Catalytic digestion of human tumor necrosis factor-a by antibody heavy chain Emi Hifumi 1,2 , Kyohei Higashi 3 and Taizo Uda 2,3 1 Research Center for Applied Medical Engineering, Oita University, Japan 2 CREST of JST (Japan Science and Technology Corporation), Kawaguchi, Saitama, Japan 3 Faculty of Engineering, Oita University, Japan Introduction This decade has seen the preparation of many natural catalytic antibodies. The first natural catalytic anti- body isolated from the serum of an asthma patient was reported by Paul et al. [1]. The catalytic antibody could enzymatically cleave vasoactive intestinal peptide (VIP). Gabibov and colleagues [2] and Nevinsky and colleagues [3] found antibodies showing catalytic activ- ity capable of cleaving DNA. The two former catalytic antibodies were prepared from samples of humoral serum from individuals with autoimmune diseases (e.g. systemic lupus erythematosus) and the latter from human milk. A natural catalytic antibody produced in the serum of patients with hemophilia A reported by Kaveri and colleagues [4] was unique because it enzy- matically decomposed the molecule of factor VIII, indicating some pathological roles of catalytic antibod- ies in vivo. These antibodies exhibited catalytic activity in the form of the whole antibody. In contrast, Bence- Jones protein, which is secreted in the urine of patients with certain diseases, particularly multiple myeloma, is well known to be a human light chain of the antibody. Matsuura et al. [5], Matsuura & Sinohara [6] and Paul et al. [7] found that Bence-Jones proteins could have peptidase activity. These reports revealed that some antibodies and ⁄ or the light chains naturally produced in the patients could have catalytic activity, but their antigens are unknown. From the standpoint of new approaches to generate or characterize a catalytic anti- body, Gololobov et al. [8] reported a unique catalytic antibody cleaving gp120 of HIV, using a covalently reactive analog method. Ponomarenko et al. [9] made a catalytic anti-idiotype antibody, and examined the Keywords catalytic antibody; cytokine; proteolysis; TNF-a Correspondence T. Uda, Oita University, Faculty of Engineering, 700 Dannoharu, Oita-shi, Oita 870-1192, Japan Fax: +81 97 554 7892 Tel: +81 97 554 7892 E-mail: uda@cc.oita-u.ac.jp (Received 10 April 2010, revised 19 June 2010, accepted 21 July 2010) doi:10.1111/j.1742-4658.2010.07785.x It has long been an important task to prepare a catalytic antibody capable of digesting a targeting crucial protein that controls specific life functions. Tumor necrosis factor-a (TNF-a) is a cytokine and an important molecule concerned with autoimmune diseases such as rheumatoid arthritis, chronic obstructive pulmonary disease, and Crohn’s disease. A mAb (ETNF-6 mAb) raised against human TNF-a was prepared, and the steric conforma- tion was created by using molecular modeling after the cDNA was sequenced. The heavy chain (ETNF-6-H) of the mAb was considered to possess a catalytic triad-like structure in the complementarity determining regions (CDRs). As a result, ETNF-6-H exhibited a peptidase and a prote- ase activity. In fact, ETNF-6-H predominantly cleaved the Ser5-Arg6 bond of TNF-a at the first step, resulting in the generation of a fragment of  17 kDa. This fragment was digested to a smaller molecule of 15 kDa by scission of the Gln21-Ala22 bond. The intermediate product was further converted into a fragment of 13.3 kDa by successive cleavage of the Leu36- Leu37 and Asn39-Gly40 bonds. The heavy chain possessed a protease activ- ity against TNF-a with a multicleavage site. Abbreviations CDR, complementarity determining region; ETNF-6-H, heavy chain of ETNF-6 mAb; HSA, human serum albumin; hTNF-a, human tumor necrosis factor-a; TNF-a, tumor necrosis factor-a; TNF-b, tumor necrosis factor-b; VIP, vasoactive intestinal peptide. FEBS Journal 277 (2010) 3823–3832 ª 2010 The Authors Journal compilation ª 2010 FEBS 3823 features in detail with the use of several potential pep- tide substrates. Apart from the natural catalytic antibodies found in human subjects, Paul and colleagues [10] and Uda and colleagues [11–14] have succeeded in producing some catalytic antibodies by immunizing ground-state poly- peptides or proteins in mice. A former catalytic anti- body could cleave the antigenic peptide VIP with its antibody light chain [10]. In the latter cases, Uda et al. obtained a light chain of 41S-2 mAb cleaving an HIV- 1 env gp41 molecule. They also succeeded in the pro- duction of catalytic antibody light and ⁄ or heavy chains capable of the degradation of urease in