Final Report Summary - CD14 (Innate and adaptive immune responses to nanocell-based tumor-targeted cancer therapeutics)
A proprietary anti-cancer drug delivery system has been recently developed by EnGeneIC Ltd Cancer Therapeutics, Sydney, Australia. Engeneic delivery vehicles (EDV)s are anucleated bacterially-derived minicells that can be packaged with active drugs or small interfering ribonucleic acid (siRNA) and directed to tumour cells in vivo via bispecific antibodies. A phase I clinical study was running between 2009 and 2011 and has proven safety in human beings. A phase IIa clinical study was commenced in 2012 aiming at proving clinical benefit.
Parallel to clinical evaluation, the CD14 project has aimed at surveying the scale of sensing EDVs by peripheral blood mononuclear cell (PBMC)s. PBMC are able to recognise pathogen associated molecular pattern (PAMP)s as lipopolysaccharide (LPS)s, flagellin or nucleic acids via so-called pattern recognition receptors (PRR). Sensing of PAMPs usually activate an inflammatory response and the induction of interferon (IFN)s. At the same time, PAMPs might mediate adjuvant or even desensitising effects (e.g. tolerance).
Results obtained during year 2 outgoing period of the project showed that EDVs do not affect viability, proliferative activity, or metabolic activity of human PBMC in vitro. Data are indicative, however, that EDVs are sensed by human PBMC and do induce a response similar to that of purified bacterial components as LPS, lipoproteins or flagellin in terms of induction of tumour necrosis factors (TNF)-alpha and IFN-beta gene expression. By using a range of specific reagents, i.e. toll-like receptor (TLR) antagonists or signalling inhibitors, TLR4 was found to be involved in stimulation of PBMC by EDVs. Similar to LPS, which is known to induce a state of 'endotoxin tolerance', EDVs were found to induce a state of reduced responsiveness both towards an endotoxin and towards an EDV challenge.
Scientific training included EDVs' purification, its counting and determination of free LPS in these individual preparations. Training also included being involved in experiments evaluating the effects of differently targeted and differently loaded EDVs on human xenograft tumour cell growth in vivo. Specifically, gene expression analyses revealed induction of innate immune mechanisms as an off-target effect to be unlikely to contribute to tumour growth inhibition by a siRNA load of EDVs. Moreover, in mice treated with EDVs that were loaded with an shRNA expression plasmid, the number of plasmids within the tumour tissue was calculated on a per tumour cell basis.
The procedure to obtain the approval for conducting a phase IIa clinical study in Australia demanded:
(i) cyclic guanosine monophosphate (cGMP) production conditions and
(ii) the assurance of sufficiently large capacities in the manufacturing and the supply of EDV preparations.
These two conditions meant the implementation of changes in the production process, namely upscaling, and additional quality assessments.
During a temporary period of non-availability of quality certified EDV batches, research was directed to the clinical condition of sepsis. In collaboration with the Department of Anaesthesiology, Emergency and Intensive Care Medicine at University Medical Centre in Goettingen, patients' samples were analysed with regard to their individual sensitivity to bacterial float. Genetic polymorphisms comprised those in PRR genes as CD14 or TLR4.
This collaboration surely will last beyond the three-year project phase. It will help to decipher the interaction of bacterial molecular patterns with peripheral blood cells in a highly relevant in vivo situation in human beings. As in cancer patients, also sepsis is featured by an immuno-compromised state. Once in a finally approved and precisely defined quality, the extent of interferences between EDVs and PBMC remain to be gauged.
Project website: http://www.engeneic.com
Parallel to clinical evaluation, the CD14 project has aimed at surveying the scale of sensing EDVs by peripheral blood mononuclear cell (PBMC)s. PBMC are able to recognise pathogen associated molecular pattern (PAMP)s as lipopolysaccharide (LPS)s, flagellin or nucleic acids via so-called pattern recognition receptors (PRR). Sensing of PAMPs usually activate an inflammatory response and the induction of interferon (IFN)s. At the same time, PAMPs might mediate adjuvant or even desensitising effects (e.g. tolerance).
Results obtained during year 2 outgoing period of the project showed that EDVs do not affect viability, proliferative activity, or metabolic activity of human PBMC in vitro. Data are indicative, however, that EDVs are sensed by human PBMC and do induce a response similar to that of purified bacterial components as LPS, lipoproteins or flagellin in terms of induction of tumour necrosis factors (TNF)-alpha and IFN-beta gene expression. By using a range of specific reagents, i.e. toll-like receptor (TLR) antagonists or signalling inhibitors, TLR4 was found to be involved in stimulation of PBMC by EDVs. Similar to LPS, which is known to induce a state of 'endotoxin tolerance', EDVs were found to induce a state of reduced responsiveness both towards an endotoxin and towards an EDV challenge.
Scientific training included EDVs' purification, its counting and determination of free LPS in these individual preparations. Training also included being involved in experiments evaluating the effects of differently targeted and differently loaded EDVs on human xenograft tumour cell growth in vivo. Specifically, gene expression analyses revealed induction of innate immune mechanisms as an off-target effect to be unlikely to contribute to tumour growth inhibition by a siRNA load of EDVs. Moreover, in mice treated with EDVs that were loaded with an shRNA expression plasmid, the number of plasmids within the tumour tissue was calculated on a per tumour cell basis.
The procedure to obtain the approval for conducting a phase IIa clinical study in Australia demanded:
(i) cyclic guanosine monophosphate (cGMP) production conditions and
(ii) the assurance of sufficiently large capacities in the manufacturing and the supply of EDV preparations.
These two conditions meant the implementation of changes in the production process, namely upscaling, and additional quality assessments.
During a temporary period of non-availability of quality certified EDV batches, research was directed to the clinical condition of sepsis. In collaboration with the Department of Anaesthesiology, Emergency and Intensive Care Medicine at University Medical Centre in Goettingen, patients' samples were analysed with regard to their individual sensitivity to bacterial float. Genetic polymorphisms comprised those in PRR genes as CD14 or TLR4.
This collaboration surely will last beyond the three-year project phase. It will help to decipher the interaction of bacterial molecular patterns with peripheral blood cells in a highly relevant in vivo situation in human beings. As in cancer patients, also sepsis is featured by an immuno-compromised state. Once in a finally approved and precisely defined quality, the extent of interferences between EDVs and PBMC remain to be gauged.
Project website: http://www.engeneic.com