Resistance to Pyrrolobenzodiazepine Dimers Is Associated with SLFN11 Downregulation and Can Be Reversed through Inhibition of ATR.

Resistance to antibody-drug conjugates (ADCs) has been observed in both preclinical models and clinical studies. However, mechanisms of resistance to pyrrolobenzodiazepine (PBD)-conjugated ADCs have not been well characterized and thus, this study was designed to investigate development of resistance to PBD dimer warheads and PBD-conjugated ADCs. We established a PBD-resistant cell line, 361-PBDr, by treating human breast cancer MDA-MB-361 cells with gradually increasing concentrations of SG3199, the PBD dimer released from the PBD drug-linker tesirine. 361-PBDr cells were over 20-fold less sensitive to SG3199 compared with parental cells and were cross-resistant to other PBD warhead and ADCs conjugated with PBDs. Proteomic profiling revealed that downregulation of Schlafen family member 11 (SLFN11), a putative DNA/RNA helicase, sensitizing cancer cells to DNA-damaging agents, was associated with PBD resistance. Confirmatory studies demonstrated that siRNA knockdown of SLFN11 in multiple tumor cell lines conferred reduced sensitivity to SG3199 and PBD-conjugated ADCs. Treatment with EPZ011989, an EZH2 inhibitor, derepressed SLFN11 expression in 361-PBDr and other SLFN11-deficient tumor cells, and increased sensitivity to PBD and PBD-conjugated ADCs, indicating that the suppression of SLFN11 expression is associated with histone methylation as reported. Moreover, we demonstrated that combining an ataxia telangiectasia and Rad3-related protein (ATR) inhibitor, AZD6738, with SG3199 or PBD-based ADCs led to synergistic cytotoxicity in either resistant 361-PBDr cells or cells that SLFN11 was knocked down via siRNA. Collectively, these data provide insights into potential development of resistance to PBDs and PBD-conjugated ADCs, and more importantly, inform strategy development to overcome such resistance.

A Reactive Antibody Platform for One-Step Production of Antibody-Drug Conjugates through a Diels-Alder Reaction with Maleimide.

The normal electron-demand Diels-Alder (DA) cycloaddition is a classic transformation routinely used in synthesis; however, applications in biological systems are limited. Here, we report a spiro[2.4]hepta-4,6-diene-containing noncanonical amino acid (SCpHK) capable of efficient incorporation into antibodies and subsequent coupling with maleimide via a DA reaction. SCpHK was stable throughout protein expression in mammalian cells and enabled covalent attachment of maleimide drug-linkers yielding DA antibody-drug conjugates (DA-ADCs) with nearly quantitative conversion in a one-step process. The uncatalyzed DA reaction between SCpHK and maleimide in aqueous buffer was rapid (1.8-5.4 M-1 s-1), and the antibody-drug adduct was stable in rat serum for at least 1 week at 37 °C. Anti-EphA2 DA-ADCs containing AZ1508 or SG3249 maleimide drug-linkers were potent inhibitors of tumor growth in PC3 tumor models in vivo. The DA bioconjugation strategy described here represents a simple method to produce site-specific and stable ADCs with maleimide drug-linkers.

Synthesis and evaluation of pyrrolobenzodiazepine dimer antibody-drug conjugates with dual ß-glucuronide and dipeptide triggers.

Antibody-drug conjugates (ADCs) containing pyrrolobenzodiazepine (PBD) dimers are currently being evaluated in human oncology clinical trials with encouraging results. To further improve the therapeutic window, next-generation PBD drug-linker design has focused on the inclusion of additional tumor-selective triggers and use of lower-potency PBDs. ß-Glucuronidase is a well-known target for discovery prodrugs due to increased presence in tumor cells and microenvironment. In this study, a ß-glucuronidase cleavable cap was investigated at the PBD N10-position and compared with corresponding free imine ADCs. SG3600 (glucuronide) ADCs showed in vitro and in vivo efficacy/tolerability comparable to SG3400 (imine) ADCs, and good 50% inhibitory concentration differentials were observed in vitro between control non-antigen-targeted ADCs and targeted ADCs. Dependence on ß-glucuronidase for SG3600 activity was demonstrated through CRISPRCas9 knockdown studies and addition of exogenous ß-glucuronidase. SG3600 showed better serum stability, improved conjugation efficiency and was able to reach high drug-to-antibody ratio without aggregation.

Detecting bliss synergy in in vivo combination studies with a tumor kinetic model.

