Proc. Natl. Acad. Sci. USA Vol. 92, pp. 9348-9352, September 1995 Immunology

Rabbit monoclonal antibodies: Generating a fusion partner to produce rabbit-rabbit hybridomas (myc/abl/transgenic rabbits/plasmacytoma/B cells) HELGA SPIEKER-POLET, PERIANNAN SETHUPATHI, PI-CHEN YAM, AND KATHERINE L. KNIGHT* Department of Microbiology and Immunology, Stritch School of Medicine, Loyola University Chicago, Maywood, IL 60153 Communicated by Alfred Nisonofft Brandeis University, Waltham, MA, June 16, 1995 (received for review April 10, 1995)

ABSTRACT During the last 15 years several laboratories needed to stably produce monoclonal rabbit antibodies. We have attempted to generate rabbit monoclonal antibodies, have now developed a fusion partner from a myc/abl double- mainly because rabbits recognize antigens and epitopes that transgenic rabbit, and we report the successful production of are not immunogenic in mice or rats, two species from which stable antigen-specific rabbit-rabbit hybridomas. monoclonal antibodies are usually generated. Monoclonal antibodies from rabbits could not be generated, however, because a plasmacytoma fusion partner was not available. To MATERIALS AND METHODS obtain a rabbit plasmacytoma cell line that could be used as Transgenic Rabbits. Single-cell zygotes were injected with a a fusion partner we generated transgenic rabbits carrying two murine E,,-abl construct [kindly provided by S. Cory (11); E,, transgenes, c-myc and v-abl. These rabbits developed plasma- is the immunoglobulin heavy chain enhancer] at a concentra- cytomas, and we obtained several plasmacytoma cell lines tion of 1 ,tg/ml and implanted into the uterus of pseudopreg- from which we isolated hypoxanthine/aminopterin/thymi- nant females (12). Offspring were tested at 3-4 weeks of age dine-sensitive clones. One of these clones, when fused with by Southern blot analysis of peripheral blood lymphocyte DNA spleen cells of immunized rabbits, produced stable hybrid- for the presence of the E,,-abl transgene. Rabbits carrying the omas that secreted antibodies specific for the immunogen. The E,,-abl transgene were mated with EK-myc transgenic rabbits hybridomas can be cloned and propagated in nude mice, and established previously in our laboratory (EK is the K-chain they can be frozen without change in their ability to secrete enhancer) (13). The offspring were tested for the presence of specific monoclonal antibodies. These rabbit-rabbit hybrid- both transgenes as described above. In addition, we directly omas will be useful not only for production of monoclonal microinjected zygotes from a transgenic EK-myc rabbit with the antibodies but also for studies of immunoglobulin gene re- EK-abl transgene. arrangements and isotype switching. Generation of Plasmacytoma Cell Lines and a Hypoxan- thine/Aminopterin/Thymidine (HAT)-Sensitive Fusion Monoclonal antibodies (mAbs) from rabbits have not been Partner. Rabbits that became ill were sacrificed and cells from available because no rabbit plasmacytomas, from which a the tumorous tissues were placed in tissue culture in an attempt hybridoma fusion partner could be generated, have been to obtain plasmacytoma cell lines. Culture medium used was identified. The availability of rabbit mAbs is, however, highly RPMI 1640 enriched with the following additions: amino acids, desirable for several reasons. First, rabbits are known to nonessential amino acids, pyruvate, glutamine, vitamins, produce antibodies to many antigens that are not especially Hepes, gentamicin, penicillin, streptomycin, fungizone (all immunogenic in mice (1-5). For example, Bystryn et al. (2) components were from GIBCO and were used at concentra- directly compared rabbit and mouse antibodies directed tions suggested by the supplier), and 50 ,uM 2-mercaptoetha- against human melanoma cells and showed that they recognize nol. After 6-8 weeks in culture, stable cell lines were growing different epitopes. Second, rabbit antibodies are generally of from these tumorous tissues. high affinity. Third, because most mAbs are generated in mice To obtain a HAT-sensitive fusion partner, three