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Receptor-selective Mutants of Apoptosis-inducing Ligand 2/Tumor Necrosis Factor-related Apoptosis-inducing Ligand Reveal a Greater Contribution of Death Receptor (DR) 5 than DR4 to Apoptosis Signaling *

Receptor-selective Mutants of Apoptosis-inducing Ligand 2/Tumor Necrosis Factor-related... THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 280, No. 3, Issue of January 21, pp. 2205–2212, 2005 © 2005 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in U.S.A. Receptor-selective Mutants of Apoptosis-inducing Ligand 2/Tumor Necrosis Factor-related Apoptosis-inducing Ligand Reveal a Greater Contribution of Death Receptor (DR) 5 than DR4 to □ S Apoptosis Signaling* Received for publication, September 16, 2004, and in revised form, November 1, 2004 Published, JBC Papers in Press, November 1, 2004, DOI 10.1074/jbc.M410660200 Robert F. Kelley‡§, Klara Totpal , Stephanie H. Lindstrom‡, Mary Mathieu‡, Karen Billeci , Laura DeForge , Roger Pai**, Sarah G. Hymowitz‡, and Avi Ashkenazi From the Departments of ‡Protein Engineering, Molecular Oncology, **Process Sciences, and Assay and Automation Technology, Genentech, Inc., South San Francisco, California 94080 and OPG lack a cytoplasmic domain, whereasDcR2 has a trun- Apoptosis-inducing ligand 2 (Apo2L), also called tumor necrosis factor-related apoptosis-inducing ligand cated death domain that is non-functional for apoptosis initia- (TRAIL), triggers programmed cell death in various types tion (1, 6). Like several other members of the TNF superfamily, of cancer cells but not in most normal cells. Apo2L/TRAIL Apo2L/TRAIL is synthesized as a type II transmembrane pro- is a homotrimeric protein that interacts with five recep- tein that can be proteolytically cleaved to release a soluble, tors: death receptor 4 (DR4) and DR5 mediate apoptosis homotrimeric molecule. A recombinant version of soluble ho- activation, whereas decoy receptor 1 (DcR1), DcR2, and motrimeric Apo2L/TRAIL (residues 114 –281) induces apo- osteoprotegerin counteract this function. Many cancer ptosis in various cancer cell lines but not in normal cells (7, 8). cell lines express both DR4 and DR5, and each of these Administration of soluble Apo2L/TRAIL in mouse xenograft receptors can initiate apoptosis independently of the models of human cancer results in marked anti-tumor activity other. However, the relative contribution of DR4 and DR5 without systemic toxicity (7–9). These results have prompted to ligand-induced apoptosis is unknown. To investigate further evaluation of Apo2L/TRAIL as a potential therapeutic this question, we generated death receptor-selective agent for human cancer. Apo2L/TRAIL variants using a novel approach that en- Structural studies show that homotrimeric TNF superfamily ables phage display of mutated trimeric proteins. Selec- ligands bind three receptor molecules (10 –12), suggesting that tive binding to DR4 or DR5 was achieved with three to six-ligand amino acid substitutions. The DR4-selective the basic functional signaling unit is trimeric. This notion is Apo2L/TRAIL variants examined in this study showed a further supported by the trimeric structure of certain signaling markedly reduced ability to trigger apoptosis, whereas adaptor molecules that act downstream of the receptors, such the DR5-selective variants had minimally decreased or as the TNF receptor-associated factors (13, 14). Further cross- slightly increased apoptosis-inducing activity. These re- linking of receptors beyond the trimeric unit in some cases can sults suggest that DR5 may contribute more than DR4 to lead to stronger signal initiation; this can be modeled by anti- Apo2L/TRAIL-induced apoptosis in cancer cells that ex- body-mediated cross-linking of N-terminally tagged ligand, press both death receptors. perhaps mimicking its transmembrane form (1). Binding of Apo2L/TRAIL to DR4 and/or DR5 leads to recruitment of the adaptor FADD (Fas-associated death domain) by the cytoplas- Apo2L (or TRAIL), a member of the tumor necrosis factor mic death domain followed by recruitment and activation of the (TNF) superfamily, induces apoptosis in a broad spectrum of apoptosis initiators caspase-8 and caspase-10 (5, 15). Studies human cancer cell lines while sparing most normal cells (1). based on receptor-blocking antibodies indicate that Apo2L/ Apo2L/TRAIL triggers apoptosis through binding to the death TRAIL can induce apoptosis through either DR4 or DR5 or receptors DR4 (2) and/or DR5 (3, 4). These receptors contain a both, but the relative contribution of each death receptor to cytoplasmic death domain that recruits adaptor molecules in- apoptosis induction in cells expressing both receptors is un- volved in caspase activation (5). In addition to these two sig- known (1, 6). Moreover, whereas binding of Apo2L/TRAIL to naling receptors, Apo2L/TRAIL binds to three decoy receptors DR4 and DR5 can result in the formation of homomeric as well that inhibit apoptosis induction: DcR1, DcR2, and OPG. DcR1 as heteromeric complexes (5), the importance of heteromeric ligand-receptor complexes for apoptosis stimulation is unclear. * The costs of publication of this article were defrayed in part by the Indeed, agonistic monoclonal antibodies specific to either DR4 payment of page charges. This article must therefore be hereby marked (16) or DR5 (17) are capable of inducing apoptosis; however, the “advertisement” in accordance with 18 U.S.C. Section 1734 solely to precise molecular mechanism of death receptor activation by indicate this fact. agonistic antibodies is not fully understood. □ S The on-line version of this article (available at http://www.jbc.org) contains Supplemental Tables I–III. To investigate the relative importance of DR4 and DR5 for § To whom correspondence should be addressed: Dept. of Protein apoptosis induction by Apo2L/TRAIL, we selected ligand vari- Engineering MS#27, Genentech, Inc., 1 DNA Way, South San Fran- ants with relative binding selectivity for DR4 or DR5 through a cisco, CA 94080. Tel.: 650-225-2321; Fax: 650-224-3734; E-mail: [email protected]. phage display approach. Although phage display has been used The abbreviations used are: Apo2L, apoptosis-inducing ligand 2; previously for optimizing the target binding affinity of mono- TNF, tumor necrosis factor; TRAIL, TNF-related apoptosis-inducing meric, homodimeric, or heterodimeric proteins (18, 19), this ligand; DR, death receptor; DcR, decoy receptor; OPG, osteoprotegerin; technique has not been successfully applied as yet to trimeric ELISA, enzyme-linked immunosorbent assay; SPR, surface plasmon proteins. Our modifications enabled the display of trimeric resonance. This paper is available on line at http://www.jbc.org 2205 This is an Open Access article under the CC BY license. 2206 Apoptosis-induction by DR5-selective Apo2L/TRAIL saline, 0.05% Tween 20 to remove unbound phage, bound phage were Apo2L/TRAIL on phage and the selection of receptor-selective eluted from the wells by the addition of 100 lof10mM HCl. After variants. Investigation of the ability of these variants to induce neutralization with 2 M Tris base pH 11, half of the eluant from the apoptosis in several cancer cell lines and in normal hepatocytes receptor plate was used to infect XL-1 E. coli to propagate phage (50 ml suggests that DR5 may play a more prominent role than DR4 culture) for the next round of sorting. A portion of the eluant from the in mediating apoptosis signals emanating from Apo2L/TRAIL receptor and blank plates was used to estimate phage concentration by in cells that express both death receptors. titering colony forming units with XL-1 E. coli. The enrichment of the selection for receptor binding was calculated from the ratio of the phage EXPERIMENTAL PROCEDURES eluted from the receptor plate to that eluted from the blank plate. Construction of Apo2L/TRAIL Phage Display Vector—A phagemid Upon completion of sorting, individual clones were screened for spe- vector designed for the expression of Apo2L/TRAIL (residues 96 –281) cific binding by “spot ELISA” (23). For the DR4 library clones, the test as a fusion to the geneIII protein of M13 bacteriophage was constructed proteins were DR4-Fc, DR5-Fc, TNFR1-Fc, and bovine serum albumin. as follows. The DNA encoding the 96 –281 portion of Apo2L/TRAIL was Only the clones that gave an ELISA signal for DR4-Fc and none of the amplified by PCR from the plasmid pAPOK5 (20) and ligated into other proteins were chosen for further analysis. Test proteins for the NsiI/BamHI-cleaved pTFAA-g3 (21). The resulting plasmid (pAPOK4) DR5 library clones were DR5-Fc and a humanized anti-HER2 antibody. encodes a fragment having the stII bacterial signal sequence fused to Clones positive for DR5-Fc binding and not anti-HER2 binding were the N terminus of the 96 –281-residue fragment of Apo2L/TRAIL. A selected for further study. tripeptide linker with the sequence GSA is appended to the C terminus Characterization of Receptor-selective Clones—Single-stranded DNA of Apo2L/TRAIL followed by an in-frame amber stop codon and the was isolated from the phage particle using the Qiaprep Spin M13 kit geneIII product of M13 bacteriophage. The alkaline phosphatase pro- (Qiagen) and subjected to dideoxynucleotide sequencing using the dye moter is used to direct expression. pAPOK4.2 was constructed by re- terminator cycle kit (Beckman-Coulter) and a primer that is compli- placing the alkaline phosphatase promoter in pAPOK4 with the mentary to a portion of the tac promoter. A few of the DR4-selective tac promoter. phage clones were analyzed by competition phage ELISA to determine Library Construction—The DR4 library was constructed by oligonu- the IC for DR4-Fc binding. Phage clones were first titered for binding cleotide-directed mutagenesis (22) using a primer having NNS codons to DR4-Fc to determine the dilution giving equal signal strength for all at positions 189, 191, 193, 199, 201, and 209. The template was pAPOK4.2 the clones. A fixed concentration of phage was then mixed with an containing Y213W/S215D mutations and TAA stop codons at 189, 191, increasing concentration of DR4-Fc or DR5-Fc and added to wells 193, 199, 201, and 209. Use of the stop template ensured that any coated with DR4-Fc. After incubation to allow binding and washing to template DNA that did not become mutated and survived the Kunkel remove unbound phage, the bound phage was detected with the horse- selection would not produce functional Apo2L. For the DR5 library, radish peroxidase-coupled anti-M13 antibody. Analysis of the ELISA TAA stop codons were introduced into pAPOK4.2 at the library posi- signal as a function of receptor-Fc concentration in solution by using a tions 189, 191, 193, 264, 266, 267, and 269. Two oligonucleotides, one four-parameter fit yields the IC value. specifying NNS codons at the library positions 189, 191, and 193 and Expression and Purification of Apo2L/TRAIL Variants—Mutants of the second containing NNS codons at sites 264, 266, 267, and 269 were Apo2L (114 –281) were constructed, expressed in E. coli, and purified as used in the library mutagenesis reaction. Library mutagenesis, electro- previously described (7, 20). Plasmids designed for E. coli expression of poration, and propagation of phage were performed as described (23). FLAG-tagged Apo2L variants were constructed by oligonucleotide-di- Upon electroporation into SS-320 Escherichia coli the DR5 library gave rected mutagenesis (22) of a plasmid (pFLAG-Apo2L; Scot Marsters, 8 9 a size of 1.75  10 clones, and the DR4 library comprised 2.5  10 Genentech) having Apo2L/TRAIL (114 –281) inserted in pFLAG-MAC clones. Because SS-320 do not have an amber suppressor, the phage (Sigma). pFLAG-Apo2L directs the cytoplasmic expression of N-termi- pellet was used to infect a 500-ml culture of early log phase XL-1 E. coli. nal FLAG-tagged Apo2L/TRAIL (114 –281) under control of the tac After the addition of KO7 helper phage and carbenecillin (50 g/ml), the promoter. For expression of FLAG-tagged Apo2L/TRAIL variants, the culture was grown overnight at 30 °C, and phage were harvested by mutant plasmids were transformed into E. coli strain 43E7. The trans- precipitation with ⁄5 volume of 20% polyethylene glycol, 2.5 M NaCl. formed E. coli were grown to early log phase at 37 °C in 500 ml of 2x Phage were amplified between rounds of sorting by infection of XL-1 yeast/tryptone media containing 50 g/ml carbenicillin, and expression Blue as described above except that the culture volume used was was induced by the addition of isopropyl 1-thio--D-galactopyranoside reduced 10-fold to 50 ml. to a final concentration of 0.4 mM. The FLAG-tagged Apo2L/TRAIL For the analysis of sequence changes giving rise to DR5-selectivity, variants were purified as previously described for