Recombinant 55-kDa Tumor Necrosis Factor ( TNF ) Receptor

The extracellular domain of the 55-kDa TNF receptor (rsTNFRB) has been expressed as a secreted protein in baculovirus-infected insect cells and Chinese hamster ovary (CHO)/dhfrcells. A chimeric fusion protein (rsTNFRB-hy3) constructed by inserting the extracellular part of the receptor in front of the hinge region of the human IgG Cy3 chain has been expressed in mouse myeloma cells. The recombinant receptor proteins were purified from transfected ell culture supernatants by TNFaor protein G affinity chromatography and gel filtration. In a solid phase binding assay rsTNFRB was found to bind TNFa with high affinity comparable  with  the  membrane-bound  full-length receptor. The affinity for TNFB was slightly impaired. However, the bivalent rsTNFRB-hy3 fusion protein bound both ligands with a significantly higher affinity than monovalent rsTNFRB reflecting most likely an increased avidity of the bivalent construct. A molecular mass of about 140 kDa for both rsTNFRB.TNFa and rsTNFRB. TNFB complexes was determined in analytical ultracentrifugation studies strongly suggesting a stoichiometry of three rsTNFRB molecules bound to one TNFa or TNFB trimer. Sedimentation velocity and quasielastic light scattering measurements indicated an extended structure for rsTNFRB and its TNFa and TNFB complexes. Multiple receptor binding sites on TNFa trimers could also be demonstrated by a TNFa-induced agglutination of Latex beads coated with the rsTNFRB-hy3 fusion protein. Both rsTNFRB and rsTNFRB-hy3 were found to inhibit binding of TNFa and TNFB to native 55and 75-kDa TNF receptors and to prevent TNFa and TNFB bioactivity in a cellular  cytotoxicity assay. Concentrations of rsTNFRB-hy3 equimolar to TNFa were sufficient to neutralize TNF activity almost completely, whereas a 10100-fold excess of rsTNFRB was needed for similar inhibitory effects. In view of their potent TNF antagonizing activity, recombinant soluble TNF receptor fragments might be useful as therapeutic agents in TNF-mediated disorders.

The extracellular domain of the 55-kDa TNF receptor (rsTNFRB) has been expressed as a secreted protein in baculovirus-infected insect cells and Chinese hamster ovary (CHO)/dhfr-cells. A chimeric fusion protein (rsTNFRB-hy3) constructed by inserting the extracellular part of the receptor in front of the hinge region of the human IgG Cy3 chain has been expressed in mouse myeloma cells. The recombinant receptor proteins were purified from transfected cell culture supernatants by TNFa-or protein G affinity chromatography and gel filtration. In a solid phase binding assay rsTNFRB was found to bind TNFa with high affinity comparable with the membrane-bound full-length receptor. The affinity for TNFB was slightly impaired. However, the bivalent rsTNFRB-hy3 fusion protein bound both ligands with a significantly higher affinity than monovalent rsTNFRB reflecting most likely an increased avidity of the bivalent construct. A molecular mass of about 140 kDa for both rsTNFRB.TNFa and rsTNFRB. TNFB complexes was determined in analytical ultracentrifugation studies strongly suggesting a stoichiometry of three rsTNFRB molecules bound to one TNFa or TNFB trimer. Sedimentation velocity and quasielastic light scattering measurements indicated an extended structure for rsTNFRB and its TNFa and TNFB complexes. Multiple receptor binding sites on TNFa trimers could also be demonstrated by a TNFa-induced agglutination of Latex beads coated with the rsTNFRB-hy3 fusion protein. Both rsTNFRB and rsTNFRB-hy3 were found to inhibit binding of TNFa and TNFB to native 55-and 75-kDa TNF receptors and to prevent TNFa and TNFB bioactivity in a cellular cytotoxicity assay. Concentrations of rs-TNFRB-hy3 equimolar to TNFa were sufficient to neutralize TNF activity almost completely, whereas a 10-100-fold excess of rsTNFRB was needed for similar inhibitory effects. In view of their potent TNF antagonizing activity, recombinant soluble TNF receptor fragments might be useful as therapeutic agents in TNF-mediated disorders.
