Antibody Drug Conjugates: Nonclinical Safety Considerations

Antibody drug conjugates (ADCs) are biopharmaceutical molecules consisting of a cytotoxic small molecule covalently linked to a targeted protein carrier via a stable cleavable or noncleavable linker. The process of conjugation yields a highly complex molecule with biochemical properties that are distinct from those of the unconjugated components. The impact of these biochemical differences on the safety and pharmacokinetic (PK) profile of the conjugate must be considered when determining the types of nonclinical safety studies required to support clinical development of ADCs. The hybrid nature of ADCs highlights the need for a science-based approach to safety assessment that incorporates relevant aspects of small and large molecule testing paradigms. This thinking is reflected in current regulatory guidelines, where sections pertaining to conjugates allow for a flexible approach to nonclinical safety testing. The aim of this article is to review regulatory expectations regarding early assessment of nonclinical safety considerations and discuss how recent advances in our understanding of ADC-mediated toxicity can be used to guide the types of nonclinical safety studies needed to support ADC clinical development. The review will also explore nonclinical testing strategies that can be used to streamline ADC development by assessing the safety and efficacy of next generation ADC constructs using a rodent screen approach.KEY WORDS: antibody drug conjugates, regulatory guidance, safety assessment, therapeutic index     

Pneumomediastinum, pneumothorax and subcutaneous emphysema during radiotherapy in primary cavitating bronchogenic carcinoma

An extremely rare case of cavitating large cell carcinoma lung is reported in a middle aged man who developed pneumomediastinum, pneumothorax and subcutaneous emphysema on thirteenth day of his tele cobalt radiotherapy to chest. Possible mechanisms of such complications during radiotherapy are also discussed.     

Mainstreaming of Emergency Contraception Pill in India: Challenges and Opportunities

Background:

Emergency Contraception Pill (ECP) is an essential intervention to prevent unwanted pregnancies. However, its use has remained low due to various barriers including reservations among medical fraternity.

Materials and Methods:

This paper presents findings on barriers to ECP''s easy access for potential users from (i) a cross-sectional survey of providers'' attitudes, beliefs, and practices and interviews with key opinion leaders, (ii) three consultations organized by Population Council with policymakers and public health experts, and (iii) evidence from scientific literature.

Results:

The major barriers to easy access of ECP include misconceptions and reservations of providers (disapproval of ECP provision by CHWs, opposition to its being an OTC product, and myths, misconceptions, and moral judgments about its users) including influential gynecologists.

Conclusion:

For mainstreaming ECP, the paper recommends educational campaign focusing on gynecologists and CHWs, relaxing restrictive policy on advertisement of ECP, involving press media and strengthening supply chain to ensure its regular supply to ASHA (CHW).     

GsdmD p30 elicited by caspase-11 during pyroptosis forms pores in membranes

Gasdermin-D (GsdmD) is a critical mediator of innate immune defense because its cleavage by the inflammatory caspases 1, 4, 5, and 11 yields an N-terminal p30 fragment that induces pyroptosis, a death program important for the elimination of intracellular bacteria. Precisely how GsdmD p30 triggers pyroptosis has not been established. Here we show that human GsdmD p30 forms functional pores within membranes. When liberated from the corresponding C-terminal GsdmD p20 fragment in the presence of liposomes, GsdmD p30 localized to the lipid bilayer, whereas p20 remained in the aqueous environment. Within liposomes, p30 existed as higher-order oligomers and formed ring-like structures that were visualized by negative stain electron microscopy. These structures appeared within minutes of GsdmD cleavage and released Ca²⁺ from preloaded liposomes. Consistent with GsdmD p30 favoring association with membranes, p30 was only detected in the membrane-containing fraction of immortalized macrophages after caspase-11 activation by lipopolysaccharide. We found that the mouse I105N/human I104N mutation, which has been shown to prevent macrophage pyroptosis, attenuated both cell killing by p30 in a 293T transient overexpression system and membrane permeabilization in vitro, suggesting that the mutants are actually hypomorphs, but must be above certain concentration to exhibit activity. Collectively, our data suggest that GsdmD p30 kills cells by forming pores that compromise the integrity of the cell membrane.Pyroptosis is an inflammatory form of programmed cell death that occurs in response to microbial products in the cytoplasm or to cellular perturbations caused by diverse stimuli, including crystalline substances, toxins, and extracellular ATP (1, 2). Pyroptosis plays a critical role in the clearance of intracellular bacteria (3), but may also contribute to autoinflammatory and autoimmune disease pathology. Mechanistically, pyroptosis occurs when cytosolic nucleotide-binding oligomerization domain (NOD)-like receptors (NLRs), including NLRP1, NLRP3, and NLRC4, or the pyrin domain-containing protein AIM2, nucleate a canonical inflammasome complex that activates the protease caspase-1 (2). Alternatively, intracellular lipopolysaccharide (LPS) from Gram-negative bacteria can trigger noncanonical activation of mouse caspase-11 and human caspases 4 and 5 (4–7). Caspases 1, 4, 5, and 11 can each cleave Gasdermin-D (GsdmD) to mediate pyroptotic cell death (8, 9). It is the N-terminal p30 fragment of GsdmD that is cytotoxic to cells, but precisely how it kills cells is unknown.Here we show that the human GsdmD p30 fragment liberated by active caspase-11 forms ring-like structures within membranes that function as pores. Therefore, we propose p30 kills cells by directly compromising the integrity of cellular membranes. We also show that the GsdmD I105N mutant that was unable to mediate macrophage pyroptosis (9) is hypomorphic at cell killing in a transient overexpression system and at liposome permeabilization in vitro—conditions where p30 concentration favors oligomerization.     

