PBD Dimer Research Articles

ASCT2-Targeting Antibody-Drug Conjugate MEDI7247 in Adult Patients with Relapsed/Refractory Hematological Malignancies: A First-in-Human, Phase 1 Study.

MEDI7247 is a first-in-class antibody-drug conjugate (ADC) consisting of an anti-sodium-dependent alanine-serine-cysteine transporter 2 antibody-conjugated to a pyrrolobenzodiazepine dimer. This first-in-human phase 1 trial evaluated MEDI7247 in patients with hematological malignancies. Adults with acute myeloid leukemia (AML), multiple myeloma (MM), or diffuse large B-cell lymphoma (DLBCL) relapsed or refractory (R/R) to standard therapies, or for whom no standard therapy exists, were eligible. Primary endpoints were safety and determination of the maximum tolerated dose (MTD). Secondary endpoints included assessments of antitumor activity, pharmacokinetics (PK), and immunogenicity. As of 26 March 2020, 67 patients were treated (AML: n = 27; MM: n = 18; DLBCL: n = 22). The most common MEDI7247-related adverse events (AEs) were thrombocytopenia (41.8%), neutropenia (35.8%), and anemia (28.4%). The most common treatment-related grade 3/4 AEs were thrombocytopenia (38.8%), neutropenia (34.3%), and anemia (22.4%). Anticancer activity (number of responders/total patients evaluated) was observed in 11/67 (16.4%) patients. No correlation was observed between ASCT2 expression and clinical response. Between-patient variability of systemic exposure of MEDI7247 ADC and total antibody were high (AUCinf geometric CV%: 62.3-134.2, and 74.8-126.1, respectively). SG3199 (PBD dimer) plasma concentrations were below the limit of quantification for all patients after Study Day 8. Anti-drug antibody (ADA) prevalence was 7.7%, ADA incidence was 1.9%, and persistent-positive ADA was 5.8%. Thrombocytopenia and neutropenia limited repeat dosing. Although limited clinical activity was detected, the dose-escalation phase was stopped early without establishing an MTD. The study was registered with ClinicalTrials.gov (NCT03106428).

A Global Study of Novel Agents in Paediatric and Adolescent Relapsed and Refractory B-Cell Non-Hodgkin Lymphoma (Glo-BNHL)

Overview Glo-BNHL is an adaptive prospective international academic-led multicentre platform clinical trial designed to evaluate the efficacy and safety of the most promising novel agents as monotherapy or in combination with existing therapies for the treatment of children, adolescents, and young adults with relapsed or refractory (r/r) B-cell Non-Hodgkin Lymphoma (B-NHL). A direct output of the second ACCELERATE multi-stakeholder Paediatric Strategy Forum (Pearson, 2019), Glo-BNHL was designed in collaboration with patient advocates, is supported by the Regulatory Authorities, and the intent is for trial data to support filing for marketing authorisation. Rationale Outcomes for r/r paediatric B-NHL are extremely poor with long term cure rates lower than 30%. Glo-BNHL fulfils the critical need for a single collaborative global approach to find new treatments for these rare patients for whom current therapy is inadequate. It allows for a rational approach to investigating the array of potentially promising agents currently in development for adults. The classes of agents currently prioritised for inclusion in Glo-BNHL are: Treatment Arm I: bispecific antibodies (BsAbs)Treatment Arm II: antibody-drug conjugates (ADCs) combined with standard chemotherapyTreatment Arm III: chimeric antigen receptor (CAR) T-cell products A robust systematic assessment of each asset is undertaken before a decision to include in the platform is made (published separately). Population Glo-BNHL will enrol participants in ≥45 sites in 18 countries spanning Europe, Asia, North America and Australasia. Eligible patients are aged ≤25 years with radiologically or histologically proven relapsed (≥ first) or refractory B-NHL with evaluable disease, adequate organ function, negative pregnancy test, agreement to use effective contraception and written informed consent. Key exclusion criteria include recent stem cell transplant, craniospinal radiation or investigational treatment, on-going moderate or severe acute toxicities, uncontrolled infection or primary immunodeficiency. Patients may be enrolled into any of the available treatment arms for which they are eligible. Statistical Design In this rare population we anticipate global enrolment of 30 patients per year. The Bayesian design enables initial cohorts of 15 patients to be meaningfully evaluated. A transition analysis will estimate with ≥80% certainty whether the true efficacy of the treatment exceeds the pre-defined target response rate. Agents demonstrating sufficient promise will be further evaluated in an expansion stage, following discussion with regulators to clarify data requirements to enable subsequent approval of the agent. Confirmatory analyses of all evaluable patients will estimate with ≥95% certainty whether the true efficacy exceeds the target response rate (“GO”). During both initial and expansion stages patients will be closely monitored, with regular interim futility analyses employed, allowing a treatment arm to stop early if there is very little probability of efficacy (“NoGO”). Outcome measures Primary outcomes measures are occurrence of an objective response after 12 weeks of treatment (Treatment Arm I), occurrence of complete response within a maximum of three cycles of treatment (Treatment Arm II) and occurrence of an objective response following CAR T-cell infusion (Treatment Arm III). Secondary outcome measures include event-free survival, progression-free survival, overall survival time, best overall response, duration of response, adverse events, pharmacokinetic profile and pharmacodynamics markers. Treatment Treatment Arm I: Odronextamab (Regeneron) - a human CD20xCD3 bispecific antibody - monotherapy will be given as an intravenous infusion weekly for 12 weeks, then at a decreasing frequency until progression or up to two years for responding patients. Treatment Arm II: Loncastuximab tesirine (ADC Therapeutics) - a humanised CD19-targeting monoclonal antibody with PBD dimer cytotoxin - will be given as an intravenous infusion with each cycle of modified R-ICE chemotherapy (rituximab, ifosfamide, carboplatin, etoposide and dexamethasone) for up to three cycles. Treatment Arm III: product negotiations are on-going Conclusion Glo-BNHL addresses an urgent unmet clinical need through an innovative trial design serving as a paradigm for evaluation of novel agents in very rare diseases.

