Cleavage Of Disulfide Linkages Research Articles

Proactively Reducing Anti‐Drug Antibodies via Immunomodulatory Bioconjugation

AbstractAlthough PEGylation reduces the immunogenicity of protein drugs to some extent, its limitations for highly immunogenic biotherapeutics have been demonstrated. Herein, a proactive strategy to alleviate the development of anti‐drug antibodies (ADAs) against protein drugs by immunomodulatory bioconjugation is reported. Rapamycin was conjugated to a PEGylated protein therapeutic via a cleavable disulfide linker. The conjugated rapamycin can be released from the bioconjugate and prevent immune responses once the bioconjugate is uptaken by antigen‐presenting cells. The immunomodulatory bioconjugate significantly reduced the titers of ADAs compared with a PEGylated protein. The inhibition of immune responses was specific to the conjugated antigen, avoiding systemic immune suppression and the risk of increased susceptibility to infections. The reported approach breaks the limitations of PEGylation by the proactive prevention of ADAs.

Polyamine-Targeting Gefitinib Prodrug and its Near-Infrared Fluorescent Theranostic Derivative for Monitoring Drug Delivery and Lung Cancer Therapy.

The therapy of non-small-cell lung cancer (NSCLC) is challenging because of poor prognosis. There are urgent demands for targeting anti-tumor drugs with reliable efficacy and clear pharmacokinetics.Methods: We designed and synthesized an active tumor-targeting prodrug for the precision therapy of NSCLC. The prodrug polyamine analog Gefitinib (PPG) was derived from the conjugation between a tumor-targeting ligand polyamine analog (PA) and an epidermal growth factor receptor tyrosine kinase inhibitor Gefitinib via a cleavable disulfide linker. Furthermore, the integration of the near-infrared azo-BODIPY fluorophore into the structure of the prodrug PPG yielded an activatable fluorescent theranostics (TPG), which could be used to monitor the in real-time delivery of prodrug PPG and initiate precise medicine in vivo.Results: PPG efficiently delivered the anti-tumor drug to cancer cells and reduced the serious side effects of the drug to normal cells, thereby increasing the potent of the anti-tumor drug. PPG was not only efficacious for killing Gefitinib-sensitive PC9 cells, but also for inhibiting the growth of Gefitinib-resistant H1650 cells. We provided a new evidence that the tumor-targeting PA ligand could inhibit the Akt pathway in H1650 cells, and had a synergistic effect with Gefitinib for anticancer efficacy. The in vivo results on nude mice bearing tumors of NSCLC cell lines demonstrated that PPG could target tumor lesions and had the expected therapeutic effects. Finally, we used TPG for fluorescent labeling of transbronchial lung biopsy (TBLB) specimens. The results indicated that TPG could provide rapid diagnosis for lung cancer within 4 h.Conclusion: Our work had identified that PPG could be effectively used for the treatment of Gefitinib-resistance NSCLC in cells and in mice models. The theranostic TPG emerged as a promising fluorescent imaging tool for the application in the therapy and diagnosis of NSCLC.

Cancer-targeted design of bioresponsive prodrug with enhanced cellular uptake to achieve precise cancer therapy

Chemical drug design based on the biochemical characteristics of cancer cells has become an important strategy for discovery of novel anticancer drugs to enhance the cancer targeting effects and biocompatibility, and decrease toxic side effects. Camptothecin (CPT) demonstrated strong anticancer activity in clinical trials but also notorious adverse effects. In this study, we presented a smart targeted delivery system (Biotin-ss-CPT) that consists of cancer-targeted moiety (biotin), a cleavable disulfide linker (S-S bond) and the active drug CPT. Biotin-ss-CPT was found to exhibit potent effects on the migration of cancer cells and induced apoptosis by induction of ROS-mediated mitochondrial dysfunction and perturbation of GSH/GPXs system, as well as activation of caspases. In vivo tumor suppression investigation including toxicity evaluation and pathology analysis, accompanied by MR images showed that Biotin-ss-CPT can be recognized specifically and selectively and taken up preferentially by cancers cells, followed by localization and accumulation effectively in tumor site, then released CPT by biological response to achieve high therapeutic effect and remarkably reduced the side effects that free CPT caused, such as liver damage, renal injury, and weight loss to realize precise cancer therapy. Taken together, our results suggest that biotinylation and bioresponsive functionalization of anticancer drugs could be a good way for the discovery of next-generation cancer therapeutics.

