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Antibody–drug conjugates (ADC) are a targeted cancer therapy that utilize the specificity of antibodies to deliver potent drugs selectively to tumors. Here we define the complex interaction among factors that dictate ADC efficacy in neuroblastoma by testing both a comprehensive panel of ADC payloads in a diverse set of neuroblastoma cell lines and utilizing the glypican 2 (GPC2)-targeting D3-GPC2-PBD ADC to study the role of target antigen density and antibody internalization in ADC efficacy in neuroblastoma. We first find that DNA binding drugs are significantly more cytotoxic to neuroblastomas than payloads that bind tubulin or inhibit DNA topoisomerase 1. We additionally show that neuroblastomas with high expression of the ABCB1 drug transporter or that harbor a TP53 mutation are significantly more resistant to tubulin and DNA/DNA topoisomerase 1 binding payloads, respectively. Next, we utilized the GPC2-specific D3-GPC2-IgG1 antibody to show that neuroblastomas internalize this antibody/GPC2 complex at significantly different rates and that these antibody internalization kinetics correlate significantly with GPC2 cell surface density. However, sensitivity to pyrrolobenzodiazepine (PBD) dimers primarily dictated sensitivity to the corresponding D3-GPC2-PBD ADC, overall having a larger influence on ADC efficacy than GPC2 cell surface density or antibody internalization. Finally, we utilized GPC2 isogenic Kelly neuroblastoma cells with different levels of cell surface GPC2 expression to define the threshold of target density required for ADC efficacy. Taken together, DNA binding ADC payloads should be prioritized for development for neuroblastoma given their superior efficacy and considering that ADC payload sensitivity is a major determinant of ADC efficacy.

Benzofused seven-membered heterocycles such as 1,4-benzo[e]diazepines (1,4-BZDs) and 1,4-benzo[e]oxazepines (1,4-BZOs) were efficiently synthesized by Rh-catalyzed hydrofunctionalization of internal alkynes and allenes in good to excellent yields. The asymmetric hydroamination of (aminomethyl)anilines gave rise to 3-vinyl-1,4-BZDs with excellent enantioselectivities. Orthogonal N-deprotection of 1,4-BZDs allowed an easy entry to an advanced pyrrolobenzodiazepine metabolite of the V2-receptor antagonist Lixivaptan® This work has received financial support from MINECO (project CTQ2017-87939R and ORFEO-CINQA network RED2018-102387-T), the Xunta de Galicia (project ED431C 2018/04 and Centro singular de investigación de Galicia accreditation 2019-2022, ED431G 2019/03) and the European Union (European Regional Development Fund – ERDF). A.V.-R. thanks Xunta de Galicia for a predoctoral fel-lowship (ED481A-2018/34, 2018-2021). SI

Loncastuximab tesirine (loncastuximab tesirine-lpyl; ZYNLONTA™) is an antibody-drug conjugate being developed for the treatment of B cell lymphomas by ADC Therapeutics SA. Loncastuximab tesirine consists of a pyrrolobenzodiazepine DNA-alkylating warhead covalently attached via a cleavable linker to an anti-CD19 antibody that binds to B cells. It is currently approved in the US for the treatment of relapsed/refractory diffuse large B cell lymphoma (DLBCL), and is being developed for the treatment of mantle-cell lymphoma, follicular lymphoma and acute lymphoblastic leukaemia. This article summarizes the milestones in the development of loncastuximab tesirine leading to this first approval for relapsed or refractory large B-cell lymphoma after two or more lines of systemic therapy, including DLBCL not otherwise specified (NOS), DLBCL arising from low grade lymphoma, and high-grade B-cell lymphoma.

Pyrrolobenzodiazepine (PBD) dimers, such as that found in SG3259, are fully synthetic highly potent (subnanomolar) small molecules currently being developed as warheads for antibody–drug conjugates...

