抗CD3抗体を用いて誘導したLymphokine Activated Killer（LAK）細胞の抗腫瘍効果ならびにリンパ球表面マーカーの検討

ANALYSIS OF CYTOLYTIC ACTIVITY AND CELL SURFACE PHENOTYPES OF LYMPHOKINE ACTIVATED KILLER CELLS STIMULATED WITH R-IL 2 AND AN ANTI-CD 3 ANTIBODY

　脳腫瘍患者末梢血中のリンパ球をrecombinant interleukin−2と抗CD3抗体とで培養することによりLAK（Lymphokine-activated killer）細胞を誘導し，この抗腫瘍作用とリンパ球表面マーカーの検討を行った。対象は脳腫瘍患者12例であり，全例約40ml以下の少量の末梢血から5×10⁸個以上のLAK細胞を得ることができた。しかし，この抗腫瘍効果を4時間⁵¹Cr releasing assayで測定すると，抗CD3抗体を用いない通常のLAK細胞に比べてすべての標的細胞に対して弱い傾向を示し，リンパ球表面マーカーはCD3が高値を，CD16，Leu7が低値を示していた。抗CD3抗体を用いてLAK細胞を誘導することにより，少量の末梢血から養子免疫療法を行うのに充分な数のLAK細胞を得ることができた。この抗腫瘍効果は通常のLAK細胞に比べて弱かったが，これは測定方法にも問題があると思われる。現在我々は，抗CD3抗体とr-IL2によって誘導したLAK細胞を用いた養子免疫療法を行っており，今後長期にわたり経過を追うことにより臨床例における抗腫瘍効果を評価していきたい。

We analyzed cytolytic activity and cell surface phenotypes of LAK (lymphokine activated killer) cells which were stimulated with recombinant IL-2 and an anti-CD 3 antibody.

This study involved 9 patients with astrocyto-mas, one patient with ganglioglioma, and one patient with medulloblastoma and their ages ran-ged between 5 and 80yr. Peripheral blood lym-phocytes were separated from about 40 ml venous blood on a Ficoll-Paque gradient. After PBL's were cultured with r-IL 2 and anti-CD 3 antibody for about two weeks, more than 10⁸ LAK cells could be obtained. Their cytolytic activity was measured by standard 4 hours-⁵¹Cr release assay and K 562, Daudi, U 251 MG, MCF-7, and autotu-mor c lls were used as target cells. Their surface phenotypes (CD3, CD4, CD8, CD16, CD25, Leu 7, HLA-DR) were measured by FACS. These LAK cells showed weaker killing activity against all kinds of tumor cells than usual LAK cells (LAK cells stimulated with r-IL 2 alone) did. Their surface phenotypes were more sensitive to CD3 and less sensitive to CD16 and Leu7 than those of usual LAK cells.

One of weak points of so-called LAK therapy is that many PBL's have to be obtained by leuko-pheresis and because of that, it is difficult to undertake LAK therapy to children or elderly pa-tients. However, by using an anti-CD 3 antibody, enough LAK cells as effector of LAK therapy could be obtained more easily. Ordinarily, 4-hours ⁵¹Cr release assay is used to measure cytoly-tic activity, but it is a problem whether actual cytotoxity in vivo is reflected in this assay because we can know only the cytotoxity against floating cells or monolayer cells by this assay and it is too short to coculture target cells with effector cells for four hours. It was published that T-cell derived LAK cells were more effective than NK derived LAK cells against multi-cellular spheroid models and by using anti-CD3 antibody, more CD3-positive LAK cells could be obtained. Be-cause of that, cytolytic activity of these LAK cells was weaker than that of usual LAK cells by this assay, but these LAK cells may be effective in vivo.

We are now trying to undertake this therapy to patients with goilmas and additional follow-up of the patients will be required before its thera-peutic value can be established.