Abstract 1604: Preclinical development of ADCT-211, a novel pyrrolobenzodiazepine dimer-based antibody-drug conjugate targeting solid tumors expressing IL13RA2

Abstract Interleukin 13 receptor subunit alpha 2 (IL13RA2) is one of the two major receptors for the cytokine interleukin 13 (IL-13). While IL13RA1 has low affinity for IL-13 and it is expressed ubiquitously in humans, IL13RA2 has high binding affinity to IL-13 and its expression in normal tissues is mainly restricted to the testes. IL13RA2 was initially regarded as a decoy receptor because it has a short cytoplasmic tail with no known signaling motifs, and its binding to IL-13 does not lead to activation of the JAK/STAT6 pathway. However, recent studies demonstrated that IL-13-mediated IL13RA2 signaling occurs via STAT6-independent pathways. IL13RA2 is reported to be expressed at a high frequency in glioblastoma multiforme (GBM) as well as in other solid tumors including melanoma, renal cell carcinoma and adrenocortical carcinoma and is correlated with poor prognosis. ADCT-211 is an antibody-drug conjugate composed of HuCl47, a humanized IgG1 antibody directed against human IL13RA2, site-specifically conjugated using GlycoConnectTM technology to PL1801, which contains HydraspaceTM, a valine-alanine cleavable linker and the PBD dimer cytotoxin SG2000 (drug to antibody ratio ~ 1.8). The purpose of this study was to characterize the in vitro and in vivo anti-tumor activity of ADCT-211 in human cancer cell lines and xenograft models, to determine its safety, tolerability and pharmacokinetic (PK) in the rat and to measure IL13RA2 expression by immunohistochemistry (IHC) in human tumor specimens. HuCl47 showed specific binding to recombinant IL13RA2, while it did not bind to IL13RA1. In vitro, ADCT-211 had highly potent and targeted cytotoxicity against various IL13RA2-expressing solid cancer cell lines. In vivo, a single dose of ADCT-211 at 10 mg/kg was able to completely eradicate tumors in the subcutaneous (sc) A375 human melanoma xenograft model and resulted in 8/8 tumor-free survivors (TFS) at the end of the study on day 57. In the sc U251 human glioblastoma xenograft model, a single dose of ADCT-211 at 3.75 mg/kg showed potent and durable antitumor activity and it resulted in 5/10 partial responders and 5/10 complete responders, 2 of which were TFS at the end of the study on day 51. In an MTD study in male rats, ADCT-211 was stable and tolerated up to 24 mg/kg single dose, with exposure data being indicative of a linear PK profile with a half-life of 12-19 days. Expression of membranous IL13RA2 was confirmed by IHC in a panel of primary and refractory GBM samples and malignant melanoma, highlighting them as potential indications for the clinical development of ADCT-211. In conclusion, ADCT-211 demonstrated potent and specific in vitro and in vivo anti-tumor activity and it was stable and well tolerated in the rat, warranting further development of ADCT-211 into the clinic in IL13RA2-expressing cancers. Citation Format: Francesca Zammarchi, Karin Havenith, Ben Leatherdale, Britanny Roberts, Lara Montolio, Afroze Patel, Narinder Janghra, Pedro Alves, Kristina Zaitseva, Cecile Oblette, Ian Kirby, Lolke de Haan, Patrick H van Berkel. Preclinical development of ADCT-211, a novel pyrrolobenzodiazepine dimer-based antibody-drug conjugate targeting solid tumors expressing IL13RA2 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1604.

IKS03, a Next Generation CD19-Targeted Antibody Drug Conjugate, Shows Potent Activity in Preclinical Models of Aggressive B-Cell Lymphomas

