Narinder Janghra

A novel morpholino oligomer targeting ISS-N1 improves rescue of severe spinal muscular atrophy transgenic mice.

In the search for the most efficacious antisense oligonucleotides (AOs) aimed at inducing SMN2 exon 7 inclusion, we systematically assessed three AOs, PMO25 (-10, -34), PMO18 (-10, -27), and PMO20 (-10, -29), complementary to the SMN2 intron 7 splicing silencer (ISS-N1). PMO25 was the most efficacious in augmenting exon 7 inclusion in vitro in spinal muscular atrophy (SMA) patient fibroblasts and in vitro splicing assays. PMO25 and PMO18 were compared further in a mouse model of severe SMA. After a single intracerebroventricular (ICV) injection in neonatal mice, PMO25 increased the life span of severe SMA mice up to 30-fold, with average survival greater by 3-fold compared with PMO18 at a dose of 20u2009μg/g and 2-fold at 40u2009μg/g. Exon 7 inclusion was increased in the CNS but not in peripheral tissues. Systemic delivery of PMO25 at birth achieved a similar outcome and produced increased exon 7 inclusion both in the CNS and peripherally. Systemic administration of a 10-μg/g concentration of PMO25 conjugated to an octaguanidine dendrimer (VMO25) increased the life span only 2-fold in neonatal type I SMA mice, although it prevented tail necrosis in mild SMA mice. Higher doses and ICV injection of VMO25 were associated with toxicity. We conclude that (1) the 25-mer AO is more efficient than the 18-mer and 20-mer in modifying SMN2 splicing in vitro; (2) it is more efficient in prolonging survival in SMA mice; and (3) naked Morpholino oligomers are more efficient and safer than the Vivo-Morpholino and have potential for future SMA clinical applications.

Dystrophin quantification: Biological and translational research implications

Objective: We formed a multi-institution collaboration in order to compare dystrophin quantification methods, reach a consensus on the most reliable method, and report its biological significance in the context of clinical trials. Methods: Five laboratories with expertise in dystrophin quantification performed a data-driven comparative analysis of a single reference set of normal and dystrophinopathy muscle biopsies using quantitative immunohistochemistry and Western blotting. We developed standardized protocols and assessed inter- and intralaboratory variability over a wide range of dystrophin expression levels. Results: Results from the different laboratories were highly concordant with minimal inter- and intralaboratory variability, particularly with quantitative immunohistochemistry. There was a good level of agreement between data generated by immunohistochemistry and Western blotting, although immunohistochemistry was more sensitive. Furthermore, mean dystrophin levels determined by alternative quantitative immunohistochemistry methods were highly comparable. Conclusions: Considering the biological function of dystrophin at the sarcolemma, our data indicate that the combined use of quantitative immunohistochemistry and Western blotting are reliable biochemical outcome measures for Duchenne muscular dystrophy clinical trials, and that standardized protocols can be comparable between competent laboratories. The methodology validated in our study will facilitate the development of experimental therapies focused on dystrophin production and their regulatory approval.

Repeated low doses of morpholino antisense oligomer: an intermediate mouse model of spinal muscular atrophy to explore the window of therapeutic response

The human SMN2 transgenic mice are well-established models of spinal muscular atrophy (SMA). While the severe type I mouse model has a rapidly progressive condition mimicking type I SMA in humans, the mild type III mice do not faithfully recapitulate chronic SMA variants affecting children. A SMA mouse model that clinically mimics the features of type II and III SMA in human is therefore needed. In this study, we generated intermediately affected SMA mice by delivering low-dose morpholino oligomer (PMO25) into the existing severe SMA mice. We show that a single low-dose administration of PMO25 moderately extended the survival of severe type I SMA mice. The neuromuscular pathology is also modestly but significantly improved in these mice. A second administration of PMO25 at postnatal day 5 (PND5) demonstrated an additive effect on survival. Additional systemic administration of low-dose PMO25 at 2-week intervals suppressed the occurrence of distal necrosis beyond postnatal day 100, and induced more complete phenotypic rescue than a single bolus high-dose injection at PND0. Our study demonstrates that survival of motor neuron (SMN) is required early at a critical threshold to prevent symptoms and suggests that subsequent systemic administration of low-dose PMO25 in SMA mice can provide therapeutic benefit and phenotypic rescue, presumably via peripheral SMN restoration. Our work also provides additional insight into the time window of response to administration of antisense oligonucleotides to SMA mice with an intermediate phenotype. This information is crucial at a time when a number of therapeutic interventions are in clinical trials in SMA patients.

