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Keywords:

  • chimeric antibody;
  • Clostridium botulinum;
  • neurotoxin

ABSTRACT

  1. Top of page
  2. ABSTRACT
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. ACKNOWLEDGMENTS
  7. DISCLOSURE
  8. REFERENCES

Mouse-human chimeric monoclonal antibodies that could neutralize botulinum neurotoxins were developed and an attempt was made to establish mouse hybridoma cell clones that produced monoclonal antibodies that neutralized botulinum neurotoxin serotype A (BoNT/A). Four clones (2–4, 2–5, 9–4 and B1) were selected for chimerization on the basis of their neutralizing activity against BoNT/A and the cDNA of the variable regions of their heavy (VH) and light chains (VL) were fused with the upstream regions of the constant counterparts of human kappa light and gamma 1 heavy chain genes, respectively. CHO-DG44 cells were transfected with these plasmids and mouse-human chimeric antibodies (AC24, AC25, AC94 and ACB1) purified to examine their binding and neutralizing activities. Each chimeric antibody exhibited almost the same capability as each parent mouse mAb to bind and neutralize activities against BoNT/A. From the chimeric antibodies against BoNT/A, shuffling chimeric antibodies designed with replacement of their VH or VL domains were constructed. A shuffling antibody (AC2494) that derived its VH and VL domains from chimeric antibodies AC24 and AC94, respectively, showed much higher neutralizing activity than did other shuffling antibodies and parent counterparts. This result indicates that it is possible to build high-potency neutralizing chimeric antibodies by selecting and shuffling VH and VL domains from a variety of repertoires. A shuffling chimeric antibody might be the best candidate for replacing horse antitoxin for inducing passive immunotherapy against botulism.

List of Abbreviations: 
BoNT/A

botulinum neurotoxin serotype A

C. botulinum

Clostridium botulinum

CDR

complementarity determining region

dhfr

dihydrofolate reductase

FR

frame region

HC

C-terminal half of heavy chain in botulinum neurotoxin

HN

N-terminal half of heavy chain in botulinum neurotoxin

i.p.

intraperitoneal

i.v.

intravenous

mAb

monoclonal antibody

neo

neomycin resistance gene

VH

variable region of heavy chain of antibody

VL

variable region of light chain of antibody

Botulinum neurotoxins produced by the bacterium Clostridium botulinum cause the neuroparalytic disease botulism. Seven distinct serotypes of the toxin have been identified and designated from A through G (1). BoNT are synthesized as single-chain peptides with a molecular mass of about 150 kDa and are proteolytically activated into compounds with a light chain (50 kDa) and a heavy chain (100 kDa) linked by a disulfide bond (2). The light chain acts as a zinc-dependent endopeptidase. The heavy chain is composed of two functional domains: the N-terminal half functions as the translocation domain and the C-terminal half as the receptor-binding domain (3). Human botulism is classified into the following four types based on the mode of intoxication: food-borne, wound, infant and adult botulism from intestinal colonization. All seven types of BoNT act by similar mechanisms. BoNT binds to presynaptic nerve terminals at neuromuscular junctions and cholinergic autonomic sites, thus disrupting cellular communication at the neuromuscular junction (4), resulting in muscular weakness and paralysis (5).

Horse immune serum preparations are used to treat botulism in humans. However, because horse immunoglobulin consists of heteroproteins (heteroantibodies) for humans, its use clearly involves great risk of immune reactions such as anaphylactic shock. In infant botulism in particular, therapy with horse immune serum has been prohibited to prevent occurrence of such anaphylactic reactions. Thus, development of safe and effective treatment for botulism patients is necessary.

Neutralizing monoclonal antibodies to BoNT could also confer effective passive immunity on endangered individuals (6). However, mouse mAb are unsuitable for repeated therapeutic use in humans, largely because of their antigenicity (7, 8). One possible solution to this problem is to humanize mouse mAbs, thus making them acceptable for therapeutic applications. The simplest way to achieve this is to make mouse-human chimeric antibodies. In a chimeric antibody the original mouse variable regions are joined to human constant regions (9, 10). This approach should ensure retention of the antigen-binding properties of the original mouse mAbs plus greatly reduce any anti-mouse immune response during treatment of humans. Clinical results suggest that chimeric antibodies are better than mouse mAbs; however, therapy with the still creates immunogenicity problems. In this study, we designed and constructed mouse-human chimeric antibodies with strong antigen affinity and high neutralizing activity against BoNT/A. Shuffling of heavy and light chains from random combinational immunoglobulin libraries, one of the techniques for improving affinity, has been commonly used in phage display systems (11). Therefore, we redesigned shuffling antibodies from chimeric antibody clones established in this study. The most high-potency neutralizing antibody we produced was from one of these shuffling antibody clones.