ABBV-321 (serclutamab talirine), a next-generation EGFR-targeted antibody–drug conjugate (ADC) incorporates a potent pyrrolobenzodiazepine (PBD) dimer toxin conjugated to the EGFR-targeting ABT-806 affinity-matured AM1 antibody. ABBV-321 follows the development of related EGFR-targeted ADCs including depatuxizumab mafodotin (depatux-m, ABT-414), ABT-806 conjugated to monomethyl auristatin F (MMAF), and ABBV-221 (losatuxizumab vedotin), AM1 antibody conjugated to monomethyl auristatin E (MMAE). The distinct tumor selectivity of ABBV-321 differentiates it from many previous highly active antibody PBD conjugates that lack a therapeutic window. Potency of the PBD dimer, combined with increased binding of AM1 to EGFR-positive tumor cells, opens the possibility to target a wide array of tumors beyond those with high levels of EGFR overexpression or amplification, including those insensitive to auristatin-based ADCs. ABBV-321 exhibits potent antitumor activity in cellular and in vivo studies including xenograft cell line and patient-derived xenograft glioblastoma, colorectal, lung, head and neck, and malignant mesothelioma tumor models that are less sensitive to depatux-m or ABBV-221. Combination studies with ABBV-321 and depatux-m suggest a promising treatment option permitting suboptimal, and potentially better tolerated, doses of both ADCs while providing improved potency. Collectively, these data suggest that ABBV-321 may offer an extended breadth of efficacy relative to other EGFR ADCs while extending utility to multiple EGFR-expressing tumor indications. Despite its highly potent PBD dimer payload, the tumor selectivity of ABBV-321, coupled with its pharmacology, toxicology, and pharmacokinetic profiles, support continuation of ongoing phase I clinical trials in patients with advanced EGFR-expressing malignancies.

c-Met, as a receptor expressed on the cell membrane, contributes to the growth and metastasis of tumors, as well as angiogenesis, mainly through the hepatocyte growth factor (HGF)/c-Met axis during tumor progression. Although several c-Met inhibitors, including small molecules and monoclonal antibody inhibitors, are currently being investigated, their clinical outcomes have not been promising. Development of an antibody–drug conjugate (ADC) against c-Met could be an attractive therapeutic strategy that would provide superior antitumor efficacy with broad-spectrum c-Met expression levels. In the present study, site-specific drug–conjugate technology was applied to develop an ADC using the human-mouse cross-reactive c-Met antibody and a prodrug pyrrolobenzodiazepine (PBD). The toxin payload was uniformly conjugated to the light-chain C-terminus of the native cIRCR201 antibody (drug-to-antibody ratio = 2), as confirmed using LC–MS. Using a high-throughput screening system, we found that cIRCR201-dPBD exhibited varying sensitivities depending on the expression levels of c-Met, and it induced receptor-mediated endocytosis and toxin-mediated apoptosis in 47 different cancer cell lines. cIRCR201-dPBD also showed significant antitumor activity on the MET-amplified cancer cells using in vivo xenograft models. Therefore, cIRCR201-dPBD could be a promising therapeutic strategy for tumors with c-Met expression.

Tilimycin is an enterotoxin produced by the opportunistic pathogen Klebsiella oxytoca that causes antibiotic-associated hemorrhagic colitis (AAHC). This pyrrolobenzodiazepine (PBD) natural product is synthesized by a bimodular nonribosomal peptide synthetase (NRPS) pathway comprised of three proteins: NpsA, ThdA and NpsB. We describe the functional and structural characterization of the fully reconstituted NRPS system and report the steady-state kinetic analysis of all natural substrates and cofactors as well as the structural characterization of both NpsA and ThdA. The mechanism of action of tilimycin was confirmed using DNA adductomics techniques through the detection of putative N-2 guanine alkylation after tilimycin exposure to eukaryotic cells, providing the first structural characterization of a PBD-DNA adduct formed in cells. Finally, we report the rational design of small-molecule inhibitors that block tilimycin biosynthesis in whole cell K. oxytoca (IC(50) = 29 ± 4 μM) through the inhibition of NpsA (K(D) = 29 ± 4 nM).