Linear models are generally reliable methods for analyzing tumor growth in vivo, with drug effectiveness being represented by the steepness of the regression slope. With immunotherapy, however, not all tumor growth follows a linear pattern, even after log transformation. Tumor kinetics models are mechanistic models that describe tumor proliferation and tumor killing macroscopically, through a set of differential equations. In drug combination studies, although an additional drug-drug interaction term can be added to such models, however, the drug interactions suggested by tumor kinetics models cannot be translated directly into synergistic effects. We have developed a novel statistical approach that simultaneously models tumor growth in control, monotherapy, and combination therapy groups. This approach makes it possible to test for synergistic effects directly and to compare such effects among different studies.

A Diene-Containing Noncanonical Amino Acid Enables Dual Functionality in Proteins: Rapid Diels-Alder Reaction with Maleimide or Proximity-Based Dimerization.

Here, we describe a diene-containing noncanonical amino acid (ncAA) capable of undergoing fast and selective normal electron-demand Diels-Alder (DA) reactions following its incorporation into antibodies. A cyclopentadiene derivative of lysine (CpHK) served as the reactive handle for DA transformations and the substrate for genetic incorporation. CpHK incorporated into antibodies with high efficiency and was available for maleimide conjugation or self-reaction depending on position in the amino acid sequence. CpHK at position K274 reacted with the maleimide drug-linker AZ1508 at a rate of ≈79 m-1 s-1 to produce functional antibody-drug conjugates (ADCs) in a one-step process. Incorporation of CpHK at position S239 resulted in dimerization, which covalently linked antibody heavy chains together. The diene ncAA described here is capable of producing therapeutic protein conjugates with clinically validated and widely available maleimide compounds, while also enabling proximity-based stapling through a DA dimerization reaction.

Potentiation of PBD Dimers by Lipophilicity Manipulation.

Background & Introduction: Pyrrolobenzodiazepine (PBD) dimers are highly potent DNA cross-linking agents used as warheads in Antibody Drug Conjugates (ADCs) for cancer therapy. We propose to investigate the correlation existing between the lipophilicity of those molecules and their activity (both in vitro and in vivo) as well as any effect observed during conjugation. MATERIALS AND METHODS: Reaction progress was monitored by Thin-Layer Chromatography (TLC) using Merck Kieselgel 60 F254 silica gel, with a fluorescent indicator on aluminium plates. Visualisation of TLC was achieved with UV light or iodine vapour unless otherwise stated. Flash chromatography was performed using Merck Kieselgel 60 F254 silica gel. RESULTS: We have successfully designed and synthesized a novel PBD warhead (SG3312) with enhanced physicochemical properties. The warhead also displayed increased potency in vitro. After overcoming some epimerization issues, the synthesis of enantiomerically pure payload was achieved (SG3259) and fulfilled our criteria for a simplified and more efficient conjugation. No addition of propylene glycol was required, and high DAR and excellent monomeric purity were achieved. CONCLUSION: The ADC (Herceptin-maia-SG3259) has been shown to release the active warhead (SG3312) upon exposure to Cathepsin B and demonstrated encouraging activity both in vitro and in vivo.

Improved Inhibition of Tumor Growth by Diabody-Drug Conjugates via Half-Life Extension.

Despite some clinical success with antibody-drug conjugates (ADCs) in patients with solid tumors and hematological malignancies, improvements in ADC design are still desirable due to the narrow therapeutic window of these compounds. Tumor-targeting antibody fragments have distinct advantages over monoclonal antibodies, including more rapid tumor accumulation and enhanced penetration, but are subject to rapid clearance. Half-life extension technologies such as PEGylation and albumin-binding domains (ABDs) have been widely used to improve the pharmacokinetics of many different types of biologics. PEGylation improves pharmacokinetics by increasing hydrodynamic size to reduce renal clearance, whereas ABDs extend half-life via FcRn-mediated recycling. In this study, we used an anti-oncofetal antigen 5T4 diabody conjugated with a highly potent cytotoxic pyrrolobenzodiazepine (PBD) warhead to assess and compare the effects of PEGylation and albumin binding on the in vivo efficacy of antibody fragment drug conjugates. Conjugation of 2× PEG20K to a diabody improved half-life from 40 min to 33 h, and an ABD-diabody fusion protein exhibited a half-life of 45 h in mice. In a xenograft model of breast cancer MDA-MB-436, the ABD-diabody-PBD showed greater tumor growth suppression and better tolerability than either PEG-diabody-PBD or diabody-PBD. These results suggest that the mechanism of half-life extension is an important consideration for designing cytotoxic antitumor agents.

