Julia Mantaj

From Anthramycin to Pyrrolobenzodiazepine (PBD)-Containing Antibody-Drug Conjugates (ADCs)

Abstract The pyrrolo[2,1‐c][1,4]benzodiazepines (PBDs) are a family of sequence‐selective DNA minor‐groove binding agents that form a covalent aminal bond between their C11‐position and the C2‐NH2 groups of guanine bases. The first example of a PBD monomer, the natural product anthramycin, was discovered in the 1960s, and the best known PBD dimer, SJG‐136 (also known as SG2000, NSC 694501 or BN2629), was synthesized in the 1990s and has recently completed Phase II clinical trials in patients with leukaemia and ovarian cancer. More recently, PBD dimer analogues are being attached to tumor‐targeting antibodies to create antibody–drug conjugates (ADCs), a number of which are now in clinical trials, with many others in pre‐clinical development. This Review maps the development from anthramycin to the first PBD dimers, and then to PBD‐containing ADCs, and explores both structure–activity relationships (SARs) and the biology of PBDs, and the strategies for their use as payloads for ADCs.

Covalent Bonding of Pyrrolobenzodiazepines (PBDs) to Terminal Guanine Residues within Duplex and Hairpin DNA Fragments

Pyrrolobenzodiazepines (PBDs) are covalent-binding DNA-interactive agents with growing importance as payloads in Antibody Drug Conjugates (ADCs). Until now, PBDs were thought to covalently bond to C2-NH2 groups of guanines in the DNA-minor groove across a three-base-pair recognition sequence. Using HPLC/MS methodology with designed hairpin and duplex oligonucleotides, we have now demonstrated that the PBD Dimer SJG-136 and the C8-conjugated PBD Monomer GWL-78 can covalently bond to a terminal guanine of DNA, with the PBD skeleton spanning only two base pairs. Control experiments with the non-C8-conjugated anthramycin along with molecular dynamics simulations suggest that the C8-substituent of a PBD Monomer, or one-half of a PBD Dimer, may provide stability for the adduct. This observation highlights the importance of PBD C8-substituents, and also suggests that PBDs may bind to terminal guanines within stretches of DNA in cells, thus representing a potentially novel mechanism of action at the end of DNA strand breaks.

Abstract 4779: In silico design, synthesis and evaluation of a new family of C1-substituted pyrrolobenzodiazepines (PBDs)

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-EB 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.

Abstract 2230: Interaction of SJG-136 with cognate sequences of oncogenic transcription factors.

Proceedings: AACR 104th Annual Meeting 2013; Apr 6-10, 2013; Washington, DC The pyrrolo\[2,1-c\]\[1,4\]benzodiazepines (PBDs) are sequence-selective DNA minor-groove interacting agents. They have a chiral centre at their C11a(S)-position which provides them with the appropriate 3-dimensional shape to fit securely within the DNA minor-groove. They also possess a “soft” electrophilic imine moiety at their N10-C11 position which can form an aminal linkage with the C2-NH2 group of a guanine base. The PBD dimer SJG-136 is presently in Phase II clinical trials in ovarian cancer and leukaemia. Transcription factors act through direct or indirect binding to specific DNA consensus sequences within gene regulatory regions, or by forming a complex with other proteins which then act as transcription factors. Covalent or non-covalent interaction of small molecules with key consensus DNA bases can prevent a transcription factor from recognizing its cognate sequence, thus preventing expression of genes critical for the survival and proliferation of cancer cells. The cognate sequences of a number of oncogenic transcription factors are unusually rich in GC sequences and provide ideal alkylation substrates (for both inter- and intrastrand cross-links and mono-alkylation) for both PBD dimers (e.g. SJG-136) and PBD monomers, and there is growing evidence that PBDs exert their pharmacological effects through this mechanism. We have developed a reversed-phase ion pair HPLC/MS method as a tool to evaluate the interaction of DNA-binding molecules with oligonucleotides of varying length and sequences. Using this methodology along with custom-designed oligonucleotides containing the cognate sequences of the transcription factors NFκB, AP-1, EGR-1 and Stat3, all of which contain ideal GC-rich binding sequences for SJG-136, we have observed both kinetic and thermodynamic adduct formation with SJG-136. Crucially, we observed a significant difference in rate and extent of adduct formation between the different cognate sequences which could partly explain the differences in activity of SJG-136 toward different tumour cell lines. For example, SJG-136 formed an adduct rapidly with the NFκB sequence (>50% adduct within 3 hours), moderately with the STAT3 (>35% adduct within 3 hours) and EGR-1 cognate sequences (>30% adduct within 3 hours) while showing insignificant adduct formation with the AP-1 cognate sequence (< 5% adduct within 3 hours) even though their GC contents are comparable. These findings add to knowledge of the possible mechanism of SJG-136, and could help with interpretation of the activity of SJG-136 in various biochemical and pharmacological assays. Citation Format: Julia Mantaj, David E. Thurston, Khondaker M. Rahman. Interaction of SJG-136 with cognate sequences of oncogenic transcription factors. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2230. doi:10.1158/1538-7445.AM2013-2230

