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

Rabbit monoclonal antibodies: Generating a fusion partner toproduce 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 laboratorieshave attempted to generate rabbit monoclonal antibodies,mainly because rabbits recognize antigens and epitopes thatare not immunogenic in mice or rats, two species from whichmonoclonal antibodies are usually generated. Monoclonalantibodies from rabbits could not be generated, however,because a plasmacytoma fusion partner was not available. Toobtain a rabbit plasmacytoma cell line that could be used asa fusion partner we generated transgenic rabbits carrying twotransgenes, c-myc and v-abl. These rabbits developed plasma-cytomas, and we obtained several plasmacytoma cell linesfrom which we isolated hypoxanthine/aminopterin/thymi-dine-sensitive clones. One of these clones, when fused withspleen cells of immunized rabbits, produced stable hybrid-omas that secreted antibodies specific for the immunogen. Thehybridomas can be cloned and propagated in nude mice, andthey can be frozen without change in their ability to secretespecific monoclonal antibodies. These rabbit-rabbit hybrid-omas will be useful not only for production of monoclonalantibodies but also for studies of immunoglobulin gene re-arrangements and isotype switching.

Monoclonal antibodies (mAbs) from rabbits have not beenavailable because no rabbit plasmacytomas, from which ahybridoma fusion partner could be generated, have beenidentified. The availability of rabbit mAbs is, however, highlydesirable for several reasons. First, rabbits are known toproduce antibodies to many antigens that are not especiallyimmunogenic in mice (1-5). For example, Bystryn et al. (2)directly compared rabbit and mouse antibodies directedagainst human melanoma cells and showed that they recognizedifferent epitopes. Second, rabbit antibodies are generally ofhigh affinity. Third, because most mAbs are generated in miceand rats, relatively few mAbs are available that react withmouse or rat immunogens. Because of this desire for rabbitmAbs several laboratories developed mouse-rabbit heterohy-bridomas, but this technology has had limited success. Theearliest mouse-rabbit heterohybridomas were unstable and/orsecreted only light (L) chain (6-9). Raybould and Takahashi(5) reportedly overcame this problem by using normal rabbitserum (NRS) instead of fetal calf serum (FCS) as a supplementto the culture medium. However, Verbanac et al. (10) de-scribed major problems with this method. For example, theyfound that the heterohybridomas were highly unstable and hadto be subcloned every 4-6 weeks to avoid loss of antibodysecretion. In our laboratory, we obtained no more than two tofive hybridomas per fusion when using the method describedby Raybould and Takahashi (5). Further, these heterohybrid-omas were difficult to clone, and the clones were generallyunstable and did not secrete antibody over a prolonged periodof time. Thus it became clear that heterohybridomas were nota satisfying solution and that rabbit-rabbit hybridomas were

needed to stably produce monoclonal rabbit antibodies. Wehave now developed a fusion partner from a myc/abl double-transgenic rabbit, and we report the successful production ofstable antigen-specific rabbit-rabbit hybridomas.

MATERIALS AND METHODSTransgenic Rabbits. Single-cell zygotes were injected with a

murine E,,-abl construct [kindly provided by S. Cory (11); E,,is the immunoglobulin heavy chain enhancer] at a concentra-tion of 1 ,tg/ml and implanted into the uterus of pseudopreg-nant females (12). Offspring were tested at 3-4 weeks of ageby Southern blot analysis of peripheral blood lymphocyte DNAfor the presence of the E,,-abl transgene. Rabbits carrying theE,,-abl transgene were mated with EK-myc transgenic rabbitsestablished previously in our laboratory (EK is the K-chainenhancer) (13). The offspring were tested for the presence ofboth transgenes as described above. In addition, we directlymicroinjected zygotes from a transgenic EK-myc rabbit with theEK-abl transgene.

