A novel monoclonal antibody cross-reactive with both human and mouse α9 integrin useful for therapy against rheumatoid arthritis.

This study introduces a novel monoclonal anti-α9 integrin antibody (MA9-413) with human variable regions, isolated by phage display technology. MA9-413 specifically binds to both human and mouse α9 integrin by recognizing a conserved loop region designated as L1 (amino acids 104-122 of human α9 integrin). MA9-413 inhibits human and mouse α9 integrin-dependent cell adhesion to ligands and suppresses synovial inflammation and osteoclast activation in a mouse model of arthritis. This is the first monoclonal anti-α9 integrin antibody that can react with and functionally inhibit both human and mouse α9 integrin. MA9-413 allows data acquisition both in animal and human pharmacological studies without resorting to surrogate antibodies. Since MA9-413 showed certain therapeutic effects in the mouse arthritis model, it can be considered as a useful therapy against rheumatoid arthritis and other α9 integrin-associated diseases.

Improved Brain Expression of Anti-Amyloid ß scFv by Complexation of mRNA Including a Secretion Sequence with PEG-based Block Catiomer.

BACKGROUND: The ever-increasing number of people living with Alzheimer's disease urges to develop more effective therapies. Despite considerable success, anti-Alzheimer immunotherapy still faces the challenge of intracerebral and intracellular delivery. This work introduces in situ production of anti-amyloid beta (Aß) antibody after intracerebral injection of PEG-PAsp(DET)/mRNA polyplexes as a novel immunotherapy approach and a safer alternative compared to high systemic antibodies doses or administration of adenovirus encoding anti- Aß antibodies. METHODS: We used mRNA encoding three different Aß-specific scFV with a secretion signal for passive immunotherapy. scFv contained a 6xHis-tag for immuno-detection. The secretion signal from IL2 (IL2ss) was added to allow extracellular engagement of senile plaques. Aß affinity of scFv was measured by surface plasmon resonance. To allow intracellular delivery, scFv were administered as polyplexes formed with our smart copolymer polyethylene glycol-poly[N'-[N-(2-aminoethyl)-2-aminoethyl] aspartamide] [PEG-PAsp (DET)]. We evaluated scFv expression in cellulo by Western blot and ELISA, their ability to disaggregate amyloid aggregates by thioflavine T assay. Moreover, in vivo expression and therapeutic activity were evaluated in a murine amyloidosis model, by anti-6xHis-tag ELISA and anti- Aß ELISA, respectively. RESULTS: The selected anti-amyloid beta scFv showed affinity towards Aß and disaggregated Aß fibers in vitro. Whereas both DNA and mRNA transfection led to scFV expression in cancer cells, only mRNA led to detectable scFv expression in primary neurons. In addition, the use of IL2ss increased by 3.4-fold scFv secretion by primary neurons over mRNA polyplexes devoid of secretion signal. In vivo, a 3 to 11- fold of intracranial scFv levels was measured for mRNA compared to DNA polyplexes and higher in vivo scFv levels were obtained with mRNA containing IL2ss over non-secreted mRNA. Intracranial injection of anti-Aß mRNA polyplexes with IL2ss resulted in 40 % Aß decrease in an acute amyloidosis model; with no decrease detected with control scFv mRNA nor DNA polyplexes. However, no Aß decrease was detected in a more challenging transgenic model of Alzheimer's disease. CONCLUSION: Our results introduce a concerted approach not only for Alzheimer's disease treatment but also for immunotherapy against neurological diseases. The effectivity of our platform required the intracranial delivery of anti-Aß scFv as mRNA not DNA, as mRNA with an IL2ss secretion sequence to favor engagement of Aß in the amyloidosis model, complexation with a smart copolymer for efficient transfection of primary neurons and to achieve detectable mRNA expression in the brain during 48h. Amyloid burden decrease in an acute amyloidosis model was only achieved when these three factors (mRNA coding scFv, smart copolymer, IL2ss) were integrated into a single formulation.

Novel Antibody for the Treatment of Transthyretin Amyloidosis.