Diffuse large B-cell lymphoma (DLBCL) is a common subtype of non-Hodgkin lymphoma (NHL). Although many patients respond to frontline chemoimmunotherapy, and treatment options for patients with relapsed or refractory DLBCL have improved with the success of CAR T cell therapies, clinical outcome for many patients is still suboptimal. CD19 is widely expressed in DLBCL tumor cells, with normal tissue expression limited to B-cells, making it an attractive target for targeted therapies including antibody drug conjugates (ADC). IKS03 is a CD19-targeting ADC with a PBD dimer pro-drug payload that induces DNA crosslinking and blocks DNA replication ultimately leading to cell death. Following tumor selective binding and uptake, IKS03 requires intracellular lysosomal processing of beta-glucuronidase protecting groups to fully activate the payload which minimizes systemic release of the PBD dimer in human plasma. IKS03 is derived from a human antibody that binds with nanomolar affinity to human and cynomolgus monkey CD19. It is generated by site-specific bioconjugation yielding a homogeneous conjugate with a drug to antibody ratio of 2. In vitro, IKS03 induces specific and dose dependent cell killing of diverse CD19+ lymphoma cell lines with sub-nanomolar EC50 values. Conversely, in vitro cytotoxicity of the conjugate against normal human primary peripheral blood mononuclear cells was attenuated with an EC50 of approximately 1 μM, similar to a non-PBMC binding ADC bearing the same PBD pro-drug. The unconjugated payload more potently reduced PBMC viability with a sub-nanomolar EC50. This is consistent with low non-specific payload release from the conjugate underscoring the improved properties of the PBD pro-drug design. Pharmacokinetics of IKS03 was assessed in a repeat dose toxicity study in monkeys and the plasma concentrations of total ADC and total antibody were determined by LC-MS/MS across the 3-week dosing interval. Overall mean half-life for total ADC was approximately 8 days and clearance rate was slow (mean 0.2 mL/h/kg). Total ADC peak concentration, total plasma exposure and half-life were comparable to that of total antibody demonstrating favorable ADC stability in plasma with limited systemic payload release. In vivo efficacy was evaluated in CD19-expressing preclinical human xenograft models representing different subtypes of B-NHL. Granta-519 cells contain the CCND1 t(11;14) translocation often present in mantle cell lymphoma (MCL). IKS03 was highly active against Granta-519 xenografts in SCID mice with complete tumor regressions observed with a single dose of 0.1 mg/kg. Efficacy was further evaluated in CD19-positive, low passage DLBCL patient-derived xenograft (PDX) models of known genomic profiles. IKS03 was highly active yielding complete tumor regressions following a single dose of 0.3 mg/kg in a refractory lymphoma Bcl2+ PDX model derived from a patient with no response to prior CHOP treatment. Post tumor collection, this patient was also refractory to EPOCH chemotherapy. A second xenograft model was derived from a treatment naïve high-grade 'triple hit' lymphoma patient (BCL2+; BCL6+; MYC+; XP01 amplified). Post tumor collection, this patient was refractory to R-CHOP therapy with disease progression within 2 months. Tumors rapidly progressed in this model, yet single dose treatment with IKS03 resulted in significant tumor growth inhibition at 0.3 mg/kg and complete tumor regressions at 1 mg/kg. Likewise, in a pre-treated MYC-amplified GCB lymphoma model, a single IKS03 dose of 0.3 mg/kg inhibited tumor growth and 1 mg/kg yielded complete inhibition of tumor growth. In conclusion, IKS03 is highly effective in preclinical MCL and DLBCL xenograft models that contain some of the genetic alterations commonly found in relapsed/refractory NHL patients in need of treatment. Preclinical data further demonstrates that IKS03's advanced ADC design results in specific target-dependent in vitro activity, excellent plasma stability and allows for effective targeting of B-cell tumors while sparing normal CD19-negative cells.

240 (PB020) - Antitumor activity of Antibody-Drug Conjugates targeting cancer-expressed EGFR in Preclinical Models

Background: The epidermal growth factor receptor (EGFR) plays a key role in the growth and survival of many human tumors of epithelial origin. The monoclonal antibody, 40H3, targeting overexpressed EGFR and the truncated form EGFR variant III (EGFRvIII) on tumor cells, was conjugated to small molecules with potent cytotoxic activity, generating five antibody-drug conjugates (ADCs). Their lethality for tumor cells was evaluated in vitro and in vivo. Materials and methods: ADC construction: Purified 40H3 antibody was conjugated to five different payloads: two tubulin inhibitors, monomethyl auristatin E (MMAE) and DM1, two topoisomerase inhibitors, SN38 and deruxtecan (DXd1) and a PBD dimer (SG-3199). Binding assay: The binding affinity constants of the various 40H3-based ADCs were determined against the His-tagged EGFR peptide loop (aa 287– 302) immobilized on Ni-NTA biosensors with the Octet Red96 analyzer. Bystander assay: Cocultured F98npEGFRvIII and F98 cells were treated with media, free payload (SG-3199), 40H3 antibody, 40H3-tesirine or IgG-tesirine at the indicated concentrations. After 48 hours, the cells were labeled with cetuximab-PE and SYTOXTM Red viability dye and then analyzed for bystander killing by flow cytometry. In vivo studies: MDA-MB-468 or BT-20 tumor xenografts were treated with unmodified 40H3 antibody or 40H3-tesirine. Tumor volumes and mouse weights were measured at least three times weekly. Results: The five ADCs retained antigen binding activity of the unmodified 40H3 antibody. They showed a potent cytotoxicity on a panel of EGFR-expressing cells including three triple negative breast cancer lines. Cell killing was correlated to the number of binding sites for the 40H3 antibody. The 40H3 conjugate with the PBD dimer (40H3-tesirine) was the most active killing agent and it also exhibited bystander killing of cells not expressing human EGFR. Moreover, in vivo, on two different models of tumor xenografts, treatment with 40H3-tesirine achieved complete remissions. Conclusions: The 40H3 antibody is a valid delivery agent for toxic payloads. Among the five ADCs, 40H3-tesirine showed the highest cytotoxic effect toward EGFR-expressing tumor cells in vitro and in vivo. No conflict of interest.

Non-Genetic Generation of Antibody Conjugates Based on Chemoenzymatic Tyrosine Click Chemistry.

The availability of tools to generate homogeneous and stable antibody conjugates without recombinant DNA technology is a valuable asset in fields spanning from in vitro diagnostics to in vivo imaging and therapeutics. We present here a general approach for the conjugation to human IgG1 antibodies, by employing a straightforward two-stage protocol based on antibody deglycosylation followed by tyrosinase-mediated ortho-quinone strain-promoted click chemistry. The technology is validated by the efficient and clean generation of highly potent DAR2 and DAR4 antibody–drug conjugates (ADCs) with cytotoxic payloads MMAE or PBD dimer, and their in vitro evaluation.

A novel BCMA PBD-ADC with ATM/ATR/WEE1 inhibitors or bortezomib induce synergistic lethality in multiple myeloma.

