Direct conversion of a general antibody to its catalytic antibody and corresponding applications —Importance and role of Pro95 in CDR-3—

Emi HIFUMI; Hiroaki TAGUCHI; Tamami NONAKA; Taizo UDA

doi:10.2183/pjab.99.010

Abstract

Catalytic antibodies possess unique features capable of both recognizing and enzymatically degrading antigens. Therefore, they are more beneficial than monoclonal antibodies (mAbs). Catalytic antibodies exhibit the ability to degrade peptides, antigenic proteins, DNA, and physiologically active molecules. However, they have a significant drawback in terms of their production. The production of a desired catalytic antibody has extensive costs, in terms of time and effort. We herein describe an evolutionary method to produce a desired catalytic antibody via conversion of a general antibody by the deletion of Pro95, which resides in complementarity-determining region-3. As over thousands of mAbs have been produced since 1975, using the novel technology discussed herein, the catalytic feature cleaving the antigen can be conferred to the mAb. In this review article, we discussed in detail not only the role of Pro95 but also the unique features of the converted catalytic antibodies. This technique will accelerate research on therapeutic application of catalytic antibodies.

1. Introduction

To date, naturally occurring catalytic antibodies (natural catalytic antibodies) have been reported to show unique features for enzymatically digesting antigens, such as peptides,¹⁾^–⁵⁾ antigenic proteins,⁶⁾^–¹⁴⁾ DNA,¹⁵⁾^–¹⁷⁾ and physiologically active molecules.¹⁸⁾^–²¹⁾ They are superior to monoclonal antibodies in terms of possessing catalytic features, in addition to recognizing the antigen, as stated in the abstract.

However, it should be noted that catalytic antibodies have a big drawback in their production. They must be screened using many monoclonal antibodies (mAbs) or their subunits. This consumes a lot of time and takes a dedicated effort. No drastic developments in the preparation of catalytic antibodies have been achieved for a long time, although many studies on catalytic antibodies have been reported.

For the application of catalytic antibodies in many fields, it is necessary to produce a catalytic antibody capable of cleaving a desired antigen with ease, in a short time period.

Recently, we found a promising preparation method for catalytic antibodies, wherein an antibody light chain acquires catalytic function by deleting the Pro95 residue of CDR-3 (in the light chain). Nowadays, over thousands of monoclonal antibodies have been made since the report by Köhler–Milstein.²²⁾ If these monoclonal antibodies produced so far can be directly converted to the catalytic antibodies, it is the best way to date. The authors found an innovative method to make this possible.²³⁾ This technique allows for the enzymization of antibody light chains. The key technology for converting the general antibody to its catalytic antibody is the deletion of Pro95 in CDR-3. In this review article, we would like to explain this new concept relating the easy and short time preparation method of catalytic antibodies, where we will describe about detail data and results obtained from our study along with citing relevant references.

2. Importance of Pro95 in antibody kappa light chain

2-1. Antibody structure.

Antibody is an attractive protein capable of highly recognizing and attacking the antigen such as virus, bacterium, cancer cell, etc. through the immune response. Figure 1A shows the whole structure of an antibody. Figure 1B shows the cartoon frequently used for introducing the relationship between structure and role. An antibody (IgG) is consisted of two heavy and light chains. To recognize the antigen, the variable regions (the pink colored region in light chain and the blue colored region of heavy chain) play the important role with cooperatively work. The heavy chain has a Fc region (green colored background in the figure), which is a constant region and can bind to Fc receptor and complements.

Fig. 1.

Antibody structure. A) Whole structure of antibody (IgG) (From the website of Dr. Mike Clark, http://www.antibody.me.uk). B) Cartoon of antibody structure. VH: variable region of heavy chain; CH1: constant region 1 of heavy chain; CH2: constant region 2 of heavy chain; CH3: constant region 3 of heavy chain; VL: variable region of light chain; CL: constant region of light chain; VL is composed of V- and J-region. C) Structure of variable region of light chain (VL). FR: framework region; CDR: complementarity-determining region (or hypervariable region). (Colors of CDR-1, CDR-2, and CDR-3 correspond to those of amino acid sequences in Fig. 2A, etc.)

In the variable region, there are two kinds of regions, framework region (FR) and CDR or hypervariable region, as shown in Fig. 1C. The amino acid sequences of CDR are hugely varied along with the antigen. On the other hand, the sequences of FR are not so changed. CDR is consisted of CDR-1, CDR-2, and CDR-3 in both light and heavy chains. CDR has important amino acid sequences to work as the antibody binding to the antigen. Pro95 residue being focused on this review resides in CDR-3 in light chain.

2-2. Highly conserved Pro95 in the CDR-3 of antibody light chain.

Although many studies related to antibodies have been performed, reports on the role of the Pro95, which resides in the CDR-3 region of kappa-type light chains (CDRL-3) and is highly conserved residue among many antibodies, are hardly seen. Fortunately, we found that the deletion of Pro95 residue in CDR-3 should be an important factor in acquiring and/or enhancing the catalytic feature of the antibody.²³⁾

We prepared hundreds of purified kappa-type human and mouse antibody light chains whose amino acid sequences were determined. Figure 2A and 2B shows the amino acid sequences of CDR-3 in the kappa-type human antibody light chains. Figure 2A shows the amino acid sequences of CDR-3 for the 21 clones examined in our study; the human light chain is encoded by the germline gene IGKV2-29*02 (IMGT classification, subgroup II). All clones, except the 2-2902-4 clone, had the Pro95 residue. This suggests that Pro95 is highly conserved (20 out of 21 clones: 95% of the clones). This tendency was also observed in other Vκ germline genes, from IGKV1 to IGKV6 (data not shown herein; see Ref. 24/Fig. S1a–c). Most clones retained a proline residue, located at the position of 95th amino acid (or neighbor) in CDR-3. In contrast, the light chain encoded by the germline gene IGKV1-5*03 did not contain Pro95, and the Ser residue was placed at the position of 95th on this protein (Fig. 2B). The amino acid sequences of 11 clones of the human light chain encoded by the germline gene IGVK1-5*03 were randomly analyzed in our study (1-503-5 clone was named H34wt clone (hereafter H34)). Of 11 clones, 7 possessed somatic mutations, resulting in the replacement of Pro at the 95th position. The rate of this replacement was 64%, which was very high considering that 20 amino acids naturally occur in humans. This implied that Ser95 of the germline was predominantly mutated to Pro. Those observations indicated that antibody light chains (kappa) tend to contain Pro95 in the CDR-3. Note that Pro95 is a crucial amino acid in the antibody light chain, although the reason remains unknown.²⁴⁾

Fig. 2.

