antibody-secreting human-human hybridomas

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MARK C. GLASSY*tt, HAROLD H. HANDLEY*tt, HIDEAKI HAGIWARAt, AND IVOR ROYSTON*tt .... the lung T293, squamous cell carcinomas of the cervix, HeLa.
Proc. Nati. Acad. Sci. USA Vol. 80, pp. 6327-6331, October 1983

Immunology

UC 729-6, a human lymphoblastoid B-cell line useful for generating antibody-secreting human-human hybridomas (human monoclonal antibody/tumor-associated antigen)

MARK C. GLASSY*tt, HAROLD H. HANDLEY*tt, HIDEAKI HAGIWARAt, AND IVOR ROYSTON*tt San *Department of Medicine, Division of Hematology/Oncology, tUniversity of California, San Diego, School of Medicine, and tThe University of California, Diego, Cancer Center and the Veterans Administration Medical Center, La Jolla, CA 92103

Communicated by W. D. McElroy, June 10, 1983

human tumor-associated antigens would require the availability of a human B lymphocyte sensitized to the host's own tumor. Recently, investigators have shown that B lymphocytes isolated from regional draining lymph nodes of cancer patients secrete antibodies that recognize the host's tumor (3, 4). These B cells, immortalized through fusion, may serve as a constant source of antibody reactive with human tumor-associated antigens. In this report, we describe a 6-thioguanine-resistant human lymphoblastoid B-cell line, termed UC 729-6, that reproducibly fuses with malignant and normal human lymphocytes resulting in stable pseudotraploid human-human hybridomas that have secreted Ig for >9 months in continuous culture. In addition, two human IgM mAbs are described that show reactivity to human tumor cell surface antigens.

ABSTRACT UC 729-6, a 6-thioguanine-resistant human lymphoblastoid B-cell line, was fused with normal and malignant human lymphocytes. Parent UC 729-6 cells were diploid with a 21p+ marker chromosome, expressed surface and cytoplasmic IgM Kc, and doubled every 17 hr. The resulting human-human hybridomas were pseudotetraploid containing the 21p+ marker, doubled every 20-30 hr, and secreted 3-9 jig of human Ig per ml per 106 hybrid cells for >9 months in continuous culture. Human Ig-secreting hybridomas were generated in 88% (14/16) of the fusions carried out and were cloned by limiting dilution (one cell per three wells) without the use of feeder layers. The mean fusion frequencies (number of wells, plated at 105 cells per well, showing hybrid growth per 106 lymphocytes fused) of UC 729-6 with normal lymphocytes ranged from 0.45 to 2.9 and with malignant B lymphocytes, from 3 to 10. Analysis of the human-human hybridomas derived from lymphocytes isolated from regional draining lymph nodes of cancer patients revealed several that secreted human monoclonal antibody that reacted by an enzyme immunoassay with some carcinoma cell lines but not with normal fibroblast cell lines. These data suggest that (i) UC 729-6 can be fused with human lymphocytes to generate stable human-human hybridomas, some of which secrete antibody reactive to human cell surface antigens, and (ii) UC 729-6 can be used to rescue Ig from nonsecretory malignant B cells and thereby allow for the production of anti-idiotype antibodies.

MATERIALS AND METHODS UC 729-6. UC 729-6 is a 6-thioguanine-resistant human lymphoblastoid B-cell line isolated by treating parent WIL-2 cells (14, 15) with 25 ,uM 6-thioguanine (16). UC 729-6 is surface and cytoplasmic IgM K+, diploid with a 21p+ marker chromosome, and Fc fragment receptor negative (17, 18). Prior to fusion, the cells were incubated with 100 p.M 6-thioguanine (Sigma). UC 729-6 cells were grown in RPMI 1640 medium/10% fetal calf serum/2 mM L-glutamine at 370C in humidified 5% C02/95% air. Cell Lines. Squamous cell carcinomas of the lung, Calu-1 and SK-MES-1, were obtained from J. Fogh (Sloan-Kettering Institute for Cancer Research, New York); oat cell carcinoma of the lung T293, squamous cell carcinomas of the cervix, HeLa and CaSki, and passage 35 of WI-38 fibroblasts were from H. Masui (University of California, San Diego, Nude Mouse Facility); prostate carcinoma Ln-Cap was obtained from H. Lowe (Department of Surgery, University of California, San Diego); and foreskin fibroblast 350Q was obtained from D. Richman (Veterans Administration Medical Center, La Jolla, CA). All cell lines were grown in RPMI 1640 medium/10% fetal calf serum/2 mM L-glutamine at 370C in humidified 5% C02/