Familial amyloidotic polyneuropathy (FAP) is a systemic amyloidosis mainly caused by amyloidogenic transthyretin (ATTR). This incurable disease causes death ∼10 years after onset. Although it has been widely accepted that conformational change of the monomeric form of transthyretin (TTR) is very important for amyloid formation and deposition in the organs, no effective therapy targeting this step is available. In this study, we generated a mouse monoclonal antibody, T24, that recognized the cryptic epitope of conformationally changed TTR. T24 inhibited TTR accumulation in FAP model rats, which expressed human ATTR V30M in various tissues and exhibited non-fibrillar deposits of ATTR in the gastrointestinal tracts. Additionally, humanized T24 (RT24) inhibited TTR fibrillation and promoted macrophage phagocytosis of aggregated TTR. This antibody did not recognize normal serum TTR functioning properly in the blood. These results demonstrate that RT24 would be an effective novel therapeutic antibody for FAP.

cis-3-Hexenol and trans-2-hexenal mixture prevents development of PTSD-like phenotype in rats.

Several green leaf volatiles have anxiolytic/antidepressant properties and attenuate adrenocortical stress response in rodents. However, it remains unknown whether a mixture of cis-3-hexenol and trans-2-hexenal so-called 'green odor (GO)' affects fear-associated post-traumatic stress disorder (PTSD)-like behavior. In the present study, fear memory of the initial conditioning stimulus was stably maintained by weekly presentation of conditioned tone. Examination of open field behavior, acoustic startle response, prepulse inhibition, and immobility in the forced swim test for 2 weeks after initial conditioning revealed that conditioned rats sustained anxiety, enhanced startle response, hypervigilance, depression-like behavior, and hypocortisolism, which is consistent with PTSD symptoms. Daily, not acute, GO presentation facilitated fear extinction and reduced PTSD-like behavioral and endocrinal responses. To further investigate the mechanism of effect of GO, we examined the effect of paroxetine (a selective serotonin reuptake inhibitor), p-chlorophenylalanine (PCPA, an irreversible serotonin synthesis inhibitor), alone or in combination of GO on PTSD-like phenotype. The alleviative effects of GO were masked by simultaneous paroxetine administration. PCPA-induced serotonin depletion abolished the effects of GO. Our results suggest that daily GO presentation facilitates fear extinction and prevents development of PTSD-like symptoms.

High-level expression and preparation of recombinant human fibrinogen as biopharmaceuticals.

Fibrinogen is a large and complex glycoprotein containing two sets of each of three different chains (α, ß and γ). There have been no reports of high-level expression of fibrinogen at commercial levels using mammalian cultured cells such as CHO cells because of the difficulty in highly expressing a protein with such a complex structure. We achieved high-level (1.3 g/l or higher) expression of recombinant human fibrinogen using CHO DG44 cells by optimizing the expression system and culture conditions. We also succeeded in establishing a high-recovery preparation method for recombinant fibrinogen that rarely yields degraded products. To characterize the properties of the recombinant human fibrinogen, we performed SDS-PAGE; western blotting of the α, ß and γ chains using specific antibodies and scanning electron microscopy observations of fibrin fibres. We also evaluated the functional equivalence between recombinant fibrinogen and plasma fibrinogen with respect to the release of fibrinopeptides initiated by thrombin and its cross-linking properties. The basic properties of recombinant fibrinogen showed no apparent differences from those of plasma fibrinogen. Here, we report the development of methods for the culture and preparation of recombinant human fibrinogen of satisfactory quality that can be scaled up to the commercial level.

Robust increase of microglia proliferation in the fornix of hippocampal axonal pathway after a single LPS stimulation.

Microglia are resident immunocompetent cells having important roles in innate immunity in the brains. In the present study, we found that a single lipopolysaccharide (LPS) administration significantly increased microglial proliferation in the fornix and dentate gyrus (DG) but not the cerebral cortex and corpus callosum of adult mice. LPS-induced microglial proliferation was especially robust at the white matter of the fornix. The density of microglia increased in the fornix and DG for roughly one week and returned to basal levels at least 20days after a single LPS administration. Consecutive LPS administration did not induce such dramatic increase of microglial proliferation in the fornix. The inhibition of vascular endothelial growth factor signaling by AZD2171 largely suppressed LPS-induced increase of microglial proliferation in the fornix. In conclusion, the present study indicates that the hippocampal neuronal system has a higher proliferative microglial capability against LPS-induced inflammatory administration compared with other brain regions.

High-yield preparation of recombinant human α-thrombin for therapeutic use.

