Catalytic Antibody Light Chain Research Articles

Obtaining Highly Active Catalytic Antibodies Capable of Enzymatically Cleaving Antigens.

A catalytic antibody has multiple functions compared with a monoclonal antibody because it possesses unique features to digest antigens enzymatically. Therefore, many catalytic antibodies, including their subunits, have been produced since 1989. The catalytic activities often depend on the preparation methods and conditions. In order to elicit the high catalytic activity of the antibodies, the most preferable methods and conditions, which can be generally applicable, must be explored. Based on this view, systematic experiments using two catalytic antibody light chains, #7TR and H34, were performed by varying the purification methods, pH, and chemical reagents. The experimental results obtained by peptidase activity tests and kinetic analysis, revealed that the light chain's high catalytic activity was observed when it was prepared under a basic condition. These data imply that a small structural modulation of the catalytic antibody occurs during the purification process to increase the catalytic activity while the antigen recognition ability is kept constant. The presence of NaCl enhanced the catalytic activity. When the catalytic light chain was prepared with these preferable conditions, #7TR and H34 hugely enhanced the degradation ability of Amyloid-beta and PD-1 peptide, respectively.

A new catalytic site functioning in antigen cleavage by H34 catalytic antibody light chain

The cleavage reactions of catalytic antibodies are mediated by a serine protease mechanism involving a catalytic triad composed of His, Ser, and Asp residues, which reside in the variable region. Recently, we discovered a catalytic antibody, H34 wild type (H34wt), that is capable of enzymatically cleaving an immune-check point PD-1 peptide and recombinant PD-1; however, H34wt does not contain His residues in the variable region. To clarify the reason behind the catalytic features of H34wt and the amino acid residues involved in the catalytic reaction, we performed site-directed mutagenesis focusing on the amino acid residues involved in the cleavage reaction, followed by catalytic activity tests, immunological reactivity evaluation, and molecular modeling. The results revealed that the cleavage reaction by H34wt proceeds through the action of a new catalytic site composed of Arg, Thr, and Gln. This new scheme differs from that of the serine protease mechanism of catalytic antibodies.

Finding and characterizing a catalytic antibody light chain, H34, capable of degrading the PD-1 molecule.

Programmed cell death 1 (PD-1) is an immune checkpoint molecule regulating T-cell function. Preventing PD-1 binding to its ligand PD-L1 has emerged as an important tool in immunotherapy. Here, we describe a unique human catalytic antibody light chain, H34, which mediates enzymatic degradation of human PD-1 peptides and recombinant human PD-1 protein and thus functions to prevent the binding of PD-1 with PD-L1. H34 degraded one half of the PD-1 molecules within about 6 h under the experimental conditions. Investigating the acquisition of the catalytic function by H34, which belongs to subgroup I and lacks a Pro95 residue in CDR-3, revealed the importance of this sequence, as a Pro95-reconstituted mutant (H34-Pro95(+)) exhibited very little catalytic activity to cleave PD-1. Interestingly, EDTA inhibited the catalytic activity of H34, which could work as a metallo-protease. Zn2+ or Co2+ ions may work as a cofactor. It is meaningfull that H34 was obtained from the human antibody gene taken from a healthy volunteer, suggesting that we potentially have such unique molecules in our body.

New technologies to introduce a catalytic function into antibodies: A unique human catalytic antibody light chain showing degradation of β-amyloid molecule along with the peptidase activity.