Accurate DNA quantitation is a prerequisite in many biomedical and pharmaceutical studies. Here we established a new DNA quantitation method by nuclease P1 digestion and UPLC-MS/MS analysis. DNA fragments can be efficiently hydrolyzed to single deoxyribonucleotides by nuclease P1 in a short time. The decent stabilities of all the four deoxyribonucleotides were confirmed under different conditions. Deoxyadenosine monophosphate (dAMP) was selected as the surrogate for DNA quantitation because dAMP showed the highest sensitivity among the four deoxyribonucleotides in the UPLC-MS/MS analysis. The linear range in DNA quantitation by this method is 1.2–5000 ng/mL. In the validation, the inter-day and intra-day accuracies were within 90%–110%, and the inter-day and intra-day precision were acceptable (RSD, Graphical abstract Image 1, Highlights • A novel method to evaluate DNA binding efficiency of the DNA alkylator PBD in tumors of mouse models is reported. • The DNA isolation, DNA digestion and the LC/MS quantitation of both DNA and PBD are involved. • The method with an enhanced sensitivity allows for the accurate quantitation of isolated DNA from various tissues.

A comprehensive screening of known methods for the synthesis of coumarins was performed in order to obtain a new type of pyrrolobenzodiazepine–coumarin hybrids. A Pechmann condensation turned out to be the method of choice for successful synthesis of the desired hybrid starting from the corresponding phenol derivative and malic acid. Overall, the desired pyrrolobenzodiazepine–coumarin hybrid was obtained in a 3 step sequence starting from readily available starting materials – L-proline methyl ester and 4-methoxyanthranilic acid.

The synthesis of three novel families of pyrrolo[2,1-c][1,4]benzodiazepine-5-ones is described. The compounds were prepared according to a three-step sequence, involving an Ugi reaction, building of the pyrrolo nucleus, and reduction–cyclization to the corresponding diazepine. Depending on the amine employed in the synthesis of the Ugi adducts, different unsaturation degrees could be obtained in the pyrrolo ring (saturated or with endo or exo unsaturations), a key feature determining their biological activity, as it affects the affinity of the pyrrolobenzodiazepines toward DNA and thus their cytotoxicity. This synthetic methodology represents a significant improvement with respect to those described in the literature so far, as it uses inexpensive and commercially available starting materials without needing derivatization or the use of protecting groups., Consejería de Educación de la Junta de Castilla y León (project BU075G19)

The development of new antitubercular agents for the treatment of infections caused by multidrug-resistant (MDR) Mycobacterium tuberculosis is an urgent priority. Pyrrolobenzodiazepines (PBDs) are a promising class of antibacterial agents that were initially discovered and isolated from a range of Streptomyces species. Recently, C8-linked PBD monomers have been shown to work by inhibiting DNA gyrase and have demonstrated activity against M. tuberculosis. However, both PBD monomers and dimers are toxic to eukaryotic cells, limiting their development as antibacterial agents. To eliminate the toxicity associated with PBDs and explore the effect of C8-modification with a known antibacterial agent with the same mechanism of action (i.e., ciprofloxacin, a gyrase inhibitor), we synthesized a C8-linked PBD-ciprofloxacin (PBD-CIP, 3) hybrid. The hybrid compound displayed minimum inhibitory concentration values of 0.4 or 2.1 μg/mL against drug-sensitive and drug-resistant M. tuberculosis strains, respectively. A molecular modeling study showed good interaction of compound 3 with wild-type M. tuberculosis DNA gyrase, suggesting gyrase inhibition as a possible mechanism of action. Compound 3 is a nontoxic combination hybrid that can be utilized as a new scaffold and further optimized to develop new antitubercular agents.