In this study, we established stable cell lines producing 1.5 mg/mL recombinant human prothrombin in 400-L fed-batch culture, using CHO DG44 cells as a host cell line. And we also established a recombinant human α-thrombin purification process that produces a purified product suitable for use as a biopharmaceutical, by using recombinant ecarin from CHO DG44 cells, achieving a total yield of approximately 27% of prothrombin in culture medium. The establishment of stable cell lines with high expression levels, long-term passage stability and satisfactory scale-up are essential to ensure the stable supply of biopharmaceuticals. Furthermore, biopharmaceuticals must be of high quality to assure safety and effectiveness in target applications. We had previously reported that recombinant human prethrombin-2 expression level in a stable cell line established using the mouse myeloma cells, Sp2/0-Ag14, reached 200 µg/mL using animal-free materials in 50-L fed-batch culture. However, the productivity was insufficient to completely replace α-thrombin in human plasma preparations. By employing CHO DG44 cells as a host cell line, we had established a stable cell line and achieved significant improvements in quality, productivity of recombinant human α-thrombin manufacture suitable for use as a biopharmaceutical.

Astrocytic TLR4 expression and LPS-induced nuclear translocation of STAT3 in the sensory circumventricular organs of adult mouse brain.

The sensory circumventricular organs (CVOs) comprise the organum vasculosum of the lamina terminalis (OVLT), subfornical organ (SFO), and area postrema (AP) and lack the blood-brain barrier. The expression of Toll-like receptor 4 (TLR4) was seen at astrocytes throughout the sensory CVOs and at microglia in the AP and solitary nucleus around the central canal. The peripheral and central administration of lipopolysaccharide induced a similar pattern of nuclear translocation of STAT3. A microglia inhibitor minocycline largely suppressed lipopolysaccharide-induced astrocytic nuclear translocation of STAT3 in the OVLT and AP, but its effect was less in the SFO.

Order of exposure to pleasant and unpleasant odors affects autonomic nervous system response.

When mammals are exposed to an odor, that odor is expected to elicit a physiological response in the autonomic nervous system. An unpleasant aversive odor causes non-invasive stress, while a pleasant odor promotes healing and relaxation in mammals. We hypothesized that pleasant odors might reduce a stress response previously induced by an aversive predator odor. Rats were thus exposed to pleasant and unpleasant odors in different orders to determine whether the order of odor exposure had an effect on the physiological response in the autonomic nervous system. The first trial examined autonomic nerve activity via sympathetic and parasympathetic nerve response while the second trial examined body temperature response. Initial exposure to a pleasant odor elicited a positive response and secondary exposure to an unpleasant odor elicited a negative response, as expected. However, we found that while initial exposure to an unpleasant odor elicited a negative stress response, subsequent secondary exposure to a pleasant odor not only did not alleviate that negative response, but actually amplified it. These findings were consistent for both the autonomic nerve activity response trial and the body temperature response trial. The trial results suggest that exposure to specific odors does not necessarily result in the expected physiological response and that the specific order of exposure plays an important role. Our study should provide new insights into our understanding of the physiological response in the autonomic nervous system related to odor memory and discrimination and point to areas that require further research.

Identificaton of 2,3-dihydro-3,5-dihydroxy-6-methyl-4H-pyran-4-one isolated from Lactobacillus pentosus strain S-PT84 culture supernatants as a compound that stimulates autonomic nerve activities in rats.

Intestinal administration of various lactobacilli has been reported to affect autonomic neurotransmission, blood pressure, and body weight in rats. In this study, three molecules (peaks A, B, and C) were isolated from Lactobacillus pentosus strain S-PT84 (S-PT 84) culture supernatants. Intraduodenal (ID) injection of these molecules increased or inhibited renal sympathetic nerve activity (RSNA) in rats as follows: peak A, 134%; peak B, 40.1%; peak C, 408%. Furthermore, we identified peak C as 2,3-dihydro-3,5-dihydroxy-6-methyl-4H-pyran-4-one (DDMP). ID injection of DDMP increased brown adipose tissue sympathetic nerve activity (BAT-SNA; 118 ± 15.3%), whereas intraoral injection of DDMP increased the body temperature above the interscapular brown adipose tissue (BAT-T; 0.72 ± 0.13 °C) in rats. These data suggest that S-PT84 produces molecules that modulate autonomic nerve activity. In addition, DDMP increased BAT-SNA and BAT-T, and these changes in BAT-T may be caused by changes in BAT-SNA.

Effects of culture supernatant from Lactobacillus pentosus strain S-PT84 on autonomic nerve activity in rats.