cell lines and rats, relatively few mAbs are available that react with were first x-irradiated with 200 rad (1 rad = 0.01 Gy) and then mouse or rat immunogens. Because of this desire for rabbit cultured in the presence of 8-azaguanine. (The concentration mAbs several laboratories developed mouse-rabbit heterohy- of 8-azaguanine was initially 0.2 ,ug/ml and was slowly in- bridomas, but this technology has had limited success. The creased to 20 ,ug/ml over a 10-month period.) We obtained earliest mouse-rabbit heterohybridomas were unstable and/or three 8-azaguanine-resistant clones: 20337-7 after one month secreted only light (L) chain (6-9). Raybould and Takahashi and 240E1-1-1 and 240E1-1-2 after 8 months in culture. Cells (5) reportedly overcame this problem by using normal rabbit of these three clones were sensitive to medium containing serum (NRS) instead of fetal calf serum (FCS) as a supplement HAT. to the culture medium. However, Verbanac et al. (10) de- Fusions. Rabbits received a primary immunization by in- scribed major problems with this method. For example, they jection of antigen (a total of 2 mg of protein or 2 x 107 cells found that the heterohybridomas were highly unstable and had per immunization) in complete Freund's adjuvant subcutane- to be subcloned every 4-6 weeks to avoid loss of antibody ously, intramuscularly, and intraperitoneally. The animals secretion. In our laboratory, we obtained no more than two to were boosted once or twice in the same manner but with five hybridomas per fusion when using the method described incomplete Freund's adjuvant. The final boost was given by Raybould and Takahashi (5). Further, these heterohybrid- intraperitoneally and intravenously with saline 4 days before omas were difficult to clone, and the clones were generally the fusion. Fusions were performed using conventional meth- unstable and did not secrete antibody over a prolonged period odology (14): spleen cells (1.5-3 x 108) of immunized rabbits of time. Thus it became clear that heterohybridomas were not and the fusion partner 240E 1-1-2 were fused at a ratio of 2:1 a satisfying solution and that rabbit-rabbit hybridomas were Abbreviations: mAb, monoclonal antibody; L chain, light chain; NRS, The publication costs of this article were defrayed in part by page charge normal rabbit serum; FCS, fetal calf serum; HAT, hypoxanthine/ payment. This article must therefore be hereby marked "advertisement" inI aminopterin/thymidine; FITC, fluorescein isothiocyanate. accordance with 18 U.S.C. §1734 solely to indicate this fact. *To whom reprint requests should be addressed. 9348 Downloaded by guest on September 29, 2021 Immunology: Spieker-Polet et aL Proc. Natl. Acad. Sci. USA 92 (1995) 9349 with 50% PEG 4000 (EM Science, Cherry Hill, NJ 08304) at Color development was read at 405 nm in an ELISA plate 37°C in serum-free medium. The cells were plated in 48-well reader. microtiter plates, at approximately 2 x 105 spleen cells per well, in medium with 15% FCS. After 72 h, HAT was added. Medium was changed every 5-6 days. Clones usually were RESULTS AND DISCUSSION observed after 2-5 weeks. Supernatants were tested for the Generation of Double-Transgenic Rabbits. Because Rosen- presence of antibody specific for the immunogen, either by baum et al. (11) obtained mice with plasmacytomas in myc/abi immunofluorescence with Jurkat cells [using fluorescein iso- double-transgenic mice, we decided to generate myc/abl dou- thiocyanate (FITC)-conjugated goat anti-rabbit L-chain anti- ble-transgenic rabbits in an effort to obtain rabbits with body as secondary reagent] (fusion 1) or by ELISA (fusions 2 plasmacytoma. A family of transgenic rabbits that carried the and 3). Hybridomas were cloned by limiting dilution in 48-well c-myc oncogene linked to the K-chain enhancer was developed microtiter plates. For feeder cells, we used the fusion partner, previously in our laboratory (13). We now generated a second 240E1-1-2, at 5 x 104 cells per well. These feeder cells were family of transgenic rabbits with the v-abl oncogene linked to killed 5-6 days later by the addition of