Incorporation of an Isoleucine Zipper Motif Enhances the Biological Activity of Soluble CD40L (CD154)*

Arvia E. MorrisDagger , Richard L. Remmele Jr., Ralph Klinke, Brian M. Macduff, William C. Fanslow, and Richard J. Armitage

From the Immunex Corporation, Seattle, Washington 98101

    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
REFERENCES

Recent progress in the understanding of immune function indicates that the interaction of CD40L with its receptor, CD40, plays a pivotal role in both humoral immunity and cell-mediated defense against pathogens. Functional studies of this interaction on both dendritic cells and malignant cells have demonstrated that CD40L also plays an important role in immune surveillance and anti-tumor immunity. CD40L exists in nature predominantly as a membrane-anchored molecule. To develop CD40L as a potential therapeutic, it is important to optimize soluble forms of this molecule that could be used in a clinical setting. Several reports have shown that soluble forms of CD40L, like CD40 antibodies, are biologically active. In the present report we demonstrate that the incorporation of an isoleucine zipper trimerization motif significantly enhances the biological activity of soluble CD40L.

    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
REFERENCES

Interaction of CD40L (CD154) with its receptor, CD40, provides essential signals for the development of a protective humoral immune response and is a key mechanism in the regulation of defense against pathogenic assault (for review see Ref. 1). CD40, a 50-kDa transmembrane glycoprotein, is a member of the tumor necrosis factor (TNF)1 receptor superfamily (for review see Refs. 2 and 3) and is expressed on antigen-presenting cells including dendritic cells, B cells, macrophages, follicular dendritic cells, and fibroblasts. CD40 is also expressed on a number of other cell types including endothelial cells and a significant proportion of carcinomas.

CD40L is predominantly expressed on activated CD4+ T cells, but variable expression has also been reported on CD8+ T cells, mast cells, basophils, B cells, monocytes, NK cells, and activated platelets (1-4). Generally, CD40L does not exist naturally in a soluble form, although exceptions have been reported (5, 6). Initial studies focusing on the interaction of CD40L with its receptor on B cells show that CD40L is largely responsible for the helper T cell function that drives B cell proliferation and Ig class switching and protects B cells from apoptotic cell death. More recent studies on other cell types have indicated an important role for CD40L in the induction of T cell-mediated effector functions, antigen presentation, and costimulatory activity of antigen-presenting cells and in the production of many cytokines (for review see Ref. 1).

CD40L is a type II membrane glycoprotein and is a member of the TNF superfamily. CD40L has an extracellular domain consisting of a 75-amino acid spacer region immediately adjacent to the membrane spanning region that is not shared by other family members and a receptor-binding domain that consists of two stacked beta -sheets. The receptor-binding domain has low amino acid sequence homology with TNF family members (<30%) but has high structural homology (7). Soluble forms of CD40L have been expressed that are able to induce B cell proliferation, costimulate Ig class switching, and suppress the induction of apoptosis (8-10). As with cell surface expressed CD40L, maximal activity of soluble forms of the molecule is dependent on the presence of costimulatory cytokines. Interestingly, in contrast to its generally stimulatory effects on normal B cells, interaction of soluble recombinant CD40L with CD40 on neoplastic B cells can lead to growth inhibition in vitro and in vivo. The in vivo effects appear to be mediated via both direct and indirect mechanisms. These findings suggest that soluble CD40L may have therapeutic potential for the treatment of some B cell lymphomas (3, 11, 12).

Earlier studies with CD40 monoclonal antibodies (mAbs), both whole antibody and F(ab)'2 fragments, and various forms of soluble CD40L have demonstrated that each reagent has a unique ability to signal through the receptor. In general, the more multimeric the signaling agent, the more sustained is the subsequent signaling. Thus, bivalent mAb and dimeric or trimeric soluble forms of CD40L have more potent biological activity than either F(ab)'2 antibody fragments or soluble CD40L lacking multimerization domains (8). Moreover, although monovalent F(ab) fragments of CD40 mAbs can bind to CD40, they are not stimulatory and in fact antagonize signaling mediated via soluble trimeric CD40L (8). These data indicate not only that CD40 cross-linking is necessary for signaling but also that the degree of cross-linking determines the magnitude of the response. Recent investigations have shown that soluble forms of recombinant CD40L consisting only of the TNF homologous region spontaneously associate as trimers (13). It has not been clear, therefore, why the addition of a multimerization domain would increase the activity of soluble CD40L.