Generation of Plasmacytoma Cell Lines and a Hypoxan-thine/Aminopterin/Thymidine (HAT)-Sensitive FusionPartner. Rabbits that became ill were sacrificed and cells fromthe tumorous tissues were placed in tissue culture in an attemptto obtain plasmacytoma cell lines. Culture medium used wasRPMI 1640 enriched with the following additions: amino acids,nonessential amino acids, pyruvate, glutamine, vitamins,Hepes, gentamicin, penicillin, streptomycin, fungizone (allcomponents were from GIBCO and were used at concentra-tions suggested by the supplier), and 50 ,uM 2-mercaptoetha-nol. After 6-8 weeks in culture, stable cell lines were growingfrom these tumorous tissues.To obtain a HAT-sensitive fusion partner, three cell lines

were first x-irradiated with 200 rad (1 rad = 0.01 Gy) and thencultured in the presence of 8-azaguanine. (The concentrationof 8-azaguanine was initially 0.2 ,ug/ml and was slowly in-creased to 20 ,ug/ml over a 10-month period.) We obtainedthree 8-azaguanine-resistant clones: 20337-7 after one monthand 240E1-1-1 and 240E1-1-2 after 8 months in culture. Cellsof these three clones were sensitive to medium containingHAT.

Fusions. Rabbits received a primary immunization by in-jection of antigen (a total of 2 mg of protein or 2 x 107 cellsper immunization) in complete Freund's adjuvant subcutane-ously, intramuscularly, and intraperitoneally. The animalswere boosted once or twice in the same manner but withincomplete Freund's adjuvant. The final boost was givenintraperitoneally and intravenously with saline 4 days beforethe fusion. Fusions were performed using conventional meth-odology (14): spleen cells (1.5-3 x 108) of immunized rabbitsand the fusion partner 240E 1-1-2 were fused at a ratio of 2:1

Abbreviations: mAb, monoclonal antibody; L chain, light chain; NRS,normal rabbit serum; FCS, fetal calf serum; HAT, hypoxanthine/

I aminopterin/thymidine; FITC, fluorescein isothiocyanate.*To whom reprint requests should be addressed.

9348

The publication costs of this article were defrayed in part by page chargepayment. This article must therefore be hereby marked "advertisement" inaccordance with 18 U.S.C. §1734 solely to indicate this fact.

Dow

nloa

ded

by g

uest

on

June

5, 2

020

Proc. Natl. Acad. Sci. USA 92 (1995) 9349

with 50% PEG 4000 (EM Science, Cherry Hill, NJ 08304) at37°C in serum-free medium. The cells were plated in 48-wellmicrotiter plates, at approximately 2 x 105 spleen cells perwell, in medium with 15% FCS. After 72 h, HAT was added.Medium was changed every 5-6 days. Clones usually wereobserved after 2-5 weeks. Supernatants were tested for thepresence of antibody specific for the immunogen, either byimmunofluorescence with Jurkat cells [using fluorescein iso-thiocyanate (FITC)-conjugated goat anti-rabbit L-chain anti-body as secondary reagent] (fusion 1) or by ELISA (fusions 2and 3). Hybridomas were cloned by limiting dilution in 48-wellmicrotiter plates. For feeder cells, we used the fusion partner,240E1-1-2, at 5 x 104 cells per well. These feeder cells werekilled 5-6 days later by the addition of HAT.

In preliminary fusion experiments we noticed excessivegrowth of adherent cells in several wells, which prevented thehybridomas from establishing themselves. Such growth ofadherent cells, which interferes with the growth of fused cells,has been reported by other investigators (5, 15). The extent ofthis growth varied from one experiment to another, and itcould be partially prevented if FCS was totally or partlyreplaced by NRS. However, the hybridomas appeared to growmore slowly in the absence of FCS. In one experiment, weattempted to remove the adherent cells by incubating thespleen cell suspension on plastic dishes for 6 h at 37°C beforefusing them. Although this method did not eliminate adherentcell growth, it did reduce the number of wells with adherentcells.ELISA. ELISA (16) was performed in 96-well microtiter