To target mechanisms critical for multiple myeloma (MM) plasma cell adaptations to genomic instabilities and further sustain MM cell killing, we here specifically trigger DNA damage response (DDR) in MM cells by a novel BCMA antibody drug conjugate (ADC) delivering the DNA cross-linking PBD dimer tesirine, MEDI2228. MEDI2228, more effectively than its anti-tubulin MMAF-ADC homolog, induces cytotoxicity against MM cells regardless of drug resistance, BCMA levels, p53 status, and the protection conferred by bone marrow stromal cells and IL-6. Distinctly, prior to apoptosis, MEDI2228 activates DDRs in MM cells via phosphorylation of ATM/ATR kinases, CHK1/2, CDK1/2, and H2AX, associated with expression of DDR-related genes. Significantly, MEDI2228 synergizes with DDR inhibitors (DDRi s) targeting ATM/ATR/WEE1 checkpoints to induce MM cell lethality. Moreover, suboptimal doses of MEDI2228 and bortezomib (btz) synergistically trigger apoptosis of even drug-resistant MM cells partly via modulation of RAD51 and accumulation of impaired DNA. Such combination further induces superior in vivo efficacy than monotherapy via increased nuclear γH2AX-expressing foci, irreversible DNA damages and tumor cell death, leading to significantly prolonged host survival. These results indicate leveraging MEDI2228 with DDRi s or btz as novel combination strategies, further supporting ongoing clinical development of MEDI2228 in patients with relapsed and refractory MM.

Abstract 211: A new class of sequence-selective DNA cross-linking ADC payloads with increased in vivotolerability

Abstract 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.

Abstract 736: Pyridinobenzodiazepines (PDDs): A new class of sequence-selective DNA mono-alkylating ADC payloads with low hydrophobicity

Abstract 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.

ADCT-402, a PBD dimer–containing antibody drug conjugate targeting CD19-expressing malignancies

AbstractHuman CD19 antigen is a 95-kDa type I membrane glycoprotein in the immunoglobulin superfamily whose expression is limited to the various stages of B-cell development and differentiation and is maintained in the majority of B-cell malignancies, including leukemias and non-Hodgkin lymphomas of B-cell origin. Coupled with its differential and favorable expression profile, CD19 has rapid internalization kinetics and is not shed into the circulation, making it an ideal target for the development of antibody-drug conjugates (ADCs) to treat B-cell malignancies. ADCT-402 (loncastuximab tesirine) is a novel CD19-targeted ADC delivering SG3199, a highly cytotoxic DNA minor groove interstrand crosslinking pyrrolobenzodiazepine (PDB) dimer warhead. It showed potent and highly targeted in vitro cytotoxicity in CD19-expressing human cell lines. ADCT-402 was specifically bound, internalized, and trafficked to lysosomes in CD19-expressing cells and, following release of the PBD warhead, resulted in formation of DNA crosslinks that persisted for 36 hours. Bystander killing of CD19− cells by ADCT-402 was also observed. In vivo, single doses of ADCT-402 resulted in highly potent, dose-dependent antitumor activity in several subcutaneous and disseminated human tumor models with marked superiority to comparator ADCs delivering tubulin inhibitors. Dose-dependent DNA crosslinks and γ-H2AX DNA damage response were measured in tumors by 24 hours after single dose administration, whereas matched peripheral blood mononuclear cells showed no evidence of DNA damage. Pharmacokinetic analysis in rat and cynomolgus monkey showed excellent stability and tolerability of ADCT-402 in vivo. Together, these impressive data were used to support the clinical testing of this novel ADC in patients with CD19-expressing B-cell malignancies.

HLL2-Cys-PBD, a New Site-Specifically Conjugated, Pyrrolobenzodiazepine (PBD) Dimer-Based Antibody Drug Conjugate (ADC) Targeting CD22-Expressing B-Cell Malignancies.

We previously described the development of hLL2-PBD, an ADC composed of the humanized monoclonal antibody epratuzumab (hLL2) against human CD22, stochastically conjugated to SG3249, a cathepsin B-cleavable valine-alanine PBD payload. SG3249, also known as tesirine, is a potent and clinically validated PBD payload currently being employed in the clinic in three ADCs: the CD25-targeted ADCT-301, the CD19-targeted ADCT-402, both for the treatment of lymphoma and leukemia, and the DDL3-targeted rovalpituzumab tesirine for the treatment of small cell lung cancer. hLL2-PBD demonstrated potent and specific in vitro and in vivo activity in the CD22-positive human Burkitt's lymphoma-derived cell lines Ramos, Daudi and WSU-DLCL2. Here, we describe the development and pre-clinical characterization of hLL2-cys-PBD, an ADC composed of an engineered version of epratuzumab (hLL2-cys), site-specifically conjugated to the PBD payload tesirine. The average drug to antibody ratio of hLL2-cys-PBD is 1.74.In vitro, hLL2-cys-PBD showed potent and specific cytoxtoxic activity against a panel of CD22-expressing human lymphoma cell lines, including Ramos, Daudi, WSU-DLCL2 and SU-DHL-4. Moreover, hLL2-cys-PBD was efficiently internalized by CD22-expressing cells and, consistent with the proposed mode of action of PBD dimer warheads, it induced DNA inter-strand cross-linking after a two hour exposure period, as measured by the modified single cell gel electrophoresis (comet) assay. In contrast, a non-CD22-targeted, site-specific, PBD-containing control ADC did not yield appreciable DNA cross-links.In vivo, hLL2-PBD showed potent, dose-dependent, and durable anti-tumor activity in subcutaneously implanted Ramos and WSU-DLCL2 xenograft models. In the Ramos model, a single dose of hLL2-cys-PBD at 1 mg/kg induced complete regression (CR) in all ten treated mice, with 9/10 tumor free survivors (TFS) at the end of the study on day 60. In the WSU-DLCL2 model, a single dose of hLL2-cys-PBD at 3 mg/kg resulted in 3/10 partial regression, 7/10 CR and 2/10 TFS at the end of the study on day 60. The toxicity of hLL2-cys-PBD was evaluated in rat (single dose study) and in cynomolgus monkey (repeat dose study). In rat, hLL2-cys-PBD was well tolerated at 2 mg/kg. In cynomolgus monkey, hLL2-cys-PBD elicited the expected rapid B-cell depletion and it was well tolerated at 0.6 mg/kg.Together, these data demonstrate that hLL2-cys-PBD has potent anti-tumor activity against CD22-expressing hematological tumors and warrants further development of this ADC into the clinic. DisclosuresZammarchi:ADC Therapeutics: Employment. Corbett:ADC Therapeutics: Research Funding. Adams:Medimmune: Employment. Mellinas-Gomez:Medimmune: Employment. Tyrer:Medimmune: Employment. Dissanayake:ADC Therapeutics: Employment. Sims:ADC Therapeutics: Employment. Havenith:ADC Therapeutics: Employment. Chivers:ADC Therapeutics: Employment. Willimas:MedImmune: Employment. Howard:MedImmune/ ADC Therapeutics: Employment, Equity Ownership, Patents & Royalties. Hartley:Spirogen Ltd/ADC Therapeutics: Employment, Equity Ownership, Research Funding. van Berkel:ADC Therapeutics: Employment, Equity Ownership, Patents & Royalties.

