Eur. J. Immunol. 1992. 22: 3045-3048
An anti-idiotypic TNF agonist
3045
Short paper Cynthia J. Galloway, Melanie S. Madanat, Timothy SamA, Terje EspevikO, Michael L. Dumas, George Mitra and Gerald E. RangesA Miles, Incorporated, (Inc.) Berkeley, Miles, Inc.A,West Haven, University of TrondheimO, Trondheim
Anti-tumor necrosis factor receptor and tumor necrosis factor agonist activity by an anti-idiotypic antibody Tumor necrosis factor (TNF) is a cytokine which, among other properties, is a principle mediator of inflammation and septic shock. It acts upon target cells by binding to specific cell surface receptors. AlOGlO is a murine monoclonal antibody which recognizes human TNF and neutralizes its activity. A rabbit polyclonal antibody directed at the antigen-binding site of AlOGlO was raised and affinity purified over an AlOGlO column. The resultant anti-idiotypic antibody recognized not onlyAlOGl0 but also bothTNF receptors. It showed TNFagonist activity in two different TNF bioassays, and competed with several anti-TNF receptor monoclonal antibodies and TNF itself for binding to cells. These results represent an example of a method for obtaining antibodies to a ligand-specific receptor in the absence of the receptor itself.
1 Introduction
2 Materials and methods
TNF is a major inflammatory cytokine which has been implicated in a variety of physiological responses. Among these are included anti-tumor activity, cachexia, septic shock, and cell proliferation [l].The consequences of TNF action are mediated by binding to two distinct cellular receptors called TNF receptor-a (p75, or utr antigen) or TNF receptor-@ (p55, or htr antigen) [2]. While the mechanism of receptor activation is still unknown, activation leads to multiple signal transduction pathways and activates a large array of cellular genes [3]. Antibodies directed to these receptors have been useful to study receptor expression, regulation, and structure [4, 51.
2.1 Reagents and cell lines
Because the unique abilities of the immune system allow molecular complementation of protein structure, the immune response to an anti-ligand antibody can produce an antibody which mimics the biological activity of the original ligand antigen. Anti-idiotypic antibodies have been used to identify receptors, act as receptor antagonists or agonists, and may also be useful as vaccines [6]. We have raised a rabbit polyclonal antibody which reacts with mouse and human TNF receptors by immunizing rabbits with a mouse IgG, monoclonal antibody directed against human recombinant TNF-a (AlOGlO). The anti-TNF antibody has been shown to neutralize TNF cytotoxic activity on mouse and human cells [7] and to protect baboons from E. coliinduced septic shock [8]. The anti-idiotypic antibody mimics the effects of TNF-a by its ability to kill cells, to compete for binding with other anti-receptor antibodies, and to activate NFxB. These data suggest that the anti-idiotypic antibody (FT2) contains a spatial and molecular similarity to TNF, and furthermore, that the antigen-binding site of AlOGlO may have structural similarity to TNF receptors.
Human recombinant TNF-a (specific activity 2 x lo8 U/mg) was prepared by Bayer AG (Wuppertal, FRG). Antibodies to TNF receptor-a (Utrl) and to TNF receptor6 (Htr5) were gift of Manfred Brockhaus and Werner Lesslauer of Hoffmann-La Roche (Basel, Switzerland). C7F7 is a murine IgGl mAb to human Factor VIII. U937 cells (ATCC no. CRL 1593) were grown in DMEM with 10% FBS. WEHI 164 clone 13 cells (T. Espevik, University of Trondheim, Norway) were grown in RPMI with 10%FBS. SW480 is a human adenocarcinoma cell line (ATCC no. CCL 228). The cell line SW480 pcNEO/P-gal is the result of stably transfecting SW480 with a reporter construct. A promoterless fi-galactosidase fragment was inserted downstream of the CMV promoter. The CMV promoter contains four consensus NFxB-binding sites upstream of the transcription start site. The construct contains the neomycin (G418) resistance gene for selection of transfectants. SW480 cells predominantly express the human TNF receptor-a. 2.2 Rabbit immunizations New Zealand white rabbits were injected intradermally with AlOGlO in complete Freund’s adjuvant. After 1 month, the rabbits were boosted intramuscularly. The rabbits were boosted intravenously with AlOGlO periodically over a l-year time period. 2.3 Purification of FT2
[I 105671 Correspondence: Cynthia, J. Galloway, Miles, Inc., Fourth and Parker Streets, Berkeley, CA 94701. USA
0 VCH Verlagsgesellschaft mbH, D-6940 Weinheim, 1992
Total immunoglubulin was isolated from rabbit serum by precipitation with caprylic acid and ammonium sulfate [9]. Antibodies directed toward all determinants of AlOGlO were isolated from total immunoglobulin by affinity purification over an 8 x 3 cm column of AlOGlO coupled to 0014-2980/92/1111-3045$3.50 + .25/0