* The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "adoertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
We have identified two human TNF receptors of about 75and 55-kDa apparent molecular masses (in the present paper called TNFRa and TNFRP, respectively) by chemical crosslinking with radiolabeled TNFa (24) and by binding of monoclonal antibodies generated against isolates of the receptors (25). Subsequently, both receptors have been purified from HL60 cells and partial amino acid sequences were determined (26). More recently, the cDNAs of TNFRa and TNFRP were isolated by us (27,28) and several other groups (29)(30)(31)(32)(33)(34). The two receptors show similar cysteine-rich motifs in their extracellular domains and belong to a new cytokine receptor gene family which includes the nerve growth factor receptor, CD40, and OX40 antigens (28,35).
Soluble fragments of both T N F receptors have been found to be present in human serum and urine (36)(37)(38)(39)(40). In certain disease states receptor shedding appears to be increased (40,41). Soluble T N F receptors have also been identified in cell culture medium of some transformed cell lines (32,42) and of stimulated polymorphonuclear leukocytes (43). In functional studies the natural T N F receptor fragments have been shown to protect cells from TNFa-induced cytotoxicity (36)(37)(38)(39) and, in a recent report, to prevent TNFa-induced hemorrhagic necrosis of a transplanted Meth A sarcoma in BALB/c mice (40). The TNF-antagonizing effects of the soluble receptor fragments in vitro and in uiuo imply a specific interaction with TNFa and TNFP which might be an important regulatory mechanism of T N F action. In the present work a recombinant soluble form of the 55-kDa TNF receptor (rsTNFRP) was produced in high yields in different eukaryotic expression systems. The rsTNFRP was also expressed as a human IgG C73 fusion protein (rsTNFRP-h73) in myeloma cells. The recombinant receptor molecules were found to bind stoichiometrically to TNFa and TNFP trimers and to neutralize TNF bioactivity in different assay systems.

EXPERIMENTAL PROChDURES
Cell Lines and Reagents-The Spodoptera frugiperda (Sf9) cell line was obtained from American Type Culture Collection (ATCC CRL 1711). The baculovirus Autographa California (AcNP virus) was obtained from M. Summers, Texas A & M University, the Chinese hamster ovary (CHO)/dhfr-cell line from P. Familetti, Hoffmann-LaRoche Ltd., Nutley, NJ, and the WEHI164 (clone 2A3) cell line from J. R. Frey (51). The mouse myeloma cell line J558L was kindly provided by A. Traunecker, Basel Institute of Immunology. The expression vector used to construct the rsTNFRP-hy3 fusion protein was modified from a CD4-immunoglobulin construct obtained from K. Karjalainen and A. Traunecker (44). Recombinant human T N F a and TNFp and mouse T N F a produced in Escherichia coli were kindly provided by W. Hunziker, H.J. Schoenfeld, and E. Hochuli (Hoffmann-LaRoche Ltd.,Basel). Radioiodination of TNFa and TNFP was performed with N a Y and Iodo-Gen (Pierce Chemical Co.) as described (25). For affinity column chromatography TNFn was coupled to CNBr-activated Sepharose 4B (Pharmacia LKB Biotechnology Inc.) according to the guidelines of the manufacturer. Protein G-Sepharose 4 Fast Flow was purchased from Pharmacia. Latex beads (polystyrene microspheres, 0.48 pm diameter) were originally obtained from Polysciences, Inc., Warrington, PA and kindly provided by R. Spinnler and M. Caravatti (Hoffmann-LaRoche Ltd., Basel, Diagnostic Division).
Construction of Vectors, Expression, and Purification-The cDNA encoding the extracellular domain of TNFRp, including the signal peptide, was amplified by the polymerase chain reaction. Unique restriction sites were introduced at both ends of the fragment. In addition, a translational stop codon was introduced behind the last amino acid of the extracellular domain (Thr+"*, numbering according t o Ref. 27). The engineered fragment was cloned into an expression vector for mammalian cells. The plasmid contained the Rous sarcoma virus long terminal repeat and the 3' intron plus the polyadenylation site from the rat preproinsulin gene. The expression cassette was finally inserted into the PvuII restriction site of plasmid pSV2-DHFR. Transfected CHO/dhfr-cells were initially selected by the neomycin analogue (2418 in a-medium containing 200 nmol/ml methotrexate. Thereafter, the concentration of methotrexate was sequentially increased by 2-5-fold increments up to 150 pmol/ml. For expression in the baculovirus system, homologous recombination was used to introduce the amplified cDNA fragment into the genome of the AcNP virus. Sf9 cells were grown a t 27 "C in EX-CELL 400 medium (J. R. Scientific, Woodland, CA) containing 2% fetal bovine serum. Cell culture and viral infection were carried out as described (45). The recombinant viruses were purified by limited dilutions in microtiter plates followed by dot blot hybridization. The rsTNFR6-hy3 fusion protein was constructed by exchanging the CD4 sequence in the pCD4-hy3-4 vector (44) with the TNFRp extracellular domain sequence using Sst restriction sites. This procedure yielded a chimeric protein in which the TNFRp sequence was inserted in front of the hinge region of the human IgG Cy3 chain. J558L mouse myeloma cells transfected with the rsTNFRP-hy3 construct by protoplast fusion were cultured in DHI medium (Dulbecco's modified Eagle's medium/Ham's F-l2/Iscove's modified Dulbecco's medium, 25/25/ 50) supplemented with selenite (20 nM), ethanolamine (20 p~) , insulin (5 pg/ml), human transferrin (6 pg/ml), Primatone RL (2.5 mg/ml), Pluronic F68 (0.1 mg/ml), and 0-2% fetal calf serum (46). Expression of rsTNFRP and rsTNFRp-hy3 was analyzed by a sandwich-type binding assay using radiolabeled or peroxidase-labeled TNFa and the non-neutralizing monoclonal antibody htr-20 (25).