Mechanistic and Pharmacodynamic Studies of Adct-301, a Pyrrolobenzodiazepine (PBD) Dimer-Containing Antibody Drug Conjugate (ADC) Targeting CD25-Expressing Hematological Malignancies

ADCT-301, currently in Phase I clinical trial, is an ADC composed of a recombinant human IgG1, HuMax®-TAC against human IL-2R-α (CD25) conjugated through a cleavable linker to a PBD dimer warhead with a drug-antibody ratio of 2.3. In vitro and ex vivo, ADCT-301 binds human CD25 with picomolar affinity. ADCT-301 has highly potent and targeted cytotoxicity against a panel of human lymphoma cell lines. On release, PBD dimers bind in the DNA minor groove and exert their cytotoxic action via the formation of DNA interstrand cross-links. In vivo, ADCT-301 demonstrates dose-dependent antitumor activity against subcutaneous and disseminated lymphoma models. For example, in the Karpas 299 xenograft model, 10/10 tumor-free survivors are observed following a single dose of 0.5 mg/kg, whereas Adcetris® gives only a modest delay in mean tumor growth at 0.5 mg/kg, despite this tumor expressing three-fold higher target antigen levels for this drug. The current study aimed to further define the mechanism of action of ADCT-301 and validate pharmacodynamic assays for clinical development.In Karpas 299 cells, evidence for internalization of ADCT-301 was shown by a reduction of CD25 molecules on the cell surface over the first three hours post-treatment followed by a return to pre-treatment levels by 16 hours. This is consistent with the documented rapid recycling of CD25 to the membrane after exposure to IL-2 (Hemar et al Journal of Cell Biology 1995). Furthermore, ADCT-301 on the cell surface declined by >70% over four hours. Following a two-hour exposure to ADCT-301, DNA interstrand cross-linking, measured using a modification of the single cell gel electrophoresis (comet) assay, reached a peak between 4 and 8 hours after which cross-links persisted up to 36 hours. In contrast, the peak of cross-link formation for an equimolar concentration of warhead was immediately following drug exposure and a non-targeted PBD-containing ADC did not produce crosslinks in these cells. A strong correlation (r = 0.97) between loss of viability and DNA cross-link formation provides support for this DNA damage being the critical initiating mechanism of cytotoxicity of ADCT-301.We have previously shown that PBD-induced DNA interstrand cross-links elicit a robust, but delayed γ-H2AX response (Wu et al Clinical Cancer Research 2013). In Karpas 299 cells phosphorylation of H2AX was observed 24 hours after a two-hour exposure to sub-GI50 concentrations of ADCT-301. In these cells continuous exposure to ADCT-301 resulted in a dose-dependent G2/M arrest, peaking at 48 hours, later than for the naked warhead. The peak of the early apoptosis marker annexin-V on the cell surface of Karpas 299 cells was observed between 60 and 72 hours and maximal loss of viability was at 96 hours.Significant bystander killing of CD25-negative human Burkitt's lymphoma-derived Ramos cells was demonstrated for ADCT-301 both by co-culture experiments with CD25-positive Karpas 299 cells, and by media transfer from Karpas 299 cells treated with ADCT-301. This is important as many lymphomas are heterogeneous in their CD25 expression profile (Strauchen et al American Journal of Pathology 1987).In SCID mice with Karpas 299 subcutaneous tumors a single dose of ADCT-301 was administered at 0.2 or 0.6 mg/kg. 24 hours after treatment, excised tumors showed a dose proportional increase in intensity of membrane and cytoplasmic staining by an anti-PBD payload antibody. Cross-linking was determined as 23% (0.2 mg/kg) vs 49% (0.6 mg/kg) (p ≤ 0.01) reduction in Tail Moment using the comet assay and dose-dependent γ-H2AX formation measured by immunohistochemistry was observed. No cross-linking was observed in matched lymphocyte samples.These data confirm the mechanism of cell killing of ADCT-301 and provide relevant pharmacodynamic assays for use in the clinical development of PBD-based ADCs. DisclosuresFlynn:Spirogen/Medimmune: Employment. van Berkel:ADC Therapeutics: Employment, Equity Ownership, Patents & Royalties. Zammarchi:ADC Therapeutics: Employment. Tyrer:Spirogen/Medimmune: Employment. Williams:Spirogen/Medimmune: Employment. Howard:ADCT Spirogen/Medimmune: Employment, Equity Ownership, Patents & Royalties. Hartley:ADCT Spirogen/Medimmune: Employment, Equity Ownership, Membership on an entity's Board of Directors or advisory committees.

