novel antibody for the treatment of transthyretin …acidic treatment (fig. 5c), similar to that...

Novel Antibody for the Treatment of TransthyretinAmyloidosis*

Received for publication, May 13, 2016, and in revised form, October 5, 2016 Published, JBC Papers in Press, October 7, 2016, DOI 10.1074/jbc.M116.738138

Akihiko Hosoi‡1, Yu Su§1, Masaharu Torikai‡, Hirofumi Jono¶�, Daisuke Ishikawa‡, Kenji Soejima‡, Hirofumi Higuchi‡,Jianying Guo§, Mitsuharu Ueda§, Genki Suenaga§, Hiroaki Motokawa§, Tokunori Ikeda**, Satoru Senju**,Toshihiro Nakashima‡2 , and Yukio Ando§3

From the ‡Chemo-Sero-Therapeutic Research Institute (KAKETSUKEN), 1314-1 Kyokushi Kawabe Kikuchi Kumamoto, 869-1298, theDepartments of §Neurology and **Immunogenetics, Graduate School of Medical Sciences, and the ¶Department of ClinicalPharmaceutical Sciences, Graduate School of Pharmaceutical Sciences, Kumamoto University, and the �Department of Pharmacy,Kumamoto University Hospital, 1-1-1 Honjo, Chuo-ku, Kumamoto 860-8556, Japan

Edited by Paul Fraser

Familial amyloidotic polyneuropathy (FAP) is a systemicamyloidosis mainly caused by amyloidogenic transthyretin(ATTR). This incurable disease causes death �10 years afteronset. Although it has been widely accepted that conformationalchange of the monomeric form of transthyretin (TTR) is veryimportant 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 recog-nized the cryptic epitope of conformationally changed TTR.T24 inhibited TTR accumulation in FAP model rats, whichexpressed human ATTR V30M in various tissues and exhibitednon-fibrillar deposits of ATTR in the gastrointestinal tracts.Additionally, humanized T24 (RT24) inhibited TTR fibrillationand promoted macrophage phagocytosis of aggregated TTR.This antibody did not recognize normal serum TTR functioningproperly in the blood. These results demonstrate that RT24would be an effective novel therapeutic antibody for FAP.

Amyloidosis is a group of diseases causing functional disor-ders because of deposits of fibrillated protein in various tissuesand includes Alzheimer’s and prion disease (1). Familial amy-loidotic polyneuropathy (FAP)4 is an autosomal dominant

hereditary amyloidosis that primarily occurs as a result ofmutation of the transthyretin (TTR) gene (2). FAP is caused bythe accumulation of TTR amyloid around the heart, kidneys,gastrointestinal tract, eyes, and peripheral nerves (2). This dis-ease generally appears during middle age and exhibits dysfunc-tion in the affected areas. FAP causes death �10 years afteronset (2). Thus far, more than 130 mutations have beenreported in the TTR gene (3). Val-30 mutated to Met (V30M)TTR is the most common mutation (4). Patients with amy-loidogenic TTR (ATTR) V30M FAP have mainly been reportedin Portugal, Sweden, and Japan (4).

Native TTR behaves as a tetramer comprising four identicalsubunits, each of which contains 127 amino acid residues andhas a molecular mass of 14 kDa (5). Each monomer containseight �-strands, which are formed by antiparallel four-strand�-sheets (5). This protein binds to the retinol-binding proteinand thyroxine in the blood (5). TTR is primarily produced in theliver and choroid plexus. Although blood levels can be as high as0.25 g/liter, its half-life is only 2 days (2, 4, 5). Dissociation of thetetramer into monomers and conformational change of themonomers are very important steps for TTR fibrillization (Fig.1) (6, 7). It is suggested that the rate-limiting step may accountfor dissociation into monomers (8). However, all toxic speciesof TTR have not been identified, and therapeutic approachesfor FAP targeting various species of TTR have yet to be estab-lished (3).

Liver transplantation (LT) is the most common treatment forFAP. By disconnecting the supply of mutant TTR from the liver,LT suppresses the progression of symptoms (2). Althoughpathology progression delay is observed after LT, deposits ofTTR to organs, including the eyes and heart, may still progress;there have been many reports of progression of symptoms tothese organs (9, 10). Recently, “TTR stabilizers” have beenapproved as therapeutic drugs (3). These drugs kinetically sta-bilize TTR and are expected to inhibit the dissociation oftetramers into monomers of TTR. Tafamidis was approved inthe European Union and Japan (11), and clinical trials of difl-unisal have been conducted worldwide (3). These TTR stabiliz-ers delay peripheral neuropathy but cannot completely sup-press pathology progression. To completely suppress pathologyprogression, it is necessary to suppress TTR fibrillization and

* This work was supported in part by the Adaptable and Seamless TechnologyTransfer Program through Target-driven R&D, Japan Science and TechnologyAgency, (AS2311336G), grants from the Surveys and Research on Specific Dis-ease, the Ministry of Health and Welfare of Japan, Grants-in-Aid for ScientificResearch from the Ministry of Education, Sports, Science and Technology ofJapan: (A) 24249036 and (B) 15H04841, and Challenging Exploratory Research15K15195 (to Y. A.) and 15K15006 (to H. J.). The authors declare that they haveno conflicts of interest with the contents of this article.