Evaluation of Pyrrolobenzodiazepine-Loaded Nanoparticles: A Targeted Drug Delivery Approach.

We describe the development and evaluation of pyrrolobenzodiazepines (PBDs) in poly(dl-lactide-co-glycolide) and lipid nanoparticle drug delivery systems. We have established that the partition coefficient (LogP) of PBD is a key influencer of the encapsulation efficiency in nanoparticle systems, with higher LogP values associated with higher encapsulation efficiencies toward increased drug payload delivery and better antitumor efficacy. Cytotoxicity assays demonstrated that compounds with higher LogP values demonstrated higher 50% inhibitory concentration values than the free drug. In vivo efficacy studies in mice demonstrated that a single injection of nanoparticle PBD formulations could inhibit tumor growth for nearly 3 weeks, whereas the free drug failed to inhibit growth. Importantly, mice treated with PBD-loaded nanoparticles did not experience significant loss of body weight. These data demonstrate that nanoparticles containing PBD molecules can be used as an alternative to the widely used antibody drug conjugate approach in delivering cytotoxic PBDs.

Improved Therapeutic Window in BRCA-mutant Tumors with Antibody-linked Pyrrolobenzodiazepine Dimers with and without PARP Inhibition.

Pyrrolobenzodiazepine dimers (PBD) form cross-links within the minor groove of DNA causing double-strand breaks (DSB). DNA repair genes such as BRCA1 and BRCA2 play important roles in homologous recombination repair of DSB. We hypothesized that PBD-based antibody-drug conjugates (ADC) will have enhanced killing of cells in which homologous recombination processes are defective by inactivation of BRCA1 or BRCA2 genes. To support this hypothesis, we found 5T4-PBD, a PBD-dimer conjugated to anti-5T4 antibody, elicited more potent antitumor activity in tumor xenografts that carry defects in DNA repair due to BRCA mutations compared with BRCA wild-type xenografts. To delineate the role of BRCA1/2 mutations in determining sensitivity to PBD, we used siRNA knockdown and isogenic BRCA1/2 knockout models to demonstrate that BRCA deficiency markedly increased cell sensitivity to PBD-based ADCs. To understand the translational potential of treating patients with BRCA deficiency using PBD-based ADCs, we conducted a "mouse clinical trial" on 23 patient-derived xenograft (PDX) models bearing mutations in BRCA1 or BRCA2 Of these PDX models, 61% to 74% had tumor stasis or regression when treated with a single dose of 0.3 mg/kg or three fractionated doses of 0.1 mg/kg of a PBD-based ADC. Furthermore, a suboptimal dose of PBD-based ADC in combination with olaparib resulted in significantly improved antitumor effects, was not associated with myelotoxicity, and was well tolerated. In conclusion, PBD-based ADC alone or in combination with a PARP inhibitor may have improved therapeutic window in patients with cancer carrying BRCA mutations.

Pre-clinical pharmacology and mechanism of action of SG3199, the pyrrolobenzodiazepine (PBD) dimer warhead component of antibody-drug conjugate (ADC) payload tesirine.

Synthetic pyrrolobenzodiazepine (PBD) dimers, where two PBD monomers are linked through their aromatic A-ring phenolic C8-positions via a flexible propyldioxy tether, are highly efficient DNA minor groove cross-linking agents with potent cytotoxicity. PBD dimer SG3199 is the released warhead component of the antibody-drug conjugate (ADC) payload tesirine (SG3249), currently being evaluated in several ADC clinical trials. SG3199 was potently cytotoxic against a panel of human solid tumour and haematological cancer cell lines with a mean GI50 of 151.5 pM. Cells defective in DNA repair protein ERCC1 or homologous recombination repair showed increased sensitivity to SG3199 and the drug was only moderately susceptible to multidrug resistance mechanisms. SG3199 was highly efficient at producing DNA interstrand cross-links in naked linear plasmid DNA and dose-dependent cross-linking was observed in cells. Cross-links formed rapidly in cells and persisted over 36 hours. Following intravenous (iv) administration to rats SG3199 showed a very rapid clearance with a half life as short as 8 minutes. These combined properties of cytotoxic potency, rapid formation and persistence of DNA interstrand cross-links and very short half-life contribute to the emerging success of SG3199 as a warhead in clinical stage ADCs.