MATERIALS AND METHODS

  1. Top of page
  2. ABSTRACT
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. ACKNOWLEDGMENTS
  7. DISCLOSURE
  8. REFERENCES

Botulinum neurotoxin serotype A and its toxoid

Clostridium botulinum type A strain 62A was used for the purification of toxin. BoNT/A was prepared as previously reported and stored in 0.05 M phosphate buffer, pH 7.5, at −80°C until use (12). For an immunogen, BoNT (0.2 mg/mL) was detoxified by dialyzing against 100 mM phosphate buffer, pH 7.0, containing 0.4% formalin at 30°C for 7 days.

Preparation of hybridomas producing monoclonal antibodies against botulinum neurotoxin serotype A

BALB/c mice were immunized three times by i.p. injection with toxoid at two weekly intervals, the first time with Freund's complete adjuvant (Wako Pure Chemicals, Osaka, Japan), and the other times with Freund's incomplete adjuvant (Wako Pure Chemicals). Three days prior to fusion, the mice were boosted with an i.v. injection of BoNT/A (10 μg, 0.1 mL). Their spleen cells were fused with myeloma cells (P3 × 63Ag8U1, P3U1) in 50% polyethylene glycol 400 (Merck, Darmstadt, Germany) containing 10% dimethyl sulfoxide as previously described (13). The resulting hybridomas were selected in HAT medium and cloned twice by limiting dilution.

The subclass of each mAb was determined using a Mouse MonoAb ID Kit (Zymed, Carlsbad, CA, USA). Each hybridoma was cultured in serum-free medium (ASF104; Ajinomoto, Tokyo, Japan). Their supernatants were collected by centrifugation at 180 g for 10 min, and mixed with an equal volume of saturated ammonium sulfate (pH 7.0). The mixture was allowed to stand overnight at 4°C and then centrifuged at 27,000 g for 30 min at 4°C. The pellets were dissolved in 0.02 M phosphate buffer, pH 7.0, and dialyzed with the same buffer. Each mAb was purified using a HiTap Protein G HP column (GE Healthcare, Buckinghamshire, UK) and dialyzed PBS(−). The mAbs were stored at -30°C until use.

Construction of chimeric light and heavy chains and preparation of mouse-human chimeric antibodies

The cDNA encoding murine immunoglobulin VH and VL regions were isolated by RT-PCR from hybridomas producing mAb against BoNT/A. The VH region was amplified using TCG AAG CTT GCC GCC ACC ATG as the 5′ primer and GAA GAT CTG GAT CCA CTC ACC as the 3′ primer. The VL region was amplified using CCG AAG CTT GCC GCC ACC ATG as the 5′ primer and CTA GAT CTG GAT CCA CTT ACG as the 3′ primer. Each 5′ and 3′ primer was tagged with HindIII and BamHI sites, respectively. After subcloning of cDNA into TOPO TA cloning vector (Invitrogen, Carlsbad, CA, USA), the nucleotide sequences were determined using an automated sequencer (Applied Biosystems, Foster City, CA, USA) and the fluoresceinated dye terminator cycle sequencing method. The subcloned PCR products were digested with HindIII and BamHI. The fragments of mouse VH and VL cDNAs (VHbont and VLbont) were then cloned into the upstream regions of the human immunoglobulin constant region genes in pCAG-γ1 and pCAG-κ, respectively (14). pCAG-γ1 and pCAG-κ are expression vectors containing the human cytomegalovirus enhancer and promoter to drive transcription of the recombinant immunoglobulin gene, and the bacterial neomycin resistance gene (neo) and dihydrofolate reductase (DHFR) gene (dhfr), respectively. DHFR-deficient CHO-DG44 cells were transfected with both pCAG-γ1 and pCAG-κ carrying VHbont and VLbont, respectively, using TransIT-LT-1 (Mirus Bio LLC, Madison, WI, USA). Transfected cells were cultured in YMM medium (nucleic acid-free minimum essential medium alpha medium with enriched amino acids/vitamins containing insulin, transferrin, ethanolamine and sodium selenite) developed in the Chemo-Sero-Therapeutic Research Institute, Kumamoto, Japan. After selection using neomycin and methotrexate (Sigma-Aldrich, St. Louis, MO), chimeric antibodies against BoNT/A were purified using a protein G column from culture supernatants.

