Optimization of Photoactive Protein Z for Fast and Efficient Site-Specific Conjugation of Native IgG

Antibody conjugates have been used in a variety of applications from immunoassays to drug conjugates. However, it is becoming increasingly clear that in order to maximize an antibody’s antigen binding ability and to produce homogeneous antibody-conjugates, the conjugated molecule should be attached onto IgG site-specifically. We previously developed a facile method for the site-specific modification of full length, native IgGs by engineering a recombinant Protein Z that forms a covalent link to the Fc domain of IgG upon exposure to long wavelength UV light. To further improve the efficiency of Protein Z production and IgG conjugation, we constructed a panel of 13 different Protein Z variants with the UV-active amino acid benzoylphenylalanine (BPA) in different locations. By using this panel of Protein Z to cross-link a range of IgGs from different hosts, including human, mouse, and rat, we discovered two previously unknown Protein Z variants, L17BPA and K35BPA, that are capable of cross-linking many commonly used IgG isotypes with efficiencies ranging from 60% to 95% after only 1 h of UV exposure. When compared to existing site-specific methods, which often require cloning or enzymatic reactions, the Protein Z-based method described here, utilizing the L17BPA, K35BPA, and the previously described Q32BPA variants, represents a vastly more accessible and efficient approach that is compatible with nearly all native IgGs, thus making site-specific conjugation more accessible to the general research community

Gd-Labeled Glycol Chitosan as a pH-Responsive Magnetic Resonance Imaging Agent for Detecting Acidic Tumor Microenvironments

Neoplastic lesions can create a hostile tumor microenvironment with low extracellular pH. It is commonly believed that these conditions can contribute to tumor progression as well as resistance to therapy. We report the development and characterization of a pH-responsive magnetic resonance imaging contrast agent for imaging the acidic tumor microenvironment. The preparation included the conjugation of 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid 1-(2,5-dioxo-1-pyrrolidinyl) ester (DOTA-NHS) to the surface of a water-soluble glycol chitosan (GC) polymer, which contains pH-titrable primary amines, followed by gadolinium complexation (GC-NH₂-GdDOTA). GC-NH₂-GdDOTA had a chelate-to-polymer ratio of approximately1:24 and a molar relaxivity of 9.1 mM^–1 s^–1. GC-NH₂-GdDOTA demonstrated pH-dependent cellular association in vitro compared to the control. It also generated a 2.4-fold enhancement in signal in tumor-bearing mice 2 h postinjection. These findings suggest that glycol chitosan coupled with contrast agents can provide important diagnostic information about the tumor microenvironment

LASIC: Light Activated Site-Specific Conjugation of Native IgGs

Numerous biological applications, from diagnostic assays to immunotherapies, rely on the use of antibody-conjugates. The efficacy of these conjugates can be significantly influenced by the site at which Immunoglobulin G (IgG) is modified. Current methods that provide control over the conjugation site, however, suffer from a number of shortfalls and often require large investments of time and cost. We have developed a novel adapter protein that, when activated by long wavelength UV light, can covalently and site-specifically label the Fc region of nearly any native, full-length IgG, including all human IgG subclasses. Labeling occurs with unprecedented efficiency and speed (>90% after 30 min), with no effect on IgG affinity. The adapter domain can be bacterially expressed and customized to contain a variety of moieties (e.g., biotin, azide, fluorophores), making reliable and efficient conjugation of antibodies widely accessible to researchers at large

Assessing the Sensitivity of Commercially Available Fluorophores to the Intracellular Environment

The use of fluorescence has become commonplace in the biological sciences, with many studies utilizing probes based on commercially available fluorophores to provide insight into cell function and behavior. As these imaging applications become more advanced, it becomes increasingly important to acquire accurate quantitative measurements of the fluorescence signal. Absolute quantification of fluorescence, however, requires the fluorophores themselves to be insensitive to environmental factors such as nonspecific protein interactions and pH. Here, we present a method for characterizing the sensitivity of fluorophores to the cytosolic environment by comparing their fluorescent intensity to an environment-insensitive reference signal before and after intracellular delivery. Results indicated that although the fluorescent intensity of a few fluorophores, e.g., fluorescein, were highly susceptible to the intracellular environment, other fluorophores, e.g., Dylight 649, Alexa647, and Alexa750, were insensitive to the intracellular environment. It was also observed that the sensitivity of the fluorophore could be dependent on the biomolecule to which it was attached. In addition to assessing the environmental sensitivity of fluorophores, a method for quantifying the amount of fluorophores within living cells is also introduced. Overall, the present study provides a means to select fluorophores for studies that require an absolute quantification of fluorescence in the intracellular environment

Sortase-Tag Expressed Protein Ligation: Combining Protein Purification and Site-Specific Bioconjugation into a Single Step

Efficient labeling of protein-based targeting ligands with various cargos (drugs, imaging agents, nanoparticles, etc.) is essential to the fields of molecular imaging and targeted therapeutics. Many common bioconjugation techniques, however, are inefficient, nonstoichiometric, not site-specific, and/or incompatible with certain classes of protein scaffolds. Additionally, these techniques can result in a mixture of conjugated and unconjugated products, which are often difficult to separate. In this study, a bacterial sortase enzyme was utilized to condense targeting ligand purification and site-specific conjugation at the C-terminus into a single step. A model was produced to determine optimal reaction conditions for high conjugate purity and efficient utilization of cargo. As proof-of-principle, the sortase-tag expressed protein ligation (STEPL) technique was used to generate tumor-specific affinity ligands with fluorescent labels and/or azide modifications at high purity (>95%) such that it was not necessary to remove unconjugated impurities. Click chemistry was then used for the highly efficient and site-specific attachment of the azide-modified targeting ligands onto nanoparticles

