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. 2018 Jul 11;8(1):10479.
doi: 10.1038/s41598-018-28533-4.

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

John A Hartley  1   2 , Michael J Flynn  3 , John P Bingham  3 , Simon Corbett  3   4 , Halla Reinert  3 , Arnaud Tiberghien  4 , Luke A Masterson  4 , Dyeison Antonow  4 , Lauren Adams  4 , Sajidah Chowdhury  4 , David G Williams  4 , Shenlan Mao  5 , Jay Harper  5 , Carin E G Havenith  6 , Francesca Zammarchi  6 , Simon Chivers  6 , Patrick H van Berkel  6 , Philip W Howard  4
Affiliations

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

John A Hartley et al. Sci Rep. .

Abstract

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.

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Conflict of interest statement

J.A.H., A.T., L.A.M., L.A., S.Cho., D.G.W. and P.W.H. are employees of Spirogen/Medimmune Limited. S.M. and J.H. are employees of MedImmune. P.H.v.B., F.Z., S.C.hi. and C.E.G.H. are employees of A.D.C. Therapeutics and P.H.v.B., J.A.H. and P.W.H. are also shareholders. The authors declare no non-financial competing interests.

Figures

Figure 1
Figure 1
(A) Structures of SG2000, SG3199 and antibody-drug conjugate payload tesirine (SG3249). (B) Synthesis of SG3199 in two steps from phenol 1 via bis-alloc carbamate 2. (a) Diiodopentane, K2CO3, Acetone, 60 °C, 18 h; (b) Pd(PPh3)4, pyrrolidine, DCM, rt, 15 min.
Figure 2
Figure 2
Sensitivity of a panel of human tumour haematological (red) and solid tumour (blue) cell lines to SG3199. AML-acute myeloid leukaemia, ALL – acute lymphoblastic leukaemia, CLL – chronic lymphocytic leukaemia, CML – chronic myelogenous leukaemia, ALCL – anaplastic large cell lymphoma, HL – Hodgkin lymphoma, BL- Burkitt lymphoma, CTCL – cutaneous T-cell lymphoma, NHL – non-Hodgkin lymphoma, MCL – mantle cell lymphoma, BCL – B-cell lymphoma, TCL – T-cell lymphoma.
Figure 3
Figure 3
Sensitivity of SG3199 in DNA repair defective and multidrug resistant cell lines. (A) Chinese hamster ovarian wild type (AA8), ERCC1 defective (UV96) and homologous recombination defective (IRS1SF) cells. (B) Human ovarian cancer SKOV3 and SKOV3-TR cells. (C) Human breast cancer MDA-MB-231 and MDA-MB-231-MDR1 cells. VP is co-treatment with verapamil.
Figure 4
Figure 4
DNA interstrand cross-linking by SG3199 in naked DNA. (A) Representative autoradiograph showing double stranded (DS) and single stranded (SS) DNA. 0(DS) is non-denatured, untreated DNA. All other samples treated with the indicated dose of SG3199 are denatured prior to loading on the neutral agarose gel. (B) Quantitation of the % double stranded (cross-linked) DNA following SG3199 treatment.
Figure 5
Figure 5
DNA interstrand cross-linking in cells by SG3199. (A) Dose response of cross-linking following a 2 h treatment of LNCaP cells with SG3199 followed by 24 h post-incubation in drug-free medium. (B) Time course of cross-linking following a 2 h SG3199 treatment in LNCaP cells at 2.5 nM. (C) Dose response of cross-linking following a 2 h treatment of NCI-N87 cells with SG3199 followed by 24 h post-incubation in drug-free medium. (D) Time course of cross-linking following a 2 h SG3199 treatment in NCI-N87 cells at 1.7 nM.
Figure 6
Figure 6
Pharmacokinetics of SG3199 in the rat following a single administration at the doses indicated. LLOQ is the lower limit of quantification.
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