Enzyme-linked immunosorbent assay

Enzyme-linked immunosorbent assay was carried out according to a previously described method with some modifications (13). Briefly, each well was coated with 0.1 mL of BoNT/A (5 μg/mL) in 10 mM phosphate buffer, pH 7.4, containing 0.15 M NaCl (PBS for ELISA) for 2 hr at 37°C, and blocked overnight with 0.2 mL of 0.2% BSA at 4°C. After washing with PBS for ELISA containing 0.01% Tween 20, 0.1 mL of supernatants or purified antibodies was added to each well and incubated for 2 hr at 37°C. After washing, 0.1 mL of goat anti-mouse (1:10,000) or anti-human IgG (1:5000) conjugated with horseradish peroxidase (both from Bio-Rad, Hercules, CA, USA) was added to each well and incubated for 2 hr at 37°C. After washing, 0.15 mL of o-phenylenediamine solution (0.4 mg/mL; Nacalai Tesque, Kyoto, Japan) as a chromogenic substrate was added to each well. After incubation for 30 min at 37°C, the reaction was stopped with 5N H2SO4 and the absorbance at 450 nm measured with a microplate reader.

Competitive enzyme-linked immunosorbent assay

The purified chimeric antibodies were labeled with EZ-Link sulfo-NHS-biotin (Pierce, Rockford, IL, USA) according to the manufacturer's instructions. Mouse mAbs (0.025 μg/0.1 mL) were added and incubated in ELISA plates coated with BoNT/A (1 μg/mL, 0.1 mL/well) for 2 hr at 37°C. After washing, homologous and heterologous biotinylated chimeric antibodies (0.25 μg/0.1 mL) were added and incubated for 2 hr at 37°C. After washing, peroxidase-labeled avidin (Zymed) diluted to 1:1000 was added and the plates incubated for 30 min at 37°C. After washing, 0.15 mL of o-phenylenediamine solution (0.4 mg/mL) was added to each well. After incubation for 30 min at 37°C, the reaction was stopped with 5N H2SO4 and the absorbance at 450 nm measured with a microplate reader. The data were expressed as percent inhibition calculated according the following equation: (1-[OD490 in the presence of mAb/OD490 in the absence of mAb]) × 100 (%).

Neutralization test

Culture supernatants or purified antibodies (10, 5 or 1 μg/mL) were mixed with an equal volume of BoNT/A (40 LD50/mL) and incubated for 30 min at room temperature. Mice (four weeks old, ddY, Japan SLC, Hamamatsu, Japan) were injected i.p. with 0.5 mL (BoNT/A; 10 LD50/mouse) of the above mixture and observed for 4 days. All mice injected with the mixture of BoNT/A (10 LD50/mouse) with an equal volume of PBS(−) as control died within 24 hr.

Immunoblotting

Botulinum neurotoxin serotype A was transferred electrophoretically to a nitrocellulose membrane (Bio-Rad) after performing SDS-PAGE with 7.5% gel under reduction with DTT by the method of Laemmli (15). The membrane was blocked with 5% skim milk in PBS (pH 7.4). The separate bands were probed with culture supernatants for 1 hr at room temperature. The bound antibodies were then reacted for 30 min at room temperature with goat anti-mouse IgG conjugated with horseradish peroxidase and visualized with 3,3′- diaminobenzidine (Dojin, Tokyo, Japan). The reaction was stopped by rinsing with distilled water.

Other methods

Protein concentrations were determined by the method of Bradford (16) with bovine gammaglobulin as standard. The toxicity of BoNT/A was determined as previously reported (13).