Helicobact- er pylori. The catalytic antibody light chain, UA15-L, could suppress the number of bacteria infecting the mouse stomach. In these catalytic antibodies, a unique structure (catalytic triad-like structure), in which three amino acids (Asp, Ser, and His) are situated close to each other, is observed in many antibodies by the use of molecular modeling. These studies suggest the possi- bility that we can prepare catalytic antibodies capable of cleaving molecules of interest. Tumor necrosis factor-a (TNF-a) is a crucial mole- cule as an inflammatory cytokine, and causes severe diseases such as rheumatoid arthritis, chronic obstruc- tive pulmonary disease, and Crohn’s disease [15–20]. In this study, we prepared a mAb (ETNF-6 mAb) raised against human TNF-a by the immunization of the ground-state molecule into Balb ⁄ c mice. The heavy chain isolated from the parent whole antibody showed the unique catalytic ability to degrade the TNF-a mol- ecule. In this article, the features of the heavy chain will be described in detail from the immunochemical and biological points of view. Results Immunological features of the antibodies By normal cell fusion [21] after the immunization of human TNF-a (hTNF-a) into mice, ETNF-6 mAb binding with hTNF-a was prepared. ETNF-6 mAb did not show any cross-reactivity to other proteins, such as human serum albumin (HSA), BSA, human IgA, human IgM, human IgE, human hemoglobin, KLH and tumor necrosis factor-b (TNF-b)(Fig. 1). ETNF-6 mAb possessed very high specificity against hTNF-a. The apparent affinity constants of intact ETNF-6 mAb and its heavy chain were evaluated by using ELISA. From A 50 of the ELISA, apparent affinity constants of ETNF-6 mAb and its heavy chain for hTNF-a were estimated to be 1.1 · 10 9 m )1 and 4.2 · 10 6 m, respectively [22]. Sequences and steric conformation of ETNF-6 mAb Sequencing of the cDNA of the variable region of ETNF-6 mAb was performed, and this was followed by molecular modeling of its three-dimensional struc- ture. The heavy chain of ETNF-6 (ETNF-6-H) seemed to encode a catalytic triad-like structure in the CDRs. The cDNA and amino acid sequences that were deduced are presented in Fig. 2A,B. Figure 3A shows the three-dimensional structure of the variable region of ETNF-6-H. In the heavy chain, three amino acids, His35, Ser95, and Asp97, are located closely together in CDR1 and CDR3. The distance between the Ca atoms of His and Ser is 7.17 A ˚ , and that between the Ca atoms of His and Asp is 9.87 A ˚ . In Fig. 3B–E, other catalytic triads, composed of Asp1, Ser27e, and His93, which are mostly observed in catalytic light chains such as VIPase [10], ECL2B-2-L [22], i41SL1-2-L [23], HpU-9-L [24], and UA15-L [25], are presented along with that of ETNF-6-H. In all cases, it is inter- esting that the three amino acids are located in the CDRs and are positioned closely together. In the case of ECL2B-L, the distance between the Ca atoms of His93 and Ser27e is 7.25 A ˚ , and that between the Ca atoms of His93 and Asp1 is 12.75 A ˚ . Among the struc- tures mentioned above, the three amino acids of ETNF-6-H are close to those of ECL2B-2-L. Cleavage assay For a peptide To avoid contamination, most glassware, plasticware and buffer solutions used in this experiment were ster- ilized as much as possible by heating (180 °C, 2 h), autoclaving (121 °C, 20 min), or filtration through a 0 TNF-α TNF-β h-IgA h-IgM h-IgE HSA h-Hb BSA KLH 0.2 0.4 0.6 0.8 1 1.2 OD 405 nm 1.4 1.6 1.8 Fig. 1. Results of cross-reactivities of ETNF-6 mAb with irrelevant proteins by ELISA. ETNF-6 mAb showed high specificity for hTNF-a. The mAb did not react at all with human TNF-b at all. Catalytic digestion of TNF-a by the antibody subunit E. Hifumi et al. 3824 FEBS Journal 277 (2010) 3823–3832 ª 2010 The Authors Journal compilation ª 2010 FEBS 0.20-lm sterilized filter. The experiments were mostly performed in a biological safety cabinet, to avoid airborne contamination. The epitope of TNF-a recognized by ETNF-6 mAb was not determined. In this study, TP41-1 (TPRGPD RPEGIEEEGGERDRD), which has mostly been used for monitoring the catalytic activity of the antibody and ⁄ or its subunits [11–14,26], was employed to inves- tigate whether or not the antibody heavy chain pos- sesses peptidase activity. An 800-lL volume of a solution containing purified ETNF-6-H (0.4 lm) and TP41-1 (60 lm) was incu- bated in 15 mm NaCl ⁄ P i at 25 °C in a sterilized test tube. RP-HPLC was used to monitor the time course of the cleavage of TP41-1. As shown in Fig. 4A, degra- dation of TP41-1 began about 24 h after ETNF-6-H and TP41-1 were