Since the discovery of a natural catalytic antibody in 1989, many catalytic antibodies targeting peptides, nucleotides, virus and bacterial proteins, and many molecules have been prepared. Although catalytic antibodies should have features superior to non‐catalytic monoclonal antibodies, the reports on catalytic antibodies are far fewer than those on normal (non‐catalytic) antibodies. Nowadays, we can obtain any monoclonal antibody we want, which is not the case for catalytic antibodies. To overcome this drawback of catalytic antibodies, much basic research must be done. As one way to attain this purpose, we have been making a protein bank of human antibody light chains, in which the light chains were expressed, purified, and stored for use in screening against many kinds of antigen, to then get clues to introducing a catalytic function in normal antibodies. As the number of stored light chains in the above protein bank has reached the hundreds, in this study, we screened them against amyloid‐beta (Aβ), which is well‐known as one of the molecules causing Alzheimer's disease. We found two interesting light chains, #7TR and #7GY. The former could degrade both a fluorescence resonance energy transfer‐Aβ substrate and Aβ1‐40 full peptide. In contrast, #7GY, whose sequence is identical to that of #7TR except for the amino acids at the 29th and 30th positions, did not degrade the FRET‐Aβ substrate at all. By using a synthetic substrate, Arg‐pNA, the difference between the chemical features of the two light chains was investigated in detail. In addition, we found that the presence of Zn(II) ion hugely influenced the catalytic activity of the #7TR light chain but not #7GY. Through these facts and the discussion, we propose one of the clues to how to put a catalytic function in a normal (non‐catalytic) antibody.

A novel method of preparing the monoform structure of catalytic antibody light chain.

Along with the development of antibody drugs and catalytic antibodies, the structural diversity (heterogeneity) of antibodies has been given attention. For >20 yr, detailed studies on the subject have not been conducted, because the phenomenon presents many difficult and complex problems. Structural diversity provides some (or many) isoforms of an antibody distinguished by different charges, different molecular sizes, and modifications of amino acid residues. For practical use, the antibody and the subunits must have a defined structure. In recent work, we have found that the copper (Cu) ion plays a substantial role in solving the diversity problem. In the current study, we used several catalytic antibody light chains to examine the effect of the Cu ion. In all cases, the different electrical charges of the molecule converged to a single charge, giving 1 peak in cation-exchange chromatography, as well as a single spot in 2-dimensional gel electrophoresis. The Cu-binding site was investigated by using mutagenesis, ultraviolet-visible spectroscopy, atomic force microscope analysis, and molecular modeling, which suggested that histidine and cysteine residues close to the C-terminus are involved with the binding site. The constant region domain of the antibody light chain played an important role in the heterogeneity of the light chain. Our findings may be a significant tool for preparing a single defined, not multiple, isoform structure.

Biochemical features and antiviral activity of a monomeric catalytic antibody light-chain 23D4 against influenza A virus.

Catalytic antibodies have exhibited interesting functions against some infectious viruses such as HIV, rabies virus, and influenza virus in vitro as well as in vivo. In some cases, a catalytic antibody light chain takes on several structures from the standpoint of molecular size (monomer, dimer, etc.) and/or isoelectronic point. In this study, we prepared a monomeric 23D4 light chain by mutating the C-terminal Cys to Ala of the wild-type. The mutated 23D4 molecule took a simple monomeric form, which could hydrolyze synthetic 4-methyl-coumaryl-7-amide substrates and a plasmid DNA. Because the monomeric 23D4 light chain suppressed the infection of influenza virus A/Hiroshima/37/2001 in an in vitro assay, the corresponding experiments were conducted in vivo, after the virus strain (which was taken from a human patient) was successfully adapted into BALB/cN Sea mice. In the experiments, a mixture of the monomeric 23D4 and the virus was nasally administered 1) with preincubation and 2) without preincubation. As a result, the monomeric 23D4 clearly exhibited the ability to suppress the influenza virus infection in both cases, indicating a potential drug for preventing infection of the influenza A virus.