Degradable Microgel Wet-Strength Adhesives: A Route to Enhanced Paper Recycling

Demonstrated is a new approach to cellulose fiber-based materials that are strong when wet, yet can be recycled after exposure to a weak reducing agent. Poly(N-isopropylacrylamide-co-acrylic acid) microgels were transformed into wet cellulose adhesives by incorporation of hydrazide groups that can form hydrazone linkages to oxidized cellulose. Reductant responsivity was obtained by introducing cleavable disulfide linkages, either in the chains tethering the adhesive hydrazide groups, or by using disulfide cross-links in the microgels. Both types of disulfide derivatives gave about 75% reduction in cellulose wet adhesion after exposure to a reductant. Truly sustainable wood-fiber replacements for plastic packaging must be insensitive to water while being fully recyclable; this work demonstrates two routes to reversible wet cellulose adhesives, facilitating recycling.

Abstract 2651: A novel CD19 targeting antibody-drug conjugate, huB4-DGN462, shows promising in vitro and in vivo activity in CD19-positive lymphoma models

Abstract Background. CD19 is a cell surface membrane protein expressed in most mature and immature B cell neoplasms, making it a promising target for antibody-drug conjugate (ADC) therapy for B cell malignancies. Here we describe the preclinical activity of a novel CD19-targeting ADC, based on the potent indolinobenzodiazepine DNA-alkylating payload DGN462. Methods. The humanized anti-CD19 antibody, huB4, was conjugated to DGN462 via a cleavable disulfide linker, sulfo-SPDB. In vitro activity of the huB4-DGN462 ADC or the unconjugated DGN462 toxin was evaluated in 54 lymphoma cell lines [27 diffuse large B cell lymphomas (DLBCL); 10 mantle cell lymphomas; 6 marginal zone lymphomas; 5 anaplastic large T-cell lymphomas; 6 others]. Cell proliferation/viability after 72h of exposure was measured using a MTT assay. Apoptosis activation was defined by at least 1.5-fold increase in caspase 3/7 signal activation in respect to controls using the Promega ApoTox-Glo Triplex Assay. Gene expression profiling (GEP) was performed with the Illumina HumanHT-12 Expression BeadChips on untreated cell lines followed by GSEA (NES > |2|, P<0.05, FDR<0.25) and limma t-test (FC> |1.2|; P< 0.05; top 200 up and top 200 down). In vivo efficacy of huB4-DGN462 was evaluated in CD19-expressing xenograft tumor models. Results. huB4-DGN462 was cytotoxic against a broad panel of 48 B cell lymphoma cell lines (median IC50 100 pM; 95%CI, 38-214). The cytotoxic activity was not limited by P53, BCL2, MYC or CDKN2A status, or associated with DLBCL cell of origin. Consistent with overall lower CD19 expression, huB4-DGN462 was significantly (p-value=0.007) less active in eight T cell-derived lymphomas (median IC50 of 1.75 nM (95%CI, 0.5-5.75)) than in B cell lymphomas. huB4-DGN462 induced caspase 3/7 activation in 48/54 cell lines (89%) consistent with an apoptotic mechanism of action. huB4-DGN462 demonstrated compelling anti-tumor activity after a single intravenous dose in two diffuse large B-cell lymphoma cell line xenograft models: DoHH2 (a subcutaneous model) and Farage (a disseminated model). In the DoHH2 model, huB4-DGN462 resulted in a significant, dose-dependent tumor growth delay and survival benefit at 1.7 mg Ab/kg compared to a non-targeted control DGN462 ADC. In the disseminated Farage model, a significant dose-dependent increase in survival was observed in mice treated with as low as 0.17 mg Ab/kg of huB4-DGN462. At 1.7 mg Ab/kg, the life span was increased >400% compared to untreated mice. Conclusions. The novel ADC huB4-DGN462 presented strong preclinical anti-lymphoma activity, which provides evidence for further study. Citation Format: Eugenio Gaudio, Chiara Tarantelli, Alberto J. Arribas, Roberta Pittau Bordone, Andrea Rinaldi, Georg Stussi, Emanuele Zucca, Davide Rossi, Anastasios Stathis, Min Li, Alan Wilhem, Kate Lai, Qifeng Qiu, Stuart Hicks, Callum Sloss, Francesco Bertoni. A novel CD19 targeting antibody-drug conjugate, huB4-DGN462, shows promising in vitro and in vivo activity in CD19-positive lymphoma models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2651. doi:10.1158/1538-7445.AM2017-2651

