Characterization of Monoclonal Antibodies Directed Against the Envelope Proteins of Feline Leukemia Virus1

(CANCER RESEARCH 44, 3512-3517, August 1984]

Characterization of Monoclonal Antibodies Directed against the Envelope Proteins of Feline Leukemia Virus1

Susan D. Youngren, Alex P. Vukasin, and Fernando de Noronha2

Department of Veterinary Microbiology, New York State College of Veterinary Medicine, Cornell University, Ithaca, New York 14853

ABSTRACT Viral neutralizing antibody is directed against the external protein, gp70, which is distinct from complement-dependent activity (9). Monoclonal antibodies directed against the feline leukemia The envelope of FeLV is composed of a closely associated virus (FeLV) envelope proteins, gp70 and p15E, were identified complex of gp70 and p15E molecules. They appear to exist in by radioimmunoprecipitation and sodium dodecyl sulfate-poly- stable associations of 4 to 6 gp70s disulfide-bonded to a p15E acrylamide gel electrophoresis. Six of these monoclonal antibod (18). Subtyping of FeLV isolates has been carried out by taking ies were specific for the gp70; two for the p15E. Enzyme-linked advantage of the antigenic variation in these external glycopro- immunosorbent assay binding assays against FeLV subtypes A, teins. Isolates have been subtyped A, B, and C on the basis of B, and C showed that most of the monoclonal antibodies bound infectivity interference and serum neutralization assays (21, 22). to more than one subtype but have a greater affinity for subtype Natural distribution and host range of FeLV subtypes (11) indi B. One monoclonal bound exclusively to the FL74 isolate. These cates that variation does exist between isolates of the same studies also indicate that antigenic variability exists between subtype. FeLV isolates previously classified as being the same subtype. In order for a serotherapy program to be effective for field use, One antibody was found to bind the gp70s of all FeLV isolates it must include an efficient means of eliminating infectious circu tested and to be directed against a viral neutralizing site. A p15E- lating FeLV in the animal. Since multiple subtypes are involved specific monoclonal antibody, in addition to binding all the FeLV in the generation of FeLV (and FeSV)-induced diseases, the subtypes, also bound to Moloney and Rauscher murine leukemia administration of a FeLV viral neutralizing antibody, capable of viruses, suggesting a group determinant is involved. No binding reacting with all the subtypes, would be desirable. MAbs directed was seen to human T-cell leukemia virus, bovine leukemia virus, against such a determinant would also provide us with the means equine infectious anemia virus, or RD114V proteins, however. to determine which site(s) on the gp70 molecule is involved in the generation of viral neutralizing antibodies in the cat. INTRODUCTION In order to advance both studies, MAbs directed to the FeLV envelope proteins, gp70 and p15E, have been produced and FeLV3 is a type C retrovirus responsible for a wide range of characterized as to their binding avidity, subtype specificity, and both nonneoplastic diseases, such as immunosuppression and viral neutralizing activity. anemia, and malignant diseases which result in a high feline mortality rate. Lymphosarcomas account for the vast majority of MATERIALS AND METHODS the tumors, most of these being FeLV positive, (for review, see Ref. 10). The replication-defective FeSV, which causes multicen- Virus. FL74 virus obtained from Dr. John S. Cole III (Research Re tric fibrosarcomas in cats, is generated by genetic recombination sources, Biological Carcinogenesis Branch, NIH, Bethesda, MD) and of a FeLV genome with cat cellular oncogenes. The resulting from cells maintained in tissue culture were both used in these studies. virus is a pseudotype, the sarcoma virus genome encapsulated Other FeLV isolates included F422-A and subtypes A, B, and C cloned by helper leukemia virus proteins. The helper virus confers the in feline embryo cell lines (FEA/A, FEA/B, and FEA/C). The RD-114 virus, subtype and infectious properties to the sarcoma virus (19). Moloney-MuLV, equine infectious anemia virus, bovine leukemia virus, and human T-cell leukemia virus were purified from cultures maintained Successful passive immune serotherapy of diseases induced by these viruses has been variable due to the outbred nature of in this laboratory. All the viruses were purified from tissue culture supernatants by a differential centrifugation method described previously the cat. It has been shown that heterologous sera directed (4). The viral pellets were either resuspended in appropriate buffer for against the major external glycoprotein protects cats against immediate use or stored dry in liquid nitrogen. Rauscher-MuLV was FeSV-induced sarcomas and persistent viremia (5, 6). An ab obtained from NIH (Bethesda, MD). sence of efficient humoral response, in the presence of a strong Virus Labeling. FL74-FeLV was labeled in vivo with [3H]leucine by cellular response in fibrosarcoma rÃ©gressercats that retain a suspending the cells in leucine-free McCoy's media (Grand Island Biolog persistent viremia (7), suggests the importance of a multi-fold ical Co.) containing [3H]leucine (25 ^Ci/ml; 130 to 190 Ci/mmol; Amer- approach to eliminating the diseases caused by these viruses. sham) supplemented with 5% fetal calf serum. The cells were maintained for 4 days in culture and the virus purified as described above. For 'Supported by USPHS Contract N01-CP91007 from the Division of Cancer immunoprecipitations, the labeled virus was lysed in 0.01 M Tris-HCI, pH Cause and Prevention, National Cancer Institute. 7.4, 1 M KCI, 10 HIM disodium EDTA, 0.03 M /i-2-mercaptoethanol, 1% 2 To whom requests for reprints should be addressed. Triton X-100 for 30 min at 37Â°. 3The abbreviations used are: FeLV, feline leukemia virus; FeSV, feline sarcoma virus; gp70, a glycoprotein with a molecular weight of 70,000; p15E, a viral Fusion Procedure for the Production of Hybridoma Cell Lines. On glycoprotein with a molecular weight of approximately 15,000; MAb, monoclonal Day 1, 3-month-old BALB/c mice were inoculated i.p. with undisrupted antibody; PBS, phosphate-buffered saline [0.01 M Na2HP04:NaH2PO4 (pH 7.0):0.01 FL74-FeLV suspended in a 1:1 dilution of PBS and complete Freund's M NaCIJ; ELISA, enzyme-linked immunosorbent assay; BSA, bovine serum albumin; adjuvant. Each mouse received 50 Â¿murine leukemia virus; SDS-PAGE. sodium dodecyl sulfate-polyacrylamide subsequent injections contained the same w/v ratio. The inoculations gel etectrophoresis; FEA, feline embryo cell line. were repeated on Day 15 using incomplete Freund's adjuvant and on Received September 9,1983; accepted April 30,1984.