1 Both authors contributed equally to this work.2 To whom correspondence may be addressed: The Chemo-Sero-Therapeutic

Research Institute (KAKETSUKEN), 1314-1 Kyokushi Kawabe KikuchiKumamoto, 869-1298, Japan. Tel.: 81-968-37-3100; Fax: 81-968-37-3616;E-mail: [email protected].

3 To whom correspondence may be addressed: Dept. of Neurology, Grad-uate School of Medical Sciences, Kumamoto University, 1-1-1 Honjo,Chuo-ku, Kumamoto 860-8556, Japan. Tel./Fax: 81-96-373-5686; E-mail:[email protected].

4 The abbreviations used are: FAP, familial amyloidotic polyneuropathy;ATTR, amyloidogenic transthyretin; IPTG, isopropyl �-D-1-thiogalactopyra-noside; TTR, transthyretin; RT24, humanized T24; LT, liver transplantation;CT24, chimera T24 antibody; A�, amyloid �; SPR, surface plasmon reso-nance; KLH, keyhole limpet hemocyanin; ML, macrophage line; CDR, com-plementarity determining region; Tg, transgenic.

crossmarkTHE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 291, NO. 48, pp. 25096 –25105, November 25, 2016

© 2016 by The American Society for Biochemistry and Molecular Biology, Inc. Published in the U.S.A.

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also to remove the amyloid deposits. Several therapeutic drugs,such as siRNA, which target the wild type (WT) TTR RNA (12),are currently undergoing clinical development, but their effecton TTR deposits in organs is unclear.

It is known that during TTR fibrillization, new epitopes(cryptic epitopes) are exposed on the molecular surface with aconformational change of TTR (13). It has been reported thatby immunizing FAP model mice with a mutant TTR that hasexposed cryptic epitopes, antibodies that recognize the crypticepitopes of TTR are generated, and these antibodies can reducethe deposition of TTR amyloids (14, 15). Likewise, Gustavssonet al. (16) reported that when they prepared 10 anti-peptidesera (antisera) of TTR, only antisera against the TTR(115–124)region strongly reacted with the amyloids in FAP patients anddid not react to serum TTR or normal (non-FAP) pancreas.Furthermore, analysis of the steric structures surroundingTTR114 has been reported to show that they are located in theintermolecular interaction interface when in the tetramer state(17). From these reports, we consider the TTR(115–124) regionto be a “cryptic epitope” that is hidden from the protein surfacewhen in the tetramer form and is exposed on the surface whenit changes into the monomer form. Bergstrom et al. (18) gener-ated rabbit polyclonal antibodies against residues 115–124 ofthe human TTR, a cryptic epitope. When these antibodies wereadministered to FAP model rats (19), TTR deposits in the intes-tinal tract were significantly reduced (20). The above suggeststhat the antibody recognizes the cryptic epitope of TTR, inter-acts with conformationally changed TTR and TTR amyloid,may inhibit fibrillization, and remove the TTR amyloids.Therefore, the antibody that recognizes the cryptic epitope ofTTR may have the potential to become a novel therapeutic drugfor FAP.

In this study, with the aim of developing a new therapeuticdrug for FAP, we report on the novel monoclonal antibody T24,which recognizes the residues 115–124 (cryptic epitope) ofTTR, and we introduce the biological activities and efficacies ofT24 and humanized T24 (RT24) antibodies.

Results

T24 Recognized 118-122 Region of Human TTR—The anti-gen binding activity of the antibodies of the obtained hybrido-mas was evaluated by ELISA, and an excellent binding activityto the TTR(115–124) peptides was found in hybridoma cloneT24. To completely analyze the epitope of the T24 antibody, thereactivity analysis of T24 was performed using the alanine sub-

stitution variant of the TTR(115–124) region. Coomassie Bril-liant Blue stain analysis and Western blotting analysis, usingT24 antibody, alanine substitution mutants from residues 114to 124 (except for Y114C, A120S, and V122I, which were muta-tions reported in patients) of the human TTR expressed inEscherichia coli and the supernatants from disrupted cell pelletswhen applied to SDS-PAGE are shown in Fig. 2, A and B (West-ern blotting analysis), respectively. Reactivity of T24 antibodywas reduced or eliminated in the TTR mutant from residues118 to 122; therefore, the epitope of T24 was thought to beTTR(118 –122).

T24 Specifically Recognized TTR Amyloid in FAP Patients—To clarify the binding specificity of T24, we analyzed bindingreactivity to human blood and amyloids in FAP patients usingELISA, Western blotting, and immunohistological staining.

In ELISA, T24 specifically reacted with the TTR(115–124)peptide and acid-treated WT TTR fibrils from the humanserum (Fig. 3A). Conversely, this antibody did not react with thehuman serum from healthy volunteers or V30M TTR FAPpatients. In Western blotting, the anti-prealbumin antibodyreacted with the TTR monomer and some of the proteins in thehuman serum, but T24 did not react with the human serumfrom healthy volunteers or V30M TTR FAP patients (Fig. 3B).Additionally, we performed Western blotting to reveal the T24specificity against TTR amyloids extracted from the kidney andheart of FAP patients. T24 specifically recognized TTR amy-loids from the kidney (Fig. 3C). In immunohistochemical stain-ing using paraffin and frozen sections, T24 specifically reactedwith amyloids in the heart (Fig. 4, A and G) and amyloids in thekidney (Fig. 4, M and S) from FAP V30M TTR patients. Theseareas showed high consistency with positive areas of anti-pre-albumin antibody (Fig. 4, B, H, N, and T), Congo red stain (Fig.4, C, I, O, and U), and Congo red polarized light (Fig. 4, D, J, P,and V). There were no specific immunoreactivities using theisotype control (Fig. 4, E, K, Q, and W) and secondary antibodyalone (Fig. 4, F, L, R, and X).

