Anti-transthyretin human antibody

Abstract

A human antibody which comprises a complementarity determining region of an H chain consisting of the amino acid sequence as shown in SEQ ID NOs: 1 to 3 and a complementarity determining region of an L chain consisting of the amino acid sequence as shown in SEQ ID NOs: 4 to 6. The human antibody of the present invention has the activity to specifically bind to transthyretin (TTR) with structural change and the activity to inhibit fibrillization of TTR and is a human antibody suitable for application to human body.

Claims

1. A method for measuring the activity to inhibit transthyretin-fibrillization which comprises a step of reacting a variant transthyretin (TTR) with a sample to be tested in the presence of sodium (Na) deoxycholate, and detecting the presence of TTR amyloid indicating inhibition of TTR-fibrillization, wherein the variant TTR is V30M TTR.

2. The method of claim 1, wherein fibrillization is let to proceed under neutral conditions.

3. The method of claim 1, wherein the concentration of Na deoxycholate is from 0.1% to 1%.

4. The method of claim 1, wherein the sample to be tested is an antibody to TTR.

Description

BRIEF DESCRIPTION OF DRAWINGS

(1) FIG. 1 shows maps of (a) chimeric 371 antibody H chain expression vector pKMA010-371-mCg1, (b) chimeric 371 antibody L chain expression vector pKMA009-371-mCλ, (c) chimeric 313 antibody H chain expression vector pKMA010-313-mCg1, and (d) chimeric 313 antibody L chain expression vector pKMA009-313-mCλ.

(2) FIG. 2 shows the results of epitope analysis for (a) positions 76-85 of TTR; and (b) positions 84-93 of TTR, respectively.

(3) FIG. 3 shows the results of reaction specificity analysis using surface plasmon resonance for (a) 371M antibody, (b) 313M antibody, (c) polyclonal antibody manufactured by Dako, and (d) negative control antibody, respectively.

(4) FIG. 4 shows the results of reactivity analysis to patient sera for (a) 371 antibody, and (b) 313 antibody, respectively.

(5) FIG. 5(a) shows the results of reactivity analysis to patient tissue (paraffin section).

(6) FIG. 5(b) shows the results of reactivity analysis to patient tissue (frozen section).

(7) FIG. 6 shows the results of reactivity analysis to TTR fibril for (a) silver stain, (b) Western blotting with 371M antibody, and (c) Western blotting with 313M antibody, respectively.

(8) FIG. 7 shows the results of preliminary examination of TTR-fibrillization inhibition test.

(9) FIG. 8 shows the results of preliminary examination of TTR-fibrillization inhibition test.

(10) FIG. 9 shows the results of TTR-fibrillization inhibition test for (a) 371 antibody and 313 antibody, and (b) 371M antibody and 313M antibody, respectively.

(11) FIG. 10 shows the results of macrophage phagocytic ability test for (a) untreated purified V30M, and (b) TTR fibril, respectively.

(12) FIG. 11 shows the results of drug efficacy evaluation test using V30M Tg rat.

BEST MODE FOR CARRYING OUT THE INVENTION

(13) The specific embodiments of the present invention are explained hereinbelow. The present invention is not construed to be limited to these embodiments.

(14) 1. Recombinant Human Antibody of the Present Invention and a Fragment Thereof

(15) In accordance with the present invention, for obtaining a human antibody that specifically binds to TTRs with structural change and inhibits TTR-fibrillization, focusing on TTR S112I which undergoes structural change and exists in a dimer, S112I was selected as an antigen for preparing an antibody and phage display was used for preparing a human antibody. Technique for preparing an antibody with phage display includes “Phage Display of Peptides and Proteins: A Laboratory Manual Edited by Brian K. Kay et al”, “Antibody Engineering: A PRACTICAL APPROACH Edited by J. McCAFFERTY et al”, and “ANTIBODY ENGINEERING second edition edited by Carl A. K. BORREBAECK”. In accordance with the present invention, S112I fibrillized by acid treatment is immobilized on a plate for panning and then reacted with phage library. For the acid treatment, temperature, time, and pH may suitably be selected for fibrillization of S112I. The presence or absence of fibrillization may be checked by ThioflavinT assay. For instance, S112I may be treated under conditions of pH 3.0 to pH 4.4 at 37° C. for 16 hours. The plate is washed to remove unbound phages and then phages bound to the target molecule are collected and infected to E. coli. After E. coli is cultured, the phages are collected and reacted again with the plate. After performing such a series of panning cycles three to five times, those phages which show excellent reactivity with S112I are selected by ELISA test.

(16) From E. coli producing the selected phages, plasmid DNAs from the phages are collected and nucleotide sequences of VH region (or VL region) are analyzed. The nucleotide sequence of VH region or VL region is inserted into an expression vector containing a nucleotide sequence coding for H chain or L chain constant region to prepare an expression vector of H chain or L chain. The resulting expression vector is introduced into a suitable host (animal cell) and a human antibody is expressed using the host.

(17) In accordance with the present invention, two kinds of human antibodies with different amino acid sequences of CDRs were obtained and the amino acid sequences of framework region of these antibodies were mutated to prepare improved human antibodies (371M, 313M).

(18) A method for analyzing an epitope for a human antibody is illustrated. With a peptide of an amino acid sequence of positions 76-93 of human TTR where one amino acid residue is altered to alanine (the amino acid residue at position 91 is alanine and thus is altered to serine), modified human TTRs are prepared. Conventional site-directed mutagenesis is used to prepare genes of the modified TTRs. The genes are inserted into an expression vector and then expressed and purified with a suitable host (preferably E. coli). With conventional Western blotting, the modified TTRs are electrophoresed on SDS-PAGE and are reacted with an antibody of the analysis object to detect reactivity between the variants and the antibody. When modified TTRs with reduced binding to the antibody are found, the modified portion may be considered to be an epitope. The antibody of the present invention has an epitope at positions 78-89 which is presumed to be present in a hidden portion of wild-type TTR tetramer. This epitope was a novel epitope.

(19) The above human antibody may be subject to tests for the specific reactivity to TTRs with structural change, the reactivity with fibrillized TTR, immunostaining to tissues derived from TTR patients, the inhibitory activity to fibrillization by variant TTR, the promoting activity to the macrophage phagocytic ability to TTR amyloid, and the drug efficacy evaluation using FAP animal model.

(20) A method for analyzing specific reactivity to TTRs with structural change includes a method using surface plasmon resonance. Wild-type TTR tetramer, variant TTR (V30M etc.) tetramer, or variant TTR amyloid are prepared. A method for preparing wild-type TTR tetramer and variant TTR tetramer includes a method of Matsubara et al. (Non-patent reference 13). A method for preparing variant TTR amyloid includes acid treatment as described above. Next, these preparations are let to bind to sensor chips, to which an antibody of the analysis object is added for reaction with the sensor chips, thereby indicating the binding of TTR with the antibody as response unit (RU). Here, those antibodies that have RU for variant TTR amyloid significantly higher than RU for wild-type TTR tetramer and variant TTR tetramer are thought to specifically recognize TTRs with structural change. As compared to such tetrameric TTRs, an antibody that specifically recognizes TTRs with a bigger size of molecular structure than tetramer (e.g. TTR amyloid) may also be regarded as an antibody that specifically recognizes TTRs with structural change.

(21) A method for analyzing the activity to inhibit TTR-fibrillization is exemplified below. A solution containing TTR and an antibody to be evaluated is mixed with a surfactant at a final concentration of 0.01-1% and left to stand at such a temperature for such a period of time that allows for TTR to form fibril. Fluorescence intensity is measured by ThioflavinT assay (excitation wavelength 440 nm, fluorescent wavelength 480 nm) to evaluate a degree of TTR-fibrillization. A surfactant includes benzalkonium chloride, sodium deoxycholate, Zwittergent3-16 and NP-40. Most preferable is deoxycholate. A final concentration includes 0.01-1%, and is most preferably 0.1%. Time and temperature includes at 37° C. for 3 to 4 days but may suitably be arranged for their combination. This method of analysis is an excellent evaluation system where denaturation of an antibody to be evaluated may be prohibited since the analysis can be performed at pH close to neutrality.

(22) A method for analyzing the macrophage phagocytic ability to TTR amyloid is exemplified below. Human iPS cells are prepared from skin tissue from FAP patients by the conventional method and are further differentiated to macrophages by the conventional method. TTR fibril and 5×10.sup.4 cells of the differentiated macrophages are mixed together. An antibody to be evaluated is added and the mixture is cultured for a fixed period of time (e.g. 3 days). A residual quantity of TTR after culture is measured by ELISA to evaluate the phagocytic ability of macrophage.

