Pyrrolobenzodiazepine Research Articles

ABBV-176, a PRLR antibody drug conjugate with a potent DNA-damaging PBD cytotoxin and enhanced activity with PARP inhibition

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

Benzodiazepine Derivatives from Marine-Derived Streptomyces cacaoi 14CM034

7-methoxy-8-hydroxy cycloanthranilylproline (2), a new natural product with pyrrolobenzodiazepine (PBD) framework, was isolated from marine-derived actinobacterium Streptomyces cacaoi 14CM034, together with cycloanthranilylproline (1). Structural elucidation of the compounds was based on FTIR, 1D- (1H and 13C NMR), 2D-NMR (COSY, HMBC and NOESY) and HR-MS analyses. Compounds 1 and 2 exhibited notable antimicrobial activity. The presence of PBD derivatives in S. cacaoi was first demonstrated with this study.

Early Development, Scale-Up, and Reverse-Phase Purification of a Highly Potent Pyrrolobenzodiazepine Dimer, SG3259, for Use in Antibody–Drug Conjugates

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

Resistance to Pyrrolobenzodiazepine Dimers Is Associated with SLFN11 Downregulation and Can Be Reversed through Inhibition of ATR.

Resistance to antibody-drug conjugates (ADCs) has been observed in both preclinical models and clinical studies. However, mechanisms of resistance to pyrrolobenzodiazepine (PBD)-conjugated ADCs have not been well characterized and thus, this study was designed to investigate development of resistance to PBD dimer warheads and PBD-conjugated ADCs. We established a PBD-resistant cell line, 361-PBDr, by treating human breast cancer MDA-MB-361 cells with gradually increasing concentrations of SG3199, the PBD dimer released from the PBD drug-linker tesirine. 361-PBDr cells were over 20-fold less sensitive to SG3199 compared with parental cells and were cross-resistant to other PBD warhead and ADCs conjugated with PBDs. Proteomic profiling revealed that downregulation of Schlafen family member 11 (SLFN11), a putative DNA/RNA helicase, sensitizing cancer cells to DNA-damaging agents, was associated with PBD resistance. Confirmatory studies demonstrated that siRNA knockdown of SLFN11 in multiple tumor cell lines conferred reduced sensitivity to SG3199 and PBD-conjugated ADCs. Treatment with EPZ011989, an EZH2 inhibitor, derepressed SLFN11 expression in 361-PBDr and other SLFN11-deficient tumor cells, and increased sensitivity to PBD and PBD-conjugated ADCs, indicating that the suppression of SLFN11 expression is associated with histone methylation as reported. Moreover, we demonstrated that combining an ataxia telangiectasia and Rad3-related protein (ATR) inhibitor, AZD6738, with SG3199 or PBD-based ADCs led to synergistic cytotoxicity in either resistant 361-PBDr cells or cells that SLFN11 was knocked down via siRNA. Collectively, these data provide insights into potential development of resistance to PBDs and PBD-conjugated ADCs, and more importantly, inform strategy development to overcome such resistance.

Combination of Camidanlumab Tesirine, a CD25-Targeted ADC, with Gemcitabine Elicits Synergistic Anti-Tumor Activity in Preclinical Tumor Models