3046
Eur. J. Immunol. 1992. 22: 3045-3048
C. J. Galloway, M . S. Madanat,T. Sarr et al.
cells/ml in PBS, 2% BSA, 1mM NaN3) were incubated for 30 min with FT2 or control antibody followed by the addition of either biotinylated recombinant human TNF-a (50 ng/ml) or anti-TNF receptor antibodies and incubated at 4°C for an aditional 30 min or 2 h in the case of TNF. After incubation, the cells were washed twice and analyzed on a FACScan (Becton Dickinson, Sunnyvale, CA).
Hydrazide AvidGel F (Bioprobe International) in PBS, 0.05% NaN3, pH 7.2 buffer and eluted with 0.1 M glycine, pH 2.5, Eluted antibody was neutralized by diluting tenfold into PBS and concentrated using an Amicon (Danvers, MA) PM 30 membrane. Anti-isotypic antibodies were removed by absorption to a column of C7F7 (a mouse monoclonal anti-factor VIII antibody of the same subclass) coupled to Hydrazide AvidGel F. The unbound antiidiotypic antibody (FT2) was pooled, concentrated, and reapplied to the C7M column, then concentrated, dialyzed against PBS, pH 7.2, sterile filtered, and stored at 4°C. Antibody bound to the C7M column, directed to framework determinants of mouse IgG1, was also eluted and used as a control antibody.
3 Results 3.1 FT2 mimics the cytolytic activity of TNF According to the idiotype theory of immune regulation, anti-idiotypic antibodies have the potential to mimic the ligand of the target antibody. Therefore, we tested the ability of FT2 to mimicTNF biological activity. As shown in Fig. 1 a, FT2 was toxic to WEHI 164 cells with an ED50 of 2.0 ng/ml. TNF itself was toxic with an EDSOof 0.2 ng/ml. The lytic activity of FT2 was specifically due to antibody because neither a nonspecific rabbit antibody nor rabbit antibody directed to framework determinants of mouse IgGl were toxic, and because the addition of AlOG10, the antibody to which FT2 was raised, inhibited the cytolytic activity of FT2 (Fig. 1b). Similarly, the cytolytic activity of TNFagainst the human myeloid cell line U937 was imitated by FT2 and this activity was partially inhibited by antibodies to either TNF receptor-a (Utrl) or TNF receptor$ (Htr5) or more completely by a combination of these antibodies (Fig. 2 ) .
2.4 Assays
The cytolytic activity of FT2 and TNF was measured using WEHI 164 clone 13 and U937 cells according to standard protocols [5, 101. The assay for activation of NFxB was carried out as follows. SW480/pcNEO cells were seeded into 96-well microtiter plates (2 x lo4 cells/well) and allowed to adhere. Prior to testing, plates were washed twice with PBS and then 100 yl of medium with or waithout test material was added to cells. After a 4-h incubation period, the medium on the plates was discarded and cells lysed by addition of 0.5% NP40 which also included 3.5 mM o-nitrophenyl galactopyranoside (a 0-galactosidase substrate which turns yellow when acted upon by the enzyme). The reaction was stopped by addition of 15 yl of 1M Na2C03 and the absorbance read at 405 nm.
