Additional file 1: Figure S1. The N-terminal Ab lock and VpreB were unable to mask the binding activity of CTLA4Ig. (A) Binding activity of Ab lock-CTLA4Ig (0.5 μg/ml, blue line) and conventional CTLA4Ig (0.5 μg/ml, black line) to HEK-293 cells overexpressing CD80 (CD80 cells), detected by FITC-conjugated goat anti-mouse Fcγ in flow cytometry. Gray line: unstained cells. (B) Binding activity of VpreB-CTLA4Ig (0.5 μg/ml, blue line) and conventional CTLA4Ig (0.5 μg/ml, black line) to CD80 cells, detected by FITC-conjugated goat anti-mouse Fcγ antibodies by flow cytometry. Gray line: unstained cells. VpreB: immunoglobulin iota chain. (C) Simulation of Ab lock-mCTLA4Ig by the computer software BIOVIA Discovery Studio 2019 (Discovery Studio v19.1.0.18287). The structures of CTLA-4, the CDR3-like domain and the Ab lock are shown in magenta, yellow and light blue, respectively. Figure S2. Full recovery of the binding activity of mAlb-CTLA4Ig after MMP2/9 digestion. Nondigested, MMP-digested mAlb-CTLA44Ig, and conventional mCTLA4Ig (all at 1 nM) were added to the ELISA. Binding of the fusion proteins on the plate was detected by an HRP-conjugated anti-mouse IgG Fcγ secondary antibody. Figure S3. Characterization of an alternative Alb-CTLA4Ig with MMP substrate linker between albumin and CTLA4Ig (mAlb-MMP-CTLA4Ig). (A) Schematic representations of mAlb-MMP-CTLA4Ig constructs. MMP: MMP substrate sequence (GPLGMWSR) linker, eCTLA4: extracellular domain of CTLA4. P: promoter in the expression vector. (B) Reducing SDS-PAGE (left) and western blot analysis (right) of purified mAlb-MMP-CTLA4Ig. (C) The stability of mAlb-MMP-CTLA4Ig in DMEM containing 10% fetal bovine sera for seven days. (D) mAlb-MMP-CTLA4Ig were digested with the indicated amount of MMP2/9 and analyzed by western blot. (E) mAlb-MMP-CTLA4Ig were subjected to varying degrees of digestion by MMP2/9. Part of the digestion was analyzed by western blot to determine the degree of cleavage. The percent (%) cleaved Alb-MMP-CTLA4Ig was quantitated and is indicated below each lane. The digestion was added to the cell-based ELISA. The binding of CD80 by 1 nM conventional mCTLA4Ig was used as a positive control. (F) Binding kinetics of mAlb-MMP-CTLA4Ig before (blue curve) and after MMP digestion (orange curve). The binding kinetics of conventional mCTLA4Ig are shown in the black curve. Figure S4. Lacking an N-terminal albumin and a C-terminal Fc decreases masking efficiency and stability. (A) Competitive binding of Ig-CTLA4 ECD (0.5 μg/ml, blue line) or conventional CTLA4Ig (0.5 μg/ml, black line) against PE-conjugated anti-mouse CD80 antibodies (1 μg/ml) to HEK-293 cells overexpressing CD80 (CD80 cells). Red line: CD80-expressing cells stained with PE-conjugated anti-CD 80 antibodies alone (1 μg/ml). Gray line: unstained cells. Digestion products of the IgG1 Fc-CTLA4 ECD by the indicated amounts of MMP2/9 are shown in the western blot using anti-mouse Fcγ antibodies (right panel). (B) Competitive binding activity of Alb-CTLA4 ECD (0.5 μg/ml, blue line) and conventional CTLA4Ig (0.5 μg/ml, black line) to CD80 cells in the presence of PE-conjugated anti-mouse CD80 antibodies (1 μg/ml) by flow cytometry. Red line: CD80 cells stained with PE-conjugated anti-CD80 antibody alone (1 μg/ml). Gray line: unstained cells. Digestion products of the Alb-CTLA4 ECD by the indicated amounts of MMP2/9 are shown in the western blot using anti-mouse CTLA4 antibodies (right panel). (C) Digestion products of conventional human CTLA4Ig (3.6 picomole) by 2 units of MMP2/9 is shown in the western blot using anti-human Fcγ antibodies. (D) Overdigestion of hAlb-CTLA4Ig (1.35 picomole) in higher amounts (9 units) of MMP2/9. Figure S5. The