RT24 Antibody Showed Reactivity Similar to T24 —Next, weperformed the humanization of T24 murine antibody. The Vregion gene of T24 was isolated by T24-producing hybridoma,and T24 was humanized by CDR grafting as described under“Experimental Procedures.” To clarify the binding specificity ofRT24, we analyzed binding reactivity using ELISA. Accordingto ELISA, RT24 specifically recognized the immunized antigen(TTR(115–124) peptide) (Fig. 5A) and the TTR fibril formed by

FIGURE 1. Pathway of TTR fibrillization. Native TTR behaves as a tetramer in the blood. The blood of heterologous patients with both WT TTR and TTR mutantproteins contains five tetramer patterns. If the TTR mutants are included in a tetramer, the tetramer easily dissociates into monomers compared with the WTTTR tetramer. It is considered that TTR monomers cause conformational change, the fibrillization step gradually progresses via oligomer state, and then TTRamyloids deposit in the various organs such as peripheral nerves. Dissociation into monomers and conformational change of the monomers are very crucialsteps for TTR fibrillization.

RT24, a Therapeutic Antibody for FAP

NOVEMBER 25, 2016 • VOLUME 291 • NUMBER 48 JOURNAL OF BIOLOGICAL CHEMISTRY 25097


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acidic treatment (Fig. 5C), similar to that with chimera T24antibody (CT24). A very weak reaction against WT TTR (Fig.5D) and no response in the absence of the peptide or recombi-nant TTR (Fig. 5, B and E) were observed. It was found thatRT24 and T24 have the same binding activity.

RT24 Specifically Interacted with Recombinant TTR-derivedFibrils but Not WT TTR Tetramer—We analyzed whetherRT24 recognizes various TTR fibrils, which has been reportedin patients, using recombinant TTR-derived fibrils. Conse-quently, it was found that RT24 recognized various TTR fibrils,which were reported in patients but not purified native (non-aggregated) WT TTR (Fig. 6B).

RT24 Specifically Interacted with Conformationally ChangedTTR but Not with the TTR Tetramer in Liquid Phase—To clar-ify the binding specificity of liquid-phase system, we analyzedbinding activity using the SPR method. RT24 specifically inter-acted with the WT TTR fibril and V30M TTR fibril formed byacidic treatment, and no interactions were observed withrecombinant WT TTR and V30M TTR (Fig. 7). Isotype con-

trols had no interaction. It was found that RT24 specificallyrecognized conformationally changed TTR in the liquid phase.

RT24 Inhibited V30M TTR Fibrillization—A fibrillizationinhibition assay was performed to evaluate whether RT24inhibits V30M TTR fibrillization. We found that RT24 signifi-cantly inhibited V30M TTR fibrillization in an antibodyconcentration-dependent manner (Fig. 8) and that IC50 ofRT24 is �150 nM. However, SPR analysis shows that the disso-ciation constant (KD) of RT24 is 300 – 420 nM (data not shown),so this result for the evaluation of RT24’s activity is reasonable.

RT24 Promoted Macrophage Phagocytosis of the TTR Fibril—The macrophage phagocytic ability test was performed toinvestigate whether RT24 antibody promotes the ability ofmacrophage to phagocytose the TTR fibril. This test mimics theprocess wherein macrophages remove TTRs deposited in the

FIGURE 2. T24 recognized the TTR residue in the 118 –122 region. A, SDS-PAGE analysis of recombinant TTR mutants. E. coli strain M15 was transfectedwith the recombinant TTR mutant from residue 114 to 124 expression vectorsand cultured. After IPTG induction of recombinant TTR expression, the cellfraction was electrophoresed under reducing conditions on 8 –16% SDS-polyacrylamide gel, and the gel was stained by Coomassie Brilliant Blue. B,reactivity of T24 against recombinant TTR mutants. The solubilized cell sus-pension was subjected to SDS-PAGE, followed by Western blotting (WB) anal-ysis using T24 antibody. E. coli, M15 without TTR expression vector, Di, TTRdimer; Mo, TTR monomer.

FIGURE 3. T24 antibody specifically reacted with TTR amyloid, but notserum TTR, from FAP patients. A, T24 reactivity was assessed by ELISA.Human serum-derived WT-TTR fibril by acidic treatment (black circle), BSA-conjugated TTR peptide (white circle), serum from a healthy volunteer (blacksquare), and serum from a V30M FAP patient (white square) were immobilizedon an immunoplate, and the reactivity of T24 against each antigen was ana-lyzed by ELISA. B and C, reactivity of T24 was assessed by Western blotting(WB). B, serum from a healthy volunteer or V30M FAP patient was applied toSDS-PAGE under non-reducing conditions and then analyzed by Westernblotting with polyclonal anti-prealbumin or T24 antibody. C, serum orextracted amyloids from the heart and kidney from a V30M FAP patient wereapplied to SDS-PAGE, and then analyzed by Western blotting with T24antibody.




