Production of neutralizing human monoclonal antibody directed to tetanus toxin in CHO cell

BIOLOGICALS Biologicals 31 (2003) 45–53 www.elsevier.com/locate/biologicals

Production of neutralizing human monoclonal antibody directed to tetanus toxin in CHO cell Jaeho Chin a,b,c, Yeowon Sohn c, Seok Ho Lee c, Young In Park b, Myung Ja Choi a* a

Center of Bioanalysis and Biotransformation Research Center, Korea Institute of Science and Technology, Seoul 130-650, South Korea b Laboratory of Molecular Biology, Graduate School of Biotechnology, Korea University, Seoul 136-701, South Korea c Center of Biological Evaluation, Korea Food and Drug Administration, Seoul 122-704, South Korea Received 24 April 2002; revised 20 September 2002; accepted 4 October 2002

Abstract By the fusion of lymphocytes from hyperimmunized people with heteromyeloma cells, 600 human hybridoma cell lines were generated. Even though seven cell lines produced antibodies against tetanus toxoid, only two antibodies from hybrid CH8 and CH5 only neutralized the tetanus toxin and completely protected the mice that had been challenged with the toxin even at the level of 90 mean lethal dose. The cDNA of light (L) chain and heavy (H) chain variable region was isolated, and then inserted into expression vectors containing human IgG constant regions. After transfection of the recombinant human IgG gene into Chinese Hamster Ovary (CHO) cells, transformants secreting the complete human antibody were selected. The recombinant human antibodies produced from CHO cells possessed neutralizing activity against tetanus toxin just like the original human antibodies produced from human hybridoma cell lines. Western blot analysis showed that rCH8 and rCH5 antibodies recognized the H chain of tetanus toxin and did not bind to its L chain. The neutralizing test showed that HmAb rCH5 had 4.55 IU and HmAb rCH8 had 1.09 IU/100 µg of IgG, respectively. Mixing of the two HmAbs resulted in synergistic effects. On a weight basis (IU/100 µg IgG), the highest potency values were obtained when the two HmAbs were combined in equal quantity. The neutralizing activity of rCH8 and rCH5 mixture was 6.94 IU/100 µg IgG.  2003 The International Association for Biologicals. Published by Elsevier Science Ltd. All rights reserved. Keywords: Toxin-neutralization; Anti-tetanus monoclonal antibodies; Protection against tetanus; Chinese Hamster Ovary cells; Recombinant human IgG

1. Introduction Tetanus is caused by the toxin of Clostridium tetani and is a highly fatal, infectious disease among all species of domestic animals. Tetanus toxin (TT) is synthesized as a single polypeptide chain of 150,000 Da, and undergoes proteolytic cleavage to produce a two chain toxin consisting of the N-terminal 50,000 Da fragment (light (L) chain or fragment A) linked by a disulfide bond to the 100,000 Da C-terminal fragment (heavy (H) chain or fragment B). Digestion of the holotoxin with papain results in fragment B that is composed of the TT light chain and the N-terminal half of the TT heavy chain and the fragment C that contains the C-terminal half of the H chain [1]. * Corresponding author. Tel.: +82-2-958-5062; fax: +82-2-958-5059 E-mail address: [email protected] (a. Choi).

TT toxicity requires several steps; binding to neurons, internalization, retrograde axonal transport, and transynaptic transport prior to its intoxicating action. These various functions have been assigned to different domains of TT. The results of in vitro experiments have suggested that fragment C is responsible for binding to neurons through gangliosides, while the N-terminal of H chain plays an important role in internalization and membrane translocation. The TT light chain (fragment A) contains the catalytic domain, a Zn-dependent metallo-protease that cleaves synaptobrevin, a protein found in synaptic vesicles that are present in nerve terminals [2]. Humoral immunity against tetanus toxin confers total protection from tetanus. It is efficiently induced by the administration of formalin-inactivated toxin or nontoxic fragments of the toxin. Despite the availability of a

1045-1056/03/$30.00  2003 The International Association for Biologicals. Published by Elsevier Science Ltd. All rights reserved. doi:1 0 . 1 0 1 6 / S 1 0 4 5 - 1 0 5 6 ( 0 2 ) 0 0 0 9 2 - 1