Comparison of amino acid sequences of the CDR-3 among germline genes (Sci. Rep. (2022) 12, 19185).²⁴⁾ A) IGKV2-29*02: The amino acid sequences of germline gene IGKV2-29*02 and 21 human light chain clones. Among 21 clones, 20 possess a Pro95 residue at the 95th position, indicating that Pro95 is highly conserved. B) IGKV1-5*03: IGKV1-5*03 does not contain Pro95; a Ser residue is placed at this position. Although the residue is replaced by somatic mutation, the Ser is replaced with Pro in 64% of the clones (7 out of 11 clones). Antibody light chains tend to contain Pro at the 95th position in CDR-3.

We believe that one of the important roles of the Pro95 residue in CDR-3 has been described in subsequent studies.

3. Conversion to catalytic antibody by deleting Pro95 in CDR-3

In this section, a drastic change (acquisition of catalytic features) of the antibody, by deleting the Pro95 residue, is described. This is where a general antibody can be converted to a catalytic antibody.

3-1. Amino acid sequences of S35 and S38.

We have prepared hundreds of human and mouse kappa light chains, expressed and purified (we named it a “bank” of antibody light chains). Out of the bank, we found two unique clones, S35 and S38, which belong to subgroup II of the human kappa light chain. The sequence of 219 amino acid residues of S35 light chain perfectly matched that of the germline 2/2D-28*01 of the Vκ region (Vκ: aa 1–95, see Fig. 3A). This indicated that there were no somatic mutations in S35 light chain. In contrast, S38 light chain possessed amino acid sequences identical to S35, except for the Pro95 residue that was deleted from S38 during the mutation process.²³⁾

Fig. 3.

Human light chains: S35 and S38 (Sci. Adv. (2020) 6, eaay6441).²³⁾ A) Amino acid sequences; comparison of Vκ germline gene (IGKV2/2D-28*01), S35, and S38. The sequence of the Vκ region (amino acids 1 to 95) of the S35 having Pro95 is identical to the germline gene (2/2D-28*01). On the other hand, the S38 lacks the Pro95. B) Time course of the cleavage reaction. The substrate: R-pNA (200 µM). Light chains: S38 (10 µM: open circle) and S35 (10 µM: closed circle). Out of five substrates (R-, E-, L-, A-, and FL-pNA), only one (R-pNA) was cleaved by S38. S35 did not cleave any substrates.

3-1-1. Peptidase activity (Hydrolysis of synthetic substrates).

We examined, using synthetic substrates, how the light chains (with or without Pro95) affected the catalytic properties. Matsuura et al.²⁵⁾ and Durova et al.⁸⁾ employed the substrate Arg-pNA to evaluate catalytic activity and synthetic substrates, such as Arg-pNA (R-pNA), Glu-pNA (E-pNA), Leu-pNA (L-pNA), Ala-pNA (A-pNA), and Phe-Leu-pNA (FL-pNA). Figure 3B shows the reaction profiles for R-pNA cleavage by S38 and S35 light chains. Among the five different substrates, only one (R-pNA) was cleaved by S38 light chain, suggesting that the light chain exhibited trypsin-like features. In contrast, S35 did not exhibit any catalytic activity for any of the five synthetic substrates.

3-1-2. Kinetics for the cleavage of R-pNA by S38.

The cleavage reaction for R-pNA by S38 light chain obeyed the Michaelis–Menten equation. The kcat and Km values were 4 × 10⁻³ min⁻¹ and 3.6 × 10⁻⁴ M, respectively. These values are comparable to those obtained by Matsuura et al., using the Bence–Jones protein (MOR) taken from a multiple myeloma patient for the R-pNA substrate (kcat = 7 × 10⁻² min⁻¹, Km = 0.21 × 10⁻⁴ M).²⁵⁾ They are also comparable to those obtained by Durova et al. using L12 light chain for the PRF-MCA substrate (kcat = 1.55 × 10⁻³ min⁻¹, Km = 5.3 × 10⁻⁴ M).⁸⁾

3-2. Amino acid sequences of T99 and T99-Pro95(−).

T99 light chain belongs to the Vκ germline gene 2-29*02 in subgroup II (IGKV2-29*02). A significant difference between T99 and S35 light chains was the number of somatic mutations that occurred (Fig. 4A). The former has many somatic mutations in its amino acid sequence, while the latter does not. T99 light chain exhibited little catalytic activity for the cleavage of R-pNA. Consequently, we employed T99 and a Pro95-deleted mutant (T99-Pro95(−)) to ascertain whether the Pro95 is a key amino acid residue in the possession of a catalytic function.

Fig. 4.

Human light chain T99 and mutant T99-Pro95(−) (Sci. Adv. (2020) 6, eaay6441).²³⁾ A) Comparison of Vκ germline gene (IGKV2-29*02) and the T99t; the sequence of the Vκ region (amino acids 1 to 95) of the T99 is compared with that of a germline gene (2-29*02). Amino acids of the sequence of the T99 are different at many positions from those of the germline gene, indicating the presence of many somatic mutations. B) Time course for the cleavage reaction of R-pNA; T99-Pro95(−) (10 µM: open circle) clearly exhibited a much higher catalytic activity than for the T99 (10 µM: closed circle) cleaving the R-pNA substrate. The reaction was carried out in triplicate, at 37 °C.

3-2-1. Peptidase activity of the synthetic substrate R-pNA.

In T99 light chain (subgroup II), there were many mutations in the light chain, as shown in Fig. 4A, which is significantly different from the case of S35. Therefore, we genetically deleted the Pro95 from T99. Interestingly, when compared to T99, T99-Pro95(−) exhibited a higher catalytic activity for R-pNA cleavage (Fig. 4B). Note that deletion of the Pro95 enhances the catalytic activity of the light chain. T99-Pro95(−) did not exhibit any catalytic activity for E-, A-, L-, and FL-pNA.

Kinetic analysis using R-pNA was performed for both T99 and T99-Pro95(−) light chains. The cleavage reactions for both light chains obeyed the Michaelis–Menten equation. The kcat and Km values of T99 were 2 × 10⁻³ min⁻¹ and 3.2 × 10⁻³ M, respectively. On the other hand, the kcat and Km values of T99-Pro95(−) were 8 × 10⁻³ min⁻¹ and 3.8 × 10⁻³ M, respectively. The turnover (kcat) value of T99-Pro95(−) was higher than that of T99 by a factor of four, while the Km values did not change. This suggests that the catalytic site (kcat) and the binding site (Km) may be different. The values are comparable to those of 22F6 catalytic light chain.¹²⁾ In contrast, the constant region domain, a common and identical amino acid sequence in both light chains,²⁶⁾ did not cleave the substrate (data not shown).

3-2-2. Molecular modeling.