SGN-CD33A in Combination with Hypomethylating Agents Is Highly Efficacious in Preclinical Models of AML

Acute myeloid leukemia (AML) remains a therapeutic challenge. Long-term survival rates, in particular for older AML patients, remain poor, highlighting the need for improved and well-tolerated treatment options. AML patients who are unfit for high dose chemotherapy in the US are often prescribed hypomethylating agents (azacitidine or decitabine) although the efficacy of these agents in this population was modest (Estey, Leukemia 2013).SGN-CD33A is a CD33-directed antibody drug conjugate (ADC) that is comprised of a cysteine-engineered anti-CD33 antibody, a cleavable dipeptide linker that is highly stable in circulation, and a PBD dimer that binds DNA with high intrinsic affinity. The ADC is active as a single agent in preclinical models of AML that are characteristically resistant to chemotherapy (multi-drug-resistant, MDR-positive) (Sutherland et al. Blood 2013). In the present study, we tested the activity of SGN-CD33A in combination with hypomethylating agents (HMA), azacitidine or decitabine. We hypothesized that the combination of SGN-CD33A with an HMA will have greater impact on the DNA repair pathway in leukemic cells, furthering the processes of apoptosis and cell death. MDR-positive AML cells were treated for 96 hours with SGN-CD33A and each of the HMAs alone and in combination, evaluating both simultaneous as well as sequential administration. Enhanced tumor cell killing was observed when AML cells were treated concomitantly with the combination of SGN-CD33A and an HMA or pre-treated with HMAs prior to the addition of SGN-CD33A.Superior anti-leukemic activity of subtherapeutic doses of SGN-CD33A in combination with HMAs was observed in mouse xenograft models generated with MDR-positive AML cell lines. In the difficult to treat HEL9217 model, decitabine or a single dose of 200 mcg/kg SGN-CD33A delayed tumor growth, while significant reductions in tumor growth were observed for the combination treatment (p=0.0001). Improved antitumor activity in this model was also observed for SGN-CD33A in combination with azacitidine. SGN-CD33A in combination with decitabine significantly reduced tumor burden compared to either agent alone in the TF1-α AML model (p=0.0002). Similarly, sequenced dosing of azacitidine followed by a subtherapeutic dose of 30 mcg/kg SGN-CD33A in mice bearing KG-1 xenografts delivered greater antitumor activity compared to the individual agents (p=0.0001).To investigate the mechanism underlying the enhancement in antileukemic activity, we looked at the impact of HMAs alone and in combination with SGN-CD33A on myeloid marker expression, PBD drug release, and impact on the DNA damage and apoptotic pathways. In AML cell lines that did not show improved cytotoxic activity with the combination of SGN-CD33A and HMA, HMA treatment appeared to have no positive effect on the cell surface levels of CD33. However in responsive cell lines such as TF1-α, HMA treatment resulted in time- and dose-dependent increases in the levels of CD33. Significant increases in expression were observed between 2 and 4 days with decitabine and after 4 days with azacitidine. In addition, more PBD dimer drug from SGN-CD33A was incorporated into DNA in HMA treated cells. Concomitant, the treatment of AML cells with the combination of SGN-CD33A and HMA resulted in greater DNA damage and apoptosis as shown by the increased levels of the early DNA damage marker, γH2AX, and the formation of cleaved PARP, an apoptosis marker, compared to either agent alone. Studies are in progress to investigate the effects on other components of the DNA repair and cell cycle pathways.These findings demonstrate that SGN-CD33A can be successfully combined with hypomethylating agents to deliver marked antitumor activity in preclinical drug-resistant models of AML. DisclosuresSutherland:Seattle Genetics, Inc.: Employment. Yu:Seattle Genetics, Inc.: Employment. O'Day:Seattle Genetics,Inc: Employment. Alley:Seattle Genetics,Inc.: Employment. Anderson:Seattle Genetics, Inc.: Employment. Emmerton:Seattle Genetics, Inc.: Employment. Zeng:Seattle Genetics, Inc.: Employment. O'Meara:Seattle Genetics, Inc: Employment, Equity Ownership. Feldman:Seattle Genetics,Inc: Employment. Kennedy:Seattle Genetics,Inc: Employment, Equity Ownership, Honoraria, Speakers Bureau. Ryan:Seattle Genetics, Inc.: Employment. Benjamin:Seattle Genetics, Inc: Employment.

Abstract 637: Preclinical activity of hLL2-PBD, a novel anti-CD22 antibody-pyrrolobenzodiazepine (PBD) conjugate in models of non-Hodgkin lymphoma