untagged proteins two additional libraries were constructed. Library DR5.B used the (20). For both the untagged and flag-tagged proteins, an additional following codons: 189 (YAS), encoding Tyr, amber, Gln, His; 191 (ARR), purification step consisting of gel filtration on a 2.6  100 column of encoding Arg and Lys; 193 (CRA), encoding Gln or Arg; 264 (CRC), Sephacryl S-200 HR equilibrated and eluted with 0.4 M sodium sulfate, encoding His and Arg; 266 (MTT), encoding Ile and Leu; 267 (SAS), 25 mM Tris-HCl, pH 7.5, was used to remove aggregated protein from encoding His, Gln, Asp, and Glu. Library DR5.C used these codons: 189 the preparations. (BVS), encoding Cys, Trp, Arg, Gly, Tyr, Ser, His, Gln, Pro, Ala, Glu; Assay of Apoptosis Induction—Apoptosis induction in tumor cell lines 191 (RVR), encoding Glu, Ala, Gly, Lys, Thr, Arg; 193 (VVA), encoding upon the addition of Apo2L/TRAIL or variants was measured using a Glu, Gly, Ala, Gln, Arg, Pro, Lys, Thr; 264 (VVC), encoding Asp, Gly, fluorescence assay of metabolic activity as previously described (20). Ala, His, Arg, Pro, Asn, Ser, Thr; 266 (VYT), encoding Ile, Thr, Leu, Pro, Caspase activation in normal cells upon Apo2L/TRAIL addition was Val, Ala; 267 (NMS), encoding Tyr, Ser, His, Pro, Gln, Asp, Glu, Ala, determined by using a fluorescent caspase substrate as previously Asn, Lys, Thr. Kunkel mutagenesis was used to construct these librar- described (24). ies as described above except that XL-1 Blue E. coli (Stratagene, Inc.) Receptor Binding by AlphaQuest® Assay—The binding of the vari- was used for electroporation. Library DR5.B gave an actual library size ants to the five known Apo2L/TRAIL receptors (DcR1, DcR2, OPG, 10 9 of 1  10 , and library DR5.C had a size of 1.6  10 . DR4, DR5) was examined by using an AlphaQuest® assay. This is a Sorting of Phage Libraries for Receptor Selectivity—Phage sorting for proximity-based assay technology in which emission of singlet oxygen receptor binding was performed using receptor-Fc fusion proteins ad- from “donor” beads yields a fluorescent signal from “acceptor” beads sorbed on the wells of microtiter plates (Nunc-Maxisorp). Purified re- brought into proximity by binding interactions. The donor bead was ceptor-Fc proteins were kindly provided by Scot Marsters (Genentech). coated with streptavidin and was used to capture biotinylated Apo2L/ Receptor-Fc proteins were diluted to 2–10 g/ml in coating buffer (50 TRAIL. The acceptor bead was coated with Staphylococcal protein A mM sodium carbonate, pH 9.6), and 100 l of this solution was added to and was used to capture the receptor-Fc protein. IC values were several wells of a 96-well plate. After a 2-h incubation the wells were calculated from binding curves obtained by displacing the biotinylated blocked with 200 l of phosphate-buffered saline containing 0.05% ligand with unbiotinylated ligand. Biotinylated Apo2L/TRAIL was pre- Tween 20 and 5% powdered skim milk (blocking buffer). Blocking was pared by reaction of a surface cysteine mutant of Apo2L/TRAIL (R170C) for1hat room temperature, and then the wells were rinsed with with biotin-1-biotinamido-4-[4(maleimidomethyl)-cyclohexane-carbox- phosphate-buffered saline, 0.05% Tween 20 (wash buffer). amido]butane (Pierce) followed by removal of excess biotin on a PD-10 The Apo2L/TRAIL library phage solution was diluted 10-fold in (Amersham Biosciences) desalting column. The various Apo2L/TRAIL blocking buffer, and then 100 l of this solution was added to the wells receptors were diluted to 250 ng/ml in assay buffer (phosphate-buffered of the receptor-Fc-coated plate. In addition, the diluted phage were also saline, pH 7.4, containing 0.5% bovine serum albumin and 0.05% Tween added to an equal number of wells of a blank plate that was prepared by 20). Apo2L/TRAIL samples were serially diluted starting at a concen- blocking the wells with blocking buffer without prior protein coating. tration of 4 –20 g/ml. Receptor (10 l) and Apo2L/TRAIL (5 l) were After incubation to allow binding and washing with phosphate-buffered combined in 384-well white Opti-Plates (PerkinElmer Life Sciences) Apoptosis-induction by DR5-selective Apo2L/TRAIL 2207 and incubated for 30 min at room temperature. Biotinylated Apo2L/ TRAIL competitor was then added (100 ng/ml, 5 l per well). After a further 2-h incubation of the plates at room temperature, a mixture of AlphaScreen streptavidin donor beads and protein A acceptor beads (PerkinElmer Life Sciences) was added ( ⁄100 dilution of each bead in assay buffer, 10 l/well). The plates were incubated for1hat room temperature protected from light and subsequently read using an AlphaQuest® plate reader. Surface Plasmon Resonance Measurements of Receptor Binding— Dissociation constants for the direct binding of Apo2L/TRAIL variants to immobilized receptors were determined by SPR measurements on a BIAcore 3000 instrument as previously described (20). Affinities were also assessed by using a competition assay in which binding of the ligand to immobilized DR5-Fc was competed with receptor in solution. These experiments employed a flow cell having a high density (15,000 resonance units) of immobilized DR5-Fc such that the initial rate of binding was linearly dependent on the concentration of free ligand. Competition experiments were conducted by preparing a series of solu- tions having a fixed concentration of ligand but a varied concentration FIG.1. Part of the contact (patch A) observed in x-ray crystal of receptor. After incubating for2hto allow equilibration to occur, these structure of the Apo2L/TRAIL DR5 complex (Hymowitz et al. solutions were injected over the DR5-Fc surface. A linear fit of the (11); Protein Data Bank ID code 1D0G). The backbone of DR5 observed sensorgrams was used to extract the binding rate. Analysis of (residues 67– 69, 91-104) is shown as the green tube with selected side the binding rate as a function of competing receptor concentration chains. Apo2L/TRAIL is shown as the space-filling model. Apo2L/ enabled calculation of IC values by using a four-parameter curve fit. TRAIL residues included in the libraries are colored aqua or pink, with the residues mutated in Apo2L.DR5– 8 colored pink. Labels for DR5 RESULTS residues are underlined. Phage Display of Apo2L/TRAIL—The successful application of the phage display approach to the probing of receptor bind- in the phage display libraries on the basis of an examination of ing determinants requires proper assembly of trimeric Apo2L/ the x-ray structure determined for the Apo2LDR5-ECD com- TRAIL on the phage surface. To this end we used a construct plex (11). “Patch A” on DR5 involves the receptor 60 (residues having the Apo2L/TRAIL gene fused to the geneIII protein of 67– 69) and 90 (residues 91–104) loops, which interact with a M13 bacteriophage with an in-frame amber stop codon. In cluster of Apo2L/TRAIL residues centered on Gln-205. “Patch strains of E. coli capable of suppressing amber stop codons B” on DR5 involves the receptor 50 loop (residues 51– 65), (supE genotype), a Gln is inserted at the amber stop resulting which makes contact with residues 155–162 and 215–218 of the in secretion of an Apo2L/TRAIL-geneIII fusion protein into the ligand. Only residues within or near the patch A contact with periplasm. Because suppression of amber stop codons is only the receptor (Fig. 1) were considered for mutagenesis. Because 10 –30% efficient, free Apo2L/TRAIL is secreted as well. When patch B is smaller than patch A, is more hydrophobic, and is supplied with assembly proteins by co-infection with helper composed of ligand residues that are more conserved across phage, phage particles that incorporate the Apo2L/TRAIL- TNF superfamily members, it has been proposed that patch A geneIII fusion protein into their surface can be produced. Tri- is more important for specificity (11). However, alanine substi- meric Apo2L/TRAIL can be produced by assembly with either tution showed that several Apo2L/TRAIL residues in patch A another fusion protein or with free Apo2L/TRAIL. However, (i.e. Gln-205, Tyr-237, Leu-239) make a large contribution to because each phage particle will display only a few copies of the the free energy of binding for all receptors tested (20). These geneIII fusion protein (18), it seems likely that trimerization is sites contact residues on DR5 that are conserved in DR4 and, driven by the free Apo2L/TRAIL produced when the amber stop thus, seemed unlikely to yield receptor-selective variants. is not suppressed. Therefore, we excluded these residues from the libraries. In Initial tests of the pAPOK4 vector suggested poor display on contrast, sites that gave only modest changes in affinity (5- the phage of correctly assembled Apo2L/TRAIL. Specific bind- fold) when replaced with alanine appeared more likely to in- ing increased if the phage were produced by growth at 30 °C crease receptor selectivity when mutated. An example of this rather than 37 °C and by replacement of the phoA promoter type of site is Gln-193, where alanine substitution causes a with the tac promoter (phagemid pAPOK4.2). Optimum display 1.7-fold decrease in affinity for DR4 but has no effect on binding (100-fold enrichment) was obtained for phage production at to DR5 and DcR2 (20). Further changes in receptor specificity 30 °C with induction of the tac promoter by addition of 1 M might be obtained by substitution of Gln-193 with a residue isopropyl 1-thio--D-galactopyranoside. other than alanine. This rationale was used to design two Phage displaying Apo2L/TRAIL showed increased nonspe- tailored Apo2L/TRAIL libraries; one including sites 189, 191, cific binding to a panel of control proteins relative to the non- 193, 199, 201, and 209 randomized (“DR4 library”), and the specific binding observed for KO7 helper phage (data not other with sites 189, 191, 193, 264, 266, 267, and 269 random- shown). In addition, KO7 helper phage displayed high nonspe- ized (“DR5 library”). As shown in Fig. 1, these residues are on cific binding to purified Apo2L/TRAIL. Sorting of a preliminary the periphery of the patch A contact observed in the x-ray Apo2L/TRAIL library having residues 213, 215, 216, 218, 220, crystal structure (11). and 222 randomized yielded a mutant of Apo2L/TRAIL Sorting for Receptor-selective Variants—The DR4 library (Y213W/S215D) that showed decreased helper phage binding. was sorted for two rounds against DR4-Fc coated on microtiter The Y213W/S215D mutant also gave increased display on wells followed by three rounds of sorting for DR4 binding in the phage, as measured by binding to an anti-Apo2L/TRAIL anti- presence of competing DR5-Fc. For sorting rounds 3, 4, and 5, body, with comparable affinity for DR4-Fc but a 10-fold reduc- the phage were incubated with 50, 250, and 750 n M DR5-Fc, tion in apparent affinity for DR5-Fc. The Y213W/S215D sub- respectively, for 30 min before the addition of these solutions to stitutions were incorporated into libraries designed for DR4-Fc-coated plates. With the DR5 library, phage were sorted improved affinity against DR4. for four rounds against DR5-Fc-coated wells followed by four Design and Production of Apo2L/TRAIL Phage Display Li- rounds with DR4-Fc as competitor. In rounds 5, 6, 7, and 8, braries—Residues in Apo2L/TRAIL were chosen for inclusion phage were incubated with 1, 10, 100, and 500 nM DR4-Fc, 2208 Apoptosis-induction by DR5-selective Apo2L/TRAIL FIG.2. Competitive phage ELISA of phage clones selected from the DR4 library. Binding to immobilized DR4-Fc was competed by increasing solution concentrations of DR4-Fc (panel A) or DR5-Fc (panel B). Phage clones Apo2L.DR4 – 8 (f), Apo2L.DR4 –9 (‚), Apo2L.DR4 –11 (Œ), Apo2L.DR4 –13 (), and wild-type () were tested. respectively, before selection for DR5-Fc binding. Both the DR4 The AlphaQuest® assay indicated that the two DR4-selective variants retained high affinity binding to DR4-Fc (Table I). and DR5 libraries gave strong, specific enrichment even in the presence of the competing receptor. Binding to both DR5 and OPG was significantly reduced, whereas the reduction in affinity for DcR1 and DcR2 was more Individual clones were isolated from the fifth round (DR4 library) or eighth round (DR5 library) of sorting and tested for modest. Both of the DR4-selective proteins showed a large decrease in potency for apoptosis induction relative to wild- receptor specificity by phage ELISA (see “Experimental Proce- dures”), and then the DNA sequence of receptor-selective type Apo2L/TRAIL (ED  12.9  5.2 ng/ml) or FLAG-Apo2L/ clones was determined. The amino acid identities deduced from TRAIL (ED  48 ng/ml). FLAG-Apo2L.DR4 – 8 showed no the DNA sequence for the library positions are shown in Sup- increase in potency upon cross-linking, whereas FLAG- plemental Tables I (DR4-selective) and II (DR5-selective). No Apo2L.DR4 –9 showed a relatively modest 6-fold gain in po- spurious sequence changes outside of the library positions were tency upon cross-linking. The apoptosis-inducing activity of detected. Analysis of four of the clones from the DR4 library by FLAG-Apo2L.DR4 – 8 on SK-MES could be inhibited by a neu- competition phage ELISA (Fig. 2) confirmed that the sorting tralizing anti-DR4 monoclonal antibody but not by a neutral- strategy yielded receptor-selective variants. These four clones izing antibody specific for DR5 (data not shown). This finding bound DR4-Fc with apparent high affinity, whereas binding to suggests that the residual activity of FLAG-Apo2L.DR4 – 8 re- DR5-Fc was undetectable. These DR4-selective variants all sults from binding to DR4 and does not reflect a weak interac- differed from the wild-type sequence at four of six library tion with DR5. positions. Substitution of Tyr-189 with Ala appeared to be All of the tested DR5-selective variants showed significant important for DR4 selectivity, since all four clones had reduction in affinity for DcR1, OPG, and DR4 while maintain- this mutation. ing high affinity for DR5 (Table I). The variation in affinity for Variants with selectivity for DR5 (Supplemental Table II) all DR5 ranged from a slight improvement (FLAG-Apo2L.DR5– 8) had at least three amino acid changes, and clones with six to about a 3-fold decrease (FLAG-Apo2L.DR5–2). The change in substitutions occurred most frequently. Although this library affinity for DcR2 was variable and, with the exception of FLAG- was subjected to eight rounds of sorting, a consensus sequence Apo2L.DR5– 8, was smaller than observed for the other recep- was not obtained, and some positions, most notably 264, re- tors. FLAG-Apo2L.DR5– 8 was the most selective variant tained high diversity. Substitution of Gln-193 with positively showing good binding to DR5 but significantly reduced affinity charged Lys or Arg appeared to be important for DR5 selectiv- for the other four receptors. Surprisingly, all of the DR5-selec- ity. A negatively charged residue at either position 267 or 269, tive variants retained a high level of apoptosis-inducing activ- but not both, was favored. Replacement of Ile-266 with Leu was ity (Table I). Changes in activity varied from a 2.6-fold increase a frequent change in these variants. Interestingly, the Y189A in ED for FLAG-Apo2L.DR5–1 to a 6.4-fold decrease for mutation was also observed in the DR5-selective variants. FLAG-Apo2L.DR5– 8. The potency of all of the DR5-selective Apoptosis Induction by Receptor-selective Variants—To char- variants increased significantly upon anti-FLAG cross-linking acterize the receptor-selective variants further, we produced to the same level as measured for the cross-linked wild-type FLAG-tagged versions of two of the DR4-selective and three of protein. There was not a strict 1:1 correspondence between the the DR5-selective mutants. These variants were profiled for affinity measured for DR5 and the potency measured for apo- receptor binding by using an AlphaQuest® assay and assayed ptosis induction. This likely reflects the use of Fc fusion pro- for apoptosis induction on SK-MES lung carcinoma cells. SK- teins to determine affinities, since the bivalency may suppress MES cells express DR4 and DR5 at similar levels (data not affinity differences. shown) and are sensitive to apoptosis induction by wild-type The specificity of a few of the variants was further confirmed Apo2L/TRAIL. Inclusion of the FLAG tag enables testing of by using SPR to measure the dissociation constants for binding oligomerized forms of Apo2L/TRAIL or its variants formed by to DR4 and DR5. As shown in Table II, FLAG-Apo2L.DR4 – 8 cross-linking the tagged protein with anti-FLAG antibody. An- and FLAG-Apo2L.DR4 –9 bind to DR4 with affinity nearly ti-FLAG cross-linking of tagged wild-type Apo2L/TRAIL re- equivalent to that measured for the wild-type protein, whereas sulted in an 70-fold increased potency for apoptosis induction binding to DR5 was significantly reduced. The apparent affin- in SK-MES (Table I). ity of FLAG-Apo2L.DR4 – 8 for DR5 was too weak to measure Apoptosis-induction by DR5-selective Apo2L/TRAIL 2209 TABLE I Receptor binding and apoptosis induction of FLAG-tagged Apo2L/TRAIL IC values from an AlphaQuest® assay of receptor binding are reported as the ratio of mutant to wild-type FLAG-Apo2L/TRAIL. “” indicates that the IC value was greater than the highest mutant concentration tested in the assay. Concentrations of Apo2L/TRAIL variants required to induce 50% apoptosis (ED ) on SK-MES cells are shown for the absence () or presence ( )of2 g/ml M2 anti-FLAG antibody. IC ratio (mutant/wt) ED (ng/ml) 50 50 Protein DcR1 DcR2 OPG DR4 DR5 FLAG-Apo2L/TRAIL 1 1 1 1 1 48.4 0.7 FLAG-Apo2L.DR4–8 5.5 15 28 1 25 4000 4100 FLAG-Apo2L.DR4–9 4.0 18 28 1.1 25 1000 170 FLAG-Apo2L.DR5–1 300 32 700 30 2.4 126 1.3 FLAG-Apo2L.DR5–2 300 5.0 700 30 2.8 67.8 1.0 FLAG-Apo2L.DR5–8 300 1900 700 30 0.7 7.6 0.3 TABLE II SPR measurement of DR4 and DR5 binding affinity K ratios were calculated relative to wild-type FLAG-Apo2L/TRAIL. K ratio (mutant/wt) Variant Amino acid changes DR4 DR5 FLAG-Apo2L.DR4–8 Trp-213; Asp-215; Ala-189; Ser-193; Val-199;Arg-201 2.3 NB FLAG-Apo2L.DR4–9 Trp-213; Asp-215; Ala-189; Ser-193; Arg-199; Arg-201 1.5 61 FLAG-Apo2L.DR5–8 Gln-189; Lys-191; Arg-193; Arg-264; Leu-266; Gln-267 NB 0.8 Amino acid changes are relative to the wild-type protein. No binding (NB) was detected at the highest concentration (500 nM) used in the SPR measurements. by SPR. In contrast, the DR5-selective variant (FLAG- Apo2L.DR5– 8) did not appear capable of binding to DR4 but, consistent with the AlphaQuest® data, displayed high affinity binding to DR5. A very slow on-rate was measured for interac- tion of FLAG-Apo2L.DR4 – 8 with DR5 and also for binding of FLAG-Apo2L.DR5– 8 to DR4. The slow binding kinetics pre- cluded accurate determination of the binding constants by SPR for these weak interactions. Because FLAG-Apo2L.DR4 – 8 and FLAG-Apo2L.DR5– 8 gave the greatest receptor selectivity, these variants were used for testing the sensitivity of additional cancer cell lines to receptor-specific ligands. Colo205, a colon carcinoma cell line, expresses slightly more DR5 than DR4 and was quite sensitive to trimeric Apo2L/TRAIL (ED  4.2  1.8 ng/ml). FLAG- Apo2L.DR4 – 8 induced apoptosis only weakly (ED  1150 ng/ml) in Colo205 (Fig. 3A). The addition of anti-FLAG anti- body increased the potency of this variant about 8-fold (ED 150 ng/ml). FLAG-Apo2L.DR5– 8 showed increased potency against Colo205 cells (ED  0.9 ng/ml; Fig. 3B), and its activity was increased 6-fold by anti-FLAG cross-linking. A similar pattern of results was obtained against Colo320, a colon carcinoma cell line that expresses similar amounts of DR4 and DR5 (data not shown). FLAG-Apo2L.DR4 – 8 was inactive against Colo320 cells even if cross-linked with anti-FLAG an- tibody. FLAG-Apo2L.DR5– 8 weakly induced apoptosis in Colo320 cells, but the activity of the cross-linked form was equivalent to that observed for anti-FLAG cross-linked wild- type Apo2L/TRAIL. The breast carcinoma cell line MDA-MB-231 expresses high levels of both DR4 and DR5 yet displays a weaker apoptotic response to Apo2L/TRAIL treatment than observed with Colo205 cells (5). FLAG-Apo2L.DR4 – 8 was completely inactive against MDA-MB-231 (Fig. 4A). FLAG-Apo2L.DR5– 8 showed modest activity against MDA-MB-231, and the activity was significantly increased upon anti-FLAG cross-linking (Fig. 4B). The Jurkat T leukemia cell line expresses DR5 but not DR4. As expected, FLAG-Apo2L.DR4 – 8 did not induce apoptosis in this cell line FIG.3. Assay of apoptosis-induction on Colo205 colon carci- even upon cross-linking (Fig. 5A). FLAG-Apo2L.DR5– 8 showed noma cells by FLAG-tagged Apo2L/TRAIL mutants. A fluores- cence assay (20) was used to test DR4-selective (panel A) or DR5- modest activity against Jurkat cells, and the activity was dra- selective (panel B) mutants. antiFLAG indicates that 2 g/ml M2 matically increased (ED  0.05 ng/ml) by cross-linking (Fig. antibody (Sigma) was added along with the specified concentration of 5B). The small increase in DR5 affinity observed for FLAG- Apo2L/TRAIL mutant. Curves represent fitting using the four-param- Apo2L.DR5– 8 appears to be amplified into a larger apoptotic eter equation. RFU, relative fluorescence units. 2210 Apoptosis-induction by DR5-selective Apo2L/TRAIL FIG.5. Apoptosis induction on Jurkat cells by receptor-selec- tive mutants of Apo2L/TRAIL. A fluorescence (20) assay was used to test DR4-selective (panel A) or DR5-selective (panel B) mutants. anti- FIG.4. Assay of apoptosis induction on MDA-MB-231 breast FLAG indicates that 2 g/ml M2 antibody (Sigma) was added along carcinoma cells by FLAG-tagged Apo2L/TRAIL mutants. A fluo- with the specified concentration of Apo2L/TRAIL variant. Curves rep- rescence assay (20) was used to test DR4-selective (panel A) or DR5- resent fitting using the four-parameter equation. RFU, relative fluores- selective (panel B) mutants. antiFLAG indicates that 2 g/ml M2 anti- cence units. body (Sigma) was added along with the specified concentration of Apo2L/TRAIL mutant. Curves represent fitting using the four-param- eter equation. RFU, relative fluorescence units. signal on Jurkat cells upon cross-linking. Normal hepatocytes are resistant to Apo2L/TRAIL unless the ligand is aggregated (8). The receptor-selective variants were evaluated for their ability to induce apoptosis in normal hepatocytes using an assay that measures activation of caspase-3 and -7. FLAG-Apo2L.DR4 – 8 did not induce caspase activation even upon cross-linking with anti-FLAG antibody (Fig. 6). Consistent with previous results (8), Apo2L/TRAIL did not increase caspase activity except after cross-linking. Similar to the wild-type ligand, FLAG- Apo2L.DR5– 8 was inactive as a trimer and induced caspase activation upon anti-FLAG cross-linking. Selection of Substitutions Necessary to Give Selectivity for DR5 Binding—To determine the minimum number of amino FIG.6. Caspase activation in cynomolgus monkey hepatocytes acid changes required for DR5 selectivity while maintaining upon the addition of the indicated concentrations of FLAG- Apo2L/TRAIL in the presence and absence of 2 g/ml M2 anti- high bioactivity, we did additional phage display experiments FLAG antibody. RFU, relative fluorescence units. followed by testing of selected variants. Two phage libraries were constructed in which the residues at 189, 191, 193, 264, 266, and 267 were allowed to vary as either the wild-type of competing DR4-Fc. By the third round of sorting, 1000-fold residue, the amino acid found in Apo2L.DR5– 8, or a limited set enrichment was obtained, and individual clones were selected of similar amino acids. These libraries were pooled and sorted for further analysis. Spot ELISA indicated that 28 of 28 clones for one round against DR5-Fc coated on microtiter wells fol- tested were positive for DR5 binding, whereas only one was lowed by two rounds of sorting for DR5 binding in the presence functional for DR4 binding. The amino acid identities deduced Apoptosis-induction by DR5-selective Apo2L/TRAIL 2211 TABLE III Relative receptor binding affinity and apoptosis-induction by variants of Apo2L.DR5–8 DR4 binding DR5 binding Variant Amino acid changes Apoptosis, ED K IC K IC D 50 D 50 Apo2L.DR5–8 Gln-189; Lys-191; Arg-193; Arg-264; Leu-266; Gln-267 NB 333 0.3 0.5 0.6 Apo2L.DR5–8E Gln-189; Lys-191 19 333 1.1 1.4 16 Apo2L.DR5–8B Gln-189; Lys-191; Arg-193 56 333 1.4 0.8 15 Apo2L.DR5–8D Gln-189; Lys-191; Leu-266 NB 333 NB NB ND Apo2L.DR5–8C Gln-189; Lys-191; Arg-193; Leu-266 59 72 0.5 0.7 0.9 Amino acid changes are relative to wild-type Apo2L/TRAIL. Variants were produced as untagged 114 –281 Apo2L/TRAIL. Values for receptor binding and apoptosis induction (ED ) on SK-MES are the ratio relative to wild-type Apo2L/TRAIL. NB indicates no detectable binding. Not determined. from the DNA sequence at the library positions for the clones most TNF superfamily members bind to their receptors at the positive for DR5 binding, but not DR4 binding, are shown in interfaces between subunits of the ligand homotrimer, a func- Supplemental Table III. These results suggest that the residue tional receptor binding site can be formed only when a ligand identity at positions 264 and 267 may not be important for DR5 trimer is properly assembled. Trimer assembly was promoted selectivity since there was no preference at these positions for by using a construct having an amber stop codon since both the amino acid found in variant DR5– 8. In contrast, the amino monomer Apo2L/TRAIL, and a geneIII fusion protein could be acids found in variant DR5– 8 predominated at positions 189, produced in the same cell. An excess