stability of hAlb-CTLA4Ig in sera. hAlb-CTLA4Ig incubated in RPMI 1640 containing 10% fetal bovine sera for seven days was analyzed by western blot using an anti-human Fcγ secondary antibody. Med: medium alone. Figure S6. Differential levels of MMP9 and MMP3 in normal control mice and CIA mice. Protein levels of MMP9 (A) and MMP3 (B) in the synovial fluid lavages, sera, and paws of normal control mice and CIA mice were measured by ELISA. Protein levels are expressed as ng/ml in synovial fluid lavages and sera, ng/mg protein in protein extracts of the paws. Figure S7. Histopathological microphotographs of the digits of a normal mouse or CIA mice. The area enclosed by red rectangles at low magnification (5X objective, upper row) is shown at higher magnification (20X objective, lower row) for inflammatory cells. Figure S8. Splenocyte proliferation in response to different concentrations of M. tuberculosis restimulation. Splenocytes from a normal (preimmune) mouse and a CIA mouse were stimulated with 0 μg/ml 5μg/ml or 25 μg/ml M. tuberculosis extracts for 72 h. BrdU was added at the final 2 hours of stimulation. Proliferation (BrdU incorporation) of the CD45+ splenocytes was analyzed by flow cytometry.
Technologies that screen multiple single-nucleotide polymorphisms (SNPs) could be very valuable in predicting patients' susceptibilities to diseases or responses to therapeutic interventions. In this study, we developed a chip that can accurately detect four SNPs at same time. This chip is cost-effective and user-friendly because it uses a detection protocol analogous to dot blotting and does not require sophisticated instruments. To establish this chip, we designed and blotted onto a nylon membrane SNP-specific oligonucleotide probes for human angiotensinogen, cholesteryl ester transfer protein, and apolipoprotein E. This chip detected the corresponding SNPs harbored within the angiotensinogen, cholesteryl ester transfer protein, and apolipoprotein E sequences from 20 donors. Importantly, the SNPs detected by our chip matched exactly with the direct sequencing results, thereby highlighting the accuracy of this chip. In conclusion, our chip is a robust tool for multiple SNP screening and holds the potential to future refinement in detecting diseases-associating genes in patients.
Abstract Systemic injection of therapeutic antibodies may cause serious adverse effects due to on-target toxicity to the antigens expressed in normal tissues. To improve the targeting selectivity to the region of disease sites, we developed protease-activated pro-antibodies by masking the binding sites of antibodies with inhibitory domains that can be removed by proteases that are highly expressed at the disease sites. The latency-associated peptide (LAP), C2b or CBa of complement factor 2/B were linked, through a substrate peptide of matrix metalloproteinase-2 (MMP-2), to an anti-epidermal growth factor receptor (EGFR) antibody and an anti-tumor necrosis factor-α (TNF-α) antibody. Results showed that all the inhibitory domains could be removed by MMP-2 to restore the binding activities of the antibodies. LAP substantially reduced (53.8%) the binding activity of the anti-EGFR antibody on EGFR-expressing cells, whereas C2b and CBa were ineffective (21% and 9.3% reduction, respectively). Similarly, LAP also blocked 53.9% of the binding activity of the anti-TNF-α antibody. Finally, molecular dynamic simulation showed that the masking efficiency of LAP, C2b and CBa was 33.7%, 10.3% and −5.4%, respectively, over the binding sites of the antibodies. This strategy may aid in designing new protease-activated pro-antibodies that attain high therapeutic potency yet reduced systemic on-target toxicity.
