Large-scale preparation of adherent lymphokine-activated killer (A-LAK) cells for adoptive immunotherapy in man (original) (raw)

Summary

Stepwise counterflow centrifugal elutriation of leukapheresed human mononuclear cells (MNC) in a Beckman JE-6B rotor and J6-M/E centrifuge yielded a population highly enriched in natural killer (NK) cells (70–75% large granular lymphocytes with 10–13 times greater NK activity) at a flow rate of 38–44 ml/min using a fixed rotor speed of 3000 rpm at 27° C. However, the mean cell recovery was <1%. To obtain sufficient numbers of purified NK cells for adoptive immunotherapy, a strategy combining counterflow centrifugal elutriation with adherence of recombinant interleukin-2(rIL-2)-activated NK cells to plastic was developed. First, MNC were elutriated to give a twofold enrichment in NK cells, containing 22% Leu19+ cells, 18% large granular lymphocytes and 51 lytic units of activity against K562 targets as opposed to the unfractionated MNC containing 10% Leu19+ cells, 7% large granular lymphocytes and 26 lytic units of activity. The mean recovery was 80±15% (_n_=10). Further enrichment was obtained by isolation of the elutriated cells that adhered to plastic after culture for 24 h in the presence of 1000 U/ml rIL-2. The initial adherent lymphokine-activated killer (A-LAK) cells represented 1–4% of total MNC, but their subsequent expansion was at least 10–22-fold during 8–14 days in culture with 1000 U/ml rIL-2. Using this strategy, 2 × 109 normal MNC, obtained by leukapheresis, yielded 5 × 108 A-LAK cells with a total of 5.7 × 105 lytic units of cytotoxicity against K562 and a total of 3.3 × 105 lytic units against Daudi targets. This enrichment method has yielded sufficient numbers of A-LAK cells to form the basis for a phase I clinical trial of adoptive immunotherapy in patients with advanced cancer.

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Authors and Affiliations

  1. Department of Pathology, University of Pittsburgh School of Medicine and Pittsburgh Cancer Institute, 15213, Pittsburgh, PA, USA
    Robert J. Melder, Ronald B. Herberman & Theresa L. Whiteside
  2. Division of Clinical Immunopathology, University of Pittsburgh School of Medicine and Pittsburgh Cancer Institute, 15213, Pittsburgh, PA, USA
    Theresa L. Whiteside
  3. Department of Medicine, University of Pittsburgh School of Medicine and Pittsburgh Cancer Institute, 15213, Pittsburgh, PA, USA
    Craig S. Rosenfeld & Ronald B. Herberman
  4. Montefiore Hospital, University of Pittsburgh School of Medicine and Pittsburgh Cancer Institute, 15213, Pittsburgh, PA, USA
    Robert J. Melder, Craig S. Rosenfeld, Ronald B. Herberman & Theresa L. Whiteside

Authors

  1. Robert J. Melder
  2. Craig S. Rosenfeld
  3. Ronald B. Herberman
  4. Theresa L. Whiteside

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Melder, R.J., Rosenfeld, C.S., Herberman, R.B. et al. Large-scale preparation of adherent lymphokine-activated killer (A-LAK) cells for adoptive immunotherapy in man.Cancer Immunol Immunother 29, 67–73 (1989). https://doi.org/10.1007/BF00199919

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