Comparison of the Inhibition Mechanisms of Adalimumab and Infliximab in Treating Tumor Necrosis Factor α-Associated Diseases from a Molecular View*

Background: The epitope and the TNFα inhabitation mechanism of Adalimumab remain unclear. Results: The crystal structure of the TNFα in complex with Adalimumab is reported at a resolution of 3.1 Å. Conclusion: The epitope of Adalimumab provided information that Adalimumab may have clinical advantage compared with Infliximab. Significance: These data reveal the Adalimumab's mechanism of TNFα inhibition and its advantages compared with other TNF inhibitors in clinical practice. TNFα-targeting therapy with the use of the drugs Etanercept, Infliximab, and Adalimumab is used in the clinical treatment of various inflammatory and immune diseases. Although all of these reagents function to disrupt the interaction between TNFα and its receptors, clinical investigations showed the advantages of Adalimumab treatment compared with Etanercept and Infliximab. However, the underlying molecular mechanism of action of Adalimumab remains unclear. In our previous work, we presented structural data on how Infliximab binds with the E-F loop of TNFα and functions as a TNFα receptor-binding blocker. To further elucidate the variations between TNFα inhibitors, we solved the crystal structure of TNFα in complex with Adalimumab Fab. The structural observation and the mutagenesis analysis provided direct evidence for identifying the Adalimumab epitope on TNFα and revealed the mechanism of Adalimumab inhibition of TNFα by occupying the TNFα receptor-binding site. The larger antigen-antibody interface in TNFα Adalimumab also provided information at a molecular level for further understanding the clinical advantages of Adalimumab therapy compared with Infliximab.


TNF␣-targeting therapy with the use of the drugs Etanercept, Infliximab, and Adalimumab is used in the clinical treatment of various inflammatory and immune diseases. Although all of these reagents function to disrupt the interaction between TNF␣ and its receptors, clinical investigations showed the advantages of Adalimumab treatment compared with
Etanercept and Infliximab. However, the underlying molecular mechanism of action of Adalimumab remains unclear. In our previous work, we presented structural data on how Infliximab binds with the E-F loop of TNF␣ and functions as a TNF␣ receptorbinding blocker. To further elucidate the variations between TNF␣ inhibitors, we solved the crystal structure of TNF␣ in complex with Adalimumab Fab. The structural observation and the mutagenesis analysis provided direct evidence for identifying the Adalimumab epitope on TNF␣ and revealed the mechanism of Adalimumab inhibition of TNF␣ by occupying the TNF␣ receptor-binding site. The larger antigenantibody interface in TNF␣ Adalimumab also provided information at a molecular level for further understanding the clinical advantages of Adalimumab therapy compared with Infliximab.
TNF is an immunity-modulating cytokine required for immune processes. The unregulated activities of TNFs can lead to the development of inflammatory diseases. Excess amounts of TNF␣ expressed in cells are associated with the development of immune diseases, including rheumatoid arthritis, Crohn's disease, psoriatic arthritis, and inflammatory bowel disease (1,2). The function of TNF␣ requires smooth interaction with its two receptors, TNF receptor 1 (TNFR1) 4 and TNF receptor 2 (TNFR2). Blocking the interaction between TNF␣ and TNFRs has successfully been developed as a therapy in treating inflammatory or autoimmune diseases (3,4). TNF␣ neutralization therapies, including the use of a soluble TNFR2-Fc recombinant (Etanercept), a mouse-human chimera mAb (Infliximab), or a human mAb (Adalimumab), have been introduced in the past decades for the management of rheumatoid arthritis and other immune diseases (5).