The use of somatic cell hybridization to produce monoclonal antibodies (mAbs) of predefined specificity, as pioneered by Kohler and Milstein (1), has revolutionized many areas of human biology. However, since most of these mAbs are of rodent origin their application to in vivo use in humans may be limited. Therefore, the production of human mAbs could potentially be of considerable value and in many instances may offer advantages over mAbs of other species. Currently available human mAbs have been obtained by mouse-human hybridizations (2-4), by Epstein-Barr virus (EBV) transformation of human immune lymphocytes (5-7), and by human-human hybridization (8-10) and has recently been reviewed (11). However, each of these approaches suffers from individual difficulties and each has met with limited success. Mouse-human hybrids preferentially lose human chromosomes and, therefore, are genetically unstable (12, 13). EBV transformation yields cell lines that produce small quantities of Ig (5-7). The reproducibility of generating human-human hybridomas is limited by the lack of a suitable fusion partner in that currently available cell lines for fusion (8-10) are difficult to work with, have low fusion frequencies, and occasionally yield revertants. The production of human mAbs of predefined specificity to

95% air. Human Tissues. Lymphocytes fused with UC 729-6 were obtained from lymph nodes, peripheral blood, spleen, and tonsils. All lymph nodes used for this study were obtained from cancer patients and patients with lymphoma. Normal and malignant peripheral blood lymphocytes were isolated by FicollHypaque centrifugation (19). Immune organs, usually received within 3 hr after surgery, were aseptically transferred to a Petri dish containing 70% ethanol for 10-30 sec, then immersed and rinsed in 5% penicillin/streptomycin (GIBCO) in serum-free

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Abbreviations: mAb, monoclonal antibody; CLL, chronic lymphocytic leukemia; EBV, Epstein-Barr virus; ELISA, enzyme-linked immunosorbent assay; Pi/NaCl, phosphate-buffered saline.

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RPMI 1640 medium and trimmed free of extraneous tissue and capsular components. After transfer to a fresh solution of serumfree RPMI 1640 medium, the tissue was teased with nugent forceps to make a single cell suspension. After large tissue aggregates had settled, the cells in suspension were removed and washed for two 5-min periods at 500 x g. All dissections and cell preparations were carried out at room temperature. All isolated lymphocytes were resuspended at 5 X 106/ml in RPMI 1640 medium/10% fetal calf serum and incubated overnight at 37°C in humidified 5% C02/95% air. Fusion and Cloning of Hybridomas. UC 729-6 was fused with isolated lymphocytes at a ratio of 1:2 according to the technique of Kohler and Milstein (1) as modified by Gefter et al. (20). Briefly, UC 729-6 cells were mixed with the isolated lymphocytes in separate 50-ml conical centrifuge tubes (Corning) and washed for two 5-min periods at 500 x g. The supernatant was completely aspirated and 1.0 ml of 35% polyethylene glycol 1500 (BDH; lot no. 6229890) was added dropwise over a 30-sec interval to a dry cell pellet and allowed to stand at room temperature for 2 min. At 2-min intervals, the following volumes of serum-free RPMI 1640 medium were added: 1.0 ml, 2.0 ml, 4.0 ml, and 8.0 ml. After addition of the final 8.0-ml volume of medium, the cells were spun at 300 X g for 5 min, the supernatant was aspirated, and the pellet was carefully suspended in medium supplemented with 10% fetal calf serum, glutamine, and 0.2 mM hypoxanthine/0.2 ,uM amethopterin/32 gM thymidine (HAT; ref. 21; Sigma). Cells were plated at 1.0 x 105 per well in Costar 96-well microtiter plates without the use of feeder layer cells. Approximately 2-5 wk after fusion, the wells were screened microscopically for hybrid growth. The supernatants of the wells showing positive hybrid growth were screened by an enzyme-linked immunosorbent assay (ELISA) for human Ig production (see below). Those human hybridomas secreting human Ig were cloned by limiting dilution (one cell per three wells) without the use of feeder layer cells. Detection and Quantitation of Human Ig. Cell surface and cytoplasmic Ig was detected by indirect immunofluorescence microscopy using techniques and reagents previously described (22). Human Ig-secretinghybridomas were detected by an ELISA as described (23). Reactivity of Human mAbs with Cell Lines. Washed logarithmic phase cells (1 x 105) were incubated for 30 min with human mAbs in an immunofiltration manifold plate, washed three times with 0.1% gelatin in NaH2PO4/Na2HPO4/0.9% saline, pH 7.4 (Pi/NaCI), incubated an additional 30 min with horseradish peroxidase conjugated goat anti-human Ig, washed, and developed with o-phenylenediamine as described (23). Assays were carried out in triplicate and read at 490 nm on a micro-ELISA reader (Dynatech, Alexandria, VA). Controls consisted of Pi/NaCl, supernatant from UC 729-6 cultures, and irrelevant affinity-purified human Igs (Cappell Laboratories, Cochranville, PA). Chromosome Analysis. Cells were incubated for 3.5 hr at 370C with colcemid at 10 Ag/ml (Sigma) in Pi/NaCl. After an additional 30-min incubation in 75 mM KCl, cells were washed (500 x g for 10 min) and fixed with methanol/acetic acid (3:1). Cells were then treated with trypsin (24) and stained with Giemsa for 10 min. Representative chromosome spreads were counted. Frequency Distribution of Cell DNA Content. Cells and hybridomas were stained by the propidium iodide method for the determination of relative cell DNA content and analyzed by cytofluorometry, as described (25). Briefly, 1.0 x 106 cells were fixed in 70% ethanol for 2 hr, washed (500 X g for 10 min), and then suspended in 100 p1 of Pi/NaCl. One milliliter of propidium iodide (0.05 mg/ml; Sigma) was added to the cell suspension, and the mixture was incubated for 15 min at 40C and

