Metuximab is the generic name of Licartin, a new drug for radioimmunotherapy of hepatocellular carcinoma. Although it is known to be a mouse monoclonal antibody against CD147, the complete epitope mediating the binding of metuximab to CD147 remains unknown. We panned the Ph.D.-12 phage display peptide library against metuximab and got six mimotopes. The following bioinformatics analysis based on mimotopes suggested that metuximab recognizes a conformational epitope composed of more than 20 residues. The residues of its epitope may include T28, V30, K36, L38, K57, F74, D77, S78, D79, D80, Q81, G83, S86, N98, Q100, L101, H102, G103, P104, V131, P132, and K191. The homology modeling of metuximab and the docking of CD147 to metuximab were also performed. Based on the top one docking model, the epitope was predicted to contain 28 residues: AGTVFTTV (23–30), I37, D45, E84, V88, EPMGTANIQLH (92–102), VPP (131–133), Q164, and K191. Almost half of the residues predicted on the basis of mimotope analysis also appear in the docking result, indicating that both results are reliable. As the predicted epitopes of metuximab largely overlap with interfaces of CD147-CD147 interactions, a structural mechanism of metuximab is proposed as blocking the formation of CD147 dimer.
Metuximab is the generic name of HAb18, a mouse monoclonal antibody of IgG1 class developed by Chen et al. in 1989 [
CD147 is a transmembrane glycoprotein of the immunoglobulin superfamily. It has been involved in various physiological functions such as spermatogenesis [
Where does metuximab bind to CD147? The answer will help us understand the mechanism of CD147 function and benefit the development of new drugs targeting CD147. Using binding assays to a series of truncated fragments of CD147 ectodomain, Ku et al. reported that the segment of 39LTCSLNDSATEV50 was the epitope on CD147 recognized by metuximab [
Crystallographic analysis of antigen-antibody complex is the most accurate approach to mapping an epitope. However, it is time consuming and sometimes technically difficult or even impossible to get an antigen-antibody complex crystallized. As an alternative choice for epitope mapping with lower but acceptable precision, mimotope analysis is becoming to be an increasingly popular method for its cheapness and quickness [
The F(ab′)2 fragment of metuximab as freeze-dried powder with purity above 97% was provided by Chengdu Huasun Bio-Tech Co. LTD. The Ph.D.-12 phage display peptide library that displays 2.7 × 109 unique 12-amino acid peptides fused to the pIII minor coat protein of the M13 filamentous phage was purchased from New England BioLabs. Three successive rounds of biopannings were performed with the F(ab′)2 fragment of metuximab as the capture reagent coated on 96-well microtiter plates, as described in the manufacturer’s manual with modifications.
In brief, the sample was diluted to a concentration of 200
The peptides displayed on the selected phages were deduced from the results of DNA sequencing. The data was firstly cleaned using the tools in the SAROTUP suite to exclude any possible target-unrelated peptides [
The sequences of the variable heavy chain (VH) and light chain (VL) of metuximab were extracted from the United State patent with the number US7638619 [
Templates used in modeling metuximab.
Region* | PDB | Identity | Align length | Mismatches | Gap openings |
|
---|---|---|---|---|---|---|
HFR | 1SBS | 95.52 | 67 | 3 | 0 |
|
LFR | 1IAI | 85.48 | 62 | 9 | 0 |
|
H1 | 2DLF | 100 | 10 | 0 | 0 |
|
H2 | 2DLF | 75 | 20 | 3 | 2 |
|
H3 | 1A0Q | 0 | 0 | 0 | 0 |
|
L1 | 1QNZ | 100 | 6 | 0 | 0 |
|
L2 | 3FCT | 83.33 | 6 | 1 | 0 |
|
L3 | 1NCD | 77.78 | 9 | 2 | 0 |
|
As shown in Table
Fv model of metuximab.
The 3D structure of CD147 monomer (3B5H, chain C) was then docked to the Fv model of metuximab using the program ZDOCK with framework region blocked [
The titer of the eluate after each round of pannings increased from 103, 104 to 106 PFU/mL, indicating an efficient enrichment of phages specifically binding to metuximab. After the third panning, twenty phage clones were randomly picked and sent to DNA sequencing. The deduced amino acids sequences are listed in Table
Peptides selected from Ph.D.-12 phage display peptide library using metuximab.
