Clinical-grade generation of NK cells from cord blood hematopoietic progenitors – protocol evaluation

by Alexey Bersenev on July 17, 2011 · 0 comments

in cell product, clinical lab, cord blood, hematopoietic

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Today, I’m doing my second evaluation at SCA. So, please comment.

The study I’ve picked for evaluation – Clinical-Grade Generation of Active NK Cells from Cord Blood Hematopoietic Progenitor Cells for Immunotherapy Using a Closed-System Culture Process.

To date only a few trials have investigated adoptive NK cell infusions in patients with cancer primarily due to difficulties in isolating high numbers of NK cells from regular leukapheresis products.

The overall aim of this study was to develop a closed ex vivo culture system for the expansion and differentiation of CD34+ UCB cells into NK cells followed by the subsequent log-scale generation of CD56+CD3− NK cells.

SCA evaluation:

  1. First of all, I’d start from the statement: I like this study! It is important work, guys! The authors presented clinically-grade protocol for generation of functional NK cells from CD34+ cord blood-derived cells. All of reagents, materials and devices they used, approved for clinical use. Cell manufacturing process was done in GMP conditions, I believe this product was use for the clinical trial. Ok, the first point – simple protocol ready to go into the clinic!
  2. Secondly, comparison-comparison-comparison! They carefully calculate all of cell culture parameters and expansion yield in order to compare few techniques. Cell culture bags versus bioreactor, fresh cord blood versus thawed, rate of expansion after 1 week versus 2 and so on. I love it!
  3. Third, functional tests. NK cell functionality was tested in standard cytotoxicity assay – ability to lyse tumor cell line. I’d only add maybe one more important test – perforin and granzyme B expression as markers for efficient killing. I’m still not sure what would be the best potency assay for this product. I’d compare 2-3 and pick the most rapid, significant and cheap.
  4. Fourth, the quality of cell product. I like the composition of NK cell product tested by flow cytometry. We can see markers, which express on different stages of NK cell development, so the product represent pretty much whole lineage. The authors paid significant attention to release criteria, such as: purity, stability, sterility, toxicity, karyotyping and so on. In my opinion, the study illustrates the art of making cell product.
  5. Fifth, the study assesses some clinical parameters, which usually missing in other pre-clinical protocols. For example, volume reduction and washing after 4 weeks of expansion before cell product injection. How would you go from liters of medium to 100 ml of suspension? Would it affect purity and functionality of your product? This study addresses these issues.
  6. Here is one of figures: Flow cytometry analysis of ex vivo bioreactor-expanded NK cells before and after washing (Jan Spanholtz et al, PLoS ONE 6(6): e20740)

    Overall, this study provides an excellent example of how to tune up your cell product, which is entering clinical trials!

  7. Things which I’d add or try: NK generation directly from freshly isolated CD34+ without prior 2 weeks expansion. 2 step MACS for better purity of NK progenitors: (1) positive for CD34 (2) negative for CD45RA. It could allow to cut a time of cell product preparation (let’s say from 4 weeks to 2) and therefore the cost of procedure. I’d try to cut on cytokines from 4 to 3 (it will make protocol cheaper).
  8. What part is missing: The cost calculation for each step, IMHO.

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If you would like to evaluate some interesting studies, please let me know!

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