Cell-free supernatants of cell transfectant cultures containing rsTNFRp or rsTNFRP-hy3 were concentrated 5-10-fold by ultrafiltration (molecular mass cutoff of 10 kDa) and clarified by centrifugation and filtration through a 0.45-wm filter. The clear filtrate was applied to a T N F a affinity column (Sf9 and CHO/dhfr-supernatants) or protein G affinity column (J558L supernatants). After extensive washing with phosphate-buffered saline (PBS) the columns were eluted with 100 mM glycine, 100 mM NaCl, pH 2.6, buffer. The fractions containing the recombinant proteins were concentrated and subjected to gel filtration chromatography on TSK3000SW or Superose 12 (Pharmacia) columns with PBS as solvent. The amount of protein was determined by amino acid analysis or BCA assay (Pierce). Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was performed as described (47) using the mini-gel system of Bio-Rad.
Binding Assay and Scatchard Analysis-A 96-well microtiter plate coated with the TNFRP-specific non-neutralizing monoclonal antibody htr-20 (25) was incubated with 10 ng/ml rsTNFRP or rsTNFRP-hy3 in 1% defatted milk powder for 3 h at room temperature. Under these conditions only about 10% of the total binding sites were occupied by the receptor protein as determined from a tiration curve (low density packing). In some experiments the antibody-coated plate was incubated with 3 pg/ml soluble receptor to saturate all receptor binding sites (high or maximum density packing). After washing with PBS the wells were incubated with different concentrations of radiolabeled T N F a or TNFP (1-250 ng/ml) in the presence or absence of a 200-fold excess unlabeled ligand for 16 h at 4 "C. The radioactivity bound to single wells was directly counted in a y-counter. Nonspecific binding was subtracted. Kd values were determined from Scatchard plots.
Quasielastic Light Scattering and Ultracentrifugation Analysis-Quasielastic light scattering experiments were performed with the system ALV-300 (ALV Laservertriebsges m.b.H., Langen, Germany). Samples of 300 gl were filtered through 0.2-pm filters in closed cylindrical quartz cells. The protein concentration was 0.5-1 mg/ml. Correlation functions were analyzed with the program CONTIN (48) that yields a distribution of relaxations. Mean values for the diffusion coefficient D were calculated assuming either an extended, i.e. rodlike (0 moment of the observed distribution) or roughly spherical structure (3rd moment) of the particles.
A Beckman Model E centrifuge with a AnD rotor and a 12-mm double sector Epon cell was used for analytical centrifugation studies. The rotor was run at 56,000 rpm in the sedimentation velocity experiments and a t 24,000 or 11,000 rpm in the sedimentation equilibrium experiments. All runs were performed a t room temperature using aliquots of the solutions investigated by quasielastic light scattering. Relative mole masses were calculated from the observed sedimentation velocities by the Svedberg equation using the mean values of the diffusion coefficients as described above. The partial specific volume of rsTNFR(3 was assumed to be 0.68 ml/g taking into account 30% glycosylation (w/w) (49). Alternatively, the molecular masses were also obtained from the sedimentation equilibrium runs by analyzing the absorption as a function of the square radius (50).
Competitive Inhibition of Ligand Binding to Native TNFRP and TNFRa Holoreceptors-1-2 ng of native TNFRP and TNFRa purified from HL60 cells (26) were spotted to prewetted nitrocellulose membranes. After blocking with a soldtion of 1% defatted milk powder, the membrane was incubated with human radiolabeled TNFn or TNFP (1 pmol/ml) in the presence of different concentrations of rsTNFR0 or rsTNFRP-hy3 for 2 h at room temperature. The membrane was then thoroughly rinsed with PBS and counted in a ycounter.