Molecular modeling is not appropriate for an understanding with high accuracy. However, it is a useful tool for interpreting the present results without the need for X-ray diffraction analysis.

Modeling with T99 and T99-Pro95(−) was performed, and the results are shown in Fig. 5A and 5B. In this case, a catalytic triad composed of Asp1, Ser27a, and His93 is preferable. The distance between Ser27a(O) and His93(N) is 7.46 Å in T99 and 6.20 Å in T99-Pro95(−). In contrast, the carboxyl group of Asp1 in T99-Pro95(−) faces the amino group of His93. The distance between His93(N) and Asp1(O) changed from 12.26 Å in T99 to 6.21 Å in T99-Pro95(−). This is a crucial change, from which the catalytic triad can be derived. His27d may not be relevant to the construction of the catalytic triad in this case.²³⁾

Fig. 5.

Molecular modeling of T99 and T99-Pro95(−) light chain (Sci. Adv. (2020) 6, eaay6441).²³⁾ A) T99. B) T99-Pro95(−). A catalytic triad composed of Asp1, Ser27a, and His93 is considered in this case. A big change occurred for the distance between His93(N) and Asp1(O) from 12.26 Å in T99 to 6.21 Å in T99-Pro95(−).

3-3. Amino acid sequences of #7TR and #7TR-Pro95(−).

In screening tests from the bank, we found another catalytic antibody (#7TR) capable of cleaving the R-pNA substrate.²⁷⁾ Therefore, the Pro95 deletion mutant was constructed for confirming the catalytic feature of the Pro95 residue. Figure 6A shows the amino acid sequences of #7TR and #7TR-Pro95(−) antibody light chains.

Fig. 6.

Human light chain #7TR and #7TR-Pro95(−). A) Amino acid sequences of human light chains #7TR and #7TR-Pro95(−). #7TR had a proline residue at the 95th position in CDR-3. Pro95 was deleted in the #7TR-Pro95(−) mutant. B) Time courses for R-pNA cleavage by #7TR and #7TR-Pro95(−). Substrate: R-pNA (200 µM). Light chain: #7TR-Pro95(−) (8 µM: open circle) and #7TR (8 µM: closed circle). Buffer: 100 mM glycine and 0.025% Tween 20/50 mM Tris-HCl (TGT). Reaction temperature: 37 °C.

3-3-1. Catalytic activity for R-pNA substrate by #7TR and #7TR-Pro95(−).

Figure 6B shows the experimental results for the catalytic activities of #7TR and #7TR-Pro95(−) light chains. Both light chains cleaved the peptide bond between the Arg and pNA of the substrate.

Kinetic studies were performed on the two light chains. The cleavage reactions by both light chains obeyed the Michaelis–Menten equation, indicating that the reaction was enzymatic. The kcat and Km values of #7TR were 2.3 × 10⁻³ min⁻¹ and 8.3 × 10⁻⁴ M, respectively. On the other hand, the kcat and Km values of #7TR-Pro95(−) were 4.3 × 10⁻² min⁻¹ and 3.6 × 10⁻³ M, respectively. These values are comparable to those of 22F6 catalytic light chain (wild type).¹²⁾ The turnover (kcat) value of #7TR-Pro95(−) was higher than that of #7TR by a factor of 19-fold. Note that the kcat value was greatly improved by deleting the Pro95 residue, while the Km value slightly changed (within fivefold).

3-3-2. Cleavage for FRET-Aβ by #7TR and #7TR-Pro95(−).

Alzheimer’s disease is the most common form of dementia worldwide. One of its causes is amyloidosis, due to the formation of amyloid-β (Aβ oligomers). The sequence of the Aβ peptide (1–40) is DAEFRHDSGYEVHHQKLVFFAEDVGSNKGAIIGLMVGGVV. For the region used as the FRET substrate (for cleavage reaction), the sequence designated was ²⁶SNKGAIIG³³. This sequence is an important region for fibril generation in the brain. Taguchi et al. found an interesting human autoantibody from the IgM class, wherein hydrolysis of Aβ at the Lys²⁸-Gly²⁹ bond leads to a decrease in the formation of Aβ aggregates and protection of neuronal cells from Aβ-induced neurotoxicity.⁵⁾

In our study, two reagents, 7-MCA and DNP, were introduced at the N- and C-terminus of the designated peptide, respectively. (7-MCA-SNKGAIIG-K(DNP)-rrr-NH₂; rrr-means (D-Arg)₃: These amino acids were introduced for enhancement of hydrophilicity of the peptide) (Fig. 7A). The cleavage reactions of #7TR and #7TR-Pro95(−) were performed using the FRET substrate, and the time course of the reaction is shown in Fig. 7B. Both #7TR and #7TR-Pro95(−) cleaved the FRET-Aβ substrate. Regarding the catalytic activity, #7TR clone showed ∼20 Fu at 72 h of reaction time. In contrast, #7TR-Pro95(−) exhibited ∼100 Fu at the same reaction time. #7TR-Pro95(−) had a higher catalytic activity than #7TR, by a factor of five. The deletion of Pro95 significantly enhanced the catalytic activity of the mutant in this case. In the cleavage reaction of the FRET-Aβ (26–33) substrate by #7TR, the scissile peptide bond was investigated by high-performance liquid chromatography (HPLC) and mass spectroscopy (MS). Figure 7C shows the results. Several fragments were observed. The large peaks corresponding to the fragments 7-MCA-S-N-K-G-OH (19.6 min) and NH₂-A-I-I-G-K(DNP)-r-r-r-NH₂ (28.4 min) indicated that the peptide bond between Gly and Ala was cleaved. The peaks of 7-MCA-S-N-K-OH (19.3 min) and NH₂-G-A-I-I-G-K(DNP)-r-r-r-NH₂ (28.4 min) indicated that the bond between Lys and Gly was cleaved. Multiple sites were cleaved by #7TR, which is a characteristic feature of catalytic antibodies.

Fig. 7.