Intestinal administration of various lactobacilli has been reported to affect autonomic neurotransmission, blood pressure, blood glucose, and body weight in rats, however, the mechanisms of action of the lactobacilli remain to be clarified. Therefore, the effect of the culture supernatant of Lactobacillus pentosus strain S-PT84 on the autonomic nerve activity in urethane-anesthetized rats was investigated. Intraduodenal injection of the low-molecular-weight (LMW) fraction (molecules less than 10,000 Da) of the S-PT84 culture supernatant elevated the brown adipose tissue sympathetic nerve activity and reduced the gastric vagal nerve activity. Moreover, intraoral administration of this LMW fraction increased the body temperature of rats above the interscapular brown adipose tissue. These results suggest that the LMW fraction of the S-PT84 culture supernatant affects the autonomic nerve activity and thermogenesis, and that the change in thermogenesis may be caused by the change in the sympathetic nerve activity of brown adipose tissue.

Antibody therapy for familial amyloidotic polyneuropathy.

Although it is believed that altered conformations exposing cryptic regions are intermediary and critical steps in the mechanism of transthyretin (TTR) amyloid formation, no effective therapy targeting this step is available. In this study, to establish the antibody therapy for familial amyloidotic polyneuropathy (FAP), we generated a monoclonal anti-TTR antibody, which specifically reacts with surface epitopes of TTR (MAb ATTR) and evaluated its binding affinity and specificity for TTR amyloid fibrils. MAb ATTR showed specific binding affinity for TTR amyloid fibrils, but not for native form of TTR. Moreover, MAb ATTR indeed showed the high consistency with Congo red positive areas in tissue specimens from FAP ATTR V30M patients, indicating that MAb ATTR showed binding affinity and specificity for TTR amyloid fibrils in vitro and in vivo. MAb ATTR may have a potential to suppress TTR amyloid deposition and become a candidate for the antibody therapy for FAP.

Direct evidence of generation and accumulation of ß-sheet-rich prion protein in scrapie-infected neuroblastoma cells with human IgG1 antibody specific for ß-form prion protein.

We prepared ß-sheet-rich recombinant full-length prion protein (ß-form PrP) (Jackson, G. S., Hosszu, L. L., Power, A., Hill, A. F., Kenney, J., Saibil, H., Craven, C. J., Waltho, J. P., Clarke, A. R., and Collinge, J. (1999) Science 283, 1935-1937). Using this ß-form PrP and a human single chain Fv-displaying phage library, we have established a human IgG1 antibody specific to ß-form but not α-form PrP, PRB7 IgG. When prion-infected ScN2a cells were cultured with PRB7 IgG, they generated and accumulated PRB7-binding granules in the cytoplasm with time, consequently becoming apoptotic cells bearing very large PRB7-bound aggregates. The SAF32 antibody recognizing the N-terminal octarepeat region of full-length PrP stained distinct granules in these cells as determined by confocal laser microscopy observation. When the accumulation of proteinase K-resistant PrP was examined in prion-infected ScN2a cells cultured in the presence of PRB7 IgG or SAF32, it was strongly inhibited by SAF32 but not at all by PRB7 IgG. Thus, we demonstrated direct evidence of the generation and accumulation of ß-sheet-rich PrP in ScN2a cells de novo. These results suggest first that PRB7-bound PrP is not responsible for the accumulation of ß-form PrP aggregates, which are rather an end product resulting in the triggering of apoptotic cell death, and second that SAF32-bound PrP lacking the PRB7-recognizing ß-form may represent so-called PrP(Sc) with prion propagation activity. PRB7 is the first human antibody specific to ß-form PrP and has become a powerful tool for the characterization of the biochemical nature of prion and its pathology.

Skin application of urea-containing cream affected cutaneous arterial sympathetic nerve activity, blood flow, and water evaporation.

BACKGROUND/PURPOSE: We observed that olfactory stimulation with scent of grapefruit oil elevated the activities of sympathetic nerves, and increased the plasma glycerol concentration and blood pressure. In contrast, olfactory stimulation with scent of lavender oil had opposite effects in rats. These suggest that changes in autonomic activities cause physiological functions via histaminergic H1 and H3 receptor. Moreover, it has been reported that somatic sensory stimulation affected autonomic neurotransmission. To examine effects of skin application of urea-containing cream on cutaneous arterial sympathetic nerve activity (CASNA), blood flow, and transepidermal water loss (TEWL). METHOD: The activity of CASNA was determined by electrophysiological method, and cutaneous blood flow was determined using laser flowmeter in urethane-anesthetized rats, TEWL was measured using VapoMeter in the back skin of HWY hairless rats. RESULTS: CASNA was markedly and significantly inhibited by skin application of 10% urea-containing cream, whereas cutaneous blood flow was significantly elevated via histaminergic H3-receptor. In conscious hairless rats, TEWL was significantly decreased 24 h after application of 10% urea-containing cream to the back skin. CONCLUSION: These findings suggest that skin application of 10% urea-containing cream increases the cutaneous blood flow and water retaining ability, and that histaminergic H3-receptors may mediate these effects.