Biochemical Features of a Catalytic Antibody Light Chain, 22F6, Prepared from Human Lymphocytes

Human antibody light chains belonging to subgroup II of germ line genes were amplified by a seminested PCR technique using B-lymphocytes taken from a human adult infected with influenza virus. Each gene of the human light chains was transferred into the Escherichia coli system. The recovered light chain was highly purified using a two-step purification system. Light chain 22F6 showed interesting catalytic features. The light chain cleaved a peptide bond of synthetic peptidyl-4-methyl-coumaryl-7-amide (MCA) substrates, such as QAR-MCA and EAR-MCA, indicating amidase activity. It also hydrolyzed a phosphodiester bond of both DNA and RNA. From the analysis of amino acid sequences and molecular modeling, the 22F6 light chain possesses two kinds of active sites as amidase and nuclease in close distances. The 22F6 catalytic light chain could suppress the infection of influenza virus type A (H1N1) of Madin-Darby canine kidney cells in an in vitro assay. In addition, the catalytic light chain clearly inhibited the infection of the influenza virus of BALB/c mice via nasal administration in an in vivo assay. In the experiment, the titer in the serum of the mice coinfected with the 22F6 light chain and H1N1 virus became considerably lowered compared with that of 22F6-non-coinfected mice. Note that the catalytic light chain was prepared from human peripheral lymphocyte and plays an important role in preventing infection by influenza virus. Considering the fact that the human light chain did not show any acute toxicity for mice, our procedure developed in this study must be unique and noteworthy for developing new drugs.

Catalytic and Biological Features of Human Light Chains Suppressing of Infection of Influenza Virus A Type

By immunizing ground‐state peptides or proteins into mice, the authors have established murine catalytic antibody light chains (antigenases) that could destroy such as HIV‐1 gp41, H. pylori urease, TNFa etc. We proposed a new concept that the antigenase posseses a catalytic‐triad structure composed Ser, His and Asp.Based on above concept, we cloned cDNAs (from a volunteer infected with influenza virus) encoding human antibody kappa light chain belonging to subgroup II of germline genes, because the group encodes a catalytic triad‐like structure with high possibility. The cDNAs were transformed into E. coli and each human light chain was expressed as the protein. The highly purified (>95%) human light chains were firstly submitted to the catalytic activity tests and to in vitro assays for examining the infectivity of influenza virus. Some clones showed the amidase and DNase activity. Surprisingly, #4 clone exhibited a strong activity to digest pBR322 plasmid. And the #4 clone suppressed the infection of influenza virus (H1N1) in vitro assay. In addition, we performed in vivo assays. The administered mice with the influenza virus treated with #4 antigenase enhanced the number of survival mice compared with non‐treated mice. Furthermore, the weigh of the antigenase‐treated mice did not decrease at all, while the antigenase‐not‐treated mice showed the marked decrease of the weigh.

Biological features of human catalytic antibody light chains showing anti‐cancer activity

The authors have reported about the human catalytic antibody light chains possessing catalytic activity, which was mostly encoded in Subgroup II in kappa type. In this study, the highly purified human light chains were submitted to investigate the function of the toxicity against cancer cells.We designed two primer sets and performed a semi‐nested PCR using cDNA prepared from human leukocyte. As the results, eighteen clones of subgroup II germline gene of human kappa light chain were established. The mutants of C220A corresponding to the 18 clones were made. And the wild types and mutants were expressed in E. coli and the light chains recovered were highly purified by two‐step purification system. Out of 18 clones, #1 clone showed the cytotoxicity to both human lung (A548) and human stomach cancer cells (SNU‐1) but not to pancreas cancer cells (PANC‐1). Interestingly, the dimeric form showed the stronger effect than the monomeric form (C220A). The #1 clone could hydrolyze QAR‐MCA synthetic peptide showing the amidase activity. In addition, #1 clone has a unique amino acid sequence compared with that of other clones. Although it is still unclear that the relationship between catalytic activity and cytotoxicity of human light chain (#1 clone) at this moment, the catalytic triad structure of the clone may contribute the anti‐cancer effect.