Introduction: Novel approaches to treating patients (pts) with relapsed or refractory (R/R) classical Hodgkin lymphoma (cHL) have improved outcomes but some pts do not respond or, despite initial response, develop progressive disease and have limited treatment options. Camidanlumab tesirine (ADCT-301; Cami) is an antibody-drug conjugate composed of a human IgG1 anti-CD25 monoclonal antibody stochastically conjugated to a potent pyrrolobenzodiazepine (PBD) dimer warhead, which triggers cell death via formation of highly cytotoxic interstrand cross-links. Data from a Phase (Ph) 1 dose-escalation, dose-expansion trial demonstrated an overall response rate (ORR) in pts with cHL of 86.5% (48.6% complete response [CR] rate) at the 45 μg/kg dose. Cami had a generally acceptable safety profile at Ph 1 but there were 5/77 cases (6.5%) of Guillain-Barré syndrome (GBS)/polyradiculopathy (Preferred Terms: 4 GBS and 1 radiculopathy) (Collins et al, ICML June 18-22, 2019, Lugano, Switzerland, Abstract 055). Here, we present preliminary efficacy and safety results of a Ph 2 trial of single-agent Cami in pts with R/R cHL (NCT04052997). Methods: A single-arm, multi-center, open-label, Ph 2 trial is currently enrolling pts ≥16 yrs (US) and ≥18 yrs (outside US) with R/R cHL following ≥3 prior treatment lines (or ≥2 lines in pts ineligible for hematopoietic stem cell transplantation). Eligible pts had prior treatment with brentuximab vedotin and PD-1 blockade, measurable disease per 2014 Lugano Classification, and Eastern Cooperative Oncology Group performance status 0-2. The primary objective is to evaluate efficacy of single-agent Cami by ORR as determined by central review. Secondary objectives include further characterization of additional efficacy endpoints and safety. Pts receive 30-min IV infusions of Cami on Day 1 of each 3-week cycle at a dose of 45 μg/kg for 2 cycles, followed by 30 μg/kg for subsequent cycles for up to 1 yr or until discontinuation due to disease progression, unacceptable toxicity, or other reasons. Pts deriving clinical benefit at 1 yr may be able to continue treatment on a case-by-case basis. Treatment-emergent adverse events (TEAEs) were defined as AEs occurring/worsening from time of first dose to either 30 days post last dose or to start of new anticancer therapy/procedure, whichever occurs first. This analysis was conducted after meeting a protocol-specified criterion for pausing enrollment (≥2 cases of GBS or other relevant severe neurologic toxicity). Results: As of June 15, 2020, 47 pts with R/R cHL were enrolled and are included in this analysis. Median age was 36 (range 23-74) yrs and pts had received a median of 7 (range 3-20) lines of prior therapy, including transplant (Table 1). Pts received a median of 5 (range 2-10; mean 4.9 [SD 1.86]) cycles of Cami. ORR was 80.9% (38/47 pts), with 18 (38.3%) and 20 (42.6%) pts attaining CR and partial response, respectively; 6 pts (12.8%) had stable disease (Figure 1). TEAEs were experienced by all 47 pts; the most common (≥20% of pts) were fatigue (22, 46.8%); nausea, pyrexia, and maculopapular rash (18, 38.3% each); anemia and headache (12, 25.5% each); pruritus (11, 23.4%); arthralgia, constipation, diarrhea, hypophosphatemia, and rash (10, 21.3% each). TEAEs thought to be PBD-associated included skin reactions and nail disorders (36, 76.6%), liver function test abnormalities (14, 29.8%), and edema or effusion (7, 14.9%). There were 3 (6.4%) pts with GBS/polyradiculopathy (Preferred Terms: grade 4 subacute inflammatory demyelinating polyneuropathy, grade 2 radiculopathy, and grade 2 peripheral motor and sensory neuropathy updated to GBS after data cut-off date). In total, 27 (57.4%) pts had grade ≥3 TEAEs; the most common (≥5% of pts) were hypophosphatemia (6, 12.8%) and gamma-glutamyltransferase increased (3, 6.4%). Overall, 3 (6.4%) pts had TEAEs leading to dose reduction/delay and 6 (12.8%) pts had TEAEs leading to treatment discontinuation. Conclusions: Current data show that therapy with Cami has encouraging anti-tumor activity in heavily pretreated pts with R/R cHL. Safety was consistent with that reported at Ph 1, with no new safety concerns identified and similar incidence of GBS/polyradiculopathy. Following a positive risk-benefit assessment, the enrollment pause was lifted, and pts continue to be enrolled. Updated efficacy and safety results will be presented at the meeting. Funding: Study funded by ADC Therapeutics SA. Disclosures Herrera: Pharmacyclics: Research Funding; Bristol Myers Squibb: Consultancy, Other: Travel, Accomodations, Expenses, Research Funding; Merck: Consultancy, Research Funding; Gilead Sciences: Consultancy, Research Funding; Genentech, Inc./F. Hoffmann-La Roche Ltd: Consultancy, Research Funding; Seattle Genetics: Consultancy, Research Funding; Karyopharm: Consultancy; AstraZeneca: