CPT-11 is an anticancer prodrug that is clinically used for the treatment of metastatic colorectal cancer. Hydrolysis of CPT-11 by human carboxylesterase 2 (CE2) generates SN-38, a topoisomerase I inhibitor that is the active anti-tumor agent. Expression of CE2 in cancer cells is under investigation for the tumor-localized activation of CPT-11. CE2 is normally expressed in the endoplasmic reticulum of cells but can be engineered to direct expression of active enzyme on the plasma membrane or as a secreted form. Although previous studies have investigated different locations of CE2 expression in cancer cells, it remains unclear if CE2 cellular location affects CPT-11 anticancer activity. In the present study, we directly compared the influence of CE2 cellular location on substrate hydrolysis and CPT-11 cytotoxicity. We linked expression of CE2 and enhanced green fluorescence protein (eGFP) via a foot-and-mouth disease virus 2A (F2A) peptide to facilitate fluorescence-activated cell sorting to achieve similar expression levels of ER-located, secreted or membrane-anchored CE2. Soluble CE2 was detected in the medium of cells that expressed secreted and membrane-anchored CE2, but not in cells that expressed ER-retained CE2. Cancer cells that expressed all three forms of CE2 were more sensitive to CPT-11 as compared to unmodified cancer cells, but the membrane-anchored and ER-retained forms of CE2 were consistently more effective than secreted CE2. We conclude that expression of CE2 in the ER or on the membrane of cancer cells is suitable for enhancing CPT-11 anticancer activity.

Extracellular vesicles (EVs) are attractive candidate drug delivery systems due to their ability to functionally transport biological cargo to recipient cells. However, the apparent lack of target cell specificity of exogenously administered EVs limits their therapeutic applicability. In this study, we propose a novel method to equip EVs with targeting properties, in order to improve their interaction with tumour cells.

How T cell receptors (TCRs) are triggered to start signaling is still not fully understood. It has been proposed that segregation of the large membrane tyrosine phosphatase CD45 from engaged TCRs initiates signaling by favoring phosphorylation of immunoreceptor tyrosine-based activation motifs (ITAMs) in the cytoplasmic domains of CD3 molecules. However, whether CD45 segregation is important to initiate triggering is still uncertain. We examined CD45 segregation from TCRs engaged to anti-CD3 scFv with high or low affinity and with defined molecular lengths on glass-supported lipid bilayers using total internal reflection microscopy. Both short and elongated high-affinity anti-CD3 scFv effectively induced similar calcium mobilization, Zap70 phosphorylation, and cytokine secretion in Jurkat T cells but CD45 segregated from activated TCR microclusters significantly less for elongated versus short anti-CD3 ligands. In addition, at early times, triggering cells with both high and low affinity elongated anti-CD3 scFv resulted in similar degrees of CD3 co-localization with CD45, but only the high-affinity scFv induced T cell activation. The lack of correlation between CD45 segregation and early markers of T cell activation suggests that segregation of CD45 from engaged TCRs is not mandatory for initial triggering of TCR signaling by elongated high-affinity ligands.

