490 / THIRD INTERNATIONAL WORKSHOP ON CYTOKINES

241

INTERLEUKIN-2 - TRUNCATED HUMAN IgM HEAVY CHAIN FUSION PROTEIN: A CYTOTOXIC AGENT FOR INTERLEUKIN 2 RECEPTOR BEARING CELLS. M

~.eYIE*mBONNEVILLE. Mane-Al& PEYRAT, & GODARD and

INSERM U211 and LYNATECH. Plateau technique CHR, Quai Moncousu, 44035 NANTRS Cedex 01.

Reagents directed against the high-affinity binding site of the lL2 receptor (monoclonal antibodies, ILZbacterial toxin conshucts) have been shown to be efficient immunosuppressants in animal models and in humans. However, such reagents elicit a vigourous host anti-xenoprotein immune response in vivo. We present here a new hybrid cDNA (lL2-Mu) comprising the IL2 cDNA and a truncated human mu chain cDNA (CH2KH4 exons). Cells transfected with the hybrid cDNA (Cos-1 or CHO cells) were shown to secrete multimeric forms of the fusion protein ranging from 120 to 680 kDa molecular mass. The immunoaffinity purified molecules bound specifically to high-affinity IL2 receptor bearing cells. Furthermore, it displayed complement-mediated killing of these cells, without any effect on receptor negative targets. We beleive that this new artificial molecules (Cytomoduline) which exhibit 1) a high affinity for the IL2 receptors, 2) specific cytotoxic properties, 3) “humanized” characteristics, may represent a further advance in the strategy of IL2 receptor targeting.

242

FGF AND FGF PROTECT FROM CYTARABINE-INDUCED ALOPECIA IN THE BAT MODEL. J.J. Jimenez, H.S. Huang, and A.A. Yunis, Depts. of Medicine, Biochemistry and Molecular Biology, and Oncology, Univ. of Miami School of Medicine, Miami, FL %iOl.

-.

Chemotherapy-induced alopecia is a distressing problem to the cancer patient. Using the young rat (7-day old) model we have demonstrated that the biologic response modifier ImuVert, and more recently IL-I, protect rats from alopecia induced by cytarabine (ARA-C). In our continued screening using this model, we have discovered that both EGF and FGF are also effective. Rxcept for topical use, all growth factors were given S.C. once daily for seven days. ARA-C, 50 mglkgfday, was given i.p. for seven days. Alopecia was scored on day 10 from 0 (no alopecia) t" 3+ (total body alopecia). In the first experiment, of 17 rate treated with ARA-C alone, 13 had 3+ alopecia. I" contrast, all 17 rats treated with AR&C + murine EGF had only 0 t" l+ alopecia. Murine EGF applied topically protected at the site of application. rHu-EGF was also protective. In separate experiments, all 14 rats treated with ABA-C alone developed 3+ alopecia. I" contrast, 11 of 14 rats treated with ARA-C + FGF had local protection at the site of injection. In other experiments using 39 rats, neither growth hormone "or TGFa protected from ARA-C-induced alopecia. The mechanism of protection from alopecia by EGF and FGF and possible relationship to the protective effect observed with ImuVert or IL-l remains to be determined. Whatever the mechanisms, these novel observations could prove applicable to chemotherapy-induced alopecia in the clinical setting.

243

COMPARATIVE EFFECTS OF IMUVERT AND IL-l ON CYTARABINE-INDUCED ALOPECIA IN THE RAT MODEL. J.J. Jimenez, M. Sawaya, and A.A. Yunis. Depts. of Medicine, Dermatology, Biochemistry and Molecular Biology, and Oncology. Univ. of Miami School of Medicine, Miami, FL 33101.

We have previously reported on the protection by ImuVert and I&l from cytarabine-induced alopecia in the young rat model. The present study was designed to compare the efficacy of ImuVert vs. IL-l under the same conditions and examine their effect on DNA synthesis in hair follicles isolated from treated animals. Twenty eeve" rats were randomized into three groups of 9 rats each. All rats received ARA-C 50 mglkglday i.p. for 7 days. In addition, group I received IL-l daily; group II received ImuVert; group III received buffer (control). All rats in the control group developed total body alopecia. I" contrast, none of the rats in group I or II developed any alopecia. In other experiments, animals were similarly treat- ed and hair follicles isolated from their skin 3 hours after one treatment dose. DNA synthesis in the isolated follicles was examined by measuring the uptake of tritiated thymidine after 24 hours incubation. DNA synthesis in follicles iso- lated from ARA-C-treated animals was 15-20% of untreated con- trols. In contrast, DNA synthesis in follicles obtained from animals treated with either ARA-C + ImuVert or AR&C + IL-l approached control levels. These results demonstrate that ImuVert and IL-l are equally effective in protecting rats from the alopecia and that protection from AU-C-induced damage can be observed early at the level of the hair follicle.

244 IMMUNORODULATORY ACTIVITY OF LIPOSONAL IL-2 (Lip-IL-2) AND "PEGILATED" IL-?. (PEG-IL-Z) IN NICE. E. Kedar Y. Rutkowski. E Braun N. Emanuel Y. Barenholz. HebrewUr;iversity-Hadass~;lSchaol.~rusalem. Israel.