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FIGURE 4. T24 antibody recognized TTR amyloids in the kidney and heart from FAP patients. T24 reactivity was evaluated by immunohistochemicalanalysis. Paraffin sections (A–F and M–R) or frozen sections (G–L and S–X) of the kidney glomerulus tissue (A–L) or the heart tissue (M–X) from a V30M FAP patient(67-year-old man) were prepared. After Congo red and hematoxylin staining (C, I, O, and U), the presence of apple-green birefringence under polarized lightconfirmed the presence of amyloid deposits (D, J, P, and V). For immunohistochemical staining, paraffin-embedded and frozen slides were treated with periodicacid, followed by immunostaining using T24 antibody (A, G, M, and S), anti-prealbumin antibody (B, H, N, and T), isotype control (E, K, Q, and W), or no primaryantibody (secondary antibody only) (F, L, R, and X). Areas that are stained brown are deposits of TTR amyloids. Bars, 200 �m.




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tissues of TTR patients. RT24 specifically promoted phagocy-tosis of the TTR fibril (Fig. 9A). No phagocytic activity changeswere observed against native (non-aggregated) TTR (Fig. 9B).Thus, RT24 promotes the phagocytic activity of macrophagestoward the TTR fibril.

T24 Inhibited TTR Deposits in FAP Model Rats—Using FAPmodel rats, we performed an efficacy evaluation test to testwhether T24 suppresses the deposition of TTR amyloid. Com-paring the stained images after administration of T24 or PBS,the group administered with T24 (T24 group, Fig. 10A) showedgreater reduction of TTR-positive areas than that administeredPBS (PBS group, Fig. 10B). Semi-quantitatively, the T24 grouptended to have a greater reduction in the amount of TTR depos-its than in the PBS group (Fig. 10C). Sera were collected once aweek, and analysis of blood biochemistry was performed. Noabnormal values were observed. Long-term administration ofT24 did not affect renal, liver, or pancreatic function (Table 1).

Discussion

On the basis of the concept that an antibody that specificallyrecognizes the cryptic epitope of TTR could be a novel thera-peutic drug of FAP, we generated monoclonal antibody T24,which recognized residues 118 –122 of the human TTR. Thisantibody recognizes various TTR species from conformation-ally changed monomers to fibrils. Humanized antibody, RT24,also has the same specificity with T24.

These antibodies have the following characteristics. First,RT24 inhibited V30M TTR fibrillization in an antibodyconcentration-dependent manner (Fig. 8). This activity is veryimportant for preventing TTR from being deposited in the tis-sue. Second, RT24’s activity promotes the phagocytic capacityagainst the TTR fibril (Fig. 9). This activity is thought to beeffective in removing TTR amyloids deposited in varioustissues. Third, T24 specifically recognized TTR amyloidsextracted from patients and did not react with the serum fromFAP patients (Fig. 3). This specificity is expected to be effectivein the serum and cause few side effects. Finally, T24 tended toreduce TTR deposits in FAP model rats (Fig. 10). For the effi-cacy study, we performed long-term administration (6 months)of the T24 antibody, which is assumed to have less immunoge-nicity, instead of RT24. During the administration period, theincrease in anti-T24 antibodies and abnormal biochemical val-ues were not observed (Table 1 and data not shown). Therefore,the long-term administration of T24 is assumed to be safe. Inthis study, a difference in TTR deposits in the muscle layer ofthe large intestine was observed between the groups that wereadministered T24 and PBS; however, it was not statistically sig-nificant (Fig. 10C). This result may be due to individual differ-ences in the amount of TTR deposits in FAP model rats.

FIGURE 5. RT24 antibody showed reactivity similar to T24. Reactivity ofRT24 was assessed by ELISA. A and B, biotin-conjugated TTR(115–124) pep-tide (A) or no peptide (B) immobilized to the streptavidin-coated plate. C–E,V30M TTR fibril formed by acidic treatment (C), purified recombinant WT TTR(D), and no TTR (E) immobilized to the nickel-chelate plate. The reactivity ofCT24 (solid line) or RT24 (broken line) against each antigen was analyzed byELISA. FIGURE 6. RT24 specifically interacted with the TTR fibrils that have been

reported in patients. Native-PAGE analysis using TTR fibrils that have beenreported in patients. Purified recombinant WT TTR or purified recombinantWT/mutant TTR fibrils treated at pH 3.0 were applied onto 4% native-poly-acrylamide gel, and the gel was stained by silver staining (A), followed byWestern blotting (WB) using RT24 antibody (B).




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Although TTR deposits increase until 3–9 months, there arelarge differences between individuals (data not shown). Theeffects of T24 may be masked by these individual differences.

TTR antibodies generated using the same concept have beenreported by Goldsteins et al. (13) and Phay et al. (21). Gold-steins et al. (13) generated mAb39-44 and mAb56-61 antibod-ies that reacted with TTR, which caused conformationalchanges, and these antibodies also recognized the V30M TTRamyloid. However, these antibodies did not inhibit TTR fibril-

FIGURE 8. RT24 inhibits V30M TTR fibrillization. Fibrillization inhibitionassay was performed. RT24 (solid line) or isotype control antibody (broken line)were mixed with recombinant purified V30M TTR protein in PBS containing0.1% sodium deoxycholate and incubated at 37 °C for 3 days (n � 3). Thefibrillization level was measured by thioflavinT assay. Data are presented asmean � S.D. Student’s t test was used for evaluation. A p value less than 0.05(*) was considered statistically significant.