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potent vaccine for tetanus, i.e. tetanus toxoid, there has been a high rate of mortality caused by tetanus in developing countries, probably due to incomplete vaccination schedules. Furthermore, passive immunization with antitoxin antibodies is imperative when sufficient time is not available for the development of immunity as a result of an active immunization with tetanus toxoid. Passive immunotheraphy with polyclonal Ig has been used effectively for tetanus toxin. Since these Ig are produced from human plasma, there are numerous drawbacks concerning cost, the need to immunize donors to obtain hyperimmune plasma, lot-to-lot heterogeneity, the possibility of transmitting adventitious viral agents, and the adverse reactions associated with the administration of a relatively large amount of proteins [1]. The application of human monoclonal antibody (HmAb) provides an essentially unlimited supply of homogeneous product that could entirely circumvent most of the above-mentioned problems. Several laboratories have described the generation of hybridoma cell lines secreting anti-TT HmAb. However, there is little information available on the long-term clonal stability and loss of antibody secretion. In several instances, the neutralizing capacity of such HmAb was not clearly demonstrated. In two studies, the potency of anti-TT HmAb was found to be inferior to that of human polyclonal tetanus immunoglobulin (TIG) and the potency of anti-TT HmAb was similar to TIG in one of the two studies [6]. Despite these advantages described above, human hybridoma technology poses several limitations. The human monoclonal cell line is derived from mouse: human heteromyeloma cell line and thus the inherent genetic instability causes the loss of productivity of human antibody in long-term cultivation. There is also a possibility of transmitting adventitious viral agents from mouse:human heteromyeloma cell lines. Production of HmAb from Chinese Hamster Ovary (CHO) cell line was allowed to overcome the above-mentioned limitations of HmAb from hybrid cell lines. We report here that human hybridoma cell lines producing human monoclonal antibodies to tetanus toxin were developed by the fusions of heteromyeloma cells with lymphocytes derived from actively immunized individuals. Two HmAbs showed in vivo neutralizing activity in mice. We identified the DNA sequences of the variable regions of two HmAb genes using RT-PCR reaction and constructed the full IgG gene. We also developed recombinant CHO cell lines that were transfected with expression vector containing full IgG genes. 2. Materials and methods 2.1. Production of HmAb in hybrid cell lines Peripheral blood cells (PBCs) were collected from the whole blood, which was obtained from tetanus

toxoid-primed volunteers. Human hybridomas were generated as previously described elsewhere with some modifications [3]: PBCs were separated on a ficoll gradient and were fused with a heteromyeloma partner, HNC20. The HNC20 cells were harvested from actively growing cultures and washed twice with a plain medium. The PBCs and HNC20 cells were pooled at a ratio of 1:1 and pelleted in a 50 ml conical tube. One millilitre of polyethylene glycol 1500 (50% w/v in the plain medium) was added with slow shaking of the tube over a period of 1 min in order to disperse the pellet. The suspension was mixed gently for additional 30 sec. Two millilitre of the plain medium was added to this suspension over a period of 2 min, followed by 8 ml over another 2 min. The suspension was left at room temperature for 5 min and cells were washed twice with 10 ml of plain medium. Fused cells were suspended in the growth medium at a concentration of 5105 cells/ml and then dispensed at 100 µl/well in 96-well plates. PBCs and HNC20 cells were dispensed at the same density into control wells. One hundred microlitre of a selection medium was added to each well after an incubation of 24 h at 37 (C. Cells were fed with fresh selection medium once every 4 days, until all the cells in control wells died, while colonies of hybrid cells appeared. Hybrids, growing as small colonies, were gradually adapted to growth medium devoid of HAT and ouabain. Putative hybrids were visible 3 weeks after the fusion and were screened for anti-tetanus toxoid antibodies by ELISA. Antibodysecreting cells were cloned and repeated five times at 0.5–1 cell/well to ensure monoclonality and to stabilize antibody secretion. 2.2. ELISA for screening anti-tetanus toxoid antibodies The assay was carried out as reported earlier [3,5]: 96-well microplates were coated with 10 µg/ml of tetanus toxoid (KGC, South Korea) diluted in 50 mM carbonate buffer, pH 9.6, and incubated 18–20 h at 4 (C. The toxoid was decanted and 200 µl of the blocking buffer (1% skim milk in PBS) was added to each well. The plates were incubated for 2.5 h at 37 (C, then washed five times with 200 µl of PBS containing 0.05% Tween 20, pH 7.2. After washing, 100 µl of culture supernatant was added to the well of a blocked plate. Human antibodies to unrelated antigens were added to the well as controls. The plates were incubated for 2 h at 37 (C, then washed as previously described. Polyclonal antibody (anti-human IgG coupled to horseradish peroxidase, Sigma, USA) was diluted 1:2500 with PBS and added (100 µl/well) to each well and incubated for 2 h at 37 (C. After washing five times with PBS containing 0.05% Tween 20, pH 7.2, was 100 µl of TMB substrate (KPL, USA) added to each well and incubated at room temperature for 10 min. The colorimetric reaction was stopped with 5 N sulfuric acid. The optical density of