Fractionated Dosing Improves Preclinical Therapeutic Index of Pyrrolobenzodiazepine-Containing Antibody Drug Conjugates.

Purpose: To use preclinical models to identify a dosing schedule that improves tolerability of highly potent pyrrolobenzodiazepine dimers (PBDs) antibody drug conjugates (ADCs) without compromising antitumor activity.Experimental Design: A series of dose-fractionation studies were conducted to investigate the pharmacokinetic drivers of safety and efficacy of PBD ADCs in animal models. The exposure-activity relationship was investigated in mouse xenograft models of human prostate cancer, breast cancer, and gastric cancer by comparing antitumor activity after single and fractionated dosing with tumor-targeting ADCs conjugated to SG3249, a potent PBD dimer. The exposure-tolerability relationship was similarly investigated in rat and monkey toxicology studies by comparing tolerability, as assessed by survival, body weight, and organ-specific toxicities, after single and fractionated dosing with ADCs conjugated to SG3249 (rats) or SG3400, a structurally related PBD (monkeys).Results: Observations of similar antitumor activity in mice treated with single or fractionated dosing suggests that antitumor activity of PBD ADCs is more closely related to total exposure (AUC) than peak drug concentrations (Cmax). In contrast, improved survival and reduced toxicity in rats and monkeys treated with a fractionated dosing schedule suggests that tolerability of PBD ADCs is more closely associated with Cmax than AUC.Conclusions: We provide the first evidence that fractionated dosing can improve preclinical tolerability of at least some PBD ADCs without compromising efficacy. These findings suggest that preclinical exploration of dosing schedule could be an important clinical strategy to improve the therapeutic window of highly potent ADCs and should be investigated further. Clin Cancer Res; 23(19); 5858-68. ©2017 AACR.

Preclinical Evaluation of MEDI0641, a Pyrrolobenzodiazepine-Conjugated Antibody-Drug Conjugate Targeting 5T4.

Antibody-drug conjugates (ADC) are used to selectively deliver cytotoxic agents to tumors and have the potential for increased clinical benefit to cancer patients. 5T4 is an oncofetal antigen overexpressed on the cell surface in many carcinomas on both bulk tumor cells as well as cancer stem cells (CSC), has very limited normal tissue expression, and can internalize when bound by an antibody. An anti-5T4 antibody was identified and optimized for efficient binding and internalization in a target-specific manner, and engineered cysteines were incorporated into the molecule for site-specific conjugation. ADCs targeting 5T4 were constructed by site-specifically conjugating the antibody with payloads that possess different mechanisms of action, either a DNA cross-linking pyrrolobenzodiazepine (PBD) dimer or a microtubule-destabilizing tubulysin, so that each ADC had a drug:antibody ratio of 2. The resulting ADCs demonstrated significant target-dependent activity in vitro and in vivo; however, the ADC conjugated with a PBD payload (5T4-PBD) elicited more durable antitumor responses in vivo than the tubulysin conjugate in xenograft models. Likewise, the 5T4-PBD more potently inhibited the growth of 5T4-positive CSCs in vivo, which likely contributed to its superior antitumor activity. Given that the 5T4-PBD possessed both potent antitumor activity as well as anti-CSC activity, and thus could potentially target bulk tumor cells and CSCs in target-positive indications, it was further evaluated in non-GLP rat toxicology studies that demonstrated excellent in vivo stability with an acceptable safety profile. Taken together, these preclinical data support further development of 5T4-PBD, also known as MEDI0641, against 5T4+ cancer indications. Mol Cancer Ther; 16(8); 1576-87. ©2017 AACR.

Seroconversion rates among health care workers exposed to hepatitis C virus-contaminated body fluids: The University of Pittsburgh 13-year experience.

BACKGROUND: Hepatitis C virus (HCV) transmission to health care personnel (HCP) after exposure to a HCV-positive source has been reported to occur at an average rate of 1.8% (range, 0%-10%). We aimed to determine the seroconversion rate after exposure to HCV-contaminated body fluid in a major U.S. academic medical center. METHODS: A longitudinal analysis of a prospectively maintained database of reported occupational injuries occurring between 2002 and 2015 at the University of Pittsburgh Medical Center was performed. Data collected include type of injury and fluid, injured body part, contamination of sharps, resident physicians' involvement, and patients' hepatitis B virus (HBV), HCV, and HIV status. RESULTS: A total of 1,361 cases were included in the study. Most exposures were caused by percutaneous injuries (65.0%), followed by mucocutaneous injuries (33.7%). Most (63.3%) were injuries to the hand, followed by the face and neck (27.6%). Blood exposure accounted for 72.7%, and blood-containing saliva accounted for 3.4%. A total of 6.9% and 3.7% of source patients were coinfected with HIV and HBV, respectively. The HCV seroconversion rate was 0.1% (n = 2) because of blood exposure secondary to percutaneous injuries. CONCLUSIONS: This study provides the largest and most recent cohort from a major U.S. academic medical center. The seroconversion rates among HCP exposed to HCV-contaminated body fluids was found to be lower than most of the data found in the literature.