Chitosan based nanogels stepwise response to intracellular delivery kinetics for enhanced delivery of doxorubicin

Chitosan based nanogels with pH/redox sensitivities tunable to stepwise response to intracellular delivery kinetics were developed. The nanogels were simply constructed by ionic gelation first, between O-Carboxymethyl-chitosan (CMCS) and thiolated chitosan (TCS), and then oxidation to form disulfide bonds for CMCS-TCS nanogels (CTNGs). Doxorubicin loaded nanogels (DOX/CTNGs) exhibited desirable stability under physiological pH with a mean size of 150.5nm, and quickly aggregated at pH 5.5 (mimic endo/lysosomes) due to protonation of the carboxyl groups on CMCS. DOX/CTNGs would maintain their TCS skeleton in acidic pH and compromised as treated with 10mM glutathione (mimic cytosol). In agreement with the structural variation, release of DOX was dramatically enhanced by the synergetic effects of acidic pH and reductive potential. Stepwise responses to intracellular delivery kinetics were evidenced by laser confocal images showing that DOX/CTNGs underwent efficient cellular internalization through endocytosis, endo/lysomse escape via self-precipitation, cleavage of disulfide linkage in cytosol and disintegration in nucleus, achieving enhanced nuclear delivery and rapid release of doxorubicin. DOX/CTNGs exerted comparable or higher anticancer efficacies than that of free DOX against hela cells. The simple construction of the nanogels and their capacity of enhancing anticancer activities of DOX are potential for translational applications in cancer chemotherapy.

Redox-responsive molecular gels based on camptothecin prodrug with disulfide linkage for controlled and sustained drug release

A novel camptothecin (CPT) prodrug was successfully synthesized by conjugating CPT to adamantanecarboxylic acid (AD) via a cleavable disulfide linkage. The resulting CPT-ss-AD prodrug could act as a low molecular weight gelator to form molecular gels in water/water-miscible organic solvent mixture. Meanwhile, biodegradable amphiphilic block copolymer mPEG-b-P (MAC-co-DTC) (PPMD) was also employed as an organic framework together with CPT-ss-AD to form gel structure. CPT-ss-AD/PPMD gel exhibited less compact molecular arrangement but much more stability than CPT-ss-AD gel. The two kinds of gels could effectively release the original CPT under reductive condition at a near-constant rate without any initial burst. As compared to CPT-ss-AD single-component gel, the two-component gel, CPT-ss-AD/PPMD, had a significantly higher release rate of CPT, while 3-[4,5-dimethylthiazol-2-yl]-2, 5-diphenyltetrazolium bromide (MTT) assays also indicated highly potent cytotoxic activity against HeLa cells.

Cleavable conjugation of CpG oligodeoxynucleotides onto microparticles for facile release and cytokine induction in macrophages

The development of biomaterials for efficient intracellular delivery of the nucleic acid-based immune-stimulating molecule, CpG oligodeoxynucleotides (CpG), is crucial for their biological activity. In this study, we successfully fabricated polydopamine-coated porous poly(lactic-co-glycolic acid) microparticles (PPM) for the delivery of CpGs. After conjugation of CpGs to PPMs via cleavable disulfide linkages, CpGs were readily released from CpG-conjugated PPM (PPM-s–s-CpGs) in reductive conditions. Released CpGs exhibited significantly enhanced induction of two cytokines, TNF-α and IL-6, in RAW264.7 cells. GpC-conjugated PPMs showed negligible cytokine induction, whereas CpG-conjugated PPMs exhibited strong induction of the two cytokines in RAW264.7 cells. The PPM-s–s-CpGs can serve as immune-stimulating adjuvants to enhance the immune responses of vaccines.