Although all of these TNF␣ blockers function by interrupting the TNF␣-TNFR interaction, information on whether the different TNF␣ inhibitors have similar clinical efficacy remains controversial because of the lack of randomized clinical trial meta-analyses. In the early stages of clinical usage of Infliximab, its discontinuation was reported to result in loss of response. This largely affected patients who received long term treatment and later discontinued use (6). Approximately 10% of the patients discontinued the use Infliximab because of the loss of response. The discontinued use caused remissions, representing an additional 13% of patients. This practice pattern still occurs, but the frequency of Infliximab discontinuation in present day clinical practice is considerably lower because of the enhanced understanding that drug holidays may lead to loss of efficacy, attenuation of response, and acute and delayed hypersensitivity reactions through retreatment (7 study involving 93 Crohn's disease patients was performed to compare the effectiveness of Infliximab and Adalimumab. The study suggested that no obvious differences could be found in obtaining and maintaining remission (8). Another study drew a similar conclusion, claiming that the difference between Infliximab and Adalimumab is not obvious because the long term maintenance of the clinical remission between the two antibodies and their effectiveness are similar in both primary and secondary-tertiary centers (9). However, evidence from a Dutch observational study involving 770 patients with rheumatoid arthritis suggested that more patients achieve moderate responses to Adalimumab and Etanercept compared with Infliximab, but the potential baseline biases corrected for in the Dutch study were not specified, and strict response criteria were not included (10). Another study also directly compared the treatment responses, remission rates, and drug adherence in patients with rheumatoid arthritis treated with Adalimumab, Etanercept, or Infliximab using the nationwide DANBIO registry, which has been designed to capture operational clinical data as part of routine clinical care. The results showed that Adalimumab had the highest rates in treatment response and disease remission compared with the other two TNF␣ inhibitors (11). Furthermore, a very recent study implemented a matching adjusted indirect comparison technique, showing that Adalimumab is associated with the higher American College of Rheumatology 70% improvement criteria and Psoriasis Area and Severity Index 50/75/90 response rates compared with Etanercept at week 24 and a higher American College of Rheumatology 70% improvement criteria response rate than that of Infliximab at week 14 (12). These clinical investigations suggested that Adalimumab is more advantageous in TNF␣blocking therapy of autoimmune diseases. However, the underlying inhibition mechanisms of these TNF blockers with regard to clinical efficacy remain elusive. In particular, no reports have compared the binding epitopes of these drugs that have been widely used, despite the fact that the affinity and the epitope are the crucial elements in evaluating antibody drugs. In a previous work, we reported the crystal structure of the TNF␣-Infliximab Fab complex, presenting the inhibition mechanism of Infliximab of the TNF␣-TNFR interaction through partial overlap with the TNF␣-TNFR interface. We also revealed the pivotal role of the E-F loop of TNF␣ in Infliximab recognition (5). To further elucidate the inhibition mechanism of Adalimumab, in particular the variation on the mechanism of Infliximab and Adalimumab targeting at the TNF␣-TNFR contact, we launched a crystallographic and mutagenesis analysis on the TNF␣-Adalimumab Fab complex.

EXPERIMENTAL PROCEDURES
Protein Expression, Purification, and Characterization-Residues 77-233 of human TNF␣ followed by a His 6 tag were expressed in Escherichia coli BL21 (DE3) cells (Novagen) using the pET-22b(ϩ) vector (Novagen). The cells were grown in LB medium at 37°C until the A 600 reached 0.6 -0.8, and the expression of the protein was induced with 0.5 mM isopropyl ␤-D-thiogalactopyranoside for 4 h. The cells were incubated in PBS containing 20 mM phosphate (pH 8.0) and 150 mM NaCl with an additional 1 mg/ml lysozyme, 1 mM PMSF, and 1% Triton X-100 for 20 min on ice and then sonicated. The cell lysate was removed through centrifugation (10,000 ϫ g) and filtration (0.45 m). Solid ammonium sulfate was added to the supernatant at a 35% final concentration and was immediately mixed and incubated on a roller at 4°C for 2 h. The precipitant was discarded, and solid ammonium sulfate was continuously added to the supernatant until a final concentration of 60% was achieved. The concentration was immediately mixed and incubated on a roller at 4°C for 4 h. The precipitated protein was pelleted through centrifugation, and then the supernatant was discarded. The precipitant was dissolved in PBS buffer and separated through gel filtration using a Superdex 75 column (GE Healthcare). The precipitant was desalted to 20 mM Tris-HCl (pH 8.0), and the target fraction was further purified with a 20-column volume linear NaCl gradient elution from a high performance Q-Sepharose (GE Healthcare). The purity was confirmed to be Ͼ95% through SDS-PAGE analysis. The bioactivity was measured through a cytotoxicity assay using the TNF-susceptible murine L-929 cell line in the presence of the metabolic inhibitor actinomycin D, as described in Ref. 13.