RESULTS

  1. Top of page
  2. ABSTRACT
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. ACKNOWLEDGMENTS
  7. DISCLOSURE
  8. REFERENCES

Selection of mouse hybridoma clones for construction of mouse-human chimeric antibody to botulinum neurotoxin serotype A

We selected mouse hybridoma clones from which we constructed mouse-human chimeric antibodies. We obtained 37 positive clones for BoNT/A by ELISA. The neutralizing test against BoNT/A showed that 13 clones resulted in delayed death or reduced symptoms in mice. The six clones (2–2, 2–4, 2–5, 4–3, 9–4 and B-1) shown in Table 1 exhibited particular neutralizing activity against BoNT/A. In the neutralizing test, all mice inoculated with culture supernatants from three of these clones 92–4, 2–5 and 9–4) survived for more than 24 hr. We evaluated the reactivity of mAbs from each clone to BoNT/A by ELISA (Table 1). The mAbs from 2–4 and 9–4 exhibited as high reactivity in terms of neutralizing activity to BoNT/A as did the other mAb clones. Although the mAbs from 2–5 possessed high neutralizing activity, their reactivity in ELISA was less than that of the other clones. In the neutralizing test, the mAbs from 4–3 and B-1 delayed death more than did other neutralizing mAbs despite their lower reactivity in ELISA. The mAbs from 2–2 had both the lowest neutralizing activity and reactivity of the mAbs from the six chosen clones. We determined the recognition region of each mAb in these six clones by immunoblotting (Fig. 1). Clones 2–2, 4–3 and 9–4 reacted with heavy chains, but the other mAbs reacted with neither heavy nor light chains. According to the above results, we preferentially selected clones 2–4, 2–5, 9–4 and B-1 from which to construct mouse-human chimeric antibodies because they exhibited high neutralizing activities as templates of VH and VL.

Table 1.  Characteristics of mouse mAbs against BoNT/A
Clone No.*Neutralizing** activityELISA*** (OD490)BlottingSubclass
  1. *, culture supernatants from each hybridoma cell clone

  2. **, BoNT/A (10 LD50/mouse) was incubated with equal volumes of each supernatant or medium alone as a control group for 30 min at room temperature. Mice were inoculated i.p. with 0.5 mL of the mixture. The differences in times of death between each culture supernatants and medium alone are shown as follows:

  3. −, in 1 hr or less; +, between 1 and 12hrs.; ++, between 12 and 24 hrs.; +++, more than 24 hrs.

  4. ***, values for optical density at 490 nm were obtained with culture supernatants from each hybridoma cell clones.

  5. n.d., not detected; OD, optical density.

2-2+0.605H chainIgG2a
2-4+++1.864n.d.IgG1
2-5+++0.628n.d.IgG1
4-3++1.270H chainIgG2a
9-4+++3.067H chainIgG2b
B-1++0.881n.d.IgG1
image

Figure 1. Immunoblotting analysis of mAb bound to BoNT/A. BoNT/A was electrophoresed in 7.5% SDS-PAGE under reducing conditions and transferred to a membrane. After transfer, the membrane was incubated with mAbs (lane 1, 2–2; lane 2, 2–4; lane 3, 2–5; lane 4, 4–3; lane 5, 9–4; lane 6, B-1) and polyclonal mouse anti-BoNT/A antibody (lane 7), detected with HRP-conjugated anti-mouse IgG and colored using 4-chloro-1-naphthol.

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Preparation and evaluation of mouse-human chimeric antibodies to botulinum neurotoxin serotype A

We constructed chimeric antibodies from the four mouse hybridoma clones producing neutralizing antibodies against BoNT/A. Using PCR technology, we created HindIII and BamHI sites at the 5′- and 3′-ends of the VH and VL cDNA derived from mouse hybridoma cells. We then cloned these modified mouse VH and VL regions into expression vectors containing human constant regions. We transfected the vectors into CHO-DG44 cells and cultured the transfected cells with EX-CELL 302 under selection using methotrexate and geneticin. After screening with ELISA and performing cloning with limiting dilution, we cultured the cells without FCS and purified each IgG from the culture supernatants.

We examined the neutralizing activities and reactivity of chimeric antibodies to BoNT/A. Every chimeric antibody resulted in significantly later morbidity or mortality after challenge with BoNT/A (data not shown). As shown in Table 2, doses of 10 μg/mL of every chimeric antibody and 5 μg/mL AC24 partially protected the mice when we challenged them with a lethal injection of 10 LD50/mouse. A dose of 5 μg/mL AC25, AC94 and ACB1 did not protect any mice challenged with BoNT/A. Each chimeric antibody exhibited the same level of neutralizing activity against BoNT/A as did all of the mouse mAbs that were sources of VH and VL. We analyzed the binding activities of chimeric antibodies by ELISA (Table 2). Every chimeric antibody showed detectable binding to BoNT/A. The binding activities of the chimeric antibodies were almost as high as those of mouse anti-BoNT/A mAbs. These results indicate that the engineered antibody genes and expression vectors were correctly constructed and functioning in CHO-DG44 cells.