Redox-Activatable ATP-Depleting Micelles with Dual Modulation Characteristics for Multidrug-Resistant Cancer Therapy.

A fast adenosine triphosphate (ATP)-depleting micellar system that is activated by intracellular redox for the codelivery of anticancer drug paclitaxel (PTX) and small interference RNA (siRNA) targeting polo-like kinase1 (PLK1) is developed to address the key challenges of multidrug-resistant (MDR) cancer therapy. The ATP-depleting micelle is self-assembled from a redox-responsive amphiphilic polymer (termed as bPEG-SS-P123-PEI (PSPP)) that is composed of biocompatible branched polyethylene glycol (PEG) with 8 arms (bPEG), ATP-depleting Pluronic P123 (P123), and cationic low molecular weight polyethylenimine (PEI) blocks. Upon critical micelle concentration, the PSPP unimer self-assembles into a well-ordered multilayered nanostructure and is able to load PTX and siRNA targeting PLK1. The cleavage of disulfide linkages at intracellular glutathione-rich reduction milieu not only promotes PTX and siRNA release, but also activates the fast ATP-depletion action that is critical in preventing intracellular PTX efflux by multidrug-resistant cancer cells. The combination of ATP depletion and siRNA inhibition by PSPP micelles is found to provide dual modulations for resensitizing multidrug-resistant cancer cells for PTX treatment. As a result, the codelivery of PTX and PLK1 siRNA exerts a stronger combinational effect against tumor growth in MDR tumor models in vivo. The development of fast ATP-depleting nanomicelle represents an original delivery strategy for the distinctive dual modulation of cancer MDR with spatial and temporal control.

Biodegradable Copolymer for Stimuli-Responsive Sustained Release of Doxorubicin

Pendent functionalization of biodegradable polymers provides unique importance in biological applications. In this work, we have synthesized a polymeric nanocarrier for the controlled release of the anticancer drug doxorubicin (DOXI). Inspired by the pH responsiveness of acylhydrazine bonds along with the interesting self-assembly behavior of amphiphilic copolymers, this report delineates the development of a PEG-SS-PCL-DOXI copolymer consisting of DOXI, PEG, and a caprolactone backbone. First, the inclusion of a PEG moiety in the copolymer helps to achieve biocompatibility and aqueous solubility as well as a prolonged circulation time of the nanocarrier. Second, an acid-sensitive acylhydrazine-based linkage is chosen to attach DOXI to trigger sustained drug release, whereas the inclusion of an enzymatically cleavable disulfide linkage in the backbone adds to the advantage of backbone biodegradability at the intracellular level.

Abstract 2967: In vitro and in vivo activity of site-specific antibody-drug conjugates (ADCs) with 2 and 4 maytansinoid molecules per antibody prepared through conjugation to SeriMabs (N-terminal serine engineered Abs)