plates (Falcon 3912, Fisher) that were coated overnight withpurified goat anti-rabbit L-chain antibody, 1,g/ml, or with theimmunogen, 2 Ag/ml. The following solutions were added,sequentially, for 1-2 h at room temperature: first, the super-natant to be tested, then biotinylated goat anti-rabbit L chainor, when assaying for rabbit immunoglobulin isotypes, biotin-ylated goat anti-rabbit g-, y-, or a-chain antibodies, 1 jig/ml.This was followed by incubation with avidin-biotin-horseradishperoxidase complex (Vectastain ABC Kit, Vector Laborato-ries) and finally with the substrate 2,2'-azinobis(3-ethylbenzthiazolinesulfonic acid) (ABTS) as suggested by the supplier.

Color development was read at 405 nm in an ELISA platereader.

RESULTS AND DISCUSSIONGeneration of Double-Transgenic Rabbits. Because Rosen-

baum et al. (11) obtained mice with plasmacytomas in myc/abidouble-transgenic mice, we decided to generate myc/abl dou-ble-transgenic rabbits in an effort to obtain rabbits withplasmacytoma. A family of transgenic rabbits that carried thec-myc oncogene linked to the K-chain enhancer was developedpreviously in our laboratory (13). We now generated a secondfamily of transgenic rabbits with the v-abl oncogene linked tothe immunoglobulin heavy chain enhancer (E,,) as a transgene.A total of 665 zygotes were microinjected and implanted in 31pseudopregnant females. From 11 pregnant females we ob-tained 19 offspring, of which 2 carried the v-abl transgene. Toobtain double-transgenic rabbits we used two methods. In thefirst method, EK-myc transgenic rabbits were mated with theE,,-abl transgenic rabbits. From four matings 22 offspring wereobtained, and of these, 5 carried both transgenes. The plas-macytomas 81E5-1 and 300F1-2 developed in offspring oftransgenic rabbits developed in our laboratory, whereas plas-macytomas 20337-7, 20337-8, and 0022-3 developed in off-spring for which the E,-abl transgenic parent (obtained withthe same E,IL-abl construct) was kindly provided by AndrewKelus and Klaus Karjalainen (Basel Institute of Immunology,Basel, Switzerland). In the second method EK-myc zygoteswere microinjected with E,,-abl DNA. In this case one,240E1-1, of five offspring carried both transgenes and devel-oped plasmacytoma. All offspring that carried both trans-genes, c-myc and v-abl, became ill between the ages of 8 and19 months. Tumors had developed in these rabbits in variouslocations. Histologic analysis of these tumors revealed that therabbits had developed immunoblastic lymphoma or earlyplasmacytoma.Development of a Rabbit Fusion Partner. From the tumor-

ous tissue of five of the six rabbits with plasmacytoma(300F1-2, 0022-3, 20337-7, 20337-8, and 240E1-1) stable celllines were obtained. From these lines, three HAT-sensitiveclones 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, McGawPark, IL ). (x1200.)

Immunology: Spieker-Polet et aL

Dow

nloa

ded

by g

uest

on

June

5, 2

020

9350 Immunology: Spieker-Polet et al.

Table 1. Frequency and stability of hybridomas obtained in three fusions of the rabbit fusion partner with spleen cells fromhyperimmunized rabbits

Wells with Hybrids secreting Hybrids yieldinghybrids/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) ND2 Ovalbumin 38/980* 0.25 9/36 (25) ND3 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 tobe removed from the adherent cells, and this was done only with 38 clones.