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each well was measured as absorbance (Molecular Devices, USA), and the repeated measurements for three times were averaged. 2.3. Isotyping of monoclonal antibodies The isotype of HmAb was determined by ELISA using H- and L chain-specific antisera (Tag, Burlingam, CA, USA). The IgG subclasses were detected using murine mAb specific for human IgG1, IgG2, IgG3, and IgG4 (Unipath Ltd, UK). 2.4. Amplication of V genes by PCR Total cellular RNA was extracted from human hybridoma cell line according to the method described previously [7]. cDNA was synthesized according to the manufacturer’s instructions using an oligo (dT) primer (Invitrogen, USA). cDNAs of H- and L chains of HmAb were amplified by PCR using degenerated primers complimentary to the signal sequences and constant regions, which were modified to eliminate the cloning site. The degenerated primer (VH) for H chain signal sequence was 5#-ATGGAT(CT)TTGGGCTGA(CG) CTGG(CG)TTT(CT)T-3#. The primer for the H chain constant region (GVH3#-1) was 5#-TTCGGGGAAGT AGTCCTTGACCAGGCAGCCC-3#. The primer (VK1) for L chain signal sequence was 5#ATGGTGTTGCAGACCCAGGTC-3#. The primer for the L chain constant region (KVL3#-1) was 5#TCATCAGATGGCGGGAAGAT-3#. PCR products were cloned into TA cloning vector (pCRII, Invitrogen) via TA complementary ligation. Ligation products were used to transform competent Escherichia coli DH5a. Clones containing 0.5 kb EcoRI fragments were subjected to double strand DNA sequencing with sequenase (U.S. Biochemicals, USA). 2.5. Insertion of full IgG genes into expression vectors Amplification of the constant region of H and L chain from vectors containing human IgG gene was performed with primers as follows: HC1 (5#-GCCTCCACCAAGGGCCCATCG-3#) HC3-XbaI (5#-ACTCTAGAGTCGACCTGACCCG TGGA-3#) LC1 (5#-ACTGTGGCTGCACCATCTGTCTTCAT CTTCCCGCCA-3#) LC3-ApaI (5#-TCCGGGCCCCCCCTCGAGGTCG ACGA-3#) Amplified constant genes were combined with the variable region of H chain and L chain employing recombinant PCR technology [12]. Combined PCR products were digested with appropriate restriction enzymes and directionally cloned into expression vectors.

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Not I and Xba I cloning sites were used for insertion of the H chain genes into Rc/CMV expression vector. For the L chain, the HindIII and Apa I sites were used for the insertion of Ig genes into Rc/CMV-dehydrofolate reductase gene (dhfr) expression vector. 2.6. Transfection H and L chain expression vectors were prepared with a plasmid isolation kit (QIAGEN, Germany). DG44 cells were transfected by the method using liposome (Lipofection, GibcoBRL, USA) as described in the manufacturer’s manual. In a 25-mm tissue culture flask, 1–2105 cells were seeded and cultured until the cells were 40–60% confluent. Solution A which had 1–2 µg DNA in 100 µl serum-free medium (OPTIMEM, GibcoBRL) and solution B which had 20 µl Lipofection (GibcoBRL) into 100 µl serum-free medium were prepared. The two solutions were combined, mixed gently, and overplayed onto washed cells. After incubation for 24 h at 37 (C in a CO2 incubator, the DNA-containing medium was replaced with 5 ml of normal growth medium containing serum. Cells were incubated at 37 (C in a CO2 incubator for 72 h. After incubation, cells were plated in 96-well microtiter plates at densities between 2104 and 1103 cells/well in the culture medium containing G418 (0.6 mg/ml, GibcoBRL). The medium in wells was removed and replaced with 200 µl every medium every third day until screening by ELISA. 2.7. Amplification Selected cells for ELISA were placed in 25-cm culture dishes at densities between 2104 and 1105 cells/ml in a culture medium containing MTX (20 nM, Sigma). The medium in each well was removed with suction and replaced with 200 µl of fresh medium for every third day for 2 weeks, then survived cells were subcultured in 25-cm culture dishes. The concentration of HmAb in culture supernatants was determined by ELISA method described below and the high producing cell lines were selected. 2.8. ELISA analysis of human antibody expression Expression of HmAb was evaluated by determining the amount of HmAb present in the culture medium. This was performed using microtiter plates coated with 10 mg/ml goat anti-human IgG (H+L) (Calbiochem, La Jolla, CA, USA) for 18–20 h at 4 (C. A peroxidaseconjugated goat anti-human IgG (Calbiochem) was used to follow the reactions and pure human IgG1 (Calbiochem) was employed as the reference material.

