Diego A. Gianolio

Site-Specific Antibody–Drug Conjugation through Glycoengineering

Antibody-drug conjugates (ADCs) have been proven clinically to be more effective anti-cancer agents than native antibodies. However, the classical conjugation chemistries to prepare ADCs by targeting primary amines or hinge disulfides have a number of shortcomings including heterogeneous product profiles and linkage instability. We have developed a novel site-specific conjugation method by targeting the native glycosylation site on antibodies as an approach to address these limitations. The native glycans on Asn-297 of antibodies were enzymatically remodeled in vitro using galactosyl and sialyltransferases to introduce terminal sialic acids. Periodate oxidation of these sialic acids yielded aldehyde groups which were subsequently used to conjugate aminooxy functionalized cytotoxic agents via oxime ligation. The process has been successfully demonstrated with three antibodies including trastuzumab and two cytotoxic agents. Hydrophobic interaction chromatography and LC-MS analyses revealed the incorporation of ~1.6 cytotoxic agents per antibody molecule, approximating the number of sialic acid residues. These glyco-conjugated ADCs exhibited target-dependent antiproliferative activity toward antigen-positive tumor cells and significantly greater antitumor efficacy than naked antibody in a Her2-positive tumor xenograft model. These findings suggest that enzymatic remodeling combined with oxime ligation of the native glycans of antibodies offers an attractive approach to generate ADCs with well-defined product profiles. The site-specific conjugation approach presented here provides a viable alternative to other methods, which involve a need to either re-engineer the antibody sequence or develop a highly controlled chemical process to ensure reproducible drug loading.

Anti-Endosialin Antibody–Drug Conjugate: Potential in Sarcoma and Other Malignancies

Endosialin/TEM1/CD248 is a cell surface protein expressed at high levels by the malignant cells of about 50% of sarcomas and neuroblastomas. The antibody–drug conjugate (ADC) anti-endosialin-MC-VC-PABC-MMAE was selectively cytotoxic to endosialin-positive cells in vitro and achieved profound and durable antitumor efficacy in preclinical human tumor xenograft models of endosialin-positive disease. MC-VC-PABC-MMAE was conjugated with anti-endosialin with 3–4 MMAE molecules per ADC. The anti-endosialin-MC-VC-PABC-MMAE conjugate was tested for activity in four human cell lines with varied endosialin levels. The HT-1080 fibrosarcoma cells do not express endosialin, A-673 Ewing sarcoma cells and SK-N-AS neuroblastoma cells are moderate expressers of endosialin, and SJSA-1 osteosarcoma cells express very high levels of endosialin. To determine whether endosialin expression was maintained in vivo, A-673 Ewing sarcoma, SK-N-AS neuroblastoma, and SJSA-1 osteosarcoma cells were grown as xenograft tumors in nude mice. The SK-N-AS neuroblastoma and the A-673 Ewing sarcoma lines were selected for in vivo efficacy testing of the anti-endosialin-MC-VC-PABC-MMAE conjugate. The treatment groups included a vehicle control, unconjugated anti-endosialin, an admix control consisting of anti-endosialin and a dose of free MMAE equivalent to the dose administered as the ADC, and the anti-endosialin-MC-VC-PABC-MMAE conjugate. The unconjugated anti-endosialin had no antitumor activity and resulted in similar tumor growth as the vehicle control. The admix control produced a modest tumor growth delay. Administration of the anti-endosialin-MC-VC-PABC-MMAE conjugate resulted in a marked prolonged tumor response of both xenograts. These proof-of-concept results break new ground and open a promising drug discovery approach to these rare and neglected tumors. Mol Cancer Ther; 14(9); 2081–9. ©2015 AACR.

Well-defined aminooxy terminated N-(2-hydroxypropyl) methacrylamide macromers for site specific bioconjugation of glycoproteins.

Syntheses and characterization of aminooxy terminated polymers of N-(2-hydroxyproyl) methacrylamide (HPMA) of controlled molecular weight and narrow molecular weight distribution are presented here. Design of a chain transfer agent (CTA) containing N-tert-butoxycarbonyl (t-Boc) protected aminooxy group enabled us to use reversible addition-fragmentation (RAFT) polymerization technique to polymerize the HPMA monomer. An amide bond was utilized to link the aminooxy group and the CTA through a triethylene glycol spacer. As a result, the aminooxy group is linked to the poly(HPMA) backbone through a hydrolytically stable amide bond. By varying the monomer to initiator ratios, polymers with targeted molecular weights were obtained. The molecular weights of the polymers were determined by gel permeation chromatography (GPC) and mass spectrometry (ESI and MALDI-TOF). The t-Boc protecting group was quantitatively removed to generate aminooxy terminated poly(HPMA) macromers. These macromers were converted to rhodamine B terminated poly(HPMA) by reacting N-hydroxysuccinimide (NHS) ester of the dye with the terminal aminooxy group to form a stable alkoxyamide bond. Utility of these dye-labeled polymers as molecular probes was evaluated by fluorescence microscopy by studying their intracellular uptake by renal epithelial cells. These aminooxy terminated poly(HPMA) were also tested as biocompatible carriers to prepare chemoselective bioconjugates of proteins using transferrin (Tf) as the protein. Oxidation of the sialic acid side chains of Tf generated aldehyde functionalized protein that was reacted with aminooxy terminated poly(HPMA), which resulted in protein-polymer bioconjugates carrying oxime linkages. These bioconjugates were characterized by gel electrophoresis and MALDI-TOF mass spectrometry.