Synthesis of fluorescence resonance energy transfer (FRET) substrate and the cleavage reaction. The amino acid sequence of the Aβ peptide (1–40) was DAEFRHDSGYEVHHQKLVFFAEDVGSNKGAIIGLMVGGVV. The sequence ²⁶SNKGAIIG³³ was designated as the FRET substrate for the Aβ peptide (FRET-Aβ). A) Chemical structures for FRET-Aβ. (FASEB Bioadv. (2018) 1, 93–104).²⁷⁾ B) Time course of the cleavage for FRET-Aβ by #7TR and #7TR-Pro95(−) light chains. FRET-Aβ: 25 µM #7TR or #7TR-Pro95(−): 5 µM. Buffer: TGT containing 0.02% NaN₃. Reaction temperature: 37 °C. N = 2. C) Scissile bonds: The scissile peptide bonds were investigated by HPLC and MS. The peptide bonds between Gly²⁹-Ala³⁰ and Lys²⁸-Gly²⁹ were cleaved. D) Cleavage reaction for Aβ1–40 peptide by #7TR. #7TR; 10 µM, Aβ1–40; 100 µM, in 100 µL PBS, pH 7.4. Incubation time: 144 h. Reaction temperature: 37 °C. Column: Cosmosil type, 5C18-AR-2 (4.6 × 250). Eluent: Milli-Q water in 0.05% TFA: acetonitrile in 0.05% TFA from 90:10 to 40:60 in 50 min (1.0 ml/min). (FASEB Bioadv. (2018) 1, 93–104).²⁷⁾ The Aβ1–40 peptide was detected at approximately 35 min. The peak at a reaction time of 18 min indicated that the fragment cleaved at the H¹⁴-Q¹⁵ peptide bond.

The cleavage reaction for the Aβ1–40 peptide (100 µM) was also carried out using #7TR clone (10 µM), and the reaction products were investigated at 144 h of reaction time. Figure 7D shows the results. The Aβ1–40 peptide peak was detected at approximately 35 min. For the reaction products, a clear peak was observed at a retention time of 18 min. It was analyzed using MS, wherein divalent and trivalent masses were detected at 849.9 m/2z and 566.94 m/3z, respectively. This fragment was identified as-DAEFRHDSGYEVHH, from the Aβ1–40 peptide, indicating that the H¹⁴-Q¹⁵ peptide bond was cleaved. The #7TR light chain cleaved the K²⁸-G²⁹ and/or G²⁹-A³⁰ peptide bond of FRET-Aβ. For the Aβ1–40 peptide, the light chain preferentially cleaved the H¹⁴-Q¹⁵ peptide bond. The scissile bond was not the same, assuming that the H¹⁴-Q¹⁵ peptide bond had a tendency to be easily cleaved, unlike the K²⁸-G²⁹ and/or G²⁹-A³⁰ peptide bonds in the FRET-Aβ substrate.²⁷⁾

4. H34 catalytic antibody and the features

The immune checkpoint molecule programmed cell death protein-1 (PD-1), found by Honjo et al.,²⁸⁾ can control T-cell function, and the monoclonal antibody against PD-1 acts as an anti-cancer drug by inhibiting the binding of PD-L1 to PD-1.²⁹⁾^–³¹⁾

4-1. Catalytic antibody capable of cleaving PD-1 molecule.

4-1-1. H34 clone.

Figure 8A shows the amino acid sequence of human PD-1. In the figure, the sequence of the epitope recognized by nivolumab (antiPD-1 mAb) is indicated in magenta. Based on these sequences, we synthesized a FRET-PD-1 peptide (aa 124–141 (7-MCA-GAISLAPKAQIKESLRAE-K(DNP)-NH₂)), for screening our antibody bank. Out of our antibody bank, we found a human antibody light chain, H34, which possessed a catalytic activity against FRET-PD-1.³²⁾ Figure 8B shows the reaction profile of H34 (5 µM) against FRET-PD-1 (25 µM).

Fig. 8.

Catalytic antibody capable of cleaving PD-1 molecule (RSC Chem. Biol. (2021) 2, 220–229).³²⁾ A) Amino acid sequence of human PD-1 molecule, the epitope of nivolumab (antipd-1 mAb), and the chemical structure of FRET-PD-1. B) Reaction profiles of H34 against FRET-PD-1, FRET-Aβ, and FRET-Tau. H34: 5 µM. FRET substrates: 25 µM. Reaction temperature: 37 °C. C) Cleaved bond for FRET-PD1. H34 reaction products were analyzed via HPLC and MS. The fragments were identified as 7-MCA-GAISLAPKAQ-OH (31.7 min) and NH₂-IKESLRAEK(DNP)-NH₂ (29.6 min), indicating cleavage of the peptide bond between Gln¹³³ and Ile¹³⁴. D) Amino acid sequence of H34. H34 belongs to subgroup I and has no proline residues in the CDR-3 region. E) Cleavage of rPD-1. SDS-PAGE analysis with silver staining under reduced conditions. rPD-1: 1 µM. HSA: 1 µM. H34 light chain: 0.5 µM. Buffer: TGT containing 0.02% sodium azide. Reaction temperature: 37 °C. F) Binding behavior of digested rPD-1 with rPD-L1 (ELISA-1). Digestion reaction of (rPD-1/0.25 µM + H34/0.5 µM) was carried out in TGT buffer containing 0.02% sodium azide for 48 h. The solution was then submitted to ELISA coated with PD-L1. Coated rPD-L1: 2 µg/mL (50 µL/well). G) Time dependency of the digestion of rPD-1 by H34 (ELISA-2). Digestion reaction: (rPD-1/0.18 µM + H34/0.5 µM). Reaction time: 0, 6, 24, and 48 h. Each reaction solution was submitted to ELISA coated with rPD-L1 as stated above.

H34 rapidly cleaved the FRET-PD-1 substrate but had little or no effect on non-relevant substrates, such as FRET-Aβ (aa 26–33 (7-MCA-SNKGAIIGK(DNP)rrr-NH₂)) and FRET-Tau (aa 391–408 (7-MCA-EIVYKpSPVVSGDTpSPRHLK(DNP)-NH₂: pS, phosphorylated serine)). In the cleavage reaction of FRET-PD-1, the peptide bond between Gln¹³³ and Ile¹³⁴ was cleaved by H34 (Fig. 8C). From the kinetic study, Km was 3.32 × 10⁻⁶ M and kcat was 1.3 × 10⁻² min⁻¹. The kcat/Km was 1.96 × 10³/M⁻¹ min⁻¹. These kinetic values for H34 were comparable to those obtained for other catalytic antibodies reported to date.³⁾^,⁶⁾^,²¹⁾ For the mouse PD-1 peptide, H34 did not cleave it at all, suggesting a high degree of specificity for human PD-1.

4-1-2. Amino acid sequence of H34.

Figure 8D shows that the amino acid sequence of H34 belongs to subgroup I and has no proline residue in the CDR-3 region.

4-1-3. Cleavage of rPD-1.