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2.9. Purification The antibody was purified by chromatography on a Hi-trap protein A-Sepharose affinity column (Amersham Pharmacia, Sweden). Elution was performed using 0.1 M citrate buffer, pH 3.0. Eluted materials were pooled and dialyzed in PBS. Dialyzed antibody was collected via aseptic filtration and froze at
70 (C. 2.10. Toxin-neutralizing assay in mice The ability of HmAb to neutralize TT in mice was evaluated (Ph. Eur. 1981) using TT and tetanus antitoxin standards (tetanus antitoxin, human, immunoglobulin, NIBSC 76/589, 9.2 IU/ampoule). The mean lethal dose (MLD) of the standard test toxin was determined from a dose–response curve (Ph. Eur. 1981). For the initial screening of toxin-neutralizing activity of HmAb in hybridoma culture, culture supernatant was mixed with 90 MLD of TT and incubated at 37 (C for 60 min. Then 0.4 ml of mixtures were injected intramuscularly (IM) into the hind legs of mice. When mice had survived 4 days after the challenge, HmAb was considered to have neutralized the toxin. The toxinneutralizing activity of HmAb was quantified using doses of toxin at L+/20 levels. Purified HmAb were titrated and mixed with 450 MLD toxin. The mixtures (0.4 ml) were injected into NMRI mice (Charles Rivers, USA) as previously described. The neutralizing activity, expressed as IU/100 µg IgG, was determined using groups of five mice weighing 20–30 g each. The SD of multiple experiments never exceeded 6%.

3. Results 3.1. Development of human monoclonal cell lines to produce antibodies directed against tetanus toxoid From the fusion of lymphocytes of hyperimmunized humans with heteromyeloma, 600 human hybridoma cell lines were generated. The results of initial ELISA showed that seven cell lines produced IgG antibodies directed against tetanus toxoid (Table 1). All the positive cell lines were initially cloned, then recloned four times to generate monoclonal hybridomas such as CH5, CH6, CH8, CH18, CH26, CH42, and CH74. 3.2. Characterization of antibodies Competitive ELISA was employed in order to compare the affinity of antibodies to tetanus toxoid. The result indicated that one cell line (CH8) had the highest binding affinity to TT and four cell lines (CH42, CH74, CH5, and CH26) had moderate binding affinity, while two cell lines (CH18, and CH6) had lower binding affinity (Fig. 1). The

Table 1 Binding properties and toxin-neutralizing activity of anti-tetanus toxoid HmAb Clone

Isotype IgG

Survival ratesa

CH42 CH6 CH8 CH5 CH18 CH26 CH74

G1/k G1/k G1/k G1/k G1/k G1/k G1/k

0 0 5 5 0 0 0

a Survival rates indicate the number of mice alive on day 4 per five mice challenge. See Section 2 for details.