Abstract Site-specific attachment of cell-killing agents to antibodies directed against tumor-associated antigens has continued to be an active area of innovation in the field of ADCs. Most reports focus on homogeneous ADCs that have a DAR (cytotoxic molecules per antibody ratio) of 2. Here we describe the preparation, biochemical characterization, and biological evaluation of ADCs made through conjugation of maytansinoids (DM1, DM4) to aldehydes derived from chemically oxidized N-terminal serines (SeriMab) engineered onto the antibody heavy chain (2 DAR) or both light and heavy chain simultaneously (4 DAR). ADCs prepared with a non-cleavable linker or a cleavable disulfide linker were homogeneous 2 or 4 DAR by MS analysis, and were produced in high yield with a monomer content of >98%. Despite conjugation at the N-termini of both the light and heavy chain variable regions, FACS analysis showed the 4 DAR SeriMab conjugates maintained binding to the target antigen. The ADCs showed antigen-specific potency in vitro on a panel of target-expressing cancer cell lines. In the disulfide cleavable linker series, the 2 DAR SeriMab conjugate was 2-5 fold less active than lysine-conjugated Ab-SPDB-DM4 (3.4 DAR), while the 4 DAR SeriMab conjugate was comparably active on an antibody basis. The SeriMab conjugates also displayed strong bystander killing. Surprisingly, in the non-cleavable linker series, the 2 DAR SeriMab conjugate was up to 17-fold more active (depending on cell line) than lysine-conjugated Ab-SMCC-DM1 (3.5 DAR), and the 4 DAR SeriMab conjugate was up to 100-fold more potent than the SMCC-DM1 conjugate on an antibody concentration basis. In a P-gp-positive multi-drug resistant cell line, the non-cleavable 4 DAR SeriMab-maytansinoid conjugate was highly active while the 2 DAR SeriMab ADCs and lysine-conjugated maytansinoid ADCs were >100-fold less potent. The unique oxime bond formed with the non-cleavable SeriMab-maytansinoid conjugate was found to be stable in circulation in mice for >3 days as assayed by affinity capture LC-MS. Polar carboxylic acid containing metabolites were identified which may lead to high cellular retention of maytansinoid species in cancer cells, yielding higher in vitro potency than lysine-linked ADCs in some cell lines. The in vivo anti-tumor activity of disulfide cleavable 2 and 4 DAR SeriMab-DM4 ADCs was evaluated in a clinically relevant cancer xenograft model. The 4 DAR conjugate was active at 60 μg/kg (maytansinoid payload dose) and was more active than the 2 DAR conjugate at this same payload dose. Using the SeriMab conjugation platform we show that in vitro and in vivo activity of site-specific ADCs can be dependent on amount of cytotoxic agent attached per antibody. Citation Format: Luke Harris, Leanne Lanieri, Jose Ponte, Erin Maloney, Laura Bartle, Olga Ab, Juliet Costoplus, Lingyun Rui, Jan Pinkas, Ravi Chari, Thomas Keating, Daniel Tavares, Nathan Fishkin. In vitro and in vivo activity of site-specific antibody-drug conjugates (ADCs) with 2 and 4 maytansinoid molecules per antibody prepared through conjugation to SeriMabs (N-terminal serine engineered Abs). [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 2967.

Tumor Selective Silencing Using an RNAi-Conjugated Polymeric Nanopharmaceutical.

Small interfering RNA (siRNA) therapeutics have potential advantages over traditional small molecule drugs such as high specificity and the ability to inhibit otherwise "undruggable" targets. However, siRNAs have short plasma half-lives in vivo, can induce a cytokine response, and show poor cellular uptake. Formulating siRNA into nanoparticles offers two advantages: enhanced siRNA stability against nuclease degradation beyond what chemical modification alone can provide; and improved site-specific delivery that takes advantage of the enhanced permeability and retention (EPR) effect. Existing delivery systems generally suffer from poor delivery to tumors. Here we describe the formation and biological activity of polymeric nanopharmaceuticals (PNPs) based on biocompatible and biodegradable poly(lactic-co-glycolic acid) (PLGA) conjugated to siRNA via an intracellular cleavable disulfide linker (PLGA-siRNA). Additionally, these PNPs contain (1) PLGA conjugated to polyethylene glycol (PEG) for enhanced pharmacokinetics of the nanocarrier; (2) a cation for complexation of siRNA and charge compensation to avoid high negative zeta potential; and (3) neutral poly(vinyl alcohol) (PVA) to stabilize the PNPs and support the PEG shell to prevent particle aggregation and protein adsorption. The biological data demonstrate that these PNPs achieve prolonged circulation, tumor accumulation that is uniform throughout the tumor, and prolonged tumor-specific knockdown. PNPs employed in this study had no effect on body weight, blood cell count, serum chemistry, or cytokine response at doses >10 times the effective dose. PNPs, therefore, constitute a promising solution for achieving durable siRNA delivery and gene silencing in tumors.

Photodynamic Graphene Quantum Dot: Reduction Condition Regulated Photoactivity and Size Dependent Efficacy.

