Arenavirus RNA genomes are initiated by a "prime and realign" mechanism, such that the initiating GTP is found as a single unpaired (overhanging) nucleotide when the complementary genome ends anneal to form double-stranded (ds) RNA panhandle structures. dsRNAs modeled on these structures do not induce interferon (IFN), as opposed to bluntended 5 ppp dsRNA. This study examines whether these viral structures can also act as decoys, by trapping RIG-I in inactive dsRNA complexes. We examined the ability of various dsRNAs to activate the RIG-I ATPase (presumably a measure of helicase translocation on dsRNA) relative to their ability to induce IFN. We found that there is no simple relationship between these two properties, as if RIG-I can translocate on short dsRNAs without inducing IFN. Moreover, we found that 5 ppp dsRNAs with a single unpaired 5 ppp-nucleotide can in fact competitively inhibit the ability of blunt-ended 5 ppp dsRNAs to induce IFN when co-transfected into cells and that this inhibition is strongly dependent on the presence of the 5 ppp. In contrast, 5 ppp dsRNAs with a single unpaired 5 ppp-nucleotide does not inhibit poly(I-C)-induced IFN activation, which is independent of the presence of a 5 ppp group.
Although four ADCs have been approved and over sixty others are in development, the majority contain payloads belonging to two classes; tubulin inhibitors and DNA cross-linkers. Challenges in the development of ADCs include managing off-target toxicity and hydrophobicity. Some payload classes (e.g., PBD dimers) are notably hydrophobic leading to problems (e.g., aggregation) during conjugation. Thus, there is interest in developing novel payloads which retain the potency of DNA cross-linkers but have lower hydrophobicity and a wider therapeutic window when part of an ADC. The pyridinobenzodiazepines (PDDs) are a new class of sequence-selective, DNA mono-alkylating ADC payload which contain a polyheterocyclic chain with sufficient span to guide them to specific DNA sequences (e.g., transcription factor binding sites). The lead PDD payload, FGX-2-62, has a different sequence-selectivity profile to other DNA-interactive agents, spanning 8-9 base-pairs compared to 6-7 for a PBD dimer, and DNA footprinting experiments indicate a preference for 5'-XGXWWWWXX-3' sequences (X is any base; W is A/T). Transcription factor array studies have shown that the molecule inhibits DNA-binding of oncogenic transcription factors (e.g., NF-κB and GATA). In in vitro cell line studies, FGX-2-62 has low pM cytotoxicity in a diverse cell line panel, including stem cells, cells from both solid and blood cancers (e.g., 9 pM in HL-60) and MDR-resistant tumours, and arrests the cell cycle at the G0/G1 phase compared to G2-M arrest for PBD dimers. It is compatible with attachment to most linker technologies, and is significantly less hydrophobic than other payload classes. Initial MTD studies were carried out by separately conjugating (with negligible aggregation) FGX-2-62 and the PBD dimer Talirine to a THIOMAB® version of trastuzumab (DAR = 2). In female athymic nude mice, a greater tolerance was observed for the THIOMAB®-(FGX-2-62) ADC compared to the THIOMAB®-PBD dimer (i.e, MTD >8 mg.kg-1 versus 4 mg.kg-1). In an efficacy study, FGX-2-62 was conjugated to a cancer stem cell-targeting IgG1 antibody (Bstrongximab) with DAR 1.9. Initial evaluation afforded IC50 values of 0.67 nM and 0.47 nM in two antigen positive cell-lines, and an MTD of 6 mg.kg-1 in mice. In an antigen-positive embryonal carcinoma stem cell CDX mouse model, complete regression was observed at a dose of 2 mg.kg-1 (Q7Dx3). In a cholangiocarcinoma PDX model, complete tumour regression was observed out to 80 days (when experiment was terminated) at a dose of 5 mg.kg-1 (Q7Dx3), with no observed toxicity. The favourable hydrophobicity profile of the PDDs and ease of conjugation, along with their novel mechanism of action, significant in vitro cytotoxicity, in vivo efficacy and tolerability in MTD studies suggest that they represent a promising new class of ADC payloads. Citation Format: Nicolas Veillard, Paolo Andriollo, Julia Mantaj, Keith R. Fox, K Miraz Rahman, George Procopiou, Francesco Cascio, David B. Corcoran, Ilona Pysz, Patricia A. Cooper, Steven D. Shnyder, Yawen Ju, Edwin Tan, William M. Schopperle, Paul J. Jackson, David E. Thurston. Pyridinobenzodiazepines (PDDs): A new class of sequence-selective DNA mono-alkylating ADC payloads with low hydrophobicity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 736.