of free monomer most 191, 193, and 266, indicating that one or more of these sites likely drives oligomerization with the fusion protein. Indeed, in may be important for DR5 selectivity. At position 189 there was sorting of the second DR5 library (Supplemental Table III) a strong preference for Gln, which can be expressed through a there was selection for an additional amber stop codon at Gln codon or through suppression of an amber stop codon. residue 189 where a Gln residue was compatible with DR5 A few variants (Apo2L.DR5– 8B-E) were produced as an un- binding. A second amber stop would increase the ratio of mon- tagged 114 –281 fragment (7) of Apo2L/TRAIL and tested for omer to fusion protein because two stops must be suppressed to receptor binding and bioactivity. Apo2L.DR5– 8E differs from express the geneIII fusion protein. Functional display of wild-type Apo2L/TRAIL by Y189Q and R191K. Apo2L.DR5– 8B Apo2L/TRAIL was also aided by lowering the growth temper- has the additional substitution of Q193R, and Apo2L.DR5– 8C ature and by using a strong, inducible promoter. Selection of also has I266L besides these substitutions. Apo2L.DR5– 8D has the DR4 library was enhanced through incorporation of the Y189Q, R191K, and I266L. Dissociation constants for receptor amino acid substitutions Y213W/S215D, which reduced non- binding were determined from SPR data for direct binding of specific binding of Apo2L/TRAIL. For both the DR4 and DR5 each variant to immobilized receptor. Because all of these vari- libraries, nonspecific binding was further reduced by using ants had decreased binding to DR4-Fc, it was necessary to use competitive selection. As with other protein-protein interac- a sensor chip with a high density of immobilized DR4-Fc to tions (25), competitive selection proved to be a powerful ap- detect sufficient signal in direct binding measurements. For proach for increasing the specificity of interaction between measurements with wild-type Apo2L/TRAIL, a 4-fold greater Apo2L/TRAIL and its death receptors. binding affinity was calculated from high density as opposed to Alanine-scanning mutagenesis has shown that the binding low density binding surfaces (data not shown). Thus, a compet- sites on Apo2L/TRAIL for the various receptors are highly itive binding assay in which Apo2L/TRAIL binding to immobi- similar, with certain key residues (e.g. Gln-205, Tyr-216) mak- lized DR5-Fc was competed with DR4-Fc or DR5-Fc in solution ing a large free energy contribution to the binding of each of the was also used to assess receptor affinities. As shown in Table five receptors (20). Although only the structure of the III, all of the variants showed decreased binding to DR4. The Apo2LDR5 complex is known, many of the residues on DR5 competition assay suggested a larger magnitude decrease in that are in contact with Apo2L/TRAIL are conserved in DR4. affinity for DR4 than the direct binding assay. With the excep- Thus, it is perhaps not surprising that multiple amino acid tion of Apo2L.DR5– 8D, these proteins displayed high affinity substitutions were required to achieve receptor selectivity. By binding to DR5. Binding of Apo2L.DR5– 8D to either DR4 or comparison, TNF mutants functional for binding to the p55 DR5 was not detectable, suggesting that this variant was mis- receptor, but not the p75 receptor, could be obtained by com- folded. Indeed, this sequence was found only in 1 of 51 clones bining two or three amino acid changes (26). Examination of from phage sorting. The two amino acid changes in the Apo2LDR5 structure (Fig. 1) suggests that the amino acid Apo2L.DR5– 8E resulted in a significant decrease in affinity for changes in Apo2L.DR5– 8 would disfavor interactions with DR4 and a slight decrease in DR5 binding, but a 16-fold reduc- DR4 residues Lys-67, Asp-69, and Asn-95. The equivalent res- tion in apoptosis-inducing activity. The addition of Q193R idues in DR5 are Asp-67, Gly-69, and Asp-95. The R191K (Apo2L.DR5– 8B) had a slight effect on DR5 affinity and did not substitution may oppose interaction with Lys-67 on DR4. Re- restore bioactivity. Incorporation of I266L (Apo2L.DR5– 8C) placement of Gln-193 with a positively charged residue may resulted in a modest improvement in DR5 affinity and restored interfere sterically with engagement of DR4 residue Asp-69, full bioactivity. The results for Apo2L.DR5– 8 indicate that the whereas the Gly in DR5 may accommodate a larger side chain. H264R and/or D267Q substitutions give further selectivity Ile-266 is a buried residue in Apo2L/TRAIL, and thus, the effect against DR4 binding while providing modest improvements in of the I266L substitution must be indirect, perhaps occurring DR5 affinity and bioactivity. through a change in the conformation of residue 267. D267Q DISCUSSION may remove a favorable electrostatic interaction with the pos- We have shown here that Apo2L/TRAIL can be expressed on itive charge on DR4 residue Lys-67. H264R would be positioned to provide a positive charge for interaction with DR5 Asp-95 M13 bacteriophage in functional form, allowing selection of clones from mutant libraries for receptor-specific binding. To while discriminating against neutral residue Asn-95 in DR4. our knowledge this is the first successful application of phage The effect of the Y189Q mutation is difficult to explain, since display technology to a TNF-superfamily member. Because Tyr-189 interacts with the backbone of DR5 and with residues 2212 Apoptosis-induction by DR5-selective Apo2L/TRAIL REFERENCES that are identical between DR4 and DR5. This substitution 1. Ashkenazi, A. (2002) Nat. Rev. Cancer 2, 420 – 430 may facilitate a conformational change in the binding site that 2. Pan, G., O’Rourke, K., Chinnaiyan, A. M., Gentz, R., Ebner, R., Ni, J., and promotes receptor selectivity. A more complete understanding Dixit, V. M. (1997) Science 276, 111–113 3. Sheridan, J. P., Marsters, S. A., Pitti, R. M., Gurney, A., Skubatch, M., of these results will require the determination of free and Baldwin, D., Ramakrishnan, L., Gray, C. L., Baker, K., Wood, W. I., God- receptor-bound structures of Apo2L.DR5– 8. dard, A. D., Godowski, P., and Ashkenazi, A. (1997) Science 277, 818 – 821 The DR5-selective variants retained a high level of apo- 4. Pan, G., Ni, J., Wei, Y. F., Yu, G., Gentz, R., and Dixit, V. M. (1997) Science 277, 815– 818 ptosis-inducing activity against cancer cells, and the activity 5. Kischkel, F. C., Lawrence, D. A., Chuntharapai, A., Schow, P., Kim, K. J., and was significantly increased when the ligand was cross-linked Ashkenazi, A. (2000) Immunity 12, 611– 620 6. Ashkenazi, A., and Dixit, V. M. (1998) Science 281, 1305–1308 through an N-terminal FLAG tag. In contrast, the DR4-selec- 7. Ashkenazi, A., Pai, R. C., Fong, S., Leung, S., Lawrence, D. A., Marsters, S. A., tive variants had significantly reduced apoptosis-inducing ac- Blackie, C., Chang, L., McMurtrey, A. E., Hebert, A., DeForge, L., Koume- nis, I. L., Lewis, D., Harris, L., Bussiere, J., Koeppen, H., Shahrokh, Z., and tivity, and their potency was only modestly increased by anti- Schwall, R. H. (1999) J. Clin. Investig. 104, 155–162 FLAG cross-linking. These results are inconsistent with the 8. Lawrence, D., Shahrokh, Z., Marsters, S., Achilles, K., Shih, D., Mounho, B., conclusions of Muhlenbeck et al. (27), who proposed that DR5 Hillan, K., Totpal, K., DeForge, L., Schow, P., Hooley, J., Sherwood, S., Pai, R., Leung, S., Khan, L., Gliniak, B., Bussiere, J., Smith, C. A., Strom, S. S., (TRAIL-R2) signals apoptosis only in response to cross-linked Kelley, S., Fox, J. A., Thomas, D., and Ashkenazi, A. (2001) Nat. Med. 7, Apo2L/TRAIL, whereas DR4 (TRAIL-R1) can respond either 383–385 9. Walczak, H., Miller, R. E., Ariail, K., Gliniak, B., Griffith, T. S., Kubin, M., to non-cross-linked or cross-linked ligand. A possible expla- Chin, W., Jones, J., Woodward, A., Le, T., Smith, C., Smolak, P., Goodwin, nation for this discrepancy is that in the Muhlenbeck study R. G., Rauch, C. T., Schuh, J. C., and Lynch, D. H. (1999) Nat. Med. 5, 157–163 some of the cell lines were treated with cycloheximide to 10. Banner, D. W., D’Arcy, A., Janes, W., Gentz, R., Schoenfeld, H. J., Broger, C., enhance sensitivity to apoptosis induction. Cycloheximide Loetscher, H., and Lesslauer, W. (1993) Cell 73, 431– 445 may decrease levels of cytoplasmic factors that repress death 11. Hymowitz, S. G., Christinger, H. W., Fuh, G., Ultsch, M., O’Connell, M., Kelley, R. F., Ashkenazi, A., and de Vos, A. M. (1999) Mol. Cell 4, 563–571 receptor signaling (28). This treatment was not required to 12. Mongkolsapaya, J., Grimes, J. M., Chen, N., Xu, X. N., Stuart, D. I., Jones, render the cancer cell lines we studied sensitive to the DR5- E. Y., and Screaton, G. R. (1999) Nat. Struct. Biol. 6, 1048 –1053 13. Park, Y. C., Burkitt, V., Villa, A. R., Tong, L., and Wu, H. (1999) Nature 398, selective variants. 533–538 Although the SK-MES and Colo205 cancer cell lines express 14. McWhirter, S. M., Pullen, S. S., Holton, J. M., Crute, J. J., Kehry, M. R., and Alber, T. (1999) Proc. Natl. Acad. Sci. U. S. A. 96, 8408 – 8413 both DR4 and DR5 at comparable levels, the Apo2L.DR5– 8 15. Kischkel, F. C., Lawrence, D. A., Tinel, A., LeBlanc, H., Virmani, A., Schow, P., variant showed increased apoptosis-inducing activity com- Gazdar, A., Blenis, J., Arnott, D., and Ashkenazi, A. (2001) J. Biol. Chem. 276, 46639 – 46646 pared with wild-type Apo2L/TRAIL against these cell lines, 16. Chuntharapai, A., Dodge, K., Grimmer, K., Schroeder, K., Marsters, S. A., whereas the Apo2L.DR4 – 8 variant was much less active. In Koeppen, H., Ashkenazi, A., and Kim, K. J. (2001) J. Immunol. 166, MDA-MB-231 cells, which also express both DR4 and DR5 and 4891– 4898 17. Ichikawa, K., Liu, W., Zhao, L., Wang, Z., Liu, D., Ohtsuka, T., Zhang, H., are sensitive to wild-type Apo2L/TRAIL, the Apo2L.DR4 – 8 Mountz, J. D., Koopman, W. J., Kimberly, R. P., and Zhou, T. (2001) Nat. mutant was inactive even upon cross-linking, whereas Med. 7, 954 –960 18. Lowman, H. B., and Wells, J. A. (1993) J. Mol. Biol. 234, 564 –578 Apo2L.DR5– 8 showed weak activity without cross-linking and 19. Sidhu, S. S., Li, B., Chen, Y., Fellouse, F. A., Eigenbrot, C., and Fuh, G. (2004) full activity upon cross-linking. In hepatocytes, which are re- J. Mol. Biol. 338, 299 –310 20. Hymowitz, S. G., O’Connell, M. P., Ultsch, M. H., Hurst, A., Totpal, K., sistant to non-cross-linked Apo2L/TRAIL but sensitive to the Ashkenazi, A., de Vos, A. M., and Kelley, R. F. (2000) Biochemistry 39, cross-linked ligand, the DR5-selective variant induced the 633– 640 21. Lee, G. F., and Kelley, R. F. (1998) J. Biol. Chem. 273, 4149 – 4154 same responses as the wild-type ligand, whereas the DR4- 22. Kunkel, T. A., Roberts, J. D., and Zakour, R. A. (1987) Methods Enzymol. 154, selective mutant was inactive. Taken together, these results 367–382 suggest that DR5 may contribute more than DR4 to Apo2L/ 23. Sidhu, S. S., Lowman, H. B., Cunningham, B. C., and Wells, J. A. (2000) Methods Enzymol. 328, 333–363 TRAIL-induced apoptosis in cancer or normal cells that express 24. LeBlanc, H., Lawrence, D., Varfolomeev, E., Totpal, K., Morlan, J., Schow, P., both death receptors. Thus, it may be possible to enhance the Fong, S., Schwall, R., Sinicropi, D., and Ashkenazi, A. (2002) Nat. Med. 8, 274 –281 anti-tumor activity of soluble trimeric Apo2L/TRAIL further by 25. Dennis, M. S., and Lazarus, R. A. (1994) J. Biol. Chem. 269, 22137–22144 engineering mutants of this protein that have increased affin- 26. Loetscher, H., Stueber, D., Banner, D., Mackay, F., and Lesslauer, W. (1993) J. Biol. Chem. 268, 26350 –26357 ity for DR5 but retain a stable trimeric structure. 27. Muhlenbeck, F., Schneider, P., Bodmer, J. L., Schwenzer, R., Hauser, A., Schubert, G., Scheurich, P., Moosmayer, D., Tschopp, J., and Wajant, H. Acknowledgments—We thank the oligonucleotide chemistry group at (2000) J. Biol. Chem. 275, 32208 –32213 Genentech for synthesis of oligonucleotides, the DNA sequencing group 28. Wajant, H., Haas, E., Schwenzer, R., Muhlenbeck, F., Kreuz, S., Schubert, G., for sequencing of phage clones, and Scot Marsters for supplying recep- Grell, M., Smith, C., and Scheurich, P. (2000) J. Biol. Chem. 275, tor-Fc fusion proteins. 24357–24366 http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Journal of Biological Chemistry American Society for Biochemistry and Molecular Biology

Receptor-selective Mutants of Apoptosis-inducing Ligand 2/Tumor Necrosis Factor-related Apoptosis-inducing Ligand Reveal a Greater Contribution of Death Receptor (DR) 5 than DR4 to Apoptosis Signaling *

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American Society for Biochemistry and Molecular Biology
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Copyright © 2005 Elsevier Inc.