The bidirectional interaction between carcinogens and gut microbiota that contributes to colorectal cancer is complicated. Reactivation of carcinogen metabolites by microbial β-glucuronidase (βG) in the gut potentially plays an important role in colorectal carcinogenesis. We assessed the chemoprotective effects and associated changes in gut microbiota induced by pre-administration of bacterial-specific βG inhibitor TCH-3511 in carcinogen azoxymethane (AOM)-treated APCMin/+ mice. AOM induced intestinal βG activity, which was reflected in increases in the incidence, formation, and number of tumors in the intestine. Notably, inhibition of gut microbial βG by TCH-3511 significantly reduced AOM-induced intestinal βG activity, decreased the number of polyps in both the small and large intestine to a frequency that was similar in mice without AOM exposure. AOM also led to lower diversity and altered composition in the gut microbiota with a significant increase in mucin-degrading Akkermansia genus. Conversely, mice treated with TCH-3511 and AOM exhibited a more similar gut microbiota structure as mice without AOM administration. Importantly, TCH-3511 treatment significant decreased Akkermansia genus and produced a concomitant increase in short-chain fatty acid butyrate-producing gut commensal microbes Lachnoospiraceae NK4A136 group genus in AOM-treated mice. Taken together, our results reveal a key role of gut microbial βG in promoting AOM-induced gut microbial dysbiosis and intestinal tumorigenesis, indicating the chemoprotective benefit of gut microbial βG inhibition against carcinogens via maintaining the gut microbiota balance and preventing cancer-associated gut microbial dysbiosis. Thus, the bacterial-specific βG inhibitor TCH-3511 is a potential chemoprevention agent for colorectal cancer.

The generation of anti-PEG antibodies in response to PEGylated proteins, peptides, and carriers significantly limits their clinical applicability. IgM antibodies mediate the clearance of these therapeutics upon repeat injection, resulting in toxicity and hindered therapeutic efficacy. We observed this phenomenon in our polymer platform, virus-inspired polymer for endosomal release (VIPER), which employs pH-sensitive triggered display of a lytic peptide, melittin, to facilitate endosomal escape. While the polymer-peptide conjugate was well tolerated after a single injection, we observed unexpected mortality upon repeat injection. Thus, the goal of this work was to enhance the safety and tolerability of VIPER for frequent dosing. Based on previous reports on anti-PEG antibodies and the adjuvant activity of melittin, we characterized the antibody response to polymer, peptide, and polymer-peptide conjugates after repeat-dosing and measured high IgM titers that bound PEG. By substituting the L-amino acid peptide for its D-amino acid enantiomer, we significantly attenuated the anti-PEG antibody generation and toxicity, permitting repeat-injections. We attempted to rescue mice from L-melittin induced toxicity by prophylactic injection of platelet activating factor (PAF) antagonist CV-6209, but observed minimal effect, suggesting that PAF is not the primary mediator of the observed hypersensitivity response. Overall, we demonstrated that the D-amino acid polymer-peptide conjugates, unlike L-amino acid polymer-peptide conjugates, exhibit good tolerability in vivo, even upon repeat administration, and do not elicit the generation of anti-PEG antibodies.

Polyethylene glycol (PEG) is widely used in industry and medicine. Anti-PEG antibodies have been developed for characterizing PEGylated drugs and other applications. However, the underlying mechanism for specific PEG binding has not been elucidated.

Immunotherapy is blooming in recent years. However, this therapy needs to overcome off-target effects, cytokine release syndrome, and low responses in the 'cold' tumor environment. Herein, various combinations of immunotherapies and chemotherapies were proposed to transform 'cold' tumors into 'hot' tumors to enhance the efficacy of immunotherapies. In this study, we prepared a biocompatible ganetespib (GSP)-loaded PEGylated nanocarriers (NCs) with a thin-film method, which exhibited a small particle size (~220.6 nm), high drug loading (~5.8%), and good stability. We designed and produced the cluster of differentiation 3 (CD3)/programmed death ligand 1 (PD-L1)/methoxy-polyethylene glycol (mPEG) trispecific antibodies (TsAbs) as bispecific T-cell engagers (BiTEs) to non-covalently bind the GSP-NCs via anti-mPEG fragment and endowed the GSP-NCs with a targeting ability and immunotherapeutic potential to activate cytotoxic T cells. Decoration of the GSP-NCs with TsAbs (BiTEs-GSP-NCs) significantly promoted the cellular uptake and showed synergistic effects through respective anti-PD-L1 and anti-CD3 activation of T cell-mediated cytotoxicity. In vivo tumor-inhibition studies also showed that the BiTEs-GSP-NCs could inhibit tumor growth with the GSP chemodrug and increase T-cell infiltration. This study provides a promising drug delivery strategy for cancer immunochemotherapy.