3512 CANCER RESEARCH VOL. 44

7.2, 2% Triton X-100, and 2 x 10~4 M toluene sulfonyl fluoride, and Day 30 with only sterile PBS. Approximately 60 days later, the mice were given Â¡.p.and i.v. injections of a virus suspension. The spleen was sonicated at high frequency for 15 sec (Model 185F; Heat Systems removed 4 days later and the cells teased out in 10 ml of Dulbecco's Ultrasonics). Undisrupted virions were removed by centrifugation at 30K modified Eagle's medium (Grand Island Biological Co.). The cells were rpm/30 min/4Â°(Beckman type 30 rotor). The detergent was removed by washed twice in Dulbecco's modified Eagle's medium and then resus- ether extraction, and the protein concentration was determined by the pended in the same plus 1.5 x 107 P3x63 Ag8 myeloma cells. This method of Lowry ef al. (14). Disrupted virus at 1 to 2 ^g/100 ^ were mixture was pelleted and resuspended gently in 1 ml of 50% w:v (J. T. incubated overnight at 4Â°in carbonate buffer, pH 9.6, for absorption to Baker) polyethylene glycol 1500 in Dulbecco's modified Eagle's medium. Immulon wells (Dynatech Laboratories, Inc.). The wells were subse The volume was slowly brought to 25 ml with complete hybridoma media quently coated with 5% BSA (Calbiochem) and stored at 4Â°until use. at 37Â°and allowed to incubate at this temperature for 1 hr. The cells Antibody binding was carried out overnight at 4Â°in Buffer 3 (50 mw Tris- were then pelleted at 800 rpm for 5 min, resuspended in 100 ml of HCI, pH 7.4, 1 rnw disodium EDTA, 150 mw NaCI, 0.1% BSA, 0.05% complete hybridoma media supplemented with hypoxanthine, aminop- Tween 20). After a washing with PBS plus 0.02% Tween 80, rabbit anti- terin, and thymidine (Sigma), and distributed into 96-well tissue culture mouse IgG peroxidase (100 Ml/well; Sigma) were added, and the plates plates (Costar) containing a fresh prepared feeder layer of mouse thy- were incubated at room temperature for 2 hr. The colorimetrie reaction mocytes and macrophages. After 5 days, two-thirds of the media was was initiated by the addition of 150 ^ of substrate buffer (o-phenylene- removed, and fresh complete media plus hypoxanthine and thymidine diamine in 0.025 M citric acid, 0.05 M sodium phosphate, and 0.04% was added. Viable hybrid cell clones were tested for antibody production H2O2) and terminated after 20 min by the addition of 150 /il of 2.5 M by ELISA using disrupted FL74-FeLV. Cell lines producing antibody H2SCv against FeLV were expanded and cloned 3 times on agar semisolid Absorbance was read in a Gilford Staser II spectrophotometer at 492 media. nm, a background absorbance being obtained from wells coated only Production and Purification of MAbs. Magnified hybridoma lines were with BSA. In order to ensure detection of binding to the various viral maintained in hybridoma media at 37Â°in a 5% CO2 buffered incubator. subtypes, the MAbs were used at a concentration giving an approxi Antibody was recovered from tissue culture media by precipitation with mately 1.0-A reading for the FL74 virus wells. 50) (v/v) ammonium sulfate at 4Â°.The precipitate was resuspended and Assays for Viral Neutralization. Viral neutralizations were performed dialyzed overnight versus PBS at 4Â°.The IgG was then purified using a as described previously using C81 sacroma virus-positive leukemia virus- Protein A-Sepharose column (Pharmacia) equilibrated with 0.05 M Tris- negative cells or CCL64 cells (4, 8). Filter-sterilized MAbs were added to HCI, pH 8.3, and 0.15 M NaCI. Bound antibody was eluted with 0.05 M the virus at 0.1 mg/ml, incubated at room temperature for 1 hr, and then sodium citrate-citrate, pH 3.5, plus 0.15 M NaCI, and adjusted immedi inoculated onto Petri dishes containing the appropriate indicator cells. ately to the proper pH using 1.0 M Tris-HCI, pH 8.2. Antibody was also collected by injecting 1 x 107 hybridoma cells i.p. into BALB/c mice that had been treated 10 to 21 days previously with RESULTS pristane. Seven to 10 days later, 0.5 to 5.0 ml of ascitic fluid were extracted from the peritoneal cavity of each mouse. The ascitic fluid was Identification of Monoclonal