Overcoming Target Driven Fratricide for T Cell Therapy.

Chimeric Antigen Receptor (CAR) T cells expressing the fusion of the NKG2D protein with CD3ζ (NKG2D-CAR T Cells) acquire a specificity for stress-induced ligands expressed on hematological and solid cancers. However, these stress ligands are also transiently expressed by activated T cells implying that NKG2D-based T cells may undergo self-killing (fratricide) during cell manufacturing or during the freeze thaw cycle prior to infusion in patients. To avoid target-driven fratricide and enable the production of NKG2D-CAR T cells for clinical application, two distinct approaches were investigated. The first focused upon the inclusion of a Phosphoinositol-3-Kinase inhibitor (LY294002) into the production process. A second strategy involved the inclusion of antibody blockade of NKG2D itself. Both processes impacted T cell fratricide, albeit at different levels with the antibody process being the most effective in terms of cell yield. While both approaches generated comparable NKG2D-CAR T cells, there were subtle differences, for example in differentiation status, that were fine-tuned through the phasing of the inhibitor and antibody during culture in order to generate a highly potent NKG2D-CAR T cell product. By means of targeted inhibition of NKG2D expression or generic inhibition of enzyme function, target-driven CAR T fratricide can be overcome. These strategies have been incorporated into on-going clinical trials to enable a highly efficient and reproducible manufacturing process for NKG2D-CAR T cells.

Cardiopoietic index predicts heart repair fitness of patient-derived stem cells.

BACKGROUND: Stem cell therapy shows promise for regeneration in heart disease, yet interpatient variability challenges implementation into practice. AIM: To establish a biomarker profile, predictive of reparative potential in patient-derived progenitors, human mesenchymal stem cells were isolated from patients undergoing coronary artery bypass grafting. MATERIALS & METHODS: Stem cell delivery postinfarction translated into divergent benefit, distinguishing reparative from nonreparative populations. RESULTS: While the nonreparative subtype was characterized by low expression of cardiac transcription factors, reparative human mesenchymal stem cells demonstrated high expression of cardiac transcription factors. CONCLUSION: This index of factors (cardiopoietic index) was found sensitive and specific in predicting impact of stem cell benefit on left ventricular function. The cardiopoietic index thus offers a tool to screen stem cell fitness for heart repair prior to intervention.

Guidelines for translational research in heart failure.

Heart failure (HF) remains a major cause of death and hospitalization worldwide. Despite medical advances, the prognosis of HF remains poor and new therapeutic approaches are urgently needed. The development of new therapies for HF is hindered by inappropriate or incomplete preclinical studies. In these guidelines, we present a number of recommendations to enhance similarity between HF animal models and the human condition in order to reduce the chances of failure in subsequent clinical trials. We propose different approaches to address safety as well as efficacy of new therapeutic products. We also propose that good practice rules are followed from the outset so that the chances of eventual approval by regulatory agencies increase. We hope that these guidelines will help improve the translation of results from animal models to humans and thereby contribute to more successful clinical trials and development of new therapies for HF.

Proof of principle: quality control of therapeutic cell preparations using senescence-associated DNA-methylation changes.

BACKGROUND: Tracking of replicative senescence is of fundamental relevance in cellular therapy. Cell preparations - such as mesenchymal stromal cells (MSCs) - undergo continuous changes during culture expansion, which is reflected by impaired proliferation and loss of differentiation potential. This process is associated with epigenetic modifications: during in vitro culture, cells acquire senescence-associated DNA methylation (SA-DNAm) changes at specific sites in the genome. We have recently described an Epigenetic-Senescence-Signature that facilitates prediction of the state of cellular aging by analysis of DNAm at six CpG sites (associated with the genes GRM7, CASR, PRAMEF2, SELP, CASP14 and KRTAP13-3), but this has not yet been proven over subsequent passages and with MSCs isolated under good manufacturing practice (GMP) conditions. FINDINGS: MSCs were isolated from human bone marrow and GMP-conform expanded for up to 11 passages. Cumulative population doublings (cPDs) and long-term growth curves were calculated based on cell numbers at each passage. Furthermore, 32 cryopreserved aliquots of these cell preparations were retrospectively analyzed using our Epigenetic-Senescence-Signature: DNAm-level was analyzed at six specific CpGs, and the results were used to estimate cPDs, time of culture expansion, and passage numbers. Overall, predicted and real parameters revealed a good correlation, particularly in cPDs. Based on predicted cPDs we could reconstruct long-term growth curves and demonstrated the continuous increase in replicative senescence on molecular level. CONCLUSION: Epigenetic analysis of specific CpG sites in the genome can be used to estimate the state of cellular aging for quality control of therapeutic cell products.

Toward cell therapy using placenta-derived cells: disease mechanisms, cell biology, preclinical studies, and regulatory aspects at the round table.