HAT. the immunoglobulin heavy chain enhancer (E,,) as a transgene. In preliminary fusion experiments we noticed excessive A total of 665 zygotes were microinjected and implanted in 31 growth of adherent cells in several wells, which prevented the pseudopregnant females. From 11 pregnant females we ob- hybridomas from establishing themselves. Such growth of tained 19 offspring, of which 2 carried the v-abl transgene. To adherent cells, which interferes with the growth of fused cells, obtain double-transgenic rabbits we used two methods. In the has been reported by other investigators (5, 15). The extent of first method, EK-myc transgenic rabbits were mated with the this growth varied from one experiment to another, and it E,,-abl transgenic rabbits. From four matings 22 offspring were could be partially prevented if FCS was totally or partly obtained, and of these, 5 carried both transgenes. The plas- replaced by NRS. However, the hybridomas appeared to grow macytomas 81E5-1 and 300F1-2 developed in offspring of more slowly in the absence of FCS. In one experiment, we transgenic rabbits developed in our laboratory, whereas plas- attempted to remove the macytomas 20337-7, 20337-8, and 0022-3 developed in off- adherent cells by incubating the spring for which the E,-abl transgenic parent (obtained with spleen cell suspension on plastic dishes for 6 h at 37°C before the same E,IL-abl construct) was kindly provided by Andrew fusing them. Although this method did not eliminate adherent Kelus and Klaus cell it did Karjalainen (Basel Institute of Immunology, growth, reduce the number of wells with adherent Basel, Switzerland). In the second method EK-myc zygotes cells. were microinjected with E,,-abl DNA. In this case one, ELISA. ELISA (16) was performed in 96-well microtiter 240E1-1, of five offspring carried both transgenes and devel- plates (Falcon 3912, Fisher) that were coated overnight with oped plasmacytoma. All offspring that carried both trans- purified goat anti-rabbit L-chain antibody, 1,g/ml, or with the genes, c-myc and v-abl, became ill between the ages of 8 and immunogen, 2 Ag/ml. The following solutions were added, 19 months. Tumors had developed in these rabbits in various sequentially, for 1-2 h at room temperature: first, the super- locations. Histologic analysis of these tumors revealed that the natant to be tested, then biotinylated goat anti-rabbit L chain rabbits had developed immunoblastic lymphoma or early or, when assaying for rabbit immunoglobulin isotypes, biotin- plasmacytoma. ylated goat anti-rabbit g-, y-, or a-chain antibodies, 1 jig/ml. Development of a Rabbit Fusion Partner. From the tumor- This was followed by incubation with avidin-biotin-horseradish ous tissue of five of the six rabbits with plasmacytoma peroxidase complex (Vectastain ABC Kit, Vector Laborato- (300F1-2, 0022-3, 20337-7, 20337-8, and 240E1-1) stable cell ries) and finally with the substrate 2,2'-azinobis(3-ethylbenz lines were obtained. From these lines, three HAT-sensitive thiazolinesulfonic acid) (ABTS) as suggested by the supplier. clones were established by selection with 8-azaguanine and

FIG. 1. Rabbit plasmacytoma fusion partner, 240E1-1-2, stained with Wright-Giemsa stain (Diff-Quick, American Scientific Products, McGaw Park, IL ). (x1200.) Downloaded by guest on September 29, 2021 9350 Immunology: Spieker-Polet et al. Proc. Natl. Acad. Sci. USA 92 (1995) Table 1. Frequency and stability of hybridomas obtained in three fusions of the rabbit fusion partner with spleen cells from hyperimmunized rabbits Wells with Hybrids secreting Hybrids yielding hybrids/wells Hybrids per 106 specific mAb/total stable clones/total Fusion Immunogen plated (%) cells fused hybrids tested (%) hybrids cloned (%) 1 Jurkat cells 200/400(50) 0.7 10/104 (10) ND 2 Ovalbumin 38/980* 0.25 9/36 (25) ND 3 Mouse serum proteins 242/980(25) 1.2 43/187 (23) 7/7 (100) ND, not determined. *In many wells adherent cells were growing that prevented the growth of the upcoming hybridomas. In such cases the hybridoma clones had to be removed from the adherent cells, and this was done only with 38 clones.