Camidanlumab tesirine (ADCT-301) is an antibody-drug conjugate (ADC) comprised of HuMax®-TAC, a monoclonal antibody directed against human CD25, conjugated to the pyrrolobenzodiazepine dimer payload tesirine[1]. Currently, camidanlumab tesirine is being evaluated in a pivotal Phase 2 clinical trial in patients with relapsed or refractory Hodgkin lymphoma (HL) (NCT04052997) and in a Phase 1b clinical trial in patients with advanced solid tumors (NCT03621982). In pre-clinical studies, camidanlumab tesirine demonstrated strong and durable single agent activity in CD25-expressing lymphoma xenograft models[1] and in vitro it synergised with selected targeted agents[2]. Moreover, CD25-ADC, a mouse CD25 cross-reactive surrogate of camidanlumab tesirine, induced potent anti-tumor immunity against established syngeneic solid tumor models by depleting CD25-positive tumor-infiltrating T regulatory cells (Tregs) and it showed synergistic activity when combined with PD-1 blockade[3]. Here, we investigated the in vitro and in vivo anti-tumor activity of camidanlumab tesirine combined with gemcitabine, a common standard-of-care chemotherapeutic agent used both in a hematological and solid tumor clinical setting. In vitro, the combination of camidanlumab tesirine and gemcitabine was evaluated in three human-derived cancer cell lines (two HL and one anaplastic large cell lymphoma, ALCL) and resulted in synergistic activity as determined by the Chou-Talalay method. In vivo, camidanlumab tesirine was tested either alone (0.05 or 0.1 mg/kg, single dose) or in combination with gemcitabine (80 mg/kg, q3dx4) in the CD25-expressing ALCL Karpas299 xenograft model. At both ADC dose levels, combination with gemcitabine resulted in synergistic anti-tumor activity (coefficient of drug interaction (CDI) 0.51 and 0.17, respectively), better response rates and increased survival compared to monotherapy with camidanlumab tesirine. In order to extend the investigation to solid tumor models, CD25-ADC was tested in the CT26 syngeneic model, a colorectal cancer model with CD25-expressing tumor-infiltrating Tregs. CD25-ADC was administered either alone (0.1, 0.5 or 1 mg/kg, single dose) or in combination with gemcitabine (80 mg/kg, q3dx4). At the 0.1 mg/kg dose of CD25-ADC, combination with gemcitabine resulted in synergistic anti-tumor activity (CDI 0.68). Moreover, at 0.5 and 1 mg/kg, the combination of CD25-ADC and gemcitabine resulted in more durable anti-tumor activity and better response rates compared to both monotherapy treatments. In conclusion, the combination of camidanlumab tesirine and gemcitabine was synergistic both in vitro and in vivo in lymphoma preclinical models. Synergistic anti-tumor activity was also demonstrated in a colorectal cancer model using CD25-ADC, a mouse-cross-reactive version of camidanlumab tesirine, in combination with gemcitabine. Altogether, these novel pre-clinical data warrant translation of the combination between camidanlumab tesirine and gemcitabine into the clinic. 1.Flynn, M.J., et al., ADCT-301, a Pyrrolobenzodiazepine (PBD) Dimer-Containing Antibody-Drug Conjugate (ADC) Targeting CD25-Expressing Hematological Malignancies. Mol Cancer Ther, 2016. 15(11): p. 2709-2721. 2.Spriano, F., et al., The anti-CD25 antibody-drug conjugate camidanlumab tesirine (ADCT-301) presents a strong preclinical activity both as single agent and in combination in lymphoma cell lines. Hematological Oncology, 2019. 37(S2): p. 323-324. 3.Zammarchi, F., et al., A CD25-targeted antibody-drug conjugate depletes regulatory T cells and eliminates established syngeneic tumors via antitumor immunity. Journal for ImmunoTherapy of Cancer, 2020. In press. Disclosures Jabeen: ADC Therapeutics: Current Employment. Hartley:ADC Therapeutics: Consultancy, Current equity holder in publicly-traded company, Research Funding. Van Berkel:ADC-Therapeutics: Current Employment, Current equity holder in publicly-traded company. Zammarchi:ADC-Therapeutics: Current Employment, Current equity holder in publicly-traded company.

Analysis of Adct-602 Pre-Clinical Activity in B-Cell Lymphoma Models and Identification of Potential Biomarkers for Its Activity

Analysis of Adct-602 Pre-Clinical Activity in B-Cell Lymphoma Models and Identification of Potential Biomarkers for Its Activity

Pharmacokinetic and Pharmacodynamic Correlates from the Phase 1 Study of Camidanlumab Tesirine (Cami) in Patients with Relapsed or Refractory Hodgkin Lymphoma and Non-Hodgkin Lymphoma