from one of these clones, 240E1-1-2, stable hybridomas couldbe obtained. In characterizing this clone, we determined thedoubling time to be 48 h, and by staining with Wright-Giemsastain (Diff-Quick) we found that the cells had features char-acteristic of early plasma cells-i.e., they are large cells withabundant cytoplasm, and the nuclei frequently contain"lumpy" chromatin. The cells have many vacuoles (Fig. 1),which indicates that they may be proplasmocytes. These cellsdo not secrete immunoglobulin (<10 ng/ml) as determined byELISA with goat anti-L-chain and goat anti-Fcy antibodies.This finding was surprising, since the parental cell line didsecrete low levels of IgG. To determine the cause for the lossof secretion we assayed for intracellular IgG. Using the sameantibodies for an ELISA with cell lysates (5 x 106 cells per ml),we did not detect intracellular IgG. However, when we usedeither anti-Fcy or anti-L chain for both capture and detectionwe found intracellular free y chain but no L chain (< 10 ng per5 x 106 cells). We conclude that our fusion partner producesfy chains but not appreciable amounts of L chain. Consequentlythe mAb secreted by hybridomas will not associate with Lchains of the fusion partner but some of them may associatewith y chains of the fusion partner.

Production of Specific mAbs. Three separate fusions wereperformed with the newly established cell line, 240E1-1-2, andspleen cells from rabbits immunized with one of three anti-gens. We chose the following as antigens: fusion 1, the humanT-cell line Jurkat, because we wanted to test whether mAbdirected to cell surface antigens can be obtained; fusion 2,ovalbumin, because it is a well-known antigen for rabbits; and

80-Control

co

Ra anti-Jurkat

10° 10, 102 103 104Fluorescence intensity

FIG. 2. Immunofluorescence labeling of human T cells, Jurkat,with a rabbit monoclonal anti-Jurkat antibody. Jurkat cells wereincubated with the supernatant of an IgG-secreting rabbit-rabbit (Ra)hybridoma anti-Jurkat antibody (fusion 1). In the control, supernatantof an IgG-secreting rabbit-rabbit hybridoma that recognizes an irrel-evant antigen (mouse IgG2) was used. The secondary antibody wasFITC-conjugated goat anti-rabbit L chain.

fusion 3, mouse serum proteins (including immunoglobulins)that were precipitated with 45% saturated (NH4)2SO4, becausemonoclonal isotype-specific antibodies to mouse immuno-globulins would be valuable reagents. From all three fusions weobtained hybridomas that secreted mAbs specific for theimmunogen (Table 1). When we tested supernatants of hybrid-omas from fusion 1 we found that 10 of 104 hybridomassecreted antibodies that bound to Jurkat cells (Fig. 2). Fromfusion 2 we tested the supernatants of 36 hybridomas by anELISA and found that 9 of these had antibodies specific forovalbumin. Finally, we found, also using an ELISA, that 43 of187 hybridoma supernatants tested from fusion 3 containedantibodies that recognized antigens of the mouse serum pro-teins that were used to immunize the rabbit. Of these 43mAb-secreting clones, we subcloned 7, all of which continuedto secrete mAb (Table 1).

Several of the cloned mAbs from fusion 3 were found, byELISA, to recognize one or more of the mouse immunoglob-ulin isotypes: one was specific for IgG2a (53-5), one for IgG2b(10-6), and two recognized both IgG2a and IgG2b (80-1 and45-9) but none of the other immunoglobulins (Table 2).Several additional mAbs recognized multiple mouse immu-noglobulins. To examine the utility of these mAbs in immu-nofluorescence experiments we tested two of the mAbs thatrecognize IgG2a for binding to IgG2a-expressing B-lymphoma cells, A20, and we found that both mAbs bind toA20 cells (Fig. 3), while a mAb that was shown by ELISA torecognize only IgG2b did not bind to the A20 cells (Fig. 3).We conclude that the rabbit mAbs will be valuable immu-nofluorescent reagents.We determined the isotype and the concentration of the

antibodies secreted by the hybridomas specific for mouseserum proteins by ELISA. We found that all 25 of the mAbsthat were tested were of the IgG isotype (Table 3). Theconcentration of mAb in the supernatant was determined byELISA 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 ascitesfluid of nude mice. Since most or all of the rabbit hybridomasobtained from fusions with spleen cells secrete mAb of the IgGisotype, and since rabbit IgG binds staphylococcal proteinA aswell 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 mAbssecreted by hybridomas from a fusion of the rabbit fusion partner240E1-1-2 with spleen cells from a rabbit immunized with mouseserum proteins

Reaction with mouse immunoglobulin isotypes

Clone -yl, K y2a, K y2b, K y3, K , A

53-5 - + - - -10-6 - - + - -80-1 - + + _ _45-9 - + +

The specificity was assayed by ELISA: the plates were coated withmouse immunoglobulin of the indicated isotypes (all obtained fromSigma) at 1 ,ug/ml.