Research Funding; Immune Design: Research Funding. Carlo-Stella:Bristol-Myers Squibb, Merck Sharp & Dohme, Janssen Oncology, AstraZeneca: Honoraria; ADC Therapeutics and Rhizen Pharmaceuticals: Research Funding; Boehringer Ingelheim and Sanofi: Consultancy; Servier, Novartis, Genenta Science srl, ADC Therapeutics, F. Hoffmann-La Roche, Karyopharm, Jazz Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees. Collins:Taekda: Consultancy, Honoraria, Other: travel, accommodations, expenses, Speakers Bureau; Novartis: Consultancy, Honoraria, Speakers Bureau; Pfizer: Honoraria; Gilead: Consultancy, Honoraria, Speakers Bureau; ADC Therapeutics: Consultancy, Honoraria; BMS: Consultancy, Honoraria, Research Funding, Speakers Bureau; MSD: Consultancy, Honoraria, Research Funding; Celleron: Consultancy, Honoraria, Research Funding; Celgene: Research Funding; Amgen: Research Funding; BeiGene: Consultancy; Roche: Consultancy, Honoraria, Other: travel, accommodations, expenses , Speakers Bureau. Maddocks:Morphosys: Consultancy, Honoraria; BMS: Consultancy, Research Funding; Karyopharm: Consultancy; Seattle Genetics: Consultancy, Honoraria; ADC Therapeutics, AstraZeneca: Consultancy; Pharmacyclics: Consultancy, Honoraria; Celgene: Consultancy, Honoraria. Bartlett:Autolus: Research Funding; BMS/Celgene: Research Funding; Forty Seven: Research Funding; Immune Design: Research Funding; Seattle Genetics: Membership on an entity's Board of Directors or advisory committees, Research Funding; Janssen: Research Funding; Kite, a Gilead Company: Research Funding; Merck: Research Funding; Millennium: Research Funding; Pfizer: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Pharmacyclics: Research Funding; Seattle Genetics: Consultancy, Research Funding; Roche/Genentech: Consultancy, Research Funding; ADC Therapeutics: Consultancy; BTG: Consultancy; Acerta: Consultancy; Affimed Therapeutics: Research Funding. Savage:BeiGene: Other: Steering Committee; Roche (institutional): Research Funding; Merck, BMS, Seattle Genetics, Gilead, AstraZeneca, AbbVie: Honoraria; Merck, BMS, Seattle Genetics, Gilead, AstraZeneca, AbbVie, Servier: Consultancy. Caimi:ADCT, Kite Therapeutics, Genentech, Amgen, Verastem, TG Therapeutics, Bayer: Consultancy; ADC Therapeutics: Membership on an entity's Board of Directors or advisory committees; Genentech, ADC Therapeutics: Research Funding; Celgene: Speakers Bureau. Hess:ADC Therapeutics: Consultancy, Membership on an entity's Board of Directors or advisory committees; BMS, AstraZeneca: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau. Zinzani:ADC Therapeutics: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Sanofi: Consultancy, Membership on an entity's Board of Directors or advisory committees; Sandoz: Consultancy, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; EUSA Pharma: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Celltrion: Consultancy, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Verastem: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Janssen-Cilag: Consultancy, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Kirin Kyowa: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Merck: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Celgene: Consultancy, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Portola: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Janssen: Consultancy, Honoraria, Speakers Bureau; Servier: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Roche: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; TG Therapeutics, Inc.: Honoraria, Speakers Bureau; BMS: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Incyte: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Takeda: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Gilead: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; AbbVie: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; MSD: Consultancy, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Eusapharma: Consultancy, Speakers Bureau; Kyowa Kirin: Consultancy, Speakers Bureau; Immune Design: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Immune Design: Consultancy, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau. Cruz:ADC Therapeutics SA: Current Employment, Current equity holder in publicly-traded company. Wang:ADC Therapeutics America, inc: Current Employment, Current equity holder in publicly-traded company. Feingold:ADC Therapeutics: Current Employment, Current equity holder in publicly-traded company. Wuerthner:ADC Therapeutics SA: Current Employment, Current equity holder in publicly-traded company. Ansell:Takeda: Research Funding; Bristol Myers Squibb: Research Funding; Seattle Genetics: Research Funding; Regeneron: Research Funding; Trillium: Research Funding; ADC Therapeutics: Research Funding; Affimed: Research Funding; AI Therapeutics: Research Funding.

