies

Abstract Group B Streptococcus type III (GBSIII) is the most relevant serotype among GBS strains causing infections and the potential of its capsular polysaccharide conjugated to a protein carrier as vaccine is well documented. Polysaccharide from GBSIII (PSIII) can form helical structures in solution where negatively charged sialic acid residues would be disposed externally providing stabilization to the helix. A peculiar high affinity to specific monoclonal antibodies (mAbs) has been reported for PSIII, and fragments of diverse size bind to mAbs in a length dependent manner. These data have been rationalized in terms of conformational epitopes that would be formed by fragments with >4 saccharidic repeating units. Saturation Transfer Difference NMR experiments have demonstrated that the sialic acid residue is not involved in antibody recognition. However the molecular basis of the interaction between PSIII and mAbs has not been fully elucidated. An important prerequisite to achieve this would be the availability of the three possible sugar sequences representing the pentasaccharide PSIII repeating unit. Herein we established a [2+3] convergent approach leading to these three pentasaccharides (1–3) with the end terminal sugar bearing a linker for possible conjugation. The PSIII fragments were coupled to the genetically detoxified diphtheria toxin CRM197 to prove by ELISA that the three pentasaccharides are recognized by polyclonal anti-PSIII serum. The presence of the branching formed by a Glc residue β-(1→6) linked to GlcNAc was proven an important motif for antibody recognition.

C-Terminal Modification and Multimerization Increase the Efficacy of a Proline-Rich Antimicrobial Peptide.

Two series of branched tetramers of the proline-rich antimicrobial peptide (PrAMP), Chex1-Arg20, were prepared to improve antibacterial selectivity and potency against a panel of Gram-negative nosocomial pathogens including Escherichia coli, Klebsiella pneumoniae, Acinetobacter baumannii and Pseudomonas aeruginosa. First, tetramerization was achieved by dithiomaleimide (DTM) conjugation of two C-terminal-cysteine bearing dimers that also incorporated C-terminal peptide chemical modification. DTM-linked tetrameric peptides containing a C-terminal hydrazide moiety on each dimer exhibited highly potent activities in the minimum inhibitory concentration (MIC) range of 0.49-2.33 μm. A second series of tetrameric analogues with C-terminal hydrazide modification was prepared by using alternative conjugation linkers including trans-1,4-dibromo-2-butene, α,α'-dibromo-p-xylene, or 6-bismaleimidohexane to determine the effect of length on activity. Each displayed potent and broadened activity against Gram-negative nosocomial pathogens, particularly the butene-linked tetrameric hydrazide. Remarkably, the greatest MIC activity is against P. aeruginosa (0.77 μm/8 μg mL-1 ) where the monomer is inactive. None of these peptides showed any cytotoxicity to mammalian cells up to 25 times the MIC. A diffusion NMR study of the tetrameric hydrazides showed that the more active antibacterial analogues were those with a more compact structure having smaller hydrodynamic radii. The results show that C-terminal PrAMP hydrazidation together with its rational tetramerization is an effective means for increasing both diversity and potency of PrAMP action.

Through-bond energy transfer based dyad and triad shape fluorescence “OFF-ON-OFF” probes for Hg2+ ions and their application in live HeLa cells and Zebrafish

Two novel through-bond energy transfer (TBET) based naphthoquinone derivatives, R1 and R2, have been designed and synthesized with characteristics of a dyad (rhodamine-quinone) and a triad (rhodamine-quinone-rhodamine), respectively. Probes R1 and R2 selectively sense Hg2+ ions over other common interference metal ions in aqueous-methanol medium with a broad pH range (6.0–8.0). By the combination of dual functions, (i.e., fluorescence off-on (rhodamine) and TBET off-on (through a conjugation linker) processes), the proposed TBET-probes exhibit very large pseudo-Stokes shifts, with a wavelength difference ≈210nm, and high energy transfer efficiency for the detection of Hg2+ with a detection limit of 41nM. The fluorescence intensity of the triad (R2) is nearly 2 orders of magnitude greater than that of the dyad R1 due to the existence of two rhodamine fragments. The OFF-ON fluorescence effect of the TBET cassettes regains the ON-OFF mode by subsequent addition of the I− ion. The optical restoring behavior of the R1/R2-Hg2+ complex with I− could be potentially be applied as an INHIBIT molecular logic gate with the combination of YES and NOT functions. Finally, the Hg2+ ion sensing ability of TBET probes R1 and R2 was utilized in a HeLa cell imaging application, and the probe R1 was used to monitor the Hg2+ ion in live zebrafish.