WEHI164 Cytotoxicity Assay"WEHI164 cells (clone 2A3, kindly provided by J. R. Frey (51)) were cultured in a microtiter plate a t lo4 cells/well in a RPMI-based medium in the presence of human TNFlv or TNFp and different concentrations of rsTNFRp or rsTNFRB-hy3 for 48 hours at 37 "C. Cell viability was determined by a dye uptake method as described earlier (9). Agglutination of Latex Beads-5 mg of Latex beads washed with PBS, pH 5.0, buffer and H20 were incubated with 250 pg of rsTNFRp-hy3 in 0.5 ml of PBS, pH 5.0, overnight at 4 "C on a rotating wheel. The beads were then treated with a solution of 1% defatted milk powder to block any remaining binding sites and washed with PBS buffer. T o induce agglutination the beads were suspended at 0.2-1.0 mg/ml in PBS, pH 7.4, containing 0.1 mg/ml bovine serum albumin and 0.1% NaN3. Human TNFa was added a t different concentrations and after overnight incubation a t room temperature agglutination was analyzed in a light microscope a t X 400 maginfication.

Expression, Purification, and Ligand Binding Affinities of rsTNFRP"sf9 insect cells infected with
the recombinant baculovirus secreted 5-10 pg/ml of soluble receptor into the medium after 3-5 days in culture. Transfected CHO/dhfrcells produced up to 30 pg/ml of the recombinant protein after amplification in the presence of increasing methotrexate concentrations. The TNFRP-hr3 fusion protein was expressed and secreted in mouse myeloma cells with a yield of about 0.5-1 pg/ml.
The recombinant soluble TNF receptors were purified by TNFa or protein G affinity chromatography and gel filtration.
SDS-PAGE analysis revealed for the baculovirus expressed protein three to four discrete bands between 21 and 25 kDa.
When virus-infected Sf9 cells were cultured in the presence of tunicamycin, however, a single protein species of 21 kDa was obtained (see Fig. 1) which also was the only TNFareacting band in a ligand blot experiment (not shown). Nterminal sequence analysis of the glycosylated baculovirusproduced material revealed a single sequence starting with Leu+' of the mature TNFRP (not shown). rsTNFRP produced in CHO/dhfr-cells yielded two bands migrating on SDS gels a t around 28 and 32 kDa. Sequence analysis of this material confirmed the expected N terminus, but a second N-terminal sequence starting at Asp+" was also present in a roughly 1:1 ratio. Interestingly, Asp+" has previously been found to be the N terminus of the naturally occurring TNFRP fragment (36). The TNFRB-hy3 fusion protein was expressed as a disulfide-linked homodimer indicating an antibody-like structure of this molecule. As shown in Fig. 1 reduced samples of baculovirus-or CHO/dhfr--derived rsTNFRP migrated at a slightly lower rate on SDS gels. This is most likely due to the high content of cysteines in these proteins. A similar observation has been made earlier with the native 55-kDa TNFRP purified from HL60 cells (26). The soluble receptor fragments produced in either expression system showed a high affinity for TNFa and a slightly lower affinity for TNFP (see Fig. 2). The difference in the apparent K d values of rsTNFRP for TNFa and TNFP was most prominent with the CHO/dhfr--derived material. This finding is in contrast to the native cell surface-bound 55-kDa TNFRP, which has been shown to bind both TNFa and TNFP with about the same affinity, i.e. K d values of 326 and 351 pM, respectively (24,52)). Interestingly, fully deglycosylated rs-TNFRP as expressed in baculovirus-infected Sf9 cells in the presence of tunicamycin displayed similar binding characteristics as the glycosylated form (data not shown), confirming that the carbohydrate moieties are not essential for ligand

FIG. 2. Binding of TNFa and TNFD to rsTNFRD and rs-TNFRB-hy3: binding curves and Scatchard analysis. Binding of ""I-TNFn ( A ) and '2sII-TNFB ( B ) to baculovirus-produced rs-TNFRB (circles), CHO/dhfr--produced rsTNFR0 (squares), and rsTNFRB-hy3 fusion protein (triangles) was measured in a solid phase assay under low density packing conditions (see "Experimental
Procedures"). The K d values were determined from Scatchard analysis of the binding curves as indicated.
binding (24, 26). The apparent affinity of the bivalent rs-TNFRP-hy3 fusion protein for TNFa and TNFP was found to be significantly higher than the affinity of baculovirus-or CHO/dhfr--derived monovalent rsTNFRB (Fig. 2). It is interesting to note that K d values determined in the solid phase assay under high receptor density conditions (see "Experimental Procedures") were generally higher and did not show a marked difference in the apparent affinities between the fusion protein and rsTNFRP (data not shown). It therefore appears that at maximum dense packing of the solid phase some interactions of receptor molecules leading to multiple valency and/or steric constrains cannot be excluded.