The isomerization of a pyrrolobenzodiazepine (PBD) containing an exo-alkene to one containing an endo-alkene has been demonstrated to prepare a key intermediate for the synthesis of tesirine. This ...

In cells, catalytic disulfide cleavage is an essential mechanism in protein folding and synthesis. However, detailed enzymatic catalytic mechanism relating cleavage of disulfide bonds in xenobiotics is not well understood. This study reports an enzymatic mechanism of cleavage of disulfide bonds in xenobiotic small molecules and antibody conjugate (ADC) linkers. The chemically stable disulfide bonds in substituted disulfide-containing pyrrolobenzodiazepine (PBD, pyrrolo[2,1-c][1,4]benzodiazepine) monomer prodrugs in presence of glutathione or cysteine were found to be unstable in incubations in whole blood of humans and rats. It was shown the enzymes involved were thioredoxin (TRX) and glutaredoxin (GRX). For a diverse set of drug-linker conjugates, we determined that TRX in the presence of TRX-reductase and NADPH generated the cleaved products that are consistent with catalytic disulfide cleavage and linker immolation. GRX was less rigorously studied; in the set of compounds studied, its role in the catalytic cleavage was also confirmed. Collectively, these in vitro experiments demonstrate that TRX as well as GRX can catalyze the cleavage of disulfide bonds in both small molecules and linkers of ADCs.

We describe the characterization of antigen binding fragments (Fab)-drug conjugates prepared using a dual maleimide pyrrolobenzodiazepine dimer cytotoxic payload (SG3710). Pyrrolobenzodiazepine dimers, which are DNA cross-linkers, are a class of payloads used in antibody-drug conjugates (ADCs). SG3710 was designed to rebridge two adjacent cysteines, such as those that form the canonical interchain disulfide bond between the light and heavy chain in Fab fragments. The rebridging generated homogenous Fab conjugates, with a drug-to-Fab ratio of one, as demonstrated by the preparation of rebridged Fabs derived from the anti-HER2 trastuzumab antibody and from a negative control antibody both prepared using recombinant expression and papain digestion. The resulting anti-HER2 trastuzumab Fab-rebridged conjugate retained antigen binding, was stable in rat serum, and demonstrated potent and antigen-dependent cancer cell-killing ability. Disulfide rebridging with SG3710 is a generic approach to prepare Fab-pyrrolobenzodiazepine dimer conjugates, which does not require the Fabs to be engineered for conjugation. Thus, SG3710 offers a flexible and straightforward platform for the controlled assembly of pyrrolobenzodiazepine dimer conjugates from any Fab for oncology applications.

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.

Most strategies used to prepare homogeneous site-specific antibody-drug conjugates (ADCs) result in ADCs with a drug-to-antibody ratio (DAR) of two. Here, we report a disulfide re-bridging strategy to prepare homogeneous ADCs with DAR of one using a dual-maleimide pyrrolobenzodiazepine (PBD) dimer (SG3710) and an engineered antibody (Flexmab), which has only one intrachain disulfide bridge at the hinge. We demonstrate that SG3710 efficiently re-bridge a Flexmab targeting human epidermal growth factor receptor 2 (HER2), and the resulting ADC was highly resistant to payload loss in serum and exhibited potent anti-tumor activity in a HER2-positive gastric carcinoma xenograft model. Moreover, this ADC was tolerated in rats at twice the dose compared to a site-specific ADC with DAR of two prepared using a single-maleimide PBD dimer (SG3249). Flexmab technologies, in combination with SG3710, provide a platform for generating site-specific homogenous PBD-based ADCs with DAR of one, which have improved biophysical properties and tolerability compared to conventional site-specific PBD-based ADCs with DAR of two.