Exploring the Sensitivity of Antibody–Drug Conjugate Efficacy to the Selection of Payload, Antibody, and Cell line

Antibody–drug conjugates (ADCs) make up a growing class of targeted therapeutics with important applications in cancer treatment. ADCs are highly modular in nature and thus can be engineered to target any cancer type, but their efficacy is strongly influenced by the specific choice of payload, antibody, and target cell. Considering the number of possible antibody–payload combinations, ADC development would benefit from an efficient method to narrow the number of ADC compositions to those with the highest and most universal potency prior to assessing pharmacokinetics and pharmacodynamics in animal models. To facilitate the identification of optimal ADC compositions, we describe the use of photoreactive antibody-binding domain-drug conjugates (known commercially as oYo-Link) to enable the site-specific labeling of off-the-shelf antibodies. This approach allows for the rapid generation of ADCs with a drug-to-antibody ratio of ∼2 with no subsequent purification required. As a demonstration of this approach, ADCs were generated with different combinations of tubulin-inhibitor drugs (DM1, DM4, VcMMAE, and VcMMAF) and anti-EGFR antibodies (cetuximab, panitumumab, anti-EGFR clone 425, and anti-EGFR clone 528) and were delivered to three EGFR-expressing cell lines (A431, A549, and MDA-MB-231). Real-time cytolysis assays indicated that the most effective antibody varied based on the choice of cell line: cetuximab was most potent against A431 cells, while 425 and 528 led to the greatest cytotoxicity against A549 and MDA-MB-231 cells. These results did not correlate with differences in measured anti-EGFR binding affinity as cetuximab had the highest affinity across all three cell lines, while 425 and 528 had the lowest affinities for all three cell lines. Panitumumab, which had the second-highest anti-EGFR affinity, exhibited the least effective cytolysis across A431, A549, and MDA-MB-231 cells. By demonstrating that ADC potency toward a given target is dependent on both the antibody and drug chosen, these findings can guide the selection of ADCs for further in vivo analysis

Analysis of RNA motion in living cells.

<p>(A) Montage of RNA transcripts classified as confined, diffusive or directed based on mean squared displacement analysis (MSD) analysis. The trajectories (far left) for the transcripts that are indicated by the arrows are color-coded for time/frames. Scale bar: 2 µm. The motion of RNA transcripts was assessed using (B) MSD analysis and (C) motion scaling spectrum (MSS) analysis. The MSD and MSS plots represent 10 tracks per category (mean ± SEM). (D) Analysis of the speed and distance covered by directed particles. After smoothing the position over 5 frames, the instantaneous frame-to-frame velocity was calculated for all directed tracks (N = 10). A histogram of the speed distribution for all tracks is shown with a gray bar indicating speeds below our resolution limit. The inset shows the maximum speed achieved in each directed track (mean = red line, 1.4 µm/s), and the net displacement per track (mean = blue line, 7 µm).</p

Schematic of RBMBs and the methodology used to detect individual RNA transcripts in living cells.

<p>(A) RBMBs are hairpin-forming oligonucleotide probes that are labeled with a reporter dye, quencher, and reference dye. The close proximity of the reporter dye and quencher in the absence of target RNA results in a low fluorescent state. Upon hybridization to complementary RNA, the fluorescent dye and quencher are forced apart, resulting in the restoration of fluorescence. The reference dye remains unquenched regardless of the conformation of the RBMB. The double-stranded domain with a 3′-UU overhang drives nuclear export. (B) To detect individual RNA transcripts, cells were engineered to stably express RNA with 96-tandem repeats of the RBMB target site in the 3′-untranslated region. Binding of up to 96 RBMBs to each RNA transcript results in discrete bright fluorescent spots that can be readily visualized and tracked in real-time by wide-field fluorescence microscopy.</p

Biodegradable Polydisulfide Dendrimer Nanoclusters as MRI Contrast Agents

Gadolinium-conjugated dendrimer nanoclusters (DNCs) are a promising platform for the early detection of disease; however, their clinical utility is potentially limited due to safety concerns related to nephrogenic systemic fibrosis (NSF). In this paper, biodegradable DNCs were prepared with polydisulfide linkages between the individual dendrimers to facilitate excretion. Further, DNCs were labeled with premetalated Gd chelates to eliminate the risk of free Gd becoming entrapped in dendrimer cavities. The biodegradable polydisulfide DNCs possessed a circulation half-life of >1.6 h in mice and produced significant contrast enhancement in the abdominal aorta and kidneys for as long as 4 h. The DNCs were reduced in circulation as a result of thiol–disulfide exchange, and the degradation products were rapidly excreted <i>via</i> renal filtration. These agents demonstrated effective and prolonged <i>in vivo</i> contrast enhancement and yet minimized Gd tissue retention. Biodegradable polydisulfide DNCs represent a promising biodegradable macromolecular MRI contrast agent for magnetic resonance angiography and can potentially be further developed into target-specific MRI contrast agents