(23) A method for analyzing the reactivity between the human antibody of the present invention and TTR amyloid is exemplified below. Wild-type TTR and variant TTR are treated under acidic conditions for a period of time sufficient for TTR-fibrillization to prepare TTR amyloid. Time for fibrillization may suitably be selected depending on pH or the kinds of TTR. The samples after the acid treatment are electrophoresed on Native PAGE and subject to silver staining. A broad band at a higher position than 60 kDa may be an index for TTR-fibrillization. Using the conventional Western blotting, the TTR amyloid is electrophoresed on SDS-PAGE and antibodies of analysis object are reacted thereto for detection. An antibody that has a higher reactivity with TTR amyloid as compared to TTR with no acid treatment (TTR not subject to fibrillization) may be regarded as an antibody having the binding activity to TTR amyloid.

(24) A method for the drug efficacy evaluation using FAP animal model is exemplified below. Using V30M Tg rat (Non-patent reference 14; transgenic rat where a gene of human TTR with mutation of valine at position 30 to methionine in the amino acid sequence of TTR is introduced), a fixed amount (e.g. 10 mg/kg) of an antibody to be evaluated is administered for a fixed period of time (e.g. for 6 months) at a fixed frequency (e.g. once per week). After administration, the large intestine is taken out by autopsy and formalin fixed. The fixed tissue of the large intestine is embedded in a paraffin block to prepare tissue section. The tissue section is subject to immunostaining using Polyclonal Rabbit Anti-Human Prealbumin (Dako), HRP-labelled Goat anti-Rabbit IgG (Dako) and a degree of TTR deposition in the muscular layer of the large intestine is digitized and compared between the groups.

(25) The human antibody of the present invention has the inhibitory activity to TTR-fibrillization, the specific binding activity to TTRs with structural change, the effect of promoting the phagocytic ability of macrophage to TTR amyloid, the binding activity to TTR amyloid, and the effect to FAP animal model. As a result of analysis of an epitope for the antibody of the present invention, it was present at TTR78-89. Thus, the present invention includes the human antibodies as follows:

(26) (1) A human antibody having the activity to inhibit fibrillization of TTR;

(27) (2) A human antibody which specifically recognizes TTRs with structural change and does not recognize tetrameric functional TTR;

(28) (3) A human antibody which specifically binds to TTR amyloid;

(29) (4) A human antibody which promotes removal of TTR amyloid;

(30) (5) A human antibody which promotes the phagocytic ability of macrophages to TTR amyloid;

(31) (6) A human antibody which has a therapeutic effect and/or a preventive effect to TTR amyloidosis;

(32) (7) A human antibody which has an epitope of TTR78-89.

(33) The antibodies of (1) to (7) above may have one characteristic feature as shown in each of (1) to (7) or may have a combination of characteristic features as shown in (1) to (7).

(34) For the human antibody (371M) of the present invention, the amino acid and nucleotide sequences of CDR1-3 of VH region or VL region are shown in the following table.

(35) TABLE-US-00001 TABLE 1 VH CDR1 SYAMS SEQ ID NO: 1 region agctatgccatgagc SEQ ID NO: 26 CDR2 AISGSGGSTYYADSVKG SEQ ID NO: 2 gctattagtggtagtggtggtagcacatac SEQ ID NO: 27 tacgcagactccgtgaagggc CDR3 GTRTNWYFDL SEQ ID NO: 3 gggacccggacgaactggtacttcgatctc SEQ ID NO: 28 VL CDR1 SGSRSNIGSNTVN SEQ ID NO: 4 region tctggaagtagatccaacatcgggagtaat SEQ ID NO: 29 actgttaac CDR2 SNNQRPS SEQ ID NO: 5 agtaataatcagcggccctca SEQ ID NO: 30 CDR3 AAWDDSLYGPV SEQ ID NO: 6 gcagcatgggatgacagtctgtatggtcct SEQ ID NO: 31 gtg

(36) Thus, the present invention includes the human antibody having the following characteristic features of the amino acid sequence:

(37) (8) A human antibody which comprises a complementarity determining region of an H chain consisting of the polypeptide of (a) or (b) below and a complementarity determining region of an L chain consisting of the polypeptide of (c) or (d) below:

(38) (a) a polypeptide consisting of the amino acid sequence as shown in SEQ ID NOs: 1 to 3; (b) a polypeptide which consists of the amino acid sequence as shown in SEQ ID NOs: 1 to 3 wherein one or several amino acid residue(s) is/are substituted, deleted, inserted and/or added and which can be a complementarity determining region of an H chain to TTR; (c) a polypeptide consisting of the amino acid sequence as shown in SEQ ID NOs: 4 to 6; (d) a polypeptide which consists of the amino acid sequence as shown in SEQ ID NOs: 4 to 6 wherein one or several amino acid residue(s) is/are substituted, deleted, inserted and/or added and which can be a complementarity determining region of an L chain to TTR.

(39) The above antibody may also have characteristic features as shown in each of (1) to (7).

(40) For the human antibody (313M) of the present invention, the amino acid and nucleotide sequences of CDR1-3 of VH region or VL region are shown in the following table.

(41) TABLE-US-00002 TABLE 2 VH CDR1 SYYMH SEQ ID NO: 7 region agctactatatgcac SEQ ID NO: 32 CDR2 IINPSGGSTSYAQKFQG SEQ ID NO: 8 ataatcaaccctagtggtggtagcacaagc SEQ ID NO: 33 tacgcacagaagttccagggc CDR3 FGSSSRGNDAFDI SEQ ID NO: 9 ttcgggtcttctagcagggggaatgatgct SEQ ID NO: 34 tttgatatc VL CDR1 SGDVLAKKYAR SEQ ID NO: 10 region tcaggagatgtactggcaaaaaaatatgct SEQ ID NO: 35 cgg CDR2 KDSERPS SEQ ID NO: 11 aaagaagtgagcggccctca SEQ ID NO: 36 CDR3 YSAADNKEAV SEQ ID NO: 12 tactctgcggctgacaacaaggaggctgtg SEQ ID NO: 37

(42) Thus, the present invention includes the human antibody having the following characteristic features of the amino acid sequence:

(43) (9) A human antibody which comprises a complementarity determining region of an H chain consisting of the polypeptide of (e) or (f) below and a complementarity determining region of an L chain consisting of the polypeptide of (g) or (h) below:

(44) (e) a polypeptide consisting of the amino acid sequence as shown in SEQ ID NOs: 7 to 9; (f) a polypeptide which consists of the amino acid sequence as shown in SEQ ID NOs: 7 to 9 wherein one or several amino acid residue(s) is/are substituted, deleted, inserted and/or added and which can be a complementarity determining region of an H chain to TTR; (g) a polypeptide consisting of the amino acid sequence as shown in SEQ ID NOs: 10 to 12; (h) a polypeptide which consists of the amino acid sequence as shown in SEQ ID NOs: 10 to 12 wherein one or several amino acid residue(s) is/are substituted, deleted, inserted and/or added and which can be a complementarity determining region of an L chain to TTR.

(45) The above antibody may also have characteristic features as shown in each of (1) to (7).

(46) For the human antibody (371M), the amino acid and nucleotide sequences of VH region or VL region are shown in the following table.

(47) TABLE-US-00003 TABLE 3 VH EVQLVESGGGVVQPGGSLRLSCAASGFTFSSYAMSWVRQA SEQ ID region PGKGLEWVSAISGSGGSTYYADSVKGRFTISRDNSKNTLY NO: 13 LQMNSLRAEDTAVYYCAKGTRTNWYFDLWGRGTLVTVSS gaggtgcagctggtggagtccgggggaggcgtggtccagc SEQ ID ctggggggtccctgagactctcctgtgcagcctctggatt NO: 38 cacctttagcagctatgccatgagctgggtccgccaggct ccagggaaggggctggagtgggtctcagctattagtggta gtggtggtagcacatactacgcagactccgtgaagggccg gttcaccatctccagagacaattccaagaacacgctgtat ctgcaaatgaacagcctgagagccgaggacacggccgtat attactgtgcgaaagggacccggacgaactggtacttcga tctctggggccgtggcaccctggtcaccgtctcctca VL SYELTQPPSASGTPGQRVTISCSGSRSNIGSNTVNWYQQV SEQ ID region PGTAPKLLIYSNNQRPSGVPDRFSGSKSGTSASLAISGLQ NO: 14 SEDEAEYYCAAWDDSLYGPVFGGGTQLTVL tcctatgagctgacacagccaccctcagcgtctgggaccc SEQ ID ccgggcagagggtcaccatctcttgttctggaagtagatc NO: 39 caacatcgggagtaatactgttaactggtaccaacaggtc ccaggaacggcccccaaactcctcatttatagtaataatc agcggccctcaggggtccctgaccgattctctggctccaa gtctggcacctcagcctccctggccatcagtggactccag tctgaggatgaggctgaatattattgtgcagcatgggatg acagtctgtatggtcctgtgttcggaggaggcacccagct gaccgtccta

(48) Thus, the present invention includes the human antibody having the following characteristic features of the amino acid sequence:

(49) (10) A human antibody which comprises an H chain variable region consisting of the polypeptide of (i) or (j) below and an L chain variable region consisting of the polypeptide of (k) or (l) below:

(50) (i) a polypeptide consisting of the amino acid sequence as shown in SEQ ID NO: 13; (j) a polypeptide which consists of the amino acid sequence as shown in SEQ ID NO: 13 wherein one or several amino acid residue(s) is/are substituted, deleted, inserted and/or added and which can be a complementarity determining region of an H chain to TTR; (k) a polypeptide consisting of the amino acid sequence as shown in SEQ ID NO: 14; (l) a polypeptide which consists of the amino acid sequence as shown in SEQ ID NO: 14 wherein one or several amino acid residue(s) is/are substituted, deleted, inserted and/or added and which can be a complementarity determining region of an L chain to TTR.