Drug Delivery and Medical Applications of Chemically Modified Hyaluronan

Publisher Summary Hyaluronan (HA) is a linear naturally occurring polyanionic polysaccharide that is ubiquitous in nature and is produced virtually by every tissue in higher organisms and some bacteria. HA has excellent biocompatibility and is readily catabolized and cleared in vivo. For these reasons, there has been a significant commercial focus on the development of products either from HA or from chemically modified derivatives of HA. HA and HA plus chemically cross-linked HA preparations are the principal components in several viscosupplements for patients with early-stage osteoarthritis of the knee. Conjugation of drugs to HA holds great promise for the generation of a new class of polymer-based therapeutics. These polymer-based systems can serve not only a biomaterials-based function, such as the separation or bulking of tissue, but also as concomitant drug delivery systems for the local delivery of therapeutics. Drugs conjugated to HA could also serve to target the drug to cells or tissues in the body that are rich in HA-binding receptors, such as CD44, RHAMM, TLR2, and TLR4. One of the most promising uses of chemically modified HA is in the area of tissue engineering. HA is found in the extracellular matrix of virtually all tissues so its use as a cell delivery vehicle is obvious. The combination of cell delivery and drug attachment to HA offers a very versatile material for the development of sophisticated products to address complex and unmet medical needs.

Hyaluronan-Tethered Opioid Depots: Synthetic Strategies and Release Kinetics In Vitro and In Vivo

We proposed the use of opioid drug bound covalently to hyaluronan (HA) via ester linkages as a method to prolong drug delivery and to possibly increase the quality of perioperative pain management. The in vitro release profile of morphine conjugated to HA (1.3 million MW) was studied. The influence of parameters such as conjugation site and steric protection of the labile ester bonds was investigated in phosphate buffered saline (PBS) medium. HA--codeine and HA--naloxone conjugates were used as structural controls. Codeine and morphine conjugated via the allylic hydroxyl group had a release half-life of 14.0 days in PBS. Naloxone conjugated via the phenolic hydroxyl group showed a half-life of 0.3 days, and all drugs admixed in HA showed half-lives of 0.1 days. Methyl, ethyl, or n-propyl introduced in vicinal position to the ester bond prolonged release of naloxone with half-lives of 0.5, 4.0, and 4.0 days in PBS, respectively. Incorporation of a methyl group prolonged codeine release with a half-life of 55.0 days in PBS. Drugs were released chemically unaltered from the conjugates as confirmed by LC-MS/MS. Further, morphine was conjugated to divinylsulfone cross-linked HA (Hylan B) particles and the release profiles in rat plasma were studied in vitro and in vivo. Release in rat plasma was faster than in PBS with a half-life of 2.5 days, but the release was similar (ca. 12 days) when a cocktail of protease inhibitors was added to the plasma. Sustained release of morphine was observed in a rat surgical model over 30 h. Morphine was released chemically unaltered from the conjugate and morphine intermediates were not detected in significant amounts as confirmed by LC-MS/MS. These results suggest that the morphine release profile from the HA conjugates depends on the alkyl groups vicinal to the ester and the nature of the leaving group. In rat plasma, hydrolysis seems to be controlled by esterase activity.

Bio)polymeric Hydrogels as Therapeutic Agents

Hydrogels derived from both natural and synthetic polymers have gained significant scientific attention in recent years for their potential use as biomedical materials to treat human diseases. While a great deal of research efforts have been directed towards investigating polymeric hydrogels as matrices for drug delivery systems, examples of such hydrogels exhibiting intrinsic therapeutic properties are relatively less common. Characteristics of synthetic and natural polymers such as high molecular weight, diverse molecular architecture, chemical compositions, and modulated molecular weight distribution are unique to polymers. These characteristics of polymers can be utilized to discover a new generation of drugs and medical devices. For example, polymeric hydrogels can be restricted to the gastrointestinal tract, where they can selectively recognize, bind, and remove the targeted disease-causing substances from the body without causing any systemic toxicity that are associated with traditional small molecule drugs. Similarly hydrogels can be implanted at specific locations (such as knee and abdomen) to impart localized therapeutic benefits. The present article provides an overview of certain recent developments in the design and synthesis of functional hydrogels that have led to several polymer derived drugs and biomedical devices. Some of these examples include FDA-approved marketed products.