Although five ADCs have been approved and over sixty others are in development, the majority contain payloads belonging to two classes: tubulin inhibitors and DNA-interactive agents. Challenges in the development of ADCs include managing off-target toxicity and hydrophobicity. Some DNA-interactive payload classes [e.g. pyrolobenzodiazepine (PBD] dimers) are notably hydrophobic, leading to problems such as aggregation during conjugation, and systemic toxicities of the resultant ADCs are also beginning to emerge in clinical settings. Thus, there is interest in developing novel payloads which retain the potency of DNA cross-linking agents but have lower hydrophobicity and a wider therapeutic window when part of an ADC. The pyridinobenzodiazepines (PDDs) are a new class of sequence-selective, DNA mono-alkylating ADC payload, which contain a polyheterocyclic chain with sufficient molecular span and DNA base-pair recognition properties to guide them to specific DNA sequences (e.g. transcription factor binding sites). The favourable hydrophobicity profile of the PDDs and ease of conjugation, along with their novel mechanism of action, significant in vitro cytotoxicity and in vivo tolerability and efficacy when in an ADC format, indicate that they represent a promising new class of ADC payloads.
The pyrrolobenzodiazepines (PBDs) are a class of naturally occurring and synthetic DNA minor-groove binding agents. They covalently bind to the C2-amino group of guanines, and can form mono-alkylated adducts and cross-links depending on the structure of the molecule. PBDs can be extremely cytotoxic, and there are reports of IC50 values in cell lines in the high femtomolar ranges. Synthetic A-ring-linked PBD dimers have found use as payloads in antibody drug conjugates (ADCs) due to their potent cytoxicity, and a number of ADCs are now in clinical trials. The C-ring of the PBD structure has been under-exploited as a means to improve cytotoxicity, and for joining PBD units together to create novel dimers. This lack of interest in the C-ring may have been due to literature reports that related C2-linked PBD dimers are poorly DNA interactive and cytotoxic. Using our proprietary molecular dynamics (MD) protocols, we undertook a series of MD simulations to establish a rationale for the poor DNA-binding affinity of C2-linked PBD dimers. We were able to determine a number of characteristics preventing efficient DNA-interaction including (a) their shape does not follow the curvature of the DNA minor groove, (b) if a molecule is attached to DNA via one imine moiety, the other imine moiety is not oriented in an appropriate position to effect the second covalent cross-linking event, (c) if a cross-linked model is produced, significant DNA distortion is observed, suggesting that rapid repair would occur in cells, perhaps contributing to the low cytotoxicity. During this study, our models and simulations suggested that, after covalent adduct formation, C1-substituents should extend along the minor groove in an appropriate orientation to form extensive van der Waals interactions with functional groups on the minor groove floor. This was reflected in free energy of binding calculations. For example, in the case of one C2-linked dimer, calculations suggested a weak binding affinity (i.e., -42.74 kcal/mol) with the DNA sequence 5’-GCGATACTCGC-3’, whereas under identical experimental conditions, an equivalent C1-linked dimer had a much higher binding affinity (i.e., -50.42 kcal/mol). Based on these promising in silico data, a small library of C1-PBD molecules was synthesised, and biophysical results indicated that they possessed significant DNA-binding affinity. HPLC-MS experiments using DNA sequences based on the STAT3 and NF-ÊB transcription factor consensus sequences showed that some library members converted up to 60% of the DNA to covalent adducts within 3 hours. Furthermore, in preliminary experiments in cell lines such as MDA-MB231, the same molecules were highly cytotoxic with IC50 values in the nanomolar region (e.g., 36 nM after 72 hour incubation for one compound). Studies are now underway to further functionalise the C1-position of PBDs to enhance cytotoxicity, and to assess their viability as novel payloads for use in ADCs. Citation Format: Paul J. M. Jackson, George Procopiou, Nicolas Veillard, Julia Mantaj, K Miraz Rahman, David E. Thurston. In silico design, synthesis and evaluation of a new family of C1-substituted pyrrolobenzodiazepines (PBDs). [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 4779.