(51) The above antibody may also have characteristic features as shown in each of (1) to (7).

(52) For the human antibody (313M), the amino acid and nucleotide sequences of VH region or VL region are shown in the following table.

(53) TABLE-US-00004 TABLE 4 VH QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYYMHWVRQAP SEQ ID region GQGLEWMGIINPSGGSTSYAQKFQGRVTMTRDTSTSTVYME NO: 15 LSSLRSEDTAVYYCASFGSSSRGNDAFDIWGQGTMVTVSS caggtccagctggtacagtctggggctgaggtgaagaagcc SEQ ID tggggcctcagtgaaggtttcctgcaaggcatctggataca NO: 40 ccttcaccagctactatatgcactgggtgcgacaggcccct ggacaagggcttgagtggatgggaataatcaaccctagtgg tggtagcacaagctacacacagaagttccagggcagagtca ccatgaccagggacacgtccacgagcacagtctacatggag ctgagcagcctgagatctgaggacacggccgtgtattactg tgcgagtttcgggtcttctagcagggggaatgatgcttttg atatctggggccaagggacaatggtcaccgtctcttca VL SYELTQPSSVSVSPGQTARITCSGDVLAKKYARWFQQKPGQ SEQ ID region APVLVIYKDSERPSGIPERFSGSSSGTTVTLTISGAQVEDE NO: 16 ADYYCYSAADNKEAVEGGGTQLTVL tcctatgagctgacacagccatcctcagtgtcagtgtctcc SEQ ID gggacagacagccaggatcacctgctcaggagatgtactgg NO: 41 caaaaaaatatgctcggtggttccagcagaagccaggccag gcccctgtgctggtgatttataaagacagtgagcggccctc agggatccctgagcgattctccggctccagctcagggacca cagtcaccttgaccatcagcggggcccaggttgaggatgag gctgactattactgttactctgcggctgacaacaaggaggc tgtgttcggaggaggcacccagctgaccgtccta

(54) Thus, the present invention includes the human antibody having the following characteristic features of the amino acid sequence:

(55) (11) A human antibody which comprises an H chain variable region consisting of the polypeptide of (m) or (n) below and an L chain variable region consisting of the polypeptide of (o) or (p) below:

(56) (m) a polypeptide consisting of the amino acid sequence as shown in SEQ ID NO: 15; (n) a polypeptide which consists of the amino acid sequence as shown in SEQ ID NO: 15 wherein one or several amino acid residue(s) is/are substituted, deleted, inserted and/or added and which can be a complementarity determining region of an H chain to TTR; (o) a polypeptide consisting of the amino acid sequence as shown in SEQ ID NO: 16; (p) a polypeptide which consists of the amino acid sequence as shown in SEQ ID NO: 16 wherein one or several amino acid residue(s) is/are substituted, deleted, inserted and/or added and which can be a complementarity determining region of an L chain to TTR.

(57) The above antibody may also have characteristic features as shown in each of (1) to (7).

(58) The present invention includes the H chain variable region fragment comprising the following CDR of H chain:

(59) (12) An H chain variable region fragment comprising a complementarity determining region of an H chain consisting of the polypeptide of (a) or (b) below:

(60) (a) a polypeptide consisting of the amino acid sequence as shown in SEQ ID NOs: 1 to 3; (b) a polypeptide which consists of the amino acid sequence as shown in SEQ ID NOs: 1 to 3 wherein one or several amino acid residue(s) is/are substituted, deleted, inserted and/or added and which can be a complementarity determining region of an H chain to TTR.
(13) An H chain variable region fragment comprising a complementarity determining region of an H chain consisting of the polypeptide of (e) or (f) below: (e) a polypeptide consisting of the amino acid sequence as shown in SEQ ID NOs: 7 to 9; (f) a polypeptide which consists of the amino acid sequence as shown in SEQ ID NOs: 7 to 9 wherein one or several amino acid residue(s) is/are substituted, deleted, inserted and/or added and which can be a complementarity determining region of an H chain to TTR.

(61) The present invention includes the L chain variable region fragment comprising the following CDR of L chain:

(62) (14) An L chain variable region fragment comprising a complementarity determining region of an L chain consisting of the polypeptide of (c) or (d) below:

(63) (c) a polypeptide consisting of the amino acid sequence as shown in SEQ ID NOs: 4 to 6; (d) a polypeptide which consists of the amino acid sequence as shown in SEQ ID NOs: 4 to 6 wherein one or several amino acid residue(s) is/are substituted, deleted, inserted and/or added and which can be a complementarity determining region of an L chain to TTR.
(15) An L chain variable region fragment comprising a complementarity determining region of an L chain consisting of the polypeptide of (g) or (h) below: (g) a polypeptide consisting of the amino acid sequence as shown in SEQ ID NOs: 10 to 12; (h) a polypeptide which consists of the amino acid sequence as shown in SEQ ID NOs: 10 to 12 wherein one or several amino acid residue(s) is/are substituted, deleted, inserted and/or added and which can be a complementarity determining region of an L chain to TTR.

(64) The present invention includes the following H chain variable region fragment:

(65) (16) An H chain variable region fragment consisting of the polypeptide of (i) or (j) below:

(66) (i) a polypeptide consisting of the amino acid sequence as shown in SEQ ID NO: 13; (j) a polypeptide which consists of the amino acid sequence as shown in SEQ ID NO: 13 wherein one or several amino acid residue(s) is/are substituted, deleted, inserted and/or added and which can be a complementarity determining region of an H chain to TTR.
(17) An H chain variable region fragment consisting of the polypeptide of (m) or (n) below: (m) a polypeptide consisting of the amino acid sequence as shown in SEQ ID NO: 15; (n) a polypeptide which consists of the amino acid sequence as shown in SEQ ID NO: 15 wherein one or several amino acid residue(s) is/are substituted, deleted, inserted and/or added and which can be a complementarity determining region of an H chain to TTR.

(67) The present invention includes the following L chain variable region fragment:

(68) (18) An L chain variable region fragment consisting of the polypeptide of (k) or (l) below:

(69) (k) a polypeptide consisting of the amino acid sequence as shown in SEQ ID NO: 14; (l) a polypeptide which consists of the amino acid sequence as shown in SEQ ID NO: 14 wherein one or several amino acid residue(s) is/are substituted, deleted, inserted and/or added and which can be a complementarity determining region of an L chain to TTR.
(19) An L chain variable region fragment consisting of the polypeptide of (o) or (p) below: (o) a polypeptide consisting of the amino acid sequence as shown in SEQ ID NO: 16; (p) a polypeptide which consists of the amino acid sequence as shown in SEQ ID NO: 16 wherein one or several amino acid residue(s) is/are substituted, deleted, inserted and/or added and which can be a complementarity determining region of an L chain to TTR.

(70) The present invention includes the following single-chain variable region fragment:

(71) (20) A single-chain variable region fragment of an antibody to TTR, which is formed by linking the H chain variable region fragment comprising a complementarity determining region of an H chain of (12) or the H chain variable region fragment of (16) and the L chain variable region fragment comprising a complementarity determining region of an L chain of (14) or the L chain variable region fragment of (18).
(21) A single-chain variable region fragment of an antibody to TTR, which is formed by linking the H chain variable region fragment comprising a complementarity determining region of an H chain of (13) or the H chain variable region fragment of (17) and the L chain variable region fragment comprising a complementarity determining region of an L chain of (15) or the L chain variable region fragment of (19).