In the current report we have explored further the role of the trimerization domain in enhancing the activity of soluble human CD40L. We have expressed soluble forms of both the TNF homologous region and the entire extracellular domain of human CD40L and compared the activity of these molecules with that of a hybrid molecule consisting of the CD40L TNF homologous region fused to an isoleucine zipper (IZ) trimerization domain at its N terminus. We have found that the soluble CD40L molecules containing either the spacer region or the trimerization domain have significantly higher biological activity than constructs containing just the TNF homologous region. Furthermore, the CD40L construct consisting of only the TNF homologous region is considerably less stable than the form containing the trimerization domain. Taken together, the findings presented here suggest that the superior biological activity seen with the IZ-containing form of soluble CD40L is likely to be due to the ability of this trimerization motif to stabilize soluble CD40L into an orientation that is more favorable for receptor signaling.

    MATERIALS AND METHODS

Expression of Soluble CD40L Variants in Chinese Hamster Ovary Cells-- The TNF homologous region of human CD40L (amino acids 113-261) (14) was polymerase chain reaction-amplified, cloned 3' to the human growth hormone leader sequence (15) in Bluescript (Stratagene, La Jolla, CA) and sequence verified. The final protein sequence included a Thr-Ser dipeptide that is retained in the N terminus after processing of the growth hormone leader. The CD40L molecule encoded by this plasmid is termed CD40L receptor-binding domain (RBD). Similarly the entire extracellular region of CD40L (amino acids 52-261) was polymerase chain reaction-amplified and cloned 3' to the human growth hormone leader sequence, and this CD40L molecule was designated CD40L FL (full length). CD40L FL also contained a Thr-Ser at the N terminus after processing of the growth hormone leader sequence. Soluble human CD40L was also expressed as a hybrid protein containing a trimerization domain fused to the N terminus of the TNF homologous region (amino acids 112-261). The trimerization domain is based upon a leucine zipper motif (16) modified to form a trimer (17). This soluble CD40L molecule, designated CD40L IZ, includes a Thr-Ser-Ser-Asp sequence at its N terminus and Leu-Leu at the junction between the IZ and RBD regions of the molecule. For expression in CHO cells, the various CD40L constructs were cloned into the expression vector 2a5I (18).

The final CD40L constructs were transfected into DXB11 DHFR- CHO cells (19) using LipofectAMINETM (Life Technologies, Inc.). After 48 h, the cells were subcultivated into DHFR selection medium lacking glycine, hypoxanthine, and thymidine (-GHT) and grown to confluence. The cells were then selected in -GHT medium containing 50 µM methotrexate. Cell clones from each transfection were pooled and used in production cultures to generate milligram quantities of each soluble CD40L molecule.

Purification-- One molar Tris, pH 8.5, was added to the harvested cell culture supernatant to a final molarity of 80 mM and adjusted to pH 8.5 with 2 N NaOH. The adjusted cell culture supernatant was then diluted 1:5 with water before loading onto a Macro-Prep High Q column (4.4cmd × 4.2cmh) at a flow rate of 70 ml/min that was previously equilibrated in 25 mM Tris, pH 8.5. The resulting unbound fraction was then directly loaded at a flow rate of 70 ml/min onto a Fractogel SO3- 650 M column (4.4-cm diameter × 1.8-cm height) that was previously equilibrated in 25 mM Tris, pH 8. The column was then washed in sequence with equilibration buffer followed by 50 mM NaCl, 25 mM Tris, pH 8.5. The recombinant CD40L was then eluted from the column with 0.3 M NaCl, 25 mM Tris, pH 8.5.

The eluted CD40L was then dialyzed into 25 mM Tris, 4% mannitol, 1% sucrose, pH 7.4 buffer and subsequently 0.2µ filtered. The final concentration of CD40L in the purified preparations was determined by amino acid analysis (20).