Synthesis of Site-Specific Radiolabeled Antibodies for Radioimmunotherapy via Genetic Code Expansion.

Radioimmunotherapy (RIT) delivers radioisotopes to antigen-expressing cells via monoantibodies for the imaging of lesions or medical therapy. The chelates are typically conjugated to the antibody through cysteine or lysine residues, resulting in heterogeneous chelate-to-antibody ratios and various conjugation sites. To overcome this heterogeneity, we have developed an approach for site-specific radiolabeling of antibodies by combination of genetic code expansion and click chemistry. As a proof-of-concept study, model systems including anti-CD20 antibody rituximab, positron-emitting isotope 64Cu, and a newly synthesized bifunctional linker (4-dibenzocyclooctynol-1,4,7,10-tetraazacyclotetradecane-1,4,7,10-tetraacetic acid, DIBO-DOTA) were used. The approach consists of three steps: (1) site-specific incorporation of an azido group-bearing amino acid (NEAK) via the genetic code expansion technique at the defined sites of the antibody as a "chemical handle"; (2) site-specific and quantitative conjugation of bifunctional linkers with the antibodies under a mild condition; and (3) radiolabeling of the chelate-modified antibodies with the appropriate isotope. We used heavy-chain A122NEAK rituximab as proof-of-concept and obtained a homogeneous radioconjugate with precisely two chelates per antibody, incorporated only at the chosen sites. The conjugation did not alter the binding and pharmacokinetics of the rituximab, as indicated by in vitro assays and in vivo PET imaging. We believe our research is a good supplement to the genetic code expansion technique for the development of novel radioimmunoconjugates.

Abstract 1217: Preclinical evaluation of a next-generation, EGFR targeting ADC that promotes regression in KRAS or BRAF mutant tumors

Abstract Cancers with downstream activating KRAS or BRAF mutations in the EGFR pathway are resistant to EGFR targeting agents such as cetuximab and correspond to a significant unmet need. We hypothesized that an anti-EGFR ADC could be effective against KRAS or BRAF mutated tumors due to the cytotoxic mechanism of the ADC warhead. In an effort to eliminate the known dermal toxicity associated with anti-EGFR therapy, and to mitigate potential toxicities associated with treatment by an anti-EGFR ADC, a mAb was engineered with increased tumor microenvironment (TME) specificity for EGFR. The lead mAb demonstrated undetectable in vivo binding to human donor foreskins grafted onto nude mice, while binding to human A431 tumor xenografts with similar intensity to cetuximab (P < 0.005, detected using DyLight-755 conjugated versions of each mAb, measured with a Caliper IVIS system). The lead mAb was further optimized and conjugated to the potent cytotoxic drug MMAE using a novel bis-alkylating conjugation linker, which covalently re-bridged the inter-chain disulfide bonds, creating a stable and defined ADC. The resulting ADC, HTI-1511, incorporated a vc-PAB cleavable moiety and a short linear PEG (24 ethylene glycol units) in a side-chain configuration. Analytical HIC revealed that HTI-1511 possessed a nearly homogenous drug:antibody ratio (DAR) of 4 (>99.7%). Approximately 70% of this compound was rapidly internalized by human tumor cells grown in vitro over 4 hours, overlapping the internalization kinetics of the unconjugated mAb. HTI-1511 was evaluated for efficacy against two human EGFR overexpressing tumor models, MDA-MB-231M (triple-negative breast cancer, KRAS-G13D) and HT-29 (colorectal cancer, BRAF-V600E), and dosed at 5, 10, and 15 mg/kg, (qw, IV). A clear dose dependent anti-tumor response was observed with complete tumor regressions observed at the 15 mg/kg dose in both models, which were resistant to treatment by cetuximab. In addition, HTI-1511 was well-tolerated at 2 and 8 mg/kg in a cynomolgus monkey toxicity study (n = 3 per group), with limited dermal findings that were comparable with the vehicle control group. No adverse findings were observed at either dose. HTI-1511 showed a high degree of circulating stability in cynomolgus monkeys, and lacked in vivo degradation and instability that was observed in a control ADC conjugated using maleimide chemistry. HTI-1511 demonstrated significantly attenuated binding to FcγRIIa, FcγIIb, FcγIIIa 158V, and FcγIIIa 158F receptors, but not attenuated binding to FcγR1, in a FACS based assay format specific for each receptor, suggesting that HTI-1511 might have improved tolerability due to lack of binding by FcγRII-III receptors, possibly due steric hindrance from the PEG side chain. Thus, HTI-1511 holds promise as a potentially safe and effective treatment of EGFR overexpressing tumors with KRAS or BRAF mutations. Citation Format: Lei Huang, Bob Veneziale, Mark Frigerio, George Badescu, Xiaoming Li, Qiping Zhao, Jesse Bahn, Jennifer Souratha, Ryan Osgood, Chunmei Zhao, Kim Phan, Jessica Cowell, Sanna Rosengren, Jason Parise, Martin Pabst, Mathew Bird, William McDowell, Gina Wei, Curtis Thompson, Antony Godwin, Michael Shepard, Christopher Thanos. Preclinical evaluation of a next-generation, EGFR targeting ADC that promotes regression in KRAS or BRAF mutant tumors. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1217.