(72) For a single-chain variable region fragment, an H chain variable region fragment and an L chain variable region fragment are usually linked to each other via a suitable peptide linker and the like. For the peptide linker, any single-chain peptide consisting of e.g. 10 to 25 amino acid residues is used.

(73) The present invention includes the following antibody or a fragment thereof, which is formed by linking a human-derived constant region to the H chain variable region fragment and/or to the L chain variable region fragment:

(74) (22) A human antibody or a fragment thereof, which is formed by linking a human-derived constant region to the H chain variable region fragment comprising a complementarity determining region of an H chain of (12) or the H chain variable region fragment of (16) and/or to the L chain variable region fragment comprising a complementarity determining region of an L chain of (14) or the L chain variable region fragment of (18).
(23) A human antibody or a fragment thereof, which is formed by linking a human-derived constant region to the H chain variable region fragment comprising a complementarity determining region of an H chain of (13) or the H chain variable region fragment of (17) and/or to the L chain variable region fragment comprising a complementarity determining region of an L chain of (15) or the L chain variable region fragment of (19).

(75) The above antibody or a fragment thereof where a human-derived constant region is bound may be Fab, Fab′, F(ab′).sub.2, scAb having at least a portion of Fc region, or scFvFc, or even a complete antibody. As used herein, scAb is that which is formed by linking a portion of domain (c domain) of L chain or H chain constant region to scFv whereas scFvFc is that which is formed by linking a portion of constant region of H chain (Fc region) to scFv.

(76) The antibody as mentioned above also includes a protein structurally relevant to an antibody and refers to an immunoglobulin. Besides, the antibody of the present invention may be of any class of IgA, IgD, IgE, IgG or IgM. In other words, the antibody of the present invention may be a monomer or a polymer such as a dimer, a trimer, a tetramer or a pentamer.

(77) As used herein, the phrase “wherein one or several amino acid residue(s) is/are substituted, deleted, inserted and/or added” means that such a number of amino acid residue(s) that can afford to substitution, deletion, insertion and/or addition is/are substituted, deleted, inserted and/or added by a known method for preparing a mutant protein such as site-directed mutagenesis. Thus, for instance, the above polypeptide (b) is a mutant peptide of the above polypeptide (a). As used herein, the term “mutation” means principally mutation artificially introduced by a known method for preparing a mutant protein but may also be the similar mutant protein which is present in nature (e.g. human) and isolated and purified.

(78) The “mutation”, when the antibody of the present invention or a fragment thereof is used as a pharmaceutical composition (i.e. administered to human), is done within such a range that a human-derived structure or human does not induce immune reaction and, when the antibody of the present invention or a fragment thereof is used as a detection device or a diagnostic agent (i.e. not administered to human), is not particularly limited. Besides, when the antibody of the present invention or a fragment thereof is administered to human, mutation is performed preferably within such a range that a higher order structure of CDR recognizing an antigen is maintained.

(79) The antibody of the present invention or a fragment thereof may comprise an additional polypeptide. Such addition of a polypeptide includes epitope labelling of the protein of the present invention with e.g. His, Myc, Flag, etc.

(80) Besides, the antibody of the present invention or a fragment thereof may be bound with a modifier so as to improve its stability or antibody titer. Namely, the antibody of the present invention or a fragment thereof may be a modified antibody. A modifier includes, for instance, a sugar chain, a macromolecule, and the like. When modification is performed with a sugar chain, the sugar chain may possibly have a certain physiological activity. However, when modification is performed with a simple macromolecule such as polyethylene glycol (PEG), the molecule per se does not show a physiological activity. Besides, it is possible that PEGylation suppresses absorption in the liver or improve stability in blood. Thus, a modifier is preferably a simple macromolecule such as PEG.

(81) As is the case with the preparation of a mutant peptide, modification of the antibody of the present invention or a fragment thereof with a modifier, when the antibody of the present invention or a fragment thereof is used as a therapeutic agent, is done within such a range that human does not induce immune reaction and, when the antibody of the present invention or a fragment thereof is used as a detection device or a diagnostic agent, is not particularly limited. Besides, when the antibody of the present invention or a fragment thereof is administered to human, modification is performed preferably within such a range that a higher order structure of CDR recognizing an antigen is maintained.

(82) 2. Gene of the Present Invention

(83) The present invention includes a gene coding for the antibody or a fragment thereof of the above item 1. For instance, the present invention includes a gene including the following nucleotide sequences as an open reading frame (ORF) region and a modified gene with these nucleotide sequences partially modified:

(84) (1) nucleotide sequence comprising SEQ ID NOs:1-3 and/or SEQ ID NOs:4-6;

(85) (2) nucleotide sequence comprising SEQ ID NOs:7-9 and/or SEQ ID NOs:10-12;

(86) (3) nucleotide sequence comprising SEQ ID NO:13 and/or SEQ ID NO:14;

(87) (4) nucleotide sequence comprising SEQ ID NO:15 and/or SEQ ID NO:16.

(88) The above gene, coding for the antibody of the present invention or a fragment thereof, may be introduced into a suitable host (e.g. bacteria, yeast) for expression of the antibody of the present invention or a fragment thereof.

(89) Besides, the above gene may be one further comprising an untranslated region (UTR) or a sequence of a vector including an expression vector in addition to a nucleotide sequence coding for the antibody or a fragment thereof. For instance, the sequence of SEQ ID NO: 13 or 14 is linked to a sequence of a vector to form the gene of the present invention. The resultant gene may then be amplified in a suitable host to amplify the gene of the present invention as desired. Also, a portion of the gene of the present invention may be used as a probe.

(90) The gene of the present invention may be utilized as a gene therapy agent in the diseases associated with TTR amyloid. The gene therapy agent may be designed to express the antibody of the present invention or a fragment thereof within the living body after administration thereof so that the antibody of the present invention or a fragment thereof is formed within the living body after ingestion thereof to thereby exhibit the similar effect to that of the above inhibitor.

(91) 3. Recombinant Expression Vector of the Present Invention

(92) The present invention includes a recombinant expression vector comprising the gene of the above item 2, i.e. the gene coding for the antibody or a fragment thereof of the above item 1. For instance, the recombinant expression vector of the present invention includes the one where cDNA having the nucleotide sequence of SEQ ID NO: 13 or 14 is inserted. The recombinant expression vector may be prepared with, but not particularly limited to, plasmid, phage, cosmid and the like.

(93) A concrete sort of a vector is not particularly limited but such a vector that allows for expression in a host cell may suitably be selected. Namely, a promoter sequence may suitably be selected so as to ensure gene expression depending on the kind of a host cell and a variety of plasmids etc. into which the promoter and the gene of the present invention are inserted may be used as an expression vector.

(94) A variety of markers may be used for confirming if the gene of the present invention is introduced into a host cell or if the gene of the present invention is surely expressed in a host cell. For instance, a gene deficient in a host cell is used as a marker and plasmid etc. comprising the marker and the gene of the present invention is introduced as an expression vector into a host cell. Thereby, the introduction of the gene of the present invention may be verified by the expression of the marker gene. Alternatively, the antibody of the present invention or a fragment thereof and a marker protein may be expressed as a fusion protein. For instance, Green Fluorescent Protein (GFP) derived from Aequorea victoria may be used as a marker and the antibody of the present invention or a fragment thereof may be expressed as a GFP fusion protein.

(95) The above host cell is not particularly limited but a variety of known cells may suitably be used. Specifically, the host cell includes, but not particularly limited to, an animal cell including cells from human or mouse, Caenorhabditis elegans, an oocyte of Xenopas laevis, a culture cell of a variety of mammals (rat, rabbit, pig, monkey, etc.), a culture cell of insects such as Drosophila melanogaster or silkworm moth, bacteria such as Escherichia coli, yeast (budding yeast (Saccharomyces cerevisiae) and fission yeast (Schizosaccharomyces pombe)) and the like.

(96) A method for introducing a recombinant expression vector into a host cell, i.e. a method for transfection, is not particularly limited but the conventional known methods such as electroporation, calcium phosphate method, liposome method and DEAE-dextran method may suitably be used.

(97) A transformant of the present invention is a transformant where the gene of the above item 2, i.e. the gene coding for the antibody or a fragment thereof of the above item 1, is introduced. As used herein, “a gene is introduced” means that a gene is introduced expressibly into a cell of interest (host cell) by known genetic engineering techniques (gene manipulation techniques). The term “transformant” refers to not only a cell, a tissue or an organ but also an animal individual. An animal of interest is not particularly limited but includes mammals such as cow, pig, sheep, goat, rabbit, dog, cat, guinea pig, hamster, mouse and rat. In particular, rodents such as mouse and rat are widely used as an experimental animal and a disease animal model. Among them, mouse is preferable as an experimental animal and a disease animal model since many inbred strains have been created and techniques of culture of fertilized eggs and in vitro fertilization have been completed.