Prequenching and selective activation of photosensitizer (PS) are highly desired in photodynamic therapy (PDT) to avoid off-target effect due to nonspecific activation and poor targeting selectivity of PS. In this study, nanographene materials as a unique π-conjugated planar system for electronic transfer were employed as the robust platform for temporarily quenching of PS. Photosensitizer chlorin e6 (Ce6) was integrated onto planar structure of graphene quantum dot (GQD) or graphene oxide (GO) via a reduction cleavable disulfide linker. The formed hybrid nanosystem displayed considerable fluorescence quenching and slight phototoxicity, even under the condition of light irradiation, while the photoactivity of PS could be selectively recovered in the presence of the reducing agent. Compared with graphene oxide system with larger size (around 200 nm), GQD nanosystem exhibited significantly improved tumor accumulation via enhanced permeation and retention effect (EPR effect). The in vivo study demonstrated extremely effective suppression of tumor growth for the group treated with the GQD nanosystem with cleavable linker, revealing the promising application of the presented novel strategy.

MMP2-Targeting and Redox-Responsive PEGylated Chlorin e6 Nanoparticles for Cancer Near-Infrared Imaging and Photodynamic Therapy.

A unique matrix metalloproteinase 2-targeted photosensitizer delivery platform was developed in this study for tumor-targeting imaging and photodynamic therapy. The model photosensitizer therapeutic agent chlorin e6 (Ce6) was first covalently conjugated with matrix metalloproteinase 2-cleavable polypeptide and then modified with polyethylene glycol via a redox-responsive cleavable disulfide linker. The resultant matrix metalloproteinase 2-cleavable polypeptide modified PEGylated Ce6 (PEG-SS-Ce6-MMP2) nanoparticles, which formed via self-assembly, were observed to be monodisperse and significantly stable in aqueous solution. In addition, owing to their cellular redox-responsiveness at the cleavable disulfide linker, the PEG-SS-Ce6-MMP2 nanoparticles were able to release Ce6 rapidly. Despite displaying enhanced intracellular internalization, the synthesized PEG-SS-Ce6-MMP2 nanoparticles did not compromise their phototoxic effects toward A549 cancer cells when compared with free Ce6 and PEGylated Ce6 nanoparticles. In vivo experiments further revealed that, in contrast with the free Ce6 or with the PEGylated Ce6 nanoparticles, the PEG-SS-Ce6-MMP2 nanoparticles showed a remarkable increase in tumor-targeting ability and a significantly improved photodynamic therapeutic efficiency in A549 tumor-bearing mice. These results suggest that the PEG-SS-Ce6-MMP2 nanoparticles hold great potential for tumor-targeting imaging and photodynamic therapy.

IMGN779, a CD33-Targeted Antibody-Drug Conjugate (ADC) with a Novel DNA-Alkylating Effector Molecule, Induces DNA Damage, Cell Cycle Arrest, and Apoptosis in AML Cells

IMGN779 is a CD33-targeting ADC consisting of a humanized anti-CD33 antibody, Z4681A, conjugated to DGN462, a novel DNA-alkylating agent, through a cleavable disulfide linker, sulfo-SPDB. CD33 is broadly expressed on leukemic blasts of patients with AML, making it a promising target for AML therapy. DGN462 is a member of the novel IGN class of DNA-acting cytotoxic agents, that consists of an indolino-benzodiazepine dimer containing a mono-imine moiety.Potent killing of AML tumor cells by DGN462 and IMGN779 has previously been demonstrated in vitro (K. Whiteman et. al, ASH 2014 #2321). Here we describe studies elucidating the mechanism of action of this novel payload in the AML cell lines: MV4-11, HL60, and EOL-1. The ability of DGN462 to alkylate DNA was demonstrated in an AML cell line by isolation and analysis of the genomic DNA from cells. DGN462 was found to co-purify with DNA, demonstrating that it covalently reacts with cellular DNA. A comet assay was performed confirming that DGN462 does not cross-link DNA.H2AX is known to be phosphorylated on serine 139 in response to DNA damage. Using flow cytometry, we assessed the phosphorylation of H2AX in AML cell lines following exposure to DGN462 and IMGN779. Increases in phosphorylated H2AX levels were detected as early as 4 hours following exposure to DGN462-SMe and 12 hours following exposure to IMGN779. Additional flow cytometry analysis showed that exposure to DGN462-SMe and IMGN779 leads to S-phase accumulation, G2/M arrest, followed by induction of apoptotic markers (cleaved PARP and cleaved Caspase-3) at later time points. Ex vivo studies using AML patient samples also showed elevation of phosphorylated H2AX and an increase in apoptosis in myeloid blasts following exposure to DGN462-SMe and IMGN779.As a demonstration of proof-of-mechanism in AML disease models, our results suggest that cell killing by IMGN779 is mediated by DNA damage, as a consequence of DNA alkylation. The DNA damage response is accompanied by cell cycle arrest, which leads to apoptosis. This relationship will be further explored in additional AML preclinical models to support the use of phosphorylated H2AX as a pharmacodynamic biomarker for IMGN779 activity in future clinical studies. DisclosuresKrystal:ImmunoGen, Inc.: Employment, Equity Ownership. Walker:ImmunoGen, Inc.: Employment, Equity Ownership. Fishkin:ImmunoGen, Inc.: Employment, Equity Ownership. Audette:ImmunoGen, Inc.: Employment, Equity Ownership. Kovtun:ImmunoGen, Inc.: Employment, Equity Ownership. Romanelli:ImmunoGen, Inc.: Employment, Equity Ownership.