The cleavage reaction for the recombinant PD-1 molecule (rPD-1; 1 µM) was performed using a H34 light chain (0.5 µM), under the reaction conditions of a TGT buffer containing 0.02% sodium azide, and was incubated at 37 °C. The reactions were analyzed using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and silver staining. Figure 8E shows the time period for the reaction of the rPD-1 with H34 light chains, where human serum albumin (HSA) was used as a negative control. In the figure (PD1 + H34), two major rPD1 bands were observed at approximately 38 and 43 kDa. H34 was present as a single band at ∼30 kDa. After 24 h of incubation, bands at ∼28 kDa and ∼17 kDa were clearly observable and became more prominent after 72 h of incubation. In contrast, the band at ∼38 kDa gradually faded over time up to 72 h. In the control experiments, no fragmented bands were detected in HSA and H34 mixture after 72 h of incubation. The bands corresponding to H34 (30 kDa), PD-1 (∼36 kDa and ∼42 kDa), and HSA (mainly 66 kDa) did not change during incubation. These findings indicated that H34 cleaves rPD-1 in a time-dependent manner. Considering the molecular sizes, the band at 28 kDa may have been a dimer of 17 kDa fragment.

4-1-4. Binding behavior.

The binding behavior of rPD-1 to rPD-L1 was examined using ELISA. The rPD-1 (having V5 epitope tag) was incubated with the H34 catalytic antibody, at 37 °C for 48 h. In addition, the rPD-1 or H34 alone was incubated under the same conditions to serve as controls. Thereafter, each was transferred to the wells of ELISA plates coated with rPD-L1, and binding was visualized using HRP-conjugated anti-V5 epitope tag antibodies. Figure 8F shows the results (ELISA-1). When rPD-1 was incubated alone, the absorbance at 492 nm was 0.569. For the H34 alone, the absorbance was 0.003, suggesting that cross-reactivity of the antibody with rPD-L1 was negligible. In contrast, incubation of rPD-1 with H34 for 48 h yielded an absorbance of 0.151, i.e., only a quarter of the value obtained with the incubation of rPD-1 alone. Thus, we concluded that the binding of rPD-1 to rPD-L1 was inhibited or blocked by the degradation of rPD-1.

In another experiment, a mixture of rPD-1 (0.18 µM) and H34 (0.5 µM) was incubated for 0, 6, 24, and 48 h. Each sample was then subjected to an ELISA. Approximately half of the rPD-1 molecules were degraded after 6 h of incubation. After incubation for 48 h, they almost disappeared, suggesting that the binding was inhibited by a degradation event, instead of a competitive reaction (Fig. 8G (ELISA-2)).

5. Insertion of Pro95

We have stated the importance of the deletion of Pro95 residue, which is concerned with the catalytic activity of the antibody. H34 did not contain the Pro95 residue in the CDR-3 region, as stated above.

5-1. Insertion of Pro95.

We constructed a mutant of H34, to which Pro95 was then inserted (H34-Pro95(+)), and we investigated its effect on catalytic activity. Figure 9 shows the reaction time periods, where H34 (without Pro95) began to cleave the FRET-PD-1 substrate soon after initial mixing, which gradually progressed up to 48 h. In contrast, the mutant with an inserted Pro95 only weakly cleaved FRET-PD-1 after 48 h. Note that the insertion of Pro95 significantly suppressed peptidase activity, indicating that the lack of the Pro95 residue is closely related to the catalytic activity of the antibody.

Fig. 9.

Comparison of catalytic activity for H34 and the mutant (RSC Chem. Biol. (2021) 2, 220–229).³²⁾ Open circle: H34. Closed circle: H34-Pro95(+). The catalytic activity of the H34-Pro95(+) mutant was substantially reduced relative to H34 lacking Pro95.

5-2. Molecular modeling.

The crystallization of only light chains is very difficult, perhaps due to their structural diversity.³³⁾ Molecular modeling of both H34 and H34-Pro95(+) was performed. Figure 10A and 10B shows comparisons of the structural models. The Asp¹ residue in H34 (ball and stick shown in pink) is more oriented toward the inner side, compared to the mutant H34-Pro95(+) (ball and stick shown in red). In fact, the distances between the carboxyl oxygen of the Asp¹ and the guanidinium nitrogen of the Arg⁹⁶ residue (ball and stick, light blue) in CDR-3 are less in the H34 (9.65 Å) than in the mutant (13.54 Å) (ball and stick, blue), by a factor of ∼4 Å. The presence of Pro95 hinders the approach of Asp¹ to the CDR-3 loop.²³⁾

Fig. 10.

Molecular modeling (RSC Chem. Biol. (2021) 2, 220–229).³²⁾ A) H34. B) H34-Pro95(+). Computational analysis was performed using the deduced antibody light chain amino acid sequences by Discovery Studio (Accelrys Software, San Diego, CA, U.S.A.). For the homology modeling, the template structures were made by a BLAST search, following the minimization of the total energy of the molecule by using the CHARMM algorithm. The resulting Protein Data Bank (PDB) data were used for modification of the complementarity-determining region (CDR) structures defined by the Kabat numbering system.

6. Bifunctional catalytic antibody

Although Alzheimer’s disease (AD) is the most common form of dementia, as previously stated, the cause of AD remains unclear. The amyloid hypothesis, which considers amyloids as a causative factor, is the mainstream idea. According to the amyloid hypothesis, Aβ is first generated from an amyloid-β precursor protein (APP), by β- and δ-secretase. When Aβ is polymerized, senile plaques are formed via the formation of Aβ oligomers. Aβ causes abnormal phosphorylation of the “Tau” protein. In addition, abnormally phosphorylated Tau proteins cause neurofibrillary tangles. Thus, Aβ and abnormally phosphorylated Tau proteins are considered to promote progression of AD by causing neuronal cell death (see Fig. 11A).

Fig. 11.

Amyloid-β and Tau protein. A) Reaction scheme of two molecules (amyloid-β (Aβ) and Tau protein) associated with Alzheimer’s disease (AD). B) Amino acid sequence of Tau 441 protein. In this study, three types of peptides from the sequence were designated as the FRET substrates: 1) ¹⁹GLGDRKDQGGYT³⁰ (Tau19–30; HA-12); 2) ²⁹²GSKDNIKHVPGGGS³⁰⁵ (Tau292–305; HA-11); 3) ³⁹¹EIVYKSPVVS GDTSPRHL⁴⁰⁸ (Tau391–408; YI-7). C) Results of screening of catalytic antibodies against FRET-Tau (Tau19–30). Light chain: 5 µM. FRET substrate (Tau19–30 peptide): 25 µM. Incubation time: 3 days. PBS background: 1.5 ± 2.4 DFu/h. Threshold of PBS background: +3SD (8.7 DFu/h). A total of 98 clones from our protein bank were tested. Among these clones, six showed more than 8.7 DFu/h. Sample #48, which was the S5 clone, showed the highest catalytic activity. D) SDS-PAGE analysis of purified S5 clone. Visualized with CBB staining. Under nonreduced conditions, the band of the monomer at ∼26 kDa was faintly detected, indicating that the S5 clone was mostly present as a dimer in solution. E) Reaction time courses for three kinds of FRET-Tau peptides by S5 clone. FRET-Tau: 25 µM. S5 clone: 2.5 µM. Red circle: ²⁹²GSKDNIKHVPGGGS³⁰⁵ (Tau292–305; HA-11). Green square: ¹⁹GLGDRKDQGGYT³⁰ (Tau19–30; HA-12). Blue rhombus: ³⁹¹EIVYKSPVVS GDTSPRHL⁴⁰⁸ (Tau391–408; YI-7). Arrows show cleaved sites. F) Identification of fragmented peptide of Tau peptide by S5 catalytic antibody. HPLC and MS analyses were performed to identify the fragments. Asp²⁹⁵-Asn²⁹⁶, His²⁹⁹-Val³⁰⁰, and Pro³⁰¹-Gly³⁰² were identified as the cleaved peptide bonds. G) Degradation test of recombinant Tau protein (rTau). Visualized with silver staining. Tau protein: 15 µg/mL. S5 clone: 15 µg/mL. Reaction temperature: 37 °C. Reaction solution: TGT buffer containing 0.02% NaN₃. The band of rTau mixed with S5 was gradually faded in a time-dependent manner, indicating that S5 clone cleaved rTau.