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0021-9258
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10.1074/jbc.m410660200
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Abstract

THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 280, No. 3, Issue of January 21, pp. 2205–2212, 2005 © 2005 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in U.S.A. Receptor-selective Mutants of Apoptosis-inducing Ligand 2/Tumor Necrosis Factor-related Apoptosis-inducing Ligand Reveal a Greater Contribution of Death Receptor (DR) 5 than DR4 to □ S Apoptosis Signaling* Received for publication, September 16, 2004, and in revised form, November 1, 2004 Published, JBC Papers in Press, November 1, 2004, DOI 10.1074/jbc.M410660200 Robert F. Kelley‡§, Klara Totpal , Stephanie H. Lindstrom‡, Mary Mathieu‡, Karen Billeci , Laura DeForge , Roger Pai**, Sarah G. Hymowitz‡, and Avi Ashkenazi From the Departments of ‡Protein Engineering, Molecular Oncology, **Process Sciences, and Assay and Automation Technology, Genentech, Inc., South San Francisco, California 94080 and OPG lack a cytoplasmic domain, whereasDcR2 has a trun- Apoptosis-inducing ligand 2 (Apo2L), also called tumor necrosis factor-related apoptosis-inducing ligand cated death domain that is non-functional for apoptosis initia- (TRAIL), triggers programmed cell death in various types tion (1, 6). Like several other members of the TNF superfamily, of cancer cells but not in most normal cells. Apo2L/TRAIL Apo2L/TRAIL is synthesized as a type II transmembrane pro- is a homotrimeric protein that interacts with five recep- tein that can be proteolytically cleaved to release a soluble, tors: death receptor 4 (DR4) and DR5 mediate apoptosis homotrimeric molecule. A recombinant version of soluble ho- activation, whereas decoy receptor 1 (DcR1), DcR2, and motrimeric Apo2L/TRAIL (residues 114 –281) induces apo- osteoprotegerin counteract this function. Many cancer ptosis in various cancer cell lines but not in normal cells (7, 8). cell lines express both DR4 and DR5, and each of these Administration of soluble Apo2L/TRAIL in mouse xenograft receptors can initiate apoptosis independently of the models of human cancer results in marked anti-tumor activity other. However, the relative contribution of DR4 and DR5 without systemic toxicity (7–9). These results have prompted to ligand-induced apoptosis is unknown. To investigate further evaluation of Apo2L/TRAIL as a potential therapeutic this question, we generated death receptor-selective agent for human cancer. Apo2L/TRAIL variants using a novel approach that en- Structural studies show that homotrimeric TNF superfamily ables phage display of mutated trimeric proteins. Selec- ligands bind three receptor molecules (10 –12), suggesting that tive binding to DR4 or DR5 was achieved with three to six-ligand amino acid substitutions. The DR4-selective the basic functional signaling unit is trimeric. This notion is Apo2L/TRAIL variants examined in this study showed a further supported by the trimeric structure of certain signaling markedly reduced ability to trigger apoptosis, whereas adaptor molecules that act downstream of the receptors, such the DR5-selective variants had minimally decreased or as the TNF receptor-associated factors (13, 14). Further cross- slightly increased apoptosis-inducing activity. These re- linking of receptors beyond the trimeric unit in some cases can sults suggest that DR5 may contribute more than DR4 to lead to stronger signal initiation; this can be modeled by anti- Apo2L/TRAIL-induced apoptosis in cancer cells that ex- body-mediated cross-linking of N-terminally tagged ligand, press both death receptors. perhaps mimicking its transmembrane form (1). Binding of Apo2L/TRAIL to DR4 and/or DR5 leads to recruitment of the adaptor FADD (Fas-associated death domain) by the cytoplas- Apo2L (or TRAIL), a member of the tumor necrosis factor mic death domain followed by recruitment and activation of the (TNF) superfamily, induces apoptosis in a broad spectrum of apoptosis initiators caspase-8 and caspase-10 (5, 15). Studies human cancer cell lines while sparing most normal cells (1). based on receptor-blocking antibodies indicate that Apo2L/ Apo2L/TRAIL triggers apoptosis through binding to the death TRAIL can induce apoptosis through either DR4 or DR5 or receptors DR4 (2) and/or DR5 (3, 4). These receptors contain a both, but the relative contribution of each death receptor to cytoplasmic death domain that recruits adaptor molecules in- apoptosis induction in cells expressing both receptors is un- volved in caspase activation (5). In addition to these two sig- known (1, 6). Moreover, whereas binding of Apo2L/TRAIL to naling receptors, Apo2L/TRAIL binds to three decoy receptors DR4 and DR5 can result in the formation of homomeric as well that inhibit apoptosis induction: DcR1, DcR2, and OPG. DcR1 as heteromeric complexes (5), the importance of heteromeric ligand-receptor complexes for apoptosis stimulation is unclear. * The costs of publication of this article were defrayed in part by the Indeed, agonistic monoclonal antibodies specific to either DR4 payment of page charges. This article must therefore be hereby marked (16) or DR5 (17) are capable of inducing apoptosis; however, the “advertisement” in accordance with 18 U.S.C. Section 1734 solely to precise molecular mechanism of death receptor activation by indicate this fact. agonistic antibodies is not fully understood. □ S The on-line version of this article (available at http://www.jbc.org) contains Supplemental Tables I–III. To investigate the relative importance of DR4 and DR5 for § To whom correspondence should be addressed: Dept. of Protein apoptosis induction by Apo2L/TRAIL, we selected ligand vari- Engineering MS#27, Genentech, Inc., 1 DNA Way, South San Fran- ants with relative binding selectivity for DR4 or DR5 through a cisco, CA 94080. Tel.: 650-225-2321; Fax: 650-224-3734; E-mail: [email protected]. phage display approach. Although phage display has been used The abbreviations used are: Apo2L, apoptosis-inducing ligand 2; previously for optimizing the target binding affinity of mono- TNF, tumor necrosis factor; TRAIL, TNF-related apoptosis-inducing meric, homodimeric, or heterodimeric proteins (18, 19), this ligand; DR, death receptor; DcR, decoy receptor; OPG, osteoprotegerin; technique has not been successfully applied as yet to trimeric ELISA, enzyme-linked immunosorbent assay; SPR, surface plasmon proteins. Our modifications enabled the display of trimeric resonance. This paper is available on line at http://www.jbc.org 2205 This is an Open Access article under the CC BY license. 2206 Apoptosis-induction by DR5-selective Apo2L/TRAIL saline, 0.05% Tween 20 to remove unbound phage, bound phage were Apo2L/TRAIL on phage and the selection of receptor-selective eluted from the wells by the addition of 100 lof10mM HCl. After variants. Investigation of the ability of these variants to induce neutralization with 2 M Tris base pH 11, half of the eluant from the apoptosis in several cancer cell lines and in normal hepatocytes receptor plate was used to infect XL-1 E. coli to propagate phage (50 ml suggests that DR5 may play a more prominent role than DR4 culture) for the next round of sorting. A portion of the eluant from the in mediating apoptosis signals emanating from Apo2L/TRAIL receptor and blank plates was used to estimate phage concentration by in cells that express both death receptors. titering colony forming units with XL-1 E. coli. The enrichment of the selection for receptor binding was calculated from the ratio of the phage EXPERIMENTAL PROCEDURES eluted from the receptor plate to that eluted from the blank plate. Construction of Apo2L/TRAIL Phage Display Vector—A phagemid Upon completion of sorting, individual clones were screened for spe- vector designed for the expression of Apo2L/TRAIL (residues 96 –281) cific binding by “spot ELISA” (23). For the DR4 library clones, the test as a fusion to the geneIII protein of M13 bacteriophage was constructed proteins were DR4-Fc, DR5-Fc, TNFR1-Fc, and bovine serum albumin. as follows. The DNA encoding the 96 –281 portion of Apo2L/TRAIL was Only the clones that gave an ELISA signal for DR4-Fc and none of the amplified by PCR from the plasmid pAPOK5 (20) and ligated into other proteins were chosen for further analysis. Test proteins for the NsiI/BamHI-cleaved pTFAA-g3 (21). The resulting plasmid (pAPOK4) DR5 library clones were DR5-Fc and a humanized anti-HER2 antibody. encodes a fragment having the stII bacterial signal sequence fused to Clones positive for DR5-Fc binding and not anti-HER2 binding were the N terminus of the 96 –281-residue fragment of Apo2L/TRAIL. A selected for further study. tripeptide linker with the sequence GSA is appended to the C terminus Characterization of Receptor-selective Clones—Single-stranded DNA of Apo2L/TRAIL followed by an in-frame amber stop codon and the was isolated from the phage particle using the Qiaprep Spin M13 kit geneIII product of M13 bacteriophage. The alkaline phosphatase pro- (Qiagen) and subjected to dideoxynucleotide sequencing using the dye moter is used to direct expression. pAPOK4.2 was constructed by re- terminator cycle kit (Beckman-Coulter) and a primer that is compli- placing the alkaline phosphatase promoter in pAPOK4 with the mentary to a portion of the tac promoter. A few of the DR4-selective tac promoter. phage clones were analyzed by competition phage ELISA to determine Library Construction—The DR4 library was constructed by oligonu- the IC for DR4-Fc binding. Phage clones were first titered for binding cleotide-directed mutagenesis (22) using a primer having NNS codons to DR4-Fc to determine the dilution giving equal signal strength for all at positions 189, 191, 193, 199, 201, and 209. The template was pAPOK4.2 the clones. A fixed concentration of phage was then mixed with an containing Y213W/S215D mutations and TAA stop codons at 189, 191, increasing concentration of DR4-Fc or DR5-Fc and added to wells 193, 199, 201, and 209. Use of the stop template ensured that any coated with DR4-Fc. After incubation to allow binding and washing to template DNA that did not become mutated and survived the Kunkel remove unbound phage, the bound phage was detected with the horse- selection would not produce functional Apo2L. For the DR5 library, radish peroxidase-coupled anti-M13 antibody. Analysis of the ELISA TAA stop codons were introduced into pAPOK4.2 at the library posi- signal as a function of receptor-Fc concentration in solution by using a tions 189, 191, 193, 264, 266, 267, and 269. Two oligonucleotides, one four-parameter fit yields the IC value. specifying NNS codons at the library positions 189, 191, and 193 and Expression and Purification of Apo2L/TRAIL Variants—Mutants of the second containing NNS codons at sites 264, 266, 267, and 269 were Apo2L (114 –281) were constructed, expressed in E. coli, and purified as used in the library mutagenesis reaction. Library mutagenesis, electro- previously described (7, 20). Plasmids designed for E. coli expression of poration, and propagation of phage were performed as described (23). FLAG-tagged Apo2L variants were constructed by oligonucleotide-di- Upon electroporation into SS-320 Escherichia coli the DR5 library gave rected mutagenesis (22) of a plasmid (pFLAG-Apo2L; Scot Marsters, 8 9 a size of 1.75  10 clones, and the DR4 library comprised 2.5  10 Genentech) having Apo2L/TRAIL (114 –281) inserted in