Introduction: Cami is an antibody (Ab)-drug conjugate composed of the human monoclonal Ab, HuMax®-TAC, directed against human CD25, and conjugated through a cleavable linker to SG3199, a pyrrolobenzodiazepine (PBD) dimer cytotoxin. Cami has shown notable single-agent antitumor activity and manageable toxicity in a phase 1 trial in patients (pts) with relapsed or refractory (R/R) Hodgkin lymphoma (HL) or non-Hodgkin lymphoma (NHL) (Collins et al, ICML Jun 18-22, 2019;https://onlinelibrary.wiley.com/doi/10.1002/hon.61_2629; Collins et al, ASH Dec 1-4, 2018;https://doi.org/10.1182/blood-2018-99-115986). Methods: This open-label, single-arm, dose escalation and expansion trial (NCT04052997) enrolled pts aged ≥18 years with histologically confirmed R/R HL or R/R NHL. The primary objectives included characterizing the safety and tolerability of Cami. The secondary objectives addressed the pharmacokinetic (PK) profile of Cami, PBD-conjugated Ab (cAb, drug-to-Ab ratio [DAR] ≥1), total Ab (tAb, DAR≥0) and unconjugated warhead SG3199, and assessed the anti-drug Ab (ADA) response in serum. Also reported are exploratory PK/pharmacodynamic correlates to concentrations of soluble CD25 (sCD25), time course of peripheral blood B cells, NK cells, and T-cell subsets: Treg(CD25+/CD127low/FoxP3+[CD3+/CD4+]), Teff (CD8+), and Teff:Treg ratio, using TruCount flow cytometry (Covance, Indianapolis, IN, USA), circulating cytokines, and immunohistochemistry (IHC) on archival or pre-treatment tumor biopsies for CD25 expression. Results: Data from 133 pts with lymphoma (HL and NHL) were available for PK analysis following Cami doses of 3-300 µg/kg administered every 3 weeks. Mean exposures increased with dose. Apparent clearance for doses of 30-80 µg/kg was relatively constant (for cAb, 1.66 L/day [CV=64%] in the 45 µg/kg dose group during Cycle 1). Good linker stability of the immunoconjugate was demonstrated by close similarity of profiles between cAb and tAb. By Cycle 2, similar respective exposures with lower inter-patient variability were noted as relative to Cycle 1. Apparent half-life of cAb was 2.7 days (CV=40%), with no accumulation observed by the end of the 3-week cycle. Levels of SG3199 were generally too low for characterization. There were no instances of positive pre- or post-dose ADA response. For pts receiving the 30-60 µg/kg doses, preferential decreases over time for IL-6, IL-8, and IL-10 were noted for pts achieving clinical response, with decreases most evident by end of first cycle. For total lymphocytes, CD19+ B cells, CD3+ and CD4+ T cells, CD8+ Teff and CD16+/CD56+ NK cell subsets, similar modulations by time were observed at 30 and 45 µg/kg doses, comprising transient increases following dosing (Figure 1). Small absolute decreases in Treg cell concentrations over time were seen following the 30 and 45 µg/kg doses, but with no distinction by best overall clinical response (complete response [CR] + partial response vs. non-responders). However, most pts had clear increases in Teff:Treg ratios over time, with greater effects in Cycle 2 relative to Cycle 1. Mean sCD25 concentrations displayed apparent decrease from baseline over time following the 45 µg/kg dose. For pts with HL, higher baseline levels of sCD25 were associated with lower Cami exposure in both responders and non-responders. Particularly for pts exhibiting CR, clustering was apparent with sCD25 concentrations <~10,000 ng/mL at baseline (Figure 2). Data from IHC showed no relationship between CD25 histoscore and clinical response. Conclusion: While Treg modulation was seen with all populations, these data support further study, particularly of the Cami 45 µg/kg dose for pts with HL. Given that CD25 is the cognate target of Cami, these data suggest pts with HL achieving CR have higher drug exposure, resulting from lower baseline sCD25, and potential association with lower tumor burden. Although exposure at the 45 µg/kg dose showed moderate inter-patient variability, it was associated with noteworthy, cycle-related modulation in circulating Treg and clear increases in Teff:Treg ratios, thought to favor clinical response. Funding: Study funded by ADC Therapeutics SA. Disclosures Boni: ADC Therapeutics America, Inc: Current Employment, Current equity holder in publicly-traded company. Havenith:ADC Therapeutics (UK) Ltd: Current Employment, Current equity holder in publicly-traded company. Hamadani:Janssen R&D; Incyte Corporation; ADC Therapeutics; Celgene Corporation; Pharmacyclics, Omeros, AbGenomics, Verastem, TeneoBio: Consultancy; ADC Therapeutics: Membership on an entity's Board of Directors or advisory committees; Takeda Pharmaceutical Company; Spectrum Pharmaceuticals; Astellas Pharma: Research Funding; Sanofi Genzyme, AstraZeneca: Speakers Bureau. Caimi:Amgen: Other: Advisory Board; Bayer: Other: Advisory Board; Kite Pharma: Other: Advisory Board; ADC Therapeutics: Other: Advisory Board, Research Funding; Verastem: Other: Advisory Board; Celgene: Speakers Bureau. Anderson:ADC Therapeutics (UK) Ltd: Current Employment, Current equity holder in publicly-traded company. Kopotsha:ADC Therapeutics (UK) Ltd: Current Employment, Current equity holder in publicly-traded company. Cruz:ADC Therapeutics SA: Current Employment, Current equity holder in publicly-traded company. Wuerthner:ADC Therapeutics SA: Current Employment, Current equity holder in publicly-traded company.