Reactivities. Viable hybridoma centrifuged for 30 min at 30,000 to remove cell debris. cells were initially selected which produced sufficient titers of Immunoglobulin Typing. The MAbs were screened for their immuno- antibody to obtain a positive binding in an ELISA with coupled globulin class and subtype by agar gel diffusion for 24 to 48 hr at room FL74-FeLV antigens. After 3 successive clonings to ensure pure temperature in 0.5% agarose in PBS. Rabbit anti-mouse sera were hybridoma lines, the antigenic reactivities were identified by obtained from Litton Bionetics. radioimmunoprecipitation using 3H-labeled FL74 virus. Radioimmunoprecipitation and SDS-PAGE. A modification of the immunoprecipitation techniques of Chen ef al. (3) was used. After disrup Heterologous seras raised against one or more of the FeLV tion, the virus was cleared for nonspecific reactivities with normal mouse proteins were used to establish the conditions for immunoprecip sera; 2 x 105 cpm of labeled virus were then combined with 10 to 20 ^ itation assay (Fig. 1). Multispecific goat sera precipitates all the of either purified MAb or ascites fluid and 100 ^l of 10% (w/v) Protein A- viral proteins. It is clear from the results that monospecific sera Sepharose (Sigma Chemical Co, St. Louis, MO) in Buffer B (0.01 M Tris- raised against the major glycoprotein, gp70, also precipitate the HCI, pH 7.4, 0.1 M KCI, 1 HIM EDTA Na2,1% Triton X-100, 0.5% sodium p15E molecule. This is most probably due to the presence of deoxycholate). All reactions were incubated overnight at 4Â°and then gp70-p15E complexes in the inoculum, since these are difficult extensively washed in Buffer B. Samples were prepared for analysis in to completely dissociate. When goat anti-p10 sera was tested, 5 to 20% sodium dodecyl sulfate polyacrylamide gels by dissociation in all the low-molecular weight proteins of FeLV were seen. In reducing and nonreducing buffers (12). Under nonreducing conditions, contrast to the anti-gp70 sera, these results are due to the the /S-2-mercaptoethanol was excluded from the buffer prior to boiling. The gels containing tritiated virus were treated with EN3HANCE (New difficulty in separating proteins that have molecular weights England Nuclear) prior to drying and exposed against Kodak XAR X-ray within a small range. film for 10 to 14 days; 14C- labeled molecular weight markers were Eight of the hybridoma lines produced MAbs which precipi included in the gels. tated the external FeLV proteins, gp70 and p15E (Table 1). Six Western Blot Procedure. Unlabeled FL74-FeLV proteins were sepa of these MAbs bound gp70: (a) 6G3; (b) 1C1; (c) 4B1; (d) 6D4; rated by SDS-PAGE and transferred to nitrocellulose (0.2 /Â¿m;Schleicher (e) 8F3; and (f) 7B3. 1C12 and 6B2 are unique to the p15E 6 Schuell) in 20 HIM NaPB, pH 6.5. After transfer, the filters were soaked molecule. Radioimmunoprecipitations of 6G3, 1C12, and 6B2 in 50 mw Tris-HCI (pH 7.4), 0.15 M NaCI, 5 mw disodium EDTA, 0.25% are shown in Fig. 2. M101 is the mouse anti-FeLV sera obtained gelatin, 0.05% Triton X-100 buffer. Sera or the MAb-supplemented buffer from the inoculated mice prior to the removal of the spleen. described above was then placed on the filters for a minimum of 4 hr at room temperature. After extensive washing, peroxidase-labeled antibody Pinter and Fleissner (17) found that about 10% of the gp71 in intact virions of MuLV were in a disulfide-linked complex with was added, and incubation carried out for 2 hr at room temperature. The colorimetrie reaction was performed using 4-chloro-1-naphthol and p15E. In FeLV, this envelope complex can be seen on nonred stopped with distilled water. ucing SDS-PAGE at approximately 90,000. Due to this strong Binding Studies by ELISA. The initial screening of the hybridomas, interaction of these molecules, it is difficult to dissociate all the comparative avidity, and viral subtype binding assays were carried out complexes prior to immunoprecipitation. To delineate the binding by ELISA. Purified viral pellets were suspended in 0.05 M Tris-HCI, pH characteristics of several MAbs, it was necessary to dissociate

