Delivery of a GMP cryogenic cold chain for clinical delivery of regenerative medicine therapeutics.

1. Peripheral blood mononuclear cells (PBMC) will be collected from normal healthy donors or from buffy coats (NHSBT) and frozen using either the LVCRF or standard laboratory procedures. Subsequently, cells will be defrosted using e-thaw or
standard methods with the viability ( 7-AAD/ annexin-V) and phenotype of PBMC (i.e. T-cell, B-cell, NK cell frequency, activation marker expression) determined by flow cytometry. The in vitro function of defrosted T cells within the PBMC population will be determined by mitogenic activation (e.g. anti-CD3/CD28 mabs) and the balance of Th1 / Th2 cytokines determined by multiplex array after twenty-four hours of stimulation. The relative expansion of anti-CD3/CD28 expanded Tcells from defrosted PBMC will also be determined over a 10 day period in order to assess the proliferative potential of these cells. Finally, defrosted PBMC will be adoptively transferred into immuno-compromised NSG (NOD/SCID IL-2R-/-) mice by tail-vein injection and T-cell engraftment determined in peripheral blood by the analysis of serial tail bleeds taken every 7/14 days (analysed by flow cytometry for human markers - CD45, CD4, CD8 and enumerated using countbright
beads). The relative number of engrafted T cells will be compared between the test systems and standard cryopreservation protocols. The successful engraftment of human T cells in NSG mice eventually results in xenogeneic graft versus host disease (xGvHD) usually within 35-50 days after transfer. The effect is driven by donor human T cells and will be used as a surrogate measure for T cell function. In all tests, comparison between EF600 and standard freezing methods will be statistically tested using non-parametric methods (e.g. Mann-Whitney U test; ANOVA).

2. Gene-modified T cells expressing a CD19 specific chimeric receptor will be generated and tested as above for function including in vivo activity against CD19 xenografted tumours in the NSG mouse.

3. Tumour tissue. There is an increasing understanding that tumours can be used to generate patient-specific treatments including the use of disaggregated tumour for vaccine generation and to isolate viable T cells (tumour infiltrating lymphocytes, TIL) which can then be expanded and returned to the patient. We have the facilities to deliver these therapies in Manchester (CTU). The key issue is collecting tumour from the operating theatre and the subsequent isolation of TIL's. We will collect melanoma and renal cell carcinoma tumours (ethical approval in place) which will be either frozen (standard or EF600 base system) or used fresh to generate TIL's using our standard procedure that has been developed to comply with ATMP regulations. Importantly, frozen tumour samples will then be defrosted and tested for their ability to generate
TIL's. It is expected that 10-20 tumours will be collected during the course of the project; the clinical delivery of TIL therapy in Manchester is about to start and it is anticipated that the large tumours to be collected for this therapy will provide enough tumour for this research study. However, we have excellent on-going collaborations with surgeons at the Christie Hospital and Manchester Royal Infirmary to provide access for further tumour material. Non-parametric statistical tests will be used to examine any observed differences in TIL production between fresh and frozen tumour samples. Furthermore, the phenotype and cytokine responses to mitogenic stimuli will be tested as described above. Also will also examine tumour materials frozen by the different methodologies for tissue integrity (H&E staining, tumour antigen expression) and will also determine the integrity of nucleic acids extracted from these samples (Agilent Bioanalyser for genomic DNA integrity and real-time PCR for house-keeping genes) to determine mRNA integrity.

4. Engraftment of EF600 frozen HSC will be tested should sufficient sample be available for engraftment into NSG mice; analysis as in 1.

A key objective of this project is to develop varying sizes of cryocoolers in order to provide the flexibility to deal with the
range of sample sizes that occur in cell/tissue bio-processing. To this end, the project aims to deliver:

1. GMP compliant ultra-low temperature transportation and cold-storage of cells and tissues.
2. Reduction in the infrastructure required to freeze and store cells/tissues, thereby widening the range of institutions able
to offer RM based therapies.
3. The rapid freezing of viable tissue at source (i.e. within an operating theatre) to optimise procurement and tissue quality.

Who will benefit from this research?
1. Academic groups working with cells and tissues.
2. Clinical service providers in the transplant
3. Biobanks
4. Clinicans.
5. Patients.
6. Regulators.

How will they benefit from this research?

1. Academic groups will benefit for all the reasons detailed in the academic beneficiaries section.

2. Clinicial service providers. These providers include hospital / NHSBT providers and private corporate service providers. Avoiding HSE issues with liquid nitrogen freezing and improving the flexibility of cold chain delivery will improve and simplify processes used by service providers including stem cell transplantation and other cell therapies such as T cell therapies.

3. Biobanks. Collecting tissue at source and rapidly preserving tissue viability is critical for biobanking. In particular, maintaining nucleic acid viability is critical for many new diagnostic processes such as PCR amplification of gene mutations. Although this is possible with fixed tissue, there are major issues with the quality of nucleic acid isolated from these samples hence freezing is the prefered method to preserve samples for nucleic acid-based studies.

4. Clinicians. With clinical service providers able to provide more flexibility in current services and the potential to deliver new therapies, clinicians will be potentially able to modify treatments or offer treatments to patients who were not suitable as a result of improved tissue collection methods and improved diagnostic methods based upon the use of fresh frozen viable tissue.

5. Patients. The patient experience will be improved since timing of surgery can be made more flexible and not dictated by the needs for cell collection / processing. Patients would also benefit from an improved ability to deliver current therapies such as stem cell and T cell therapies and will also benefit from new therapies that should develop as a result of the development of a GMP compliant cold chain.

6. Regulators. The implementation of GMP compliant cold chain will improve overall compliance of bio-processing and thereby facilitate regulation.