Phase 1, First-in-Human Study of MEDI2228, a BCMA-Targeted ADC in Patients with Relapsed/Refractory Multiple Myeloma

Phase 1, First-in-Human Study of MEDI2228, a BCMA-Targeted ADC in Patients with Relapsed/Refractory Multiple Myeloma

Evaluating 225Ac and 177Lu Radioimmunoconjugates against Antibody-Drug Conjugates for Small-Cell Lung Cancer.

Interest in the use of 225Ac for targeted alpha therapies has increased dramatically over the past few years, resulting in a multitude of new isotope production and translational research efforts. However, 225Ac radioimmunoconjugate (RIC) research is still in its infancy, with most prior experience in hematologic malignancies and only one reported preclinical solid tumor study using 225Ac RICs. In an effort to compare 225Ac RICs to other current antibody conjugates, a variety of RICs are tested against intractable small-cell lung cancer (SCLC). We directly compare, in vitro and in vivo, two promising candidates of each α or β- category, 225Ac and 177Lu, versus pyrrolobenzodiazepine (PBD) nonradioactive benchmarks. The monoclonal antibody constructs are targeted to either delta like 3 protein (DLL3), a recently discovered SCLC target, or CD46 as a positive control. An immunocompromised maximum tolerated dose assay is performed on NOD SCID mice, along with tumor efficacy proof-of-concept studies in vivo. We overview the conjugation techniques required to create serum-stable RICs and characterize and compare in vitro cell killing with RICs conjugated to nonspecific antibodies (huIgG1) with either native or site-specific thiol loci against tumor antigen DLL3-expressing and nonexpressing cell lines. Using patient-derived xenografts of SCLC onto NOD SCID mice, solid tumor growth was controlled throughout 3 weeks before growth appeared, in comparison to PBD conjugate controls. NOD SCID mice showed lengthened survival using 225Ac compared to 177Lu RICs, and PBD dimers showed full tumor suppression with nine out of ten mice. The exploration of RICs on a variety of antibody-antigen systems is necessary to direct efforts in cancer research toward promising candidates. However, the anti-DLL3-RIC system with 225Ac and 177Lu appears to be not as effective as the anti-DLL3-PBD counterpart in SCLC therapy with matched antibodies and portrays the challenges in both SCLC therapy as well as the specialized utility of RICs in cancer treatment.

The Role of Specific ATP-Binding Cassette Transporters in the Acquired Resistance to Pyrrolobenzodiazepine Dimer-Containing Antibody-Drug Conjugates.

Antibody-drug conjugates (ADC) containing pyrrolobenzodiazepine (PBD) dimers are being evaluated clinically in both hematologic and solid tumors. These include ADCT-301 (camidanlumab tesirine) and ADCT-402 (loncastuximab tesirine) in pivotal phase II trials that contain the payload tesirine, which releases the PBD dimer warhead SG3199. An important consideration in future clinical development is acquired resistance. The aim was to generate and characterize PBD acquired resistant cell lines in both hematologic and solid tumor settings. Human Karpas-299 (ALCL) and NCI-N87 (gastric cancer) cells were incubated with increasing IC50 doses of ADC (targeting CD25 and HER2, respectively) or SG3199 in a pulsed manner until stable acquired resistance was established. The level of resistance achieved was approximately 3,000-fold for ADCT-301 and 3-fold for SG3199 in Karpas-299, and 8-fold for ADCT-502 and 4-fold for SG3199 in NCI-N87. Cross-resistance between ADC and SG3199, and with an alternative PBD-containing ADC or PBD dimer was observed. The acquired resistant lines produced fewer DNA interstrand cross-links, indicating an upstream mechanism of resistance. Loss of antibody binding or internalization was not observed. A human drug transporter PCR Array revealed several genes upregulated in all the resistant cell lines, including ABCG2 and ABCC2, but not ABCB1(MDR1). These findings were confirmed by RT-PCR and Western blot, and inhibitors and siRNA knockdown of ABCG2 and ABCC2 recovered drug sensitivity. These data show that acquired resistance to PBD-ADCs and SG3199 can involve specific ATP-binding cassette drug transporters. This has clinical implications as potential biomarkers of resistance and for the rational design of drug combinations.