3.2 FT2 activates NFxB 2.5 FACS analysis
The expression of many genes coding for immunoregulatory proteins is modulated by TNE The activation of the transcription factor, NFxB, by TNF is an intermediate step in the activation of many of these genes. We have created a cell line stably transfected with a reporter gene construct
Recombinant human TNF-a and antibodies to TNF receptors and CD44 (antibody 2.3) were biotinylated according to previously described methods [ l l ] . U937 cells (1 x lo6
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Figure 1 . Cytolytic activity of FT2 and TNFon WEHI 164 cells, loss of absorbance is correlated with cell death. In (a) TNF (V) and FT2 (0)caused the death of WEHI 164 cells. Neither a rabbit potyclonal nonspecific antibody ( 0 )nor an anti-framework antibody (V) had any similar toxic activity. In (b) at a constant toxic concentration of FT2 (1 Kglml), addition of AlOGlO (0)neutralized toxicity, while a nonspecific mouse IgGl ( 0 )antibody did not.
An anti-idiotypic TNF agonist
Eur. J. Immunol. 1992. 22: 3045-3048
3047
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Fzgure 2. FT2 mimicked the toxic effect of TNFon human U937 cells. (a) The toxic activity of FT2 ( 0 )was partially inhibited by either an anti-TNF receptor-i5 antibody (V, Htr5) or an anti-TNF receptor-u antibody (V,Utrl). A combination of both anti-receptor antibodies (0)produced a higher level of protection. (b) Anti-TNF receptor-fi (V) or -a (V)antibodies, or a combination of the two (0) also inhibited TNF ( 0 )toxicity. In bath panels, the final concentration of anti-receptor antibody was 10 pg/ml. Percentage specific lysis is defined in [lO].
composed of a CMV promoter which contains four NKxBbinding sites [12] and a structural gene for @-galactosidase. This cell line responded to both PMA and TNF by displaying [3-galactosidaseactivity. As shown in Fig. 3, FT2 but not a control antibody imitated TNF, demonstrating that the TNF-like activity of FT2 was not restricted to cytolytic activity. It is interesting to note that, in this model PMA stimulation resulted in approximately twice the P-galactosidase activity as TNF and that FT2 stimulated activity comparable to TNF.
1-1
.
TNF+Anti AlOGlO
1-1
Utrl+Anti AlOGlO
11
2.3+Anti AlOClO
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3.3 FT2 competitively inhibits binding of‘TNF receptor-specific antibodies and TNF Biotinylated TNF or biotinylated antibodies to the p55 TNF receptor (Htr5) or p75 TNF receptor (Utrl) were used to label U937 cells in the presence or absence of 30 pg/ml FT2 (anti-AlOG10) and analyzed by the FACScan. Receptor binding by both antibodies as well as TNF itself was
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I;igure 3. Using the NFxB-reporter cell line SW480 pcNEO@-gdl, FT2 ( 0 )caused activation of KKxB. In the same assay, 50 ml of TNF-tx.gave an absorbance value of 0.39. PMA treatment (0.10 nglml) produced an absorbance of 0.75.
Figure4. Anti-AlOG10 (FT2) competed with TNF and antireceptor antibodies for binding to U937 cells. In (a) FT2 inhibited binding of biotinylated TNF. In (b) FT2 inhibited binding of biotinylated anti-TNF receptor-a (Utrl). In (c) FT2 inhibited binding of biotinylated anti-TNF receptor-f3 (Htr5). In (d) FT2 did not inhibit the binding of antibody 2.3 (anti-CD44). An affinitypurified nonspecific rabbit antibody (Kab Ab) did not inhibit binding of TW, anti-receptor antibodies, or antibody 2.3.
3048
C. J. Galloway, M. S. Madanat,T. Sarr et a1
inhibited by the presence of FT2 (Fig. 4). Cell binding by antibody 2.3 (anti-CD44) was not affected by FT2. A control rabbit antibody also had no effect on binding of either TNF or anti-receptor antibodies.