Polyacrylamide Gel Electrophoresis and Western Blotting-- Western blots were performed using polyacrylamide gel electrophoresis (4-20% Tris-Glycine gels, Novex, San Diego, CA). After electrophoresis, the gels were either stained with Coomassie Blue or blotted onto nitrocellulose paper (Novex, San Diego, CA.) for Western blotting to visualize the protein bands. Western blots were performed using the mouse anti-human CD40L mAb M90 (21), which was generated at Immunex using recombinant human CD40L as immunogen. Horseradish peroxidase-conjugated anti-mouse antibody (Bio-Rad) was used as the secondary reagent, and Western blots were developed using a chemiluminecent reagent (LumiGlo, Kirkegaard, and Perry Labs, Gaithersburg, MA).

Bioassays-- Human peripheral blood mononuclear cells were isolated from heparinized blood of healthy donors by centrifugation over Isolymph (Gallard-Schlesinger Industries Inc., Carle Place, NY). Purification of B cells was achieved by removal of cells rosetting with 2-aminoethylisothiouronium bromide-treated sheep red blood cells followed by positive enrichment by magnetic cell separation technology (Miltenyi Biotec, Auburn, CA) using CD19-coated magnetic beads. The resultant B cell population was >98% CD20+ with no detectable CD3+ T cells as determined by flow cytometric analysis on a FACScan (Becton Dickinson, Mountain View, CA).

B cell proliferation assays were conducted in RPMI medium + 10% heat-inactivated fetal bovine serum at 37 °C in a humidified atmosphere of 5% CO2. 50,000 cells/well were cultured in triplicate in round-bottomed 96-well microtiter plates (Corning, Corning, NY) for 90 h in the presence of the appropriate additives as described under "Results." Cells were pulsed with 1 µCi/well tritiated thymidine (25 Ci/mmol, Amersham Pharmacia Biotech) for the final 18 h of culture. Cells were harvested, and the number of cpm incorporated was determined by tritium-sensitive avalanche gas ionization detection on a Matrix 96 Direct Beta Counter (Packard, Meriden, CT). For IgE secretion, cells (5 × 104/well) were cultured in Iscove's modified Dulbecco's medium + 10% heat-inactivated fetal bovine serum, 50 µg/ml transferrin (Sigma), 5 µg/ml bovine insulin (Sigma), 100 units/ml penicillin, and 100 µg/ml streptomycin at 37 °C in a humidified atmosphere of 10% CO2. Quantitation of IgE levels in supernatants after 10 days of culture was performed by enzyme-linked immunosorbent assay as described (22).

Three-detector Light Scattering Assessment of Molecular Mass-- The nonglycosylated polypeptide molecular masses of the CD40L IZ and CD40L RBD constructs were determined using the SEC-UV/LS/RI method (on-line SEC followed by detection using ultraviolet absorbance at 280 nm, light scattering at 90 °, and refractive index detectors in series). Light scattering and refractive index measurements were collected using a DAWN DSP and Optilab differential refractometer (products of Wyatt Technology Corporation, Santa Barbara, CA). The experiments were carried out using an Integral high pressure liquid chromatography system (PerSeptive Biosystems, Inc., Farmingham, MA) with a Superdex 200 HR 10/30 column (Amersham Pharmacia Biotech). Column loads for the two cases studied ranged from 150 to 200 µg. The flow rate was 1 ml/min with a mobile phase of 200 mM sodium phosphate at pH 8. All detector systems were calibrated as described (23-25) using ovalbumin (Sigma), bovine serum albumin monomer (Sigma), and bovine serum albumin dimer (Sigma) at a nominal concentration of 1 mg/ml in each case. The resulting plot of (LS × UV)/[epsilon  × (RI)2] as a function of molecular mass was linear, passing through the origin and having an r2 = 0.994. LS is the light scattering intensity, RI is the refractive index, and UV is the absorbance. The slope of this plot provided the combined instrumental calibration constant k" used in the determination of the molecular mass of the CD40L IZ and CD40L RBD. Theoretical molar extinction coefficients (epsilon ) were determined from the amino acid composition (26) for the CD40L RBD and CD40L IZ versions of soluble CD40L and were found to be 0.76 and 0.65, respectively. Data were acquired using ASTRA (accompanying software from Wyatt Technology Corporation).