AUGUST 1984 3513

O m 04 o Ã• 8 5 co i u- Ã¶fl GO cv C9 CU

70 p15E>

Fig. 2. SDS-PAGE analysis of 3H-labeled FeLV proteins precipitated by MAbs 6G3,1C12, and 6B2. M101 is mouse anti-FeLV sera which precipitates all the viral proteins. Location of viral proteins gp70 (M, 70,000) and p15E (M, 16,000) are noted.

15= 12,1,10:

Fig. 1. Immunoprecipitation of 3H-labeled FeLV proteins using goat sera raised against viral proteins. Left, molecular weights (x 10"3).

Table 1 Characterization ol MAbs sub MAb6G3 antigengp70 classIQQ1igGi

1C14B1 9P70 gp70 igGi 6D4 gp70gp70 igGi 8F3 lgG1 7B3 gp70p15E IgGi 6B2 igGi 1C12FeLV p15EImmunoglobulin Ig2a

and electrophoretically separate the gp70 and pi 5E molecules prior to antibody binding. Fig. 3 shows the results of a Western blot carried out to determine against which envelope proteins 8F3 and 7B3 were directed. It is clear that both of these mono- Fig. 3. Western blot analysis of the reactivities of MAbs GG3, 7B3, and 8F3. clonals bind the gp70 molecule. No difference in the radioimmu- Normal goat sera (NGS) and goat anti-FeLV (G101) were used as controls. The location of the gp70 (M, 70,000) viral envelope protein is indicated. noprecipitation patterns of the monoclonals was seen using either hybridoma cell supernatant or Protein A-chromatography- purified MAbs. All the antibodies discussed here were 761 except 1C12, which The results of 7-class typing are also presented in Table 1. was 7G2a. Rabbit anti-mouse sera directed against specific globulin classes Relative Avidities of the MAbs to FL74-FeLV Antigens. were used to type the MAbs in agarose immunodiffusion plates. Serial dilutions of the Protein A-purified monoclonals were tested