mixed together. After the lag phase, TP41-1 was rapidly cleaved. At about 80 h, the peptide had completely disappeared, indicating complete degradation. Decomposition of TP41-1 exhibited a double-phase reaction profile, as mostly observed in [11–14,26]. Without the presence of ETNF-6-H, TP41-1 was not degraded. Figure 4B also shows HPLC chromatograms. After a reaction time of 47.2 h, the amount of TP41-1 at a retention time of 11 min decreased, because of frag- mentation. The fragment was observed at a retention time of 13 min (indicated by an arrow) as a small peak. The peak at 13 min is considered to be a frag- ment cleaved at the Glu14-Gly15 bond of TP41-1, because the retention time was consistent with that observed with 41S-2-L [13] and i41-7 subunits [27]. At a reaction time of 66.9 h, the peaks at both 11 and 13 min decreased further. Finally, at 78.5 h, TP41-1 and its fragment almost disappeared from the reaction system. The intact mAb exhibited no catalytic activity (data not shown). For human TNF-a It has already been shown that catalytic antibody subunits assume the preferable conformations in the induction period [13] or in a reaction mixture [28]. Once the conformation has been assumed, the catalytic activity becomes stable, showing no induction time [13,22–27]. Thus, prior to the cleavage test for hTNF-a, TP41-1 was completely digested by the catalytic reac- tion of ETNF-6-H. To determine whether ETNF-6-H can digest intact hTNF-a, 12% gel SDS ⁄ PAGE with silver staining was performed to monitor the time course of the cleavage of hTNF-a at 0, 8, 20, 50 and 94 h of incubation (Fig. 5A). (In this case, nonreduc- ing conditions were employed, because, when proteins are treated under reducing conditions with 2-mercapto- ethanol at 95 °C, there is a possibility that protein cleavage will occur, and we wanted to prevent this from happening.) Lanes 1–5 in Fig. 5A represent the bands obtained by mixing ETNF-6-H (0.1 lm) and hTNF-a (6.6 lm) during the incubation. Lanes 6–10 show the controls. As shown in lane 1, some bands were detected at 0 h of incubation. A clear and strong band appearing at  17 kDa corresponds to mono- meric hTNF-a. The dimeric form was observed at 33.6 kDa. Two small bands at 19 and 20 kDa might be ascribed to isomers of hTNF-a or some adducts to the hTNF-a molecule. (For these two bands, N-termi- nal amino acid sequencing was performed, but the analysis failed, because the N-terminus might have Fig. 2. Nucleotide sequences of cDNA and deduced amino acid sequences for ETNF-6-H. (CDR-1, green; CDR-2, pink; CDR-3, blue). E. Hifumi et al. Catalytic digestion of TNF-a by the antibody subunit FEBS Journal 277 (2010) 3823–3832 ª 2010 The Authors Journal compilation ª 2010 FEBS 3825 been blocked. Therefore, we could not identify the molecules; see later results regarding N-terminal sequencing.) At 8 h of incubation (lane 2), the bands at 19 and 20 kDa had disappeared, and two clear bands appeared at 15.0 and 13.3 kDa. These bands are thought to be fragments of hTNF-a.At20hof incubation (lane 3), a faint band was observed at 30.6 kDa. This is considered to be a fragment gener- ated from a dimer of hTNF-a. The strong band at 17 kDa and the two bands at 15.0 and 13.3 kDa became clearer with incubation. At 94 h of incuba- tion, the band at 15 kDa became faint, suggesting that the fragment was successively degraded into smaller molecular fragments, presumably of 13.3 kDa. In contrast, there were no changes with the incuba- tion times in the controls without ETNF-6-H (Fig. 5A, lanes 6–10). For myoglobin and BSA In order to examine substrate specificity, ETNF-6-H (0.2 lm) was incubated with an irrelevant protein, myoglobin (0.9 lm), which is of a similar molecular size as hTNF-a, under conditions identical to those employed in the above experiment. Myoglobin was hardly cleaved after 74 h of incubation, as shown in Fig. 5B. BSA (0.3 lm) was also used for up to 74 h for a degradation test, but no change occurred. Analysis of the N-terminal sequence We characterized the cleavage sites of hTNF-a by N-terminal amino acid sequencing of the fragments produced by the cleavage with ETNF-6-H. The results are summarized in Table 1. First, we sequenced hTNF-a itself. From band 1 at 20 kDa and band 2 at 19 kDa, the sequencing failed, because of the blocking of the N-terminus by molecules. Band 3 at  17 kDa, corresponding to hTNF-a, gave one main and two minor sequences. The main band (fragment A in Table 1) was VRSSS, which is consistent with the N-terminal sequence of hTNF-a. The two other bands were RSSS (fragment B) and SRTPS (fragment