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 favourable expression profile, CD19 has rapid internalization kinetics and it 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 cross-linking pyrrolobenzodiazepine (PBD) 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 warhead, resulted in formation of DNA cross-links which persisted for 36 h. Bystander killing of CD19-negative cells by ADCT-402 was also observed. In vivo, single doses of ADCT-402 resulted in highly potent, dose-dependent anti-tumor activity in several subcutaneous and disseminated human tumor models with marked superiority to comparator ADCs delivering tubulin inhibitors. Dose-dependent DNA cross-links and γ-H2AX DNA damage response were measured in tumors by 24 h after single dose administration, while matched PBMCs 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

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

Synthetic pyrrolobenzodiazepine (PBD) dimers, where two PBD monomers are linked through their aromatic A-ring phenolic C8-positions via a flexible propyldioxy tether, are highly efficient DNA minor groove cross-linking agents with potent cytotoxicity. PBD dimer SG3199 is the released warhead component of the antibody-drug conjugate (ADC) payload tesirine (SG3249), currently being evaluated in several ADC clinical trials. SG3199 was potently cytotoxic against a panel of human solid tumour and haematological cancer cell lines with a mean GI50 of 151.5 pM. Cells defective in DNA repair protein ERCC1 or homologous recombination repair showed increased sensitivity to SG3199 and the drug was only moderately susceptible to multidrug resistance mechanisms. SG3199 was highly efficient at producing DNA interstrand cross-links in naked linear plasmid DNA and dose-dependent cross-linking was observed in cells. Cross-links formed rapidly in cells and persisted over 36 hours. Following intravenous (iv) administration to rats SG3199 showed a very rapid clearance with a half life as short as 8 minutes. These combined properties of cytotoxic potency, rapid formation and persistence of DNA interstrand cross-links and very short half-life contribute to the emerging success of SG3199 as a warhead in clinical stage ADCs

ADCT-301, a Pyrrolobenzodiazepine (PBD) Dimer-Containing Antibody-Drug Conjugate (ADC) Targeting CD25-Expressing Hematological Malignancies

Despite the many advances in the treatment of hematologic malignancies over the past decade, outcomes in refractory lymphomas remain poor. One potential strategy in this patient population is the specific targeting of IL2R-α (CD25), which is overexpressed on many lymphoma and leukemic cells, using antibody–drug conjugates (ADC). ADCT-301 is an ADC composed of human IgG1 HuMax-TAC against CD25, stochastically conjugated through a dipeptide cleavable linker to a pyrrolobenzodiazepine (PBD) dimer warhead with a drug–antibody ratio (DAR) of 2.3. ADCT-301 binds human CD25 with picomolar affinity. ADCT-301 has highly potent and selective cytotoxicity against a panel of CD25-expressing human lymphoma cell lines. Once internalized, the released warhead binds in the DNA minor groove and exerts its potent cytotoxic action via the formation of DNA interstrand cross-links. A strong correlation between loss of viability and DNA cross-link formation is demonstrated. DNA damage persists, resulting in phosphorylation of histone H2AX, cell-cycle arrest in G2–M, and apoptosis. Bystander killing of CD25-negative cells by ADCT-301 is also observed. In vivo, a single dose of ADCT-301 results in dose-dependent and targeted antitumor activity against both subcutaneous and disseminated CD25-positive lymphoma models. In xenografts of Karpas 299, which expressed both CD25 and CD30, marked superiority over brentuximab vedotin (Adcetris) is observed. Dose-dependent increases in DNA cross-linking, γ-H2AX, and PBD payload staining were observed in tumors in vivo indicating a role as relevant pharmacodynamic assays. Together, these data support the clinical testing of this novel ADC in patients with CD25-expressing tumors

Experimental models in peritoneal dialysis: A European experience.