Catch and release of concanavalin A by a mannose-immobilized photoaffinity PEGA resin coupled with a cleavable disulfide linker.

A photoaffinity PEGA resin containing mannose as a ligand and disulfide as a cleavable linker was prepared. The resin was crosslinked to concanavalin A, a binding protein of mannose, by UV irradiation, and the protein was subsequently released by cleavage of the disulfide linker.

In vitro controlled release of cisplatin from gold-carbon nanobottles via cleavable linkages.

Carbon nanotubes’ (CNTs) hollow interior space has been explored for biomedical applications, such as drug repository against undesirable inactivation. To further devise CNTs as smart material for controlled release of cargo molecules, we propose the concept of “gold-carbon nanobottles”. After encapsulating cis-diammineplatinum(II) dichloride (cisplatin, CDDP) in CNTs, we covalently attached gold nanoparticles (AuNPs) at the open-tips of CNTs via different cleavable linkages, namely hydrazine, ester, and disulfide-containing linkages. Compared with our previous study in which more than 80% of CDDP leaked from CNTs in 2 hours, AuNPs were found to significantly decrease such spontaneous release to <40%. In addition, CDDP release from AuNP-capped CNTs via disulfide linkage was selectively enhanced by twofolds in reducing conditions (namely with 1 mM dithiothreitol [DTT]), which mimic the intracellular environment. We treated human colon adenocarcinoma cells HCT116 with our CDDP-loaded gold-carbon nanobottles and examined the cell viability using lactate dehydrogenase assay. Interestingly, we found that our nanobottles with cleavable disulfide linkage exerted stronger cytotoxic effect in HCT116 compared with normal human fetal lung fibroblast cells IMR-90. Therefore, we infer that our nanobottles strategy with inbuilt disulfide linkage could attain selective release of payload in highly reductive tumor tissues while avoiding collateral damage to normal tissues.

Bioreducible Micelles Self-Assembled from Poly(ethylene glycol)-Cholesteryl Conjugate As a Drug Delivery Platform

The ability of polymeric micelles to self-assemble into nanosized particles has created interest in their application as potential anticancer drug delivery systems. A poly(ethylene glycol)-cholesteryl conjugate (Chol-ss-PEG-ss-Chol) connected by cleavable disulfide linkages was synthesized and used as a nanocarrier for in vitro release of doxorubicin (DOX). Owing to its amphiphilic structure, Chol-ss-PEG-ss-Chol was able to self-assemble into micelles with an average diameter 18.6 nm in aqueous solution. The micelles formed large aggregates due to the shedding of the PEG shell through cleavage of disulfide bonds in a reductive environment. The in vitro release studies revealed that Chol-ss-PEG-ss-Chol micelles released 80% and approximately 9% of the encapsulated DOX within 6 h under reductive and non-reductive conditions, respectively. The glutathione (GSH)-mediated intracellular drug delivery was investigated in a KB cell line. The cytotoxicity of DOX-loaded micelles indicated a higher cellular anti-proliferative effect against GSH-pretreated than untreated KB cells. Furthermore, confocal laser scanning microscopy (CLSM) measurement demonstrated that Chol-ss-PEG-ss-Chol micelles exhibited faster drug release in GSH-pretreated KB cells than untreated KB cells. These results suggest the potential usefulness of disulfide-based polymeric micelles as controlled drug delivery carriers.