Localization of plasminogen in mouse hippocampus, cerebral cortex, and hypothalamus.

Although the tissue plasminogen activator/plasminogen system contributes to numerous brain functions, such as learning, memory, and anxiety behavior, little attention has as yet been given to the localization of plasminogen in the brain. We have investigated the localization of plasminogen in the adult mouse brain by using immunohistochemistry. In the hippocampus, plasminogen immunoreactivity was seen in the pyramidal cell layer as numerous punctate structures in neuronal somata. An electron-microscopic study further demonstrated that the plasminogen-immunoreactive punctate structures represented secretory vesicles and/or vesicle clusters. In the cerebral cortex, plasminogen immunoreactivity was evident in the somata of the layer II/III and V neurons. A quantitative analysis revealed that parvalbumin (PV)-positive neurons had more plasminogen-immunoreactive puncta compared with those of PV-negative neurons in the hippocampus and cerebral cortex. Plasminogen immunoreactivity was present throughout the hypothalamus, being particularly prominent in the neuronal somata of the organum vasculosum laminae terminalis, ventromedial preoptic nucleus, supraoptic nucleus, subfornical organ, medial part of the paraventricular nucleus (PVN), posterior part of the PVN, and arcuate hypothalamic nucleus. Thus, plasminogen is highly expressed in specific populations of hippocampal, cortical, and hypothalamic neurons, and plasminogen-containing vesicles are mainly observed at neuronal somata.

Base of molecular mimicry between human ribosomal protein S19 dimer and human C5a anaphylatoxin.

The crosslinked homodimer of human ribosomal protein S19 (hRP S19) but not hRP S19 monomer shares the hC5a receptor ligation capacity with anaphylatoxin hC5a. The hRP S19 dimer engages hC5a receptor-bearing monocytes in chemotactic movement and secretion as does hC5a. Two submolecular regions essential for the receptor ligation were already identified in hRP S19 as well as in hC5a. Using the tertiary structure data base of an archaeobacterial RP S19 as template, we made a tertiary structure model of hRP S19. The obtained structure was almost entirely α-helical with two short ß-sheet regions, and folds a five α-helix bundle organized around a central amphipathic α-helix. While the secondary structure components were similar to those of hC5a, the gross tertiary structure of hRP S19 was loose and the distance between the two receptor binding regions was rather big in comparison to that of hC5a. Anti-recombinant hC5a rabbit antibodies cross-recognized not only the crosslinked hRP S19 dimer but also the guinea pig (gp) RP S19 dimer, however, these antibodies reacted hRP S19 monomer and crosslinked Gln137Asn-hRP S19 mutant dimer at significantly less extents. These antibodies neutralized the monocyte attracting capacity of the hRP S19 dimer in vitro and that of the gpRP S19 dimer in vivo. We assume that the crosslinkage between Lys122 of one hRP S19 molecule and Gln137 of the other one would assemble the hC5a-like structure probably providing one of two receptor binding regions by each hRP S19 subunit.

Characterization of human single-chain antibodies against highly pathogenic avian influenza H5N1 viruses: mimotope and neutralizing activity.

The development of new therapeutic targets and strategies to control highly pathogenic avian influenza (HPAI) H5N1 virus infection in humans is urgently needed. Neutralizing recombinant human antibodies would provide important agents for immunotherapy on human H5N1 virus infection and definition of the critical mimotope for vaccine development. In this study, we have characterized an anti-H5-specific scFv clone, 3D1 from the human-scFv-displaying phage library. 3D1 blocked the binding of H5-Fc to MDCK cells in flow cytometry and neutralized H5N1 subtype influenza A viruses in a microneutralization assay. Employing a peptide-displaying phage library, Ph.D-12, the mimotope was determined to be at #128-131 and #204-211 of H5, which are silic acid-binding regions. In consistency with this result, 3D1 binds the recombinant sugar-binding domain (#50G-#272E) produced by a baculovirus vector. The 3D1 antibody employs the germline gene VH1-23. As this antibody is the first human anti-H5 scFv clearly defined on the sugar-binding epitope, it allows us to investigate the influence of amino acid substitutions in this region on the determination of the binding specificity to either sialic acid α2,6-galactose (SA α2,6Gal) or sialic acid α2,3-galactose (SA α2,3Gal) providing new insight for the development of effective H5N1 pandemic vaccines.

