How many cells should a therapeutic cell sorter sort? (Part 1)

Part 1: How many cells are needed in the therapeutic dose?

The general scheme of an autologous cell therapy is as follows: (A) extract cells, (B) modify cells in vitro, (C) expand in vitro and (D) re-inject cells into the patient. So, working backwards from the injected cell number, the question of how many cells to sort depends on:

1. How many cells are needed in the therapeutic dose?
2. To what extent can the cells be allowed to proliferate in vitro?
3. What is the yield of the in-vitro activation or genetic transduction step?
4. How many cells (of the correct type) can be practically and safely extracted?

All of these questions are vast and scientifically open. So we’ll treat them one by one in our blog posts. In this post we’ll start with the first (or last) question: How many cells are needed in the therapeutic dose?

Now, firstly, to simplify the field of application, we’ll focus on T-cell therapies. (Stem cell therapies are arguably far more varied: a recent review by Samuel Golpanian in Stem Cellls Translational Medicine for heart disease alone, gives examples of doses in the range of 1 – 500 x 10⁶ cells (Golpanian et al. 2015). We’ll leave these aside, and perhaps return in a later blog post.)

The excitement today is about CAR T-cells for cancer immunotherapy. The high profile results of the past couple of years follow decades of research into T cells. So to discover the reasons why the therapies are designed the way they are, one has to look back to the classic papers and textbooks.

The meeting in Bethesda, Maryland of 10-11 September 2013 resulted in some documents that are well worth reading. Jacqueline Corrigan-Curay reviewed the meeting in Nature Molecular Therapy (Corrigan-Curay et al. 2014) and a presentation of Renier Brentjens “Dosing Strategies: Goals and Options” is still available online (http://osp.od.nih.gov/sites/default/files/3_Brentjens_s3.pdf). The summary is that almost all current T-cell therapy doses are delivered as follows:

• Total dose in the range of 3 × 10⁶ to 3 × 10⁷ cells/kg
• Delivered in escalating doses starting as low as 106 cells and progressing to target doses of 10⁹ or 10¹⁰ cells

Only one recent outlier is highlighted: the far lower dose of 105 cells/kg by Porter et al. in N Engl J Med. (Porter et al. 2011) which expanded x1000 in vivo. Naive T cells and Memory T cells are known for this ability to expand vastly in response to activation, so this would obviously be an attractive property in a therapy, if it could be well controlled.

Brentjens highlights that there is no established correlation between dose and clinical outcome, or between dose and toxicity. And lower doses would obviously be attractive from a process/manufacturing point-of-view.

So in view of these observations, it seems puzzling that today’s T-cell therapies in late clinical trials all require such vast numbers of cells. They might be much cheaper to produce if they only required a million cells (rather than a billion), and in that case, they wouldn’t require a huge advance in cell sorting technology.

Perhaps the main reason relates to activation of T cells. Cancers are known to inhibit T cells, so a therapy is better controlled if the T cells are already very numerous when presented to the tumour. Doses of billions of CAR T cells therefore represent comparable numbers to those generated by systemic infections. T cells are quickly anergised in an immune reaction, so the CAR T cell number is difficult to control unless it starts out large.

It remains possible that significantly lower cell doses may be effective, but might require advances in the control of immunological activation and inhibition. Therefore, it seems T cell therapies will still require huge doses of 109 – 1010 cells for the foreseeable future.

In addition to the above, there are many other variables that might affect the required dose, such as which disease is treated and the extent of the tumours, CAR design, conditioning chemotherapy, and the yield of in vitro genetic transduction. These are all fruitful subjects for future blog posts… watch this space.

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References

01. Corrigan-Curay, J., Kiem, H.-P., Baltimore, D., O’Reilly, M., Brentjens, R.J., Cooper, L., Forman, S., Gottschalk, S., Greenberg, P., Junghans, R., et al. (2014). T-Cell Immunotherapy: Looking Forward. Mol. Ther. 22, 1564–1574.

02. Golpanian, S., Schulman, I.H., Ebert, R.F., Heldman, A.W., DiFede, D.L., Yang, P.C., Wu, J.C., Bolli, R., Perin, E.C., Moyé, L., et al. (2015). Concise Review: Review and Perspective of Cell Dosage and Routes of Administration From Preclinical and Clinical Studies of Stem Cell Therapy for Heart Disease. Stem Cells Transl. Med. sctm.2015-0101.

03. Porter, D.L., Levine, B.L., Kalos, M., Bagg, A., and June, C.H. (2011). Chimeric Antigen Receptor–Modified T Cells in Chronic Lymphoid Leukemia. N. Engl. J. Med. 365, 725–733.

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