Proc. Natl. Acad. Sci. USA 92 (1995)

Dow

nloa

ded

by g

uest

on

June

5, 2

020

Proc. Natl. Acad. Sci. USA 92 (1995) 9351

701C

Control andRa anti-Ms IgG2b

I-~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ Ju'1

100 101 102 100 101 102 103 100 101 102Fluorescence intensity

FIG. 3. Immunofluorescence labeling of mouse A20 B-lymphoma cells with monoclonal rabbit (Ra) anti-mouse (Ms) IgG2 antibodies. A20 cellswere incubated with the supernatants of IgG-secreting rabbit-rabbit hybridomas (fusion 3) that were shown by ELISA (Table 2) to recognize mouseIgG2a (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 mAbper ml) (C) and none of the other mouse immunoglobulin isotypes. In control samples, A20 cells were incubated with the supernatant of anIgG-secreting rabbit-rabbit hybridoma that recognizes an irrelevant antigen-i.e., a surface antigen of Jurkat cells (fusion 1). As secondary antibodywe used FITC-conjugated goat anti-rabbit L chain.

efficiency for the three fusions performed was between 0.25and 1.2 in 106 cells, which is comparable to the efficiencygenerally obtained in mouse-mouse fusions. Of the hybrid-omas produced in the three fusions, 10%, 23%, and 25%secreted mAbs that were specific for the immunogens (Table1). Again, this percentage of hybridomas that secretes specificmAb is comparable to that obtained in mouse-mouse fusions.The hybridomas have been subcloned and they were frozenand thawed without loss in their ability to secrete mAb. Thesedata indicate that the hybridomas are stable and that frequentcloning, which had been necessary for the heterohybridomas,is not needed for the rabbit-rabbit hybridomas.

Production of IgA-Secreting Hybridomas. Most of thehybridomas from spleen secreted IgG and none were foundthat secreted IgA (Table 3). Because IgA-producing hybri-domas would be valuable reagents we decided to performfusions with cells from Peyer's patch (PP) and mesentericlymph node (MLN) to obtain IgA-secreting hybridomas.From two separate fusions 34% of the hybridomas fromMLN and 81% of the hybridomas from PP secreted IgA(Table 3). Similar results were obtained in two additionalexperiments-i.e., 35% of the hybridomas from MLN and38% of the hybridomas from PP secreted IgA. (The otherisotypes were not determined in these experiments.) A high

Table 3. mAbs produced by rabbit hybridomas obtained fromfusions of 240E1-1-2 with mesenteric lymph node (MLN), Peyer'spatch (PP), or spleen cells

No. of clones (% of total clones)

Cells fused Total IgG IgM IgASpleen* 25 25 (100) 0 0MLNt 82 14 (17) 34 (41) 28 (34)ppI 48 2 (4) 2 (4) 39 (81)

*Data from fusion 3, Table 1: 25 of 43 specific mAbs were analyzed.tMLN cells of an unimmunized rabbit were activated by murineCD40-ligand-transfected CHO cells (generously provided by MelanieSpriggs, Immunex Research and Development Corp., Seattle) for 48h prior to fusion. The fused cells were plated in 1000 wells.tPP cells were treated as described above for MLN cells and plated in400 wells.