C), which are the sequences of amino acids 2–6 and 5–9 from the N-terminus of hTNF-a. This means that the recombinant hTNF-a contains impurities lacking some amino acids near the N-terminus. For the reacted sam- ple at 24 h of incubation, the band corresponding to hTNF-a at 17.0 kDa gave two sequences. One was a strong signal, RTPSD (fragment D), and the other was weak, SSSRT (fragment E). The former suggests that the cleavage occurred at the bond between Ser5 and Arg6. The latter weak band suggests a cleaved bond between Arg2 and Ser3. A faint band at 15 kDa gave two signals, AEGQL (fragment F) and RTPSD (frag- ment G). The former indicates the scission of the bond between Gln21 and Ala22. The latter means that a UA15-L His93 Asp1 Ser27a Asp99 Ser58 Asp97 Ser95 His3 5 Asp72 Asp102 ETNF-6-H A CD E B Fig. 3. Three-dimensional structure of the variable region of several catalytic antibodies as determined by molecular modeling. Red: Asp. Violet space: His. Green: Ser. A circle shows the catalytic triad-like structure com- posed of Asp, His, and Ser. (A) shows the structure of ETNF-6-H. In (B), (C), (D), and (E), there are light chains that possess an identical catalytic triad-like structure com- posed of Asp1 in FR-1, Ser27a in CDR1, and His93 in CDR3. In ETNF-6-H, Ser95 and Asp97 in CDR3 and His35 in CDR1 seem to create a catalytic triad-like structure. Catalytic digestion of TNF-a by the antibody subunit E. Hifumi et al. 3826 FEBS Journal 277 (2010) 3823–3832 ª 2010 The Authors Journal compilation ª 2010 FEBS short C-terminal peptide (presumably YLLFAES- GQVYFGIIA at the C-terminus) might be successively cut from the fragment of Ser5-Arg6, as judged from the molecular size. Band 6 at 13.3 kDa, gave two sig- nals. One was the main signal, LANGV (fragment H), and the other was a weak signal, GVELR (frag- ment I). The former suggests that the Leu36-Leu37 bond was digested, and the latter suggests that that the Asn39-Gly40 bond was digested, which must have been caused by the successive digestion of hTNF-a or its fragments. Regarding the bands at 15.0 and 13.3 kDa, it is clear that the band at 15.0 kDa faded with incubation time. In contrast, the band at 13.3 kDa became deeper with incubation time. It is plausible that the fragment generated at 15.0 kDa was converted to the fragment at 13.3 kDa by a successive reaction. It seems that no changes occurred for the band at  17 kDa during incubation. The signal must be satu- rated, because a large amount of hTNF-a 6.6 lm was added to the reaction system, and SDS ⁄ PAGE with silver staining was performed. At 33.6 kDa (dimeric form of hTNF-a), the band became faint as incubation time increased, indicating digestion of hTNF-a. 0 h 47.2 h 66.9 h 0 78.5 h 10 min 0 10 min 0 10 min 0 10 min A 80 60 40 20 0 02448 Reaction time (h) Concentration of TP41-1 peptide (µ M) 72 96 120 B Fig. 4. Peptidase activity test of ETNF-6-H. TP41-1, 60 lM; ETNF-6- H, 0.4 l M. The reaction was conducted at 25 °Cin15mM phos- phate buffer (pH 6.5). (A); (s) Curve for degradation of TP41-1 by ETNF-6-H. (h) Time course of TP41-1 peptide without ETNF-6-H, as a control. Degradation of TP41-1 by ETNF-6-H advanced after a short induction time (indicating a double-phase reaction profile). TP41-1 was quickly cleaved by ETNF-6-H after a reaction time of about 24 h, as shown in (B) by a small fragmented peak at a reten- tion time of 13 min. Finally, at about 80 h, TP41-1 disappeared from the reaction system. Intact ETNF-6 mAb exhibited no catalytic activ- ity (data not shown). A B Fig. 5. Assay for cleavage of hTNF-a by ETNF-6-H. The reaction was conducted at 25 °Cin15m M phosphate buffer (pH 6.5). (A) For hTNF-a: hTNF-a, 6.6 l M; ETNF-6-H, 0.1 lM. Lanes 1, 2, 3, 4, and 5: 0, 8, 20, 50 and 94 h of incubation, respectively, after mixing of hTNF-a and ETNF-6-H. Lanes 6, 7, 8, 9, and 10: 0, 8, 20, 50 and 94 h of incubation, respectively, of hTNF-a without ETNF-6-H (control). (B) For Myoglobin: myoglobin, 0.9 l M; ETNF-6-H, 0.2 lM. A clear, strong band appearing at 17.0 kDa corresponds to mono- meric hTNF-a. The dimeric form was observed at 33.6 kDa. Two small bands at 19 and 20 kDa might be ascribed to isomers of hTNF-a or some adducts to the hTNF-a molecule. After 8 h of incu- bation, the bands at 19 and 29 kDa had disappeared, and two clear bands appeared at 15.0 and 13.3 kDa. These bands are thought to be fragments of hTNF-a. After 20 h of incubation, a faint band was observed at 30.6 kDa, which is thought to be a fragment generated from a dimer of hTNF-a. The band and the two bands below hTNF-a became clearer as incubation time increased. After 