Adalimumab was cloned, expressed, and purified following reported procedures. EcoRV and XbaI sites were added to the 5Ј-end of the heavy chain variable region gene (V H ), and an NheI site was added to the 3Ј-end. The PCR product was cloned into the pGEM-T vector, and the sequence of the product was confirmed through DNA sequencing. V H was excised through EcoRV and NheI digestion and then inserted into the EcoRV/ NheI sites of the pAH4604 vector containing the human gamma-1 constant region gene (C H ). The resultant pAH4604-V H vector was cleaved with XbaI and BamHI. The 3.3-kb fragment containing the human antibody heavy chain gene was cloned into the pcDNA3.1(Ϫ) vector (Invitrogen), which was digested with the same restriction enzymes and yielded the heavy chain expression vector pcDNA3.1(Ϫ)V H C H . The human chain constant cDNA (C L ) was obtained as a 0.3-kb PCR product derived from pAG4622. The light chain variable region gene (V L ) of Adalimumab was fused to the 5Ј-end of C L through the overlapping PCR method. The resultant human light chain gene (V L C L ), which has a HindIII site upstream of the start codon and an EcoRI site downstream of the stop codon, was cloned into the pGEM-T vector. The sequence of the chain was then verified. V L C L was excised through HindIII and EcoRI digestion and was ligated into pcDNA3.1 The Zeo(ϩ) vector (Invitrogen) cleaved with the same restriction enzymes yielded the chimeric light chain expression vector pcDNA3.1 Zeo(ϩ)V L C L . The light and heavy chain expression vectors were co-transfected into Chinese hamster ovary K1 cells using Lipofectamine 2000 (Invitrogen). The stable transfectants were isolated through limiting dilution in the presence of 600 g/ml G418 and 300 g/ml Zeocin. The culture supernatants from the individual cell clones were analyzed for antibody production through a sandwich enzyme-linked immunosorbent assay. The assay used goat anti-human IgG Fc (KPL, Gaithersburg, MD) as the capture antibody and goat anti-human -HRP (Southern Biotechnology Associates, Birmingham, AL) as the detecting antibody. Purified human IgG1/ (Sigma) was used as the standard control. The clones producing the highest amount of recombinant antibody were selected and grown in serum-free medium. The recombinant antibodies were purified through protein A affinity chromatography from the serum-free culture supernatant. The antibody concentrations were determined by absorbance at 280 nm, and the purity was confirmed through SDS-PAGE analysis. Bioactivity was measured in a cytotoxicity antagonist assay using the TNF-susceptible murine L-929 cell line in the presence of the metabolic inhibitors actinomycin D and TNF␣.
The Fab fragment of Adalimumab for the crystallographic investigation was obtained through papain digestion of the antibody. The digested protein sample was loaded onto a Protein A-Sepharose 4 FF column (GE Healthcare). The Fab fragment eluted in the flow through was separated from the Fc fragment and further purified through ion exchange chromatography using a Q-Sepharose FF column (GE Healthcare). The protein sample was concentrated to ϳ10 mg/ml and then exchanged to a stock buffer containing 10 mM Tris-HCl (pH 8.0) and 100 mM NaCl. TNF␣ was subsequently mixed with an excess of Adalimumab Fab, and the complex was purified through gel filtration chromatography (GE Healthcare). This complex was dialyzed against 50 mM Tris-HCl (pH 8.0) and 150 mM NaCl and was concentrated to 30 mg/ml.