from one of these clones, 240E1-1-2, stable hybridomas could fusion 3, mouse serum proteins (including immunoglobulins) be obtained. In characterizing this clone, we determined the that were precipitated with 45% saturated (NH4)2SO4, because doubling time to be 48 h, and by staining with Wright-Giemsa monoclonal isotype-specific antibodies to mouse immuno- stain (Diff-Quick) we found that the cells had features char- globulins would be valuable reagents. From all three fusions we acteristic of early plasma cells-i.e., they are large cells with obtained hybridomas that secreted mAbs specific for the abundant cytoplasm, and the nuclei frequently contain immunogen (Table 1). When we tested supernatants of hybrid- "lumpy" chromatin. The cells have many vacuoles (Fig. 1), omas from fusion 1 we found that 10 of 104 hybridomas which indicates that they may be proplasmocytes. These cells secreted antibodies that bound to Jurkat cells (Fig. 2). From do not secrete immunoglobulin (<10 ng/ml) as determined by fusion 2 we tested the supernatants of 36 hybridomas by an ELISA with goat anti-L-chain and goat anti-Fcy antibodies. ELISA and found that 9 of these had antibodies specific for This finding was surprising, since the parental cell line did ovalbumin. Finally, we found, also using an ELISA, that 43 of secrete low levels of IgG. To determine the cause for the loss 187 hybridoma supernatants tested from fusion 3 contained of secretion we assayed for intracellular IgG. Using the same antibodies that recognized antigens of the mouse serum pro- antibodies for an ELISA with cell lysates (5 x 106 cells per ml), teins that were used to immunize the rabbit. Of these 43 we did not detect intracellular IgG. However, when we used mAb-secreting clones, we subcloned 7, all of which continued either anti-Fcy or anti-L chain for both capture and detection to secrete mAb (Table 1). Several of the mAbs from were we found intracellular free y chain but no L chain (< 10 ng per cloned fusion 3 found, by ELISA, to recognize one or more of the mouse immunoglob- 5 x 106 cells). We conclude that our fusion partner produces ulin isotypes: one was specific for IgG2a (53-5), one for IgG2b fy chains but not appreciable amounts of L chain. Consequently (10-6), and two recognized both IgG2a and IgG2b (80-1 and the mAb secreted by hybridomas will not associate with L 45-9) but none of the other immunoglobulins (Table 2). chains of the fusion partner but some of them may associate Several additional mAbs recognized multiple mouse immu- with y chains of the fusion partner. noglobulins. To examine the utility of these mAbs in immu- Production of Specific mAbs. Three separate fusions were nofluorescence experiments we tested two of the mAbs that performed with the newly established cell line, 240E1-1-2, and recognize IgG2a for binding to IgG2a-expressing B- spleen cells from rabbits immunized with one of three anti- lymphoma cells, A20, and we found that both mAbs bind to gens. We chose the following as antigens: fusion 1, the human A20 cells (Fig. 3), while a mAb that was shown by ELISA to T-cell line Jurkat, because we wanted to test whether mAb recognize only IgG2b did not bind to the A20 cells (Fig. 3). directed to cell surface antigens can be obtained; fusion 2, We conclude that the rabbit mAbs will be valuable immu- ovalbumin, because it is a well-known antigen for rabbits; and nofluorescent reagents. We determined the isotype and the concentration of the 80- antibodies secreted by the hybridomas specific for mouse Con trol serum proteins by