Exposure to TMT odor affects adrenal sympathetic nerve activity and behavioral consequences in rats.

The odor of 2,5-dihydro-2,4,5-trimethylthiazoline (TMT), a synthetic compound isolated from fox feces, induces various emotional behavioral and stress responses. Here we examined the effect of TMT on behavioral responses and adrenal sympathetic nerve activity (ASNA) in rats. TMT increased freezing behavior, defensive-burying and defensive-attack, and decreased exploration, grooming and approach behaviors. On the other hand, butyric acid (BA), a pungent but non-predatory odor, increased defensive-burying only. TMT increased ASNA strongly, whereas the effects of BA increased ASNA extremely weakly. Furthermore, pre-treatment with the histaminergic H1-receptor antagonist diphenhydramine eliminated the effects of TMT on ASNA. These findings suggest that TMT odor affects autonomic neurotransmission via histaminergic neurons. Exposure to TMT odor likely regulates the controlling autonomic function and output to a motor system simultaneously, evoking behavioral stress responses.

Contribution of IL-18 to eosinophilic airway inflammation induced by immunization and challenge with Staphylococcus aureus proteins.

We previously reported that intranasal challenge with ovalbumin (OVA) plus IL-18 induces airway hyperresponsiveness (AHR) and eosinophilic airway inflammation in mice with OVA-specific T(h)1 cells. These two conditions can be prevented by neutralizing anti-IFN-gamma and anti-IL-13 antibodies, respectively. The mice develop AHR and eosinophilic airway inflammation after challenge with OVA plus LPS instead of IL-18 and endogenous IL-18 is known to be involved. In contrast, IL-18 does not facilitate these changes in mice possessing OVA-specific T(h)2 cells. Here, we investigated whether IL-18 is involved in the development of asthma in mice immunized and challenged with bacterial proteins. Upon intranasal exposure to protein A (SpA) derived from Staphylococcus aureus, mice immunized with SpA exhibited AHR and peribronchial eosinophilic inflammation if IFN-gamma or IL-13 were present, respectively. The CD4(+) T cells from draining lymph nodes (DLNs) of the SpA-immunized and -challenged mice produced a robust IFN-gamma and IL-13 in response to immobilized anti-CD3 antibodies. Treatment with neutralizing anti-IL-18 antibodies prevented asthmatic inflammation concomitant with their impaired potential to express IFN-gamma and IL-13. Furthermore, naive mice that received the CD4(+) T cells from DLNs of SpA-immunized mice developed airway inflammation depending upon the presence of IL-18. Immunodeficient mice that received human PBMCs, which had been stimulated with SpA in vitro, developed dense peribronchial accumulation of human CD4(+) T cells upon SpA challenge. Neutralizing anti-human IL-18 antibodies protected against this airway inflammation. These results suggest the importance of IL-18 for the development of asthmatic inflammation associated with airway exposure to bacterial proteins.

Contribution of the flexible loop region to the function of staphylococcal enterotoxin B.

Staphylococcal enterotoxin B (SEB), a toxin produced by Staphylococcus aureus, causes food poisoning and other fatal diseases by inducing high levels of pro-inflammatory cytokines. These cytokines are released from CD4+ T cells and major histocompatibility complex (MHC) class II antigen-presenting cells, which are activated through binding of wild-type (WT) SEB to both the MHC class II molecule and specific T-cell receptor Vbeta chains. Here, we focused on a trypsin/cathepsin cleavage site of WT SEB, which is known to be cleaved in vivo between Lys97 and Lys98, located within the loop region. To know the function of the cleavage, an SEB mutant, in which both of these Lys residues have been changed to Ser, was examined. This mutant showed prolonged tolerance to protease cleavage at a different site between Thr107 and Asp108, and structural analyses revealed no major conformational differences between WT SEB and the mutant protein. However, differential scanning calorimetric analysis showed an increase in enthalpy upon thermal denaturation of the mutant protein, which correlated with the speed of cleavage between Thr107 and Asp108. The mutant protein also had slightly increased affinity for MHC. In the in vivo experiment, the SEB mutant showed lower proliferative response in peripheral blood mononuclear cells and had lower cytokine-induction activity, compared with WT SEB. These results highlight the importance of the flexible loop region for the functional, physical and chemical properties of WT SEB, thus providing insight into the nature of WT SEB that was unrevealed previously.