percentage of IgA-producing hybridomas from MLN, PP,and other mucosal tissues had been found by other investi-gators for rat (15, 17, 18). In contrast, fusion with spleen cellsdoes not generally yield IgA-secreting hybridomas in eithermice or rats, as we now report for rabbits.Concluding Statement. The research described here, de-

tailing our search for a rabbit fusion partner, began in thelate 1970s, before transgene technology was available. Onceinvestigators showed that lymphoid tumors developed intransgenic mice carrying various oncogenes (11, 19), we usedthis technology to develop rabbit plasmacytomas. Our break-through came in 1991 when we found that the myc/abldouble-transgenic rabbits developed plasmacytomas. Sincethen, we have established plasmacytoma cell lines and wereable to develop one into a usable fusion partner. Theavailability of a rabbit fusion partner provides us with theopportunity to produce mAbs specific for mouse antigensand also for antigens or epitopes that are not immunogenicin mice. Such mAbs will be useful in diagnosis of diseases andtreatment of patients.

This work was supported by Public Health Service Grant Al 11234.

1. Krause, R. M. (1970) Adv. Immunol. 12, 12-29.2. Bystryn, J., Jacobsen, S. J., Liu, P. & Heaney-Kieras, J. (1982)

Hybridoma 1, 465-472.3. Weller, A., Meek, J. & Adamson, E. D. (1987) Development

(Cambridge, U.K) 100, 351-363.4. Norrby, E., Mufson, M. A., Alexander, H., Houghton, R. A. &

Lerner, R.-A. (1987) Proc. Natl. Acad. Sci. USA 84, 6572-6576.5. Raybould, T. J. G. & Takahashi, M. (1988) Science 240,1788-1790.6. Yarmush, M. L., Gates, F. T., III, Weisfogel, D. R. & Kindt, T. J.

(1980) Proc. Natl. Acad. Sci. USA 77, 2899-2903.7. Yarmush, M. L., Gates, F. T., III, Dreher, K. L. & Kindt, T. J.

(1981) J. Immunol. 126, 2240-2244.8. Dreher, K. L., Sogn, J. A., Gates, F. T., III, Kuo, M. & Kindt, T. J.

(1983) J. ImmunoL 130, 442-448.9. Kuo, M., Sogn, J. A., Max, E. E. & Kindt, T. J. (1985) Mol.

Immunol. 22, 351-359.10. Verbanac, K M., Gross, U. M., Rebellato, L. M. & Thomas,

J. M. (1993) Hybridoma 12, 285-295.11. Rosenbaum, H., Harris, A. W., Bath, M. L., McNeall, J., Webb,

E., Adams, J. M. & Cory, S. (1990) EMBO J. 9, 897-905.

Immunology: Spieker-Polet et aL

Dow

nloa

ded

by g

uest

on

June

5, 2

020

9352 Immunology: Spieker-Polet et al.

12. Knight, K L., Spieker-Polet, H., Kazdin, D. S. & Oi, V. T. (1988)Proc. Natl. Acad. Sci. USA 85, 3130-3134.

13. Sethupathi, P., Spieker-Polet, H., Polet, H., Yam, P., Tunyaplin,C. & Knight, K. L. (1994) Leukemia 8, 2144-2155.

14. Mishell, B. B. & Shiigi, S. M. (1980) Selected Methods in CellularImmunology (W.H. Freeman, San Francisco), pp. 354-367.

15. Oudghiri, M., Seguin, J. & Deslauriers, N. (1987)Adv. Exp. Med.Biol. 216B, 1215-1222.

Proc. Natl. Acad. Sci. USA 92 (1995)

16. Goding, J. W. (1986) MonoclonalAntibodies: Principles and Prac-tice (Academic, NY), pp. 82-85.

17. Komisar, J. L., Fuhrman, J. A. & Cebra, J. J. (1982) J. Immunol.128, 2376-2378.

18. Peppard, J. V. & Jackson, L. E. (1987)Adv. Exp. Med. Biol. 216B,1207-1213.

19. Stewart, T., Pattengale, P. K. & Leder, P. (1984) Cell 38,627-637.

Dow

nloa

ded

by g

uest

on

June

5, 2

020