Stoichiometry of rsTNFRP. TNFa and rsTNFRB. TNFP Complexes-rsTNFRP purified from CHO/dhfr-cell culture medium was incubated with TNFa or TNFP at different receptor to ligand molar ratios and fractionated according to size by gel filtration chromatography. The chromatographic conditions chosen allowed to separate receptor-ligand complexes from free receptor and free ligand. As shown in Fig. 3, at an approximate 1:l molar ratio neither free receptor nor free TNFa or TNFP could be detected in the elution profiles indicating that under these conditions complete complex formation had occurred. Amino acid composition analysis of the separated complexes evaluated by a recently described computer program (53) confirmed the 1:l stoichiometry (not shown). When the amount of TNFa added was gradually increased, a transition of the TNFRP. TNFa complex toward a slightly lower molecular mass was observed in the elution profile (Fig. 3, left panel). In contrast, adding increasing amounts of TNFP did not affect the elution behavior of the TNFRP.TNF(3 complex (Fig. 3, right panel).
To obtain a more accurate molecular mass estimate of rsTNFRP and its complexes with TNFa and TNFP, quasielastic light scattering and analytical ultracentrifugation studies were performed. The results are summarized in Table I mation (see above) yielded for both complexes a molecular mass of about 140 kDa. If a stoichiometry of three rsTNFRP molecules bound to one 49-kDa TNFa or 57-kDa TNFP trimer (9) is assumed, theoretical molecular masses of 124 and 132 kDa, respectively, are calculated which are in approximate agreement with the observed values. Sedimentation velocity analysis combined with quasielastic light scattering data confirmed the molecular masses observed in the equilibrium runs and were, in addition, indicative for a rather extended, i.e. rod-like structure of rsTNFRP and its TNFa and TNFP complexes.
Inhibition of TNFa and TNFP Binding by rsTNFRP and rsTNFRP-hy3"rsTNFRP and rsTNFRP-hy3 were tested for their ability to competitively inhibit binding of TNFa and TNFP to native TNFRa and TNFRP purified from HL60 cells. In this assay native highly purified receptors were spotted onto nitrocellulose membranes and incubated with '"I-TNFa or '2sI-TNFP in the presence of different concentrations of rsTNFRP or rsTNFRP-hy3. As shown in Fig. 4, A and C, binding of '*'I-TNFa to both TNF receptors was blocked by rsTNFRp and rsTNFRp-hy3 in a concentrationdependent manner. It is interesting to note that a roughly equimolar concentration of the fusion protein was sufficient to prevent TNFa binding almost completely. rsTNFRP was about 10-100 X less potent in inhibiting the binding. The binding of '2sI-TNF~ was also inhibited (Fig.4, B and D), but higher concentrations of rsTNFRP and rsTNFRp-hy3 were needed to achieve inhibitory effects comparable to TNFa. The 10-15% residual binding seen with iodinated TNFP at high soluble receptor concentrations is due to nonspecific binding of radioactivity to the nitrocellulose filter. The inhibition of TNF cytotoxicity by rsTNFRp and rs-TNFRP-hy3 was tested in a cellular cytotoxicity assay using the 2A3 subclone of the murine fibrosarcoma cell line WEHI164 (51). As expected from the binding studies, rs-TNFRP-hy3 very efficiently inhibited TNF activity; at a concentration of 0.1 pmol/ml, i.e. equimolar to the TNFa concentration used in the assay, rsTNFRP-hy3 prevented TNFa-induced cytolysis very efficiently (Fig. 5A). rsTNFRP also had inhibitory activity but a concentration about 100fold in excess of TNFa was needed for complete inhibition. TNFP-induced cytotoxicity was also inhibited by the fusion protein, albeit not at equimolar concentrations. The protective effects of rsTNFRP in these cytotoxicity assays were only evident at rather high concentrations (Fig. 5B).