Highly efficient method of preparing human catalytic antibody light chains and their biological characteristics

The ultimate goal of catalytic antibody research is to develop new patient therapies that use the advantages offered by human catalytic antibodies. The establishment of a high-throughput method for obtaining valuable candidate catalytic antibodies must be accelerated to achieve this objective. In this study, based on our concept that we can find antibody light chains with a high probability of success if they include a serine protease-like catalytic triad composed of Ser, His, and Asp on a variable region of the antibody structure, we amplified and cloned DNAs encoding human antibody light chains from germline genes of subgroup II by seminested PCR using two primer sets designed for this purpose. Seven DNA fragments encoding light chains in 17 clones were derived from germline gene A18b, 6 DNA fragments from A3/A19, 2 DNA fragments from A17, and a clone DNA fragment from A5 and O11/O1. All light chains expressed in Escherichia coli and highly purified under nondenaturing conditions exhibited amidolytic activity against synthetic peptides. Some of the light chains exhibited unique features that suppressed the infectious activity of the rabies virus. Furthermore, the survival rate of mice in which a lethal level of the rabies virus was coinoculated directly into the brain with light chain 18 was significantly improved. In the case of humans, these results demonstrate that high-throughput selection of light chains possessing catalytic functions and specificity for a target molecule can be attained from a light-chain DNA library amplified from germline genes belonging to subgroup II.

Effective preparation method of human catalytic antibody light chains

We have successfully produced mouse type catalytic antibody light chains (antigenases) by immunizing ground‐state peptides or proteins. The antigenase possesses the ability to enzymatically decompose a targeting molecule using the catalytic triad formed in the structure. In the case of human antibody light chains, the catalytic triad was mostly encoded in Subgroup II, suggesting that the light chains belonging to the subgroup should exhibit enzymatic functions.In order to verify the above concept, we amplified an antibody germline gene as the cDNA belonging to Subgroup II, which was taken from human leucocyte. The cDNA was inserted in pET system, followed by a transformation to express the protein (human antibody light chain) in E.coli. We could recover the protein with very high purity by passing two affinity columns. Its catalytic activity was measured using synthetic substrate (peptide‐MCA) to monitor the fluorescence generated by the cleavage of the peptide‐MCA bond, suggesting the human antibody light chain exhibited the amidase activity. Furthermore, the light chains showed other interesting enzymatic reaction in addition to the amidase activity.

Monoclonal antibodies specific to Cucumber mosaic virus coat protein possess DNA-hydrolyzing activity

Monoclonal antibodies (mAbs) specific to Cucumber mosaic virus coat protein (CMV-CP) were designed from cDNA and deduced amino acid sequences of the light chain genes of 10 out of 14 different hybridoma cell lines. Ten of these mAbs revealed a very restricted germline family VκII, within which gene bd2 has identical amino acid sequences with VIPase and an i41SL 1-2 catalytic antibody light chain, both of which possess peptidase activity. Four out of the 14 mAbs illustrated another germline family VκIA, within which gene bb1.1 had high homology with BV04-01 light chain mAb, which hydrolyses ssDNA. Interestingly, our mAbs showed DNA-hydrolytic activity at an optimum pH of 4–5, which is a typical pattern of autoimmune diseases in which autoantibodies hydrolyze supercoiled plasmid DNA. This is the first evidence ever that CMV-CP could stimulate catalytic antibodies, which have an identical sequence homology with autoantibodies. Furthermore, the CMV-CP-specific mAbs will be important for isolating antibodies specific to the CPs of bacteria, viruses, cancer cells, etc. that could be used for medical therapy.

Catalytic Features and Eradication Ability of Antibody Light-chain UA15-L against Helicobacter pylori

We have successfully developed a catalytic antibody capable of degrading the active site of the urease of Helicobacter pylori and eradicating the bacterial infection in a mouse stomach. This monoclonal antibody UA15 was generated using a designed recombinant protein UreB, which contained the crucial region of the H. pylori urease beta-subunit active site, for immunization. The light chain of this antibody (UA15-L) by itself showed a proteolytic activity to substantially degrade both UreB and the intact urease. Oral administration of UA15-L also significantly reduced the number of H. pylori in a mouse stomach. This is the first example of a monoclonal catalytic antibody capable of functioning in vivo, and such an antibody may have a therapeutic utility in the future.