IL-2 has been widely used in cancer immunotherapy. Its therapeutic efficacy is limited by high toxicity and its short plasma t1!2. Aims: To study the impact of IL-2 chemical modification (pegilation) and encapsulation in long-circulating liposomes on the pharmacokinetics and immunomodulatory activity. Methods: rHIL-2 (C&us. USA) was encapsulated in 50 nm unilamellar lipo- somes. composed of egg phosphatidylcholine, cholesterol and polyethylene glycol (PEG) phosphatidylethanolamine. In pharmacokinetics exp., 5 I 10 Cetus U of IL-2, Lip IL-2 and PEG-IL-Z (Cetus) were injected i.v. For immunological studies. untreated normal and cytoxan (CY. 100 mglkg, day-s) treated tumor-bearing mice were injected i.v. or i.p. with IL-2 (4 x IO' CU q.24h, days 1-5). Lip-IL-2 or PEG-IL-2 (I x 10' CU d.l.d.4). Tests were performed on days 6-S with WBC. splenacytes (sp.) and peritoneal cells (PC.). Results: IL-2 Lip-IL-2 PEti-IL-2 t112 plasma Durat'ion of plasma

c 2 min 20 min 50 40 min 8 hi- 12-24 hr

level L Loo U/ml % Increase in cell no.-WBC O-20 O-40 O-100 % Increase in cell sp.lpc. o-20010-80 100-350~20-400 200-400~100-600 **LAK cell activity sp. & DC. 10-20 20-30 *vs. HASS control; **fold-increase vs. IL-2.

The stimulatory effects depended on the route of inoculation and the timing of assay. They were usually stronger in CY treated mice. In such mice, Lip-IL-2 and PEG-IL-2 caused a marked decrease (5~80%) in the no. of sp. expressing the Thy 1.2 and MEL-14 antigens. Conclusion: Immunomodulation by IL-2 depends on its pharmacokinetics which can be improved by pegilation and encapsulation in long- circulating liposomes.

245

DEvEu)E'MEN'I OF ENZYME-BASED ASSAYS TO DETECT CIRCULATING LEVELS OF IL7 IN SERUMAND To DETECT TflE IMMUNE RFSFONSE TO IL7 INJECTED INTO A XENCGENEIC SPECIES. M.M. Kelley and

i 19355-1314. Sena A. Smith Sterling Ihxg, Inc., Malvern, PP We have developed enzyme-based assays to quantitate

circulating IL-7 levels in human serum. 'Ihe protocol involves coating a 96-well ELISA plate with an antibody to hm IL7 in order to capture the cytokine. Bound IL7 is detected with biotinvlated anti-IL-7 antibodv. followed bv streptavidin-conjugated alkaline phosphatase to generate an optical signal. Ihe assay is read at OD405 on an automated plate reader. The most sensitive assay uses a rabbit poly- clonal antibody, generated to human recombinant IIr7, as both the capture antibody and the biotinylated antibody. Sensitivity is about 10 pg/ml in human serum. Less sensitive assays (to about 200 pg/ml in human serum) have been developed with monoclonals as either the capture antibody, laixlled antibody or both. The reduced sensitivity obtained with monqclonal antibodies may originate from cross-reactivity with the serum matrix. Methods investigated to eliminate non-specific cross reactivity will be discussed. We have also developed an enzyme based innnmogenicity assay to detect circulating antibodies to human IL-7 injected into xenogeneic species. This assay is sensitive to about 8 pg/d.

246

IN VIVO ADMINISTRATION OF rhIL7 GENERATES A LEUKOCYTOSIS AND ANTI-TUMOR ACTIVITY. K.L. Komschlies'. G. Dan&. F. Ruscetti', O.C. Boerma"'. C. Faltv"ek3. and R.H. Wiltrout'. 'BCDP, PRI/DynCorp and 'BRMP, NCI, NCI-FCRDC, Frederick, MD 21702-1201 and 'Sterling Drug, Inc., Malvern. PA 19355.

Normal mice given >5sg of rhIL7 ip twice a day for 7 days exhibit a 3-5 fold increase in leukocytes in the spleen, lymph nodes and peripheral blood. The splenic leukocytosis was due to a" increase in mature cells especially CDS+ T cells and a disproportionate increase in pre-B cells. There was little change in bone marrcw cellularity; however. there was a 3 fold increase in pre-B cells, a 3 fold decrease in MAC-l' cells, a >90% decrease in myeloid progenitors, a 4 fold decrease in CFLI-s activity and a decrease in the ability of bone marrow to repopulate the lymphoid lineages in irradiated recipients. Conversely, there was a 5-10 fold in- crease in myeloid progenitors and a 4 fold increase in CFLJ-s activity in the spleens of rhIL7 treated mice. The repeated ip administration of ylOpg/injection of rhIL7 decreased the number of preexistent experimental lung metastases established from the 816 melanoma, Renca renal carcinoma, or MCA-38 colon adenocarcinoma by 40%, 50% oi- 95%, respectively. Further, in the rhIL7-treated MCA-38 tumor bearers, there was a 4 fold and 9 fold increase over controls in the "umber of CD4+ and CDB+ T cells, respectively, in the lungs and spleens. Thus, in viva treatment with rhIL7 may be useful in accelerating the recovery from immunosuppressed or otherwise leukopenic conditions and as a" antitumor agent.