(98) The antibody or a fragment thereof of the above item 1 can be prepared with the transformant of the present invention which is prepared using the expression vector of the present invention.

(99) 4. Utilization of Human Antibody of the Present Invention or a Fragment Thereof

(100) The human antibody of the present invention specifically recognizes TTR with structural change (e.g. TTR amyloid), inhibits fibrillization of TTR and exerts the preventive effect against FAP. Thus, the present invention includes a device for detecting structural change of TTR, a diagnostic agent for TTR amyloidosis (in particular, FAP), a medicament for inhibiting fibrillization of TTR, and a pharmaceutical composition for preventing and/or treating TTR amyloidosis (in particular, FAP).

(101) The present invention includes a device for detecting structural change of TTR comprising the antibody of (1) or a fragment thereof (a detection device for TTR amyloid). The detection device of the present invention includes, for instance, an antibody chip or an antibody column etc. in which an antibody that specifically binds to TTR with structural change or a fragment thereof is immobilized on a basement (carrier). The detection device of the present invention, for instance, may be used for detecting TTR with structural change (e.g. TTR amyloid) contained in a sample such as blood or urine. Besides, the detection device of the present invention may also be used for diagnostic or therapeutic application for determining diseases associated with TTR with structural change (e.g. TTR amyloid) or for evaluating the therapeutic effect.

(102) The present invention further includes a carrier used for removal of TTR amyloid comprising the antibody of (1) or a fragment thereof (a carrier for removal of TTR amyloid). This carrier for removal may be prepared by binding by a usual method the antibody and the like to a carrier that is normally used in chromatography. The above carrier for removal is used in such a manner that blood is taken from patients suffering from amyloidosis caused by TTR amyloid and is passed through a column filled up with the carrier for removal to thereby remove TTR amyloid in blood.

(103) Furthermore, the present invention includes a reagent for detecting TTR amyloid comprising the antibody or a fragment thereof of the above item 1 (a reagent for detecting TTR amyloid). Thus, when label immunoassay such as radioimmunoassay, enzyme immunoassay and fluorescent immunoassay is applied, TTR in a test sample can qualitatively or quantitatively be analyzed in a rapid and accurate manner. In the label immunoassay, the above antibody or a fragment thereof is used with a label of e.g. a radioactive substance, an enzyme and/or a fluorescent substance. Besides, the antibody or a fragment thereof specifically reacts with TTR amyloid to show an immune reaction and therefore the measurement of the immune reaction with the labelling substance as an index allows for detection of small quantities of TTR amyloid present in a test sample at high precision. Label immunoassay, as compared to bioassay, is characterized by that a large number of test samples can be analyzed at a time, that time and labor for analysis is small, and that analysis is at high precision.

(104) The present invention includes a diagnostic agent for TTR amyloidosis comprising the antibody or a fragment thereof of the above item 1. A method for diagnosing the disease of the present invention comprises measuring an amount of TTR amyloid in a test sample (blood, body fluid, tissue etc.) and diagnosing the disease in accordance with the results of the measurement. The disease of interest includes the one caused by TTR amyloid, including Senile Systemic Amyloidosis (SSA) and Familial Amyloidosis (FAP).

(105) The antibody of the present invention proved to show the effect to suppress fibrillization of TTR. Therefore, the present invention includes a medicament for inhibiting fibrillization of TTR comprising the antibody or a fragment thereof of the above item 1 (a fibrillization inhibitor). The fibrillization inhibitor may contain pharmaceutically acceptable additives such as one or more kinds of excipients, one or more kinds of binding agents, one or more kinds of disintegrating agents, one or more kinds of lubricants and one or more kinds of buffers.

(106) The human antibody of the present invention proved to show the effect when administered to a model animal of TTR amyloidosis. Therefore, the present invention includes a pharmaceutical composition for preventing and/or treating TTR amyloidosis comprising the antibody or a fragment thereof of the above item 1. The pharmaceutical composition may contain pharmaceutically acceptable additives such as one or more kinds of excipients, one or more kinds of binding agents, one or more kinds of disintegrating agents, one or more kinds of lubricants and one or more kinds of buffers.

(107) The present invention is further explained in more detail by means of the following Examples but is not construed to be limited thereto. When the commercially available kits or reagents are used, the experiments were performed in accordance with protocol attached thereto unless otherwise mentioned.

Example 1

(108) Preparation of Purified Recombinant S112I TTR

(109) Referring to Matsubara et al. (Non-patent reference 13 and Non-patent reference 15), a purified recombinant TTR was prepared. E. coli strain M15 was transfected with S112I TTR (mutant human TTR where serine at position 112 was altered to isoleucine) expression vector pQE30-hTTR(S112I)-His and cultured with 20 mL of LB/Ampicillin (50 μg/mL)/Kanamycin (25 μg/mL) at 37° C. At the point of O.D.600 nm=0.5, IPTG was added at a final concentration of 10 mM and culture was continued overnight. The cells were collected from the culture by centrifugation and suspended in Buffer A (50 mM PB+0.3 M NaCl+10 mM Imidazole+20 mM 2-Mercaptoethanol). The suspension was sonicated for 15 minutes and then centrifuged to collect supernatant. The supernatant was subject to His-tag purification with Ni-NTA Agarose (QIAGEN) and the eluent fraction containing the recombinant TTR was dialyzed against 20 mM NaHCO.sub.3. The recombinant TTR after dialysis was purified by gel filtration with Superdex 75 (GE Healthcare) using 10 mM PB (pH7.5) and a fraction of dimeric TTR was used as a purified recombinant TTR S112I.

Example 2

(110) Cloning of Human TTR Gene

(111) For constructing a human TTR expression vector, cloning of a human TTR gene was performed. Using Human liver Marathon-Ready cDNA (Clontech) as a template, PCR was conducted using primers (TTR-F2: SEQ ID NO:17 and TTR-R: SEQ ID NO:18), designed at the 5′-end and the 3′-end of mature TTR, and Ex-Taq (Takara). After TA cloning of the PCR products into pCR2.1-TOPO, the nucleotide sequence of a human TTR gene was confirmed by sequence analysis. After confirming that the sequence was correct, pCR2.1-TOPO where the TTR gene was inserted was treated with BamHI and HindIII to cleave a region containing the sequence coding for the TTR gene. The cleaved sequence was introduced into pQE-30 (QIAGEN) previously treated with BamHI and HindIII to construct a wild-type human TTR expression vector.

Example 3

(112) Construction of Human TTR Mutant Expression Vector

(113) Using the human TTR expression vector constructed in Example 2 as a template, point mutation of an amino acid was introduced using site-directed mutagenesis. Point mutation of an amino acid was conducted for each of 24 kinds of mutations D18G, V30M, E54K, L55P, Y114C, V122I, K76A, S77A, Y78A, W79A, K80A, A81S, L82A, G83A, I84A, S85A, P86A, F87A, H88A, E89A, H90A, A91A, E92A, and V93A. The sequences coding for the above 16 kinds of the TTR mutants were introduced into pQE-30.

Example 4

(114) Preparation of Purified Recombinant TTR

(115) E. coli strain M15 was transfected with the expression vectors constructed in Examples 2 and 3 which express the wild-type TTR or D18G, V30M, E54K, L55P, Y114C, Y116S or V122I TTR mutants and purified recombinant TTRs were prepared by the procedures of Example 1. For the above TTR mutants, a tetrameric TTR fraction was used as a purified recombinant TTR.

Example 5

(116) Isolation of Anti-Human TTR Antibody

(117) An antibody to a human TTR with structural change was isolated by screening scFv phage display library prepared with human VH and VL cDNAs from mRNA from human B cells (e.g. the lymph node and the spleen). The antibody library used is excellent one containing more than 10.sup.11 kinds of various antibody molecules.

(118) The purified S112I TTR mutant prepared in Example 1 was diluted with 10 mM PB (pH 7.5) at 3 mg/mL and mixed with an equivalent amount of 200 mM acetate buffer+100 mM NaCl (pH 4.4) at a concentration of 1.5 mg/mL. The mixture was reacted in an incubator at 37° C. for 16 hours to prepare S112I TTR fibril. For TTR after the reaction, fluorescence intensity was measured by ThioflavinT assay to confirm the progress of fibrillization. ThioflavinT assay was performed by diluting the mixture with 50 mM Glycine-NaOH Buffer (pH 9.5) so that ThioflavinT is 20 μM and TTR is 30-60 μg/mL and measuring fluorescence intensity with a spectral fluorescence photometer FP-6500 (JASCO) (excitation wavelength 440 nm, fluorescent wavelength 480 nm).