Considering the above situation, we designed a catalytic antibody, which possesses two functional characteristics (bifunctional catalytic antibody), capable of simultaneously degrading both Aβ and Tau molecules.

6-1. Screening of catalytic antibody capable of degrading Tau molecules.

6-1-1. Design of FRET-Tau peptides.

We explored a catalytic antibody capable of degrading Tau protein (composed of 441 amino acid residues). Figure 11B shows the amino acid sequence. In this study, three kinds of peptides in the sequence were designated as FRET substrates for the screening of the catalytic antibodies and investigating their properties. They are as follows:

1. ¹⁹GLGDRKDQGGYT³⁰ (Tau19–30; HA-12 peptide). Since a part of the N-terminus of the Tau protein is concerned with neurotoxicity, the molecular designing of FRET-Tau (19–30) was performed to explore the catalytic antibodies that would cleave the portion.

2. ²⁹²GSKDNIKHVPGGGS³⁰⁵ (Tau292–305; HA-11 peptide). This region is responsible for aggregation properties. Oligomeric forms dominate the toxic effects of Tau. The molecular designing of FRET-Tau (292–305) was performed to develop the catalytic antibodies that reduce the formation of aggregates.

3. ³⁹¹EIVYKSPVVS GDTSPRHL⁴⁰⁸ (Tau391–408; YI-7 peptide). Since the phosphorylation of Ser 396 and Ser 404 is closely related to pathogenicity, the molecular designing of FRET-Tau (391–408) was carried out to produce the catalytic antibodies, in order to destroy these phosphorylation sites.³⁴⁾^–³⁷⁾

For high-throughput screening, we used three kinds of FRET substrates, prepared similarly to those described in the previous sections.

Using the above versions of FRET substrates for Tau proteins, human light chains stored in our protein bank were screened in terms of a blind test. Figure 11C shows the results. Among them, sample #48, which was S5 clone, showed the highest catalytic activity for the N-terminal side peptide (Tau19–30; HA-12 peptide).

6-1-2. Features of S5 clone.

Figure 11D shows the SDS-PAGE results for the purified S5 clone. Under nonreduced conditions, the band of the monomer at ∼26 kDa was faintly detected, and S5 clone was mostly present as a dimer in solution. For the three types of FERT-Tau peptides, the cleavage reaction by S5 clone was investigated. Figure 11E shows that S5 clone exhibited the highest catalytic activity for ²⁹²GSKDNIKHVPGGGS³⁰⁵ (Tau292–305; HA-11 peptide) peptide. A partial cleavage reaction was observed for the ¹⁹GLGDRKDQGGYT³⁰ (Tau19–30) peptide. Cleavage hardly occurred for the ³⁹¹GSKDNIKHVPGGGS⁴⁰⁸ peptide (Tau391–408; YI-7 peptide). The scissile bond of the ²⁹²GSKDNIKHVPGGGS³⁰⁵ (Tau292–305) peptide was analyzed using HPLC and mass spectrometry. Figure 11F shows the results. In this case, three cleavage bonds, namely, Asp²⁹⁵-Asn²⁹⁶, His²⁹⁹-Val³⁰⁰, and Pro³⁰¹-Gly³⁰², were identified.

6-1-3. Cleavage of recombinant Tau protein.

A recombinant Tau molecule (rTau, 30 µg/mL) was mixed with S5 light chains (15 µg/mL) in the TGT buffer containing 0.02% sodium azide and then incubated at 37 °C. Samples taken after 0, 24, and 48 h were analyzed by SDS-PAGE with silver staining (Fig. 11G), wherein the band of Tau protein was observed at approximately 70 kDa. The band of S5 was detected at around 30 kDa. In the case of the sample mixed with rTau protein and S5 light chains (rTau +S5), the band of rTau protein at ∼70 kDa gradually faded from 0 to 48 h of incubation, in a time-dependent manner. The clear fragment band from rTau protein was hardly observed with the naked eye, in this case. On the other hand, the intensities of the Tau protein (rTau) and S5 light chains (S5) in the control experiments did not change.

6-1-4. scFv-type molecules (#7TR-linker-S5).

As stated in the previous section, two types of molecules, namely, Aβ peptide and Tau protein, are strongly involved in AD. It is desirable to develop a bifunctional catalytic antibody that can simultaneously cleave both the Aβ peptide and the Tau protein. Based on this idea, a bifunctional catalytic antibody was designed using clones of #7TR and S5 for the cleavage of the Aβ and Tau molecules, respectively. We employed an scFv-type catalytic antibody, where the variable regions of #7TR and S5 were connected with a linker. Figure 12A shows the model of the bifunctional scFv-type catalytic antibody.

Fig. 12.

Molecular design and preparation of the bifunctional catalytic antibody. A) scFv-type molecule. The bifunctional catalytic antibody was designed using the variable regions of #7TR and the S5 light chains. B) Cleavage reaction by the bifunctional scFv (#7TR-S5). FRET substrate (Aβ or Tau (Tau292–305; HA-11)): 25 µM. Bifunctional scFv (#7TR-S5): 2.5 µM. Closed circle: cleavage for Aβ peptide. Open triangle: cleavage for Tau peptide. C) Cleavage reaction for mixture of FRET-Aβ and FRET-Tau. Final concentrations: FRET-Aβ (12.5 µM) and FRET-Tau (12.5 µM). scFv (#7TR and S5): 2.5 µM. D) Cleavage reaction for FRET-Aβ by #7TR. FRET-Aβ: 25 µM. #7TR clone: 2.5 µM. E) Cleavage reaction for FRET-Tau by S5. FRET-Tau (HA-11): 25 µM. S5 clone: 2.5 µM.

