1. Volume 25, Issue 4, Pages 909-920.e4 (October 2018)
Limited Cross-Linking of 4-1BB by 4-1BB Ligand and the Agonist Monoclonal Antibody Utomilumab 
Yan Li, Shuguang Tan, Chang Zhang, Yan Chai, Mengnan He, Catherine W.-H. Zhang, Qihui Wang, Zhou Tong, Kefang Liu, Yifan Lei, William J. Liu, Yingxia Liu, Zhigang Tian, Xuetao Cao, Jinghua Yan, Jianxun Qi, Po Tien, Shan Gao, George F. Gao 
Cell Reports 
Volume 25, Issue 4, Pages e4 (October 2018)
DOI: /j.celrep
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2. Cell Reports 2018 25, 909-920.e4DOI: (10.1016/j.celrep.2018.09.073)
Copyright © 2018 The Authors Terms and Conditions

3. Figure 1
Overall Structure of the 4-1BB/4-1BBL Complex and the CRDs of 4-1BB
(A) The complex structure of 4-1BB bound to 4-1BBL. Three mono-4-1BBs bound to a tri-4-1BBL in the center. 4-1BB is shown in light pink, while 4-1BBL is presented in light blue.
(B) Side view of the 4-1BB/4-1BBL complex, with CRD1 to CRD4 in lemon, cyan, purple, and marine, respectively. 4-1BBL is depicted as a surface in light blue. Disulfide bonds within 4-1BB are represented as sticks, with the sulfur atoms shown as spheres. The only free cysteine, C121, and glycosylated N149 are represented as sticks.
(C) Each of the CRDs from 4-1BB was compared with those from TNFR2, OX40, CD40, and RANK to elucidate the conservation of each of the CRDs of 4-1BB in the TNFR superfamily. The CRDs of 4-1BB, TNFR2, OX40, CD40, and RANK are light pink, green, yellow, purple, and red, respectively. The intradomain disulfide bonds are depicted as lines.
See also Figure S1 and Table S1.
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4. Figure 2 Interaction between 4-1BB and 4-1BBL
(A) Overview of a 4-1BB molecule with two interacting 4-1BBLs. The 4-1BBL (4-1BBL-a) forming major contacts with 4-1BB is depicted in light blue, while the other 4-1BBL (4-1BBL-b), which forms partial contacts with 4-1BB, is orange.
(B and C) Details of the interaction involved in the binding of 4-1BB to 4-1BBL-a (B) and 4-1BBL-b (C). Residues involved in the hydrogen bond interactions are shown as sticks and labeled. Hydrogen bonds are shown as dashed black lines.
See also Figure S1 and Table S2.
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5. Figure 3 Rearrangement of the AB Region of 4-1BBL for 4-1BB Binding
(A) Superimposition of apo-4-1BBL with 4-1BBL extracted from the 4-1BB/4-1BBL complex. Apo-4-1BBL is depicted in wheat, while 4-1BBL in the 4-1BB/4-1BBL complex is light blue. The AB region of apo-4-1BBL is shown in lemon, while that of receptor-bound 4-1BBL is depicted in marine, to highlight the conformational differences of the AB region in the apo- and receptor-bound forms.
(B) Details of the differences among the three segments of the AB region.
See also Figure S1.
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6. Figure 4 Binding of Utomilumab-scFv to Dimeric 4-1BB
(A) Complex structure of di-4-1BB with two utomilumab-scFvs. Di-4-1BB is presented in light pink, with disulfide bonds shown as sticks and the sulfur atoms shown as spheres. VH and VL of utomilumab are blue and hot pink, respectively.
(B) Contribution of the CDRs of utomilumab to the binding to 4-1BB. The CDR1, CDR2, and CDR3 loops of the heavy chain (HCDR1, HCDR2, and HCDR3) are red, green, and yellow, respectively. The CDR1, CDR2, and CDR3 loops of the light chain (LCDR1, LCDR2, and LCDR3) are smudge, cyan, and lemon, respectively. 4-1BB is depicted as a light pink surface.
(C and D) Details of the binding between utomilumab and CRD2 and CRD3 (C), or CRD4 (D) of 4-1BB. Residues involved in the hydrogen bond interactions are shown as sticks and labeled. Hydrogen bonds are shown as dashed black lines.
(E–H) SPR assay-based characterization of the binding of mono- (E) or di-4-1BB (F) to 4-1BBL, and mono- (G) or di-4-1BB (H) to utomilumab-scFv, was accomplished using a single-cycle BIAcore T100 system. The association constant (Ka), dissociation constant (Kd), and equilibrium dissociation constant (KD) of the binding are labeled accordingly. The fits of the binding curves are shown in red. The data presented here are representative of two independent experiments with similar results.
See also Figure S2 and Table S3.