CD25-targeted antibody-drug conjugate depletes regulatory T cells and eliminates established syngeneic tumors via antitumor immunity.

BackgroundRegulatory T cells (Tregs) contribute to an immunosuppressive tumor microenvironment. They play an important role in the establishment and progression of tumors with high Tregs infiltration and present a major obstacle to tumor eradication by immunotherapies. Numerous strategies have been attempted to deplete or block Tregs, although their success has been limited.MethodsA CD25-targeted, pyrrolobenzodiazepine (PBD) dimer-based antibody–drug conjugate (ADC) was investigated for its ability to deplete Tregs and induce antitumor immunity. Antitumor activity of CD25-ADC either alone or in combination with an anti-programmed cell death protein 1 (PD-1) antibody was evaluated in CD25-negative syngeneic models that exhibit tumor infiltration of CD25-expressing Tregs, and its pharmacodynamics and pharmacokinetics were assessed.ResultsSingle low doses of CD25-ADC resulted in potent and durable antitumor activity in established syngeneic solid tumor models and the combination of a suboptimal dose was synergistic with PD-1 blockade. Tumor eradication by the CD25-targeted ADC was CD8+ T cell-dependent and CD25-ADC induced protective immunity. Importantly, while CD25-ADC mediated a significant and sustained intratumoral Tregs depletion, accompanied by a concomitant increase in the number of activated and proliferating tumor-infiltrating CD8+ T effector cells, systemic Tregs depletion was transient, alleviating concerns of potential autoimmune side effects.ConclusionsThis study shows that a PBD dimer-based, CD25-targeted ADC is able to deplete Tregs and eradicate established tumors via antitumor immunity. This represents a novel approach to efficiently deplete Tregs via a very potent DNA damaging toxin known to induce immunogenic cell death. Moreover, this study provides proof of concept for a completely new application of ADCs as immunotherapeutic agents, as the main mode of action relies on the ADC directly targeting immune cells, rather than tumor cells. These strong preclinical data warrant the clinical evaluation of camidanlumab tesirine (ADCT-301), a PBD-based ADC targeting human CD25, either alone or in combination with checkpoint inhibitors in solid tumors with known Tregs infiltration. A phase I trial (NCT03621982) of camidanlumab tesirine in patients with selected advanced solid tumors is ongoing.

Abstract CT124: First-in-human study of SC-004, an antibody-drug conjugate targeting CLDN6/9, in patients with epithelial ovarian cancers

Abstract Background: More than 90% of ovarian cancers are epithelial ovarian cancers (EOC), and approximately 70% of EOCs are serous histology. Despite advancements with the development of PARP inhibitors and incorporation of bevacizumab, a clear unmet need remains for patients (pts) with recurrent EOC which is no longer considered appropriate for platinum-based chemotherapy (platinum-resistant). Endometrial cancer (EC) is the most common gynecologic malignancy in the United States, and there are limited options for pts who progress after first and/or second line chemotherapy. Claudins (CLDNs) are four-pass transmembrane proteins with key roles in tight junction formation and function, regulation of paracellular transport, and maintenance of cell polarity. CLDN6 and CLDN9 are expressed on neoplastic cells in a proportion of high-grade EOC and high-grade serous EC with minimal expression in normal tissues. SC-004 is an antibody-drug conjugate (ADC), consisting of an antibody targeting CLDN6/9 linked to a pyrrolobenzodiazepine (PBD) dimer DNA cross-linking agent. In this open-label, phase 1 clinical trial, the safety, tolerability and maximum tolerated dose (MTD) of SC-004 were assessed in pts with platinum-resistant/refractory EOC and EC (NCT03138408). Methods: Enrolled pts were adults with platinum-refractory or resistant high-grade serous EOC or EC, who had previously received at least 1 platinum-based chemotherapeutic regimen. SC-004 was administered intravenously at 0.005-0.3 mg/kg every 3 weeks (0.3 mg/kg was administered every 4 weeks). Treatment emergent adverse events (TEAEs) were defined as any adverse event (AE) that worsened from the first dose of SC-004 through 90 days following discontinuation of treatment. Membranous CLDN6 expression was assessed by immunohistochemistry retrospectively. Results: A total of 24 pts were enrolled (11 with ovarian and 13 with EC). Pts received SC-004 for a median of 2.5 cycles (range:1-25). The most common TEAEs (>20% of pts, N=24) were fatigue (38%), peripheral edema (33%), vomiting (33%), pleural effusion (25%), urinary tract infection (25%), rash maculo-papular (25%), anemia (21%), abdominal pain (21%), diarrhea (21%), and decreased appetite (21%). Twenty-one pts (88%) had at least 1 AE possibly related to SC-004. Eight pts (33%) had at least 1 grade 3 or higher AE with a possibility of being related to SC-004, the most common being pericardial effusion in 2 pts (8%). Eight pts (33%) had at least 1 SAE, including pericardial effusion, pleural effusion, renal failure, respiratory failure, abdominal pain, and failure to thrive, each occurring in 2 pts (8%) each. Eight pts (33%) had at least 1 AE leading to SC-004 dose interruption. Three pts (13%) had a fatal AE considered unrelated to SC-004. Thirteen pts (54%) died during the trial, mainly due to disease progression. The MTD was 0.2 mg/kg Q3W. Among 19 evaluable pts, best overall response of stable disease occurred in 14 pts (73%), partial response in 1 pt (5%), and progressive disease in 4 pts (21%). 4/19 pts (21%) with evaluable tissue had at least 50% of tumor cells with membranous CLDN6 expression, and one of these pts had the only partial response. Conclusion: SC-004 demonstrated low tolerability with a side effect profile similar to other PBD-conjugated ADCs, along with insufficient evidence of activity to support continued investigation. Citation Format: Erika Hamilton, Gini F. Fleming, Premal H. Thaker, Vivek Subbiah, Chris Chen, Abraham Fong, Daniel Brickman, Kathleen Moore. First-in-human study of SC-004, an antibody-drug conjugate targeting CLDN6/9, in patients with epithelial ovarian cancers [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr CT124.