Evaluation of the effects of chemically different linkers on hepatic accumulations, cell tropism and gene silencing ability of cholesterol-conjugated antisense oligonucleotides

Cholesterol conjugation of oligonucleotides is an attractive way to deliver the oligonucleotides specifically to the liver. However cholesterol-conjugated antisense oligonucleotides (ASOs) mainly accumulate in non-parenchymal cells (NPCs) such as Kupffer cells. In this study, to increase the hepatic accumulation of cholesterol-conjugated ASOs, we prepared a variety of linkers for cholesterol conjugation to anti-Pcsk9 ASOs and examined their effects on pharmacological parameters. Hepatic accumulation of ASO was dramatically increased with cholesterol conjugation. The increase in hepatic accumulation depended largely on the linker chemistry of each cholesterol-conjugated ASO. In addition to hepatic accumulation, the cell tropism of each cholesterol-conjugated ASO tended to depend on their linker. Although a linker bearing a disulfide bond accumulated mainly in NPCs, hexamethylene succinimide linker accumulated mainly in hepatocytes. To estimate the benefits of releasing ASO from the conjugated cholesterol in hepatocyte, we designed another linker based on hexamethylene succinimide, which has a phosphodiester bond between the linker and the ASO. The cholesterol-conjugated ASO bearing such a phosphodiester bond showed a significantly improved Pcsk9 mRNA inhibitory effect compared to its counterpart, cholesterol-conjugated ASO with a phosphorothioate bond, while the hepatic accumulation of both cholesterol-conjugated ASOs was comparable, indicating the effectiveness of removing the conjugated cholesterol for ASO activity. In toxicity analysis, some of the linkers induced lethal toxicities when they were injected at high concentrations (>600μM). These toxicities were attributed to decreased platelet levels in the blood, suggesting an interaction between cholesterol-conjugated ASO and platelets. Our findings may provide a guideline for the design of molecule-conjugated ASOs.

Molecular design of light-harvesting photosensitizers: effect of varied linker conjugation on interfacial electron transfer.

Interfacial electron transfer dynamics of a series of photosensitizers bound to TiO2via linkers of varying conjugation strength are explored by spectroscopic and computational techniques. Injection and recombination depend on the extent of conjugation in the linker, where the LUMO delocalization determines the injection dynamics but both the HOMO and HOMO-1 are involved in recombination.