Eur. J. Immunol. 1992. 22: 3045-3048
Although the TNF receptor-a and TNF receptor-fl are distinct gene products, the extracellular regions contain four conserved domains delineated by a repetitive pattern of cysteine residues [2]. Because of the complementary nature of anti-idiotypic antibodies, FT2 most likely interacts with some shared domain of both the TNF receptor-a and TNF receptor-fl.
4 Discussion Received April 10, 1992; in revised form July 16, 1992.
Recently, a number of anti-TNF receptor antibodies have been generated [4,5]. These reagents produce a number of in vitro physiological responses, and both mimic and inhibit TNF action. For example in [4], rabbit polyclonal antibodies raised against the soluble form of TNF receptor are cytotoxic to TNF-sensitive cells, are mitogenic to fibroblasts, and induce prostaglandin synthesis. However, Fab fragments of such antibodies are incapable of these TNFlike activities until cross-linked. Rather than mimicking TNF, the antibodies of Shalaby et al. [ 5 ] , behave as TNF inhibitors. In particular the antibodies inhibit TNF cytotoxicity, inhibit TNF-dependent IL-6 production and inhibit TNF-induced increases in cell adhesiveness. Other anti-idiotypic antibodies which have biological activity have been produced. For example, one of the first anti-idiotypic antibodies had insulin-like activity [13]. Anti-idiotypic antibodies which behave as substance P agonists and antagonists have been produced [14]. The structural basis for the activity of anti-idiotypic antibodies has been studied with smaller non-protein antigens, where it was determined that the antigen binding sites maintain a spatial and molecular similarity, but not necessarily identity, to their original ligand [ 151. Because anti-idiotypic antibodies can mimic their original ligand, anti-idiotypic technology has been used to discover new ligand binding sites which could not otherwise be identified [16], and has been explored as a method of producing vaccines [17, 181. The anti-idiotypic antibody described here mimics several TNF-dependent cell functions. FT2 is toxic to TNFsensitive cells at concentrations of antibody less than 1 yglml. Treatment of cells with FT2 results in activation of NFxB, a known gene transcription factor linked to TNF action [19]. FT2 competes for binding toTNF receptor with antibodies to both the a and fl receptors. These data indicate that FT2 is capable of both binding to TNF receptors and activating TNF-dependent functions. FT2 interacts with both mouse and human TNF receptors. The ligand used to make AlOGlO was human recombinant TNF-a, which has been shown to interact with the mouse TNF receptor-fi [2O, 211 and both human receptors.
5 References 1 Beutler, B. and Cerami, A., Annu. Rev. Immunol. 1989.7: 625. 2 Loetscher, H., Steinmetz, M. and Lesslauer,W., Cancer Cells 1991. 3: 221. 3 Vilcek, J. and Lee, T. H., J. Biol. Chem. 1991. 266: 7313. 4 Engelman, H., Holtrnan, H., Brakebusch, C., Avni, Y S., Sarov, I., Nophar,Y, Hada, E., Leitner, 0. and Wallach, D., J. Biol. Chem. 1990. 265: 14497. 5 Shalaby, M. R., Sundan, A., Loetscher, H., Brockhaus, M., Lesslauer, W. and Espevik, T., J. Exp. Med. 1990. 172: 1517. 6 Kohler, H., Kaveri, S., Kieber-Emmons, T., Morrow,W. J. W., Muller, S. and Raychaudhuri, S., Methods Enzymol. 1989.178: 3. 7 Galloway, C. J., Madanat, M. S. and G. Mitra, J. Immunol. Methods 1991. 140: 37. 