Biochemical characterization of patients with in-frame or out-of-frame DMD deletions pertinent to exon 44 or 45 skipping

IMPORTANCEnIn Duchenne muscular dystrophy (DMD), the reading frame of an out-of-frame DMD deletion can be repaired by antisense oligonucleotide (AO)-mediated exon skipping. This creates a shorter dystrophin protein, similar to those expressed in the milder Becker muscular dystrophy (BMD). The skipping of some exons may be more efficacious than others. Patients with exon 44 or 45 skippable deletions (AOs in clinical development) have a less predictable phenotype than those skippable for exon 51, a group in advanced clinical trials. A way to predict the potential of AOs is the study of patients with BMD who have deletions that naturally mimic those that would be achieved by exon skipping.nnnOBJECTIVEnTo quantify dystrophin messenger RNA (mRNA) and protein expression in patients with DMD deletions treatable by, or mimicking, exon 44 or 45 skipping.nnnDESIGN, SETTING, AND PARTICIPANTSnRetrospective study of nondystrophic controls (nu2009=u20092), patients with DMD (nu2009=u20095), patients with intermediate muscular dystrophy (nu2009=u20093), and patients with BMD (nu2009=u200913) at 4 university-based academic centers and pediatric hospitals. Biochemical analysis of existing muscle biopsies was correlated with the severity of the skeletal muscle phenotype.nnnMAIN OUTCOMES AND MEASURESnDystrophin mRNA and protein expression.nnnRESULTSnPatients with DMD who have out-of-frame deletions skippable for exon 44 or 45 had an elevated number of revertant and trace dystrophin expression (approximately 19% of control, using quantitative immunohistochemistry) with 4 of 9 patients presenting with an intermediate muscular dystrophy phenotype (3 patients) or a BMD-like phenotype (1 patient). Corresponding in-frame deletions presented with predominantly mild BMD phenotypes and lower dystrophin levels (approximately 42% of control) than patients with BMD modeling exon 51 skipping (approximately 80% of control). All 12 patients with in-frame deletions had a stable transcript compared with 2 of 9 patients with out-of-frame deletions (who had intermediate muscular dystrophy and BMD phenotypes).nnnCONCLUSIONS AND RELEVANCEnExon 44 or 45 skipping will likely yield lower levels of dystrophin than exon 51 skipping, although the resulting protein is functional enough to often maintain a mild BMD phenotype. Dystrophin transcript stability is an important indicator of dystrophin expression, and transcript instability in DMD compared with BMD should be explored as a potential biomarker of response to AOs. This study is beneficial for the planning, execution, and analysis of clinical trials for exon 44 and 45 skipping.

Correlation of Utrophin Levels with the Dystrophin Protein Complex and Muscle Fibre Regeneration in Duchenne and Becker Muscular Dystrophy Muscle Biopsies.

Duchenne muscular dystrophy is a severe and currently incurable progressive neuromuscular condition, caused by mutations in the DMD gene that result in the inability to produce dystrophin. Lack of dystrophin leads to loss of muscle fibres and a reduction in muscle mass and function. There is evidence from dystrophin-deficient mouse models that increasing levels of utrophin at the muscle fibre sarcolemma by genetic or pharmacological means significantly reduces the muscular dystrophy pathology. In order to determine the efficacy of utrophin modulators in clinical trials, it is necessary to accurately measure utrophin levels and other biomarkers on a fibre by fibre basis within a biopsy section. Our aim was to develop robust and reproducible staining and imaging protocols to quantify sarcolemmal utrophin levels, sarcolemmal dystrophin complex members and numbers of regenerating fibres within a biopsy section. We quantified sarcolemmal utrophin in mature and regenerating fibres and the percentage of regenerating muscle fibres, in muscle biopsies from Duchenne, the milder Becker muscular dystrophy and controls. Fluorescent immunostaining followed by image analysis was performed to quantify utrophin intensity and β-dystrogylcan and ɣ –sarcoglycan intensity at the sarcolemma. Antibodies to fetal and developmental myosins were used to identify regenerating muscle fibres allowing the accurate calculation of percentage regeneration fibres in the biopsy. Our results indicate that muscle biopsies from Becker muscular dystrophy patients have fewer numbers of regenerating fibres and reduced utrophin intensity compared to muscle biopsies from Duchenne muscular dystrophy patients. Of particular interest, we show for the first time that the percentage of regenerating muscle fibres within the muscle biopsy correlate with the clinical severity of Becker and Duchenne muscular dystrophy patients from whom the biopsy was taken. The ongoing development of these tools to quantify sarcolemmal utrophin and muscle regeneration in muscle biopsies will be invaluable for assessing utrophin modulator activity in future clinical trials.

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

Human 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.