Systematic Variation of Pyrrolobenzodiazepine (PBD)-Dimer Payload Physicochemical Properties Impacts Efficacy and Tolerability of the Corresponding Antibody-Drug Conjugates.

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

Biosynthesis, Mechanism of Action, and Inhibition of the Enterotoxin Tilimycin Produced by the Opportunistic Pathogen Klebsiella oxytoca.

Tilimycin is an enterotoxin produced by the opportunistic pathogen Klebsiella oxytoca that causes antibiotic-associated hemorrhagic colitis (AAHC). This pyrrolobenzodiazepine (PBD) natural product is synthesized by a bimodular nonribosomal peptide synthetase (NRPS) pathway composed of three proteins: NpsA, ThdA, and NpsB. We describe the functional and structural characterization of the fully reconstituted NRPS system and report the steady-state kinetic analysis of all natural substrates and cofactors as well as the structural characterization of both NpsA and ThdA. The mechanism of action of tilimycin was confirmed using DNA adductomics techniques through the detection of putative N-2 guanine alkylation after tilimycin exposure to eukaryotic cells, providing the first structural characterization of a PBD-DNA adduct formed in cells. Finally, we report the rational design of small-molecule inhibitors that block tilimycin biosynthesis in whole cell K.oxytoca (IC50 = 29 ± 4 μM) through the inhibition of NpsA (KD = 29 ± 4 nM).

Overcoming Challenges in the Development of Anticancer ADCs

Overcoming Challenges in the Development of Anticancer ADCs

Phase I study of the antibody-drug conjugate ABBV-321 in patients with non-small cell lung cancer and squamous head and neck cancer with overexpression of the epidermal growth factor receptor.

TPS3649 Background: ABBV-321 (serclutamab talirine) is an epidermal growth factor receptor (EGFR)-targeted antibody-drug conjugate that consists of a humanized immunoglobulin G1 anti-EGFR monoclonal antibody conjugated to a pyrrolobenzodiazepine (PBD) dimer via a maleimidocaproyl-valine-alanine linker. Once bound, ABBV-321 is internalized, the maleimidocaproyl-valine-alanine linker undergoes proteolytic cleavage, and the cytotoxic PBD is released, causing DNA cross-links and cell death. Preclinical studies have shown cytotoxicity in numerous human xenograft and patient (pt)-derived tumor models. This first-in-human trial is assessing the safety, pharmacokinetic (PK), and preliminary antitumor activity of ABBV-321 in pts with advanced solid tumor types likely to exhibit elevated levels of EGFR. Methods: This is a 2-part, multicenter phase 1 study (NCT03234712) of ABBV-321 monotherapy in pts (≥18 years; Eastern Cooperative Oncology Group performance status 0–1) with advanced solid tumors associated with overexpression of EGFR. EGFR overexpression will be determined by centralized testing using an RNA-based assay. Primary objectives of the completed part 1 (dose escalation) were to determine the maximum tolerated dose (MTD)/recommended phase 2 dose (RP2D) of ABBV-321 and assess the PK and toxicity and safety profile; part 2 (dose expansion) will evaluate safety and PK profile at the RP2D in specific cohorts (NSCLC and HNSCC). Secondary objectives include assessment of preliminary antitumor activity. Pts will receive escalating doses of ABBV-321 until the MTD/RP2D is determined. Dose-limiting toxicities will be assessed during the first cycle of dosing. Adverse events (AEs) will be evaluated per National Cancer Institute Common Terminology Criteria for AEs (version 4.03). Blood samples for PK analysis (ABBV-321, total antibody, unconjugated PBD) will be collected at designated time points throughout the study. The multinational trial is active, with the first pt screened on 1 Feb 2018. The dose-escalation phase has been completed; screening and enrollment for the expansion phase of the study in NSCLC and HNSCC is underway. Clinical trial information: NCT03234712 .