FIGURE 7. RT24 recognizes the TTR fibril but not the TTR tetramer in aliquid-phase system. RT24 specificity in a liquid-phase system was analyzedby SPR analysis. A, purified recombinant WT TTR (solid line) and WT TTR fibriltreated at pH 3.0 (broken line) were immobilized on a sensor chip. RT24 andisotype control (inset) were injected during the association phase for 2 min.The dissociation phase was performed over a period of 60 min. B, purifiedrecombinant V30M TTR (solid line) and V30M TTR fibril treated at pH 3.0 (bro-ken line) were immobilized on a sensor chip. Subsequent operations wereperformed in the same manner as A. Ab, antibody.

FIGURE 9. RT24 promotes macrophage phagocytosis against aggregatedTTR. A macrophage phagocytic ability test was performed to investigate theclearance via antibodies against misfolded TTR. Aggregated TTR were gener-ated from native V30M TTR by incubating for 24 h at 37 °C under acidic con-ditions (pH 4.0). Aggregated TTR (A) or native TTR (B) or were co-cultured withiPS-MLs in medium containing antibodies (RT24, isotype control, or none, n �3). After incubation for 2 days, reduction of TTR in the medium was evaluatedby ELISA using anti-prealbumin antibody. Data are presented as mean � S.D.Student’s t test was used for evaluation. A p value less than 0.05 (*) was con-sidered statistically significant.

FIGURE 10. T24 inhibits TTR deposition in FAP model rats. Using FAPmodel rats, we performed efficacy evaluation to test whether T24 suppressesthe deposition of TTR amyloid. TTR Tg rats received intravenous administra-tion of T24 or PBS once a week for 26 weeks. Paraffin sections of rat colonswere stained using anti-prealbumin antibody; the amount of TTR deposits inthe muscle layer of the large intestine was detected and compared betweenthe groups (group T24, n � 8, and group PBS, n � 7). A and B, representativeimages of paraffin sections of group T24 (A) or PBS (B). Stained areas in brownindicate deposits of TTR fibril in the muscle layer. C, stained areas were quan-titated as the amount of TTR accumulation for each individual. The averagevalue of the group PBS was regarded as “1,” and the distribution of the relativevalue was displayed by a boxplot.




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lization, and Goldsteins et al. (13) only mentioned the possibil-ity of utilizing these antibodies for diagnosis. Phay et al. (21)generated mouse monoclonal antibody 2T5C9, which specifi-cally recognized TTR amyloids from FAP patients by ELISAand immunohistochemical analysis. However, the biologicalactivities of this antibody were not reported, and Phay et al. (21)only mentioned the possibility of utilizing this antibody fordiagnosis. Therefore, no antibody has been reported that spe-cifically recognizes conformationally changed TTR but doesnot recognize normal serum TTR functioning properly in theblood, that shows biological activities in vitro and in vivo, andthat is suitable for administration to humans.

Antibodies against amyloidosis have been actively devel-oped. In particular, knowledge regarding the treatment of Alz-heimer’s disease is beginning to expand (22). In Alzheimer’sdisease, it has been reported that removing the amyloid � (A�)fibrils deposited in the brain does not lead to an improvementin the clinical condition. Therefore, it has been suggested thatA� oligomers and not A� fibrils are the pathogenic molecules(23). For treatment, we need to target all A� intermediates fromthe conformationally changed monomer to the A� fibril (22). InTTR amyloidosis, although the expression of ATTR was inhib-ited by LT, TTR deposition progressed, and there were manycases of the disease being exacerbated (9, 10). Even if both dis-eases are recognized as amyloidosis, the effects of Alzheimer’sdisease and TTR amyloidosis on the organs may be different.For the treatment of FAP, we may need to both inhibit TTR

fibrillation and remove amyloid deposits. Thus, RT24, whichtargets conformationally changed TTR via fibrils and TTRamyloids, is expected to not only inhibit deposition of mutatedTTR but also remove amyloid deposits (Fig. 10); therefore, thisantibody has the potential to be a novel therapeutic drug forFAP.

RT24 recognized various recombinant TTR (D18G, E54K,L55P, Y114C, Y116S, and V122I) amyloids that have beenreported in patients (Fig. 6). This result suggests that RT24 maywork effectively against patients with mutations other thanV30M ATTR. Because this antibody reacted with conforma-tionally changed WT TTR (Fig. 6B), administration of RT24may be effective for senile systemic amyloidosis, which iscaused by the deposition of WT TTR amyloids and associatedwith aging (10). With regard to the TTR depositions in theheart, T24 did not detect TTR amyloids extracted from heartsobtained from FAP patients (Fig. 3C); however, specific T24immunoreactivities were observed in the heart amyloids from aFAP V30M TTR patient using immunohistochemical stainingof heart sections (Fig. 4, M and S). For this reason, it is assumedthat T24 may also recognize TTR deposits in heart tissue. There-fore, it is expected that RT24 works effectively against patientswith various TTR mutations, and this new approach has thepotential to be adapted for treatment of senile systemicamyloidosis.

Another interesting finding in this study was that we estab-lished fibrillization inhibition assay under neutral conditions.Until now, fibrillization inhibition assays have generally beenperformed in the screening stage of antibody drugs for thetreatment of amyloidosis. With respect to the V30M TTR fibril-lization assay, there are some examples that have been appliedto small molecules (24), but no examples have been applied toantibodies. This is probably because acidic conditions arerequired to fibrillize V30M TTR in a short time, and this pro-cess denatures antibodies. We treated V30M TTR by addingsodium deoxycholate to PBS and observed V30M TTR fibrilli-zation in a short time under neutral conditions, without dena-turation of antibodies. We previously confirmed that V30MTTR fibril formation kinetically continues in the presence ofPBS containing sodium deoxycholate (data not shown). There-fore, using the V30M TTR fibrillization assay, the activity ofantibodies could be evaluated (Fig. 8). To the best of our knowl-edge, this is the first report to perform fibrillization inhibitionassay under neutral conditions.