An analysis of Antibody-Drug Conjugate Payload manufacturing has revealed that the majority of the cost is associated with the use of high-containment facilities for the latter stages of the synthesis. To make a significant reduction in the Cost of Goods (CoGs), a new approach to route design has been introduced which focuses on minimizing the number of steps that require high containment. This approach has been exemplified in a new synthesis of tesirine, including the first application of a ring-closing copper(I)/TEMPO aerobic oxidation to the pyrrolobenzodiazepine ring system, affording a 60% reduction in CoGs.

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.

An efficient approach to difluoroalkylated pyrrolobenzodiazepines has been developed using a Pd-catalyzed two-component C–H difluoroalkylation/cyclization cascade reaction. This protocol constitutes a straightforward route for the synthesis of difluoroalkylated pyrrolobenzodiazepine derivatives in good yields with excellent functional group tolerance.

Chronic lymphocytic leukemia (CLL) and multiple myeloma are incurable hematologic malignancies that are pathologically linked with aberrant nuclear factor-kappa B (NF-κB) activation. In this study, we identified a group of novel C8-linked benzofused pyrrolo[2,1- c][1,4]benzodiazepine monomeric hybrids capable of sequence-selective inhibition of NF-κB with low nanomolar LD50 values in CLL (n=46) and multiple myeloma cell lines (n=5). The lead compound, DC-1-192, significantly inhibited NF-κB DNA binding after just 4 h of exposure, demonstrating inhibitory effects on both canonical and non-canonical NF-κB subunits. In primary CLL cells, sensitivity to DC-1-192 was inversely correlated with RelA subunit expression (r2=0.2) and samples with BIRC3 or NOTCH1 mutations showed increased sensitivity (P=0.001). RNAsequencing and gene set enrichment analysis confirmed the over-representation of NF-κB regulated genes in the downregulated gene list. Furthermore, in vivo efficacy studies in NOD/SCID mice, using a systemic RPMI 8226 human multiple myeloma xenograft model, showed that DC- 1-192 significantly prolonged survival (P=0.017). In addition, DC1-192 showed synergy with bortezomib and ibrutinib; synergy with ibrutinib was enhanced when CLL cells were co-cultured on CD40L-expressing fibroblasts in order to mimic the cytoprotective lymph node microenvironment (P=0.01). Given that NF-κB plays a role in both bortezomib and ibrutinib resistance mechanisms, these data provide a strong rationale for the use of DC-1-192 in the treatment of NF-κB-driven cancers, particularly in the context of relapsed/refractory disease.

7-methoxy-8-hydroxy cycloanthranilylproline (2), a new natural product with pyrrolobenzodiazepine (PBD) framework, was isolated from marine-derived actinobacterium Streptomyces cacaoi 14CM034, together with cycloanthranilylproline (1). Structural elucidation of the compounds was based on FTIR, 1D-(H-1 and C-13 NMR), 2D-NMR (COSY, HMBC and NOESY) and HR-MS analyses. Compounds 1 and 2 exhibited notable antimicrobial activity. The presence of PBD derivatives in S. cacaoi was first demonstrated with this study., Scientific and Technological Research Council of Turkey (TUBITAK) [109S361]; EBILTEM [2012/BIL/028], This work was supported by the Scientific and Technological Research Council of Turkey (TUBITAK, Project No: 109S361) and EBILTEM (2012/BIL/028).

