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

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

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

Fluorescent images of spleens following histological sectioning.

<p>(A–D) Representative fluorescent images of an excised and sectioned spleen, obtained from a mouse that was not subject to B cell transfer. (A) Aside from autofluorescence, there is little detectable fluorescent signal in the (A) GFP, (B) Alexa-680 (i.e. SPIO), and (C) NIR815 channels. (D) A composite image shows no significant co-localization between the fluo rescent images. (E–F) Representative images of a spleen that was excised and sectioned 7 days following the transfer of SPIO- and NIR815-labeled B cells into C57BL/6 mice. (E) Despite the high level of autofluorescence, distinct punctate areas of fluorescence could still be discerned. Presumably, these fluorescent signals signify the presence of GFP-positive B cells. Similar patterns of punctate fluorescent signals were also observed in the (F) SPIO and (G) NIR815 channels. (H) The composite image shows that there is considerable overlap between the fluorescent signals in all three channels (arrows). All images were acquired using a LUC PLAN FLN 40× objective (NA 0.6).</p

In vivo fluorescent imaging of contrast-labeled B cells prior to and following B cell depletion therapy.

<p>A, Representative whole animal fluorescence images of B cells loaded with SPIO and NIR815 (groups i and ii) or just NIR815 (groups iii and iv) following injection into C57BL/6 mice. Prior to injection (day 0), no signal was evident in the NIR channel. Signal accumulated within the spleen by 24 h after the cell injection. Immediately following imaging on day 1, animals were injected with either anti-CD79 antibodies or PBS. Anti-CD79 treatment led to a rapid (by day 3) loss is signal in mice injected with B cells labeled with NIR815 only. B, The abundance of B cells, labeled with SPIO and NIR815, in the spleen was quantified by measuring the spleen-to-muscle signal–to-background ratio (SBR). Quantification of fluorescence revealed only a gradual loss in SBR following treatment with PBS (group I, solid line) and anti-CD79 antibodies (group ii, dashed line). No statistical significance was observed between the two groups (p<0.05). C, NIR815-labeled B cells (i.e. no SPIO) were rapidly depleted following administration of anti-CD79 antibodies (group iv, dashed line) compared with PBS-treated controls (group iii, solid line). Statistical significance for individual time points is indicated with an asterisk (p<0.05).</p

Fluorescent images of contrast-labeled B cells in excised organs.

<p>A, Representative fluorescent images of the excised heart (H), lung (L), spleen (S), and liver (L) from animals injected with contrast labeled B cells. The B cells were either labeled with SPIO and NIR815 (groups i and ii) or just NIR815 (groups iii and iv), as indicated. Animals were either treated with PBS (groups i and iii) or anti-CD79 antibodies (ii and iv) following imaging on day 1. One animal per group was sacrificed at each time point, immediately following MR and in vivo fluorescent imaging. B and C, The mean fluorescence intensity (MFI) of the spleen, normalized to day 1 values for each group, is plotted for the length of the experiment. A gradual loss of signal from the spleen is seen in all groups, save the rapid decrease following treatment of NIR815-only labeled cells (iv).</p