Various approaches have been taken in the research anddevelopment of treatments for FAP, and several therapeuticdrugs are expected to be approved in the near future. Amongthem, antibody therapy has a unique mechanism and high spec-ificity and could meet the medical needs as a novel therapeutictreatment with excellent safety and efficacy. We hope thatRT24 may be used as an antibody therapy for FAP in the future.

Experimental Procedures

Human Samples, Animals, Peptide, Plasmids, and Anti-body—All studies with human samples were conducted inaccordance with the current version of the Declaration of Hel-sinki. The ethical committee of Kumamoto Universityapproved this study. Animals were maintained in a specific

TABLE 1Biochemical value of groups T24 and PBSDuring the efficacy study, blood was collected once a week; blood levels of creatinine(CRE indicates kidney function), total protein (TP indicates liver function), amylase(AMY indicates pancreatic function), TTR, albumin and serum amyloid A wereanalyzed. The data showed average value between individuals. SAA is serum amy-loid A and ALB is albumin.




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pathogen-free environment at the Center for Animal Resourcesand Development, Kumamoto University. Animals were han-dled according to Kumamoto University’s animal care policy.Peptides in which cysteine was added at the amino-terminalregion of the human TTR(115–124) peptide were prepared bychemical synthesis (Sigma). KLH or BSA was conjugated withcysteine residue using an Immuject� Immunogen EDC kit withmcKLH and BSA (Thermo). WT TTR DNA was cloned by PCRusing human liver Marathon-Ready cDNA (Clontech) as a tem-plate. The cloned TTR DNA fragment was introduced intopQE-30 (Qiagen) to construct a WT human TTR expressionvector. The human TTR mutant (D18G, V30M, E54K, L55P,Y114C, S115A, Y116A, Y116S, S117A, Y118A, T119A, A120S,V121A, V122A, V122I, T123A, and N124A) expression vectorswere prepared by site-directed mutagenesis using the above-mentioned WT human TTR expression vector as a template.Anti-hepatitis B surface antigen monoclonal antibody was usedas an isotype control (negative control).

Preparation of Recombinant Mutant TTRs—WT TTR andTTR mutant proteins were expressed by E. coli and purifiedusing a previously described method (25). E. coli strain M15 wastransfected with each expression vector of WT TTR or variantTTRs, such as D18G, V30M, E54K, L55P, Y114C, Y116S, orV122I. Subsequently, each E. coli clone was cultured with 20 mlof Luria-Bertani medium/ampicillin (50 �g/ml)/kanamycin (25�g/ml) at 37 °C. When A600 was 0.5, IPTG was added at a finalconcentration of 10 mM, and the culture was incubated over-night. The cells were collected from the culture by centrifuga-tion and resuspended in Buffer A (50 mM phosphate buffer(PB) � 0.3 M NaCl � 10 mM imidazole � 20 mM 2-mercapto-ethanol). The suspension was sonicated for 15 min and then cen-trifuged to collect supernatant. The supernatant was subjected toHis tag purification with nickel-nitrilotriacetic acid-agarose (Qia-gen) and the eluent fraction containing the recombinant TTRswas dialyzed against 20 mM NaHCO3. The recombinant TTRsafter dialysis were purified by gel filtration with Superdex 75 (GEHealthcare) using 10 mM PB (pH 7.5), and a fraction of tetramericTTR was used as purified recombinant TTRs.

Generation of Monoclonal Antibodies—TTR knock-out mice(26) were immunized with a 1:1 emulsification of KLH-conju-gated TTR peptide and Freund’s complete adjuvant (Difco).Over 2 weeks after the first immunization, these mice wereimmunized with a 1:1 emulsification of KLH-conjugated TTRpeptide and Freund’s incomplete adjuvant (Difco). Antibodytiter was confirmed by ELISA. After a sufficient increase inantibody titer was confirmed, the spleen cells were collectedfrom the mouse and fused with mouse myeloma cells P3U1using the PEG method (27). At days 7–11 after the inoculationin HAT medium, the binding activity to TTR(115–124) peptidewas screened via ELISA using the hybridoma supernatants.

Epitope Analysis—E. coli strain M15 was transfected witheach recombinant TTR mutant (Y114C, S115A, Y116A, S117A,Y118A, T119A, A120S, V121A, V122A, V122I, T123A, orN124A) expression vector individually and cultured in 20 ml ofLuria-Bertani medium/ampicillin (50 �g/ml)/kanamycin (25�g/ml) at 37 °C. When A600 was 0.5, IPTG was added at a finalconcentration of 10 mM, and the culture was incubated over-night. The cultured cell suspension was centrifuged, and the

precipitate fraction was resolubilized with Bugbuster (Merck).The resolubilized cell fraction was electrophoresed underreducing conditions on 8 –16% SDS-polyacrylamide gel andtransferred to Immobilon-P membrane (Millipore). The mem-brane was soaked in 2% skimmed milk/PBST (PBST, PBS con-taining 0.05% Tween 20) and shaken at room temperature for1 h to block the membrane. T24 antibody was diluted with 2%skimmed milk/PBST at a concentration of 1 �g/ml, and themembrane was soaked in 10 ml of this antibody solution andshaken at room temperature for 1 h. The membrane waswashed with PBST and soaked in HRP-labeled anti-mouse IgG(H�L) (American Qualex International) solution, which waspreviously diluted 5000-fold with 2% skimmed milk/PBST andshaken at room temperature for 1 h. After washing with PBST,color development was conducted with Ez West Blue (ATTO).