pFLAG-MAC clones. Because SS-320 do not have an amber suppressor, the phage (Sigma). pFLAG-Apo2L directs the cytoplasmic expression of N-termi- pellet was used to infect a 500-ml culture of early log phase XL-1 E. coli. nal FLAG-tagged Apo2L/TRAIL (114 –281) under control of the tac After the addition of KO7 helper phage and carbenecillin (50 g/ml), the promoter. For expression of FLAG-tagged Apo2L/TRAIL variants, the culture was grown overnight at 30 °C, and phage were harvested by mutant plasmids were transformed into E. coli strain 43E7. The trans- precipitation with ⁄5 volume of 20% polyethylene glycol, 2.5 M NaCl. formed E. coli were grown to early log phase at 37 °C in 500 ml of 2x Phage were amplified between rounds of sorting by infection of XL-1 yeast/tryptone media containing 50 g/ml carbenicillin, and expression Blue as described above except that the culture volume used was was induced by the addition of isopropyl 1-thio--D-galactopyranoside reduced 10-fold to 50 ml. to a final concentration of 0.4 mM. The FLAG-tagged Apo2L/TRAIL For the analysis of sequence changes giving rise to DR5-selectivity, variants were purified as previously described for untagged proteins two additional libraries were constructed. Library DR5.B used the (20). For both the untagged and flag-tagged proteins, an additional following codons: 189 (YAS), encoding Tyr, amber, Gln, His; 191 (ARR), purification step consisting of gel filtration on a 2.6  100 column of encoding Arg and Lys; 193 (CRA), encoding Gln or Arg; 264 (CRC), Sephacryl S-200 HR equilibrated and eluted with 0.4 M sodium sulfate, encoding His and Arg; 266 (MTT), encoding Ile and Leu; 267 (SAS), 25 mM Tris-HCl, pH 7.5, was used to remove aggregated protein from encoding His, Gln, Asp, and Glu. Library DR5.C used these codons: 189 the preparations. (BVS), encoding Cys, Trp, Arg, Gly, Tyr, Ser, His, Gln, Pro, Ala, Glu; Assay of Apoptosis Induction—Apoptosis induction in tumor cell lines 191 (RVR), encoding Glu, Ala, Gly, Lys, Thr, Arg; 193 (VVA), encoding upon the addition of Apo2L/TRAIL or variants was measured using a Glu, Gly, Ala, Gln, Arg, Pro, Lys, Thr; 264 (VVC), encoding Asp, Gly, fluorescence assay of metabolic activity as previously described (20). Ala, His, Arg, Pro, Asn, Ser, Thr; 266 (VYT), encoding Ile, Thr, Leu, Pro, Caspase activation in normal cells upon Apo2L/TRAIL addition was Val, Ala; 267 (NMS), encoding Tyr, Ser, His, Pro, Gln, Asp, Glu, Ala, determined by using a fluorescent caspase substrate as previously Asn, Lys, Thr. Kunkel mutagenesis was used to construct these librar- described (24). ies as described above except that XL-1 Blue E. coli (Stratagene, Inc.) Receptor Binding by AlphaQuest® Assay—The binding of the vari- was used for electroporation. Library DR5.B gave an actual library size ants to the five known Apo2L/TRAIL receptors (DcR1, DcR2, OPG, 10 9 of 1  10 , and library DR5.C had a size of 1.6  10 . DR4, DR5) was examined by using an AlphaQuest® assay. This is a Sorting of Phage Libraries for Receptor Selectivity—Phage sorting for proximity-based assay technology in which emission of singlet oxygen receptor binding was performed using receptor-Fc fusion proteins ad- from “donor” beads yields a fluorescent signal from “acceptor” beads sorbed on the wells of microtiter plates (Nunc-Maxisorp). Purified re- brought into proximity by binding interactions. The donor bead was ceptor-Fc proteins were kindly provided by Scot Marsters (Genentech). coated with streptavidin and was used to capture biotinylated Apo2L/ Receptor-Fc proteins were diluted to 2–10 g/ml in coating buffer (50 TRAIL. The acceptor bead was coated with Staphylococcal protein A mM sodium carbonate, pH 9.6), and 100 l of this solution was added to and was used to capture the receptor-Fc protein. IC values were several wells of a 96-well plate. After a 2-h incubation the wells were calculated from binding curves obtained by displacing the biotinylated blocked with 200 l of phosphate-buffered saline containing 0.05% ligand with unbiotinylated ligand. Biotinylated Apo2L/TRAIL was pre- Tween 20 and 5% powdered skim milk (blocking buffer). Blocking was pared by reaction of a surface cysteine mutant of Apo2L/TRAIL (R170C) for1hat room temperature, and then the wells were rinsed with with biotin-1-biotinamido-4-[4(maleimidomethyl)-cyclohexane-carbox- phosphate-buffered saline, 0.05% Tween 20 (wash buffer). amido]butane (Pierce) followed by removal of excess biotin on a PD-10 The Apo2L/TRAIL library phage solution was diluted 10-fold in (Amersham Biosciences) desalting column. The various Apo2L/TRAIL blocking buffer, and then 100 l of this solution was added to the wells receptors were diluted to 250 ng/ml in assay buffer (phosphate-buffered of the receptor-Fc-coated plate. In addition, the diluted phage were also saline, pH 7.4, containing 0.5% bovine serum albumin and 0.05% Tween added to an equal number of wells of a blank plate that was prepared by 20). Apo2L/TRAIL samples were serially diluted starting at a concen- blocking the wells with blocking buffer without prior protein coating. tration of 4 –20 g/ml. Receptor (10 l) and Apo2L/TRAIL (5 l) were After incubation to allow binding and washing with phosphate-buffered combined in 384-well white Opti-Plates (PerkinElmer Life Sciences) Apoptosis-induction by DR5-selective Apo2L/TRAIL 2207 and incubated for 30 min at room temperature. Biotinylated Apo2L/ TRAIL competitor was then added (100 ng/ml, 5 l per well). After a further 2-h incubation of the plates at room temperature, a mixture of AlphaScreen streptavidin donor beads and protein A acceptor beads (PerkinElmer Life Sciences) was added ( ⁄100 dilution of each bead in assay buffer, 10 l/well). The plates were incubated for1hat room temperature protected from light and subsequently read using an AlphaQuest® plate reader. Surface Plasmon Resonance Measurements of Receptor Binding— Dissociation constants for the direct binding of Apo2L/TRAIL variants to immobilized receptors were determined by SPR measurements on a BIAcore 3000 instrument as previously described (20). Affinities were also assessed by using a competition assay in which binding of the ligand to immobilized DR5-Fc was competed with receptor in solution. These experiments employed a flow cell having a high density (15,000 resonance units) of immobilized DR5-Fc such that the initial rate of binding was linearly dependent on the concentration of free ligand. Competition experiments were conducted by preparing a series of solu- tions having a fixed concentration of ligand but a varied concentration FIG.1. Part of the contact (patch A) observed in x-ray crystal of receptor. After incubating for2hto allow equilibration to occur, these structure of the Apo2L/TRAIL DR5 complex (Hymowitz et al. solutions were injected over the DR5-Fc surface. A linear fit of the (11); Protein Data Bank ID code 1D0G). The backbone of DR5 observed sensorgrams was used to extract the binding rate. Analysis of (residues 67– 69, 91-104) is shown as the green tube with selected side the binding rate as a function of competing receptor concentration chains. Apo2L/TRAIL is shown as the space-filling model. Apo2L/ enabled calculation of IC values by using a four-parameter curve fit. TRAIL residues included in the libraries are colored aqua or pink, with the residues mutated in Apo2L.DR5– 8 colored pink. Labels for DR5 RESULTS residues are underlined. Phage Display of Apo2L/TRAIL—The successful application of the phage display approach to the probing of receptor bind- in the phage display libraries on the basis of an examination of ing determinants requires proper assembly of trimeric Apo2L/ the x-ray structure determined for the Apo2LDR5-ECD com- TRAIL on the phage surface. To this end we used a construct plex (11). “Patch A” on DR5 involves the receptor 60 (residues having the Apo2L/TRAIL gene fused to the geneIII protein of 67– 69) and 90 (residues 91–104) loops, which interact with a M13 bacteriophage with an in-frame amber stop codon. In cluster of Apo2L/TRAIL residues centered on Gln-205. “Patch strains of E. coli capable of suppressing amber stop codons B” on DR5 involves the receptor 50 loop (residues 51– 65), (supE genotype), a Gln is inserted at the amber stop resulting which makes contact with residues 155–162 and 215–218 of the in secretion of an Apo2L/TRAIL-geneIII fusion protein into the ligand. Only residues within or near the patch A contact with periplasm. Because suppression of amber stop codons is only the receptor (Fig. 1) were considered for mutagenesis. Because 10 –30% efficient, free Apo2L/TRAIL is secreted as well. When patch B is smaller than patch A, is more hydrophobic, and is supplied with assembly proteins by co-infection with helper composed of ligand residues that are more conserved across phage, phage particles that incorporate the Apo2L/TRAIL- TNF superfamily members, it has been proposed that patch A geneIII fusion protein into their surface can be produced. Tri- is more important for specificity (11). However, alanine substi- meric Apo2L/TRAIL can be produced by assembly with either tution showed that several Apo2L/TRAIL residues in patch A another fusion protein or with free Apo2L/TRAIL. However, (i.e. Gln-205, Tyr-237, Leu-239) make a large contribution to because each phage particle will display only a few copies of the the free energy of binding for all receptors tested (20). These geneIII fusion protein (18), it seems likely that trimerization is sites contact residues on DR5 that are conserved in DR4 and, driven by the free Apo2L/TRAIL produced when the amber stop thus, seemed unlikely to yield receptor-selective variants. is not suppressed. Therefore, we excluded these residues from the libraries. In Initial tests of the pAPOK4 vector suggested poor display on contrast, sites that gave only modest changes in affinity (5- the phage of correctly assembled Apo2L/TRAIL. Specific bind- fold) when replaced with alanine appeared more likely to in- ing increased if the phage were produced by growth at 30 °C crease receptor selectivity when mutated. An example of this rather than 37 °C and by replacement of the phoA promoter type of site is Gln-193, where alanine substitution causes a with the tac promoter (phagemid pAPOK4.2). Optimum display 1.7-fold decrease in affinity for DR4 but has no effect on binding (100-fold enrichment) was obtained for phage production at to DR5 and DcR2 (20). Further changes in receptor specificity 30 °C with induction of the tac promoter by addition of 1 M might be obtained by substitution of Gln-193 with a residue isopropyl 1-thio--D-galactopyranoside. other than alanine. This rationale was used to design two Phage displaying Apo2L/TRAIL showed increased nonspe- tailored Apo2L/TRAIL libraries; one including sites 189, 191, cific binding to a panel of control proteins relative to the non- 193, 199, 201, and 209 randomized (“DR4 library”), and the specific binding observed for KO7 helper phage (data not other with sites 189, 191, 193, 264, 266, 267, and 269 random- shown). In addition, KO7 helper phage displayed high nonspe- ized (“DR5 library”). As shown in Fig. 1, these residues are on cific binding to purified Apo2L/TRAIL. Sorting of a preliminary the periphery of the patch A contact observed in the x-ray Apo2L/TRAIL library having residues 213, 215, 216, 218, 220, crystal structure (11). and 222 randomized yielded a mutant of Apo2L/TRAIL Sorting for Receptor-selective Variants—The DR4 library (Y213W/S215D) that