Among the many cell types that may prove useful to regenerative medicine, mounting evidence suggests that human term placenta-derived cells will join the list of significant contributors. In making new cell therapy-based strategies a clinical reality, it is fundamental that no a priori claims are made regarding which cell source is preferable for a particular therapeutic application. Rather, ongoing comparisons of the potentiality and characteristics of cells from different sources should be made to promote constant improvement in cell therapies, and such comparisons will likely show that individually tailored cells can address disease-specific clinical needs. The principle underlying such an approach is resistance to the notion that comprehensive characterization of any cell type has been achieved, neither in terms of phenotype nor risks-to-benefits ratio. Tailoring cell therapy approaches to specific conditions also requires an understanding of basic disease mechanisms and close collaboration between translational researchers and clinicians, to identify current needs and shortcomings in existing treatments. To this end, the international workshop entitled "Placenta-derived stem cells for treatment of inflammatory diseases: moving toward clinical application" was held in Brescia, Italy, in March 2009, and aimed to harness an understanding of basic inflammatory mechanisms inherent in human diseases with updated findings regarding biological and therapeutic properties of human placenta-derived cells, with particular emphasis on their potential for treating inflammatory diseases. Finally, steps required to allow their future clinical application according to regulatory aspects including good manufacturing practice (GMP) were also considered. In September 2009, the International Placenta Stem Cell Society (IPLASS) was founded to help strengthen the research network in this field.

Efficacy and safety of the freeze-dried cultured human keratinocyte lysate, LyphoDerm 0.9%, in the treatment of hard-to-heal venous leg ulcers.

LyphoDerm (XCELLentis, Belgium) is an end-sterilized, freeze-dried lysate from cultured allogeneic epidermal keratinocytes, formulated into a hydrophilic gel. Its efficacy and safety were evaluated, in combination with standard care (hydrocolloid dressing and compression therapy), in 194 patients suffering from hard-to-heal (lasting more than 6 weeks and not responding to conventional therapy) venous leg ulcers. Two control groups received standard care, with or without vehicle, respectively. Patients had a median age of 67.5 years and the majority were females (61%). The median duration of the ulcer was 43 weeks and in 39% of the subjects it had been present for more than 1 year. Thirty-eight percent of the patients in the standard care + LyphoDerm group had complete ulcer healing within 24 weeks (primary end point) compared to 27% of patients in the standard care + vehicle pooled groups (P = 0.114) in the "as treated" intent-to-treat cohort (37% vs. 27% in the "as randomized intent-to-treat cohort; p = 0.137). In the subgroup of patients with enlarging ulcers, the difference between the two groups was significant (30% vs. 11%; p = 0.024 in the "as treated" intent-to-treat cohort and 31% vs. 9%; p = 0.005 in the "as randomized" intent-to-treat cohort). LyphoDerm was well tolerated and safe, and no differences in the frequency of adverse events were noted between the treatment groups. Although the primary objective of the study was not achieved, the exploratory analysis carried out in patients with enlarging ulcers suggests that LyphoDerm could offer a new prospect for the treatment of patients with venous ulcers that may prove to be a significant adjunct to the overall provision of care.

Use of in Vitro Correlates for Selection of Candidate Immunosuppressive Antibodies Prior to a Primate Transplant Model.

The selection of possible candidate immunosuppressive antibodies to prevent graft rejection is performed in vitro. Additionally, due to the species specificity of these monoclonal antibodies (MABs), pre-clinical studies in non-human primates are necessary. If a positive correlation between the in vitro and in vivo findings would exist, these tests can act as a pre-screening before new reagents are tested in vivo. The correlation of the in vitro and in vivo efficacy of an anti-T-lymphocyte globulin (ATG) and an anti-CD80 MAB is evaluated in a rhesus monkey skin transplant model. The results show that lymphocytotoxic titers (NIH-test) do not predict the outcome of in vivo effectiveness of ATG in rhesus monkeys. Additionally, no evidence of tolerance to a skin allograft could be shown to correlate with inhibition of a secondary mixed lymphocyte culture (MLC) by anti-CD80 and cyclosporin A (CsA). Thus, these in vitro assay used can not predict the in vivo efficacy of new immunosuppressive antibodies.

Neuronal and mesodermal differentiation of P19 embryonal carcinoma cells is characterized by expression of specific marker genes and modulated by activin and fibroblast growth factors.

We have used the P19 embryonal carcinoma (EC) aggregation system as a model for early mouse development to study induction and modulation of mesodermal and neuronal differentiation. By studying the expression of marker genes for differentiated cells in this model we have shown that there is a good correlation between the differentiation direction induced in P19 EC aggregates and the expression of these genes. Expression of the neuronal gene midkine is exclusively upregulated when P19 EC cells are induced to form neurons while expression of early mesodermal genes such as Brachyury T, evx-1, goosecoid and nodal is elevated after induction to the mesodermal pathway. In the present study we have further shown that activin A blocks the different directions of differentiation of P19 EC cells induced by retinoic acid (RA) in a dose-dependent way. To understand the mechanism behind this inhibitory action of activin A the expression of several RA-responsive genes, including the three RA receptor genes (RARα, RARß and RARγ) was determined. Since activin has no clear effect on the expression and activity of the RAR it is very likely that this factor acts downstream of these receptors. In addition to activin, fibroblast growth factors (FGF) were shown to modulate P19 EC cell differentiation. However, in contrast to activin, FGF exclusively blocks the mesodermal differentiation of P19 EC cells by either 10-9 mol/L RA or a factor produced by visceral endoderm-like cells (END-2 factor). The FGF effect is dose-independent. These results suggest an important function for RA and the END-2 factor in the induction and for activin and FGF in the modulation of specific differentiation processes in murine development.