(119) S112I TTR fibril was immobilized to Maxisorp plate (Nunc) using the conventional procedures and scFv phages specifically binding to S112I TTR fibril were obtained using the conventional procedures (Antibody Phage Display Methods and protocols Edited by Philippa M. O'Brien and Robert Aitken). The clones of the obtained scFv-phages were named “371” and “313”. The binding activity of the obtained scFv phages was evaluated by the method as described below.

Example 6

(120) Preparation of Recombinant TTR Fibril

(121) Seven kinds of the purified mutant TTRs D18G, V30M, E54K, L55P, Y114C, Y116S and V122I prepared in Example 4 and the purified wild-type TTR were diluted with 10 mM PB (pH 7.5) at 3 mg/mL and mixed with an equivalent amount of 200 mM acetate buffer+100 mM NaCl (pH 3.0) at a concentration of 1.5 mg/mL. The mixture was reacted in an incubator at 37° C. overnight to prepare TTR fibril. For TTR after the reaction, fibrillization was confirmed by ThioflavinT assay.

Example 7

(122) Binding Activity Test of Anti-TTR Antibody

(123) The antibody binding activity of the obtained scFv phages was evaluated by ELISA. The S112I TTR fibril prepared in Example 5 and the V30M TTR fibril prepared in Example 6 were diluted with PBS (SIGMA) at 10 μg/mL. Each 50 μL/well of the diluent was added to Maxisorp Plate (Nunc) and incubated at room temperature for 1 hour to immobilize TTR. Each 300 μL/well of 1% BSA-PBS was added to the immobilized plate and incubated at room temperature for 1 hour for blocking the plate. Each 100 ML of the culture supernatant of the obtained scFv phages was added to each well of the plate and incubated at 37° C. After 1 hour, the well was washed with PBST and each 100 μL of the detection antibody anti-M13/HRP (GE Healthcare) diluted 5000-folds with 1% BSA-PBS was added to each well of the plate and incubated at 37° C. After 1 hour, the well was washed with PBST and each 100 μL of TMB (SIGMA) was added to each well of the plate for development. After 30 minutes, the reaction was quenched with IN sulfuric acid and color development value (O.D. 450 nm/650 nm) was measured with a microplate reader (Molecular Devices).

(124) As a result, scFv371 and scFv313 were found to have an excellent binding activity to S112I TTR fibril and V30M TTR fibril.

Example 8

(125) Sequence Analysis of scFv371 and scFv313

(126) The nucleotide sequences of VH region or VL region of the obtained scFv371 and scFv313 were confirmed. The DNA nucleotide sequences contained in both phages were determined with Big Dye Terminator v3.1 Cycle sequencing kit (Applied Biosystems).

(127) ScFv371 and scFv313 where several amino acid residues were modified in VH region or VL region were designed and constructed and named 371M antibody and 313M antibody, respectively. CDR1-3 in VH region or VL region of 371M and 313M were determined by Kabat numbering. The amino acid sequences and the nucleotide sequences of said CDR1-3 are shown in SEQ ID NOs: 1 to 12. The amino acid sequences and the nucleotide sequences of VH region or VL region are shown in SEQ ID NOs: 13 to 16.

Example 9

(128) Expression of Chimeric 371 Antibody and Chimeric 313 Antibody

(129) The V region sequences of the obtained scFv371 and scFv313 were introduced into mouse IgG1λ expression vector and expression of these antibodies having mouse constant regions (hereinafter referred to as “chimeric 371 antibody” and “chimeric 313 antibody”) were performed. Using as a template the plasmids contained in scFv371 and scFv313 phages obtained in Example 5, a region containing VH and VL of scFv371 and scFv313 in the combination as shown in the following table was amplified by PCR. Table 5 shows a combination of primers and SEQ ID NOs.

(130) TABLE-US-00005 TABLE 5 VH VL 5′ - 3′ - 5′ - 3′ - 371 371-VH-Fw 371-VH-Rv 371/313-VL- 371/313-VL- SEQ ID SEQ ID Fw SEQ Rv SEQ NO: 19 NO: 20 ID NO: 21 ID NO: 22 313 313-VH-Fw 313-VH-Rv 371/313-VL- 371/313-VL- SEQ ID SEQ ID Fw SEQ Rv SEQ NO: 23 NO: 24 ID NO: 21 ID NO: 22

(131) The amplified sequence of VH region was introduced into the vector pKMA010-mCg1 (previously treated with XhoI and NruI) expressing mouse Cγ1 constant region and the amplified sequence of VL region was introduced into the vector pKMA009-mCλ (previously treated with XhoI and BamHI) expressing mouse Cλ constant region, using In-fusion enzyme (Clontech) (FIG. 1). pKMA010-371/313-mCg1 vector is such that the VH region of scFv371 or scFv313 and the mouse Cγ1 constant region are inserted downstream CAG promoter and has Dhfr gene as a drug resistant gene. pKMA009-371/313-mCλ vector is such that the VL region of scFv371 or scFv313 and the mouse Cλ constant region are inserted downstream CAG promoter. pKMA010-371-mCg1 and pKMA009-371-mCλ vectors are as a whole referred to as chimeric 371 antibody expression vector. pKMA010-313-mCg1 and pKMA009-313-mCλ vectors are as a whole referred to as chimeric 313 antibody expression vector.

(132) Using VH region or VL region of scFv371 and scFv313 as a template, mutation was introduced using site-directed mutagenesis to prepare VH region and VL region of 371M antibody and 313M antibody designed in Example 8. In the same manner as described above, expression vectors of these antibodies having mouse constant region (hereinafter referred to as “chimeric 371M antibody” and “chimeric 313M antibody”) were constructed. pKMA010-371M/313M-mCg1 vector is such that the VH region of 371M or 313M and the mouse Cγ1 constant region are inserted downstream CAG promoter. pKMA009-371M/313M-mCλ vector is such that the VL region of 371M or 313M and the mouse CA constant region are inserted downstream CAG promoter. pKMA010-371M-mCg1 and pKMA009-371M-mCλ are as a whole referred to as chimeric 371M antibody expression vector. pKMA010-313M-mCg1 and pKMA009-313M-mCλ are as a whole referred to as chimeric 313M antibody expression vector.

(133) Next, vectors expressing 371M antibody and 313M antibody having human constant region (hereinafter referred to as “human 371M antibody” and “human 313M antibody”, respectively) were constructed as described below. Using as a template the chimeric 371M antibody expression vector and the chimeric 313M antibody expression vector constructed above, a region containing VH and VL of 371M and 313M in the combination as shown in the following table was amplified by PCR. Table 6 shows a combination of primers and SEQ ID NOs.

(134) TABLE-US-00006 TABLE 6 VH VL 5′ - 3′ - 5′ - 3′ - 371M 371-VE- 371-VH-Rv 371/313-VL- 371/313-VL- Fw SEQ ID SEQ ID Fw SEQ Rv SEQ ID NO: 19 NO: 20 ID NO: 21 NO: 22 313M 313M-VH- 313-VH-Rv 371/313-VL- 371/313-VL- Fw SEQ ID SEQ ID Fw SEQ Rv SEQ ID NO: 25 NO: 24 ID NO: 21 NO: 22

(135) The amplified sequence of VH region was introduced into the vector pKMA010-hCg1 (previously treated with XhoI and BamHI) expressing human Cγ1 constant region and the amplified sequence of VL region was introduced into the vector pKMA009-hCL (previously treated with XhoI and BamHI) expressing human CA constant region, using In-fusion enzyme (Clontech). pKMA010-371/313-hCg1 vector is such that the H chain sequence of 371M or 313M antibody is inserted downstream CAG promoter and has Dhfr gene as a drug resistant gene. pKMA009-371/313-hCL vector is such that the L chain sequence of 371M or 313M antibody is inserted downstream CAG promoter. pKMA010-371-hCg1 and pKMA009-371-hCL are as a whole referred to as human 371M antibody expression vector. pKMA010-313-hCg1 and pKMA009-313-hCL are as a whole referred to as human 313M antibody expression vector. Relationship between various vectors and the inserted sequences is shown in the following table.