Number | Sequence | Occurrence |
---|---|---|
P1 |
|
9 |
P2 |
|
1 |
P3 |
|
1 |
P4 |
|
1 |
P5 |
|
1 |
P6 |
|
1 |
P7 |
|
6 |
As shown in Table
These peptides were checked using tools in the SAROTUP suite. Interestingly, it was reported that the peptide DFDVSFLSARMR had also been panned out from the Ph.D.-12 phage display peptide library using the protein tonB of
As shown in Tables
Epitope mapping solution no. 1 based on mimotope analysis.
Mimotope | Predicted epitopic residues |
---|---|
|
L101, H102, G103, P104, R106, P132, P133, T135, T188, K191, G192 |
|
S78, Q81, Q100, L101, H102, G103, P104, R106, S128, S130, P132, P133, S189, S190, K191, G192, S193 |
|
L101, H102, P104, R106, P132, P133, T135, T188, K191, D194 |
|
L101, H102, P104, R106, P132, P133, T135, T188, K191 |
|
V30, S78, Q81, Q100, H102, G103, R106, S128, S130, V131, S189, S190, K191, G192, S193 |
|
V30, S78, Q81, Q100, H102, R106, S128, S130, V131, S189, S190, S193 |
| |
Union |
|
Epitope mapping solution no. 2 based on mimotope analysis.
Mimotope | Predicted epitopic residues |
---|---|
|
T28, K36, L38, K57, F74, G83, L101, H102, G103, P104, P132, K191 |
|
K36, L38, K57, F74, S78, Q81, G83, S86, Q100, L101, H102, G103, P104, P132, K191 |
|
T28, K36, L38, K57, F74, D77, D79, D80, L101, H102, P104, P132, K191 |
|
T28, K36, L38, K57, F74, L101, H102, P104, P132, K191 |
|
V30, K36, K57, S78, Q81, G83, S86, Q100, H102, G103, V131, K191 |
|
V30, F74, S78, Q81, S86, N98, Q100, H102, V131 |
| |
Union |
|
Epitope mapping results from mimotope analysis. Residues only appearing in solutions nos. 1 and 2 are colored in purple and orange, respectively; the overlapping region of the two solutions is drawn in green. All other parts of CD147 surface are presented in pale cyan.
When the parameter “Accuracy cutoff” (allowed mismatch) was set from 3 (default) to 2 (a stricter value), only solution 2 was left. Thus, solution 2 might be a more accurate prediction, and we considered this solution to be the epitope predicted by phage display and mimotope analysis.
It has been reported that the frequencies of peptides are not correlated to their binding strength and the diversity of peptides are important in analysis [
We have also used other tools such as PepMapper [
The computation of the top one model from RDOCK results revealed that metuximab might bind to a conformational epitope. As shown in Figure
Theoretical model of metuximab-CD147 complex. CD147 was displayed as cartoon in rainbow color, and the interface residues on it were drawn as sticks and labeled.
Other nine models of the top ten RDOCK poses were also used to compute the theoretical epitopes. The results show that seven of them were identical to that of top one model. The epitope derived from the 7th model is a little bit different. Besides including all residues shown in Figure
Though there are arguments about the role of molecular docking in epitope mapping, the results of molecular docking can be very accurate. In 2001, Saphire et al. solved the structure of b12, a neutralizing human IgG against HIV-1. They docked gp120 to b12 and predicted the epitope recognized by b12 [
CD147 exists in several forms, such as monomer, dimer, and polymer [
Very interestingly, the predicted epitopes of metuximab based on either phage display or molecular docking overlap the interfaces between AC, BC, and AD dimmers. Coincidentally, the nonspecific D D′ dimer is the only exception, which has no intersection with the epitope of metuximab. Thus, at least one mechanism of metuximab is to block CD147-CD147 interactions. Therefore, peptides obtained from screening the phage-displayed random peptide library might also have potential applications in blocking CD147 pathways.
According to the results from and analyses on molecular docking and phage display experiments, we conclude that metuximab recognizes a conformational epitope composed of more than 20 residues. These residues mainly locate on the N-terminal domain surface of CD147 and largely overlap with interfaces of CD147-CD147 interactions. Blocking the formation of CD147-CD147 dimer may be an important mechanism of metuximab function.
The authors are grateful to the anonymous reviewers for their valuable suggestions and comments, which have led to the improvement of this paper. This work was supported in part by the National Natural Science Foundation of China under Grant 61071177 and the Program for New Century Excellent Talents in University (NCET-12-0088).