94 h of incu- bation, the band at 15.0 kDa became faint, suggesting that the fragment was successively degraded into smaller fragments. In contrast, there were no changes at any incubation time in the controls without ETNF-6-H. E. Hifumi et al. Catalytic digestion of TNF-a by the antibody subunit FEBS Journal 277 (2010) 3823–3832 ª 2010 The Authors Journal compilation ª 2010 FEBS 3827 Discussion It is well known that the active sites of serine proteases such as trypsin, chymotrypsin and thrombin are com- posed of Ser, His and Asp residues, whose sites are referred to as catalytic triads. We have already pointed out the high probability of obtaining a catalytic anti- body light (or heavy) chain by the immunization of a ground-state peptide if a catalytic triad composed of Asp, Ser and His is generated in the antibody structure [23,26]. In this study, we produced a mAb (ETNF-6 mAb) against human TNF- a. As shown in Fig. 2A, ETNF-6-H seems to form a catalytic triad composed of Asp97, Ser95, and His35. On the topic of antibody light chains, VIPases (Asp1 in FR-1, Ser27a in CDR1, and His93 in CDR3) cleaving antigenic VIP have been reported [28]. Uda and colleagues [23] have also found several catalytic antibody light chains, such as i41SL1- 2-L for antigenic peptide RSSKSLLYSNGNTYLY, ECL2B-2-L for the chemokine receptor of CCR-5 [22], and UA15-L for H. pylori urease [24], which are light chains possessing a catalytic triad at the identical posi- tions of Asp1, Ser27a, and His93. Kolesnikov et al. [29] reported that a dyad composed of His and Ser is the active site for the hydrolysis of esters in their anti- idiotypic antibody catalyst. Note that His plays an important role in generating hydrolysis. Several reports on catalytic heavy chains have been published [12,23,26]. We considered that a similar catalytic triad- like structure as seen in the light chains described above might be generated in the CDRs of ETNF-6-H, resulting in hydrolytic activity. Up to now, we have used a peptide, TP41-1, to monitor the peptidase activity of catalytic antibodies [13,23,25,26,30], because the peptide is highly soluble and not bound to the wall of the reaction vessel, in addition to showing very little degradation in the phos- phate buffer. In this study, the peptide was degraded by ETNF-6-H within about 80 h of incubation. A lag phase that occurred within 24 h of incubation was also observed in this case. This phase is seen in many cleav- age reactions with natural catalytic antibodies [11– 14,23,26]. This sort of lag phase was also observed in proteolysis with an anti-idiotypic antibody [31]. In the lag phase, it is considered that conformational changes of catalytic antibody subunits must take place, result- ing in the active form of the antibody subunit generat- ing a multimeric form [13]. Recombinant human hTNF-a was gradually degraded by ETNF-6-H during 94 h of incubation. The cleavage sites, which were confirmed by N-termi- nal amino acid sequencing, are indicated by red arrows in Fig. 6. The commercially available recombinant hTNF-a used in this study contained a small amount of two short forms that lack Val or Val-Arg at the N-terminus. Nonetheless, in the digestion of hTNF-a by ETNF-6-H, a strong signal of RTPSD (170 pmol for Arg) for the band at  17 kDa suggests that the bond between Ser5 and Arg6 was predominantly cleaved by ETNF-6-H as the first step. The band at 15 kDa, which appeared after 8 h of incubation, gradually became faint with increased incubation time. Thus, the generated fragment is an intermediate prod- uct in the degradation of TNF-a. The strong signal of the band at 13.3 kDa, LANGV (29.9 pmol for Leu), is considered to result from a fragment generated from the polypeptide cleaved at Ser5-Arg6. ETNF-6-H could cleave several peptide bonds, such as Ser-Arg, Arg-Ser, Gln-Ala, Leu-Leu, and Asn-Gly. Many catalytic antibodies, such as those against VIP Table 1. Results of N-terminal amino acid sequence analysis for fragmented polypeptides from human TNF-a. Fragmented band Size (kDa) Name of fragment (expected mass in kDa) Detected amino acids of N-terminus (five residues) (pmol) Cleavage site (expected mass) Band 1 (only TNF-a) 20.0 Could not be analyzed No cleavage (blocked at N-terminus) Band 2 (only TNF-a) 19.0 Could not be analyzed No cleavage (blocked at N-terminus) Band 3 (only TNF-a) 17.0 A (17.3) V (18.4), R (6.7), S (12.3), S (8.4), S (8.3) N-terminus B (17.2) R (3.5), S (3.1), S (12.3), S (8.4), R (4.0) V1-R2 C (17.0) S (2.4), R (6.7), T (1.7), P (1.3), S (8.3) S4-S5 Band 4 (TNF-a + ETNF-6-H) 17.0 D (16.8) R (170), T (40), P (56), S (17), D (83) S5-R6 E (17.1) S (8.4), S (7.5), S (6.3), R (19.7), T (4.6) R2-S3 Band 5 (TNF-a + ETNF-6-H) 15.0 F (15.1) A (3.9), E (3.3), G (2.3), Q (2.7), L (2.8) Q21-A22 G (14.9) R (0.6), T (0.9), R (0.9), S (0.5), D (0.5) S5-R6 (C-terminus: YLLFAESGQVYFGIIAL may be digested) Band 6 (TNF-a + ETNF-6-H) 13.3 H (13.5) L (22.9), A (22.1), N (14.3), G (12.0), V (10.7) L36-L37 I (13.3) G (2.5), V (2.9), E (2.1), L (3.7), R (0.5) N39-G40 Catalytic digestion of TNF-a by the antibody subunit E. Hifumi et al. 3828 FEBS Journal 277 (2010) 3823–3832 ª 2010 The Authors Journal compilation ª 2010 FEBS [28], factor VIII [4], gp120 [32], and H. pylori urease [24], have been shown to possess a multicleavage site, at which several peptide bonds were hydrolyzed in the protein. It is thought that a similar phenomenon was observed in this case. The cleaved peptide bonds in the structure of hTNF-a are shown in Fig. 7. Several research groups have determined the three-dimensional structure of hTNF-a by X-ray diffraction analysis. However, in many cases, the conformation of four or five amino acids at the N-terminus of hTNF-a could not be deter- mined. The amino acids in the crystal are presumably flexible. Thus, in the structure in Fig. 6, Ser5 of hTNF-a starts as an N-terminal amino acid. The peptide bonds cleaved by ETNF-6-H are indi- cated by arrows in Fig. 6. The structure of hTNF-a is trimeric in its natural form. The cleaved Gln21-Ala22 and Asn40-Gly41 bonds are in the loops and are situ- ated on the surface of the hTNF-a protein. The Leu36-Leu37 exists on the b-sheet and is also on the surface. Specifically, it seems that all cleaved peptide bonds are on the surface of the hTNF-a protein, enabling easy access to catalytic antibodies. The acces- sibility of the TNF-a molecule to ETNF-6-H to is one of the most important factors in cleaving the molecule. A summary of the assumed process of cleavage by ETNF-6-H is shown in Fig. 8. TNF-a was mainly degraded to fragment D, which was finally cleaved to fragment I. The main route is a successive reaction. It seems that many cleavage sites are present in the suc- cessive steps. On the other hand, there must be other minor routes generating some minute fragments, such as fragment G or fragment E. The latter fragment may be converted to fragment D and undergo a similar cleavage process as that in the main route. Conclu- sively, the multicleavage sites may be generated by a successive degradation reaction and ⁄ or simultaneously occurring cleavage reactions. It is considered that the catalytic antibody heavy chain first accesses the flexible region (N-terminus) and then the loop structure (Gln21-Ala22 or Asn40-Gly41). TNF-a is a cytokine that plays an important role, causing diseases such as COPD and Crohn’s disease. Recently, a mAb, e.g. infliximab, against TNF-a has been used for the treatment of such diseases [15–20]. The difference between the antibody drug and the cat- alytic antibody is considered as follows. The antibody drug (150 kDa), such as infliximab, firmly binds to TNF-a and blocks its function, resulting in a lowering of the activity of the molecule. Two molecules of 17 kDa 15 kDa 13 kDa Fig. 7. Site of cleavage by ETNF-6-H. The cleaved peptide bonds are indicated by arrows. Cleavage at Gln21-Ala22 gave a band at 15 kDa in SDS ⁄ PAGE. Cleavages at Leu36-Leu37 and Asn40-Gly41 gave a band at 13 kDa. The cleaved Gln21-Ala22 and Asn40-Gly41 bonds are in loops and are situated on the surface of hTNF-a. The Leu36-Leu37 bond is on a b-sheet and is also on the surface. It seems that all cleaved peptide bonds are on the surface of hTNF-a, which the catalytic antibody heavy chain seems to be able to access easily to reach the cleavage sites. 1 VRSSSRTPSD KPVAHVVANP QAEGQLQWLN RRANALLANG VELRDNQLV V 51 PSEGLYLIYS QVLFKGQGCP STHVLLTHTI SRIAVSYQTK VNLLSAIKSP 101 CQRETPEGAE AKPWYEPIYL GGVFQLEKGD RLSAEINRPD YLLFAESGQV 151 YFGIIAL Fig. 6. Peptide bonds of hTNF-a cleaved by ETNF-6-H. The identified cleaved peptide bonds are indicated by red arrows in the sequence of hTNF-a. Fig. 8. An assumed cleavage scheme of hTNFa by ETNF-6-H. A main-route for the cleavage was illustrated with bold thick arrows. A sub-route was with dotted arrows. E. Hifumi et al. Catalytic digestion of TNF-a by the antibody subunit FEBS Journal 277 (2010) 3823–3832 ª 2010 The Authors Journal compilation ª 2010 FEBS 3829 TNF-a may be blocked by one antibody drug molecule. On the other hand, one catalytic antibody molecule (heavy chain in this case; 50 kDa) degraded one TNF-a molecule for 1.5 h, based on a rough esti- mation from Fig. 5. Taking