Young red blood cells; observations about the cytometric and morphologic alterations in the beginning of their life

Summary A recently described separation technique consisting of a combination of counterflow centrifugation and subsequent density (Percoll) scparation was tested for its ability to enrich red cell populations with young cells in comparison to either separation technique alone. The relative age of every fraction was determined by HbAlc measurements, resulting in the lowest HbAlc for the combination method. Conventional reticulocyte counting and floweytometric counting with thiazole orange indicated that in the youngest fractions the combination method showed the highest reticulocyte counts. There was a good correlation between manual and flowcytometric counting results. Radio-iron studies showed a two-fold enrichment with young cells in the fraction with the lowest HbAIc acquired by the combination technique in comparison to the other two methods. Cytometric measurements showed that the fractions with the lowest HbAlc were the ones with the highest MCV and MCH and the lowest MCHC. Besides loss of their RNA-material, young cells already seem to loose water and haemoglobin like older cells, resulting in a decrease of MCV and MCH and in increase in MCHC. It is concluded that combining counterflow centrifugation with subsequent density fractionation results in superior enrichment with young cells in comparison to the results of each method alone.     

Quantitative comparison of spontaneous and paced 12-lead electrocardiogram during right ventricular outflow tract ventricular tachycardia

OBJECTIVES: The purpose of this study was to objectively quantify the similarity of 12-lead electrocardiogram (ECG) waveforms using two quantitative metrics, the correlation coefficient (CORR) and the mean absolute deviation (MAD). BACKGROUND: Comparison of the 12-lead ECG morphology between ventricular tachycardia (VT) and a pace-map is frequently performed; however, there are no objective criteria for quantifying the similarity between two waveform morphologies. METHODS: During ablation of right ventricular outflow tract (RVOT) VT, 12-lead ECG pace-maps were acquired from three superior septal sites, three superior free wall sites, and before each ablation attempt in 15 patients. The 12-lead ECG waveforms of the clinical tachycardia and pace-maps were compared using both MAD and CORR at each site. RESULTS: The MAD scores were lower (i.e., more closely matched) for septal compared with free wall sites (15.9 +/- 5.3% vs. 25.3 +/- 10.2%; p < 0.001). Successful ablation sites had a significantly lower MAD score compared with unsuccessful sites (9.5 +/- 2.8% vs. 13.3 +/- 5.6%; p = 0.01), whereas there was only a trend toward a higher CORR for successful ablation sites (98.2 +/- 1.2% vs. 96 +/- 4.7%; p = 0.07). A MAD score < or =12% was 93% sensitive and 75% specific for identifying a successful ablation site. There was an inverse correlation between MAD score and distance from the site of VT origin (r = 0.63, p < 0.001). CONCLUSIONS: A MAD score >12% between RVOT VT and a pace-map at any site suggests sufficient dissimilarity to dissuade ablation at that site. The MAD score can be used to standardize 12-lead ECG waveform morphology comparisons among different laboratories, and may be useful for guiding ablation of VT.     

Clinical, haematological and histomorphological profile of adult myelodysplastic syndrome. Study of 96 cases in a single institute

Myelodysplastic syndromes (MDS) are clonal haematopoietic stem cell disorders characterised by ineffective and dyspoietic haematopoiesis. The natural history of these disorders is variable and ranges from a chronic to a rapid course towards leukaemic progression. Certain shortcomings have been encountered in the French-American-British (FAB) classification over the years, and therefore there is a need for an alternative method of classification. In 1999, the WHO published a revised classification of MDS. In the present study, we have analysed the clinical, haematological and histomorphological features in 96 cases of primary MDS seen in the department of haematology at the All India Institute of Medical Sciences (AIIMS) over a 6-yr period (1996-2001). Both FAB and WHO classifications have been incorporated and the Bournemouth scoring system applied in each case at presentation. The Bournemouth scoring system, in the absence of a cytogenetic study, offers a good prognostication and long-term survival estimate.     

A NOD2-NALP1 complex mediates caspase-1-dependent IL-1beta secretion in response to Bacillus anthracis infection and muramyl dipeptide

NOD2, a NOD-like receptor (NLR), is an intracellular sensor of bacterial muramyl dipeptide (MDP) that was suggested to promote secretion of the proinflammatory cytokine IL-1beta. Yet, the molecular mechanism by which NOD2 can stimulate IL-1beta secretion, and its biological significance were heretofore unknown. We found that NOD2 through its N-terminal caspase recruitment domain directly binds and activates caspase-1 to trigger IL-1beta processing and secretion in MDP-stimulated macrophages, whereas the C-terminal leucine-rich repeats of NOD2 prevent caspase-1 activation in nonstimulated cells. MDP challenge induces the association of NOD2 with another NLR protein, NALP1, and gel filtration analysis revealed the formation of a complex consisting of NOD2, NALP1, and caspase-1. Importantly, Bacillus anthracis infection induces IL-1beta secretion in a manner that depended on caspase-1 and NOD2. In vitro, Anthrax lethal toxin strongly potentiated IL-1beta secretion, and that response was NOD2 and caspase-1-dependent. Thus, NOD2 plays a key role in the B. anthracis-induced inflammatory response by being a critical mediator of IL-1beta secretion.