Abstract 636: Next-generation site-specific antibody-drug conjugates using the SMARTagTM technology platform

Abstract The two clinically-approved antibody-drug conjugates, AdcetrisTM and KadcylaTM, demonstrate that targeting cytotoxic payloads directly to tumors is an effective clinical approach. However, at the molecular level, these therapeutics are composed of heterogeneous mixtures resulting from the nonselective ligation of cytotoxic payload to cysteine and lysine residues, respectively. The nonselective ligation approaches used to generate these two ADCs render it difficult to optimize their biological, physical, and pharmacological properties. Redwood Bioscience creates site-selective, stable linkages that allow us to design ADCs with enhanced efficacy, safety, and pharmacokinetics. Additionally, we are able to control payload stoichiometry and placement. Redwood Bioscience creates homogeneous ADCs using the SMARTagTM technology, in which a genetically encoded 5 amino acid “aldehyde-tag” sequence contains a cysteine that is cotranslationally converted to formylglycine via an enzymatic transformation. This method affords control over the placement of the reactive formylglycine aldehyde side chain, which can then be ligated using carbonyl bioconjugation methods. Here, we will present our novel protein modification platform—including our new conjugation chemistries and linker libraries—and its application to ADC generation. We will address SMARTagTM ADC safety, pharmacokinetics, and efficacy in preclinical tumor models, and discuss how these parameters are impacted by linker selection and payload placement on the antibody. Citation Format: Romas Kudirka. Next-generation site-specific antibody-drug conjugates using the SMARTagTM technology platform. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 636. doi:10.1158/1538-7445.AM2015-636

Lead structures for applications in photodynamic therapy 7. Efficient synthesis of amphiphilic glycosylated lipid porphyrin derivatives: refining linker conjugation for potential PDT applications

Herein, we report synthetic strategies towards a library of amphiphilic tetraphenyl porphyrins anchored to synthetic saccharides and lipid modalities. The carbohydrates and lipid functionalities are covalently linked to the model photosensitizer via a copper (I) catalyzed alkyne azide cycloaddition reaction or an oxypropyl linkage achieved by nucleophilic substitution chemistry. Varying substitutions of carbohydrates and lipids allows for potential fine tuning of solubility and photophysical characteristics important for imaging and treatment applications in photomedicine, specifically in photodynamic therapy.

Stabilization of resveratrol in blood circulation by conjugation to mPEG and mPEG-PLA polymers: investigation of conjugate linker and polymer composition on stability, metabolism, antioxidant activity and pharmacokinetic profile.

Resveratrol is naturally occurring phytochemical with diverse biological activities such as chemoprevention, anti-inflammatory, anti-cancer, anti-oxidant. But undergoes rapid metabolism in the body (half life 0.13h). Hence Polymer conjugation utilizing different chemical linkers and polymer compositions was investigated for enhanced pharmacokinetic profile of resveratrol. Ester conjugates such as α-methoxy-ω-carboxylic acid poly(ethylene glycol) succinylamide resveratrol (MeO-PEGN-Succ-RSV) (2 and 20 kDa); MeO-PEG succinyl ester resveratrol (MeO-PEGO-Succ-RSV) (2 kDa); α-methoxy poly(ethylene glycol)-co-polylactide succinyl ester resveratrol (MeO-PEG-PLAO-Succ-RSV) (2 and 6.6kDa) were prepared by carbodiimide coupling reactions. Resveratrol-PEG ethers (2 and 5 kDa) were synthesized by alkali-mediated etherification. All polymer conjugates were fully characterized in vitro and the pharmacokinetic profile of selected conjugates was characterized in rats. Buffer and plasma stability of conjugates was dependent on polymer hydrophobicity, aggregation behavior and PEG corona, with MeO-PEG-PLAO-Succ-RSV (2 kDa) showing a 3h half-life in rat plasma in vitro. Polymer conjugates irrespective of linker chemistry protected resveratrol against metabolism in vitro. MeO-PEG-PLAO-Succ-RSV (2 kDa), Resveratrol-PEG ether (2 and 5 kDa) displayed improved pharmacokinetic profiles with significantly higher plasma area under curve (AUC), slower clearance and smaller volume of distribution, compared to resveratrol.

Sensitive ELISA Method for the Measurement of Catabolites of Antibody-Drug Conjugates (ADCs) in Target Cancer Cells.