Polymer Based Therapies for the Treatment of Chronic Pain

Since chronic pain manifests functional limitation, it is the leading cause of longer term disability [1, 2]. In the US alone, an estimated 75 million people suffer from chronic pain [3]. In addition to chronic pain, proper management of postoperative acute pain impacts the clinical outcome of patients undergoing surgery [4]. Opioid family of analgesics and nonsteroidal anti-inflammatory drugs (NSAIDs) are the main stays of current pharmacological agents available for the management of chronic pain [5, 6]. However, current therapies for pain management show modest efficacy and are associated with significant side effects. The major adverse effects of oral NSAIDs are gastrointestinal bleeding, gastric ulcer, renal failure and cardiovascular risks (in particular with selective COX-2 inhibitors) [7, 8]. The side effects of opioid family therapies include constipation, nausea, cognitive impairment and most importantly addiction [9, 10]. Thus, development of safer and effective treatment of chronic pain is an important goal of current pharmaceutical research. In recent years numerous efforts have been made to develop long-acting opioid analgesics and NSAIDs to modulate their pharmacokinetic profiles. Some of these include sustained release formulations and topical gels [11, 12]. Biological agents such as antibody against nerve growth factor (NGF) have also been evaluated as therapies for chronic pain. The antiNGF antibody acts by sequestering NGF and thus inhibits its interaction with the NGFreceptor on the sensory neurons [13]. Polymeric approach offers an attractive route to develop novel therapeutic agents for effective management of chronic pain. Interesting physical and chemical characteristics of synthetic and natural polymers enable them as promising materials for biomedical applications such as therapeutic agents, drug delivery carriers, and medical devices [14, 15]. A number of polymer derived therapies have been commercialized in the marketplace [16, 17]. The present article reviews the current state of research and development efforts to discover and develop biomedical polymer as therapeutic agents for the treatment of chronic pain. While use of polymer-derived agents for the treatment of different kinds of pains will be highlighted, the primary focus of the present article pertains to management of pain arising from osteoarthritis. Furthermore, role of polymers as intrinsically pain relieving agents either alone or as chemical conjugates of low molecular weight pain modulating agents are described in this article. The research and development efforts to develop control release formulations of low molecular weight pain therapies are outside the scope of this article. There are in fact a number of interesting articles that describe this aspect of pain management therapies [18, 19].

Abstract C80: Characterizing models for personalized medicine: Endosialin in sarcoma from cultured cells to mice to humans

A survey of endosialin by IHC was previously conducted in 250 clinical specimens of cancer, revealing a key difference between sarcomas and carcinomas: in sarcomas, endosialin was expressed in tumor cells, stromal fibroblasts and perivascular cells, while in carcinomas tumor cells were negative. Endosialin expression was frequent among sarcoma patients and reached high levels: 70 of 86 sarcomas (81%) were positive for endosialin, with 44 (51%) displaying at least 50% coverage of all 3 immunoreactive cell types as a whole. Staining intensity was scored on the scale 0, 1+, 2+, 3+: all 9 sarcoma subtypes surveyed included specimens reaching 2+ and 3+. Endosialin emerged from that study as a potential molecular target for sarcomas. However, despite frequent expression and high levels observed in patients, some sarcomas are endosialin‐negative and some express low levels. Therefore, targeting endosialin would mandate a personalized medicine approach where patients are tested for endosialin expression in order to determine eligibility for endosialin‐directed therapy. We characterized the expression of endosialin in human tumor cell lines in vitro and in vivo, from mRNA and protein in cell culture to protein in xenograft tumors. Thirty‐seven human sarcoma cell lines and one neuroblastoma cell line were assayed for endosialin by real‐time PCR and flow cytometry. Eighteen cell lines were positive for the protein at various levels. There was a positive correlation between transcript and protein expression, with a few exceptions: 3 cell lines expressed very low levels of the transcript yet clearly detectable protein and one cell line expressed detectable transcript yet no protein. Fifteen of the 18 cell lines positive for the protein in vitro were implanted in mice subcutaneously. Eight of the 15 cell lines implanted failed to form tumors. Seven formed tumor nodules that were excised and tested for endosialin by IHC using the same assay as that used for clinical specimens. Protein expression was not always as robust in vivo as the in vitro expression might have suggested. Staining in the xenograft tumors was scored on the same scale as the clinical specimens. Some models showed robust and homogeneous expression throughout the tumor tissue (SK‐N‐AS neuroblastoma), while others showed highly heterogeneous staining. In one model representing extreme heterogeneity (SJSA‐1 osteosarcoma), endosialin was highly regional, with areas staining positive at the 3+ intensity level and adjacent areas being negative. Both homogeneous and heterogeneous staining was seen in human clinical specimens. Our work demonstrates the importance of characterizing disease models to ascertain target expression at all stages of testing from in vitro to mice and to humans, since expression in vivo cannot be assumed from expression in cell culture and expression patterns in mice may or may not be similar to expression patterns in humans. Citation Information: Mol Cancer Ther 2009;8(12 Suppl):C80.