The scFv comprised of variable regions of #7TR and S5 light chains (scFv of #7TR-S5), which was expressed in the E. coli system and recovered using the same protocol as that described in prior studies.⁹⁾^,¹²⁾^,²³⁾^,²⁴⁾

The cleavage reaction for FRET-Aβ (25 µM) and FRET-Tau peptides (H11: 25 µM) was performed using the scFv of #7TR and S5 (2.5 µM) in TGT buffer. The cleavage reaction of FRET-Aβ was first performed using scFv of #7TR-S5. The FRET-Tau peptide was independently tested, as another cleavage reaction. Figure 12B shows the reaction time periods. Both peptides were cleaved in a time-dependent manner, up to 54 h of reaction time. Both the Aβ and Tau peptides showed similar reaction profiles. In addition, a mixture of FRET-Aβ (12.5 µM) and FRET-Tau (12.5 µM) (final concentration of the mixed peptides: 25 µM) was subjected to cleavage by the scFv of #7TR and S5. Figure 12C shows the results. The cleavage reaction gradually increased up to a time of 54 h, in the linear mode. In this case, it was impossible to distinguish which molecule decomposed to what extent, as the same fluorescence reagents were used for both peptides. Considering the results of Fig. 12C, it was assumed that approximately 50% of both peptides were cleaved. Anyhow, the scFv of #7TR and S5 was confirmed to cleave both peptides, indicating that the scFv has a bifunctional ability.

Under identical reaction conditions, the cleavage reactions for the FRET-Aβ peptide by #7TR, and for the FRET-Tau peptide by S5, were carried out. Figure 12D and 12E shows the results. In the case of the cleavage of FRET-Aβ peptide by #7TR, the catalytic activity was 20.3 Fu at 30 h of reaction time, while that of Tau by S5 was 11.4 Fu after 30 h of reaction time. At the same reaction time (regarding scFv of #7TR-linker-S5), 13.1 Fu for FRET-Aβ cleavage and 14.5 Fu for FRET-Tau cleavage were observed. In this case, the catalytic activity for Aβ cleavage was reduced by 0.65-fold. Conversely, for Tau cleavage, the catalytic activity was increased by a factor of 1.27-fold compared to that of only S5 clone. Although the catalytic activities of the bifunctional catalytic antibody were almost preserved, the catalytic features may be slightly changed.

6-2. #7TR-Pro95(−)-linker-S5-Pro95(−).

6-2-1. Improvement of bifunctional catalytic antibody.

Deletion of the Pro95 residing in CDR-3 greatly contributes to the acquisition and/or enhancement of catalytic activity of the antibody light chain, as previously stated. Hence, for scFv of #7TR-linker-S5, the deletion was performed. In this case, each Pro95 in the CDR-3 of clones #7TR and S5 was deleted (scFv #7TR/P95(−)-S5/P95(−)). Using the scFv of #7TR/P95(−)-S5/P95(−), cleavage reactions for FRET-Aβ and Tau peptides were carried out (FRET substrate; 25 µM, scFv; 5 µM). The time courses of the reaction are shown in Fig. 13A and 13B, and the same cleavage reactions by scFv of #7TR-S5 were performed, to compare the catalytic activity.

Fig. 13.

Comparison of catalytic activity of scFv with scFv-Pro95(−) mutants. A) Cleavage of FRET-Aβ. FRET-Aβ: 25 µM. scFv(#7TR-S5) or scFv(#7TR/P95(−)-S5/P95(−)): 5 µM. B) The cleavage of FRET-Tau. FRET-Tau (HA-11): 25 µM. scFv(#7TR-S5) or scFv(#7TR/P95(−)-S5/P95(−)): 5 µM.

For the Aβ peptide, at a reaction time of 30 h, the Fu value was 91.2 for scFv of #7TR/P95(−)-S5/P95(−). In contrast, the value was 27.3 for scFv #7TR-S5. The catalytic activity of scFv of #7TR/P95(−)-S5/P95(−) was enhanced, when compared with that of scFv of #7TR-S5, by a factor of 3.3-fold.

For the Tau peptide, the Fu value was 74.9 for scFv of #7TR/P95(−)-S5/P95(−), and the value was 25.6 for scFv of #7TR-S5. The catalytic activity of #7TR/P95(−)-S5/P95(−) scFv increased by a factor of threefold when compared to that of #7TR-S5 scFv. In conclusion, deletion of Pro95 was effective in enhancing the catalytic activity of the bifunctional catalytic antibody.

6-2-2. Inhibitive effect on the aggregation.

We assessed the aggregation inhibition activity of the bifunctional catalytic antibodies. The ability of scFv to inhibit the formation of fibrillar aggregates was studied, using a thioflavin T (ThT) fluorescence assay for Aβ1–40.³⁸⁾ The Aβ1–40 was treated with 1,1,1,3,3,3-hexafluoro-2-propanol to eliminate any preformed aggregates. The pretreated Aβ1–40 (25 µM) and #7TR, #7TR/P95(−), or scFv of #7TR-S5 (1 µM) was incubated in PBS containing 20 µM ThT at 37 °C for 100 h. Reaction progress was monitored by the increasing ThT fluorescence. Figure 14 shows the results. The scFv and light chains completely inhibited the fibrillization of Aβ1–40, indicating that these catalytic antibodies possess not only catalytic activity but also bioactivity.

Fig. 14.

Inhibitive effect on aggregation. Thioflavin T (ThT) fluorescence assay was performed for Aβ1–40. Aβ1–40: 25 µM. #7TR, #7TR/P95(−), or scFv #7TR-S5: 1 µM. ThT: 20 µM in PBS (the reaction was conducted at 37 °C for 100 h).

7. Other cases

In some cases, conversion of a general antibody to a catalytic antibody can be attained. Liu et al. changed several amino acids in the CDR-3 of the antibody heavy chain to Glu, Lys, and His, to create a catalytic triad.³⁹⁾ Hifumi et al. succeeded in producing a catalytic antibody by mutating the two amino acids Gly²⁹-Tyr³⁰ to Thr²⁹-Arg³⁰, in the variable region of the antibody light chain in 2018.²⁷⁾ These catalytic antibodies were obtained by trial and error. A unified consistency is not yet found.

8. Conclusions, opinions, and prospects

1. “Deletion of the Pro95 residing in CDR-3” can confer catalytic activity against antigens. In addition, the deletion can significantly enhance the catalytic activity when the light chain inherently possesses catalytic features. (There is no Pro95 in some cases. When other Pro close to 95th position resides in the CDR-3, the deletion of the Pro can afford the same effect as the Pro95.)