(136) TABLE-US-00007 TABLE 7 Chimeric 371 antibody pKMA010- 371 VH/mouse Cγ1 expression vector 371-mCg1 constant region pKMA009- 371 VL/mouse Cλ 371-mCλ constant region Chimeric 313 antibody pKMA010- 313 VH/mouse Cγ1 expression vector 313-mCg1 constant region pKMA009- 313 VL/mouse Cλ 313-mCλ constant region Chimeric 371M antibody pKMA010- 371M VH/mouse Cγ1 expression vector 371M-mCg1 constant region pKMA009- 371M VL/mouse Cλ 371M-mCλ constant region Chimeric 313M antibody pKMA010- 313M VH/mouse Cγ1 expression vector 313M-mCg1 constant region pKMA009- 313M VL/mouse Cλ 313M-mCλ constant region Human 371M antibody pKMA010- 371M VH/human Cγ1 expression vector 371M-hCg1 constant region pKMA009- 371M VL/human Cλ 371M-hCL constant region Human 313M antibody pKMA010- 313M VH/human Cγ1 expression vector 313M-hCg1 constant region pKMA009- 313M VL/human Cλ 313M-hCL constant region

(137) Freestyle293F cells (Invitrogen) were transfected with vectors expressing H chain and L chain of chimeric 371, 313, 371M and 313M antibodies and human 371M and 313M antibodies using Neofection (ASTEC Co., Ltd.) and were subject to shaking culture at 37° C. under environmental conditions of 8% CO.sub.2 at 125 rpm for expression of various antibodies. On the fifth day of culture, the culture supernatant was collected and purified by chromatography using HiTrap rProteinA FF (GE Healthcare). The elution fraction containing each antibody was dialyzed against PBS (SIGMA) to provide the purified form of chimeric 371 antibody, chimeric 313 antibody, chimeric 371M antibody, chimeric 313M antibody, human 371M antibody and human 313M antibody, respectively.

Example 10

(138) Epitope Analysis of 371M Antibody

(139) For more fully analyzing the epitope of 371M antibody, the reactivity analysis of 371M antibody was performed using the TTR alanine-substitution variant constructed in Example 3. E. coli strain M15 was transfected with the TTR variant expression vector constructed in Example 3 and cultured in 20 mL of LB/Ampicillin (50 μg/mL)/Kanamycin (25 μg/mL) at 37° C. At the point of O.D.600 nm=0.5, IPTG was added at a final concentration of 1 mM and the culture was continued overnight. The culture was centrifuged and the precipitate fraction was solubilized with Bugbuster (Merck). The solubilized cell suspension was electrophoresed on 8-16% SDS-PAGE gel and transferred to Immobilon-P (Millipore) from the gel. The transferred membrane was added with 2% Skimmilk-PBST and shaken at room temperature for 1 hour for blocking the membrane. Chimeric 371M antibody was diluted with 2% Skimmilk-PBST at a concentration of 1 μg/mL and the membrane was added with 10 mL of the diluent and shaken at room temperature for 1 hour. The membrane was washed with PBST, added with a detection antibody HRP-labelled anti-mouse IgG(H+L) (AMERICAN QUALEX INTERNATIONAL), which was previously diluted 5000-folds with 2% Skimmilk-PBST, and was shaken at room temperature for 1 hour. After washing with PBST, color development was conducted with Ez West Blue (ATTO).

(140) As a result, as shown in FIG. 2, it was found that 371M antibody had an epitope at positions 78-89 of human TTR.

Example 11

(141) Reaction Specificity Analysis of 371M Antibody and 313M Antibody

(142) For analyzing the reactivity of 371M antibody and 313M antibody to TTR tetramer, reaction specificity analysis was performed using surface plasmon resonance. Using Biacore2000 (GE Healthcare), around each 1,000 RU of WT TTR tetramer, V30M TTR tetramer and V30M TTR fibril (prepared in Examples 4 and 6; recombinant) was immobilized on Sensorchip CM5 (GE Healthcare). Immobilization of the ligand was performed with 10 mM acetate buffer (pH 6.0). Polyclonal Rabbit Anti-Human Prealbumin (Dako), chimeric 371M antibody, chimeric 313M antibody and negative control antibody, which were diluted with HBS-EP Buffer at 10 μg/mL, were migrated at 20 μL/min for 2 minutes. After migration, dissociation was carried out for 60 minutes and regeneration was performed with 10 mM Gly-NaOH (pH 9.0) for 30 seconds.

(143) As a result, as shown in FIG. 3, it was revealed that 371M antibody and 313M antibody did not react with WT TTR and V30M tetrameric TTRs but specifically recognized V30M fibril ((a) and (b)). Polyclonal antibody manufactured by Dako strongly reacted with WT TTR and V30M TTR and weakly reacted with V30M TTR fibril (c). Negative control antibody reacted with none of the TTRs (d).

Example 12

(144) Reactivity Analysis of 371 Antibody and 313 Antibody to Patient Sera

(145) Analysis was conducted to investigate whether 371 antibody and 313 antibody show the reactivity to sera from FAP patients. It is a preferable property for an antibody for FAP therapy that the administered antibody does not recognize human TTR in patient sera. Sera from healthy adults and sera from FAP patients having V30M TTR variant at 2 μg/mL and fibril of wild-type TTR from sera treated with an acid as in Example 6 and TTR amyloid extracted from the spleen of FAP patients at about 4 μg/mL were added to Maxisorp plate (Nunc) at 100 μL/well for immobilization of the antigens. Each 300 μL/well of 1% BSA-PBS was added to the immobilized plate and incubated at room temperature for 1 hour for blocking the plate. Chimeric 371 antibody and chimeric 313 antibody were serially diluted with 1% BSA-PBS and each 100 μL of the diluent was added to each well of the plate and incubated at 37° C. After 1 hour, the well was washed with PBST and each 100 μL of the detection antibody anti-mouse IgG(H+L)/HRP (Zymed) was added to each well of the plate and incubated at 37° C. After 1 hour, the well was washed with PBST and each 100 μL of TMB (SIGMA) was added to each well of the plate for development. After 30 minutes, the reaction was quenched with IN sulfuric acid and color development value (O.D. 450 nm) was measured with a microplate reader (Molecular Devices).

(146) As a result, as shown in FIG. 4, 371 antibody (a) and 313 antibody (b) clearly showed the reactivity to TTR amyloid from FAP patients and to amyloid formed by acid treatment of wild-type TTR from sera in a concentration-dependent manner but not to sera from healthy adults and from FAP patients.

Example 13

(147) Reactivity Analysis of 371M Antibody and 313M Antibody to Patient Tissues

(148) The heart was removed from FAP patients having V30M TTR and formalin fixed. The fixed heart tissue was embedded in paraffin block to prepare tissue section. After the tissue section was sliced to a thickness of 4 μm and attached to an object glass, deparaffinization treatment was conducted. After washing with PBS, the tissue section was infiltrated to 0.1% periodic acid dihydrate for 10 minutes and further washed with PBS. The tissue section was immersed in Rabbit serum (Dako) diluted 50-folds with 0.5% BSA-PBS for 1 hour for blocking. After washing with PBS, the tissue section was immersed in chimeric 371 antibody/chimeric 313 antibody/chimeric 371M antibody/chimeric 313M antibody/negative control antibody as a primary antibody, which were diluted with 0.5% BSA-PBS to 10 μg/mL, at 4° C. overnight. The tissue section was then immersed in HRP-labelled Rabbit anti-mouse IgG (Dako) as a secondary antibody, which was diluted 100-folds with 0.5% BSA-PBS, at room temperature for 1 hour. After washing with PBS, development with DAB was conducted. Hematoxylin staining was also done. For positive control, the same procedures were performed using Polyclonal Rabbit Anti-Human Prealbumin (Dako) as a primary antibody and HRP-labelled Goat anti-Rabbit IgG (Dako) as a secondary antibody. Besides, taking into consideration the possibility that TTR is denatured by formalin fixation to thereby alter its steric structure, frozen tissue section of the heart of FAP patients having V30M TTR was also subject to immunostaining in like manner. Furthermore, for confirming the presence of amyloid fibril, Congo red staining was also conducted. Congo red is known to attach to amyloid fibril to thereby cause short-wavelength shift.

(149) As a result, as shown in FIG. 5, it was confirmed that 371 antibody/313 antibody/371M antibody/313M antibody specifically recognized TTR deposited in the heart from FAP patients in both the paraffin section (FIG. 5a) and the frozen section (FIG. 5b).