Abstract 51: Characterization of the mechanism of action, pharmacodynamics and preclinical safety of ADCT-402, a pyrrolobenzodiazepine (PBD) dimer-containing antibody-drug conjugate (ADC) targeting CD19-expressing hematological malignancies

ADCT-402, currently in Phase I clinical trials for B-cell hematological malignancies, is an ADC composed of a recombinant humanized IgG1 against human CD19, stochastically conjugated via a cleavable linker to a PBD dimer cytotoxin (DAR of 2.3). PBD dimers, DNA minor groove interstrand cross-linking agents, are gaining increasing attention and are currently being tested as the ADC warheads in several clinical trials. ADCT-402 has potent and targeted cytotoxicity against a panel of human lymphoma and leukemia cell lines in vitro. In vivo, ADCT-402 demonstrates dose-dependent antitumor activity against Burkitt’s lymphoma xenograft models. Moreover, ADCT-402 is markedly superior to maytansinoid- and auristatin-based CD19-targeting ADCs in the Ramos xenograft model. In a rat toxicology study, a single dose of ADCT-402 at 2 mg/kg is well tolerated with a favorable PK profile and excellent stability in vivo. The current study aimed to further define the mechanism of action (MOA) of ADCT-402 and validate its pharmacology and preclinical safety for clinical development. CD19 is a clinically validated target with restricted normal tissue expression and a widespread expression in the majority of B-cell malignancies. Importantly, we show here the consistent expression of CD19 in matched samples (initial diagnosis and relapsed/refractory) from panels of lymphoma patients, indicating that relapsed/refractory patients are appropriate for treatment with ADCT-402. ADCT-402 was shown to be efficiently internalized by CD19+ cells in vitro. Moreover, in line with the PBD dimer MOA, following a 2 hour exposure to ADCT-402, DNA interstrand cross-links reached a peak between 8 - 12 hours and persisted for up to 36 hours post-treatment. In contrast, the peak of cross-link formation for the PBD dimer warhead alone was observed immediately after 2 hour incubation, while a non-targeted PBD-ADC did not yield any appreciable DNA cross-links. In SCID mice s.c. implanted with Ramos cells, a single dose of ADCT-402 was administered at 0.33 or 1 mg/kg. Twenty-four hours after treatment, excised tumors showed a dose proportional increase in intensity of staining by an anti-PBD payload antibody, as well as in DNA cross-linking and in γ-H2AX formation. In contrast, no DNA cross-linking was observed in matched lymphocyte samples. The toxicity of ADCT-402 was further evaluated in a repeat dose cynomolgus monkey study. ADCT-402 was clinically well tolerated with an acceptable off-target safety profile. The PK of the ADC was consistent with normal antibody clearance with a half-life of about 12 to 17 days. These data confirm the MOA of ADCT-402 and provide relevant pharmacodynamic assays and preclinical safety assessment to guide the clinical development of this promising ADC in B-cell malignancies. Citation Format: Francesca Zammarchi, Simon Corbett, Karin Havenith, Narinder Janghra, Konstantinos Kiakos, Teresa Marafioti, David G. Williams, Simon Chivers, Phil W. Howard, John A. Hartley, Patrick H. van Berkel. Characterization of the mechanism of action, pharmacodynamics and preclinical safety of ADCT-402, a pyrrolobenzodiazepine (PBD) dimer-containing antibody-drug conjugate (ADC) targeting CD19-expressing hematological malignancies [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 51. doi:10.1158/1538-7445.AM2017-51

Abstract 52: Mechanistic and benchmarking studies of ADCT-502, a pyrrolobenzodiazepine (PBD) dimer-containing antibody-drug conjugate (ADC) targeting HER2-expressing solid tumors