TNFa-induced Agglutination of rsTNFRP-hy3-coated Latex Beads-In view of the trimeric structure of TNFa and TNFP, each capable of binding three recombinant soluble receptor molecules, it is very likely that these cytokines aggregate TNF receptors on the cell surface into microclusters which may be a necessary step in signal transduction. To mimick cell sur-  face-bound TNF receptors, Latex beads were coated with rsTNFRP-hr3 fusion protein and subsequently exposed to different concentrations of TNFa. TNFa induced an oligomerization of rsTNFRP-hr3 as visualized by agglutination of the Latex beads (Fig. 6). A similar effect was seen with TNFP, but agglutination was much less pronounced (results not shown).

DISCUSSION
In this study TNF binding and inhibiting properties of the extracellular region of the human TNFRP were analyzed. The recombinant soluble receptors (rsTNFRB and rsTNFRB-hy3 fusion protein) expressed in different eukaryotic expression systems displayed high affinity binding to human TNFa similar to that of native cell surface-bound 55-kDa TNFRP.
In contrast, the binding affinity of rsTNFRP for TNFP was significantly decreased when compared with the native cell surface receptor. A similar observation, i.e. impaired neutralization of TNFP uersw TNFa, has also been made with a socalled TNF binding protein, which is a naturally occurring soluble receptor derived from TNFR/3 (33,36,39). It therefore appears that with respect to ligand binding properties, rs-TNFRP closely ressembles the natural TNF inhibitor. The apparent lower affinity of rsTNFRP (and also of the detergent-solubilized holoreceptor (9)) for TNFP might reflect a microenvironment of the ligand binding site which is slightly different from that of the cell surface-bound full-length TNF receptor. It is noteworthy that with respect to monovalent rsTNFRB the rsTNFRP-hy3 fusion protein binds bothTNFa and TNFP with a severalfold higher affinity when measured under appropriate assay conditions. This increase in affinity most probably reflects a higher avidity of the rsTNFRB-hy3 construct due to its bivalency. Comparison of rsTNFRP and the fusion protein to compete with native full-length TNF receptors for TNF binding and to protect WEHI 164 cells from TNF-induced cytotoxicity indeed confirmed the expected higher activity of the fusion protein.
The results from the ultracentrifugation analyses indicate that rsTNFRP is monomeric in solution. The complexes of rsTNFRP with TNFa or TNFP both had a molecular mass of about 140 kDa which favors a stoichiometry of three rsTNFRP monomers bound to one TNFa or TNFP trimer. It has been proposed that the receptor binding site on the TNFa trimer is located at the boundary of two monomeric units near the base of the bell-shaped structure thus implying three potential receptor binding sites (7,54). Such a model is fully compatible with the size of receptor-ligand complexes as determined in the present study. It is interesting to note that an intermediate lower molecular weight form of the rsTNFRP.TNFa complex can be partially resolved by gel filtration when a slight excess of TNFa over rsTNFRP is present. Most likely, this intermediate form represents TNFa trimers complexed to only one or two rsTNFRP molecules. Such intermediate forms are not seen with rsTNFRP-TNFP complexes. Whether these distinct binding characteristics of TNFa and TNFP are also true for cell surface-bound receptors remains to be elucidated.
The results of sedimentation velocity and quasielastic light scattering measurements indicate that rsTNFRP and its TNFa and TNFP complexes have a rather extended, i.e. rodlike structure. This conclusion is supported by the relative large apparent molecular masses of 62, 170, and 150 kDa for rsTNFRP, rsTNFRP. TNFa and rsTNFRP. TNFP complexes, respectively, determined by gel filtration chromatography. A similar relatively large apparent molecular mass (50 kDa) has been found for the natural soluble TNFRP on sizing columns (42).
Soluble fragments of both TNFRP and TNFRa are found in uiuo. They are present at relatively high concentrations in normal human serum and urine but can be drastically increased in certain disease states.* The cellular source and the mechanism of receptor shedding remain unclear. It has been speculated that soluble TNF receptor fragments might participate in the control of TNFa and/or TNFP toxicity by neutralization and rapid clearance of systemic TNFa and TNFp (33,36,37,39,42). However, the fact that at least a 10-fold excess of the soluble receptor with respect to TNFa (and more than a 100-fold excess with respect to TNFP) is needed to obtain a significant neutralization demonstrates that the neutralizing capacity of serum is restricted. The rsTNFRP-hy3 construct as described in this study, therefore, is a promising TNF antagonizing agent for neutralization of systemic TNF toxicity in certain disease states.