Table 2.  Properties of chimeric antibodies against BoNT/A
Chimeric AntibodyNeutralization*ELISA (OD490)***
105 (μg/mL)10050 (ng/mL)
  1. *, BoNT/A (10 LD50/mouse) was incubated with equal volume of each purified chimeric IgG (10 or 5 μg/mL) for 30 mins at room temperature. Five mice per antibody were inoculated i.p. with 0.5 mL of the mixture. Survival was scored at 96 hrs.

  2. **, survival number/inoculated number

  3. ***, values for optical density at 490 nm were obtained with each chimeric antibody (100 and 50 ng/mL).

  4. OD, optical density.

AC243/5**1/52.7631.897
AC252/50/51.5881.325
AC942/50/52.4951.030
ACB11/50/53.0002.919

Location of the epitope recognized by chimeric antibodies against botulinum neurotoxin serotype A

To evaluate if chimeric antibodies against BoNT/A would bind to overlapping epitopes; we tested chimeric antibodies using competitive ELISA (Table 3). For this experiment, unlabeled mouse mAbs against BoNT/A that were sources of VH and VL in chimeric antibodies were incubated with BoNT/A on ELISA plates, after which each biotinylated chimeric antibody was added as a competitor. The mAb from 2–5 and 2–4 partially competed with binding of AC24 and AC25, respectively, but not with the mAbs from 9–4 and B-1. AC94 and ACB1 did not compete with any of the other mAbs. These results indicate that AC25 and AC24 recognize partially overlapping epitopes.

Table 3.  Inhibition of the binding of biotinylated chimeric antibodies against BoNT/A
Labeled* chimeric antibodyInhibition (%)** with unlabeled mouse mAbs
2–42–59–4b–1
  1. *, after reaction of mouse mAbs (2.5 μg/mL), homologous and heterologous biotinylated chimeric antibody (2.5 μg/mL) was added.

  2. **, values are expressed as percent inhibition according to the following equation: ([1 –[OD490 in the presence of mAb/OD490 in the absence of mAb]) × 100

AC2490843759
AC2583913457
AC9421338838
ACB12517288

We determined the amino acid sequences of VH and VL of mAb from 2–4, 2–5, 9–4 and B1 by sequencing RT-PCR-generated cDNA (Fig. 2). The division into FRs and CDRs was according to Kabat et al. (17). When we compared the amino acid sequences of the VH regions in the four clones, their CDR regions varied, the homology in the FR3 regions being less than in the FR1, FR2 and FR4 regions. In the VL regions, the homology was high throughout the FR region. In particular, the only change between 2–4VL and 2–5VL was from arginine to tyrosine at position 121.

image

Figure 2. Amino acid sequences of (a) murine VH and (b) VL regions in chimeric antibodies. Amino acid sequences of VH and VL regions of murine anti-BoNT/A mAb are aligned. The CDR-1, -2 and -3 regions are boxed. The amino acid residues shared among clones are indicated by asterisks for matches of four and filled circles for matches of three of four.

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Properties of shuffling antibodies after replacement of variable regions of heavy or light chains in chimeric antibodies against botulinum neurotoxin serotype A

We designed shuffling anti-BoNT/A chimeric antibodies in which VH or VL of AC24 replaced AC25 or AC94 (Fig. 3). We transfected CHO-DG44 cells with both pCAG-γ1 carrying VHbont from AC24 and pCAG-κ carrying VLbont from AC25 or AC94. We named these clones AC2425 and AC2494, respectively. We named the clones that derived VH from AC25 or AC94 and VL from AC24 AC2524 and AC9424, respectively. We cultured all the transfected cells and selected using neomycin and methotrexate. We purified shuffling antibodies against BoNT/A from culture supernatants using a protein G column.

image

Figure 3. Shuffling of heavy and light chains among anti-BoNT/A chimeric antibodies. One side of a VH or VL cDNA was replaced with that from another clone in the expression vector and expressed in CHO-DG44 cells. After cloning, the culture supernatants were collected and purified.