ELISA. We found that all 25 of the mAbs that were tested were of the IgG isotype (Table 3). The concentration of mAb in the supernatant was determined by Ra anti-Jurkat ELISA using purified rabbit IgG as standard. The concentra- tion of antibody varied between 200 ng/ml and 5 ,ug/ml. Higher concentrations of mAb could be obtained in ascites fluid of nude mice. Since most or all of the rabbit hybridomas obtained from fusions with spleen cells secrete mAb of the IgG co isotype, and since rabbit IgG binds staphylococcal protein A as well as complement, these mAbs will be useful for immuno- precipitation and cytotoxicity assays. In summary, the fusion Table 2. Specificity of rabbit anti-mouse immunoglobulin mAbs secreted by hybridomas from a fusion of the rabbit fusion partner 240E1-1-2 with spleen cells from a rabbit immunized with mouse serum proteins Reaction with mouse immunoglobulin isotypes 10° 10, 102 103 104 Fluorescence intensity Clone -yl, K y2a, K y2b, K y3, K , A 53-5 - + - - - FIG. 2. Immunofluorescence labeling of human T cells, Jurkat, 10-6 - - + - - with a rabbit monoclonal anti-Jurkat antibody. Jurkat cells were 80-1 - + + _ _ incubated with the supernatant of an IgG-secreting rabbit-rabbit (Ra) 45-9 - + + hybridoma anti-Jurkat antibody (fusion 1). In the control, supernatant of an IgG-secreting rabbit-rabbit hybridoma that recognizes an irrel- The specificity was assayed by ELISA: the plates were coated with evant antigen (mouse IgG2) was used. The secondary antibody was mouse immunoglobulin of the indicated isotypes (all obtained from FITC-conjugated goat anti-rabbit L chain. Sigma) at 1 ,ug/ml. Downloaded by guest on September 29, 2021 Immunology: Spieker-Polet et aL Proc. Natl. Acad. Sci. USA 92 (1995) 9351 701 C

Control and Ra anti- Ms IgG2b

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100 101 102 100 101 102 103 100 101 102 Fluorescence intensity FIG. 3. Immunofluorescence labeling of mouse A20 B-lymphoma cells with monoclonal rabbit (Ra) anti-mouse (Ms) IgG2 antibodies. A20 cells were incubated with the supernatants of IgG-secreting rabbit-rabbit hybridomas (fusion 3) that were shown by ELISA (Table 2) to recognize mouse IgG2a (clone 53-5) (0.25 ,ug of mAb per ml) (A), IgG2a and IgG2b (clone 80-1) (5 iLg of mAb per ml) (B), or IgG2b (clone 10-6) (0.5 ,ug of mAb per ml) (C) and none of the other mouse immunoglobulin isotypes. In control samples, A20 cells were incubated with the supernatant of an IgG-secreting rabbit-rabbit hybridoma that recognizes an irrelevant antigen-i.e., a surface antigen ofJurkat cells (fusion 1). As secondary antibody we used FITC-conjugated goat anti-rabbit L chain. efficiency for the three fusions performed was between 0.25 percentage of IgA-producing hybridomas from MLN, PP, and 1.2 in 106 cells, which is comparable to the efficiency and other mucosal tissues had been found by other investi- generally obtained in mouse-mouse fusions. Of the hybrid- gators for rat (15, 17, 18). In contrast, fusion with spleen cells omas produced in the three fusions, 10%, 23%, and 25% does not generally yield IgA-secreting hybridomas in either secreted mAbs that were specific for the immunogens (Table mice or rats, as we now report for rabbits. 