Fig. 1. Determination of affinity rank for anti-tetanus toxoid HmAb. Competitive ELISA was employed in order to compare the affinity of antibodies to tetanus toxoid. Microplates (96-well) were coated with 10 µg/ml of tetanus toxoid (KGC). After blocking the plates, the serial diluted HmAbs were added to each well and incubated for 2 h at 37 (C. After washing, polyclonal antibody (anti-human IgG coupled to horseradish peroxidase, Sigma) was diluted 1:2500 with PBS and added (100 µl/well) to each well and incubated for 2 h at 37 (C. After washing five times with PBS containing 0.05% Tween 20, pH 7.2, 100 µl of TMB substrate (KPL) was added to each well and incubated at room temperature for 10 min. The colorimetric reaction was stopped with 5 N sulfuric acid. The optical density of each well was measured as absorbance (Molecular Devices), and the repeated measurements for three times were averaged.

results of isotyping revealed that all HmAb were of the IgG1 class possessing
– chains (Table 1). In the neutralization study, mice that were challenged with a mixture containing toxin and unrelated (control) culture supernatants died between the second and the fourth days after injections. Not only did antibodies secreted by the hybrid CH8 and CH5 neutralize the toxin but also completely protected the mice even at 90 MLD of challenges with toxin. However, antibodies secreted by the hybrid CH6, CH18, CH26, CH42, and CH74 did not neutralize the toxin and the mice in these groups died within 4 days after a challenge as those in

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Fig. 2. (a and b).

the control group (Table 1). Thus, CH8 and CH5 cell lines were selected to produce the recombinant HmAb. 3.3. Cloning and sequencing variable region genes of CH8 and CH5 Total RNAs from hybridoma cells were isolated and VH and VL cDNAs were obtained by reverse transcription followed by PCR amplification. The amplified VH and VL genes were cloned into TA cloning vector and sequenced. To classify the antibody, the partial sequences of its  and
-1 chains were determined by cDNA sequence analysis (Fig. 2). The variable regions of the L chain of mAb CH8 and CH5 were almost homologous to the subgroup V of human V region. The H chain variable regions can be classified into class III human VH regions [13]. Cloned L- and H chain variable region genes of CH8 were combined with human constant region genes using recombinant PCR technology. The combined DNA sequence was confirmed with DNA sequencing (data not

shown). The PCR primers and the details of combining H- and L chains are described in Section 2. The amplified H- and L chains were introduced into the Rc/CMV and Rc/CMV-dhfr plasmids to obtain pCH8H9, pCH5H12, pCH8L7, and pCH5L19, respectively (Fig. 3). Expression vectors were cotransfected in DG44 cells and transfectants were subjected to G418 selection. Synthesis of full HmAb was determined by screening the supernatant of transfectant clones by ELISA using anti-human IgG (H+L) and anti-human IgG HRP conjugates as described in Section 2. Stable CHO transfectants produced 2–3 µg HmAb/106 cells/24 h. This productivity can be further improved by methotrexatedependent amplification of the dhfr gene. 3.4. Characterization of rCH8 and rCH5 antibodies HmAbs were purified from the supernatant of serumfree culture by protein A sepharose chromatography followed by gel filtration on sephacryl S400. Analytical gel filtration chromatography of purified HmAbs gave a

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Fig. 2. (c and d). Fig. 2. Sequences of the HmAb CH8 and CH5 heavy and L chain variable region. The cDNA sequences are shown in the 5# to 3# direction: (a) CH8 heavy chain variable region, (b) CH8 light chain variable region, (c) CH5 heavy chain variable region, and (d) CH5 light chain variable region. Underlined nucleotides constitute the primers used for PCR amplification. The deduced amino acid sequences are presented in the standard one-letter code. The framework region (FR) and complimentarily determining region (CDR) are shown on top of each sequence.

unique peak with an apparent size of approximately 150 kDa (data not shown). The purified material was analyzed with non-reducing and reducing SDS-PAGE. It was shown that rCH8 and rCH5 migrated under non-reducing conditions as a single band of approximately 150 kDa (lanes 2 and 3) as in Fig. 4a. This band dissociated under reducing conditions into two bands of approximately 50 and 25 kDa each (lanes 4 and 5). These data appeared to support the fact that H- and L chains of rCH8 and rCH5 antibodies are associated in a tetrameric (H2L2) IgG1 molecule. Analysis by isoelectrical focusing (IEF) (Fig. 4b) showed that rCH8 and rCH5 antibodies have isoelectrical points (pI) in the range between 8.15 and 8.65. 3.5. Partial characterization of the binding site of rCH8 and rCH5 antibodies In order to determine the binding specificity, purified rCH8 and rCH5 antibodies were subjected to Western blot analysis. After an electrophoresis of tetanus toxin on 10% sodium dodecyl polyacrylamide gel, the protein was transferred to a nitrocellulose membrane. The mem-