8 Hinshaw, L. B.,TeKamp-Olson, P., Chang, A. C., Lee, I? A., Taylor, F. B., Murray, C. K., Peer, G. T., Emerson Jr., T. E., Passey, R. B. and Kuo, G. C., Circ. Shock 1990. 30: 279. 9 McKinney. M. M. and Parkinson, A., J. Immunol. Methods 1987. 96: 271. 10 Espevik, T. and Nissen-Meyer, J., J. Immunol. Methods 1986. 95: 99. 11 Ranges, G. E., Bombara, M. P., Aiyer, R. A., Rice, G. G. and Palladino Jr., M. A., J. Imrnunol. 1989. 142: 1203. 12 Boshart, M., Weber, F., Jahn, G., Dorsch-Hasler, K., Fleckenstein, B. and Schaffner,W., Cell 1985. 41: 521. 13 Sege, K. and Peterson, P. A., Proc. Natl. Acad. Sci. USA 1978. 75: 2443. 14 Couraud, J.-Y., Escher, E., Dornenico, R., Imhoff,V., Rossignol, B. and Pradelles, P., J. Biol. Chem. 1985. 260: 9461. 15 Williams,W.V.,Weiner,D. B., Kieber-Emmons,T. and Greene, M. I . , Trends Biotechnol. 1990. 8: 256. 16 Vaux, D., Tooze, J. and Fuller, S., Nature 1990. 345: 495. 17 Kennedy, R. C., Eichberg, J.W., Lanford, R. E. and Dreesman, G. R., Science 1986. 232: 220. 18 Dalgleish, A. G.,Thomson, B. J., Chanh,T. C., Malkovsky, M. and Kennedy, R. C., Lancet 1987. ii: 1047. 19 Hohman, H.-I?, Brockhaus, M., Baeuerle, P. A., Remy, R., Kolbeck, R. and van Loon, A. P. G. M., J. Biol. Chem. 1990. 265: 22409. 20 Tartaglia, L. A., Weber, R. F., Figari, I. S., Reynold, C., Palladino, Jr., M. A. and Goedell, D.V., Proc. Natl. Acad. Sci. U S A 1991. 88: 9292. 21 Barret, K.,Taylor-Fishwick, D. A., Cope, A. PL., Kissonerghis, A. M., Gray, P.W., Feldman, M. and Foxwell, B. M. J., Eur. J. Immunol. 1991. 21: 1649.
An anti-idiotypic TNF agonist
3045
Short paper Cynthia J. Galloway, Melanie S. Madanat, Timothy SamA, Terje EspevikO, Michael L. Dumas, George Mitra and Gerald E. RangesA Miles, Incorporated, (Inc.) Berkeley, Miles, Inc.A,West Haven, University of TrondheimO, Trondheim
Anti-tumor necrosis factor receptor and tumor necrosis factor agonist activity by an anti-idiotypic antibody Tumor necrosis factor (TNF) is a cytokine which, among other properties, is a principle mediator of inflammation and septic shock. It acts upon target cells by binding to specific cell surface receptors. AlOGlO is a murine monoclonal antibody which recognizes human TNF and neutralizes its activity. A rabbit polyclonal antibody directed at the antigen-binding site of AlOGlO was raised and affinity purified over an AlOGlO column. The resultant anti-idiotypic antibody recognized not onlyAlOGl0 but also bothTNF receptors. It showed TNFagonist activity in two different TNF bioassays, and competed with several anti-TNF receptor monoclonal antibodies and TNF itself for binding to cells. These results represent an example of a method for obtaining antibodies to a ligand-specific receptor in the absence of the receptor itself.
1 Introduction
2 Materials and methods
TNF is a major inflammatory cytokine which has been implicated in a variety of physiological responses. Among these are included anti-tumor activity, cachexia, septic shock, and cell proliferation [l].The consequences of TNF action are mediated by binding to two distinct cellular receptors called TNF receptor-a (p75, or utr antigen) or TNF receptor-@ (p55, or htr antigen) [2]. While the mechanism of receptor activation is still unknown, activation leads to multiple signal transduction pathways and activates a large array of cellular genes [3]. Antibodies directed to these receptors have been useful to study receptor expression, regulation, and structure [4, 51.