Nanomedicines and macromolecular drugs can induce hypersensitivity reactions (HSRs) with symptoms ranging from flushing and breathing difficulties to hypothermia, hypotension, and death in the most severe cases. Because many normal individuals have pre-existing antibodies that bind to poly(ethylene glycol) (PEG), which is often present on the surface of nanomedicines and macromolecular drugs, we examined if and how anti-PEG antibodies induce HSRs to PEGylated liposomal doxorubicin (PLD). Anti-PEG IgG but not anti-PEG IgM induced symptoms of HSRs including hypothermia, altered lung function, and hypotension after PLD administration in C57BL/6 and nonobese diabetic/severe combined immunodeficiency (NOD/SCID) mice. Hypothermia was significantly reduced by blocking FcγRII/III, by depleting basophils, monocytes, neutrophils, or mast cells, and by inhibiting secretion of histamine and platelet-activating factor. Anti-PEG IgG also induced hypothermia in mice after administration of other PEGylated liposomes, nanoparticles, or proteins. Humanized anti-PEG IgG promoted binding of PEGylated nanoparticles to human immune cells and induced secretion of histamine from human basophils in the presence of PLD. Anti-PEG IgE could also induce hypersensitivity reactions in mice after administration of PLD. Our results demonstrate an important role for IgG antibodies in induction of HSRs to PEGylated nanomedicines through interaction with Fcγ receptors on innate immune cells and provide a deeper understanding of HSRs to PEGylated nanoparticles and macromolecular drugs that may facilitate development of safer nanomedicines.

CPT-11 is a camptothecin analog used for the clinical treatment of colorectal adenocarcinoma. CPT-11 is converted into the therapeutic anti-cancer agent SN-38 by liver enzymes and can be further metabolized to a non-toxic glucuronide SN-38G, resulting in low SN-38 but high SN-38G concentrations in the circulation. We previously demonstrated that adenoviral expression of membrane-anchored beta-glucuronidase could promote conversion of SN-38G to SN-38 in tumors and increase the anticancer activity of CPT-11. Here, we identified impediments to effective tumor therapy with E. coli that were engineered to constitutively express highly active E. coli beta-glucuronidase intracellularly to enhance the anticancer activity of CPT-11. The engineered bacteria, E. coli (lux/βG), could hydrolyze SN-38G to SN-38, increased the sensitivity of cultured tumor cells to SN-38G by about 100 fold and selectively accumulated in tumors. However, E. coli (lux/βG) did not more effectively increase CPT-11 anticancer activity in human tumor xenografts as compared to non-engineered E. coli. SN-38G conversion to SN-38 by E. coli (lux/βG) appeared to be limited by slow uptake into bacteria as well as by segregation of E. coli in necrotic regions of tumors that may be relatively inaccessible to systemically-administered drug molecules. Studies using a fluorescent glucuronide probe showed that significantly greater glucuronide hydrolysis could be achieved in mice pretreated with E. coli (lux/βG) by direct intratumoral injection of the glucuronide probe or by intratumoral lysis of bacteria to release intracellular beta-glucuronidase. Our study suggests that the distribution of beta-glucuronidase, and possibly other therapeutic proteins, in the tumor microenvironment might be an important barrier for effective bacterial-based tumor therapy. Expression of secreted therapeutic proteins or induction of therapeutic protein release from bacteria might therefore be a promising strategy to enhance anti-tumor activity.

Targeted therapy of human cancers is an attractive approach and has been investigated with limited success. We have developed novel cytotoxic agents for targeted therapy of human cancers based on the extracellular cytotoxicity domain of CD178 (FasL) and the specificity offered by single chain antibodies (scFv) against dominant human tumor Ag TAG-72 (cc49scFv) and TAL6 (L6scFv).