into account the quantity of infliximab administered ( 30 mg per person per one shot), about 0.4 lmol of TNF-a in the patient should be blocked. If a catalytic antibody is present in a patient, about 0.2 mg of the catalytic antibody will degrade the TNF-a molecules (0.4 lmol) for 1 week. It is expected that the quantity of the catalytic antibody (heavy chain) administered can be decreased to 1 ⁄ 100- fold as compared with the antibody drug, indicating that the cost of the medicine and ⁄ or adverse side effects from the administration may be reduced. Con- sidering the above discussion, our finding of a catalytic antibody cleaving TNF-a is interesting. Although this is basic research at the present time, it may provide a new tool for medicinal application instead of the mAb drug in the future. Experimental procedures Antibody production The ETNF-6 mAb used in this study was prepared by using commercially available recombinant human TNF-a (Strath- man Biotech AG, Hamburg, Germany). First, two female Balb ⁄ c mice (6 weeks old) were subcu- taneously immunized with 50 lg per mouse of hTNF-a (0.5 mgÆmL )1 in NaCl ⁄ P i ), which was emulsified with an equal volume of Freund’s complete adjuvant (Difco Labo- ratories, Detroit, MI, USA). Further immunizations were subcutaneously administered by the injection of 50 lg per mouse of the emulsion in Freund’s incomplete adjuvant (Difco Laboratories) at 2-week intervals after the first immunization. A final dose of 50 lg of human hTNF-a in 100 lL of NaCl ⁄ P i was intravenously injected through the tail vein of each respondent mouse 3 or 4 days before fusion. The immunized spleen cells were removed from the mice and fused with myeloma SP ⁄ NSI ⁄ 1-Ag4-1 (NS-1) at a ratio of 5 : 1, using 50% poly(ethylene glycol) 1500 (Boeh- ringer Mannheim GmbH). The fused cells were placed into the wells of 96-well culture plates (Becton Dickinson, Franklin Lakes, NJ, USA) and cultivated in HAT medium. The fused cells were screened to find the antibody-secreting cells by means of a modified sandwich ELISA. Hybrids that were found to secrete antibodies specific for the peptide were cloned by the limiting dilution method. The isotypes of the resulting mAbs were determined with a mouse mAb isotyping kit (IsoStrip; Roche 1493027, Indianapolis, IN, USA). Ascites fluid was obtained by intraperitoneal injec- tion of the hybridoma cell lines into pristane-primed female Balb ⁄ c mice. Purification and separation of the antibody subunits ETNF-6 mAb was purified according to the purification manual from the Bio-Rad Protein A MAPS-II kit (Nippon Bio-Rad, Tokyo, Japan). First, 5 mL of ascites fluid con- taining ETNF-6 mAb was mixed with the same volume of saturated ammonium sulfate solution. The precipitate was recovered by centrifugation (9000 g, 10 min), and 5 mL of NaCl ⁄ P i was then added to the precipitate. This process was repeated twice, and was followed by two dialyses against NaCl ⁄ P i . An aliquot of the NaCl ⁄ P i solution con- taining ETNF-6 mAb was mixed with the same volume of the binding buffer of MAPS-II. This mixture was then placed on a bed packed with Affi-Gel protein A for elution of the bound mAb. The eluted mAb was dialyzed twice against the buffer (50 mm Tris, 0.15 m NaCl, pH 8.0), at 4 °C. The resulting antibody was ultrafiltered three times by the use of a Centriprep YM-10 (Amicon, MA, USA). Five milligrams of antibody was dissolved in 2.7 mL of a buffer (pH 8.0) consisting of 50 mm Tris and 0.15 m NaCl, and reduced by the addition of 0.3 mL of 2 m 2-mercapto- ethanol for 3 h at 15 °C. To this solution, 3 mL of 0.6 m iodoacetamide was added, and the pH was then adjusted to 8 with 1 m Tris. The solution was then incubated for 15 min at 15 °C. The resulting solution was ultrafiltered to 0.5 mL, after which a half-volume of the sample was injected into an HPLC column (Protein-Pak 300SW, 7.8 · 300 mm; Nippon Waters, Tokyo, Japan) at a flow rate of 0.15 mLÆmin )1 , with 6 m guanidine hydrochloride (pH 6.5) as an eluent. Heavy chain fractions were collected, and this was followed by dilu- tion with 6 m guanidine hydrochloride. The fractions were dialyzed against NaCl ⁄ P i by replacing the buffer seven times for 3–4 days at 4 °C. ELISA Four microliters of hTNF-a dissolved in NaCl ⁄ P i (5 lgÆmL )1 ) was fixed on an immunoplate (Nunc, Den- mark) at 4 °C overnight. Blocking was performed with 2% gelatin for 1 h at room temperature. After the plate had been washed, ETNF-6 mAb was immunoreacted, and this was followed by a reaction with anti-mouse Ig(G+A+M) conjugated with alkaline phosphatase. After the substrate