Differential Scanning Calorimetry-- Calorimetric measurements were carried out using a MicroCal MC-2 DSC. Samples were buffer exchanged into 25 mM Tris, pH 7.6, 400 mM NaCl, and 10% glycerol. Protein concentrations under these conditions were nominally between 2 and 3 mg/ml. The scan rate in each case tested was 68 °C/h. The thermograms were background corrected and rescans were employed to evaluate the reversibility of the unfolding transitions. In all cases examined the second upscan yielded no reversible enthalpy, suggesting that the sample was irreversibly denatured as indicated by the presence of aggregate when withdrawing the sample from the DSC. In this study, 90 °C was set as the upper temperature limit, which was beyond the completion of the major melting transition of the soluble CD40L molecules. All data manipulation was performed using the Origin software provided with the instrument.

    RESULTS

Expression and Purification of the Soluble CD40L Variants-- The three CD40L molecules compared in this study are diagrammed in Fig. 1A. After purification as described under "Materials and Methods," each soluble CD40L preparation was analyzed for purity using Western blots (Fig. 1B, lanes 1-3) and Coomassie gels (Fig. 1B, lanes 4-6). Each molecule had one main band that migrated slightly higher than the predicted molecular mass and at least one faint lower molecular mass band that migrated closer to the expected monomeric molecular mass. The calculated peptide molecular masses for CD40L FL, CD40L RBD, and CD40L IZ are 24.0, 16.4, and 20.9 kDa, respectively. The preparations of CD40L IZ and CD40L RBD showed no bands on Coomassie gels besides the CD40L bands. The Coomassie preparation of CD40L FL (Fig. 1B, lane 4) had a large number of background bands that were not detected by Western blot. This molecule did not express as well as the other two, and thus it was more difficult to obtain a highly purified final preparation. Previous analysis using N-glycanase of CD40L molecules expressed in CHO cells have indicated that the higher molecular mass bands detected on the Western blot are glycoforms of CD40L.


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Fig. 1.   Expression of recombinant human soluble forms of CD40L. A, schematic representation of soluble CD40L molecules. CD40L RBD consists of just the TNF homologous region of the extracellular domain (gray box). CD40L FL consists of the extracellular spacer between the cell membrane and the TNF homologous region (hatched box) and the TNF homologous region. CD40L IZ consists of the receptor-binding domain and an IZ motif at the amino end of the molecule (black box). B, Western blots and Coomassie stain of various soluble human CD40L constructs. Lanes 1-3, Western blot. Lane 1, CD40L FL; lane 2, CD40L RBD; lane 3, CD40L IZ. Lanes 4-6, Coomassie stained gel. Lane 4, CD40L FL; lane 5, CD40L RBD; lane 6, CD40L IZ.

Bioactivity of the CD40L Variants-- The biological activities of CD40L RBD and CD40L IZ were compared in a human B cell proliferation assay performed in the presence of IL-4 as a costimulus (Fig. 2A). Although both forms of CD40L induce B cell proliferation, the IZ construct showed significantly higher biological activity with effects seen at ligand concentrations as low as 20 ng/ml. Proliferation induced by these forms of soluble CD40L could be inhibited by addition of anti-CD40L antibodies to the cultures (data not shown). A similar distinction in the activities of the two CD40L constructs was seen in the induction of IgE secretion from IL-4-costimulated B cells (Fig. 2B).


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Fig. 2.   Biological activity of CD40L RBD and CD40L IZ. A, purified peripheral blood B cells were cultured for 90 h in the presence of IL-4 (5 ng/ml) and a titration of CD40L RBD (open circle ) or CD40L IZ (bullet ). Tritiated thymidine was added to cells for the final 18 h of culture. Results are expressed as the mean cpm of triplicate cultures and are representative of three experiments performed. B, purified peripheral blood B cells were cultured for 10 days in the presence of IL-4 (5 ng/ml) and a titration of CD40L RBD (open circle ) or CD40L IZ (bullet ). IgE levels in day 10 culture supernatants were determined by enzyme-linked immunosorbent assay. Results are expressed as the mean values from triplicate cultures and are representative of three experiments performed.