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7. Figure 5 Competitive Binding of Utomilumab and 4-1BBL to 4-1BB
(A) Superimposition of ligand-bound 4-1BB (with CRD1, CRD2, CRD3, and CRD4 shown in lemon, cyan, purple, and marine, respectively) and utomilumab-bound 4-1BB (hot pink), based on the conserved CRD2. The angle indicates the shift of CRD4 of 4-1BB from the ligand-bound to the utomilumab-bound forms.
(B) Binding surface of 4-1BBL and utomilumab on 4-1BB, with residues forming contacts with 4-1BBL alone depicted in light blue, residues forming contacts with utomilumab alone in lemon, and residues forming contacts with both 4-1BBL and utomilumab in cyan.
(C) Competitive binding of utomilumab and 4-1BBL with 4-1BB. The 4-1BBL, which forms major contacts with 4-1BB, is presented in light blue (4-1BBL-a), while the second 4-1BBL, which provides partial contacts, is shown in orange (4-1BBL-b).
(D) Octet competitive binding assay of the binding of 4-1BBL or utomilumab to mono- and di-4-1BB. AR2G sensors loaded with mono- (left) or di- (right) 4-1BB were first saturated with 4-1BBL, utomilumab, or buffer. Then, the capacity of additional binding was monitored by measuring further shifts after the injection of additional 4-1BBL in the presence of saturated utomilumab, or vice versa.
(E) Competition of utomilumab with 4-1BBL for the binding to mono- or di-4-1BB in flow cytometry assay. Mono- or di-4-1BBs were incubated with different molar ratios of utomilumab before staining with 293T cells expressing 4-1BBLs. The data presented here are representative of three independent experiments with similar results.
See also Figure S3.
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8. Figure 6 Binding Characteristics of Mono- and Di-4-1BB with 4-1BBL
(A–D) Gel filtration profiles of mono- or di-4-1BB with 4-1BBL were analyzed by size-exclusion chromatography.
(A) Gel filtration analysis of the incubated mono-4-1BB from peak 1 and tri-4-1BBL from peak 2, with a molar ratio of 2:1 and a final concentration of mixture of approximately 3 mg proteins in 2 mL volume, resulted in a complex peak 3, eluted at 66.0 mL. Further confirmation by SDS-PAGE analysis demonstrated the presence of both mono-4-1BB and tri-4-1BB in peak 3.
(B and C). Tri-4-1BBL (peak 2) was incubated with di-4-1BB (peak 1), with a di-4-1BB:4-1BBL molar ratio of 1:2 (B) or 2:1 (C), and a final amount of the mixture of 3 mg proteins in 2 mL volume. The incubation resulted in the major complex peak 3 at an elution volume of 70.3 mL. SDS-PAGE confirmed the presence of both di-4-1BB and 4-1BBL in peak 3.
(D) Di-4-1BB/tri-4-1BBL from the complex peak 3 in (B) and (C) was incubated with mono-4-1BB (peak 1) at a molar ratio of 1:2 for 4-1BBL to mono-4-1BB, and a final amount of mixture protein of approximately 5 mg proteins in 2 mL volume, and subsequently analyzed.
(E) Native PAGE analysis of the binding ratio of mono- and di-4-1BB to tri-4-1BBL. tri-4-1BBL (0.252 nmol) was mixed with mono- (lanes 2–6) or di-4-1BB (lanes 8–12) at a series of protein concentrations and analyzed by native PAGE in parallel with 0.756 nmol of mono-4-1BB (lane 1), 0.756 nmol of di-4-1BB (lane 13), and 0.252 nmol of tri-4-1BBL (lane 7). The data presented here are representative of three independent experiments with similar results.
DTT, dithiothreitol. See also Figure S4.
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9. Figure 7
C121-Mediated 4-1BB Dimerization and the Model of 4-1BB Cross-Linking
(A) Di-4-1BB was detected in PBMCs after stimulation with IL-2, anti-CD3, and CD28 mAbs for 48 hr, as determined with non-reducing SDS-PAGE. C121-mediated dimerization was confirmed by the observation that no di-4-1BB could be detected after the adoption of the C121A point mutant when full-length 4-1BB was transiently expressed in 293T cells. GAPDH was enrolled as the loading control. The data presented here are representative of three independent experiments with similar results. GAPDH, glyceraldehyde 3-phosphate dehydrogenase.
(B) Sequence alignment of the CRD4s of TNFR superfamily members that have four typical CRDs.
(C) Sequence alignment of CRD4 of 4-1BBs from different species. C121 is indicated in the green box and highlighted with the solid black triangle below.
(D) Model of the cross-linking of mono- or di-4-1BBs by ligand or agonist mAbs. The limited cross-linking observed here demonstrated that the binding of di-4-1BB to tri-4-1BBL mainly resulted in the formation of the (di-4-1BB)2/(tri-4-1BBL) complex, while oligomerization of di-4-BB by utomilumab was terminated upon binding to mono-4-1BB.
See also Figure S4.
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