Abstract 5242: Further evidence that the DNA-interactive Pyrrolobenzodiazepine (PBD) Dimer SJG-136 works through a transcription factor inhibition mechanism

The pyrrolo[2,1-c][1,4]benzodiazepines (PBDs) are sequence-selective DNA minor-groove interacting agents. The PBD dimer SJG-136 has currently been investigated in Phase II clinical trials in ovarian cancer and leukaemia in the UK and the USA. More recently, PBD dimer analogues are being attached to tumour-targeting antibodies to create Antibody-Drug Conjugates (ADCs), some which are now in Phase 3 clinical trials with many others in pre-clinical and clinical development. Transcription factors (TFs) are sequence-specific DNA-interacting proteins that bind to consensus DNA sequences, thereby controlling transcription. TFs regulate processes such as cell differentiation, proliferation and apoptosis. The interaction of a small-molecule with the consensus DNA recognition sequences of TFs can prevent a TF from interacting with its cognate sequence, thereby inhibiting the expression of genes critical for the survival and proliferation of cancer cells. There is growing evidence that PBDs may exert, at least in part, their pharmacological effect through TF inhibition in addition to the arrest of the replication fork, DNA strand breakage, and inhibition of enzymes including endonucleases and RNA polymerases. For this reason, there is now interest in using PBDs as the basis for a small-molecule strategy to target specific DNA sequences for TF inhibition as a novel anticancer therapy. We have developed a reversed-phase HPLC/MS method as a tool to evaluate the interaction of DNA-binding PBD molecules with oligonucleotides of varying lengths and sequences. Using this methodology, we have demonstrated that the PBD dimer SJG-136 binds to the cognate sequences of the oncogenic transcription factors NF-κB, EGR-1, AP-1 and STAT3. Surprisingly, significant differences in the rate and extent of adduct formation between the different cognate sequences were observed which may explain, at least in part, the differences in potency of SJG-136 in various tumour cell lines. Furthermore, an RT PCR study has been carried out using the human tumour cell lines MDA-MB-231 (breast) and HT-29 (colon) to see whether these transcription factors are affected in vitro. The results were consistent with the HPLC-MS studies in that SJG-136 was shown to significantly down-regulate a number of AP-1- and STAT3-dependent genes such as Bcl-2, VEGF, p53 and survivin. These findings add significantly to knowledge of the mechanism of action of SJG-136 and related PBD compounds, and could be relevant for the correct interpretation of clinical activity of molecules of this type both as standalone agents and as ADC payloads. Citation Format: Julia Mantaj, Paul J. Jackson, David E. Thurston, Khondaker Miraz Rahman. Further evidence that the DNA-interactive Pyrrolobenzodiazepine (PBD) Dimer SJG-136 works through a transcription factor inhibition mechanism [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5242. doi:10.1158/1538-7445.AM2017-5242