The CD40L RBD and CD40L FL were compared also for their ability to induce proliferation of peripheral blood B cells (Fig. 3). An effect of the CD40L RBD was seen at concentrations higher than 1 µg/ml, whereas the full-length form of the ligand had greater biological activity, measurable down to 300 ng/ml.


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Fig. 3.   Biological activity of CD40L RBD and CD40L FL. Purified peripheral blood B cells were cultured for 90 h in the presence of IL-4 (5 ng/ml) and a titration of CD40L RBD (open circle ) or CD40L FL (bullet ). Tritiated thymidine was added to cells for the final 18 h of culture. Results are expressed as the mean cpm of triplicate cultures and are representative of three experiments performed.

Three-detector Analysis of CD40L-- One possible explanation for the increased activity of CD40L IZ compared with that of the CD40L RBD construct could be attributed to it existing in a higher order multimeric form in solution. To evaluate the multimeric state of these two recombinant CD40L variants, SEC followed by three-detector analysis was employed. Evaluation of the molecular mass of glycosylated proteins using the SEC-UV/LS/RI method offers the advantage of being able to assess the molecular mass of the molecule as if it were nonglycosylated; glycosylation is essentially transparent at 280 nm (23). Fig. 4A shows the elution profile of the CD40L RBD with a peak elution volume of 13.6 ml. The SEC-UV/LS/RI method yielded a measured molecular mass of 49.47 ± 0.81 kDa. Utilizing the monomer polypeptide molecular mass of this molecule (16.4 kDa), it was determined that CD40L RBD is indeed a trimer. This confirms and extends the findings from sucrose gradient sedimentation experiments published earlier (10). Similarly, the polypeptide molecular mass of the CD40L IZ as measured by SEC-UV/LS/RI was 62.2 ± 0.14 kDa (Fig. 4B). This is approximately three times the monomer molecular mass (20.9 kDa). These results provide evidence that both soluble forms of CD40L are trimeric.


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Fig. 4.   The SEC elution profiles for CD40L RBD (A) and CD40L IZ (B). Labeled are the corresponding eluting components as measured by the UV absorbance at 280 nm, light scattering at 90 ° (LS), and refractometer (RI). The vertical dashed lines in each case display the bracketed region designated for molecular mass characterization (after computing the molecular mass per slice) surrounding the peak maximum.

An alternative explanation for the increased activity of CD40L IZ compared with CD40L RBD is that the latter may disassociate into monomers at lower concentrations. Disassociation of trimeric CD40L RBD at low concentrations could greatly impair its ability to cross-link and thus signal through CD40. To address this issue, CD40L RBD was diluted to 2 µg/ml, and disassociation was monitored using the SEC-UV method. No disassociation of the CD40L RBD was detected (Fig. 5), suggesting that this was not the reason for the differences observed in the biological activity of the CD40L IZ and CD40L RBD constructs. At 2 µg/ml the CD40L IZ molecule showed greater activity than CD40L RBD (Fig. 2A); thus, disassociation of CD40L RBD into monomers does not explain the differences in activity.


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Fig. 5.   SEC showing (from top to bottom) the buffer blank, lysozyme standard, CD40L RBD (1.95 mg/ml), and CD40L RBD (1.95 µg/ml). The effect of dilution does not show discernible evidence of further disassociation of the trimer.

DSC-- It is possible that the distinct activities of the CD40L IZ and CD40L RBD could be due to a difference in stability. To address this issue, differential scanning calorimetry (DSC) experiments were performed. The DSC data distinctly showed a 10 °C difference in the melting transition, with the CD40L RBD having the lower melting transition (Fig. 6). Collectively, these data suggest that in the CD40L IZ, the IZ confers appreciable conformational stability compared with that seen in CD40L RBD, despite both existing in solution as trimers. That is to say, it is more difficult thermodynamically for the IZ version of the molecule to unfold under the given solution conditions. This might be explained in terms of the local restraint offered by the IZ in proximity to the receptor-binding domain.