Crystallization-Crystallization was performed at 291 K through the hanging drop vapor diffusion technique. The crystals were obtained by mixing 1 l of the protein solution with an equal volume of a reservoir solution. The mixture drop was equilibrated against 500 l of the reservoir solution. The crystals were obtained with a reservoir solution containing 30% PEG 400, 0.1 M sodium acetate trihydrate (pH 4.6), and 0.1 M cadmium chloride hydrate, which reached the final dimensions of 100 ϫ 100 ϫ 100 m 3 with the best diffraction within 1 week. The crystals were then cryo-protected through soaking in a cryo-protectant composed of the reservoir solution and 5% glycol. The cryo-protected crystals were subsequently flashcooled in liquid nitrogen and then transferred into a dry nitrogen stream at 100 K for x-ray data collection.
X-ray Data Collection, Processing, and Structure Determination-The diffraction data for the TNF␣-Adalimumab Fab complex were collected at Beamline BL17A (Photon Factory) with a resolution of 3.1 Å. The data were processed, integrated, and scaled using the HKL2000 package (14). The crystals belong to space group I2 1 The statistics of all data collections and structure refinements are summarized in Table 1.
The TNF␣-Adalimumab Fab structure was solved through the molecular replacement method, which employs the crystal structures of apo TNF␣ (Protein Data Bank code 1TNF) and GA101 Fab (Protein Data Bank code 3PP3) as the initial searching model using the program PHASER (15). The clear solutions in both the rotation and translation functions indicated the presence of one complex molecule, including one TNF␣ and one Adalimumab Fab molecule, in one asymmetric unit. This result is consistent with the Matthews coefficient and solvent content (16). The inconsistent residues were manually rebuilt in the program Coot (17) under the guidance of the F o Ϫ F c and 2F o Ϫ F c electron density maps. The residues were refined in PHENIX (18), and the respective working R factor and R free values decreased from 0.42 and 0.48 to 0.19 and 0.28, respectively, for all data from 50.0 to 3.1 Å. The refinement was monitored by calculating R free based on a subset containing 5% of the total reflections. Model geometry was verified using the program PROCHECK (19). The data collection and refinement statistics are presented in detail in Table 1. All structure figures were prepared using PyMOL (20).
Kinetics and Affinity Assay of TNF␣ Mutants-Site-directed mutants ( TNF P20A, TNF Q21A, TNF E23A, TNF K65A, TNF Q67A   TNF T72A, TNF K90A, TNF V91A, TNF N92A, TNF E110A, TNF P113A,  TNF E135A, TNF I136A, and TNF E146A) were created through PCR. The mutants were expressed and purified as recommended for wild-type proteins. Adalimumab Fab was immobilized on the surface of a CM-5 sensor chip (GE Healthcare) through amine coupling following the manufacturer's instructions. The maximal electrostatic interaction was obtained with 10 mM sodium acetate (pH 5.0). We regularly obtained Adalimumab Fab immobilization levels ranging from ϳ1000 to 1500 resonance units. The BIAcore T100 (GE Healthcare) instrument for the binding experiments was operated at 25°C, and the assay buffer was PBS. The contact time (the period during which the analyte consisting of TNF␣ mutants 4 and 11 was perfused over the chip) was limited to 300 s. The flow rate was set at 30 l/min. A 10 mM glycine (pH 2.0) solution was used to dissociate the bound TNF at the end of each experiment while retaining the surface integrity for chip surface regeneration.