ADCT-502 is an ADC composed of an engineered version of humanized IgG1 trastuzumab, directed against human HER2, site-specifically conjugated to the highly cytotoxic PBD-based linker-drug tesirine (drug-antibody ratio of 1.7). In vitro, ADCT-502 has highly potent and targeted cytotoxicity against various solid cancer cell lines. In vivo, ADCT-502 demonstrates strong and durable antitumor activity in mouse xenografts with various levels of HER2, but is inactive in a HER2-negative xenograft. ADCT-502 is stable, well tolerated and has a favorable PK profile both in rat and cynomolgus monkey. The current study aimed to define further the mechanism of action of ADCT-502 and to benchmark its activity in xenograft models against ado-trastuzumab emtansine (T-DM1), the ADC currently approved for the treatment of HER2+ metastatic breast cancers. ADCT-502 bound and internalized efficiently in JIMT-1 cells (HER2+) and co-localized with lysosomes within 2 hours. PBD dimers bind in the DNA minor groove and exert cytotoxicity via the formation of DNA interstrand cross-links. Following a 2-hour exposure to ADCT-502, DNA interstrand cross-linking peaked between 12 and 24 hours, after which cross-links persisted at least 36 hours. In contrast, cross-link formation by an equimolar concentration of warhead alone, peaked immediately following drug exposure and a non-targeted ADC did not produce DNA crosslinks in these cells. Moreover, ADCT-502 showed indirect bystander killing activity in HER2-negative MDA-MB-468 cells incubated with conditioned medium from ADCT-502-treated HER2+ SK-BR-3 cells. In vivo, antitumor activity of ADCT-502 was compared to T-DM1 in both cell line- and patient-derived-xenograft (PDX) models. For example, in a HER2 1+, FISH- breast cancer PDX, ADCT-502 showed dose-dependent antitumor activity resulting in 1/8 and 8/8 TFS after a single dose at 0.1 and 0.2 mg/kg, respectively. Conversely, a single dose of T-DM1 at 30 mg/kg showed only marginal activity compared to the control. Similarly, in a HER2 1+, FISH- esophageal cancer PDX, while a single dose of ADCT-502 at 0.44 mg/kg resulted in strong and durable antitumor activity, single doses of T-DM1 at either 10 or 30 mg/kg showed no activity compared to the control. These data confirm that the mechanism of cell killing of ADCT-502 is via target-specific internalization and subsequent cross-linking of DNA. They also show superior in vivo antitumor activity of ADCT-502 compared to T-DM1 in various tumor xenografts, including those with low HER2 levels. Taken together, these results support the development of ADCT-502 not only in patients that have become resistant/refractory to T-DM1, but also in patients whose tumors express low levels of HER2, and are not eligible for treatment with T-DM1. Citation Format: Francesca Zammarchi, Halla W. Reinert, Narinder Janghra, Simon Corbett, Maria Mellinas-Gomez, Sajidah Chowdhury, Neha Arora, Peter Tyrer, Francois Bertelli, David G. Williams, Philip W. Howard, John A. Hartley, Patrick H. van Berkel. Mechanistic and benchmarking studies of ADCT-502, a pyrrolobenzodiazepine (PBD) dimer-containing antibody-drug conjugate (ADC) targeting HER2-expressing solid tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 52. doi:10.1158/1538-7445.AM2017-52

Biomarker development to support the clinical development of utrophin modulators for Duchenne muscular dystrophy therapy

The continual expression of utrophin protein by pharmacological maintenance of utrophin transcription in dystrophin deficient muscle fibres is a potential disease modifying treatment for Duchenne muscular dystrophy (DMD). In order for proof of concept of utrophin modulators to be demonstrated in DMD patients, a multicomponent biomarker strategy has been instigated to quantify utrophin levels and demonstrate evidence of reduced muscle fibre regeneration. To demonstrate an increase in utrophin derived from drug treatment above the natural levels resulting from regeneration, we aim to quantify both fibre numbers and levels of utrophin protein localised to mature fibres in pre- and post-dose muscle biopsies. To determine a reduction in the rate of degeneration, i.e. increase in mature fibre survival, changes in the percentage of regenerating fibres, determined by the presence of neonatal and foetal myosin, will be calculated from biopsies. Serum samples will be analysed to quantify the levels of specific microRNAs (miRs) associated with fibre leakage and peptide markers of active fibrosis which characterise fibre damage and degeneration respectively. We will present data from these candidate biomarkers tested in DMD, Becker and normal samples looking to demonstrate quantifiable changes which are indicative of benefit. In future clinical trials of utrophin modulators, and potentially other DMD therapeutic approaches, these biomarkers may be appropriate to confirm benefit to dystrophin deficient muscle.

G.P.103

The continual expression of utrophin protein by pharmacological maintenance of utrophin transcription in dystrophin deficient muscle fibres is a potential disease modifying treatment for Duchenne muscular dystrophy (DMD). In order for proof of concept of utrophin modulators to be demonstrated in DMD patients, a multicomponent biomarker strategy has been instigated to quantify utrophin levels and demonstrate evidence of reduced muscle fibre regeneration. To demonstrate an increase in utrophin derived from drug treatment above the natural levels resulting from regeneration, we aim to quantify both fibre numbers and levels of utrophin protein localised to mature fibres in pre- and post-dose muscle biopsies. To determine a reduction in the rate of degeneration, i.e. increase in mature fibre survival, changes in the percentage of regenerating fibres, determined by the presence of neonatal and foetal myosin, will be calculated from biopsies. Serum samples will be analysed to quantify the levels of specific microRNAs (miRs) associated with fibre leakage and peptide markers of active fibrosis which characterise fibre damage and degeneration respectively. We will present data from these candidate biomarkers tested in DMD, Becker and normal samples looking to demonstrate quantifiable changes which are indicative of benefit. In future clinical trials of utrophin modulators, and potentially other DMD therapeutic approaches, these biomarkers may be appropriate to confirm benefit to dystrophin deficient muscle.