Seroconversion rate among health care workers exposed to HIV-contaminated body fluids: The University of Pittsburgh 13-year experience.

BACKGROUND: The studies enumerating the risk of HIV transmission to health care workers (HCWs) as 0.3% after percutaneous exposure to HIV-positive blood, and 0.09% after a mucous membrane exposure, are weakened by dated literature. Our study aims to demonstrate the seroconversion rate after exposure to HIV-contaminated body fluids in a major academic center in the United States. METHODS: A prospectively maintained database of reported occupational injuries occurring between 2002 and 2015 at an academic medical center was analyzed. Data collected included the type of injury, injured body part, type of fluid, contamination of sharps, involvement of resident physicians, use of postexposure prophylaxis, and patients' HIV, hepatitis B virus, and hepatitis C virus status. RESULTS: A total of 266 cases were included in the study. Most exposures were caused by percutaneous injuries (52.6%), followed by 43.2% mucocutaneous injuries. Of the injuries, 52.6% were to the hand and 33.5% to the face and neck. Blood exposure accounted for 64.3% of all cases. Of the patients, 21.1% received postexposure prophylaxis. None of the HCWs exposed to HIV-contaminated body fluids seroconverted (seroconversion rate, 0%). CONCLUSIONS: HIV does not seem to be as easily transmitted by needlestick, laceration, or splash injuries as previously surmised. Further large-scale and multicenter studies are needed for a more accurate estimation of the risk of transmission of HIV in U.S. health care workers.

Antibody-Drug Conjugates Bearing Pyrrolobenzodiazepine or Tubulysin Payloads Are Immunomodulatory and Synergize with Multiple Immunotherapies.

Immunogenic cell death (ICD) is the process by which certain cytotoxic drugs induce apoptosis of tumor cells in a manner that stimulates the immune system. In this study, we investigated whether antibody-drug conjugates (ADCS) conjugated with pyrrolobenzodiazepine dimer (PBD) or tubulysin payloads induce ICD, modulate the immune microenvironment, and could combine with immuno-oncology drugs to enhance antitumor activity. We show that these payloads on their own induced an immune response that prevented the growth of tumors following subsequent tumor cell challenge. ADCs had greater antitumor activity in immunocompetent versus immunodeficient mice, demonstrating a contribution of the immune system to the antitumor activity of these ADCs. ADCs also induced immunologic memory. In the CT26 model, depletion of CD8+ T cells abrogated the activity of ADCs when used alone or in combination with a PD-L1 antibody, confirming a role for T cells in antitumor activity. Combinations of ADCs with immuno-oncology drugs, including PD-1 or PD-L1 antibodies, OX40 ligand, or GITR ligand fusion proteins, produced synergistic antitumor responses. Importantly, synergy was observed in some cases with suboptimal doses of ADCs, potentially providing an approach to achieve potent antitumor responses while minimizing ADC-induced toxicity. Immunophenotyping studies in different tumor models revealed broad immunomodulation of lymphoid and myeloid cells by ADC and ADC/immuno-oncology combinations. These results suggest that it may be possible to develop novel combinatorial therapies with PBD- and tubulysin-based ADC and immuno-oncology drugs that may increase clinical responses. Cancer Res; 77(10); 2686-98. ©2017 AACR.

Pyrrolobenzodiazepine Antibody-Drug Conjugates Designed for Stable Thiol Conjugation.