Antibody Reactivity against Patient’s Serum and ExtractedTTR Amyloid—BSA-conjugated TTR peptides (2 �g/ml), thesera obtained from either a healthy volunteer or a V30M FAPpatient or 4 �g/ml human serum-derived WT-TTR fibriltreated at pH 3.0 were immoblized on a Maxisorp plate (Nunc)at room temperature. Plates were blocked with PBS containing1% BSA (1% BSA/PBS). After the plates were coated, they wereincubated with T24 antibodies in 1% BSA/PBS at 37 °C for 1 h.The plates were washed in PBS containing 0.05% Tween 20(PBST). Bound T24 was detected with anti-mouse IgG (H�L)/HRP (Zymed Laboratories Inc.) using the standard method. Inaddition, analysis of the reactivity of T24 and polyclonal rabbitanti-human prealbumin (TTR) (Dako) was performed by West-ern blotting against the healthy patient’s serum and extractedTTR amyloid from the FAP patient’s kidney and heart.

Congo Red and Immunohistochemical Staining—Paraffin-embedded 4-�m-thick sections and frozen 10-�m-thick sec-tions of the kidney and heart from the FAP patient were sub-jected to Congo red and immunohistochemical staining. AfterCongo red and hematoxylin staining, the presence of apple-green birefringence under polarized light confirmed the pres-ence of amyloid deposits. For immunohistochemical staining,paraffin-embedded sections were prepared and deparaffinizedin xylene and rehydrated in graded alcohols. Both paraffin-em-bedded and frozen slides were treated with periodic acid for 10min at room temperature, after which they were incubated in5% normal serum for 1 h at room temperature in a moist cham-ber. Ten micrograms/ml T24 or isotype control was used as theprimary antibody and incubated with sections at 4 °C over-night. The secondary antibody was an HRP-conjugated rabbitanti-mouse immunoglobulin antibody (Dako) diluted 1:100 inbuffer. The dilution buffer used was 0.5% BSA/PBS. Reactivitywas visualized via the DAB Liquid System (Dako), according tothe manufacturer’s instructions. Sections were counterstainedwith hematoxylin.

Humanization of the T24 Antibody—cDNA encoding themurine IgVH and VL were isolated by real time PCR from T24-producing hybridoma. First, to produce CT24, the VH region ofT24 was joined with the human IgG heavy-chain constantregion gene and ligated to the SalI site of the pCAGGS/mdhfrplasmid (28), and the VL region of T24 was joined with thehuman IgG light-chain constant region gene and ligated to theSalI site of the pCAGGS plasmid (28). The humanization of T24




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was performed using the CDR-grafting method, adding theknowledge from our previous antibody humanization studiesand those of other authors (29, 30). To produce RT24, thedesigned VH and VL regions for RT24 were joined with thehuman immunoglobulin constant region gene, as describedabove. Freestyle 293-F cells (Invitrogen) were transfected withRT24 and CT24 expression plasmids using Neofection (ASTECCo., Ltd.), and the culture was shaken at 125 rpm at 37 °C underenvironmental conditions of 8% CO2 to express these antibod-ies. Five days later, transiently expressed culture supernatantswere harvested, and then antibodies were purified from thesupernatants using protein A chromatography and dialyzedagainst PBS.

Specificity of the RT24 Antibody—Specificity of the RT24antibody was assessed by ELISA and SPR using a BIAcore 2000instrument (GE Healthcare). To prepare the WT and V30MTTR fibril form, purified recombinant WT and V30M TTRprotein was adjusted to 3 mg/ml with sodium phosphate buffer(pH 7.5), mixed with an equal volume of fibrillization buffer (0.2M acetate buffer containing 0.1 M NaCl (pH 3.0)), and incubatedat 37 °C for 16 h. After the reaction, fibrillization was confirmedby thioflavinT assay (31). ThioflavinT assay was performed bydiluting TTR with 50 mM glycine/NaOH buffer (pH 9.5) so thatthe final concentrations of thioflavinT and TTR were 20 �M and15 �g/ml, respectively, and by measuring the fluorescenceintensity with spectrofluorometer FP-6500 (Jasco) (excitationwavelength, 450/442 nm; emission wavelength, 482/489 nm).For ELISA, biotin-conjugated TTR(115–124) peptide wasdiluted to 50 nM with PBS and immobilized to a Nunc Immo-bilizer Streptavidin Plate (Nunc). Purified recombinant WT (2�g/ml), V30M TTR fibril, and no proteins were immobilized tothe Nunc Immobilizer Nickel-Chelate Plate (Nunc). Plateswere blocked with 1% BSA/PBS. After coating, the plates wereincubated with RT24 or CT24 antibodies in 1% BSA/PBS at37 °C for 1 h. The plates were washed five times with PBST.Bound antibodies were detected with anti-human Fc/HRP(Rockland) according to standard procedures. For SPR anal-ysis, WT TTR, V30M TTR, WT TTR fibril, or V30M TTRfibril were immobilized in 10 mM sodium acetate (pH 6.0), at1000 response units on a CM5 sensor chip (GE Healthcare)using an amine coupling kit (GE Healthcare). RT24 (10�g/ml) or isotype control was injected during the associationphase for 2 min (20 �l/min), with HBS-EP as the runningbuffer. The dissociation phase, initiated by the passage of HBS-EPalone, was performed over a period of 60 min. Sensor chip surfaceswere regenerated by a 30-s injection of 10 mM glycine/NaOH (pH9.0).