Structure of the TNF␣-Adalimumab Fab Complex-The
TNF␣-Adalimumab Fab complex was crystallized, and the structure was determined and refined to 3.1 Å resolution with a final R work value of 19.7% (R free ϭ 28.5%) ( Table 1). One TNF␣-Adalimumab Fab complex molecule in the asymmetric unit with a Matthews coefficient of 3.5 Å 3 /Da exists, corresponding to 64% of the solvent content (21). The central TNF␣ trimer is bound by three symmetrically arranged Adalimumab Fab molecules related through a crystallographic 3-fold axis (Fig. 1), which is analogous to the structures of TNF␣-TNFR2 (22), TNF␤-TNFR1 (23), and TNF␣ in complex with other binding partners (5,24). Superimposing TNF␣ in the TNF␣-Adalimumab Fab complex with its free form yielded a root mean square deviation of 0.9 Å for all of the C␣ atoms, indicating that no significant overall structural difference occurred, except for several key residues on the antibody-antigen interface (Fig. 2). Residues TNF Leu-29, TNF Arg-31, TNF Ser-52, and TNF Tyr-56, which are crucial for TNF␣ cytotoxicity and TNFR interaction (25), were confirmed to have the correct conformation through a cytotoxicity assay.
The Adalimumab Fab presents a canonical ␤-sandwich immunoglobulin fold with the heavy chain folding into the V H and C H domains and the light chain folding into the V L and C L domains. The elbow angle, defined as the subtended angle by two pseudo 2-fold axes relating V H to V L and C H to C L of Adalimumab Fab, was ϳ140°. The complementarity-determining region (CDR) loops L1, L2, L3, H1, and H2 of Adalimumab belong to the Chothia canonical classes (26) 2, 1, 1, 1, and 3, respectively. The CDR loops L1, L2, L3, H1, H2, and H3 of Adalimumab form a large deep pocket to accommodate the epitope (Fig. 3), whereas not all of the CDR loops of Infliximab participate in the interaction with TNF␣ (5).
Interactions between TNF␣ and Adalimumab-The Adalimumab Fab binds to TNF␣ through a large and highly complementary interface, with a total buried surface area of 2,540 Å 2 (Fig. 3A), which is consistent with the high avid association between Adalimumab and TNF␣ (27). The Adalimumab epitope on TNF␣ is composed of a number of discontinuous segments, including residues TNF Pro-19, TNF Gln-20, TNF Glu-23, TNF Lys-65 to TNF Gln-67, TNF Glu-10 to TNF Pro-113, TNF Tyr-141, and TNF Ala-145, to TNF Glu-146 and TNF Thr-71, TNF His-72, TNF Thr-77, TNF Thr-79, TNF Ser-81, TNF Lys-89 to TNF Asn-91, and TNF Glu-135 to TNF Asn-137 of an adjacent TNF␣ protomer (Figs. 3 and 4). Both the heavy and light chains of Adalimumab participated in the interaction with TNF␣, with all of the contacts coming from CDRs. CDRs L2 and H2 contribute to the majority of the interactions with the antigen. Additional contributions are given from CDRs L1, L3, H1, and H3. Over 20 pairs of hydrogen bonds or salt bridges stabilized the TNF␣-Adalimumab Fab complex (Table 2), which indicates a strong and stable interface within this antigen-antibody pair.  The interface between the heavy chain of Adalimumab and TNF␣ is primarily composed of the residues in the G-H loop, several amino acids in strand D, and the D-E loop of an adjacent TNF␣ protomer. Ada His-57 in CDR H2 and Ada Ser-103 and Ada Thr-104 in CDR H3 make hydrogen bonds with TNF Ala-145 to TNF Ser-147 in the G-H loop. Moreover, the residues Ada Asn-54 and Ada Gly-56 of CDR H2 contact TNF Thr-79, TNF Ser-81, and TNF Asn-92 in strands D and E and TNF Glu-135 in strand G of another neighboring TNF␣ polypeptide.