Abstract CD22 is a type I transmembrane sialoglycoprotein, which expression is restricted to the B-cell lineage. CD22 is also found highly expressed on most malignant mature B cells, including follicular non-Hodgkin lymphoma (NHL), marginal zone lymphoma (MZL), mantle cell lymphoma (MCL), diffuse large B-cell lymphoma (DLBCL), small lymphocytic lymphoma (SLL) and chronic lymphocytic leukemia (CLL). The differential and favorable expression profile of CD22 in normal and tumor tissues, together with its rapid internalization upon binding with a ligand or antibody, make CD22 an attractive target for an antibody drug conjugate (ADC) mediated treatment of B-cell malignancies. hLL2-PBD is an ADC composed of the humanized monoclonal antibody epratuzumab (hLL2) against human CD22 stochastically conjugated via a valine-alanine cleavable, maleimide linker to a PBD dimer. PBD dimers represent a novel class of payloads, which bind in the DNA minor groove and form highly cytotoxic DNA interstrand cross-links. The average drug to antibody ratio (DAR) of hLL2-PBD is 2. In vitro, hLL2-PBD showed potent and specific cytotoxicity, as assessed by the MTS assay, in the CD22-positive human Burkitt's lymphoma-derived cell lines Ramos and Daudi and the human diffuse large B-cell lymphoma-derived cell line WSU-DLCL2. In vivo, hLL2-PBD demonstrated potent anti-tumor efficacy in subcutaneously (s.c.) implanted Daudi and Ramos xenograft models. In the Daudi model, hLL2-PBD, administered as a single dose at either 0.1 or 0.3 mg/kg, achieved dose-dependent anti-tumor activity, resulting in significant increase in survival. At 0.3 mg/kg, hLL2-PBD induced complete tumor regression in all treated animals. In the Ramos xenograft model, a single dose of 1 mg/kg of hLL2-PBD achieved strong tumor regression. Moreover, hLL2-PBD achieved a superior anti-tumor activity compared to Hu10F4-vcMMAE, a CD22-targeting ADC with the auristatin payload vcMMAE and a DAR of 4 (Genentech Inc.), when tested at 1 mg/kg, single dose. Pharmacokinetic (PK) analysis of hLL2-PBD in CB.17 SCID mice showed a favorable PK profile, with a half-life of approximately 12 days. In conclusion, these data demonstrate the potent in vitro and in vivo anti-tumor activity of hLL2-PBD against CD22-positive hematological tumors and warrant further development of this ADC into the clinic. Citation Format: Francesca Zammarchi, David Williams, Karin Havenith, Francois D'Hooge, Philip W. Howard, John A. Hartley, Patrick van Berkel. Preclinical activity of hLL2-PBD, a novel anti-CD22 antibody-pyrrolobenzodiazepine (PBD) conjugate in models of non-Hodgkin lymphoma. [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 637. doi:10.1158/1538-7445.AM2015-637

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

Abstract 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, ß 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

Pre-Clinical Activity of Adct-301, a Novel Pyrrolobenzodiazepine (PBD) Dimer-Containing Antibody Drug Conjugate (ADC) Targeting CD25-Expressing Hematological Malignancies

Interleukin-2 receptor-α (IL2R-α, CD25), one of a heterotrimer that makes up the IL2R, plays a key role in signal transduction pathways involved in the pathogenesis of autoimmunity and graft rejection (Burchill et al Immunol Lett 2007). In addition, the preponderance of CD25+ cells in hematological malignancies (Srivastava et al Leuk Lymphoma 1994) and the relationship between increased CD25 expression and poor prognosis (Yoshida et al PLoS One 2013) raise the possibility of using an anti-CD25 antibody to deliver a cytotoxin to these cells in patients. Clinical proof of concept for treatment of CD25-positive malignancies has previously been established using radio-immunoconjugates (Dancey et al Clin Cancer Res 2009) and immunotoxins (Kreitman et al J Clin Oncol 2000) utilising antibodies basiliximab and daclizumab.ADCT-301 is an ADC composed of a recombinant human IgG1, HuMax®-TAC against human CD25 attached to a PBD warhead. The drug-antibody ratio is 2.3 ± 0.3. ADCT-301 was potently cytotoxic against CD25-expressing anaplastic large cell lymphoma lines SUDHL1 (341,000 CD25 copies/cell, GI50 0.7 ng/ml) and Karpas 299 (112,000 copies/cell, GI50 3.9 ng/ml) and Hodgkin’s lymphoma line L540 (91,000 copies/cell, GI50 3.9 ng/ml). In contrast, CD25-negative Burkitt’s lymphoma line, Daudi, gave a GI50 >> 1 mg/ml. The released PBD dimer warhead induces highly cytotoxic interstrand cross-links in the DNA minor groove. Unique to this class of ADCs, the single cell gel electrophoresis (comet) assay can therefore be used as a pharmacodynamic endpoint. For ADCT-301, cross-link formation was dose dependent and the peak of cross-linking occurred 16 to 24 hours after a 2 hour exposure of Karpas 299 cells.In vivo, ADCT-301 demonstrated dose-dependent antitumor activity against SUDHL1 and Karpas 299 xenograft and disseminated models. For example, ADCT-301 at a single dose of 0.2 mg/kg significantly delayed Karpas 299 tumor growth compared to vehicle-treated and isotype control ADC-treated mice, and at 0.4 and 0.6 mg/kg gave 3/10 and 10/10 tumor-free survivors, respectively (Figure A). 10/10 tumor-free survivors were also observed at a single dose of 0.5 mg/kg, whereas Adcetris gave only a modest delay in mean tumor growth at a single dose of 0.5 mg/kg despite this tumor expressing three times the level of Adcetris target CD30 antigen compared to CD25 (Figure B). ADCT-301 was well tolerated with no signs of toxicity at 6 mg/kg, currently the highest dose tested.Together, these data clearly demonstrate the potent antitumor activity of ADCT-301 against CD25-expressing hematological tumors and warrants the rapid development of this agent into the clinic. [Display omitted] DisclosuresFlynn:Medimmune: Employment. van Berkel:ADC Therapeutics Sarl: Employment, Equity Ownership, Patents & Royalties. Zammarchi:ADC Therapeutics Sarl: Employment. Levy:Medimmune: Employment. Tiberghien:Medimmune: Employment, Patents & Royalties. Masterson:Medimmune: Employment. D’Hooge:Medimmune: Employment. Adams:Medimmune: Employment. Williams:Medimmune: Employment. Howard:ADC Therapeutics Sarl: Equity Ownership, Patents & Royalties. Hartley:Medimmune: Employment, Equity Ownership, Patents & Royalties; ADC Therapeutics Sarl: Equity Ownership, Patents & Royalties.