Specific degradation of H. pylori urease by a catalytic antibody light chain

Catalytic antibodies capable of digesting crucial proteins of pathogenic bacteria have long been sought for potential therapeutic use. Helicobacter pylori urease plays a crucial role for the survival of this bacterium in the highly acidic conditions of human stomach. The HpU-9 monoclonal antibody (mAb) raised against H. pylori urease recognized the alpha-subunit of the urease, but only slightly recognized the beta-subunit. However, when isolated both the light and the heavy chains of this antibody were mostly bound to the beta-subunit. The cleavage reaction catalyzed by HpU-9 light chain (HpU-9-L) followed the Michaelis-Menten equation with a K(m) of 1.6 x 10(-5) m and a k(cat) of 0.11 min(-1), suggesting that the cleavage reaction was enzymatic. In a cleavage test using H. pylori urease, HpU-9-L efficiently cleaved the beta-subunit but not the alpha-subunit, indicating that the degradation by HpU-9-L had a specificity. The cleaved peptide bonds in the beta-subunit were L121-A122, E124-G125, S229-A230, Y241-D242, and M262-A263. BSA was hardly cleaved by HpU-9-L, again indicating the digestion by HpU-9-L was specific. In summary, we succeeded in the preparation of a catalytic antibody light chain capable of specifically digesting the beta-subunit of H. pylori urease.

Investigation of active form of catalytic antibody light chain 41S-2-L

We have raised a monoclonal antibody (41S-2) against the conserved sequence, RGPDRPEGIEEEGGERDRD, of human immunodeficiency virus type1 (HIV-1) envelope gp41. That antibody light chain (41S-2-L) cleaves gp41-derived peptide (TPRGPDRPEGIEEEGGERDRD; TP41-1) with a characteristic biphasic profile composed of induction and active phases. It is considered that the conformation of 41S-2-L is changed, by such as induced fitting, to move to active phase to decompose the antigenic peptide during the induction phase. In order to investigate what happens to 41S-2-L in the induction and active phase, the cleavage reaction of the peptide by 41S-2-L was examined in detail from the viewpoint of kinetic and spectroscopic analysis. The kinetic data showed that the preferable conformational transition of 41S-2-L took place by the unimolecular reaction of 41S-2-L in the induction phase. UV–vis and fluorescence spectroscopic analysis suggested that the conformational transition leads to the generation of aggregates of 41S-2-L in the reacting solution, which causes the huge enhancement of the catalytic activity of 41S-2-L. The nuclei of the aggregates may be formed in the induction phase. The aggregates and soluble 41S-2-L are considered to be in chemical equilibrium during the cleavage reaction of the antigen.

Catalytic antibody light chain capable of cleaving a chemokine receptor CCR-5 peptide with a high reaction rate constant.

A monoclonal antibody (MAb), ECL2B-2, was obtained by immunizing a peptide possessing a part of a sequence of a chemokine receptor, CCR-5, which is present as a membrane protein on the macrophage surface, and which plays an important role in human immunodeficiency virus (HIV) infection. From the DNA and the deduced amino acid sequences of the light and heavy chains of ECL2B-2 MAb, molecular modeling was conducted to calculate the steric conformation of the antibody. Modeling suggested that the structure of ECL2B-2 could possess one or two catalytic triad(s), composed of Asp(1), Ser(27a) (or Ser(27e)), and His(93) (or His(27d)), in the light chain of ECL2B-2. The three amino acid residues, Asp(1), Ser(27a), and His(93), are identical to those of catalytic antibody light chains such as VIPase and i41SL1-2. The light chain of ECL2B-2 MAb degraded the antigenic peptide CCR-5 within about 100 h. Surprisingly, the light chain had a very high catalytic reaction rate constant (k(cat)) of 2.23 min(-1), which is greater by factors of tens to hundreds than those of natural catalytic antibodies obtained previously. The heavy chain of ECL2B-2 MAb, which has no catalytic triad because of a lack of His residue, did not degrade the CCR-5 peptide.