Comparison of the Interfaces between TNF␣ and TNFRs and the Infliximab Fab and the Adalimumab Fab-A comparison of the interfaces in TNF␣-TNFRs, TNF␣-Infliximab, and
TNF␣-Adalimumab Fab provides a better understanding of the mechanism of TNF␣ inhibition by blocking the communication with TNFRs, which is what allows Adalimumab to effectively inhibit the TNF␣ function more directly compared with Infliximab (Fig. 5).
In the TNF␣-TNFR2 complex, one TNFR2 molecule interacts with two adjacent TNF␣ protomers, such as in the TNF␣-Adalimumab complex. By contrast, the antigen-antibody interaction only involves one TNF␣ molecule in the TNF␣-Infliximab complex (5). The structures of the extracellular domains of the TNFR superfamily are composed of cysteinerich domains (CRDs) that typically contain six cysteine residues that form three disulfide bonds (23). In the TNF␣-TNFR2 complex, the CRD2 and CRD3 of TNFR2 play important roles in the cytokine-receptor interaction. CRD2 and CRD3 contact strand Adalimumab complex, whereas none of these residues can be observed in the TNF␣-Infliximab structure. Additionally, the Infliximab paratope consists of five CDRs, namely L2, L3, H1, H2, and H3, whereas the Adalimumab paratope is composed of all six CDRs (Fig. 6). These structural features reveal that the Adalimumab epitope directly overlaps the TNFR binding area with a larger area of the antigen-antibody interface of TNF␣-Adalimumab (2,340 Å 2 ), whereas the Infliximab epitope is distant from the receptor-binding sites with less interacting surface (1,977 Å 2 ).
The replacement of TNF Pro-21, TNF Thr-72, TNF Lys-90, TNF Val-91, TNF Glu-110, and TNF Ile-136 with alanine residues did not obviously affect the binding capacity of TNF␣ with Adalimumab, whereas the substitutions on TNF Glu-23, TNF Asn-92, and TNF Pro-113 showed 5-10-fold decreases in binding. Nota-bly, the mutant TNF Q21A presented a sharp decrease in the binding to Adalimumab with a 200-fold lower binding affinity. The same phenomenon was observed in the TNF K65A, TNF Q67A, TNF E135A, and TNF E146A mutations. All of these mutants resulted in a 100 -200-fold affinity decrease. The TNF Gln-21 of strand A, TNF Lys-65 and TNF Gln-67 of strand C, as well as TNF Glu-135 and TNF Glu-146 of the G-H loop, which are crucial for TNF␣-

FIGURE 5. A comparison of the interface between TNF␣ and receptors and Infliximab/Adalimumab Fab complexes.
A comparison of the interface between TNF␣ and receptors and mAbs is shown. TNF␣ from the complex structures is represented as a colored surface with TNFR2 and the mAb Fabs interface highlighted in red at one of three interfaces on the TNF␣ trimer. The E-F loop region, which is missing in the TNF␣-TNFR2 (A) complex because of a lack of interaction, is labeled. The TNF␤ from the TNF␤-TNFR1 (B) complex structure is shown as a colored surface with one of the TNFR1-binding sites highlighted in red. The TNF␣-Infliximab Fab (C) and the TNF␣-Adalimumab Fab (D) interfaces are shown as colored surfaces with TNF␣-binding sites highlighted in red.

DISCUSSION
Etanercept, Infliximab, and Adalimumab have remarkably enhanced the treatment of immune diseases after they were successfully developed. A number of clinical investigations have studied the current use of these TNF␣ inhibitors and revealed that Adalimumab has an advantage in therapeutic treatment. However, the cause for this distinct efficacy remains elusive, although all of these TNF␣ inhibitors function as blockers that interrupt TNF␣-TNFR communication.