2.1 Reagents and cell lines
Because the unique abilities of the immune system allow molecular complementation of protein structure, the immune response to an anti-ligand antibody can produce an antibody which mimics the biological activity of the original ligand antigen. Anti-idiotypic antibodies have been used to identify receptors, act as receptor antagonists or agonists, and may also be useful as vaccines [6]. We have raised a rabbit polyclonal antibody which reacts with mouse and human TNF receptors by immunizing rabbits with a mouse IgG, monoclonal antibody directed against human recombinant TNF-a (AlOGlO). The anti-TNF antibody has been shown to neutralize TNF cytotoxic activity on mouse and human cells [7] and to protect baboons from E. coliinduced septic shock [8]. The anti-idiotypic antibody mimics the effects of TNF-a by its ability to kill cells, to compete for binding with other anti-receptor antibodies, and to activate NFxB. These data suggest that the anti-idiotypic antibody (FT2) contains a spatial and molecular similarity to TNF, and furthermore, that the antigen-binding site of AlOGlO may have structural similarity to TNF receptors.
Human recombinant TNF-a (specific activity 2 x lo8 U/mg) was prepared by Bayer AG (Wuppertal, FRG). Antibodies to TNF receptor-a (Utrl) and to TNF receptor6 (Htr5) were gift of Manfred Brockhaus and Werner Lesslauer of Hoffmann-La Roche (Basel, Switzerland). C7F7 is a murine IgGl mAb to human Factor VIII. U937 cells (ATCC no. CRL 1593) were grown in DMEM with 10% FBS. WEHI 164 clone 13 cells (T. Espevik, University of Trondheim, Norway) were grown in RPMI with 10%FBS. SW480 is a human adenocarcinoma cell line (ATCC no. CCL 228). The cell line SW480 pcNEO/P-gal is the result of stably transfecting SW480 with a reporter construct. A promoterless fi-galactosidase fragment was inserted downstream of the CMV promoter. The CMV promoter contains four consensus NFxB-binding sites upstream of the transcription start site. The construct contains the neomycin (G418) resistance gene for selection of transfectants. SW480 cells predominantly express the human TNF receptor-a. 2.2 Rabbit immunizations New Zealand white rabbits were injected intradermally with AlOGlO in complete Freund’s adjuvant. After 1 month, the rabbits were boosted intramuscularly. The rabbits were boosted intravenously with AlOGlO periodically over a l-year time period. 2.3 Purification of FT2
[I 105671 Correspondence: Cynthia, J. Galloway, Miles, Inc., Fourth and Parker Streets, Berkeley, CA 94701. USA
0 VCH Verlagsgesellschaft mbH, D-6940 Weinheim, 1992
Total immunoglubulin was isolated from rabbit serum by precipitation with caprylic acid and ammonium sulfate [9]. Antibodies directed toward all determinants of AlOGlO were isolated from total immunoglobulin by affinity purification over an 8 x 3 cm column of AlOGlO coupled to 0014-2980/92/1111-3045$3.50 + .25/0
3046
Eur. J. Immunol. 1992. 22: 3045-3048
C. J. Galloway, M . S. Madanat,T. Sarr et al.
cells/ml in PBS, 2% BSA, 1mM NaN3) were incubated for 30 min with FT2 or control antibody followed by the addition of either biotinylated recombinant human TNF-a (50 ng/ml) or anti-TNF receptor antibodies and incubated at 4°C for an aditional 30 min or 2 h in the case of TNF. After incubation, the cells were washed twice and analyzed on a FACScan (Becton Dickinson, Sunnyvale, CA).
Hydrazide AvidGel F (Bioprobe International) in PBS, 0.05% NaN3, pH 7.2 buffer and eluted with 0.1 M glycine, pH 2.5, Eluted antibody was neutralized by diluting tenfold into PBS and concentrated using an Amicon (Danvers, MA) PM 30 membrane. Anti-isotypic antibodies were removed by absorption to a column of C7F7 (a mouse monoclonal anti-factor VIII antibody of the same subclass) coupled to Hydrazide AvidGel F. The unbound antiidiotypic antibody (FT2) was pooled, concentrated, and reapplied to the C7M column, then concentrated, dialyzed against PBS, pH 7.2, sterile filtered, and stored at 4°C. Antibody bound to the C7M column, directed to framework determinants of mouse IgG1, was also eluted and used as a control antibody.