Design, Synthesis, and in vitro Evaluation of Multivalent Drug Linkers for High‐Drug‐Load Antibody–Drug Conjugates

A series of novel multivalent drug linkers (MDLs) containing cytotoxic agents were synthesized and conjugated to antibodies to yield highly potent antibody–drug conjugates (ADCs) with drug/antibody ratios (DARs) higher than those typically reported in the literature (10 vs. ≈4). These MDLs contain two copies of a cytotoxic agent attached to biocompatible scaffolds composed of a branched peptide core and discrete polyethylene glycol (PEG) chains to enhance solubility and decrease aggregation. These drug linkers produced well‐defined ADCs, whose DARs could be accurately determined by LC–MS. Using this approach, ADCs with significantly lower aggregation and higher DAR than those of conventional drug linker design were obtained with highly hydrophobic cytotoxic agents such as monomethyldolastatin 10 (MMAD). The in vitro potencies of the MDL‐derived conjugates matched that of ADCs of similar DAR with conventional linkers, and the potency increased proportionally with drug loading. This approach may provide a means to prepare highly potent ADCs from a broader range of drugs, including those with lower cytotoxicity or poor solubility, which otherwise limits their use for antibody–drug conjugates. This may also provide a means to further improve the potency achievable with cytotoxins currently used in ADCs.

Abstract 2140: Cabazitaxel is more active than first generation taxanes in ABCB1 (+) cell lines due to its higher intracellular accumulation and reduction in ATPase stimulation

The taxanes paclitaxel (Taxol) and docetaxel (Taxotere) have substantial clinical activity in breast, ovarian, lung, and other cancers, but their clinical efficacy is limited by preexisting or acquired drug resistance, such as the expression of the ATP-dependent multidrug resistance (MDR) transporter P-glycoprotein (P-gp). Cabazitaxel (Jevtana), the dimethoxy derivative of docetaxel, was identified because of its activity in taxane-resistant models both in vitro and in vivo. Our studies confirm that cabazitaxel is more active in cell variants that express P-gp, and the objective of this study was to investigate cabazitaxel9s affinity for the transporter. Taxane accumulation patterns were studied in ABCB1(+) variants using [ 14 C]-radiolabeled docetaxel and cabazitaxel over a time course up to 1 h. Time points were collected and spun (10,000 x g, 1 min) through Nyosil M20 oil thereby terminating uptake, cell pellets lysed with 2% (w/v) SDS, and counts determined by liquid scintillation, with all measurements normalized to protein content. The kinetics of drug accumulation between the two taxanes is different, with the maximum intracellular drug concentration achieved faster with cabazitaxel (5 min) than docetaxel (15-30 min) in all cell lines. ABCB1(+) variants accumulated less taxane, and these levels could be restored to parental levels in the presence of known P-gp inhibitors (2 μM PSC-833, valspodar). However, the MDR cell models tested accumulated twice as much cabazitaxel than docetaxel under identical experimental conditions. Efflux in drug-free medium confirmed that ABCB1(+) variants retained 2.2x more cabazitaxel than docetaxel. We observed a strong association (r 2 = 0.91) between the degree of taxane resistance conferred by P-gp expression and the accumulation differences observed with the two taxanes. Several low P-gp-expressing cell models were not cross-resistant to cabazitaxel while demonstrating resistance to docetaxel. Furthermore, we determined ATPase stimulation in membranes isolated from our MDR cell models as an indirect measure of P-gp activity following taxane treatment. We observed a 1.9x reduction in sodium orthovanadate-sensitive ATPase stimulation resulting from treatment with cabazitaxel than with docetaxel (1 μM for 30 min). Future experiments will include direct photoaffinity labeling of P-gp with [ 3 H]-azido-cabazitaxel and docetaxel, and competition assays with known P-gp substrates. Our studies indicate that the improved activity of cabazitaxel in MDR models might be due to its reduced affinity for P-gp compared to docetaxel. Citation Format: George E. Duran, Dietmar Weitz, Dorothee Semiond, Diego Gianolio, Sandrine Mace, Branimir I. Sikic. Cabazitaxel is more active than first generation taxanes in ABCB1 (+) cell lines due to its higher intracellular accumulation and reduction in ATPase stimulation. [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 2140.