The Pluristem team, coordinated by R Ofir, E Lukasiewicz-Hagai, will bring in its first-in-class IP-protected manufacturing process for expansion of PLX-PAD cells, including the surrounding QC expertise.

PLX-PAD cells are manufactured, packaged and labelled by Pluristem Ltd. in GMP-compliant facilities located in Haifa, Israel. The PLX-PAD cell expansion manufacturing process is described in the figure below

WP2 targets the analysis of the molecular and functional signature of PLX-PAD product, including in depth analysis of batch-to-batch and donor-to-donor variability.

PDuring the long-lasting collaboration with Pluristem, BCRT has implemented several core technologies applied for cell characterisation, including PLX cells. The following well established platforms will be applied in PACE:

• Multiparameter flowcytometry using two selected 12-color surface and intracellular marker panels (HD Volk)
  
  
• Mass-cytometry using one selected 40 marker-Cytof panel (HD Volk)
  
  
• Elispot for detecting angiogenesis-supporting factor secreting cells (P Reinke, R Preyer GenID). The figure below describes the principle of Elispot for detecting cytokines or other soluble ligand secreted by stimulated cells (WP3+5)

• Immunomodulation of monocytes, T cells, B cells (HD Volk, P Reinke, D Strunk, Pluristem)
  
  
• Immunomodulation in a humanized NSG mouse model in vivo (M Seifert, Pluristem)
  
  
• Modulation of endothelial cell function in vitro (M Seifert, D Strunk, Pluristem)
  
  
• Modulation of vessel formation in humanized NSG mouse model in vivo (D Strunk, see the above schema)
  
  
• Modulation of survival, histology, and several soluble read-out parameters in a 5-organ (kidney, skeletal muscle, vessel, islets, immune system) “human-on-the-chip” 3D-organoid model (P Reinke in collaboration with tissuse GmbH, Berlin).
  
  
• CpG methylation analysis by Methyl-CAP-seq method using NGS (HD Volk, D Strunk, K Jürchott)
  
  
• Gene expression analysis by RNAseq using NGS and bioinformatics (HD Volk, K Jürchott)

The WP3 is a randomized, double-blind, multicenter, placebo-controlled, parallel-group phase III study to evaluate the efficacy, tolerability and safety of intramuscular injections of PLX-PAD for the treatment of subjects with critical limb ischemia (CLI) with minor tissue loss who are unsuitable for revascularization.

Protocol Number of the study: PLX-CLI-03
Investigational Treatment: PLX-PAD, allogeneic ex vivo expanded placental mesenchymal-like adherent stromal cells

The clinical study is sponsored by Pluristem. The CRO involved in the clinical trial is ICON, a global player in the field and well experienced in monitoring international multicentre studies under EMA and FDA rules. ICON has previously conducted a multinational Phase III in 800 CLI patients..

Very importantly, the clinical study is supported by excellent clinicians, who are internationally well recognized experts in clinical research and medical management of cardiovascular and diabetic diseases and have participated as PI´s in several clinical studies in the past.

Study timelines

WP4 focuses on the study of biomarker for monitoring the therapy. The panel was designed by the Pluristem team in collaboration with Charité, P1 (P Reinke, HD Volk, U Landmesser) and the main clinical investigators of the study (S Nikol, N Weiss). The panel goes far beyond typical standard laboratory markers and addresses the following questions:

• Impact of therapy on cytokine plasma profiles measured by applying ultrasensitive, and highly standardised multiplex ligand assays (Mesoscale and Ella platform) (P1 HD Volk)
  
  
• Humoral alloantibody response analysed by Luminex platform in HLA lab (P1 P Reinke)
  
  
• Impact on RNA profiles (mRNAs, miRNAs) by applying NGS (RNAseq) and bioinformatic analyses (P1, K Jürchott) with focus on immune response genes (P1, HD Volk), proangiogenic and growth factor response (P1, U Landmesser) and metabolic response (P1, J Spranger, P4 N Weiss)

WP5 utilises highly specialised, exploratory tests in mechanistic side-studies on subgroups of patients:

• Impact on metabolic function: we want to analyse the impact of PLX-PAD treatment on metabolic markers more in details. These studies will be guided by J Spranger, head of Dept. of Endocrinology, Nutrition, and Diabetes at Charité.
  
  
• Cellular Alloreactivity. It is urgent to confirm the low cellular immunogenicity of PLX-PAD, seen in previous studies. This is particularly important because the repetitive application of PLX-PAD in this study might increase the risk of allosensitisation. We will use IFN Elispot, a specific test optimized by Charité and GenID (P Reinke, R Preyer) and validated in multicentre trials. It is now in use for the first time as a stratification marker for guiding immunosuppression in transplant patients within the EU-funded project BIO-DrIM (www.biodrim.eu).
  
  
• Circulating endothelial cells (CEC) and endothelial progenitor cells (EPC). We have recently demonstrated the feasibility of detecting the rare CEC (including differentiation of microvascular CEC) and EPC in peripheral blood by using a highly standardised multiparameter flowcytometry with CVs <20% (HD Volk). We observed significantly increased levels of CEC but diminished levels of EPC in CLI patients. In this study, we will analyze whether PLX-PAD treatment can normalize the disturbed CEC/EPC ratio in CLI patients. We also want to explore whether numerous and/or functional changes of CEC/EPC can be used as biomarker for the mode-of-action of PLX-PAD.

We ensure efficient operational management including that of administrative, financial and legal issues The goal of this WP is to establish and guarantee full synergy, motivation, integration and effective interactions among PACE participants.

WP7 Communication, dissemination, training and exploitation. The focus is to set up of the proper tools and activities of communicating the project results and new achievement to the general public and patients, clinical/scientific community. The main dissemination activities are:

• 1. Participation to national scientific meeting, conference and symposia.
• 2. Organization of conferences, workshops, academic courses, meetings, patients´ forum, cultural events, discussion groups within communities. The collaboration with Deutsche Gesellschaft für Angiologie, Deutsche Gefäßliga e.V. etc will be very crucial for these activities.
• 3. Development of products as: articles in peer-review journals, articles and features in local mass media (e.g. radio, television, and newspapers), brochures, flyers, course materials, product of highly visible impact, such as posters, brochures, websites and electronic communications.

Training activities include mainly the following:

• a) Workshop on: "Advances in biomarker research for monitoring patients" organized by Charité. This workshop is open to all participating countries and beyond.
• b) Exchange of key staff members from one lab to another.
• c) Participation of young scientists involved in the project to national and international congresses for increasing their skills.
• d) Organization of training courses.

Particular attention is also devoted to the exploitation of the project results, that will be transferred to and absorbed by the project industrial stakeholders Pluristem and GenID.
Marketing and business development will mostly be managed by Pluristem, with support from the clinical and business development departments of Charité.