Abstract 3400A: Development and application of an immunohistochemistry-based clinical assay for evaluating Folate Receptor Alpha (FRα) expression in a phase I clinical trial of IMGN853

Abstract A biomarker-based patient selection strategy, coupled with the co-development of a companion diagnostic, is key to the successful development of molecularly targeted cancer therapeutics. IMGN853 is a Folate Receptor Alpha (FRα)-targeting antibody-drug conjugate (ADC) consisting of an anti-FRα antibody linked to DM4, a highly cytotoxic maytansinoid, via a cleavable disulfide linker. FRα is a glycosyl-phosphatidylinositol-linked membrane protein commonly over-expressed in several solid tumor types including epithelial ovarian cancer (EOC), endometrial cancer and lung adenocarcinoma, with limited expression in normal tissues. Previously reported preclinical data demonstrated regression or significant inhibition of tumor growth by the conjugate in tumor models with clinically relevant levels of FRα expression. IMGN853 is currently in a phase I clinical trial for safety and tolerability in patients with FRα-positive solid tumors. Preliminary evidence of clinical activity has been observed in patients receiving IMGN853 treatment. Immunohistochemistry (IHC) is one of the most widely adopted techniques in clinical labs for semi-quantitative determination of protein expression in different cellular compartments. We have developed an IHC assay to support the FRα-expression based patient selection strategy in the clinical development of IMGN853. To this end, a novel murine monoclonal antibody with high specificity for human FRα was generated. This antibody, 353-2.1, is compatible with formalin fixed and paraffin embedded (FFPE) tissue samples and can recognize the same pool of FRα molecules as IMGN853, making it an ideal candidate for a companion diagnostic for IMGN853. The antibody was used to develop and optimize an assay that covers a broad dynamic range of FRα expression, allowing discrimination among high, moderate and low levels of expression. Data from tissue microarrays that cover a broad range of normal and tumor tissue types, as well as additional prevalence data in selected tumor types, were used to validate this assay. They also demonstrate that the FRα-expression data generated using this assay are consistent with previous findings. This clinical FRα IHC assay has been successfully used to measure FRα expression in more than 100 patient tumor samples submitted for testing in the ongoing IMGN853 phase I trial. A summary of FRα staining pattern and expression level data from these patients will be presented, as well as the prevalence in each tumor type. Citation Format: Jianhua Zhao, Alyssa LaBelle, Olga Ab, Krista Acosta, Al Yates, Yinghui Zhou, Angela Romanelli. Development and application of an immunohistochemistry-based clinical assay for evaluating Folate Receptor Alpha (FRα) expression in a phase I clinical trial of IMGN853. [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 3400A. doi:10.1158/1538-7445.AM2015-3400A

Structure‐Invertible Nanoparticles for Triggered Co‐Delivery of Nucleic Acids and Hydrophobic Drugs for Combination Cancer Therapy

Here, a new type of structure‐invertible, redox‐responsive polymeric nanoparticle for the efficient co‐delivery of nucleic acids and hydrophobic drugs in vitro and in vivo is reported for the first time, to combat the major challenges facing combination cancer therapy. The co‐delivery vector, which is prepared by conjugating branched poly(ethylene glycol) with dendrimers of two generations (G2) through disulfide linkages, is able to complex nucleic acids and load hydrophobic drugs with high loading capacity through structure inversion. The cleavage of disulfide linkages at intracellular glutathione‐rich reduction environment significantly decreases the cytotoxicity, and promotes more efficient drug release and gene transfection in vitro and in vivo. The co‐delivery carrier also displays enhanced endosomal escape capability and improved serum stability in vitro as compared with G2, and exhibits prolonged residence time and stronger transfection activity in vivo. Most importantly, co‐delivery of doxorubicin (DOX) and B‐cell lymphoma 2 (Bcl‐2) small interfering RNA (siRNA) exerts a combinational effect against tumor growth in murine tumor models in vivo, which is much more effective than either DOX or Bcl‐2 siRNA‐based monotherapy. The structure‐invertible nanoparticles may constitute a promising stimuli‐responsive system for the efficacious co‐delivery of multiple cargoes in future clinical applications of combination cancer therapies.