Membrane-tethered proteins (mammalian surface display) are increasingly being used for novel therapeutic and biotechnology applications. Maximizing surface expression of chimeric proteins on mammalian cells is important for these applications. We show that the cytoplasmic domain from the B7-1 antigen, a commonly used element for mammalian surface display, can enhance the intracellular transport and surface display of chimeric proteins in a Sar1 and Rab1 dependent fashion. However, mutational, alanine scanning and deletion analysis demonstrate the absence of linear ER export motifs in the B7 cytoplasmic domain. Rather, efficient intracellular transport correlated with the presence of predicted secondary structure in the cytoplasmic tail. Examination of the cytoplasmic domains of 984 human and 782 mouse type I transmembrane proteins revealed that many previously identified ER export motifs are rarely found in the cytoplasmic tail of type I transmembrane proteins. Our results suggest that efficient intracellular transport of B7 chimeric proteins is associated with the structure rather than to the presence of a linear ER export motif in the cytoplasmic tail, and indicate that short (less than ~ 10-20 amino acids) and unstructured cytoplasmic tails should be avoided to express high levels of chimeric proteins on mammalian cells.

Developing a high-throughput method for the effecient selection of the highest producing cell is very important for the production of recombinant protein drugs. Here, we developed a novel transiently protein-anchored system coupled with fluorescence activated cell sorting (FACS) for the efficient selection of the highest producing cell. A furin cleavage peptide (RAKR) was used to join a human anti-epithelial growth factor antibody (αEGFR Ab) and the extracellular-transmembrane-cytosolic domains of the mouse B7-1 antigen (B7). The furin inhibitor can transiently switch secreted αEGFR Ab into a membrane-anchored form. After cell sorting, the level of membrane αEGFR Ab-RAKR-B7 is proportional to the amount of secreted αEGFR Ab in the medium. We further selected 23 αEGFR Ab expressing cells and demonstrated a high correlation (R2 = 0.9165) between the secretion level and surface expression levels of αEGFR Ab. These results suggested that the novel transiently protein-anchored system can easily and efficiently select the highest producing cells, reducing the cost for the production of biopharmaceuticals.

Attachment of polyethylene glycol (PEG) molecules to nanoparticles (PEGylation) is a widely-used method to improve the stability, biocompatibility and half-life of nanomedicines. However, the evaluation of the PEGylated nanomedicine pharmacokinetics (PK) requires the decomposition of particles and purification of lead compounds before analysis by high performance liquid chromatography (HPLC), mass spectrometry, etc. Therefore, a method to directly quantify un-decomposed PEGylated nanoparticles is needed. In this study, we developed anti-PEG bioparticles and combined them with anti-PEG antibodies to generate a quantitative enzyme-linked immunosorbent assay (ELISA) for direct measurement of PEGylated nanoparticles without compound purification. The anti-PEG bioparticles quantitative ELISA directly quantify PEG-quantum dots (PEG-QD), PEG-stabilizing super-paramagnetic iron oxide (PEG-SPIO), Lipo-Dox and PEGASYS and the detection limits were 0.01 nM, 0.1 nM, 15.63 ng/mL and 0.48 ng/mL, respectively. Furthermore, this anti-PEG bioparticle-based ELISA tolerated samples containing up to 10% mouse or human serum. There was no significant difference in pharmacokinetic studies of radiolabeled PEG-nanoparticles (Nano-X-111In) through anti-PEG bioparticle-based ELISA and a traditional gamma counter. These results suggest that the anti-PEG bioparticle-based ELISA may provide a direct and effective method for the quantitation of any whole PEGylated nanoparticles without sample preparation.