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labte 2 or subtype C. Comparativeavidities at MAb (0.01 mg/ml) to FL74-FeLV" In contrast to the other MAbs, which react well to at least one MAb1C126G34B18F36D47B31C16B2Absorbance*2.882.602.562.101.670.770.200RelativeavidityHighHighHighMiddleMiddleLowLowLowof the subtypes, 8F3 shows specificity for a unique FL74 deter minant. In order to further characterize 8F3, feline fibroblasts (FEA cells) were infected with either the FL74 or F422 isolates of FeLV. Virus produced by these cells was used in binding assays against 8F3 and the other MAbs described here. We saw no difference in the binding results. 8F3 again bound only the FEA/FL74 virus but not the virus produced by the FEA/F422 Virus (1.0 ng) absorbed to reaction wells. cells. No binding studies were carried out using the infected Measured at 492 nm. cells. These binding studies indicate that antigenic variability exists against a constant quantity of FL74 virus by ELISA, the absorb- between FeLV isolates previously characterized and designated ance of the reaction plotted versus the MAb concentration, and A, B, or C. The other assays used to type these isolates have on the basis of the plateau regions of the titration curves, a been based upon antigenic sites necessary for infectivity and single concentration of MAb (1 ^g/IOO n\) was taken to compare viral neutralization. Antigenic variability between isolates used binding strength. Table 2 shows the relative binding strengths of here does not seem unreasonable due to the specific nature of the 8 monoclonals. A wide range in binding strength was seen the MAbs. which was not dependent upon the target protein(s). The highl In addition to the FeLV-isolates, these monoclonals were and mid-range avidity MAbs consistently bound well in all our tested against several heterologous virus isolates. As can be assays. Since a large quantity of virus was bound to the ELISA seen in Table 4, only 3 MAbs showed interspecies reactivities, wells, and a wide range of antibody dilutions used, we feel that and only then to murine isolates. Previous immunological studies these results do not reflect the number of binding sites available using multispecific sera have indicated homologies between on the viral proteins. Due to the low values for 7B3, 1C1, and FeLV and MuLVs (23). No reactivity was seen to the endogenous 6B2, much higher concentrations were used when these mono feline virus RD114, to equine infectious anemia virus, bovine clonals were tested against the FeLV subtypes. leukemia virus, or to human T-cell leukemia virus. MAbs not The fact that the monoclonals were first purified by Protein A shown in Table 4 reacted only with FeLV, as is seen with 1C1. chromatography from cell-free hybridoma supernatants raised Viral Neutralizing Assays. Viral neutralizing activity was as the question as to whether the concentrations of protein repre sessed for each of the MAbs against the FeLV subtypes by foci sented only mouse IgG. A small constituent of the fetal bovine formation on CCC81 (sarcoma virus-positive leukemia virus- serum used in tissue culture is capable of binding to Protein-A negative) and CCL64 mink cells as previously described for and therefore may be present in these samples. To test this, an multispecific sera (7). Tissue culture-purified MAbs were used equivalent volume of hybridoma media containing fetal bovine for these studies, since murine ascites fluid produced a significant serum was passed through a Protein A-Sepharose column used for MAb purification, the bound bovine component eluted and measured. An insignificant amount of IgG was found to be Table 3 present in the eluant which would not alter the results described Determination of FeLV subtype specificities by ELISA above. ^g/wellFL741.75s at 1.0 FeLV Subtype and Viral Group Specificity. In order to deter anti MAb6G3 gengp70 mine whether these MAbs were directed against subtype spec ificities on the gp70 and p15E molecules, ELISA assays were 1C1 gp70 0.89 0.15 0.50 0.24 0.57 carried out using several FeLV isolates. These included FL74, 4B16D4 gp70 1.43 0 1.44 0.46 0.19 containing subtypes A, B, and C; virus purified from cloned FEAs gp70 0.80 0 0.84 0.14 0 8F3 gp70 0.94 0 0 0 0 producing each of the subtypes and the F422 cell line, producer 7B3 gp70 0.64 0 0.21 0.07 0 of type A FeLV. As can be seen from the results presented in 6B2 p15E 0.88 0.06 0.70 0.24 0.61 Table 3, all the monoclonals bound to FL74 viral proteins at a 1C12FeLV p15EVirus 1.80FEA/A0.050.30FEA/B0.690.92FEA/C0.080.45F4220.811.05 greater efficiency than to any of the subtypes. We found this to Absorbance values at A,M. be unusual, since we would have expected the binding to be much stronger against the same quantity of a purified subtype. Table4 Only 1C1 (anti-gp70) and 1C12 (anti-p15E) bound all the FeLV Interspeciesbinding by FeLV-directedMAbs isolates strongly. Weak binding for several of the MAbs was MAb Virus"FL74RD114M-MuLVR-MuLV consistent and above background wells coated only with BSA. 1C12+" 1C1 6B2 6G3 can be seen to interact with the gp70s of all the subtypes, +â€” + + although only weakly to FEA/A and FEA/C. Other weak inter _â€” â€” _ actions exist between 7B3 and FEA/C and F422/A, and between +! â€” â€” 6B2 and FEA/A. When the results of the binding to FEA/A and EIACBLVHTLV6G3 !â€” :! F422/A isolates are compared, we must conclude that even _- â€” _ though previous tests have indicated they are the same subtype, " Lysed virus (2 Â¿ig)absorbedto reaction wells. antigenic variation does exist between their gp70 molecules. In " +, a0.10-A A450; -, <0.10-A A450. all cases, stronger binding was seen to subtype B FeLV, whereas c EIA, equine infectious anemia; BLV, bovine leukemia virus; HTLV, human T- none of the MAbs showed specific reactivity to either subtype A cell leukemiavirus.