Proc. Natl. Acad. Sci. USA 80 (1983)

filtered through a 50-,um-pore nylon mesh. Samples were analyzed on an Ortho Cytofluorograf 50H-computer 2150 equipped with a 488-mm argon ion laser at 400 mW. Detection of T65 Antigens. T65 is a T-lymphocyte antigen found on most chronic lymphocytic leukemia (CLL) cells that are surface Ig' (22) but not on lymphoblastoid B-cell lines, including UC 729-6 (18). T65 was detected by indirect immunofluorescence using the murine mAbs T101 previously described (22).

RESULTS Characteristics of UC 729-6. UC 729-6, a hypoxanthine phosphoribosyltransferase-negative cell line derived from parental WIL-2 cells (16), was resistant to 100 ,uM 6-thioguanine and died within 10 days when exposed to HAT medium (Fig. 1). UC 729-6 has remained HAT sensitive for at least 50 passages in the presence of 100 .tM 6-thioguanine and has a doubling time of 17 hr (Fig. 1). Out of 16 fusions attempted, neither UC 729-6 nor the unfused lymphocytes grew in HAT medium. UC 729-6 was split by dilution at 1:20 or 1:40 and grown to limiting densities of >106 cells per ml in RPMI 1640 medium. Both the WIL-2 and the UC 729-6 cell lines were found to contain the EBV nuclear antigen [EBNA; ref. 26; D. Carson (Scripps Clinic and Research Foundation), personal communication]. In addition, UC 729-6 was 12% surface and cytoplasmic IgM K+, diploid with a 21p+ chromosome marker, and Fc receptor-negative. Fusion Frequencies of UC 729-6. The UC 729-6 cell line was found to reproducibly fuse with human lymphocytes when polyethylene glycol was used. Of the attempted fusions, 93% (15/16) resulted in hybrid formation and 88% (14/16) contained hybridomas secreting Ig (Table 1). Successful fusions with this cell line have been carried out over an 18-month period. The fusion frequencies (number of wells, plated at 105 cells per well, showing human hybrid growth per 106 lymphocytes fused) of UC 729-6 with lymphocytes isolated from lymph nodes, peripheral blood, spleen, and tonsil are shown in Table 1. Lymphocytes isolated from lymph nodes and tonsil gave higher fusion frequencies (means, 1.7 and 2.9, respectively) than those from peripheral blood and spleen (means, 0.45 and 0.54, respectively). The highest percentage of Ig-secreting hybridomas was obtained with lymph node lymphocytes, and IgM producers were more prevalent than IgG producers. Fusion of malignant B lymphocytes from patients with CLL and lymphoma with UC 729.-6 resulted in hybrid formation in 100

xx -4

c)

-

cd

0

2

4 6 Time, days

8

10

FIG. 1. Growth of UC 729-6 cells in normal medium (RPMI 1640/ 10% fetal calf serum/glutamine) or in HAT-containing medium (21). o, Normal; *, HAT. Viability was determined by trypan blue (0.04%) exclusion.