PEGylated nanomedicines are known to induce infusion reactions (IRs) that in some cases can be life-threatening. Herein, we report a case study in which a patient with rare mediastinal and intracardiac IgG4-related sclerosing disease received 8 treatments of intravenously administered PEGylated liposomal methylprednisolone-succinate (NSSL-MPS). Due to the ethical requirements to reduce IRs, the patient received a cocktail of premedication including low dose of steroids, acetaminophen and H2 blockers before each infusion. The treatment was well-tolerated in that IRs, complement activation, anti-PEG antibodies and accelerated blood clearance of the PEGylated drug were not detected. Prior to the clinical study, an in vitro panel of assays utilizing blood of healthy donors was used to determine the potential of a PEGylated drug to activate complement system, elicit pro-inflammatory cytokines, damage erythrocytes and affect various components of the blood coagulation system. The overall findings of the in vitro panel were negative and correlated with the results observed in the clinical phase.

Molecular weight markers that can tolerate denaturing conditions and be auto-detected by secondary antibodies offer great efficacy and convenience for Western Blotting. Here, we describe M&R LE protein markers which contain linear epitopes derived from the heavy chain constant regions of mouse and rabbit immunoglobulin G (IgG Fc LE). These markers can be directly recognized and stained by a wide range of anti-mouse and anti-rabbit secondary antibodies. We selected three mouse (M1, M2 and M3) linear IgG1 and three rabbit (R1, R2 and R3) linear IgG heavy chain epitope candidates based on their respective crystal structures. Western blot analysis indicated that M2 and R2 linear epitopes are effectively recognized by anti-mouse and anti-rabbit secondary antibodies, respectively. We fused the M2 and R2 epitopes (M&R LE) and incorporated the polypeptide in a range of 15-120 kDa auto-detecting markers (M&R LE protein marker). The M&R LE protein marker can be auto-detected by anti-mouse and anti-rabbit IgG secondary antibodies in standard immunoblots. Linear regression analysis of the M&R LE protein marker plotted as gel mobility versus the log of the marker molecular weights revealed good linearity with a correlation coefficient R2 value of 0.9965, indicating that the M&R LE protein marker displays high accuracy for determining protein molecular weights. This accurate, regular and auto-detected M&R LE protein marker may provide a simple, efficient and economical tool for protein analysis.

T lymphocytes are important mediators of adoptive immunity but the mechanism of T cell receptor (TCR) triggering remains uncertain. The interspatial distance between engaged T cells and antigen-presenting cells (APCs) is believed to be important for topological rearrangement of membrane tyrosine phosphatases and initiation of TCR signaling. We investigated the relationship between ligand topology and affinity by generating a series of artificial APCs that express membrane-tethered anti-CD3 scFv with different affinities (OKT3, BC3, and 2C11) in addition to recombinant class I and II pMHC molecules. The dimensions of membrane-tethered anti-CD3 and pMHC molecules were progressively increased by insertion of different extracellular domains. In agreement with previous studies, elongation of pMHC molecules or low-affinity anti-CD3 scFv caused progressive loss of T cell activation. However, elongation of high-affinity ligands (BC3 and OKT3 scFv) did not abolish TCR phosphorylation and T cell activation. Mutation of key amino acids in OKT3 to reduce binding affinity to CD3 resulted in restoration of topological dependence on T cell activation. Our results show that high-affinity TCR ligands can effectively induce TCR triggering even at large interspatial distances between T cells and APCs.

Triple-negative breast cancer (TNBC) lacks effective treatment options due to the absence of traditional therapeutic targets. The epidermal growth factor receptor (EGFR) has emerged as a promising target for TNBC therapy because it is overexpressed in about 50% of TNBC patients. Here we describe a PEG engager that simultaneously binds polyethylene glycol and EGFR to deliver PEGylated nanomedicines to EGFR+ TNBC. The PEG engager displays conditional internalization by remaining on the surface of TNBC cells until contact with PEGylated nanocarriers triggers rapid engulfment of nanocargos. PEG engager enhances the anti-proliferative activity of PEG-liposomal doxorubicin to EGFR+ TNBC cells by up to 100-fold with potency dependent on EGFR expression levels. The PEG engager significantly increases retention of fluorescent PEG probes and enhances the antitumour activity of PEGylated liposomal doxorubicin in human TNBC xenografts. PEG engagers with specificity for EGFR are promising for improved treatment of EGFR+ TNBC patients.