showed decreased helper phage binding. was sorted for two rounds against DR4-Fc coated on microtiter The Y213W/S215D mutant also gave increased display on wells followed by three rounds of sorting for DR4 binding in the phage, as measured by binding to an anti-Apo2L/TRAIL anti- presence of competing DR5-Fc. For sorting rounds 3, 4, and 5, body, with comparable affinity for DR4-Fc but a 10-fold reduc- the phage were incubated with 50, 250, and 750 n M DR5-Fc, tion in apparent affinity for DR5-Fc. The Y213W/S215D sub- respectively, for 30 min before the addition of these solutions to stitutions were incorporated into libraries designed for DR4-Fc-coated plates. With the DR5 library, phage were sorted improved affinity against DR4. for four rounds against DR5-Fc-coated wells followed by four Design and Production of Apo2L/TRAIL Phage Display Li- rounds with DR4-Fc as competitor. In rounds 5, 6, 7, and 8, braries—Residues in Apo2L/TRAIL were chosen for inclusion phage were incubated with 1, 10, 100, and 500 nM DR4-Fc, 2208 Apoptosis-induction by DR5-selective Apo2L/TRAIL FIG.2. Competitive phage ELISA of phage clones selected from the DR4 library. Binding to immobilized DR4-Fc was competed by increasing solution concentrations of DR4-Fc (panel A) or DR5-Fc (panel B). Phage clones Apo2L.DR4 – 8 (f), Apo2L.DR4 –9 (‚), Apo2L.DR4 –11 (Œ), Apo2L.DR4 –13 (), and wild-type () were tested. respectively, before selection for DR5-Fc binding. Both the DR4 The AlphaQuest® assay indicated that the two DR4-selective variants retained high affinity binding to DR4-Fc (Table I). and DR5 libraries gave strong, specific enrichment even in the presence of the competing receptor. Binding to both DR5 and OPG was significantly reduced, whereas the reduction in affinity for DcR1 and DcR2 was more Individual clones were isolated from the fifth round (DR4 library) or eighth round (DR5 library) of sorting and tested for modest. Both of the DR4-selective proteins showed a large decrease in potency for apoptosis induction relative to wild- receptor specificity by phage ELISA (see “Experimental Proce- dures”), and then the DNA sequence of receptor-selective type Apo2L/TRAIL (ED  12.9  5.2 ng/ml) or FLAG-Apo2L/ clones was determined. The amino acid identities deduced from TRAIL (ED  48 ng/ml). FLAG-Apo2L.DR4 – 8 showed no the DNA sequence for the library positions are shown in Sup- increase in potency upon cross-linking, whereas FLAG- plemental Tables I (DR4-selective) and II (DR5-selective). No Apo2L.DR4 –9 showed a relatively modest 6-fold gain in po- spurious sequence changes outside of the library positions were tency upon cross-linking. The apoptosis-inducing activity of detected. Analysis of four of the clones from the DR4 library by FLAG-Apo2L.DR4 – 8 on SK-MES could be inhibited by a neu- competition phage ELISA (Fig. 2) confirmed that the sorting tralizing anti-DR4 monoclonal antibody but not by a neutral- strategy yielded receptor-selective variants. These four clones izing antibody specific for DR5 (data not shown). This finding bound DR4-Fc with apparent high affinity, whereas binding to suggests that the residual activity of FLAG-Apo2L.DR4 – 8 re- DR5-Fc was undetectable. These DR4-selective variants all sults from binding to DR4 and does not reflect a weak interac- differed from the wild-type sequence at four of six library tion with DR5. positions. Substitution of Tyr-189 with Ala appeared to be All of the tested DR5-selective variants showed significant important for DR4 selectivity, since all four clones had reduction in affinity for DcR1, OPG, and DR4 while maintain- this mutation. ing high affinity for DR5 (Table I). The variation in affinity for Variants with selectivity for DR5 (Supplemental Table II) all DR5 ranged from a slight improvement (FLAG-Apo2L.DR5– 8) had at least three amino acid changes, and clones with six to about a 3-fold decrease (FLAG-Apo2L.DR5–2). The change in substitutions occurred most frequently. Although this library affinity for DcR2 was variable and, with the exception of FLAG- was subjected to eight rounds of sorting, a consensus sequence Apo2L.DR5– 8, was smaller than observed for the other recep- was not obtained, and some positions, most notably 264, re- tors. FLAG-Apo2L.DR5– 8 was the most selective variant tained high diversity. Substitution of Gln-193 with positively showing good binding to DR5 but significantly reduced affinity charged Lys or Arg appeared to be important for DR5 selectiv- for the other four receptors. Surprisingly, all of the DR5-selec- ity. A negatively charged residue at either position 267 or 269, tive variants retained a high level of apoptosis-inducing activ- but not both, was favored. Replacement of Ile-266 with Leu was ity (Table I). Changes in activity varied from a 2.6-fold increase a frequent change in these variants. Interestingly, the Y189A in ED for FLAG-Apo2L.DR5–1 to a 6.4-fold decrease for mutation was also observed in the DR5-selective variants. FLAG-Apo2L.DR5– 8. The potency of all of the DR5-selective Apoptosis Induction by Receptor-selective Variants—To char- variants increased significantly upon anti-FLAG cross-linking acterize the receptor-selective variants further, we produced to the same level as measured for the cross-linked wild-type FLAG-tagged versions of two of the DR4-selective and three of protein. There was not a strict 1:1 correspondence between the the DR5-selective mutants. These variants were profiled for affinity measured for DR5 and the potency measured for apo- receptor binding by using an AlphaQuest® assay and assayed ptosis induction. This likely reflects the use of Fc fusion pro- for apoptosis induction on SK-MES lung carcinoma cells. SK- teins to determine affinities, since the bivalency may suppress MES cells express DR4 and DR5 at similar levels (data not affinity differences. shown) and are sensitive to apoptosis induction by wild-type The specificity of a few of the variants was further confirmed Apo2L/TRAIL. Inclusion of the FLAG tag enables testing of by using SPR to measure the dissociation constants for binding oligomerized forms of Apo2L/TRAIL or its variants formed by to DR4 and DR5. As shown in Table II, FLAG-Apo2L.DR4 – 8 cross-linking the tagged protein with anti-FLAG antibody. An- and FLAG-Apo2L.DR4 –9 bind to DR4 with affinity nearly ti-FLAG cross-linking of tagged wild-type Apo2L/TRAIL re- equivalent to that measured for the wild-type protein, whereas sulted in an 70-fold increased potency for apoptosis induction binding to DR5 was significantly reduced. The apparent affin- in SK-MES (Table I). ity of FLAG-Apo2L.DR4 – 8 for DR5 was too weak to measure Apoptosis-induction by DR5-selective Apo2L/TRAIL 2209 TABLE I Receptor binding and apoptosis induction of FLAG-tagged Apo2L/TRAIL IC values from an AlphaQuest® assay of receptor binding are reported as the ratio of mutant to wild-type FLAG-Apo2L/TRAIL. “” indicates that the IC value was greater than the highest mutant concentration tested in the assay. Concentrations of Apo2L/TRAIL variants required to induce 50% apoptosis (ED ) on SK-MES cells are shown for the absence () or presence ( )of2 g/ml M2 anti-FLAG antibody. IC ratio (mutant/wt) ED (ng/ml) 50 50 Protein DcR1 DcR2 OPG DR4 DR5 FLAG-Apo2L/TRAIL 1 1 1 1 1 48.4 0.7 FLAG-Apo2L.DR4–8 5.5 15 28 1 25 4000 4100 FLAG-Apo2L.DR4–9 4.0 18 28 1.1 25 1000 170 FLAG-Apo2L.DR5–1 300 32 700 30 2.4 126 1.3 FLAG-Apo2L.DR5–2 300 5.0 700 30 2.8 67.8 1.0 FLAG-Apo2L.DR5–8 300 1900 700 30 0.7 7.6 0.3 TABLE II SPR measurement of DR4 and DR5 binding affinity K ratios were calculated relative to wild-type FLAG-Apo2L/TRAIL. K ratio (mutant/wt) Variant Amino acid changes DR4 DR5 FLAG-Apo2L.DR4–8 Trp-213; Asp-215; Ala-189; Ser-193; Val-199;Arg-201 2.3 NB FLAG-Apo2L.DR4–9 Trp-213; Asp-215; Ala-189; Ser-193; Arg-199; Arg-201 1.5 61 FLAG-Apo2L.DR5–8 Gln-189; Lys-191; Arg-193; Arg-264; Leu-266; Gln-267 NB 0.8 Amino acid changes are relative to the wild-type protein. No binding (NB) was detected at the highest concentration (500 nM) used in the SPR measurements. by SPR. In contrast, the DR5-selective variant (FLAG- Apo2L.DR5– 8) did not appear capable of binding to DR4 but, consistent with the AlphaQuest® data, displayed high affinity binding to DR5. A very slow on-rate was measured for interac- tion of FLAG-Apo2L.DR4 – 8 with DR5 and also for binding of FLAG-Apo2L.DR5– 8 to DR4. The slow binding kinetics pre- cluded accurate determination of the binding constants by SPR for these weak interactions. Because FLAG-Apo2L.DR4 – 8 and FLAG-Apo2L.DR5– 8 gave the greatest receptor selectivity, these variants were used for testing the sensitivity of additional cancer cell lines to receptor-specific ligands. Colo205, a colon carcinoma cell line, expresses slightly more DR5 than DR4 and was quite sensitive to trimeric Apo2L/TRAIL (ED  4.2  1.8 ng/ml). FLAG- Apo2L.DR4 – 8 induced apoptosis only weakly (ED  1150 ng/ml) in Colo205 (Fig. 3A). The addition of anti-FLAG anti- body increased the potency of this variant about 8-fold (ED 150 ng/ml). FLAG-Apo2L.DR5– 8 showed increased potency against Colo205 cells (ED  0.9 ng/ml; Fig. 3B), and its activity was increased 6-fold by anti-FLAG cross-linking. A similar pattern of results was obtained against Colo320, a colon carcinoma cell line that expresses similar amounts of DR4 and DR5 (data not shown). FLAG-Apo2L.DR4 – 8 was inactive against Colo320 cells even if cross-linked with anti-FLAG an- tibody. FLAG-Apo2L.DR5– 8 weakly induced apoptosis in Colo320 cells, but the activity of the cross-linked form was equivalent to that observed for anti-FLAG cross-linked wild- type Apo2L/TRAIL. The breast carcinoma cell line MDA-MB-231 expresses high levels of both DR4 and DR5 yet displays a weaker apoptotic response to Apo2L/TRAIL treatment than observed with Colo205 cells (5). FLAG-Apo2L.DR4 – 8 was completely inactive against MDA-MB-231 (Fig. 4A). FLAG-Apo2L.DR5– 8 showed modest activity against MDA-MB-231, and the activity was significantly increased upon anti-FLAG cross-linking (Fig. 4B). The Jurkat T leukemia cell line expresses DR5 but not DR4. As expected, FLAG-Apo2L.DR4 – 8 did not induce apoptosis in this cell line FIG.3. Assay of apoptosis-induction on Colo205 colon carci- even upon cross-linking (Fig. 5A). FLAG-Apo2L.DR5– 8 showed noma cells by FLAG-tagged Apo2L/TRAIL mutants. A fluores- cence assay (20) was used to test DR4-selective (panel A) or DR5- modest activity against Jurkat cells, and the activity was dra- selective (panel B) mutants. antiFLAG indicates that 2 g/ml M2 matically increased (ED  0.05 ng/ml) by cross-linking (Fig. antibody (Sigma) was added along with the specified concentration of 5B). The small increase in DR5 affinity observed for FLAG- Apo2L/TRAIL mutant. Curves represent fitting using the four-param- Apo2L.DR5– 8 appears to be amplified into a larger apoptotic eter equation. RFU, relative fluorescence units. 