3 Results 3.1 FT2 mimics the cytolytic activity of TNF According to the idiotype theory of immune regulation, anti-idiotypic antibodies have the potential to mimic the ligand of the target antibody. Therefore, we tested the ability of FT2 to mimicTNF biological activity. As shown in Fig. 1 a, FT2 was toxic to WEHI 164 cells with an ED50 of 2.0 ng/ml. TNF itself was toxic with an EDSOof 0.2 ng/ml. The lytic activity of FT2 was specifically due to antibody because neither a nonspecific rabbit antibody nor rabbit antibody directed to framework determinants of mouse IgGl were toxic, and because the addition of AlOG10, the antibody to which FT2 was raised, inhibited the cytolytic activity of FT2 (Fig. 1b). Similarly, the cytolytic activity of TNFagainst the human myeloid cell line U937 was imitated by FT2 and this activity was partially inhibited by antibodies to either TNF receptor-a (Utrl) or TNF receptor$ (Htr5) or more completely by a combination of these antibodies (Fig. 2 ) .
2.4 Assays
The cytolytic activity of FT2 and TNF was measured using WEHI 164 clone 13 and U937 cells according to standard protocols [5, 101. The assay for activation of NFxB was carried out as follows. SW480/pcNEO cells were seeded into 96-well microtiter plates (2 x lo4 cells/well) and allowed to adhere. Prior to testing, plates were washed twice with PBS and then 100 yl of medium with or waithout test material was added to cells. After a 4-h incubation period, the medium on the plates was discarded and cells lysed by addition of 0.5% NP40 which also included 3.5 mM o-nitrophenyl galactopyranoside (a 0-galactosidase substrate which turns yellow when acted upon by the enzyme). The reaction was stopped by addition of 15 yl of 1M Na2C03 and the absorbance read at 405 nm.
3.2 FT2 activates NFxB 2.5 FACS analysis
The expression of many genes coding for immunoregulatory proteins is modulated by TNE The activation of the transcription factor, NFxB, by TNF is an intermediate step in the activation of many of these genes. We have created a cell line stably transfected with a reporter gene construct
Recombinant human TNF-a and antibodies to TNF receptors and CD44 (antibody 2.3) were biotinylated according to previously described methods [ l l ] . U937 cells (1 x lo6
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Figure 1 . Cytolytic activity of FT2 and TNFon WEHI 164 cells, loss of absorbance is correlated with cell death. In (a) TNF (V) and FT2 (0)caused the death of WEHI 164 cells. Neither a rabbit potyclonal nonspecific antibody ( 0 )nor an anti-framework antibody (V) had any similar toxic activity. In (b) at a constant toxic concentration of FT2 (1 Kglml), addition of AlOGlO (0)neutralized toxicity, while a nonspecific mouse IgGl ( 0 )antibody did not.
An anti-idiotypic TNF agonist
Eur. J. Immunol. 1992. 22: 3045-3048
3047
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Fzgure 2. FT2 mimicked the toxic effect of TNFon human U937 cells. (a) The toxic activity of FT2 ( 0 )was partially inhibited by either an anti-TNF receptor-i5 antibody (V, Htr5) or an anti-TNF receptor-u antibody (V,Utrl). A combination of both anti-receptor antibodies (0)produced a higher level of protection. (b) Anti-TNF receptor-fi (V) or -a (V)antibodies, or a combination of the two (0) also inhibited TNF ( 0 )toxicity. In bath panels, the final concentration of anti-receptor antibody was 10 pg/ml. Percentage specific lysis is defined in [lO].
composed of a CMV promoter which contains four NKxBbinding sites [12] and a structural gene for @-galactosidase. This cell line responded to both PMA and TNF by displaying [3-galactosidaseactivity. As shown in Fig. 3, FT2 but not a control antibody imitated TNF, demonstrating that the TNF-like activity of FT2 was not restricted to cytolytic activity. It is interesting to note that, in this model PMA stimulation resulted in approximately twice the P-galactosidase activity as TNF and that FT2 stimulated activity comparable to TNF.
1-1
.