branes were incubated with purified rCH8 and rCH5 antibodies and washed five times with 200 µl of PBS buffer containing 0.05% Tween-20 (v/v). Then membranes were incubated with anti-human IgG (H+L) Ap conjugate and washed. The result of Western analysis showed that rCH8 and rCH5 antibodies recognized the H chain of tetanus toxin but did not bind to the L chain of tetanus toxin. (Fig. 5). 3.6. Estimation of the binding constants of HmAb rCH8 and rCH5 to tetanus toxoid The binding constants of HmAb were measured by ELISA and Scatchard analysis as described in the solid phase method previously [11]. It was shown that the binding constant of HmAb rCH8 was 3.7510
10 M and that of rCH5 was 2.510
9 M. 3.7. Measurements of neutralizing activity of rCH8 and rCH5 HmAb The neutralizing activities of the HmAb were measured by mixing the purified antibody with 450 MLD

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neutralizing activity of rCH8 and rCH5 mixture was 6.94 IU/100 µg IgG.

4. Discussion

Fig. 3. Expression vectors of HmAb CH8 and CH5 for CHO cell lines. Rc/CMV-H is the expression vector for HmAb heavy chain and Rc/CMV-dhfr-L is the expression vector for HmAb light chain: Hu-HV; variable region of H chain, Hu-LV; variable region of L chain, Hu-HC; constant region of human g1 gene, Hu-LC; constant region of human k gene, Pcmv; CMV promotor, SV40ori; origin of replication, Amp; ampicillin resistance gene, dhfr; dehydrofolate reductase gene. Plasmids are not drawn on scale.

tetanus toxin, followed by an injection to NMRI mice. HmAb rCH5 protected the mice from tetanus toxin for 16 days but HmAb rCH8 resulted in only a delay in the death of up to 6 days. The results indicated that HmAb rCH5 contained 4.55 IU/100 µg IgG and HmAb rCH8 had 1.09 IU/100 µg IgG. Mixing the two HmAb appeared to result in synergistic effects (Fig. 6). On a weight basis (IU/100 µg IgG), the highest potency value was obtained with the combination of two HmAbs. The

In this study, we established seven hybrid cell lines that produced anti-tetanus HmAbs with desired toxinneutralizing activity. However, these hybrid cell lines were not suitable for large-scale production of the monoclonal antibodies for clinical use. They did not produce the amount of antibody considered sufficient to substitute the human polyclonal TIG. Also it was revealed that they were not suitable for antibody production due to cellular instability (data not shown). The poor stability of heterohybridoma has been observed in the previous report [1]. The instability of human hybrids could be overcome by the recombinant DNA technology to generate a stable CHO cell line producing identical human antibodies. We determined the DNA sequences of variable regions of antibody and combined the variable regions with human IgG constant regions in CHO cell expression vectors. The CHO cell lines producing anti-tetanus HmAbs were selected. We determined that HmAbs presented in these CHO cell lines had the same specificity and affinity just like the antibody presented in hybrid cell lines. In addition, neutralization study showed that these antibodies could completely neutralize the toxin in vitro and could also protect mice from tetanus toxin challenge. The findings indicate that these HmAbs could be useful for the prevention and treatment of human tetanus. In previous report, Lang et al. [6] found that three commercial polyclonal TIG products had higher affinity constant to the A fragment than that of the C fragment. They also revealed that the protective capacity of all three TIG products was completely abolished by the addition of purified A fragment but was not abolished by the addition of C fragment. They reported that the majority of neutralizing antibodies present in polyclonal TIG are directed against the A fragment of the toxin. Arunachalam et al. [1] and Matsuda et al. [8] found that some HmAbs against tetanus toxin had epitopes either in the H- or in the L chain (fragment A) of the toxin. In these studies, it was demonstrated that HmAbs recognized each of the three functional domains of tetanus toxin, including the L chain having high neutralizing capacity. However, Lang et al. described that the only HmAb found to bind to both A and C fragments provided complete protection against tetanus for more than 28 days [6]. In this report, we demonstrate that the two HmAbs bind to the H chain and have moderate neutralizing capacities. We show that rCH8 HmAb has lower neutralizing capacity than rCH5 HmAb, even though rCH8 HmAb has higher binding affinity than rCH5. In the