Abstract 4778: Covalent bonding of a C8-conjugated pyrrolobenzodiazepine (PBD) monomer and dimer to a terminal guanine residue of DNA duplex and hairpin fragments

The pyrrolobenzodiazepines (PBDs) are a family of covalent-binding DNA-interactive minor-groove binding agents that are of growing interest due to their potential as stand-alone anticancer agents, and for their use as payloads in Antibody Drug Conjugates (ADCs). For example, one PBD Dimer, SJG-136 has reached Phase II evaluation in the clinic for ovarian and haematological cancers, and a number of PBD-based ADCs are now being evaluated in pre-clinical, Phase I and II clinical trials (e.g., SGN-CD19B, SGN-CD33A [vadastuximab talirine], SGN-CD70A, SGN-CD123A, SC16LD6.5 [Rova-T, rovalpituzumab tesirine] and ADCT-402). Until now, PBDs, which bind in the minor groove of DNA and covalently bond to a guanine residue through its C2-NH2 functional group, were thought to require a three-base-pair recognition sequence prior to covalent bonding to the central guanine (with a thermodynamic preference for binding to 5’-Pu-G-Pu-3’ sequences but a kinetic preference for 5’-Py-G-Py-3’; Pu = Purine, Py = Pyrimidine). Using HPLC/MS methodology with designed hairpin and duplex oligonucleotides, we have now demonstrated that the PBD dimer SJG-136 and the C8-conjugated PBD Monomer GWL-78 can covalently bond to a terminal guanine of DNA, with the PBD skeleton spanning only two base pairs. Control experiments with the non-C8-conjugated anthramycin along with molecular dynamics simulations suggest that the C8-substituent of a PBD monomer, or one-half of a PBD Dimer, may provide stability for the adduct. This observation highlights the importance of PBD C8-substituents, and also suggests that PBDs may bind to terminal guanines within stretches of DNA in cells, thus representing a potentially novel mechanism of action at the end of DNA strand breaks. Citation Format: Julia Mantaj, Paul J. M. Jackson, Khondaker Miraz Jackson, David E. Thurston. Covalent bonding of a C8-conjugated pyrrolobenzodiazepine (PBD) monomer and dimer to a terminal guanine residue of DNA duplex and hairpin fragments. [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 4778.

Abstract 1986: Effect of the PBD dimer SJG-136 on expression of STAT3 dependent genes

Proceedings: AACR 106th Annual Meeting 2015; April 18-22, 2015; Philadelphia, PA Signal Transducer and Activator of Transcription 3 (STAT3) regulates the transcription of genes involved in cell differentiation, proliferation, apoptosis, angiogenesis, metastasis, and immune responses. STAT3 plays a crucial role in cancer progression as it is known to be over-expressed in many human tumours, including leukaemia, breast cancer and ovarian carcinoma. The pyrrolo\[2,1-c\]\[1,4\]benzodiazepines (PBDs) are sequence-selective DNA minor-groove interacting agents which form an aminal linkage with the C2-NH2 group of a guanine base within the DNA minor groove. The PBD dimer SJG-136 has completed a Phase II clinical trial in ovarian cancer, and is presently being evaluated in leukaemia. There is growing evidence that PBD monomers exert their pharmacological effects through transcription factor inhibition. For example, GWL-78, a C8-linked PBD-Py-Py conjugate, has been shown to block interaction of the transcription factor NF-Y, and KMR-28-39, a GC sequence selective C8-linked PBD-MPB conjugate, inhibits transcription factors including NFκB. It has been recently observed that the PBD dimer SJG-136 can form covalent adducts with STAT3 consensus sequences. The aim of this study was to investigate whether the interaction of SJG-136 with STAT3 binding sequences is biologically relevant, and to explore whether STAT3 inhibition is one of the key mechanisms in addition to previously described DNA strand-breakage, inhibition of endonuclease and RNA polymerases, and arrest of the replication fork. We have developed an ion pair reversed phase HPLC/MS analytical methodology, and have previously demonstrated for the first time the ability of SJG-136 to bind to specific DNA consensus sequences of the transcription factors NFκB, EGR-1, AP-1 and STAT3. After completing this biophysical study, the effect of SGJ-136 on the expression of STAT3-dependent genes and proteins utilizing the breast cancer cell line MDA-MB-231 was studied using the quantitative polymerase chain reaction (qPCR) and Western blot analyses. Briefly, cells were stimulated with LPS and incubated with various concentrations of SJG-136 for 24 hours. The cells were lysed, and gene expression profiles compared with control. The results demonstrate that SJG-136 appears to produce a significant dose-dependent down-regulation of STAT3-dependent genes including Bcl-2, cyclin D1, survivin, NNMT and fascin compared to the reference genes GAPDH, s actin and α tubulin. These findings are in broad agreement with our bio-physical observations, have implications for understanding the mechanism of action of SJG-136, and may potentially explain the differences in activity of SJG-136 toward various tumour cell lines. Citation Format: Julia Mantaj, David E. Thurston, Khondaker M. Rahman. Effect of the PBD dimer SJG-136 on expression of STAT3 dependent genes. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1986. doi:10.1158/1538-7445.AM2015-1986