AUGUST 1984 3515

Tables to neither F422-FeLV nor FeLV-C. We have not carried out Viral neutralizing activity of FeLV binding MAbs binding studies using the infected cells to determine if this epitope % of neutralization is expressed on the cell surface. The possibility that this deter MAb6G3ICI4B16D48F37836B21C12FL74082.3000000FEA/A098.1000000FEA/B065.598.50050.500FEA/C062.582.100000ST-FeLVNT*90.8NTNTNTNTNTNTST-FeSV"NT37.8NTNTNTNTNTNTminant is expressed on the cell but not on mature particles of these cells cannot be ruled out. RNA-fingerprinting studies of several FeLV isolates have indi cated that genomic differences occur not only between the subtypes A, B, and C but also between the Glasgow-1 and Rickard subtype A isolates (20). Our ELISA assays also suggest that antigenic variability exists between 2 independent subtype * Titrated on CCL64 cells. A FeLVs. At the present time, we do not know the location of 6 NT, not tested. these variable regions. Studies by BrÃ¼ckefal. (1), indicating that antigenic variation may occur between bovine leukemia virus background neutralizing activity. The results of the neutralization isolates from different cell lines also lends support to our results. assays are presented in Table 5. Neutralization values of less Of special interest are the 2 MAbs that bind to all the FeLV subtypes tested; 1C12 (anti-p15E) and 1C1 (anti-gp70). 1C12, than 15% were not reproducible and were therefore considered which also binds to M-MuLV and R-MuLV in our ELISA assays, negative. Three MAbs, 6G3, 6D4, and 8F3, showed no neutral izing activity against the FL74 isolate or subtypes A, B, and C. shows no reactivity to other retroviruses tested thus far. In his studies on MuLV, Lostrum ef al. (13) found an anti-p15E mono 4B1 displayed a very strong neutralizing activity to both B clonal that was reactive with FeLV but not RD-114 virus. It would (98.5%) and C (82.1%) FeLVs, correlating well with the binding data. 7B3 showed only weak activity to type B, and complete be interesting to determine whether these 2 monoclonals are loss of viral infectivity was never seen. directed against the same region of these molecules. MAb 1C1, MAb 1C1, which bound to all the FeLV subtypes tested, also even though of low binding strength, is directed against a viral prevented foci formation by all the isolates. In addition to the neutralizing site on all the FeLV isolates tested to date. Other previously described FeLV isolates, we attempted to neutralize investigators, using MAbs directed against the external glyco the infectivity of Snyder-Theilen FeLV and FeSV. The isolate proteins of retroviruses, MMTV (15), MuLV (16), and bovine used here contained a FeSV:FeLV ratio of 1:50. Essentially leukemia virus (2), have indicated that distinct sites exist on complete neutralization of infectious FeLV was observed. This these molecules that are involved in viral neutralization. It has was not the case with FeSV, however, most probably due to the been proposed that the antibody binds close to or directly upon 50-fold excess of FeLV in the reaction. the site of the glycoprotein that is necessary for cell receptor recognition prior to infectivity. Their evidence supports a mech anism of stehe hindrance. DISCUSSION We are in the process of finishing competition assays with these monoclonals to determine if they share antigenic determi Eight MAbs raised against FL74-FeLV have been shown to nants, and shortly will begin mapping the sites on the FeLV have reactivities to the external glycoproteins of the virus by gp70 and p15E molecules to which these monoclonals corre radioimmunoprecipitation and SDS-PAGE. Six bind to the gp70 spond. Additional MAbs are being produced and characterized alone; 2 bind to the p15E molecule. so that these studies may be more extensive. Localization of A wide range of binding strengths exist between these MAbs viral neutralizing sites on the FeLV gp70 will be invaluable, which is not dependent upon the target viral antigen or immuno- advancing the production of vaccines containing only the portion globulin subclass. FeLV subtype specificity of the MAbs was of the molecule necessary to stimulate an immune response in directed predominantly against the B determinants, although in the animal. most cases weaker interactions did exist with the other sub MAbs to the external proteins of FeLV will be useful in both types. The binding studies do reveal antigenic variability between the diagnosis and serotherapy of FeLV- and FeSV-infected ani FeLVs obtained from different cell lines. Previous subtyping mals. High-avidity antibodies can be used immediately as very techniques have focused on the antigenic determinants involved specific and sensitive probes for detection of FeLV in cat sera. in viral infectivity and neutralization, while many of our mono- In the past, we have shown that viral neutralizing activity is clonals are specific for nonneutralizing sites. present in the sera of experimental regressor cats that become The unique nature of the determinant recognized by 8F3 raises virus-free. It is our hope to augment the humoral antibody the possibility that this MAb is directed against an epitope on response to FeLV and FeSV in infected animals and are currently the feline oncornavirus-associated cell membrane antigen. Early preparing a set of serotherapy experiments using several of studies suggested that this was a tumor antigen on feline lym- these MAbs. In addition to these, anti-fes MAbs kindly supplied phosarcoma and fibrosarcoma cells (for a review, see Ref. 24). by Dr. Fred Reynolds, and described elsewhere (27), will be used Recent work by Vedbrat ef al. (26) and Snyder ef al. (25) indicates to treat developing fibrosarcomas. We do not expect that the that the feline oncornavirus-associated cell membrane antigen findings from the first serotherapy will be conclusive, since reactivity in feline sera is directed against a polymorphic group tremendous variability exists in the dosage and timing of antibody of epitopes that are related to, but distinct from those on the administration to infected animals. These monoclonals have a gp70 of FeLV-C. MAb 8F3 does not appear to be directed against great potential beyond the current serotherapy studies. Tagged a feline-oncornavirus-associated cell membrane antigen compo with radioactive tracers, the MAbs may be used for localization nent as it binds to virus produced by both FL74 cells and feline of viral infected and/or dispersed tumorigenic cells. Immunotox- fibroblasts infected with FL74-FeLV (FEA/FL74). This MAb binds ins, such as ricin, which has already been used in other studies