Proc. Natl. Acad. Sci. USA 80 (1983)

Immunology: Glassy et A

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Table 1. Fusion frequencies of UC 729-6 cells

Tissue source Lymph node

Exp. 1 2 3 4 5 6

Lymphocytes fused, no. x 10-7 1.1 0.47 1.33 1.0 2.0

Wells containing hybridomas, no. per 106 lymphocytes fused* 0.36 1.28 3.16 2.10 2.35 0.86 x= 1.7

0.7

% hybrid wells secreting Ig IgG IgM 0 0 0 33 19 31 14 24 21 36 0 33

PBL

1 2 3 4

0.66 4.9 4.4 3.7

0.91 0.13 0.75 0.00 x= 0.45

50 66 9 0

0 0 0 0

Spleen

1 2

0.73 1.0

0.27 0.80 F= 0.54

50 25

0 0

Tonsil

1 2 3 4

2.0 0.6 0.6 1.33

0.45 3.48 2.42 5.34 x= 2.9

33 0 12.5 32

0 22 37 2

PBL, peripheral blood lymphocytes. * After fusion, cells were plated at 1.0 x 105 per well.

83% of the attempts (three out of three for CLL and two out of three for lymphoma) and fusion frequencies of 3-10 (data not shown). All of the resulting hybrids secreted Ig, all of which were either monoclonal K or A. Characteristics of Selected Human-Human Hybridomas. Some of the characteristics of selected human-human hybridomas produced in this study, designated 8A1, 8B1, CLNH5, CLNH1l, and WLNA6, are given in Table 2. Hybridomas 8A1 and 8B1 were the product of fusion of UC 729-6 with the peripheral blood of a patient with CLL (18) and serve as a useful model for the rescue of Ig from a non-Ig-secreting malignant B cell. Hybridomas 8A1 and 8B1 were 10-17% surface IgM K+, simultaneously positive for both cytoplasmic IgM K (28-38%) and cytoplasmic IgM A (30-48%), pseudotetraploid, contained the 21p+ chromosome marker found in UC 729-6, and were

positive for T65 antigen, a characteristic of CLL (22). CLNH5 and CLNH11 were derived from fusion of UC 7296 with pooled lymphocytes isolated from a regional draining lymph node and the peripheral blood of a patient with cervical carcinoma, whereas WLNA6 was derived from a lymph node

draining a Wilm tumor. These hybridomas were chosen for further analysis because their secreted Ig was shown by ELISA to react with a panel of cell lines (see below). CLNH5, CLNH11, and WLNA6 are cytoplasmic IgM K+, IgG K+, and IgM K+ hybridomas, respectively. All hybridomas have continued to secrete human Ig at 3-9 jig/ml per 1.0 x 106 cells for >9 months in continuous culture and have doubled in quantity every 2030 hr. The properties of these human-human hybridomas have remained unchanged after preservation in liquid nitrogen and they have reconstituted reliably.

Table 2. Cellular characteristics of UC 729-6 and hybrids Hybrids with UC 729-6 as parent CLNH11t CLNH5t 8A1, 8B1* Characteristic UC 729-6 K K IgD, IgG K IgD, IgM Surface Ig IgM IgM K IgG IgM K IgM K IgM K Cytoplasmic Ig

Chromosome marker Chromosome content % T65 reactivity§ Supernatant, ;Ag/ml per 106 cells IgA IgG

WLNA6t ND

IgM

21p+ Diploid

IgM A 21p+ Tetraploid

ND

ND

ND

Tetraploid

Tetraploid

Tetraploid

Negative

99

Negative

Negative

Negative

UD UD UD UD UD UD 3-5 UD UD UD UD 3 5 UD 6-9 IgM ND, not determined; UD, undetectable [lower limit of the ELISA (23) is