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Fig. 6.   Concentration-normalized DSC scans depicting the endothermic melting transitions of CD40L RBD and CD40L IZ. Both samples are in a common buffer system consisting of 25 mM Tris, pH 7.6, 400 mM NaCl, and 10% glycerol. The exotherm at 80 °C (lower trace) is due to the formation of aggregate.


    DISCUSSION

The results presented here demonstrate that the three forms of soluble CD40L examined, CD40L RBD, CD40L FL, and CD40L IZ, are biologically active. The presence of the IZ trimerization domain or the native extracellular spacer region significantly increased the activity of soluble CD40L in B cell proliferation assays. The presence of the IZ trimerization domain on CD40L RBD also increased IgE secretion. Significantly enhanced activity of CD40L IZ constructs, compared with that of CD40L RBD, was seen also in the induction of B cell activation antigens, CD54, CD86, and HLA-DR (data not shown). Furthermore, the SEC-UV/LS/RI method demonstrated that CD40L RBD and CD40L IZ exist as trimers in solution. Finally, the DSC study indicated that addition of the IZ serves to conformationally stabilize CD40L RBD.

Molecular modeling studies have shown (7) and crystallographic studies at 2 Å resolution have confirmed (13) that the trimerization of CD40L RBD involves Tyr170 and His224 to form an unusual cluster of two triads along the 3-fold axis. The trimerized assemblages are stabilized by hydrogen bonds and presumably salt bridge interactions. The DSC data indicate that significant differences exist in overall conformational stability when the IZ motif is incorporated at the N terminus of the RBD. Without the IZ, the CD40L RBD molecule would be more prone to unfold at the N terminus rather than the C terminus, because the latter is buried in the interior of the trimerized molecule (13). With the IZ one can begin to imagine how the N terminus would be constrained, making it more difficult to unfold from that end. The broad features in the unfolding profile of the IZ may suggest an unraveling of the molecule that begins with an overall loosening of the protein interior and ends with the sheet regions of the molecule exposed. The trimerized assemblages may thus form aggregates in the unfolded state (observations after removal of samples from the DSC have shown visible signs of aggregation). Such structural restraint of the N terminus may also affect the way in which the ligand interacts with the receptor and explain the enhanced bioactivity. Site-directed mutagenesis has shown that Lys143 and Tyr145 are critical for CD40L interaction with CD40 (27). These residues are in close proximity to the N terminus. Restraint at the N terminus conferred by the IZ may stabilize the configuration in this region and thus augment signaling.

In addition to improved conformational stability, the IZ may enhance signaling through other mechanisms. Based on previous studies using mAbs to mediate receptor ligation, it is predicted that binding of soluble CD40L to CD40 is followed by rapid cell surface localization and internalization of the receptor-ligand complex. The presence of the IZ motif in the CD40L IZ molecule may result in binding to CD40 in such a way that these processes are retarded, resulting in prolonged signaling through CD40.

It is not clear why we were able to detect activity for the CD40L FL and others were not (10). It could be that in the bacterial system it was not possible to achieve the correct folding or that important differences exist in the glycosylation achieved in bacteria compared with CHO cells. Either could affect the biological activity of CD40L FL. Furthermore, our studies with CHO cell-derived CD40L FL indicate that this molecule has a tendency to aggregate, which could explain why it has relatively good biological activity compared with CD40L RBD, because in a partially aggregated form it could more effectively mediate receptor cross-linking.

Modeling studies have pointed to structural similarities between CD40L and TNF, a prototype member of the TNF/nerve growth factor superfamily of ligands. However, it is important to note that although biologically active TNF exists naturally as a soluble homotrimer, CD40L is predominantly expressed as a membrane-anchored molecule. Results presented in this study and those of Mazzei and colleagues (10) have demonstrated that the receptor-binding domain of CD40L can associate to form homotrimers in solution. However, in contrast to TNF, such a configuration does not impart optimal biological activity to the soluble CD40L molecule, because its stability appears to be enhanced considerably by the presence of the 75-amino acid spacer region or the IZ trimerization domain. This suggests that this region between the RBD and the transmembrane domain of the CD40L molecule may be important in facilitating formation of stable CD40L complexes on the cell surface. Supporting evidence for the importance of N-terminal multimerization in the structural integrity of CD40L comes from studies on a hyper IgM patient whose CD40L gene contained a deletion of the coding region for the extracellular spacer domain (28). In this study the mutant CD40L was expressed in COS cells and found to have a reduced ability to bind to CD40 compared with a wild type CD40L.

