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.

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. Mol Cancer Ther; 15(11); 2709-21. ©2016 AACR.

Escherichia coli-derived outer membrane vesicles are genotoxic to human enterocyte-like cells.

BACKGROUND: Colorectal cancers are the third most common type in the world. The causes of the disease are poorly understood, but since the discovery of Helicobacter pylori as a causative agent of gastric cancer, attention has turned to bacteria as a possible trigger for colorectal cancer. Recently H. pylori outer membrane vesicles (OMVs) were revealed as potentially genotoxic which can be important first step in carcinogenesis. We therefore investigated whether OMVs from intestinal Escherichia coli could be genotoxic. METHODS: OMVs from the avirulent DH5α strain, a pathogenic adherent-invasive E. coli (AIEC) and an enterohaemolytic (EHEC) strain of E. coli were enriched by ultracentrifugation. The effect on the growth and viability of human enterocyte-like Caco-2 cells by OMVs was determined by trypan blue exclusion, MTT and BrdU incorporation assays. The ability of OMVs to induce DNA damage was assayed by single-cell gel electrophoresis, and 8-oxo-dG and γH2Ax immunofluorescence staining. Cytopathological changes were assessed by microscopy. The induction of aneuploidy by the OMVs was measured by flow cytometry in Caco-2 and LoVo cells. RESULTS: We found that OMVs derived were internalised by Caco-2 cells, increased cell numbers, induced double-stranded DNA breaks, recruited γH2Ax to the nucleus, initiated DNA rereplication, and produced distended multinucleate cells. DH5α and AIEC OMVs caused free radical generation as indicated by the reduction of glutathione in cells, leading to the development of mutagenic 8-oxo-dG adducts in DNA. Flow cytometry revealed that DH5α and EHEC OMVs increased aneuploidy in p53 mutant Caco-2 cells, but not in p53 wild type LoVo cells. CONCLUSION: We conclude that E. coli derived OMVs, whether from avirulent or pathogenic strains are potentially genotoxic.

Influences of enteral nutrition upon CEACAM6 expression by intestinal epithelial cells.

Exclusive enteral nutrition is established as an initial therapy to induce remission in active Crohn's disease (CD), especially in children, but the mechanisms of action of this therapy are yet to be fully defined. CEACAM6 protein is an adhesion molecule that is up-regulated in active CD and implicated in the attachment of adherent-invasive Escherichia coli (AIEC) to the gut epithelium. Using the Caco-2 human adenocarcinoma cell line, this study showed that the incubation of human cells with a polymeric formula (PF) resulted in a dose-dependent increase in the expression of CEACAM6, and that this effect was most noticeable on the cell surface. Further investigation revealed that PF doubled the release of CEACAM6 protein by Caco-2 cells exposed to PF, and that an increase in release of soluble CEACAM6 inversely correlated with the ability of AIEC to associate with the intestinal epithelial cells. Our findings suggest that the secretion of cell surface-associated proteins acting as releasable decoys may be an aspect of the gut's innate immune response to pathogenic bacteria that is strengthened by PF in the setting of CD.

Human lactoferrin increases Helicobacter pylori internalisation into AGS cells.

Helicobacter pylori has high global infection rates and can cause other undesirable clinical manifestations such as duodenal ulcer (DU) and gastric cancer (GC). Frequencies of re-infection after therapeutic clearance and rates of DU versus GC vary geographically and differ markedly between developed and developing countries, which suggests additional factors may be involved. The possibility that, in vivo, lactoferrin (Lf) may play a subtle role in modulating micronutrient availability or bacterial internalisation with implications for disease etiology is considered. Lf is an iron binding protein produced in mammals that has antimicrobial and immunomodulatory properties. Some bacteria that regularly colonise mammalian hosts have adapted to living in high Lf environments and we investigated if this included the gastric pathogen H. pylori. We found that H. pylori was able to use iron from fully iron-saturated human Lf (hLf) whereas partially iron-saturated hLf (apo) did not increase H. pylori growth. Instead, apo-hLf increased adherence to and internalisation of bacteria into cultured epithelial cells. By increasing internalisation, we speculate that apo-human lactoferrin may contribute to H. pylori's ability to persistence in the human stomach, an observation that potentially has implications for the risk of H. pylori-associated disease.

Effects of bacterial products on enterocyte-macrophage interactions in vitro.

We describe a coculture model of a human intestinal epithelial cell line and human peripheral blood monocytes in which monocytes differentiate into cells with features of resident intestinal macrophages. Caco-2 cells are grown on the lower surface of a semipermeable filter with pore size of 3 µm (Transwells) until they differentiate into enterocytes. Peripheral-blood monocytes are added and the co-culture incubated for two days. Monocytes migrate through the pores of the membrane, come into direct contact with the basolateral surfaces of the epithelial cell monolayer, and develop characteristics of resident intestinal macrophages including downregulation of CD14 expression and reduced pro-inflammatory cytokine responses (IL-8, TNF and IL-1ß) to bacterial products. The apical application of lipopolysaccharide (LPS) and muramyl dipeptide (MDP) resulted in an increased number of integrated monocytes, but abrogated the downregulation of CD14 expression and the diminished cytokine responses. MDP also reduced tight-junctional integrity, whilst LPS had no effect. These data indicate that LPS and MDP have significant pathophysiological effects on enterocyte-monocyte interactions, and confirm other studies that demonstrate that enterocytes and their products influence monocyte differentiation. This model may be useful in providing insights into the interaction between monocytes, epithelial cells and intestinal bacteria in health and disease.

Receptor mediated targeting of M-cells.

The intestinal epithelium is a complex system of highly specialised cells that provide digestive and absorptive functions as well as innate and adaptive immunity. Induction of an adaptive immune response in the intestine can occur through the interaction of antigen with M-cells that overlay the lymphoid aggregates of the intestine (Peyer's patches). This study demonstrated that specific common microbial pathogen-associated molecular patterns are recognised by pattern recognition receptors on the surface of the M-cells and this interaction initiates transcytosis through the M-cell of particulate antigen from the intestinal milieu to underlying antigen presenting cells within the Peyer's patch. The study has found that among the pattern recognition molecules that have a role in recognising bacterial components, the apical expression of alpha5beta1 integrin was important for the transcytotic function of M-cells. A proportion of intestinal enterocytes transform to an M-cell morphology in vitro, when cultured with Peyer's patch cells and our studies have demonstrated that CD4+ cells are integral for the development of M-cells in vitro.