Because Etanercept is a soluble TNFR2-Fc recombinant, the structure of TNF␣-TNFR2 explains the mechanism by which Etanercept blocks the TNF␣-TNFR interaction by occupying the receptor binding site on TNF␣ (22). One Etanercept/ TNFR2 molecule interacted with two TNF␣ molecules, and the majority of the interface was made up of CRD2 and CRD3 regions of Etanercept/TNFR2 and the interface of two adjacent TNF␣ protomers, with a buried surface of 2,500 Å 2 (22) (Fig.  5A). The epitope of Infliximab on TNF␣ is primarily composed of C-D and E-F loop residues and several key residues in strands C and D of the TNF␣ molecule, with a total buried surface of 1,977 Å 2 (Fig. 5C). Interestingly, one Infliximab Fab contacts only one TNF␣ protomer, whereas two adjacent TNF␣ protomers both contribute to the Etanercept/TNFR2 contact. Although both Etanercept and Infliximab bind to TNF␣, their affinity for binding to TNF␣ is controversial. Scallon et al. (28) suggested that Infliximab has a slightly lower K D value of 4.5 ϫ 10 Ϫ10 M compared with 1.15 ϫ 10 Ϫ9 M for Etanercept. However, Smith et al. (29) showed greater affinity of Etanercept with a K D of 2.35 ϫ 10 Ϫ11 M compared with the lower K D value of 1.17 ϫ 10 Ϫ10 M of Infliximab. The larger affinity displayed by Infliximab is believed to be a consequence of the greater stability of the TNF␣-Infliximab complex (28), whereas the greater affinity of Etanercept was attributed to the faster rate of ligand binding (29). The area of buried surfaces FIGURE 6. Sequence comparison between two TNF␣ therapeutic antibodies (Infliximab and Adalimumab). The CDRs are highlighted by black frames and labeled. The residues that play crucial roles in the antibody-antigen interaction are framed with blue frames. The residue numbers (top) refer to those in Infliximab.

TABLE 3 Kinetics and binding affinity of TNF␣ mutants with the Adalimumab Fab
Kinetics and affinity of TNF␣ mutant and the Adalimumab Fab were analyzed using a BIAcore T100. Wild type TNF␣ and the mutants were passed over the immobilized Adalimumab Fab surface, and the data were globally analyzed using a simultaneous fit for both dissociation (k d ) and association (k a ). The value for K D was calculated as k d /k a . shown in the structural information is likely consistent with the greater K D value of Etanercept. Although the binding affinities displayed by Etanercept and Infliximab are debated, Adalimumab has been reported to bind TNF␣ with a relatively higher affinity than Etanercept and Infliximab, with K D values ranging from 7.05 ϫ 10 Ϫ11 M (30) to 1.0 ϫ 10 Ϫ10 M (31). The buried surface of one Adalimumab Fab with trimeric TNF␣ is consistently 2,536 Å 2 , which is larger than those of one Infliximab Fab and one Etanercept molecule with trimeric TNF␣. The structural comparisons of the TNF␣-Adalimumab Fab with the TNF␣-Infliximab and TNF␣-TNFR2 complex reveal that the Adalimumab epitope extensively overlaps with the TNF␣-TNFR2 interface, whereas Infliximab only partially occupies the TNF␣-TNFR2 binding area and is mainly targeted at the E-F loop of TNF␣ and spatially obstructs the correct interaction with TNFRs. Furthermore, a total of 7 of 21 TNF␣ residues involved in TNFR2 binding also participate in contacting the Adalimumab Fab. The three regions, including residues TNF Gln-21, TNF Glu-23, TNF Ala-145, and TNF Glu-146 that form two negatively charged surface patches, residues TNF Pro-113 and TNF Tyr-115 that form a hydrophobic surface patch and residues TNF Val-85-TNF Tyr-87 and TNF Thr-89 of an adjacent TNF␣ that also form a negatively charged surface patch, are shared by both TNFR2 and Adalimumab Fab. Notably, although TNF Gln-21, TNF Glu-23, TNF His-73, TNF Pro-113, TNF Tyr-115, TNF Ala-145, and TNF Gln-146 play essential roles in the TNFR2 and Adalimumab interaction, they are irrelevant for TNF␣-Infliximab recognition. In the previous investigations, random mutagenesis studies on TNF␣ were performed to produce inactive molecules that lost their cytotoxic activity. Interestingly, the residues that were identified to lose most cytotoxic activity are involved in both TNFR2 and Adalimumab interactions. For example, the activity of TNF␣ dropped when alterations were introduced in TNF Glu-23, TNF Pro-113, and TNF Tyr-115 without marked changes in immunoreactivity or physicochemical characteristics, as well as TNF Q146K mutation, which causes a nearly complete loss of the cytotoxicity for TNF␣ (32). Moreover, TNF Asp-143, TNF Gln-149, and TNF Glu-24, which are important residues for the TNFR2 recognition not only in the structure analysis but also in previous mutagenesis studies (22,32,33), are involved in the TNF␣-Adalimumab interface.