Background Prolactin receptor (PRLR) is an attractive antibody therapeutic target with expression across a broad population of breast cancers. Antibody efficacy, however, may be limited to subtypes with either PRLR overexpression and/or those where estradiol no longer functions as a mitogen and are, therefore, reliant on PRLR signaling for growth. In contrast a potent PRLR antibody-drug conjugate (ADC) may provide improved therapeutic outcomes extending beyond either PRLR overexpressing or estradiol-insensitive breast cancer populations. Methods We derived a novel ADC targeting PRLR, ABBV-176, that delivers a pyrrolobenzodiazepine (PBD) dimer cytotoxin, an emerging class of warheads with enhanced potency and broader anticancer activity than the clinically validated auristatin or maytansine derivatives. This agent was tested in vitro and in vivo cell lines and patient derived xenograft models. Results In both in vitro and in vivo assays, ABBV-176 exhibits potent cytotoxicity against multiple cell line and patient-derived xenograft breast tumor models, including triple negative and low PRLR expressing models insensitive to monomethyl auristatin (MMAE) based PRLR ADCs. ABBV-176, which cross links DNA and causes DNA breaks by virtue of its PBD warhead, also demonstrates enhanced anti-tumor activity in several breast cancer models when combined with a poly-ADP ribose polymerase (PARP) inhibitor, a potentiator of DNA damage. Conclusions Collectively the efficacy and safety profile of ABBV-176 suggest it may be an effective therapy across a broad range of breast cancers and other cancer types where PRLR is expressed with the potential to combine with other therapeutics including PARP inhibitors.

Cytotoxic pyrrolobenzodiazepine (PBD)-dimer molecules are frequently utilized as payloads for antibody-drug conjugates (ADCs), and many examples are currently in clinical development. In order to further explore this ADC payload class, the physicochemical properties of various PBD-dimer molecules were modified by the systematic introduction of acidic and basic moieties into their chemical structures. The impact of these changes on DNA binding, cell membrane permeability, and in vitro antiproliferation potency was, respectively, determined using a DNA alkylation assay, PAMPA assessments, and cell-based cytotoxicity measurements conducted with a variety of cancer lines. The modified PBD-dimer compounds were subsequently incorporated into CD22-targeting ADCs, and these entities were profiled in a variety of in vitro and in vivo experiments. The introduction of a strongly basic moiety into the PBD-dimer scaffold afforded a conjugate with dramatically worsened mouse tolerability properties relative to ADCs derived from related payloads, which lacked the basic group.

The rationally designed pyrrolobenzodiazepine (PBD) dimers emerged around ten years ago as a new class of drug component for antibody-drug conjugates (ADC). They produce highly cytotoxic DNA cross-links, exploiting a completely different cellular target to the auristatin and maytansinoid tubulin inhibitor classes and a different mode of DNA damage to other DNA interacting warheads such as calicheamicin.The properties which make the PBD dimers suitable warheads for ADCs, and the development of the two main payload structures talirine and tesirine, are discussed. The clinical experience with the twenty PBD dimer-containing ADCs to enter the clinic is reviewed, with a focus on vadastuximab talirine and rovalpituzumab tesirine, both of which were discontinued following pivotal studies, and loncastuximab tesirine and camidanlumab tesirine which are progressing towards approval.Reviewing the clinical efficacy and safety data from almost forty clinical trials of PBD dimer-containing ADCs highlights the complexities and challenges of ADC early clinical development. It enables some conclusions to be made about reasons for failure and suggests strategies to optimise the future clinical development of this promising class of ADCs in a rapidly expanding field.

It is urgent to find new antibiotic classes with activity against multidrug-resistant (MDR) Gram-negative pathogens as the pipeline of antibiotics is essentially empty. Modified pyrrolobenzodiazepines with a C8-linked aliphatic heterocycle provide a new class of broad-spectrum antibacterial agents with activity against MDR Gram-negative bacteria, including WHO priority pathogens. The structure-activity relationship established that the third ring was particularly important for Gram-negative activity. Minimum inhibitory concentrations for the lead compounds ranged from 0.125 to 2 mg/L for MDR Gram-negative, excluding Pseudomonas aeruginosa, and between 0.03 and 1 mg/L for MDR Gram-positive species. The lead compounds were rapidly bactericidal with >5 log reduction in viable count within 4 h for Acinetobacter baumannii and Klebsiella pneumoniae. The lead compound inhibited DNA gyrase in gel-based assays, with an IC50 of 3.16 ± 1.36 mg/L. This study provides a new chemical scaffold for developing novel broad-spectrum antibiotics which can help replenish the pipeline of antibiotics.

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.