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We then examined the neutralizing activities and reactivity of shuffling antibodies to BoNT/A. When we shuffled the VH and VL of AC24 and AC25, the neutralization and binding activities of the shuffling antibodies (AC2425 and AC2524) were almost identical to those of the chimeric antibodies before shuffling (AC24 and AC25), as shown in Table 2 (Table 4). As determined by ELISA, the binding activities in both AC2494 and AC9424 shuffling antibodies remained as high as those of chimeric antibodies before shuffling (AC24 and AC94). Surprisingly, AC2494 that derived VH and VL from AC24 and AC94, respectively, exhibited higher neutralizing activity than did AC24 and AC94 (Table 4). In doses of more than 5 μg/mL, this shuffling antibody perfectly protected mice challenged with BoNT/A. With 1 μg/mL AC2494, four of five mice remained alive despite injections of BoNT/A. In contrast, doses of 10 μg/mL of the reverse shuffling antibody (AC9424) only partially protected the mice challenged with lethal injections. Doses of 5 μg/mL AC9424 did not protect any mice challenged with BoNT/A.

Table 4.  Properties of shuffling antibodies against BoNT/A
Chimeric antibodyNeutralization*ELISA (OD490)***
1051 (μg/mL)10050 (ng/mL)
  1. *, BoNT/A (10 LD50/mouse) was incubated with equal volumes of each purified shuffling IgG (10, 5 or 1 μg/mL) for 30 mins at room temperature. Five mice per antibody were inoculated i.p. with 0.5 mL of the mixture. Survival was scored at 96 hrs.

  2. **, survival number/inoculated number.

  3. ***, values for optical density at 490 nm were obtained with each chimeric antibody (100 and 50 ng/mL).

  4. n.d., not detected; OD, optical density.

AC24252/5**0/5n.d.1.8741.067
AC25242/50/5n.d.2.1381.176
AC24945/55/54/52.1411.497
AC94241/50/5n.d.2.3721.461

DISCUSSION

  1. Top of page
  2. ABSTRACT
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. ACKNOWLEDGMENTS
  7. DISCLOSURE
  8. REFERENCES

Because of their high lethality, BoNT have been classified by the Centers for Disease Control and Prevention into category A, which includes substances that could be used as biological weapons. However, because botulinum toxins have been utilized in recent years for treating dystonia, practical use and laboratory handling of them have become relatively commonplace. Therefore, prevention of biohazards caused by them has become increasingly important, as have techniques for adequate emergency treatment. In some countries, vaccination with botulinum toxoid has been implemented and found to be effective in prevention of infection (18, 19). However, inoculation of toxoid vaccines has been limited to individuals at particularly high risk, such as certain military personnel. Serum therapy has been successfully used for post-exposure treatment of botulism, mainly with equine antitoxin immunoglobulin (20). However, equine antitoxin can cause anaphylactic shock in humans (21).

Some disadvantages of using mouse monoclonal antibodies as immunotherapeutic agents for humans have been reported (7, 8). It is naturally essential to limit or avoid possible side effects linked to the murine origin of such antibodies. One of the approaches to achieve this is to replace most of the mouse sequences with their human counterparts. In this study, as a first step towards achieving this, we prepared and characterized mouse-human chimeric antibodies against BoNT/A.