A Novel Antibody-Drug Conjugate (ADC) Delivering a DNA Mono-Alkylating Payload to Chondroitin Sulfate Proteoglycan (CSPG4)-Expressing Melanoma.

Despite emerging targeted and immunotherapy treatments, no monoclonal antibodies or antibody-drug conjugates (ADCs) directly targeting tumor cells are currently approved for melanoma therapy. The tumor-associated antigen chondroitin sulphate proteoglycan 4 (CSPG4), a neural crest glycoprotein over-expressed on 70% of melanomas, contributes to proliferative signaling pathways, but despite highly tumor-selective expression it has not yet been targeted using ADCs. We developed a novel ADC comprising an anti-CSPG4 antibody linked to a DNA minor groove-binding agent belonging to the novel pyrridinobenzodiazepine (PDD) class. Unlike conventional DNA-interactive pyrrolobenzodiazepine (PBD) dimer payloads that cross-link DNA, PDD-based payloads are mono-alkylating agents but have similar efficacy and substantially enhanced tolerability profiles compared to PBD-based cross-linkers. We investigated the anti-tumor activity and safety of the anti-CSPG4-(PDD) ADC in vitro and in human melanoma xenografts. Anti-CSPG4-(PDD) inhibited CSPG4-expressing melanoma cell growth and colony formation and triggered apoptosis in vitro at low nanomolar to picomolar concentrations without off-target Fab-mediated or Fc-mediated toxicity. Anti-CSPG4-(PDD) restricted xenograft growth in vivo at 2 mg/kg doses. One 5 mg/kg injection triggered tumor regression in the absence of overt toxic effects or of acquired residual tumor cell resistance. This anti-CSPG4-(PDD) can deliver a highly cytotoxic DNA mono-alkylating payload to CSPG4-expressing tumors at doses tolerated in vivo.

Design and synthesis of a folate-receptor targeted diazepine-ring-opened pyrrolobenzodiazepine prodrug conjugate

Pyrrolobenzodiazepines (PBDs) and their dimers (bis-PBDs) have emerged as some of the most potent chemotherapeutic compounds and are currently under development as novel payloads in antibody-drug conjugates (ADCs). However, when used as stand-alone therapeutics or as warheads for small molecule drug conjugates (SMDCs), dose-limiting toxicities are often observed. As an elegant solution to this inherent problem, we designed and synthesized a diazepine-ring-opened bis-PBD prodrug (pro-PBD-PBD) folate conjugate lacking the one of the two imine moieties found in the corresponding free bis-PBD. Upon entering a targeted cell, cleavage of the linker system, including the hydrolysis of an oxazolidine moiety, results in the formation of a reactive intermediate which possesses a newly formed aldehyde as well as an aromatic amine. A fast and spontaneous intramolecular ring-closing reaction subsequently takes place as the aromatic amine adds to the aldehyde with the loss of water to give the imine, and as a result, the diazepine ring, thereby delivering the bis-PBD to the targeted cell. The in vitro and in vivo activity of this conjugate has been evaluated on folate receptor positive KB cells. Sub-nanomolar activity with good specificity and high cure rates with minimal toxicity have been observed.