Regardless of the mechanism responsible for the enhanced biological activity observed, the results presented here shed new light on signaling mediated by soluble forms of CD40L. In two examples, the CD40L IZ and the CD40L FL, the presence of an N-terminal multimerization domain resulted in soluble forms of the ligand with improved specific activities. This result is not predicted by protein modeling studies, which suggest that the critical region for functional association of the CD40L molecule is just contained within the TNF homologous region. In fact, within the TNF ligand superfamily enhanced biological activity of IZ-containing constructs is not confined to CD40L. The activities of soluble Fas ligand and TRAIL, the extracellular domains of which can occur naturally in soluble form, are significantly enhanced by the addition of the IZ motif at the N terminus.2 Further investigations into the mechanism of action of the IZ motif should lead to greater insight into regulation of receptor signaling and development of therapeutic constructs for this important family of molecules.

    ACKNOWLEDGEMENTS

We are grateful to Mi G. Kim for tissue culture support and Anne Aumell for editorial comments.

    FOOTNOTES

* The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Dagger To whom correspondence should be addressed: Immunex Corp., 51 University St., Seattle, WA 98101. Tel.: 206-587-0430; Fax: 206-233-9733.

2 D. Lynch, unpublished observations.

    ABBREVIATIONS

The abbreviations used are: TNF, tumor necrosis factor; mAb, monoclonal antibody; IZ, isoleucine zipper; CHO, Chinese hamster ovary; RBD, receptor-binding domain; LS, light scattering; RI, refractive index; DSC, differential scanning calorimetry; IL, interleukin; SEC, size exclusion chromatography..