A new, sensitive ELISA method has been developed which measures catabolites in cells and media upon processing of antibody-drug conjugates (ADCs) by target cancer cells. This ELISA method, exemplified for maytansinoid ADCs, uses competitive inhibition by a maytansinoid analyte of the binding of biotinylated antimaytansine antibody to an immobilized BSA-maytansinoid conjugate. Synthetic standards of several maytansinoid catabolites derived from ADCs with different linkers were tested and showed similar inhibition curves, with an EC50 of about 0.1 nM (0.03 pmol in an assay volume of 0.25 mL). This high sensitivity allowed quantification of catabolites from a methanolic cell extract and from the medium, generated from an ADC in 1 day using only about 1 million cells. The processing of anti-EpCAM and anti-CanAg ADCs with noncleavable linker (SMCC-DM1), disulfide linker (SPDB-DM4), and charged sulfonate-bearing disulfide linker (sulfo-SPDB-DM4), each containing an average of about four maytansinoid molecules per antibody, were compared in colon cancer cell lines (COLO 205 and HT-29). An 8-10-fold higher total level of catabolite was observed for anti-CanAg ADCs than for anti-EpCAM ADCs upon processing by COLO 205 cells, consistent with a higher cell-surface expression of CanAg. In a multidrug resistant HCT-15 colon cancer cell line, the anti-EpCAM-SPDB-DM4 linker conjugate was not cytotoxic and showed a significantly lower level of catabolite within cells compared to that in medium, presumably due to Pgp-mediated efflux of the nonpolar DM4 catabolite. In contrast, sulfo-SPDB-DM4 and SMCC-DM1 linker conjugates were cytotoxic, which correlated with higher amounts of catabolites found within the HCT-15 cells relative to amounts in medium. In a nonmultidrug resistant HT-29 cell line, the anti-EpCAM-SPDB-DM4 linker conjugate was cytotoxic, with most of the catabolite found in cells and little in the medium. In conclusion, this highly sensitive ELISA method for measurement of ADC catabolite is convenient for screening multiple ADC parameters such as linkers and antibodies in a number of cell lines, does not require concentration of sample or extraction of media, and is complementary to other reported methods such as radiolabeling of ADCs or mass spectrometry.

Silica Core‐based Surface‐enhanced Raman Scattering (SERS) Tag: Advances in Multifunctional SERS Nanoprobes for Bioimaging and Targeting of Biomarkers#

Surface‐enhanced Raman scattering (SERS) has attracted considerable interest as a sensitive vibration‐specific probe for bioanalytical and imaging applications. Among the various bioprobes available, Ag‐embedded SERS tags have been rigorously developed for an extensive range of biodetection applications. In this review, we look at the additional functionality that SERS tags can offer via its magnetic properties, fluorescence, and an extension of the optical region into the near‐infrared (NIR) spectrum. Such functionality can be achieved by using Ag nanoparticles (NPs) or Au/Ag hollow‐shells (HS) as a SERS signaling unit, with SiO2 nanospheres providing a back‐bone unit. This back‐bone can include a magnetic core (M‐SERS dots), but also provides an outer shell that protects the optical unit and allows for easy conjugation of linkers that can include fluorescent organic dyes for an additional optical unit (F‐SERS dots). In use, M‐SERS dots allow for the separation of target cancer or cancer stem cells with an external magnetic field, while F‐SERS dots can rapidly locate specific proteins within large areas of tissue and simultaneously analyze multiple targets based on their Raman signals. Moreover, NIR SERS dots can be detected with a high sensitivity within deep tissues, thus allowing them to be applied to in vivo multiplex detection. As none of these advanced functional SERS dots exhibit any sign of cytotoxicity for cell lines, they demonstrate a clear potential for more efficient, high‐throughput screening of biological molecules using Raman technology.

Dual-purpose linker for alpha helix stabilization and imaging agent conjugation to glucagon-like peptide-1 receptor ligands.