Although five Antibody-Drug Conjugates (ADCs) have been approved and over eighty others are in development, the majority contain payloads belonging to two classes: tubulin inhibitors and DNA interactive agents. Most DNA cross-linking payloads (e.g., the PBD dimers) have potent cytotoxicity but ADCs containing them have high hydrophobicity and a narrow therapeutic window. Thus, there is interest in developing novel payloads which benefit from a potency similar to the PBD dimers but that possess lower hydrophobicity and produce ADCs with a wider Therapeutic Index (TI). The pyridinobenzodiazepines (PDDs) are a new class of guanine-alkylating payloads, and these have been coupled to an adenine-alkylating CXI/duocarmycin pharmacophore to generate molecules that can form G-A DNA cross-links. The lead PDD-CXI payload (FGX-8-46) has a sequence-selectivity profile that differs from other DNA cross-linking agents in that it spans seven to eight base-pairs compared to six to seven for a typical PBD dimer. DNA cleavage experiments have indicated that it cleaves at discrete Adenine-containing sequences of the type 5’-XGXWWWW-3’ (X is any base; W is A/T, and the underlined bases show the cleavage points), and Transcription Factor (TF) Array studies show that it is a potent TF inhibitor, down-regulating several key oncogenic TFs (e.g., NF-κB). In in vitro cell line studies, the G-A cross-linkers have low pM cytotoxicity comparable to the PBD dimers in a wide and diverse range of cell lines, including those from both solid and haematological cancers (e.g., IC50 of ~2 pM in SW-48). This payload class is also compatible with a wide variety of linker technologies, and attachment can be made through either the PDD or CXI units. Importantly, these payloads are significantly less hydrophobic than other equivalent payload classes. ADCs have been generated by conjugating these new payloads to the EGFR-targeting antibody Cetuximab with DARs of between 1.8 and 2.2. The ADCs exhibit potent cytotoxicity in vitro, significant in vivo efficacy and substantially increased tolerability compared to other DNA cross-linking payloads (e.g., the PBD dimer Tesirine). While the G-A cross-linkers retain the cytotoxic potency of the PBD dimers, the “softer” cross-link formed compared to the G-G cross-linking PBD dimers may contribute to the enhanced tolerability profile of this molecular class. The favourable hydrophobicity profile of the PDD-CXI payloads and their ease of conjugation to antibodies, along with their significant in vitro cytotoxicity, in vivo efficacy and tolerability of ADCs produced from them, suggest that they represent a promising new class of ADC payloads. Citation Format: George Procopiou, Jennifer Auer, Daniella di Mascio, Keith R. Fox, Paolo Andriollo, Ilona Pysz, Francesco Cascio, Nicolas Veillard, K. Miraz Rahman, Paul J. Jackson, David E. Thurston. A new class of sequence-selective DNA cross-linking ADC payloads with increased in vivotolerability [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 211.
Although five Antibody-Drug Conjugates (ADCs) have been approved and over eighty others are in development, the majority contain payloads belonging to two classes: tubulin inhibitors and DNA interactive agents. Most DNA-interactive payloads (e.g., the PBD dimers and IGNs) have potent cytotoxicity but ADCs containing them have high hydrophobicity and a narrow therapeutic window. Thus, there is interest in developing novel payloads which benefit from similar in vivo efficacy but possess lower hydrophobicity and a substantially wider Therapeutic Window (TW). The pyridinobenzodiazepines (PDDs) are a new class of sequence-selective, DNA guanine monoalkylating ADC payload which contain a polyheterocyclic chain with sufficient span to guide them to specific DNA sequences (e.g., transcription factor binding sites). The lead high potency PDD payload (FGX2-62) is active in vitro in the picomolar range in a wide panel of cell-lines, is easily conjugated and has in vivo potency at sub-mg/kg levels in ADC form. Within the PDD platform, we have now developed a lower potency DNA monoalkylator with substantially superior in vivo properties to other DNA alkylating agents. FGX20-75 is active in the low nanomolar range (e.g., 2.1 nM in the gastric cell-line SW48, 72 hour incubation), but is sufficiently hydrophilic to allow efficient conjugation at DARs > 4. The increase in loading compared to other DNA-interactive payloads compensates for the drop in potency of FGX20-75, resulting in a potent ADC with substantially increased tolerability. ADCs have been generated by conjugating a maleimide-linked analogue of FGX20-75 to the EGFR-targeting antibody Cetuximab with DAR of 4.2. The ADCs exhibit significant in vivo efficacy compared to other DNA-interactive payloads, and a substantially increased tolerability profile. A maximum tolerated dose (MTD) has not yet been reached at 35 mg/kg (single dose, CD1 mice). The favourable hydrophobicity profile of the low potency alkylator (FGX20-75) and its ease of conjugation to antibodies, along with the significant in vivo efficacy and tolerability of the ADCs produced, suggest that this low potency DNA-alkylating payload represents a promising new approach in ADC development. Citation Format: Nicolas Veillard, Paolo Andriollo, Francesco Cascio, Paul J. M. Jackson, David E. Thurston. A new low potency DNA guanine monoalkylating ADC payload with enhanced in vivo tolerability [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference on Molecular Targets and Cancer Therapeutics; 2019 Oct 26-30; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2019;18(12 Suppl):Abstract nr C030. doi:10.1158/1535-7163.TARG-19-C030
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