We obtained six clones of mouse mAb that exhibited a neutralizing effect against BoNT/A. Among these clones, three clones (2–2, 4–3 and 9–4) recognized a heavy chain in BoNT/A. The other clones (2–4, 2–5 and B-1) were not detected by immunoblotting analysis (Fig. 1), suggesting that their clones may recognize conformational epitopes. We selected the four clones that exhibited the highest neutralizing activities against BoNT/A and constructed mouse-human chimeric antibodies on templates of these clones. Every chimeric antibody retained the individual properties of its murine counterpart (Table 2), showing that we had succeeded in transferring both antigen-binding and neutralizing activities from mouse mAbs to chimeric antibodies. In competitive ELISA, binding of AC24 and AC25 was partially disrupted by mouse mAbs from 2–5 and 2–4, respectively (Table 3), suggesting that AC24 and AC25 bind to a neighboring, or the same, site. After we had shuffled VH and VL of AC24 and AC25, their binding and neutralizing activities were almost identical and their levels remained the same as those of these clones (AC24 and AC25) before shuffling (Table 4). These results may be attributable to a difference between AC24 and AC25 of only one amino acid in CDR3 of the VL region (Fig. 2). The results of competitive ELISA also reflect their highly homologous sequences in the VL region. Binding and neutralizing activities of AC9424, which consists of VH of AC94 and VL of AC24, were almost identical with those of AC24 and AC94. These results indicate that we succeeded in transferring both antigen-binding and neutralizing activities from chimeric antibodies to shuffling antibodies. In AC2494, which consists of VH of AC24 and VL of AC94, the binding activity to BoNT/A was almost unchanged, but the neutralizing activity had surprisingly increased to more than tenfold those of AC24 and AC94 (Table 4). Because AC2494 (1.25 to 2.5 μg/mouse) perfectly neutralizes 10 LD50/mouse of BoNT/A, it is probable that this antibody neutralizes more than 4000 LD50/mg antibody. These data also indicate that the shuffling antibody has strong neutralizing activity that is comparable to that of BabyBIG (3000 LD50/mg antibody), which was developed as an orphan drug consisting of human-derived botulism antitoxin antibodies (22). Previous studies have reported a correlation between binding effect and neutralizing activity against bacterial toxin (23, 24). It is probable that the neutralizing activity of an antibody against toxin correlates closely with antigen-binding activity. However, in spite of the increased neutralizing activity of AC2494, its antigen-binding activity was almost identical to those of AC24 and AC94. This may be attributable to a difference between VH and VL domains in the original clones. VH and VL domains are packed together and the hypervariable loops on each domain contribute to binding antigen (25). The relative importance of VH and VL domains in creating antigen-binding sites is under much discussion. Many kinds of antigen-specific single VH domains can be isolated using a phage-displayed library (26, 27). Although there has been limited isolation of VL domains with binding activity, VL domains also exhibit antigen-specific binding activity (28). Among particular antibodies, the relative importance of VH and VL domains may vary. In our chimeric antibodies, it is not clear whether their binding activity to BoNT/A depends on VH or VL domains. However, the combination of VH and VL from AC24 and AC94, respectively, may vigorously restrict structural changes of BoNT/A for binding to surface nerve-cell receptors and translocation across the cell membrane. It is not easy to prepare hybridomas that produce high titer neutralizing monoclonal antibodies against BoNT/A. VH and VL domains have a restricted structural spectrum of antigens that they can recognize. There would be very few VH or VL domain repertoires that could perfectly neutralize BoNT/A. In this study, we succeeded in constructing a high-potency neutralizing chimeric antibody, AC2494, in vitro using DNA shuffling of VH and VL domains from another clone. The combination of VH and VL domains, as in AC2494, might not exist in natural antibodies produced by B cells. Researchers have previously designed many shuffling antibodies with strong antigen affinity from immunoglobulin libraries using phage display systems (29, 30). By shuffling 20 nonbinding clones against the hapten nitrophenyl phosphonamidate, 240 clones of antigen binding clones were generated (29). Human shuffling antibody in which the light chains binding to TNF-α were paired with a repertoire of heavy chains displayed on phage was found to have a binding affinity similar to a mouse mAb (30). These data indicate that it is possible to build more neutralizing recombinant antibodies by selecting and shuffling VH and VL domains from a variety of repertoires against BoNT/A.

When researchers tested chimeric antibodies in humans, they found that humanized rodent antibodies were less immunogenic and that this increased their half-life and improved their efficacy (31). Because of its neutralizing efficiency, our shuffling recombinant chimeric antibody (AC2494) seems to be a good candidate for passive human immunotherapy. However, as part of this antibody is still derived from mice, reshaping of a completely human antibody would ensure safer usage.

ACKNOWLEDGMENTS

  1. Top of page
  2. ABSTRACT
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. ACKNOWLEDGMENTS
  7. DISCLOSURE
  8. REFERENCES

This study was supported by a Health and Labour Sciences Research Grant (Research on Regulatory Science of Pharmaceuticals and Medical Devices) from the Ministry of Health, Labor and Welfare of Japan.

DISCLOSURE

  1. Top of page
  2. ABSTRACT
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. ACKNOWLEDGMENTS
  7. DISCLOSURE
  8. REFERENCES

The authors have no conflicts of interest associated with this study.

REFERENCES

  1. Top of page
  2. ABSTRACT
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. ACKNOWLEDGMENTS
  7. DISCLOSURE
  8. REFERENCES
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