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
REFERENCES
  1. Grewal, I. S., and Flavell, R. A. (1997) Immunol. Res. 16, 59-70[Medline] [Order article via Infotrieve]
  2. Armitage, R. J. (1994) Curr. Opin. Immunol. 6, 407-413[CrossRef][Medline] [Order article via Infotrieve]
  3. Gruss, H. J., and Dower, S. K. (1995) Blood 85, 3378-3404[Abstract/Free Full Text]
  4. Henn, V., Slupsky, J. R., Grafe, M., Anagnostopoulos, I., Forster, R., Muller-Berghaus, G., and Kroczek, R. A. (1998) Nature 391, 591-594[CrossRef][Medline] [Order article via Infotrieve]
  5. Armitage, R. J., Sato, T. A., Macduff, B. M., Clifford, K. N., Alpert, A. R., Smith, C. A., and Fanslow, W. C. (1992) Eur. J. Immunol. 22, 2071-2076[Medline] [Order article via Infotrieve]
  6. Graf, D., Muller, S., Korthauer, U., van Kooten, C., Weise, C., and Kroczek, R. A. (1995) Eur. J. Immunol. 25, 1749-1754[Medline] [Order article via Infotrieve]
  7. Peitsch, M. C., and Jongeneel, C. V. (1993) Int. Immunol. 5, 233-238[Abstract]
  8. Fanslow, W. C., Srinivasan, S., Paxton, R., Gibson, M. G., Spriggs, M. K., and Armitage, R. J. (1994) Semin. Immunol. 6, 267-278[CrossRef][Medline] [Order article via Infotrieve]
  9. Hollenbaugh, D., Grosmaire, L. S., Kullas, C. D., Chalupny, N. J., Braesch-Andersen, S., Noelle, R. J., Stamenkovic, I., Ledbetter, J. A., and Aruffo, A. (1992) EMBO J. 11, 4313-4321[Abstract]
  10. Mazzei, G. J., Edgerton, M. D., Losberger, C., Lecoanet-Henchoz, S., Graber, P., Durandy, A., Gauchat, J.-F., Bernard, A., Allet, B., and Bonnefoy, J.-Y. (1995) J. Biol. Chem. 270, 7025-7028[Abstract/Free Full Text]
  11. Funakoshi, S., Longo, D. L., Beckwith, M., Conley, D. K., Tsarfaty, G., Tsarfaty, I., Armitage, R. J., Fanslow, W. C., Spriggs, M. K., and Murphy, W. J. (1994) Blood 83, 2787-2794[Abstract/Free Full Text]
  12. Sumimoto, S., Heike, T., Kanazashi, S., Shintaku, N., Jung, E. Y., Hata, D., Katamura, K., and Mayumi, M. (1994) J. Immunol. 153, 2488-2496[Abstract/Free Full Text]
  13. Karpusas, M., Hsu, Y. M., Wang, J. H., Thompson, J., Lederman, S., Chess, L., and Thomas, D. (1995) Structure 3, 1031-1039[CrossRef][Medline] [Order article via Infotrieve]
  14. Spriggs, M. K., Armitage, R. J., Strockbine, L., Clifford, K. N., Macduff, B. M., Sato, T. A., Maliszewski, C. R., and Fanslow, W. C. (1992) J. Exp. Med. 176, 1543-1550[Abstract]
  15. Pecceu, F., Dousset, P., Shire, D., Cavrois, E., Marchese, E., Ferrara, P., Kaghad, M., Dumont, X., and Lupker, J. (1991) Gene (Amst.) 97, 253-258[CrossRef][Medline] [Order article via Infotrieve]
  16. O'Shea, E. K., Rutkowski, R., and Kim, P. S. (1989) Science 243, 538-542[Medline] [Order article via Infotrieve]
  17. Harbury, P. B., Zhang, T., Kim, P. S., and Alber, T. (1993) Science 262, 1401-1407[Medline] [Order article via Infotrieve]
  18. Morris, A. E., Lee, C. C., Hodges, K., Aldrich, T. L., Krantz, C., Smidt, P. S., and Thomas, J. N. (1997) in Animal Cell Technology (Carrondo, M. J. T., Griffiths, B., and Moreira, J. L. P., eds), pp. 529-534, Kluwer Academic Publishers, Amsterdam, The Netherlands
  19. Urlaub, G., and Chasin, L. A. (1980) Proc. Natl. Acad. Sci. U. S. A. 77, 4216-4220[Abstract]
  20. Spackman, D. H., Stein, W. H., and Moore, S. (1958) Anal. Chem. 30, 1185-1190
  21. Fanslow, W. C., Rousseau, A.-M., Lofton, T. E., Klinke, R., Ulrich, D. T., and Armitage, R. J. (1997) in Leucocyte Typing VI (Kishimoto, T. E. A., ed), p. 101, Garland Publishing, Inc., New York
  22. Armitage, R. J., Macduff, B. M., Eisenman, J., Paxton, R., and Grabstein, K. H. (1995) J. Immunol. 154, 483-490[Abstract/Free Full Text]
  23. Arakawa, T., Langley, K. E., Kameyama, K., and Takagi, T. (1992) Anal. Biochem. 203, 53-57[Medline] [Order article via Infotrieve]
  24. Wen, J., Arakawa, T., and Philo, J. S. (1996) Anal. Biochem. 240, 155-166[CrossRef][Medline] [Order article via Infotrieve]
  25. Wen, J., Arakawa, T., Talvenheimo, J., Welcher, A. A., Horan, T., Kita, Y., Tseng, J., Nicolson, M., and Philo, J. S. (1996) in Techniques in Protein Chemistry VII (Marshak, D. R., ed), pp. 23-31, Academic Press, Orlando, FL
  26. Gill, S. C., and von Hippel, P. H. (1989) Anal. Biochem. 182, 319-326[Medline] [Order article via Infotrieve]
  27. Bajorath, J., Chalupny, N. J., Marken, J. S., Siadak, A. W., Skonier, J., Gordon, M., Hollenbaugh, D., Noelle, R. J., Ochs, H. D., and Aruffo, A. (1995) Biochemistry 34, 1833-1844[Medline] [Order article via Infotrieve]
  28. Seyama, K., Nonoyama, S., Gangaas, I., Hollenbaugh, D., Pabst, H. F., Alejandro, A., and Ochs, H. D. (1998) Blood 92, 2421-2434[Abstract/Free Full Text]


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