Thiosuccinimide-linked antibody-drug conjugates (ADCs) are susceptible to drug loss over time due to a retro-Michael reaction, which can be prevented by selecting stable conjugation positions or hydrolysis of the thiosuccinimide. Here, we investigate pyrrolobenzodiazepine (PBD) ADC drug-linkers equipped with N-phenyl maleimide functionality for stable thiol conjugation via thiosuccinimide hydrolysis. Two PBD drug-linker formats (enzyme-cleavable and non-cleavable) were evaluated following site-specific conjugation to an engineered cysteine incorporated at position T289, which is known to be unstable for N-alkyl maleimide conjugates. N-phenyl maleimide PBDs conjugated to antibodies with similar efficiencies as N-alkyl maleimide PBDs and enhanced thiosuccinimide hydrolysis for N-phenyl maleimide PBDs was confirmed by mass spectrometry, capillary isoelectric focusing, and a SYPRO Orange dye binding assay. All of the PBD ADCs were highly potent in vitro regardless of maleimide- or linker-type, exhibiting low pM EC50 values. Thiol conjugation to N-phenyl maleimide PBD minimized the retro-Michael reaction in both rat and mouse serum. However, cleavage of the valine-alanine dipeptide in mouse serum for ADCs containing cleavable drug-linker led to drug loss regardless of maleimide type, which impacted ADC potency in tumor growth inhibition studies that were conducted in mouse models. Therapeutic improvement in mouse tumor models was realized for ADCs prepared with non-cleavable PBD drug-linkers that were conjugated through N-phenyl maleimide, where a stronger tumor growth inhibition (TGI) response was achieved when compared to the analogous N-alkyl maleimide drug-linker ADC. Altogether, our findings highlight the stability and efficacy benefits of N-phenyl maleimide functionality for ADCs that are produced with thiol-maleimide conjugation chemistry.

Structure-Cytotoxicity Relationships of Analogues of N14-Desacetoxytubulysin H.

Herein we report structure-cytotoxicity relationships for analogues of N14-desacetoxytubulyisn H 1. A novel synthetic approach toward 1 enabled the discovery of compounds with a range of activity. Calculated basicity of the N-terminus of tubulysins was shown to be a good predictor of cytotoxicity. The impact of structural modifications at the C-terminus of 1 upon cytotoxicity is also described. These findings will facilitate the development of new tubulysin analogues for the treatment of cancer.

Rational design, biophysical and biological characterization of site-specific antibody-tubulysin conjugates with improved stability, efficacy and pharmacokinetics.

Antibody-drug conjugates (ADCs) are among the most promising empowered biologics for cancer treatment. ADCs are commonly prepared by chemical conjugation of small molecule cytotoxic anti-cancer drugs to antibodies through either lysine side chains or cysteine thiols generated by the reduction of interchain disulfide bonds. Both methods yield heterogeneous conjugates with complex biophysical properties and suboptimal serum stability, efficacy, and pharmacokinetics. To limit the complexity of cysteine-based ADCs, we have engineered and characterized in vitro and in vivo antibody cysteine variants that allow precise control of both site of conjugation and drug load per antibody molecule. We demonstrate that the chemically-defined cysteine-engineered antibody-tubulysin conjugates have improved ex vivo and in vivo stability, efficacy, and pharmacokinetics when compared to conventional cysteine-based ADCs with similar drug-to-antibody ratios. In addition, to limit the non-target FcγRs mediated uptake of the ADCs by cells of the innate immune system, which may result in off-target toxicities, the ADCs have been engineered to lack Fc-receptor binding. The strategies described herein are broadly applicable to any full-length IgG or Fc-based ADC and have been incorporated into an ADC that is in phase I clinical development.

Stabilization of cysteine-linked antibody drug conjugates with N-aryl maleimides.

Maleimides are often used to covalently attach drugs to cysteine thiols for production of antibody-drug conjugates (ADCs). However, ADCs formed with traditional N-alkyl maleimides have variable stability in the bloodstream leading to loss of drug. Here, we report that N-aryl maleimides form stable antibody conjugates under very mild conditions while also maintaining high conjugation efficiency. Thiol-maleimide coupling and ADC stabilization via thiosuccinimide hydrolysis were accelerated by addition of N-phenyl or N-fluorophenyl groups to the ring-head nitrogen. Cysteine-linked ADCs prepared with N-aryl maleimides exhibited less than 20% deconjugation in both thiol-containing buffer and serum when incubated at 37 °C over a period of 7 days, whereas the analogous ADCs prepared with N-alkyl maleimides showed 35-67% deconjugation under the same conditions. ADCs prepared with the anticancer drug N-phenyl maleimide monomethyl-auristatin-E (MMAE) maintained high cytotoxicity following long-term exposure to serum whereas the N-alkyl maleimide MMAE ADC lost potency over time. These data demonstrate that N-aryl maleimides are a convenient and flexible platform to improve the stability of ADCs through manipulation of functional groups attached to the maleimide ring-head nitrogen.

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.