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Fig. 4. SDS-PAGE and IEF analysis of purified HmAb and rHmAb. (a) SDS-PAGE analysis of purified HmAb run on a 10% polyacrylamide gel: lane 1, molecular weight markers; lane 2, rCH8 antibody; lane 3, rCH5 under non-reducing conditions; lane 4, rCH8 antibody; and lane 5, rCH5 under reducing conditions. The gel was stained with Coomassie brilliant blue. (b) IEF analysis of purified rCH8, rCH5 antibodies. Samples were analyzed on a pH 3.5–9.3 acrylamide gel: lane 1, pI 3–10 markers; lane 2, rCH8; lane 3, rCH5; and lane 4, pI 3–10 markers. Protein bands were visualized using Coomassie brilliant blue staining.

case of rCH8, the patterns of delayed death are observed. Injection of diluted preparations of rCH8 antibody together with the toxin caused symptoms of tetanus after a delay of several days. Mice died after an additional delay. Simpson et al. [9] also observed this ‘delayed intoxification’ in mice. However, it was shown that rCH5 HmAb had complete protection for more than 16 days. Two HmAbs directed against the same fragment showed different patterns of effects on the symptomatic progression. HmAb from CHO cell lines had similar affinities to tetanus toxin as HmAb from hybrid cell lines. This finding excludes the possibility that the difference in the neutralizing capacity is due to a silent mutation in the variable region of rCH8 HmAb genes in CHO expression vectors. The difference in the neutralizing capacity of two HmAbs may have appeared from the assumption that these HmAbs recognized different epitopes in the H chain of tetanus toxin. The H chain is composed of a translocation domain and a ganglioside-binding domain (fragment C). Studies by several investigators showed that ganglioside-binding domain of H chain would be important for the specificity of toxin activity [2]. To better understand the reason of discrepancy in the neutralizing capacity of two HmAbs, further work is needed to identify the epitope of two HmAbs in H chain of toxin. Additional study should be conducted to understand the reason why two HmAbs have neutralizing activity against toxin even though they faintly recognized the denatured toxin on SDS-PAGE.

Fig. 5. Immunoblot analysis of purified antibodies of rCH8 and rCH5. After SDS-PAGE, tetanus toxin and toxoid were blotted onto nitrocellulose membranes and then the membranes were blocked by incubation with 5% skim milk, followed by incubation in solution containing purified antibodies of rCH8 and rCH5. After washing, the membrane was treated with alkaline phosphatase conjugated anti-human IgG for 1 h. Color was developed using BCIP/NBT. Lane 1, tetanus toxoid; lane 2, tetanus toxin; and SM, size marker. Panel A was result of immunoblot for rCH8 and panel B was for rCH5. Hn of TT is H chain of tetanus toxin.

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now studying that the HmAb of CHO cell lines could rescue intoxicated animals after toxin administration. It appears to support that the HmAbs are effective not only for the preventive purposes but also for the cure of tetanus. References

Fig. 6. In vivo protection with recombinant HmAbs. Mice were injected with a constant amount of toxin (450 MLD) mixed with graded doses of HmAb.

The HmAb described herein has an in vivo potency comparable with previously described anti-TT HmAb presented in human heterohybridoma cell lines. Lang et al. [6] described two HmAbs that possessed more than 10 IU/100 µg IgG. Of particular importance is the fact that such a neutralizing HmAb, when combined, acts synergistically. Others have suggested that potency increase significantly only when three or more HmAbs were combined [4,6,8,10]. The specific activity of antibody developed here was approximately 45 IU/mg which is close to the reported specific activity (60 IU/ mg) of human polyclonal preparation [1]. We also observed identical synergistical effects described above. The HmAbs of CHO cell lines can be produced in large scale and yields of 2–3 µg IgG/106cells/day can be obtained in a serum-free medium. Culturing of CHO cell lines in serum-free medium allows ease of purification with minimal loss either in yield or in potency. As a result, genetic stability of CHO cell lines appears to be suitable for a long-term cultivation. We estimate that a 250-IU dose can be obtained from <150 ml of culture supernatant at a fraction of the cost of producing a comparable dose of polyclonal TIG. Furthermore, lotto-lot variation of the HmAb products seems negligible (data not shown). For clinical use, HmAb must be shown to have not only neutralizing activity when tested as toxin– monoclonal antibody mixtures, but also protective activity when administered before toxin in vivo. We are

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