To develop a new glucuronide probe for micro-positron emission topography (PET) that can depict beta-glucuronidase (betaG)-expressing tumors in vivo.All animal experiments were preapproved by the Institutional Animal Care and Use Committee. A betaG-specific probe was generated by labeling phenolphthalein glucuronide (PTH-G) with iodine 131 ((131)I) or (124)I. To test the specificity of the probe in vitro, (124)I-PTH-G was added to CT26 and betaG-expressing CT26 (CT26/betaG) cells. Mice bearing CT26 and CT26/betaG tumors (n = 6) were injected with (124)I-PTH-G and subjected to micro-PET imaging. A betaG-specific inhibitor D-saccharic acid 1,4-lactone monohydrate was used in vitro and in vivo to ascertain the specificity of the glucuronide probes. Finally, the biodistributions of the probes were determined in selected organs after injection of (131)I-PTH-G to mice bearing CT26 and CT26/betaG tumors (n = 14). Differences in the radioactivity in CT26 and CT26/betaG tumors were analyzed with the Wilcoxon signed rank test.(124)I-PTH-G was selectively converted to (124)I-PTH (phenolphthalein), which accumulated in CT26/betaG cells and tumors in vitro. The micro-PET images demonstrated enhanced activity in CT26/betaG tumors resulting from betaG-mediated conversion and trapping of the radioactive probes. Accumulation of radioactive signals was 3.6-, 3.4-, and 3.3-fold higher in the CT26/betaG tumors than in parental CT26 tumors at 1, 3, and 20 hours, respectively, after injection of the probe (for all the three time points, P < .05).Hydrophilic-hydrophobic conversion of (124)I-PTH-G probe can aid in imaging of betaG-expressing tumors in vivo.
Fibrinogen-like protein 1 (FGL1) was recently identified as a major ligand of lymphocyte-activation gene-3 (LAG-3) on activated T cells and serves as an immune suppressive molecule for regulation of immune homeostasis. However, whether FGL1 has therapeutic potential for use in the T cell-induced the autoimmune disease, rheumatoid arthritis (RA), is still unknown. Here, we attempted to evaluate the effect of FGL1 protein on arthritis progression. We also evaluated potential adverse events in a collagen-induced arthritis (CIA) mouse model. We first confirmed that soluble Fgl1 protein could specifically bind to surface Lag-3 receptor on 3T3-Lag-3 cells and further inhibit interleukin (IL-2) and interferon gamma (IFNγ) secretion from activated primary mouse T cells by 95% and 43%, respectively. Intraperitoneal administration of Fgl1 protein significantly decreased the inflammatory cytokine level (i.e., IL-1β and IL-6) in local paw tissue, and prevented joint inflammation, cellular infiltration, bone deformation and attenuated collagen-induced arthritis progression in vivo . We further demonstrated that exogenous Fgl1 does not cause obvious adverse events during treatment by monitoring body weight and liver weight, and assessing the morphology of several organs (i.e., heart, liver, spleen, lung and kidney) by pathological studies. We expect that Fgl1 protein may be suitable to serve as a potential therapeutic agent for treatment of RA or even other types of T cell-induced autoimmune or inflammatory diseases in the future.
Autoimmune hypophysitis can be reproduced experimentally by the injection of pituitary proteins mixed with an adjuvant into susceptible mice1. Mouse models allow us to study how diseases unfold, often providing a good replica of the same processes occurring in humans. For some autoimmune diseases, like type 1A diabetes, there are models (the NOD mouse) that spontaneously develop a disease similar to the human counterpart. For many other autoimmune diseases, however, the model needs to be induced experimentally. A common approach in this regard is to inject the mouse with a dominant antigen derived from the organ being studied. For example, investigators interested in autoimmune thyroiditis inject mice with thyroglobulin2, and those interested in myasthenia gravis inject them with the acetylcholine receptor3. If the autoantigen for a particular autoimmune disease is not known, investigators inject a crude protein extract from the organ targeted by the autoimmune reaction. For autoimmune hypophysitis, the pathogenic autoantigen(s) remain to be identified4, and thus a crude pituitary protein preparation is used. In this video article we demonstrate how to induce experimental autoimmune hypophysitis in SJL mice.
Protein or peptide drugs are emerging therapeutics for treating human diseases. However, current protein drugs are typically limited to acting on extracellular/cell membrane components associated with the diseases, while intracellular delivery of recombinant proteins replaces or replenishes faulty/missing proteins and remains inadequate. In this study, we developed a convenient and efficient intracellular protein delivery vehicle.A cationic liposomal polyethylenimine and polyethylene glycol complex (LPPC) was developed to noncovalently capture proteins for protein transfer into cells via endocytosis. β-glucuronidase (βG) was used in vitro and in vivo as a model enzyme to demonstrate the enzymatic activity of the intracellular transport of a protein.The endocytosed protein/LPPC complexes escaped from lysosomes, and the bound protein dissociated from LPPC in the cytosol. The enzymatic activity of βG was well preserved after intracellular delivery in vitro and in vivo.Using LPPC as an intracellular protein transporter for protein therapeutics, we illustrated that LPPC may be an effective and convenient tool for studying diseases and developing therapeutics.
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