Example 14

(150) Reactivity Analysis of 371M Antibody and 313M Antibody to TTR Fibril

(151) Each 1.5 μg of seven kinds of the TTR fibrils prepared in Example 6 and the purified wild-type TTR was electrophoresed on 8-16% SDS-PAGE gel and transferred to Immobilon-P (Millipore) from the gel. The transferred membrane was added with 2% Skimmilk-PBST and shaken at room temperature for 1 hour for blocking the membrane. Chimeric 371M antibody or chimeric 313M antibody was diluted with 2% Skimmilk-PBST at a concentration of 1 μg/mL and the membrane was added with 10 mL of the diluent and shaken at room temperature for 1 hour. The membrane was washed with PBST, added with a detection antibody anti-mouse IgG(H+L), which was previously diluted 5000-folds with 2% Skimmilk-PBST, and was shaken at room temperature for 1 hour. After washing with PBST, color development was conducted with Ez West Blue (ATTO).

(152) As a result, as shown in FIG. 6, it was found that 371M antibody and 313M antibody recognized various TTR fibrils but on the other hand did not recognize purified wild-type TTR.

Example 15

(153) Construction of Measurement System for Inhibitory Activity to V30M TTR-Fibrillization

(154) Recombinant V30M TTR was diluted with PBS(−) to 375 μg/mL and mixed with four kinds of surfactants at a final concentration of 0.1%, 0.01% and 0.001%. The surfactants used were (1) benzalkonium chloride (Yamazen Corporation), (2) sodium deoxycholate (Nacalai Tesque), (3) Zwittergent3-16 (Carbiochem), and (4) NP-40 (Wako). The mixtures were left to stand at 37° C. for 4 days and fluorescence intensity was measured by ThioflavinT assay (excitation wavelength 440 nm, fluorescent wavelength 480 nm) to evaluate a degree of TTR-fibrillization.

(155) The results of ThioflavinT assay are shown in FIG. 7. For any of the surfactants, TTR-fibrillization progressed when used at a concentration of 0.1%. Among others, it was found that TTR-fibrillization progressed most rapidly when sodium deoxycholate was used. Next, an optimum concentration of sodium deoxycholate was investigated. Recombinant V30M TTR was diluted with PBS(−) to 375 μg/mL and mixed with sodium deoxycholate at a concentration of 1%, 0.5%, 0.2%, 0.1% and 0.01%. The mixtures were left to stand at 37° C. and, after 4 days and 7 days, ThioflavinT assay was conducted to evaluate a degree of TTR-fibrillization.

(156) The results are shown in FIG. 8. The most remarkable fibrillization was observed under the conditions of 1% of a concentration of sodium deoxycholate. However, since fibrillization was observed even at 0 hour (immediately after addition), it was thought that progress of fibrillization was too rapid. To the contrary, under the conditions of 0.1%, fibrillization was observed after the mixtures were left to stand overnight or for a longer period of time and remarkable progress of fibrillization most depending on treatment time was observed as compared to the conditions of 0.5%, 0.2% and 0.01%. From these, it was found that the optimum concentration of sodium deoxycholate was 0.1%. Up till the present, the conditions under which fibrillization of V30M TTR progresses under the circumstance of neutral pH have not yet been reported and thus fibrillization of TTR was made to progress by placing TTR under acidic pH circumstances such as pH 3.0. On the other hand, since an antibody is denatured and loses its activity when it is exposed to acidic circumstances, it has been difficult to evaluate the inhibitory ability of anti-TTR antibody to TTR-fibrillization. In accordance with the present invention, it has newly been found that fibrillization of V30M TTR progresses even under neutral circumstances by introducing sodium deoxycholate into the system to thereby succeed in constructing the system which allows for evaluation of the inhibitory ability of anti-TTR antibody to fibrillization.

Example 16

(157) V30M TTR-Fibrillization Inhibition Test of 371M Antibody and 313M Antibody

(158) Purified V30M TTR, and 371 antibody, 313 antibody, 371M antibody, 313M antibody or negative control antibody were mixed together at a molar ratio of 10 μM:0.01 to 2 μM (TTR: 550 μg/mL, antibodies: 1.5 to 300 μg/mL) and the mixture was left to stand under PBS+0.1% sodium deoxycholate at 37° C. for 3 days. Using the samples after being left to stand, ThioflavinT assay (excitation wavelength 440 nm, fluorescent wavelength 480 nm) was performed to measure fluorescence intensity.

(159) As a result, as shown in FIG. 9, it was found that 371, 313, 371M and 313M antibodies had the activity to inhibit fibrillization of V30M TTR in an antibody concentration-dependent manner.

Example 17

(160) Macrophage Phagocytic Ability Test

(161) To investigate whether 371M antibody and 313M antibody promote the ability of macrophage to phagocytose TTR fibril, macrophage phagocytic ability test was performed. This test mimics the process where macrophage removes TTRs deposited in the tissues of TTR patients. If the phagocytic ability of macrophage is promoted by the addition of these antibodies, it is expected that these antibodies have the activity to promote removal of TTR deposition in human tissues.

(162) Human iPS cells were prepared from the skin tissue from FAP patients in accordance with the method described in Non-patent reference 16 and further differentiated into macrophages (iPS-MP). iPS-MPs (1 to 2×10.sup.6 cells) were cultured in the presence of 50 ng/mL hGM-SCF and 25 μg/mL M-CSF in 10 cm dish for 24 hours. iPS-MPs were washed with PBS, then incubated in a medium containing 20 μg/mL of mitomycin C at 37° C. for 10 minutes to suspend the cell proliferative ability and added to 96-well plate at 5×10.sup.4 cells/100 μL/well. V30M TTRs, untreated or acid treated for 24 hours, were diluted with the culture medium to 3.2 μg/mL and each 50 μL of the dilution was added. In addition, PBS/human 371M antibody/human 313M antibody/negative control antibody were diluted to 40 μg/mL and each 50 μL was added. Culture was continued at 37° C. under 5% CO.sub.2 for 3 days and thereafter culture supernatant was collected.

(163) A residual quantity of TTR after culture was quantified by ELISA as described below to evaluate the phagocytic ability of macrophage. A 96-well plate was added with each 5 μL of the culture supernatant and with 100 μL of a coating solution (25 mM sodium carbonate buffer) and thereafter was left to stand at 4° C. overnight. After washing with PBST, 250 μL of a blocking solution (a solution of 0.5% gelatin dissolved in the coating solution) was added and the plate was incubated at room temperature for 1 hour. After washing with PBST, Polyclonal Rabbit Anti-Human Prealbumin (Dako) was diluted 1,000-folds with 0.05% gelatin-PBST, each 100 μL of the dilution was added and the plate was incubated at room temperature for 1 hour. After washing with PBST, HRP-labelled Goat anti-Rabbit IgG (Dako) was diluted 5,000-folds with 0.05% gelatin-PBST, each 100 μL of the dilution was added and the plate was incubated at room temperature for 1 hour. After washing with PBST, development was performed with 100 μL of SureBlue (KPL) for 5 minutes and stopped with 100 μL of 1 M hydrochloric acid. A wavelength at 450 nm was measured with xMARK microplate reader (Bio-Rad Laboratories).

(164) The results are shown in FIG. 10. For untreated purified V30M, no statically significant difference in a residual quantity of TTR could be seen between the samples (a). To the contrary, it was found that, for TTR fibril, statistically significant reduction in a residual quantity of TTR was observed for 371M antibody and 313M antibody as compared to PBS (none)(b), demonstrating that 371M antibody and 313M antibody had the activity to promote the phagocytic activity of iPS cell-differentiated macrophages to TTR fibril.

Example 18

(165) Drug Efficacy Evaluation Test Using V30M Tg Rat

(166) Using V30M Tg rat (Non-patent reference 14; transgenic rat where a gene of human TTR with mutation of valine at position 30 to methionine in the amino acid sequence of TTR is introduced), each 10 mg/kg of chimeric 371 antibody and chimeric 313 antibody or PBS was administered for 6 months, from 3-month old to 9-month old, each group consisting of 7 or 8 rats, once per week 26 times in total. After administration, the large intestine was taken out by autopsy and formalin fixed. The fixed tissue of the large intestine was embedded in a paraffin block to prepare tissue section. The tissue section was subject to immunostaining using Polyclonal Rabbit Anti-Human Prealbumin (Dako) as a primary antibody and HRP-labelled Goat anti-Rabbit IgG (Dako) as a secondary antibody and a degree of TTR deposition in the muscular layer of the large intestine was digitized and compared between the groups.

(167) As a result, as shown in FIG. 11, TTR deposition was significantly suppressed in the group of 371 antibody administration and the group of 313 antibody administration as compared to the group of PBS administration.

INDUSTRIAL APPLICABILITY

(168) The recombinant human anti-transthyretin antibody of the present invention, as being excellent in its activity (the inhibitory activity to TTR fibrillization, the activity to promote the phagocytic ability of macrophage, etc.) and/or specificity (specifically recognizes TTRs with structural change and TTR fibril), is useful as an effective medicament to various diseases associated with structural change or fibrillization of TTR.