3516 CANCER RESEARCH VOL. 44

(for a review, see Ref. 28), could be coupled to a specific 14. Lowry, O. H., Rosebrough, N. J., Farr, A. L., and Randall, R. J. Protein measurement with Folin phenol reagent. J. Biol. Chem., 793: 265-275, 1951. antitumor monoclonal, alleviating the need to administer high 15. Massey, R. J., and Schochetman, G. Topographical analysis of viral epitopes doses of toxic material to an animal. using monoclonal antibodies: mechanism of virus neutralization. Virology, 775: 20-32, 1981. 16. Nowinski, R. C., Pickering, R., O'Donnell, P. V., Pinter, A., and Hammerling. U. Selective neutralization of ecotropic murine leukemia virus by monoclonal REFERENCES antibodies: localization of a site on the gp70 protein associated with ecotrop- ism. Virology, 777: 84-92, 1981. 1. BrÃ¼ck,C., Mathot, S., Portetene, D., Berta, C., Franssen, J. D., Herion, P., and 17. Pinter, A., and Fleissner, E. Structural studies of retroviruses: characterization Burney, A. 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AUGUST 1984 3517

Downloaded from cancerres.aacrjournals.org on September 30, 2021. © 1984 American Association for Cancer Research. Characterization of Monoclonal Antibodies Directed against the Envelope Proteins of Feline Leukemia Virus

Susan D. Youngren, Alex P. Vukasin and Fernando de Noronha

Cancer Res 1984;44:3512-3517.

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