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

Abstract 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 4321: Development of pyrrolobenzodiazepine-based antibody-drug conjugates for cancer.

Abstract Pyrrolobenzodiazepine (PBD) dimers are highly cytotoxic minor groove binding DNA crosslinking agents derived from the anthramycin class of natural products. In our efforts to develop highly effective and well-tolerated treatment options for cancer patients, we have investigated several antibody-drug conjugates (ADCs) based on a PBD dimer. The fully synthetic PBD-linker, comprised of a protease-cleavable val-ala sequence and a reactive maleimide group, was conjugated via engineered cysteine residues to humanized monoclonal antibodies specific for their binding to the well-characterized cancer antigen targets, CD33 and CD70. The resulting ADCs were well-defined, monomeric and showed pronounced in vitro and in vivo activity against both hematologic and solid tumor cancer models. These included models of acute myelogenous leukemia, Hodgkin lymphoma, non-Hodgkin lymphoma and renal cell carcinoma. The ADCs were immunologically specific, and showed potent activity in MDR-positive models, including models resistant to other ADCs. These results underscore the importance of the drug component in developing highly efficacious ADCs for cancer therapy. Citation Format: Scott C. Jeffrey, Patrick J. Burke, May K. Sutherland, David W. Meyer, Robert P. Lyon, Julie A. McEarchern, Che-Leung Law, Peter D. Senter. Development of pyrrolobenzodiazepine-based antibody-drug conjugates for cancer. [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 4321. doi:10.1158/1538-7445.AM2013-4321

Inter- and intrastrand DNA crosslinks by 2-fluoro-substituted pyrrolobenzodiazepine dimers: stability, stereochemistry and drug orientation

A 2-fluoro-substituted pyrrolo[2,1-c][1,4]benzodiazepine (PBD) dimer with a 1,4-di-n-propyl piperazine linker was studied with respect to its binding and crosslinking capability towards double-helical DNA targets. Duplex thermal stabilizations upon drug binding as measured by UV melting experiments suggest that two guanine bases separated by four AT base pairs constitute the favorable binding site for the PBD dimer. Large stabilizations were observed for the self-complementary duplex d(AACAATTGTT)(2) as well as for the non-self-complementary duplex d(AAGAATTGTT)·d(AACAATTCTT) with both guanines located on the same strand. Formation of interstrand and intrastrand crosslinks by the covalent binding of both PBD moieties of the dimer to the exocyclic 2-amino group of the two guanine bases within the duplex minor groove was confirmed by NMR structural studies. In both the symmetric and non-symmetric DNA-PBD adducts the newly created stereogenic center at C11 of the tricyclic PBD subunits favors an S configuration. Different orientations of the PBD aromatic A-ring with respect to the covalently modified guanine as observed in the non-symmetric complex are shown to result in characteristic changes of PBD H11 and H11a proton chemical shifts. Based on a compilation of available NMR data on various PBD complexes, these differences may be used as valuable probes for the identification of PBD orientational preferences in DNA-PBD adducts.

Nuclear Magnetic Resonance Solution Structures of Inter- and Intrastrand Adducts of DNA Cross-Linker SJG-136

SJG-136 (1) is a sequence-selective DNA-interactive agent that is about to enter phase II clinical trials for the treatment of malignant disease. Previous studies on the pyrrolo[2,1-c][1,4]benzodiazepine (PBD) dimers, typified by SJG-136 and DSB-120 (2), have shown that these planar ligands react with the exocyclic NH(2) groups of two guanine bases in the base of the minor groove of DNA to form an irreversible interstrand cross-linked sequence-specific adduct. Using high-field NMR, we have characterized and modeled the previously predicted interstrand duplex adduct formed by SJG-136 with the self-complementary 5'-d(CICGATCICG)(2) duplex (4). This first SJG-136 NMR-refined adduct structure has been compared with previous high-field NMR studies of the adducts of the closely related PBD dimer DSB-120 with the same duplex and of the adduct of tomaymycin (3) formed with 5'-d(ATGCAT)(2). Surprisingly, the SJG-136 duplex adduct appears to be more closely related to the tomaymycin adduct than to the DSB-120 adduct with respect of the orientation and depth of insertion of the ligand within the minor groove. The intrastrand duplex adduct formed in the reaction of SJG-136 with the noncomplementary 5'-d(CTCATCAC)·(GTGATGAG) duplex (5) has also been synthesized and modeled. In this duplex adduct, the nature of the cross-link was confirmed, the central guanines were identified as the sites of alkylation, and the stereochemical configuration at C11 at both ends of the SJG-136 molecule was determined to be S. The NMR-refined solution structures produced for the intrastrand adduct confirm the previously proposed structure (which was based solely on mass spectroscopy). Both the inter- and intrastrand SJG-136 duplex adducts form with minimal distortion of the DNA duplex. These observations have an impact on the proposal for the mechanism of action of SJG-136 both in vitro and in vivo, on the repair of its adducts and mechanism of resistance in cells, and, potentially, on the type of pharmacodynamic assay to be used in clinical trials. SGJ-136 is currently in phase II clinical trials with several groups working on both dimeric cross-linking agents and monoalkylating ligands based on the PBD alkylating moiety. This study suggests subtle differences between the DNA binding of SJG-136 and the C2 unsubstituted analogue DSB-120 that are likely to be the origins of the differences in potency. Confirmation of the stereochemical configuration at the C11 position (particularly in the intrastrand adduct) provides confirmation of binding orientation that was previously only speculation in the HPLC MS study. Together, these observations are likely to be of value in the development of third-generation PBD-based cross-linkers and monoalkylating analogues.