2210 Apoptosis-induction by DR5-selective Apo2L/TRAIL FIG.5. Apoptosis induction on Jurkat cells by receptor-selec- tive mutants of Apo2L/TRAIL. A fluorescence (20) assay was used to test DR4-selective (panel A) or DR5-selective (panel B) mutants. anti- FIG.4. Assay of apoptosis induction on MDA-MB-231 breast FLAG indicates that 2 g/ml M2 antibody (Sigma) was added along carcinoma cells by FLAG-tagged Apo2L/TRAIL mutants. A fluo- with the specified concentration of Apo2L/TRAIL variant. Curves rep- rescence assay (20) was used to test DR4-selective (panel A) or DR5- resent fitting using the four-parameter equation. RFU, relative fluores- selective (panel B) mutants. antiFLAG indicates that 2 g/ml M2 anti- cence units. body (Sigma) was added along with the specified concentration of Apo2L/TRAIL mutant. Curves represent fitting using the four-param- eter equation. RFU, relative fluorescence units. signal on Jurkat cells upon cross-linking. Normal hepatocytes are resistant to Apo2L/TRAIL unless the ligand is aggregated (8). The receptor-selective variants were evaluated for their ability to induce apoptosis in normal hepatocytes using an assay that measures activation of caspase-3 and -7. FLAG-Apo2L.DR4 – 8 did not induce caspase activation even upon cross-linking with anti-FLAG antibody (Fig. 6). Consistent with previous results (8), Apo2L/TRAIL did not increase caspase activity except after cross-linking. Similar to the wild-type ligand, FLAG- Apo2L.DR5– 8 was inactive as a trimer and induced caspase activation upon anti-FLAG cross-linking. Selection of Substitutions Necessary to Give Selectivity for DR5 Binding—To determine the minimum number of amino FIG.6. Caspase activation in cynomolgus monkey hepatocytes acid changes required for DR5 selectivity while maintaining upon the addition of the indicated concentrations of FLAG- Apo2L/TRAIL in the presence and absence of 2 g/ml M2 anti- high bioactivity, we did additional phage display experiments FLAG antibody. RFU, relative fluorescence units. followed by testing of selected variants. Two phage libraries were constructed in which the residues at 189, 191, 193, 264, 266, and 267 were allowed to vary as either the wild-type of competing DR4-Fc. By the third round of sorting, 1000-fold residue, the amino acid found in Apo2L.DR5– 8, or a limited set enrichment was obtained, and individual clones were selected of similar amino acids. These libraries were pooled and sorted for further analysis. Spot ELISA indicated that 28 of 28 clones for one round against DR5-Fc coated on microtiter wells fol- tested were positive for DR5 binding, whereas only one was lowed by two rounds of sorting for DR5 binding in the presence functional for DR4 binding. The amino acid identities deduced Apoptosis-induction by DR5-selective Apo2L/TRAIL 2211 TABLE III Relative receptor binding affinity and apoptosis-induction by variants of Apo2L.DR5–8 DR4 binding DR5 binding Variant Amino acid changes Apoptosis, ED K IC K IC D 50 D 50 Apo2L.DR5–8 Gln-189; Lys-191; Arg-193; Arg-264; Leu-266; Gln-267 NB 333 0.3 0.5 0.6 Apo2L.DR5–8E Gln-189; Lys-191 19 333 1.1 1.4 16 Apo2L.DR5–8B Gln-189; Lys-191; Arg-193 56 333 1.4 0.8 15 Apo2L.DR5–8D Gln-189; Lys-191; Leu-266 NB 333 NB NB ND Apo2L.DR5–8C Gln-189; Lys-191; Arg-193; Leu-266 59 72 0.5 0.7 0.9 Amino acid changes are relative to wild-type Apo2L/TRAIL. Variants were produced as untagged 114 –281 Apo2L/TRAIL. Values for receptor binding and apoptosis induction (ED ) on SK-MES are the ratio relative to wild-type Apo2L/TRAIL. NB indicates no detectable binding. Not determined. from the DNA sequence at the library positions for the clones most TNF superfamily members bind to their receptors at the positive for DR5 binding, but not DR4 binding, are shown in interfaces between subunits of the ligand homotrimer, a func- Supplemental Table III. These results suggest that the residue tional receptor binding site can be formed only when a ligand identity at positions 264 and 267 may not be important for DR5 trimer is properly assembled. Trimer assembly was promoted selectivity since there was no preference at these positions for by using a construct having an amber stop codon since both the amino acid found in variant DR5– 8. In contrast, the amino monomer Apo2L/TRAIL, and a geneIII fusion protein could be acids found in variant DR5– 8 predominated at positions 189, produced in the same cell. An excess of free monomer most 191, 193, and 266, indicating that one or more of these sites likely drives oligomerization with the fusion protein. Indeed, in may be important for DR5 selectivity. At position 189 there was sorting of the second DR5 library (Supplemental Table III) a strong preference for Gln, which can be expressed through a there was selection for an additional amber stop codon at Gln codon or through suppression of an amber stop codon. residue 189 where a Gln residue was compatible with DR5 A few variants (Apo2L.DR5– 8B-E) were produced as an un- binding. A second amber stop would increase the ratio of mon- tagged 114 –281 fragment (7) of Apo2L/TRAIL and tested for omer to fusion protein because two stops must be suppressed to receptor binding and bioactivity. Apo2L.DR5– 8E differs from express the geneIII fusion protein. Functional display of wild-type Apo2L/TRAIL by Y189Q and R191K. Apo2L.DR5– 8B Apo2L/TRAIL was also aided by lowering the growth temper- has the additional substitution of Q193R, and Apo2L.DR5– 8C ature and by using a strong, inducible promoter. Selection of also has I266L besides these substitutions. Apo2L.DR5– 8D has the DR4 library was enhanced through incorporation of the Y189Q, R191K, and I266L. Dissociation constants for receptor amino acid substitutions Y213W/S215D, which reduced non- binding were determined from SPR data for direct binding of specific binding of Apo2L/TRAIL. For both the DR4 and DR5 each variant to immobilized receptor. Because all of these vari- libraries, nonspecific binding was further reduced by using ants had decreased binding to DR4-Fc, it was necessary to use competitive selection. As with other protein-protein interac- a sensor chip with a high density of immobilized DR4-Fc to tions (25), competitive selection proved to be a powerful ap- detect sufficient signal in direct binding measurements. For proach for increasing the specificity of interaction between measurements with wild-type Apo2L/TRAIL, a 4-fold greater Apo2L/TRAIL and its death receptors. binding affinity was calculated from high density as opposed to Alanine-scanning mutagenesis has shown that the binding low density binding surfaces (data not shown). Thus, a compet- sites on Apo2L/TRAIL for the various receptors are highly itive binding assay in which Apo2L/TRAIL binding to immobi- similar, with certain key residues (e.g. Gln-205, Tyr-216) mak- lized DR5-Fc was competed with DR4-Fc or DR5-Fc in solution ing a large free energy contribution to the binding of each of the was also used to assess receptor affinities. As shown in Table five receptors (20). Although only the structure of the III, all of the variants showed decreased binding to DR4. The Apo2LDR5 complex is known, many of the residues on DR5 competition assay suggested a larger magnitude decrease in that are in contact with Apo2L/TRAIL are conserved in DR4. affinity for DR4 than the direct binding assay. With the excep- Thus, it is perhaps not surprising that multiple amino acid tion of Apo2L.DR5– 8D, these proteins displayed high affinity substitutions were required to achieve receptor selectivity. By binding to DR5. Binding of Apo2L.DR5– 8D to either DR4 or comparison, TNF mutants functional for binding to the p55 DR5 was not detectable, suggesting that this variant was mis- receptor, but not the p75 receptor, could be obtained by com- folded. Indeed, this sequence was found only in 1 of 51 clones bining two or three amino acid changes (26). Examination of from phage sorting. The two amino acid changes in the Apo2LDR5 structure (Fig. 1) suggests that the amino acid Apo2L.DR5– 8E resulted in a significant decrease in affinity for changes in Apo2L.DR5– 8 would disfavor interactions with DR4 and a slight decrease in DR5 binding, but a 16-fold reduc- DR4 residues Lys-67, Asp-69, and Asn-95. The equivalent res- tion in apoptosis-inducing activity. The addition of Q193R idues in DR5 are Asp-67, Gly-69, and Asp-95. The R191K (Apo2L.DR5– 8B) had a slight effect on DR5 affinity and did not substitution may oppose interaction with Lys-67 on DR4. Re- restore bioactivity. Incorporation of I266L (Apo2L.DR5– 8C) placement of Gln-193 with a positively charged residue may resulted in a modest improvement in DR5 affinity and restored interfere sterically with engagement of DR4 residue Asp-69, full bioactivity. The results for Apo2L.DR5– 8 indicate that the whereas the Gly in DR5 may accommodate a larger side chain. H264R and/or D267Q substitutions give further selectivity Ile-266 is a buried residue in Apo2L/TRAIL, and thus, the effect against DR4 binding while providing modest improvements in of the I266L substitution must be indirect, perhaps occurring DR5 affinity and bioactivity. through a change in the conformation of residue 267. D267Q DISCUSSION may remove a favorable electrostatic interaction with the pos- We have shown here that Apo2L/TRAIL can be expressed on itive charge on DR4 residue Lys-67. H264R would be positioned to provide a positive charge for interaction with DR5 Asp-95 M13 bacteriophage in functional form, allowing selection of clones from mutant libraries for receptor-specific binding. To while discriminating against neutral residue Asn-95 in DR4. our knowledge this is the first successful application of phage The effect of the Y189Q mutation is difficult to explain, since display technology to a TNF-superfamily member. 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A., Eigenbrot, C., and Fuh, G. (2004) full activity upon cross-linking. In hepatocytes, which are re- J. Mol. Biol. 338, 299 –310 20. Hymowitz, S. G., O’Connell, M. P., Ultsch, M. H., Hurst, A., Totpal, K., sistant to non-cross-linked Apo2L/TRAIL but sensitive to the Ashkenazi, A., de Vos, A. M., and Kelley, R. F. (2000) Biochemistry 39, cross-linked ligand, the DR5-selective variant induced the 633– 640 21. Lee, G. F., and Kelley, R. F. (1998) J. Biol. Chem. 273, 4149 – 4154 same responses as the wild-type ligand, whereas the DR4- 22. Kunkel, T. A., Roberts, J. D., and Zakour, R. A. (1987) Methods Enzymol. 154, selective mutant was inactive. Taken together, these results 367–382 suggest that DR5 may contribute more than DR4 to Apo2L/ 23. Sidhu, S. S., Lowman, H. B., Cunningham, B. C., and Wells, J. A. (2000) Methods Enzymol. 328, 333–363 TRAIL-induced apoptosis in cancer or normal cells that express 24. LeBlanc, H., Lawrence, D., Varfolomeev, E., Totpal, K., Morlan, J., Schow, P., both death receptors. Thus, it may be possible to enhance the Fong, S., Schwall, R., Sinicropi, D., and Ashkenazi, A. (2002) Nat. Med. 8, 274 –281 anti-tumor activity of soluble trimeric Apo2L/TRAIL further by 25. Dennis, M. S., and Lazarus, R. A. (1994) J. Biol. Chem. 269, 22137–22144 engineering mutants of this protein that have increased affin- 26. Loetscher, H., Stueber, D., Banner, D., Mackay, F., and Lesslauer, W. (1993) J. Biol. Chem. 268, 26350 –26357 ity for DR5 but retain a stable trimeric structure. 27. Muhlenbeck, F., Schneider, P., Bodmer, J. L., Schwenzer, R., Hauser, A., Schubert, G., Scheurich, P., Moosmayer, D., Tschopp, J., and Wajant, H. Acknowledgments—We thank the oligonucleotide chemistry group at (2000) J. Biol. Chem. 275, 32208 –32213 Genentech for synthesis of oligonucleotides, the DNA sequencing group 28. Wajant, H., Haas, E., Schwenzer, R., Muhlenbeck, F., Kreuz, S., Schubert, G., for sequencing of phage clones, and Scot Marsters for supplying recep- Grell, M., Smith, C., and Scheurich, P. (2000) J. Biol. Chem. 275, tor-Fc fusion proteins. 24357–24366

Journal

Journal of Biological Chemistry – American Society for Biochemistry and Molecular Biology

Published: Jan 21, 2005

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