Abstract 1630: C8-linked pyrrolobenzodiazepine (pbd)-benzofused conjugates with low-picomolar in vitro cytotoxicity

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA There is significant interest in developing small-molecule ligands that can locate and bind to specific sequences of DNA as potential antitumor agents. Such molecules could be used, for example, to inhibit transcription factors and other control proteins from interacting with DNA at their consensus recognition sites. A set of DNA-interactive benzofused building blocks based on phenyl-substituted heterocycles have been developed with sufficient length to span two DNA base pairs. These building blocks have been conjugated to DNA minor-groove covalent-binding pyrrolobenzodiazepine (PBD) molecules via a four-carbon linker to produce C8-linked PBD-Benzofused hybrid molecules. These focused libraries contained structural variations where the benzofused building blocks were either directly linked to the PBD core or separated by pyrrole/imidazole heterocycles or GC-recognizing methylpyrrolebenzenamine (MPB) biaryl building blocks. The design rationale was based on the unique longitudinal curvature of these PBD conjugates which produces greater isohelicity with the DNA minor groove, and also on predicted hydrogen-bonding interactions with individual DNA bases. These molecules showed significant enhancement of melting temperatures of designed oligonucleotides in a FRET-based assay compared to the PBD core alone, highlighting the synergistic effect of joining non-covalent benzofused rings to the covalent-binding PBD unit. The cytotoxicity of these compounds was assessed in primary CLL cells and a panel of other tumor cell lines (A431, A2780, A549, MIA PaCa2, HeLa and MDA-MB-231) after 48h and 96h exposure using the Annexin V apoptosis assay and the MTT colorimetric assay, respectively. Normal lymphocytes and the non-tumor fibroblast cell line WI38 were used as controls. Some of these compounds were highly potent (i.e., low picomolar) in the primary CLL and other cell lines with at least 3-orders of magnitude selectivity compared to the normal lymphocytes or WI38 cells. Importantly, molecules with different distances of separation between the benzofused ring moieties had significant differences in cytotoxicity, thus not only providing useful SAR information but also suggesting that there was scope for optimizing activity through a medicinal chemistry approach. The effect of these conjugates on regulation of the NF-κB-associated regulatory proteins IKKα and IKKβ was studied in the CLL and breast cancer cell lines in which NF-κB is known to be active and closely correlated with the initiation and progression of malignancy. The lead PBD-Benzofused conjugate KMR-28-32 exhibited marked inhibition of both IKKα and IKKβ proteins at 4h and 24h time points at concentrations of 0.1nM and 1nM, respectively, with negligible effect on tubulin expression which was used as a housekeeping control. Citation Format: Julia Mantaj, Paul JM Jackson, Chris Pepper, David E. Thurston, Khondaker M. Rahman. C8-linked pyrrolobenzodiazepine (pbd)-benzofused conjugates with low-picomolar in vitro cytotoxicity. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1630. doi:10.1158/1538-7445.AM2014-1630