In early stage usage of Infliximab, a number of patients discontinued the use of Infliximab because of the loss of response (6). It was proposed that Infliximab is a chimeric mAb, and its usage in humans could lead to the production of "antibodies to Infliximab" in a small subset of patients (34 -37). Using more human sequence content by grafting murine CDRs may be crucial for the integral capacity of antigen binding and should be retained during humanization (38). Although this practice pattern still occurs, in present day clinical practice, the frequency of Infliximab discontinuation for this reason is low (7). However, the efficacy of Adalimumab-based TNF-blocking therapy in autoimmune diseases is higher than that of Etanercept and Infliximab. The structure shown here illustrates how Adalimumab prevents ligands from binding to TNFR2, inhibits ligand-receptor binding, and blocks TNFR activation. The primary consequence of Adalimumab binding to TNF␣ is the steric blocking of TNF␣ and the prevention of ligand binding to the receptor. The solvent-accessible surface contributed by the ligand-receptor interaction covers over 60% of the total interface between TNF␣ and the Adalimumab Fab, which indicates a straightforward overlap between the TNF␣ receptor binding sites and the Adalimumab epitope. Moreover, several residues that are crucial for TNF␣ receptor binding also participate in the TNF␣-Adalimumab Fab interface, especially the groove between the two associated TNF␣ molecules in the TNF␣ trimer. These results may partly explain the clinical data regarding the more significant effectiveness of the treatment compared with placebo in inducing remission in patients with Crohn's disease who are intolerant of or have lost response to Infliximab. Therefore, binding Adalimumab to TNF␣ can efficiently compete with binding TNFRs to TNF␣. The interface between TNF␣ and TNFRs is blocked in the presence of a sufficient amount of Adalimumab, which prevents the function of TNF␣ in the pathological process. Taken together, these data indicate that Adalimumab occupies the binding site and competitively inhibits the binding of TNFR to TNF␣. Combined with the structural information of TNF␣-Infliximab, these structures provide information to improve the interface complementarity between antibody and antigen, to strengthen the interaction, and to thereby enhance the binding affinity through altering the paratope of the antibody in targeted therapy and antibody engineering. Moreover, this information may also be useful to evaluate the antibody in the early stages of use. A therapeutic antibody whose epitope directly occupies the receptor binding position and competitively inhibits TNFR-TNF␣ communication, such as Adalimumab, may have better and more predictable clinical effects than an antibody that has a more distant epitope and uses the steric properties, such as Infliximab. Once a new antibody is identified as having promising results in preclinical studies, a crystallography study to determine its precise epitope may help us in making strategic decisions before proceeding with costly clinical drug trials.
In summary, our findings structurally explain the varying clinical observations for the anti-TNF antibodies. An evaluation of the effectiveness showed that Adalimumab had better outcomes than Infliximab from a molecular view. This finding, which agrees with another clinical report (10), highlights an opportunity for therapeutic improvement despite the significant advances in the past decade. The precise epitope revealed by our complex structure provides useful information for structurebased improvements for current TNF␣ mAbs and highlights the importance of identifying the predictors of a beneficial outcome through the structural and biological characteristics of the drug.