Catalytic features of monoclonal antibody i41SL1-2 subunits.

A monoclonal antibody (mAb), i41SL1-2, was obtained by immunizing the peptide of complementarity-determining region-1 (CDRL-1: RSSKSLLYSNGNTYLY) of a super catalytic antibody light chain, 41S-2-L, capable of enzymatically destroying the gp41 molecule of the HIV-1 envelope. From the DNA and the deduced amino acid sequences of the light and heavy chain of i41SL1-2 mAb, molecular modeling was conducted that suggested that both subunits of i41SL1-2 mAb possess catalytic triads in their structures. Especially the light chain of i41SL1-2 mAb possesses a characteristic catalytic triad composed of Asp(1), Ser(27A), and His(93), whose positions are identical to the catalytic antibody light chain, VIPase, of S. Paul and colleagues (see text). The antibody gene of i41SL1-2 light chain and VIPase belong to the same germline, bd2, suggesting that the discrete germline inherently possesses catalytic activity. Both light and heavy chains of i41SL1-2 mAb degraded the antigenic peptide CDRL-1 within 47 and 57 h, respectively. The catalytic reaction constant (kcat) of the light and heavy chain was 6.1 x 10(-1) and 6.2 x 10(-1) min(-1), respectively. These are high values for the natural catalytic antibodies reported so far. The catalytic efficiency (kcat/Km) of the light and heavy chain was 3.1 x 10(5) and 4.9 x 10(4) M(-1) min(-1), respectively. The first cleaved bond of the antigenic peptide by subunits of i41SL1-2 mAb was between Arg(1) and Ser(2) in the sequence of CDRL-1, suggesting a serine protease character.

A mechanism-based probe for gp120-Hydrolyzing antibodies

An antigenic peptide analogue consisting of HIV gp120 residues 421–431 (an antigen recognition site probe) with diphenyl amino(4-amidinophenyl)methanephosphonate located at the C-terminus (a catalytic site probe) was synthesized and its trypsin and antibody reactivity characteristics were studied. Antibodies to the peptide determinant recognized the peptidyl phosphonate probe. Trypsin was inhibited equipotently by the peptidyl phosphonate and its simple phosphonate counterpart devoid of the peptide determinant. The peptidyl phosphonate inhibited the gp120-hydrolyzing activity of a catalytic antibody light chain. It was bound covalently by the light chain and the binding was inhibited by the classical active-site directed inhibitor of serine proteinase, diisopropyl fluorophosphate. These results reveal that the peptidyl phosphonate ester can serve as a probe for the antigen recognition and catalytic subsites of proteolytic antibodies.

Targeted destruction of the HIV-1 coat protein gp41 by a catalytic antibody light chain

Generation of antibodies with the ability to destroy targeted viral coat proteins or tumor antigens is an important aim in current research aimed at developing superior catalytic antibodies. To this end, we raised a monoclonal antibody against a discrete sequence of the envelope gp41, RGPDRPEGIEEEGGERDRD, which is a highly conserved sequence in many human immunodeficiency virus (HIV)-1 strains. The light chain subunit of this antibody catalytically decomposed the targeted peptide antigen. The degradation of the immunized peptide antigen by the light chain was initiated by the hydrolytic scission of the peptide bond between Glu12–Gly13, followed by the successive cleavage reactions of the additional peptic bonds into small peptides and amino acids. The decomposition by the light chain obeyed Michaelis–Menten kinetics ( k cat/ K m=2.8×10 5 M −1 min −1). A characteristic feature of the reaction was a slow initial degradation step, followed by an increase in the rate of catalysis. Removal of the light chain by immunoadsorption at either stage of the reaction resulted in recession of catalysis. The light chain also cleaved recombinant gp41 molecule, but did not degrade proteins unrelated in the sequence to the peptide immunogen (bovine and human serum albumins).