Presence of Soluble Interleukin-2 Receptor Adult T-Cell Leukemia Lymph Node and Blood Cells in the Cytotoxicity of Recombinant Fab and Fv Immunotoxins on

Single-chain immunotoxins anti-Tac(Fv)-PE40 and anti-Tac(Fv)PE40KDEL, composed of variable domains of the anti-Tac monocional antibody and truncated forms of P s e u d o m o n a s exotoxin, have shown potent cytotoxic activity against malignant peripheral blood mononuclear cells (PBMCs) from adult T-cell leukemia (ATL) patients originating from the Caribbean. However, several clinically important issues have not previously been addressed. These include the potential of soluble interleukin 2 receptor in ATL patients to block immunotoxin effectiveness, the relative sensitivity of malignant lymph node cells (LNCs) versus PBMCs, the effect of an immunotoxin with a prolonged half-life, and finally whether ATL cells from patients in Japan have toxin sensitivity equal to those of the Caribbean patients. To resolve these questions, we studied 32 malignant PBMC and LNC samples from 30 ATL patients from Japan. PBMCs from 27 of 27 patients were very sensitive with 50% inhibition of protein synthesis achieved with 0.02-0.85 ng/ml (0.3-13 p~a) of anti-Tac(Fv)PE40KDEL or anti-Tac(Fv)-PE40. LNCs had sensitivity very similar to that of PBMCs in the five patients tested. The fully recombinant immunotoxin, anti-Tac(Fab)-PE40, which has 8-10 times the tl/2 ot and IB compared to the Fv-immunotoxins, was also very cytotoxic toward cells from 27 of 27 patients tested with 50% inhibition of protein synthesis of 0.08-25 ng/ml. It was found that purified soluble interleukin 2 receptor added to the cytotoxicity assay decreased the cytotoxic activity of anti-Tac(Fv)PE40KDEL or anti-Tac(Fab)-PE40, but that 1 x 1 0 4 units/ml or less had minimal competitive effects. It was found that ATL patients who have responded even incompletely to conventional chemotherapy have soluble interleukin 2 receptor levels lower than this at posttreatment. We conclude that recombinant immunotoxins containing anti-Tac(Fv) are effective against Japanese ATL PBMCs or LNCs and might be most effective if used in vivo after conventional chemotherapy. If it is found in humans that the effectiveness of single-chain recombinant toxins is limited by short halflife, anti-Tac(Fab)-PE40 should be considered as an alternative agent. I N T R O D U C T I O N ATL 3 a peripheral T-cell malignancy, shows some characteristic clinical and geographic features (1-4). Patients are refractory to conventional chemotherapy, and malignant cells become more resistant to various anticancer agents in terminal stages (5-8). Overexpression of P-glycoprotein may be correlated with this refractory nature in some ATL patients (9). So far, attempts to overcome this drug resistance have not succeeded, making it difficult to improve the poor prognosis of ATL patients. Received 9/1/93; accepted 12/16/93. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. This work was supported in part by a Grant-in-Aid for Cancer Research from the Ministry of Health and Welfare of Japan. 2 To whom requests for reprints should be addressed, at Laboratory of Molecular Biology, National Cancer Institute, 9000 Rockville Pike, Bldg. 37, Room 4E16, Bethesda, MD 20892. 3 The abbreviations used are: ATL, adult T-cell leukemia; (s)IL2(R), (soluble) interleukin 2 (receptor); PE, Pseudomonas exotoxin; ER, endoplasmic reticulum; LNC, lymph node cells; PBMCs, peripheral blood mononuclear cells; FCS, fetal calf serum; IC5o, 50% inhibitory concentration. ATL cells and ATL-derived T-cell lines nearly always express the low-affinity IL2R, which is the CD25 (Tac) antigen on the cell surface (10), and release a soluble form of the receptor (sIL2R) in the plasma (11-13). A recombinant immunotoxin, anti-Tac(Fv)-PE40, that is composed of the variable domains of the anti-Tac monoclonal antibody and truncated P s e u d o m o n a s exotoxin (PE40), was described previously (14). PE40 is devoid of domain Ia, which is necessary for the toxin to bind to the PE receptor on normal cell surfaces (15). This immunotoxin showed potent cytotoxic effects on malignant cells from ATL patients (16). Anti-Tac(Fv)-PE40KDEL is a derivative of antiTac(Fv)-PE40 which contains the typical ER retention sequence, KDEL, replacing the native sequence, REDLK, at the COOH-terminus. Anti-Tac(Fv)-PE40KDEL was 1.5to 9-fold more cytotoxic than anti-Tac(Fv)-PE40 toward activated T-cells, ATL cell lines, and fresh ATL cells, probably due to improved routing of the toxin to the ER from which it translocates to the cytosol (17-19). Before such agents are tested in clinical trials, several crucial issues must be addressed. For example, will high sIL2R levels in the serum of ATL patients prevent the immunotoxin from working in vivo? Do malignant cells in the LNC of ATL patients have the same sensitivity as the malignant PBMCs which have previously been tested? Do ATL cells from patients in Japan, which have not previously been tested with PE-related immunotoxins, have the same sensitivity to these molecules as cells from Caribbean ATL patients? Finally, if recombinant toxins containing anti-Tac(Fv) are found to have insufficient antitumor activity in humans due to inadequate half-life, are molecules with prolonged half-life effective on ATL cells? It has previously been found that DAB486 IL2, a recombinant toxin composed of truncated diphtheria toxin and IL2, was cytotoxic to LNC and a minority of PBMC samples from Japanese ATL patients at a concentration of 10 -s M (20). Other than this study, these issues have not been previously addressed with any recombinant toxin. To answer these clinically relevant questions, LNCs and PBMCs from 30 Japanese ATL patients were examined. We tested not only anti-Tac(Fv)-PE40 and anti-Tac(Fv)-PE40KDEL but also the new molecule anti-Tac(Fab)-PE40. This fully recombinant Fab-toxin, like the previously reported anti-Tac(Fab)-phospholipase C molecule (21), is encoded by two DNA fragments on a single plasmid. Anti-Tac(Fab)-PE40 purified from the periplasm of Escher ich ia coli is composed of the anti-Tac Fd chain ( V . + CH1) connected by a disulfide bond to a fusion of the anti-Tac light chain and PE40. Anti-Tac(Fab)PE40 had within 2-fold the cytotoxic activity of anti-Tac(Fv)-PE40. While anti-Tac(Fab)-PE40 is only slightly larger than anti-Tac(Fv)PE40 (Mr 90,000 versus 66,000), its tl/2 a of 22 min and tl/2 [3 of 430 min were about 8-10 times higher than those of the single-chain toxin in mice. 4 We also examined the potential of soluble Tac to interfere with the cytotoxic activity of anti-Tac(Fv)-PE40KDEL or anti-Tac(Fab)-PE40 toward the ATL patient cells. 4 R. J. Kreitman, in preparation.