1). Again, this percentage of hybridomas that secretes specific Concluding Statement. The research described here, de- mAb is comparable to that obtained in mouse-mouse fusions. tailing our search for a rabbit fusion partner, began in the The hybridomas have been subcloned and they were frozen late 1970s, before transgene technology was available. Once and thawed without loss in their ability to secrete mAb. These investigators showed that lymphoid tumors developed in data indicate that the hybridomas are stable and that frequent transgenic mice carrying various oncogenes (11, 19), we used cloning, which had been necessary for the heterohybridomas, this technology to develop rabbit plasmacytomas. Our break- is not needed for the rabbit-rabbit hybridomas. through came in 1991 when we found that the myc/abl Production of IgA-Secreting Hybridomas. Most of the double-transgenic rabbits developed plasmacytomas. Since hybridomas from spleen secreted IgG and none were found then, we have established plasmacytoma cell lines and were that secreted IgA (Table 3). Because IgA-producing hybri- able to develop one into a usable fusion partner. The domas would be valuable reagents we decided to perform availability of a rabbit fusion partner provides us with the fusions with cells from Peyer's patch (PP) and mesenteric opportunity to produce mAbs specific for mouse antigens lymph node (MLN) to obtain IgA-secreting hybridomas. and also for antigens or epitopes that are not immunogenic From two separate fusions 34% of the hybridomas from in mice. Such mAbs will be useful in diagnosis of diseases and MLN and 81% of the hybridomas from PP secreted IgA treatment of patients. (Table 3). Similar results were obtained in two additional experiments-i.e., 35% of the hybridomas from MLN and This work was supported by Public Health Service Grant Al 11234. PP 38% of the hybridomas from secreted IgA. (The other 1. Krause, R. M. (1970) Adv. Immunol. 12, 12-29. isotypes were not determined in these experiments.) A high 2. Bystryn, J., Jacobsen, S. J., Liu, P. & Heaney-Kieras, J. (1982) Hybridoma 1, 465-472. Table 3. mAbs produced by rabbit hybridomas obtained from 3. Weller, A., Meek, J. & Adamson, E. D. (1987) Development fusions of 240E1-1-2 with mesenteric lymph node (MLN), Peyer's (Cambridge, U.K) 100, 351-363. patch (PP), or spleen cells 4. Norrby, E., Mufson, M. A., Alexander, H., Houghton, R. A. & No. of clones (% of total clones) Lerner, R.-A. (1987) Proc. Natl. Acad. Sci. USA 84, 6572-6576. 5. Raybould, T. J. G. & Takahashi, M. (1988) Science 240,1788-1790. Cells fused Total IgG IgM IgA 6. Yarmush, M. L., Gates, F. T., III, Weisfogel, D. R. & Kindt, T. J. Spleen* 25 25 (100) 0 0 (1980) Proc. Natl. Acad. Sci. USA 77, 2899-2903. MLNt 82 14 (17) 34 (41) 28 (34) 7. Yarmush, M. L., Gates, F. T., III, Dreher, K. L. & Kindt, T. J. 48 2 (4) 2 (4) 39 (81) (1981) J. Immunol. 126, 2240-2244. ppI 8. Dreher, K. L., Sogn, J. A., Gates, F. T., III, Kuo, M. & Kindt, T. J. *Data from fusion 3, Table 1: 25 of 43 specific mAbs were analyzed. (1983) J. ImmunoL 130, 442-448. tMLN cells of an unimmunized rabbit were activated by murine 9. Kuo, M., Sogn, J. A., Max, E. E. & Kindt, T. J. (1985) Mol. CD40-ligand-transfected CHO cells (generously provided by Melanie Immunol. 22, 351-359. Spriggs, Immunex Research and Development Corp., Seattle) for 48 10. Verbanac, K M., Gross, U. M., Rebellato, L. M. & Thomas, h prior to fusion. The fused cells were plated in 1000 wells. J. M. (1993) Hybridoma 12, 285-295. tPP cells were treated as described above for MLN cells and plated in 11. Rosenbaum, H., Harris, A. W., Bath, M. L., McNeall, J., Webb, 400 wells. E., Adams, J. M. & Cory, S. (1990) EMBO J. 9, 897-905. Downloaded by guest on September 29, 2021 9352 Immunology: Spieker-Polet et al. Proc. Natl. Acad. Sci. USA 92 (1995)

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