TNF+Anti AlOGlO
1-1
Utrl+Anti AlOGlO
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2.3+Anti AlOClO
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3.3 FT2 competitively inhibits binding of‘TNF receptor-specific antibodies and TNF Biotinylated TNF or biotinylated antibodies to the p55 TNF receptor (Htr5) or p75 TNF receptor (Utrl) were used to label U937 cells in the presence or absence of 30 pg/ml FT2 (anti-AlOG10) and analyzed by the FACScan. Receptor binding by both antibodies as well as TNF itself was
,
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I;igure 3. Using the NFxB-reporter cell line SW480 pcNEO@-gdl, FT2 ( 0 )caused activation of KKxB. In the same assay, 50 ml of TNF-tx.gave an absorbance value of 0.39. PMA treatment (0.10 nglml) produced an absorbance of 0.75.
Figure4. Anti-AlOG10 (FT2) competed with TNF and antireceptor antibodies for binding to U937 cells. In (a) FT2 inhibited binding of biotinylated TNF. In (b) FT2 inhibited binding of biotinylated anti-TNF receptor-a (Utrl). In (c) FT2 inhibited binding of biotinylated anti-TNF receptor-f3 (Htr5). In (d) FT2 did not inhibit the binding of antibody 2.3 (anti-CD44). An affinitypurified nonspecific rabbit antibody (Kab Ab) did not inhibit binding of TW, anti-receptor antibodies, or antibody 2.3.
3048
C. J. Galloway, M. S. Madanat,T. Sarr et a1
inhibited by the presence of FT2 (Fig. 4). Cell binding by antibody 2.3 (anti-CD44) was not affected by FT2. A control rabbit antibody also had no effect on binding of either TNF or anti-receptor antibodies.
Eur. J. Immunol. 1992. 22: 3045-3048
Although the TNF receptor-a and TNF receptor-fl are distinct gene products, the extracellular regions contain four conserved domains delineated by a repetitive pattern of cysteine residues [2]. Because of the complementary nature of anti-idiotypic antibodies, FT2 most likely interacts with some shared domain of both the TNF receptor-a and TNF receptor-fl.
4 Discussion Received April 10, 1992; in revised form July 16, 1992.
Recently, a number of anti-TNF receptor antibodies have been generated [4,5]. These reagents produce a number of in vitro physiological responses, and both mimic and inhibit TNF action. For example in [4], rabbit polyclonal antibodies raised against the soluble form of TNF receptor are cytotoxic to TNF-sensitive cells, are mitogenic to fibroblasts, and induce prostaglandin synthesis. However, Fab fragments of such antibodies are incapable of these TNFlike activities until cross-linked. Rather than mimicking TNF, the antibodies of Shalaby et al. [ 5 ] , behave as TNF inhibitors. In particular the antibodies inhibit TNF cytotoxicity, inhibit TNF-dependent IL-6 production and inhibit TNF-induced increases in cell adhesiveness. Other anti-idiotypic antibodies which have biological activity have been produced. For example, one of the first anti-idiotypic antibodies had insulin-like activity [13]. Anti-idiotypic antibodies which behave as substance P agonists and antagonists have been produced [14]. The structural basis for the activity of anti-idiotypic antibodies has been studied with smaller non-protein antigens, where it was determined that the antigen binding sites maintain a spatial and molecular similarity, but not necessarily identity, to their original ligand [ 151. Because anti-idiotypic antibodies can mimic their original ligand, anti-idiotypic technology has been used to discover new ligand binding sites which could not otherwise be identified [16], and has been explored as a method of producing vaccines [17, 181. The anti-idiotypic antibody described here mimics several TNF-dependent cell functions. FT2 is toxic to TNFsensitive cells at concentrations of antibody less than 1 yglml. Treatment of cells with FT2 results in activation of NFxB, a known gene transcription factor linked to TNF action [19]. FT2 competes for binding toTNF receptor with antibodies to both the a and fl receptors. These data indicate that FT2 is capable of both binding to TNF receptors and activating TNF-dependent functions. FT2 interacts with both mouse and human TNF receptors. The ligand used to make AlOGlO was human recombinant TNF-a, which has been shown to interact with the mouse TNF receptor-fi [2O, 211 and both human receptors.
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