An Anti-CD300f Antibody Drug Conjugate Depletes Hematopoietic Stem Cells and Primary Acute Myeloid Leukemia (AML): Facilitating a Targeted Conditioning Regimen for Allogeneic Stem Cell Transplantation in AML

Allogeneic Hematopoietic Stem Cell Transplantation (allo-HSCT) significantly reduces the rate of relapse in Acute Myeloid Leukemia (AML) but comes at the cost of significant Treatment Related Mortality (TRM). Despite the reduction in relapse overall, it remains common, especially in high risk groups. There is increasing evidence that many cases of relapse are driven by residual AML that can be detected with minimal residual disease techniques. A large proportion of the morbidity that prevents most patients accessing allo-HSCT is due to nonspecific toxic conditioning agents which are required to remove recipient Hematopoietic Stem and Progenitor Cells (HSPC) allowing for successful engraftment. We propose that a targeted conditioning agent against AML and HSPC can facilitate a reduction in relapse and TRM in allo-HSCT. CD300f is expressed evenly across HSPC subtypes. CD300f has equivalent transcription and protein expression as CD33 on AML. We have developed an anti-CD300f antibody which efficiently internalises into target cells. Harnessing this ability to internalise, we have generated a highly potent anti-CD300f Antibody Drug Conjugate (ADC) with a pyrrolobenzodiazepine (PBD) warhead which selectively depletes AML cell lines in vitro. Colony Forming Units were inhibited when cultured in the presence of the ADC. The anti-CD300f ADC has a rapid onset of action with >99% cytotoxicity of the AML cell line HL-60 occurring within 24 hours. The ADC synergises with fludarabine, making it a natural combination to use in a minimal toxicity conditioning regimen. The growth of subcutaneous tumours in mice engrafted with the AML cell line U937 was significantly reduced by a single dose of our ADC (at 150 μg/kg or 300 μg/kg). In a bone marrow engraftment model with HL-60, our ADC prolongs the survival at a single dose of 300 μg/kg (Fig 1A). In a humanised mouse model, 85% of all CD34+ cells and 90% of the primitive CD34+ CD38- CD90+ population were depleted with a single injection of our ADC at 300 μg/kg (Fig1 B). There was significant reduction in primary AML cells with a single injection of our ADC at 300 μg/kg (Fig 1C). This proof of principle work establishes an anti-CD300f ADC as an attractive potential therapeutic to develop which will reduce the toxicity of allo-HSCT and the relapse rate afterwards. We have demonstrated that CD300f is expressed across HSPC and AML. Our antibody to CD300f is efficiently internalised and capable of delivering a PBD warhead. Our ADC synergised with fludarabine and has a rapid onset of action. We have demonstrated that our ADC prolongs survival in mouse cell line models as well as depletes both HSPC and primary AML in vivo. A targeted conditioning agent has the potential to reduce relapse risk and toxicity of allo-HSCT in AML.

Different Reaction Specificities of F420H2-Dependent Reductases Facilitate Pyrrolobenzodiazepines and Lincomycin To Fit Their Biological Targets.

Antitumor pyrrolobenzodiazepines (PBDs), lincosamide antibiotics, quorum-sensing molecule hormaomycin, and antimicrobial griselimycin are structurally and functionally diverse groups of actinobacterial metabolites. The common feature of these compounds is the incorporation of l-tyrosine- or l-leucine-derived 4-alkyl-l-proline derivatives (APDs) in their structures. Here, we report that the last reaction in the biosynthetic pathway of APDs, catalyzed by F420H2-dependent Apd6 reductases, contributes to the structural diversity of APD precursors. Specifically, the heterologous overproduction of six Apd6 enzymes demonstrated that Apd6 from the biosynthesis of PBDs and hormaomycin can reduce only an endocyclic imine double bond, whereas Apd6 LmbY and partially GriH from the biosyntheses of lincomycin and griselimycin, respectively, also reduce the more inert exocyclic double bond of the same 4-substituted Δ1-pyrroline-2-carboxylic acid substrate, making LmbY and GriH unusual, if not unique, among reductases. Furthermore, the differences in the reaction specificity of the Apd6 reductases determine the formation of the fully saturated APD moiety of lincomycin versus the unsaturated APD moiety of PBDs, providing molecules with optimal shapes to bind their distinct biological targets. Moreover, the Apd6 reductases establish the first F420H2-dependent enzymes from the luciferase-like hydride transferase protein superfamily in the biosynthesis of bioactive molecules. Finally, our bioinformatics analysis demonstrates that Apd6 and their homologues, widely distributed within several bacterial phyla, play a role in the formation of novel yet unknown natural products with incorporated l-proline-like precursors and likely in the microbial central metabolism.