Polyethylene glycol (PEG) is a biocompatible polymer used in biotherapeutics to increase bioavailability, reduce the frequency of administration, and optimize pharmacokinetics. Anti-PEG antibodies have been detected in healthy individuals and may decrease efficacy and alter the pharmacokinetics of PEGylated therapeutics; however, the prevalence of anti-PEG antibodies is unclear. In this study, a flow cytometry assay was optimized to detect anti-PEG IgG and IgM in human blood plasma. Three hundred (300) plasma samples from healthy blood donors were screened; anti-PEG IgG or IgM was detected in 65.3% of the total population, with 21.3% having anti-PEG IgG, 19.0% having anti-PEG IgM, and 25.0% having both anti-PEG IgG and IgM. The presence of anti-PEG IgG and IgM was confirmed using a 0.5% Tween-20 interference assay, a 20 kDa PEGylated polystyrene bead binding assay, and Western blotting of purified plasma from human IgG and IgM purification columns. The concentrations of anti-PEG IgG and IgM in positive samples ranged from 39 ng/mL to 18.7 μg/mL and 26 ng/mL to 11.6 μg/mL, respectively. The highest prevalence of both anti-IgG and anti-IgM was in individuals 18-24 years of age. The prevalence of anti-PEG IgG and IgM tended to be higher in women but did not differ among races. Age, sex, and race were not associated with the concentrations of anti-PEG IgG or IgM. No correlation was found between anti-PEG IgG and IgM concentrations. Our study indicates that flow cytometry can be used to detect anti-PEG IgG and IgM antibodies in human plasma.

Pancreatic cancer is one of the most common causes of death in Taiwan. Previous studies have shown that more than 90% of pancreatic cancer cells presented epidermal growth factor receptor (EGFR) cell marker, and this marker is thought to be important as it is related to activation of cancer cell proliferation, angiogenesis, and cancer progression. Moreover, tumor-associated fibroblasts were involved in tumor proliferation and progression. In this study, we fabricated an anti-EGFR and anti-fibroblast activation protein bispecific antibody-targeted liposomal irinotecan (BS-LipoIRI), which could specifically bind to pancreatic cancer cells and tumor-associated fibroblasts. The drug encapsulation efficiency of BS-LipoIRI was 80.95%, and the drug loading was 8.41%. We proved that both pancreatic cancer cells and fibroblasts could be targeted by BS-LipoIRI, which showed better cellular uptake efficacy compared to LipoIRI. Furthermore, an in vivo mouse tumor test indicated that BS-LipoIRI could inhibit pancreatic cancer growth up to 46.2% compared to phosphate-buffered saline control, suggesting that BS-LipoIRI could be useful in clinical cancer treatment.

Irinotecan inhibits cell proliferation and thus is used for the primary treatment of colorectal cancer. Metabolism of irinotecan involves incorporation of β-glucuronic acid to facilitate excretion. During transit of the glucuronidated product through the gastrointestinal tract, an induced upregulation of gut microbial β-glucuronidase (GUS) activity may cause severe diarrhea and thus force many patients to stop treatment. We herein report the development of uronic isofagomine (UIFG) derivatives that act as general, potent inhibitors of bacterial GUSs, especially those of Escherichia coli and Clostridium perfringens. The best inhibitor, C6-nonyl UIFG, is 23,300-fold more selective for E. coli GUS than for human GUS (Ki = 0.0045 and 105 μM, respectively). Structural evidence indicated that the loss of coordinated water molecules, with the consequent increase in entropy, contributes to the high affinity and selectivity for bacterial GUSs. The inhibitors also effectively reduced irinotecan-induced diarrhea in mice without damaging intestinal epithelial cells.