Peptides display many characteristics of efficient imaging agents such as rapid targeting, fast background clearance, and low non-specific cellular uptake. However, poor stability, low affinity, and loss of binding after labeling often preclude their use in vivo. Using glucagon-like peptide-1 receptor (GLP-1R) ligands exendin and GLP-1 as a model system, we designed a novel α-helix-stabilizing linker to simultaneously address these limitations. The stabilized and labeled peptides showed an increase in helicity, improved protease resistance, negligible loss or an improvement in binding affinity, and excellent in vivo targeting. The ease of incorporating azidohomoalanine in peptides and efficient reaction with the dialkyne linker enable this technique to potentially be used as a general method for labeling α helices. This strategy should be useful for imaging beta cells in diabetes research and in developing and testing other peptide targeting agents.

Molecularly linked 3D plasmonic nanoparticle core/satellite assemblies: SERS nanotags with single-particle Raman sensitivity.

A fast, generic, and suspension-based route to highly SERS-active assemblies of noble metal nanoparticles (Au, Ag) with small core-satellite gaps and single-particle Raman sensitivity is presented. Rationally designed, heterobifunctional Raman reporters serve as molecular linkers for electrostatic conjugation of the small satellites to the large core.

Probing the regeneration process of triphenylamine-based organic dyes in dye-sensitized solar cells

The regeneration processes of triphenylamine (TPA)-based dyes with cobalt redox mediator in dye-sensitized solar cells (DSSCs) have been investigated using density functional theory combined with the Marcus theory of electron transfer. Our results show that with the extension or rigidification of the oligothiophene conjugation linker the absorption spectra of TPA dyes exhibit observable red-shift in the maximum absorbance that favors light-harvesting, while the electron transfer rates for dye regeneration decrease in some degrees due to the increased activation free energies and the reduced electronic coupling energies which hampers the dye regeneration. Importantly, the undesirable influences on dye regeneration by extending the linker moiety are more significant than that by the way of rigidification. Thus, the rigidification is a better choice than the extension of the conjugated moiety for the design of D-π-A type dyes based on the properties of light-harvesting and the kinetics of dye regeneration.

Abstract 2662: Site specific ADC generation using SMARTag technology with programmable payload placement

Abstract Antibody-drug conjugates (ADCs) are an exciting, new therapeutic option for cancer treatment. ADCs offer the promise of targeted drug delivery with increased efficacy and safety. Currently approved ADCs are heterogeneous mixtures resulting from nonselective ligation of drug to either cysteine or lysine residues. This heterogeneity complicates the pharmacokinetics and pharmacodynamics of these drugs. Redwood Bioscience, Inc. has developed the SMARTagTM technology platform that enables precise, programmable, site-selective chemical protein modification and stable bioconjugate generation. Leveraging the target sequence of Formylglycine Generating Enzyme (FGE), proteins are chemoenzymatically modified to generate a precisely placed aldehyde functionality that can be chemically elaborated. Subsequently, novel ligation chemistry is employed that exploits this “aldehyde tag” site. This new reaction, the hydrazino-iso-Pictet-Spengler (HIPS) ligation, possesses two distinct advantages over current carbonyl ligations. First, the HIPS ligation proceeds quickly at near neutral pH in the absence of catalysts, allowing for one-step labeling of aldehyde-functionalized proteins under mild conditions. Second, HIPS ligation products are very stable in human plasma relative to an oxime-linked conjugates. Thus, the HIPS ligation exhibits a combination of stability and speed at near neutral pH that is unmatched by current carbonyl bioconjugation chemistries. We will present our novel protein modification platform and its application to generating ADCs, including our new conjugation chemistries and linker libraries. These technologies have been applied to the generation of a panel of site-specifically modified bioconjugates. Data from preclinical tumor models and PK studies highlighting the efficacy and safety of these ADCs and how these results are impacted by payload placement to the antibody will be presented. Citation Format: David Rabuka. Site specific ADC generation using SMARTag technology with programmable payload placement. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2662. doi:10.1158/1538-7445.AM2014-2662

Improved immunogenicity of tetanus toxoid by Brucella abortus S19 LPS adjuvant.