reaction with p-nitrophenyl phosphate, the absorption band at 405 nm was measured by use of an immunoplate reader (InterMed NJ-2001, Tokyo, Japan). Cleavage assay Before the degradation reaction was conducted, most glass- ware, plasticware and buffer solutions were sterilized by heating (180 °C, 2 h), autoclaving (121 °C, 20 min), or passing through a 0.2-lm sterilized filter. Manipulations in Catalytic digestion of TNF-a by the antibody subunit E. Hifumi et al. 3830 FEBS Journal 277 (2010) 3823–3832 ª 2010 The Authors Journal compilation ª 2010 FEBS the experiment were mostly performed in a safety cabinet, to avoid airborne contamination. The degradation reaction of TP41-1 (60 lm) by ETNF-6- H (0.4 lm) was conducted in a 15 mm phosphate buffer (pH 6.5) at 25 °C. For monitoring of the reaction, 20 lLof the reacting solution was injected into the RP-HPLC col- umn (Jasco) under isocratic conditions (0.05% trifluoroace- tic acid and 12.5% acetonitrile), with a column temperature of 40 °C. For assay of the cleavage of TNF-a, ETNF-6-H (0.1 lm) prepared as in the kinetics experiments was used along with the initial concentration of hTNF-a (6.6 lm) under the same reaction conditions as in the above experiments. The degradation of hTNF-a was monitored by SDS ⁄ PAGE (running gel: 16%) with silver staining. Sequencing and molecular modeling mRNA was isolated from the hybridoma secreting ETNF-6 mAb with an mRNA purification kit (Amersham Pharma- cia Biotech UK, UK). The cDNAs of the light and heavy chains were synthesized with a First-Strand cDNA Synthe- sis Kit (Life Science, Branford, CT, USA). The variable heavy and variable light fragments were amplified directly by adding them to a mixture containing PCR components and mouse Ig primers specific for IgG (Mouse Ig Primer kit; Novagen, Darmstadt, Germany). The amplified DNA was visualized on 2.0% agarose gel containing 0.5 lgÆmL )1 ethidium bromide. A band of approximately 450 bp was observed, corresponding to the size of the variable fragment of the antibody gene with little or no extraneous product. The PCR product was cloned into a pGEM-T Easy Vector System (Promega, Madison, WI, USA). Sequencing was conducted with an AutoRead Sequencing Kit (Amersham Pharmacia Biotech) and an automated DNA sequencing system (OpenGene System, Long-Read Tower; Amersham Pharmacia Biotech). Computational analyses of the antibody structures were performed with the deduced variable light and variable heavy amino acid sequences by a workstation (Silicon Graphics, Sunnyvale, CA, USA) running abm software (Oxford Molecular, Oxford, UK), which is used for build- ing models of three-dimensional molecules. The resulting Protein Data Bank data were applied to minimize the total energy by using discover II software (Molecular Simula- tions, Princeton, NJ, USA). This software uses the charmm-based algorithm for minimizing the energy of a molecule [33]. protein adviser Version 3.5 (FQS, Fukuoka, Japan) was used to visualize, analyze and draw the structures. N-terminal sequencing At 24 h of incubation, the reaction solution was recovered and concentrated up to 10-fold, with an ultrafiltration mem- brane (Amicon Ultra-4 5000MWCO; Millipore Corpora- tion, Bedford, MA, USA). The samples were then subjected to 16% SDS ⁄ PAGE (nonreduced condition). The bands were transferred for 1 h at 112 mA onto an Immobilon- PQS poly(vinylidene difluoride) membrane (Millipore Corporation) in 0.1 m Tris ⁄ HCl, 0.19 m glycine and 5% methanol at pH 8.7. After being stained with Coomassie Brilliant Blue, visible bands were cut and subjected to N-terminal sequence analyses (Automated Protein Sequen- cer, Prosize 494 HT; Applied Biosystems, Foster City, CA, USA), with the amount of protein used ranging from 2 to 40 pmol. For 0.5–2 pmol of the fragment, an automatic protein microsequencer, Prosize 494 cLC (Applied Biosys- tems), was used. Acknowledgements This study was supported by the Japan Science and Technology Agency (Creation of Bio-devices and Bio-systems with Chemical and Biological Molecules for Medical Use) and Grants-in-Aid for Scientific Research from the Ministry of Education, Culture, Sports, Science and Technology of Japan. We are also grateful to K. Hatiuchi and E. Terada for helping with some of the experiments. 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ETNF-6-H, heavy chain of ETNF-6 mAb; HSA, human serum albumin; hTNF-a, human tumor necrosis factor-a; TNF-a, tumor necrosis factor-a; TNF-b, tumor necrosis factor-b;. Catalytic digestion of human tumor necrosis factor-a by antibody heavy chain Emi Hifumi 1,2 , Kyohei Higashi 3 and