Experimental models in peritoneal dialysis: A European experience.BackgroundThe development of adequate animal models is important for the in vivo study of selected aspects of peritoneal dialysis (PD) that cannot be evaluated by an in vitro model, such as peritoneal membrane transport, the influence of local defense mechanisms, and for testing new osmotic agents and their biocompatibilities.MethodsOur experience with animal models for PD, including the acute Stockholm model in non-uremic rats, the acute and chronic Amsterdam model in non-uremic rats, and the chronic Gent model in uremic rats, is described.ResultsThe Stockholm model proved to be useful in understanding the normal physiology of peritoneal transport, and for testing new dialysis solutions and their biocompatibilities. It is a rather simple and inexpensive model, and thus is suitable for screening new solutions and additives. The Amsterdam model permits the study of chemokines and mesothelial cell regeneration in vivo, and is applied in a model of chronic peritonitis. The results of the Gent model suggest that chronic peritoneal dialysis in uremic rats is feasible for at least eight weeks. This model is, however, very laborious, time consuming, and expensive.ConclusionFurther improvement of the technique and increase of the dialysis dose should result in a better and more realistic model for peritoneal dialysis. It is hoped that in the future these models will be useful to test the effects of long-term intraperitoneal application of different dialysis solutions and additives in uremic animals

BspA (CyuC) in L. fermentum BR11 is a highly expressed high affinity L-cystine-binding protein

The BspA protein of Lactobacillus fermentum BR11 (BR11) is a cell envelope constituent that is similar to known solute-binding proteins and putative adhesins. BspA is required for L-cystine uptake and oxidative defense and is likely to be an L-cystine-binding protein. The aim of this study was to directly measure L-cystine-BspA binding and BspA expression. De-energized BR11 cells bound radiolabelled L-cystine with a Kd of 0.2 mgrM. A bspA mutant could not bind L-cystine. L-cystine-BR11 binding was unaffected by large excesses of L-glutamine, L-methionine, or collagen, indicating L-cystine specificity. BR11 and the bspA mutant were identical in their abilities to bind L-cysteine, indicating that L-cysteine is not a BspA ligand. BspA expression levels were deduced from radiolabelled L-cystine binding and it was found that there are 1–2 × 105 BspA molecules per cell, and that expression is slightly higher under oxidizing conditions. It is proposed that BspA be renamed CyuC

Lung dendritic cells are primed by inhaled particulate antigens, and retain MHC class II/antigenic peptide complexes in hilar lymph nodes for a prolonged period of time

Intratracheal (IT) administration of heat-killed Listeria monocytogenes (HKL) results in an influx of macrophage and dendritic cell (DC) precursors into the lung interstitium. Low-density, FcR(+), interstitial lung cells isolated from rats instilled 24 hr before with HKL or vehicle alone, were > 90% Mar1(+). After culturing with granulocyte–macrophage colony-stimulating factor (GM-CSF) for 3 days, up to 24% of the loosely adherent cells were DC that stimulated allogeneic T-cell proliferation in an mixed lymphocyte reaction (MLR) assay. After only an overnight incubation with GM-CSF, however, the capacity of interstitial Mar1(+) cells to stimulate HKL immune T-cell proliferation without exogenous antigen was low. By contrast, when DC were isolated as major histocompatibility complex (MHC) class II(+) cells from rat lungs at 1, 3, 7 and 14 days after HKL instillation and cultured overnight with GM-CSF, their antigen presentation capacity without added exogenous antigen was robust, but declined over the 2-week period. Interestingly, hilar lymph node DC maintained their HKL antigen-presenting capacity for up to 2 weeks after instillation of HKL. Following IT administration of PKH-26 labelled HKL, fluorescent or immunolabelled organisms were detected in OX62(+) DC in airway epithelium, lung interstitium and hilar lymph nodes in situ and in MHC class II(+) DC isolated from these sites. We conclude that newly immigrated Mar1(+) lung DC precursors, while efficient in endocytosing particulate antigens, are incapable of eliciting a significant proliferative response from HKL-sensitized T cells. By contrast, MHC class II(+) DC isolated from lungs and incubated overnight with GM-CSF induce vigorous antigen-specific T-cell proliferation. Antigen-loaded lung DC in hilar lymph nodes maintain their antigen presentation capacity for up to 2 weeks