Adjuvants are used to increase the immunogenicity of new generation vaccines, especially those based on recombinant proteins. Despite immunostimulatory properties, the use of bacterial lipopolysaccharide (LPS) as an adjuvant has been hampered due to its toxicity and pyrogenicity. Brucella abortus LPS is less toxic and has no pyrogenic properties compared to LPS from other gram negative bacteria. To evaluate the adjuvant effect of B. abortus (vaccine strain, S19) LPS for tetanus toxoid antigen (TT) and to investigate the protective effect of different tetanus vaccine preparations. LPS was extracted and purified from B. abortus S19 and KDO, glycan, phosphate content, and protein contamination were measured. Adipic acid dihydrazide (ADH) was used as a linker for conjugation of TT to LPS. Different amounts of B. abortus LPS, TT, TT conjugated with LPS, and TT mixed with LPS or complete Freund's adjuvant (CFA) were injected into mice and antibody production against TT was measured. The protective effect of induced antibodies was determined by LD50. Immunization of mice with TT+LPS produced the highest anti-TT antibody titer in comparison to the group immunized with TT without any adjuvant or the groups immunized with TT-LPS or TT+CFA. Tetanus toxid-S19 LPS also produced a 100% protective effect against TT in immunized mice. These data indicate that B. abortus LPS enhances the immune responses to TT and suggest the possible use of B. abortus LPS as an adjuvant in vaccine preparations.

Functionalization of gold nanorod‐based immunosensors for label‐free bio‐detection with high sensitivity and specificity (780.7)

Gold nanoparticles have shown great potential applications in biomedical diagnosis and therapeutic treatments due to their shape and size‐dependent optical properties. However, densely packed CTAB double layer (surfactant used to prevent nanoparticle aggregation) on the nanorod surfaces present challenges for effective biofunctionalization to setup a nanoparticle‐based platform with high detection sensitivity. In this study, we systematically studied the functionalization of gold nanorods (GNRs) with various bioconjugate linkers in pursuit of developing nanorod‐based immunosensors with maximal sensitivity and specificity. First, GNR chips were fabricated by chemically binding nanorods onto silanized glass substrate; then the GNRs were functionalized with carboxylic acid or amine group via thiol linkers for antibody conjugation. The effects of thiol linkers with different lengths such as 11‐mercaptoundecanic acid (MUDA), 4‐aminothiophenol (4‐ATP), 2‐mercaptopropionic acid (MPA), cysteamine hydrochloride (CAE) and 6‐mercaptohexanoic acid (MHA) were investigated. GNR nano‐biochip exhibited high detection sensitivity (0.3017/ IgGnM), low detection limit (1.3 nM) and high specificity, with the features of label free and simple detection mechanism. The optimized functionalization of GNR chip was applied to develop a nano‐biosensor to detect human serum IgG as a model system. Since the localized surface plasmon resonance is highly sensitive to the biological binding on the nanorod surface, this mechanism provides a label‐free biodetection, eliminating signaling labels such as fluorophore and radioactive agents. The miniaturized nano‐biochip is attractive for rapid medical diagnostics in clinics, due to its user‐friendly and cost‐effective features.

Applications of Copper-Catalyzed Click Chemistry in Activity-Based Protein Profiling

Activity-based protein profiling (ABPP) is a chemical proteomic technique that enables the interrogation of protein activity directly within complex proteomes. Given the dominant role of posttranslational modifications in regulating protein function in vivo, ABPP provides a direct readout of activity that is not attained through traditional proteomic methods. ABPP relies on the design of covalent binding probes that either target a specific enzyme or a class of enzymes with related function. These covalent warheads are coupled to either fluorophores or biotin groups for visualization and enrichment of these active proteins. The advent of bioorthogonal chemistries, in particular, the copper (I)-catalyzed azide-alkyne cycloaddition (CuAAC), has benefitted the field of ABPP by achieving the following: (1) replacing bulky reporter groups with smaller alkyne or azide groups to promote cell permeability; (2) adding modularity to the system such that a single probe can be diversified with a variety of reporter groups without the need to develop new synthetic routes; and (3) enabling the conjugation of complex linkers to facilitate quantitative proteomic analyses. Here, we summarize recent examples of CuAAC in ABPP that serve to illustrate the contribution of bioorthogonal chemistry to advancing discoveries in this field.