2. The Pro95 residue in CDR-3 has a rigid light chain conformation. Conversely, deletion of the Pro95 makes the conformation flexible, which may lead to the acquisition and/or enhancement of the catalytic activity of the light chain.

3. The catalytic scFv-type molecule, which was designed by combining two different variable regions, can function as a bifunctional catalytic antibody capable of simultaneously cleaving two different antigens.

4. Even in the bifunctional catalytic antibody, the deletion of the Pro95 could enhance the catalytic activity.

As stated in the introduction, many studies on naturally occurring catalytic antibodies have been conducted, since the report was released in 1989.¹⁾ This type of study has increased worldwide, and many interesting reports have been published in the USA,¹⁴⁾^,⁴⁰⁾^–⁴³⁾ Europe,⁴⁴⁾^–⁵¹⁾ and Africa/Asia.⁵²⁾^–⁵⁴⁾ The catalytic antibody has potential in future applications due to its high catalytic activity to cleave the peptide bond of targeting peptides, as well as proteins. In addition, it can cleave DNA (or RNA), which can lead to the suppression of viral infection.⁵⁵⁾

To enable the actual application of catalytic antibodies in the future, a key focus point is the development of preparation technologies, in addition to making the process for using monoclonal antibodies to produce desired catalytic antibodies easier. In many cases, the catalytic antibody can be obtained by screening from many monoclonal antibodies, or it can be separated from natural biological samples (e.g., urine, serum, and milk).

We have faced the difficulty on how to easily and shortly create the desired catalytic antibody. To overcome this problem, we have created a protein bank for 15 years, wherein hundreds of monoclonal antibody light chains (expressed and purified) have been stored. With helps of the Japanese government and many technologists and scientists, the fundamental works requiring a lot of time and effort were continued. The method for deletion of Pro95, which facilitated the ease of effort in producing catalytic antibodies, was discovered through such fundamental studies.

Since it is well known that thousands of monoclonal antibodies have been produced worldwide, the conversion of a general antibody to its catalytic antibody counterpart has the potential to become a crucial technology. In the near future, catalytic antibodies can be easily made by referencing and mutating the amino acid sequence of the monoclonal antibodies produced thus far.

One point should be showcased to the readers: “With the deletion of the Pro95, we can convert the general antibody to its catalytic antibody in many cases, but in some cases, the monoclonal antibodies cannot be converted to their catalytic antibody counterparts”. Given that some conditions remain seemingly unsolved currently, future efforts must solve for unknown factors. The inherent presence of some kinds of catalytic sites, such as triads³⁾^,⁷⁾^,⁹⁾^,¹⁰⁾^,⁵⁶⁾ and dyads,⁵⁷⁾^–⁵⁹⁾ among others,²⁴⁾^,⁵³⁾ may become an important clue to solve the issue.

Acknowledgments

The authors would like to thank Ms. Haruna Tsuda, Mr. Tetsuro Minagawa, Ms. Ayuka Tanaka, and Mr. Asuka Omura for their assistance with this study. This study was supported by KAKENHI Grant Numbers 16H02282, 18K05328, 20K21255, and 21K06486 (Grants-in-Aid for Scientific Research) from the Ministry of Education, Culture, Sports, Science and Technology of Japan. The JST-CREST Programs (Precise arrangement toward functionality: Japan Science and Technology Agency) also supported a part of this study.

The authors also express the appreciation to AAAS, Royal Society of Chemistry, Springer Nature, and Wiley, because some figures and explanations are reproduced in this review.

Notes

Edited by Takao SEKIYA, M.J.A.

Correspondence should be addressed to: E. Hifumi, Oita University, Institute for Research Management & Research Center for GLOBAL/LOCAL Infectious Diseases, 700 Dannoharu, Oita-shi, Oita 870-1192, Japan (e-mail: e-hifumi@oita-u.ac.jp).

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Non-standard abbreviation list

amino acid

arbitrary unit

CBB

Coomassie brilliant blue

CDR-3

complementarity-determining region 3

DNP

2,4-dinitrophenyl

FRET

fluorescence resonance energy transfer

fluorescence unit

mAb

monoclonal antibody

mass spectroscopy

PBS

phosphate-buffered saline

PD-1

programmed cell death protein-1

R-pNA

Arg-p-nitroaniline

TGT

50 mM/Tris-100 mM/glycine-Tween-20 buffer

Profile

Emi Hifumi is a Professor at Oita University of Institute for Research Management and Research Center for GLOBAL/LOCAL Infectious Diseases. She was born in 1964 and received her Ph.D. in Engineering from the University of Kyushu in 2001. Her initial research was to prepare and apply monoclonal antibodies for detection of biological molecules. She expanded the technology to antibody engineering and biosensing field at Hiroshima Prefectural University. In 1998, she discovered a unique antibody light chain, which could enzymatically decompose HIV-gp41 envelope protein. It was named “super catalytic antibody (or Antigenase: antigen decomposing enzyme)”. In 2007, she moved to Oita University (Research Center for Applied Medical Engineering). Until now, she has devoted 25 years to develop super catalytic antibody as a functional molecule possessing anti-viral, anti-bacterial, and anti-cancer effects. In 2020, she found a promising algorism in which one can easily and in a short time convert a monoclonal antibody to the corresponding catalytic antibody. It was enabled that an enzymatic function is conferred to a monoclonal antibody. She won the Morita prize for science and technology in 2004 and Saruhashi prize in 2014.

Hiroaki Taguchi was born in Hyogo Prefecture, Japan in 1969. He completed undergraduation from the Department of Medicinal Chemistry at Kobe Gakuin University, Faculty of Pharmaceutical Sciences, in 1992, and subsequently earned his Ph.D. from the same university in 1997. Thereafter, he worked as a researcher in Zeria Pharmaceutical Co., Ltd. In 2000, he joined Professor Sudhir Paul's laboratory at the University of Texas Health Science Center at Houston Medical School, Department of Pathology and Laboratory Medicines, as a postdoctoral researcher. He was promoted to Assistant Professor at the same institution in 2005. During his tenure, he focused on the detailed characterization of catalytic antibodies and the development of methods to generate catalytic antibodies using chemical probes. After working for 9 years at the University of Texas Health Science Center, he returned to Japan and became an Associate Professor at Suzuka University of Medical Science in 2009. His research efforts primarily revolve around the cutting edge of antibody therapeutics, with a particular focus on the use of catalytic antibodies to treat neurodegenerative diseases such as Alzheimer’s disease. In 2015, he was promoted to Professor at Suzuka University of Medical Science, Faculty of Pharmaceutical Sciences/Graduate School of Pharmaceutical Sciences.

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