RT24 Reactivity against Recombinant TTR-derived Fibrils—Purified recombinant TTRs (WT, D18G, V30M, E54K, L55P,Y114C, Y116S, and V122I) proteins were incubated for 16 h at37 °C and pH 3.0. These samples were defined as TTR fibrils.Native-PAGE was performed for 1.5 �g of each TTR fibril, fol-lowed by silver staining. Next, the TTR fibrils were analyzed byWestern blotting with the RT24 antibody.

Inhibition Assay of V30M TTR Fibrillization—RT24 anti-body or isotype control was mixed with purified recombinantV30M TTR protein in PBS containing 0.1% sodium deoxy-cholate and incubated at 37 °C for 3 days. The molar ratio of V30M

TTR and the antibodies was 10:0.01 to 10:2 �M. Fluorescenceintensity was measured by thioflavinT assay with PHERAstar(BMG Labtech) (excitation wavelength, 440 nm; emission wave-length, 480 nm) to evaluate the degree of TTR fibrillization.

Macrophage Phagocytosis Assay—The establishment andcharacterization of the iPS cell-derived myeloid/macrophageline (iPS-ML) have been previously reported (32). AggregatedTTR was generated from native V30M TTR by incubating for24 h at 37 °C under acidic conditions (pH 4.0). To investigatethe clearance of antibodies against misfolded TTR, mitomycinC-treated iPS-MLs (1 � 105 cells/well) with TTRs (800 nM)were co-cultured in 200 �l of serum-free medium (Opti-MEM)supplemented with 100 ng/ml GM-CSF and 50 ng/ml M-CSF in96-well plates. In addition, RT24 antibody (10 �l/ml) was addedto the plates. After incubation for 2 days, TTR levels in eachculture supernatant were measured by ELISA. Briefly, 96-wellplates were coated with each culture supernatant in carbonatebuffer overnight at 4 °C. The plates were blocked with coatingbuffer containing 0.5% gelatin for 1 h at room temperature. Todetect TTRs, rabbit polyclonal anti-prealbumin and goat anti-rabbit IgG antibodies conjugated with HRP (Dako) were usedfor 1 h at room temperature as the primary and secondary anti-bodies, respectively. After incubation with SureBlueTM TMBmicrowell peroxidase substrate (KPL), absorbance was de-tected at 450 nm.

Efficacy Estimation of T24 Antibody in FAP Model Rats—Tg rats possessing a human ATTR V30M gene (ATTR V30MTg rats) were generated as described previously (19). Each3-month-old Tg rat received caudal intravenous injections of10 mg/kg T24 and blood examinations every week. After 6months, blood and tissues were collected for analysis. For quan-titation of TTR deposition, paraffin-embedded 4-�m-thicksections of rat colons were stained as described. The sectionswere examined under a light microscopy, and the entire field ofthe rat colon was digitized using an Olympus DP71 camera andDP-BSW-V3.1 software at �100 magnification. Briefly, indi-vidual images of the section were captured and then merged toproduce an image of the whole colon section. The degree ofamyloid deposition was determined by computer measurementof TTR-positive areas via the public domain ImageJ programdeveloped by the National Institutes of Health and available atrsb.info.nih.gov, as described previously (33). The analysis ofblood biochemistry was performed by the central clinical labo-ratory at Kumamoto University.

Statistical Analysis—Data are presented as mean � S.D.Student’s t test was used evaluating statistical significance. pvalues � 0.05 were considered statistically significant.

Author Contributions—A. H. and M. T. generated antibody, de-signed, performed, and analyzed the experiments shown in Figs. 2and 5– 8, and wrote most of the paper. Y. S. and J. G. designed, per-formed, and analyzed the experiments shown in Figs. 3, 4, and 10 andwrote the paper. D. I. performed the experiment shown in Fig. 7.G. S., H. M., and S. S. designed, performed, and analyzed the exper-iments shown in Fig. 9. A. H. and H. H. designed the antibodyhumanization. M. U. and T. I. conducted experiments and designedresearch studies. T. N., K. S., H. J., and Y. A. conducted experiments,designed research studies, and wrote the paper.




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Acknowledgments—We thank Tomoyo Takeo and Masayo Oohori fortheir technical assistance.

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Motokawa, Tokunori Ikeda, Satoru Senju, Toshihiro Nakashima and Yukio AndoSoejima, Hirofumi Higuchi, Jianying Guo, Mitsuharu Ueda, Genki Suenaga, Hiroaki Akihiko Hosoi, Yu Su, Masaharu Torikai, Hirofumi Jono, Daisuke Ishikawa, Kenji

Novel Antibody for the Treatment of Transthyretin Amyloidosis

doi: 10.1074/jbc.M116.738138 originally published online October 7, 20162016, 291:25096-25105.J. Biol. Chem.

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novel antibody for the treatment of transthyretin …acidic treatment (fig. 5c), similar to that...

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