ADAMTS13 VARIANT HAVING INCREASED ESCAPING RATE OR ACTIVITY AGAINST AUTOANTIBODY

Abstract

An ADAMTS13 mutant protein having an improved escaping rate against an autoantibody and a composition that is suitable for preventing or treating thrombotic diseases are disclosed. By efficiently avoiding autoantibodies known to have high binding affinity to the main domain of ADAMTS13, the ADAMTS13 variant protein of the present invention can be used as an effective therapeutic composition for various thrombotic diseases, such as TTP (thrombotic thrombocytopenic purpura), in which the presence of such autoantibodies is the main etiology, and can stably maintain the biological activity thereof when administered into a body. In addition, a newly identified site within ADAMTS13, which is recognized by an autoantibody can be used in screening novel ADAMTS13 variants having an improved autoantibody escaping rate by applying a combination of various mutations within the corresponding site.

Claims

1. An ADAMTS13 (a disintegrin and metalloproteinase with a thrombospondin type 1 motif, member 13) variant protein comprising substitutions of one or more amino acid residues selected from the group consisting of the 85.sup.th, 93.sup.rd, 126.sup.th, 135.sup.th, 278.sup.th, 282.sup.nd, 308.sup.th, 314.sup.th, 317.sup.th, 334.sup.th, 364.sup.th, 376.sup.th, 413.sup.rd, 427.sup.th, 452.sup.nd, 465.sup.th, 567.sup.th, 578.sup.th, 585.sup.th, 589.sup.th, 607.sup.th, 608.sup.th, 609.sup.th, 612.sup.th, 618.sup.th, 624.sup.th, 630.sup.th, 635.sup.th, 643.sup.rd, 650.sup.th, 651.sup.st, 654.sup.th, 655.sup.th, 656.sup.th, 658.sup.th, 664.sup.th, and 672.sup.nd residues of SEQ ID NO: 1, or a functional fragment thereof.

2. The ADAMTS13 variant protein or a functional fragment thereof of claim 1, wherein the variant protein of ADAMTS13 is selected from the group consisting of each of the variant proteins comprising substitutions of the amino acid residues at the following positions: the 85.sup.th and 317.sup.th residues; the 612.sup.th residue; two or more among the 282, 465.sup.th, and 672.sup.nd residues; the 635.sup.th residue; the 452.sup.nd and 612.sup.th residues; two or more among the 278.sup.th, 334.sup.th, and 427.sup.th residues; the 618.sup.th residue; the 135.sup.th residue; two or more among the 126.sup.th, 567.sup.th, and 651.sup.st residues; the 413.sup.th residue; the 334.sup.th residue; the 314.sup.th residue; two or more among the 93.sup.rd, 364.sup.th, and 376.sup.th residues; the 308.sup.th residue; the 656.sup.th residue; the 607.sup.th residue; the 612 and 624.sup.th residues; the 589.sup.th residue; the 650.sup.th and 656.sup.th residues; the 643.sup.rd residue; the 585.sup.th and 658.sup.th residues; two or more among the 630.sup.th, 654.sup.th, and 664.sup.th residues; four or more among the 589.sup.th, 608.sup.th, 609.sup.th, 624.sup.th, and 655.sup.th residues; the 578.sup.th residue; the 585.sup.th residue; the 314.sup.th and 635.sup.th residues; and the 314.sup.th and 612.sup.th residues.

3. The ADAMTS13 variant protein or a functional fragment thereof of claim 1, wherein the substitution of the amino acid residue is at least one selected from the group consisting of substitution of the 85.sup.th residue with Phe, substitution of the 93.sup.rd residue with Val, substitution of the 126.sup.th residue with Met, substitution of the 135.sup.th residue with Ile, substitution of the 278.sup.th residue with Ile, substitution of the 282.sup.nd residue with Ala, substitution of the 308.sup.th residue with Lys, substitution of the 314.sup.th residue with Thr, substitution of the 317.sup.th residue with His, substitution of the 334.sup.th residue with Thr or Val, substitution of the 364.sup.th residue with Arg, substitution of the 376.sup.th residue with Asp, substitution of the 413.sup.th residue with Asp, substitution of the 427.sup.th residue with Asn, substitution of the 452.sup.nd residue with Ile, substitution of the 465.sup.th residue with Asp, substitution of the 567.sup.th residue with Ser, substitution of the 578.sup.th residue with Leu, substitution of the 585.sup.th residue with Asn or Met, substitution of the 589.sup.th residue with Gln, substitution of the 607.sup.th residue with Arg, substitution of the 608.sup.th with Met, substitution of the 609.sup.th residue with Leu, substitution of the 612.sup.th residue with Phe or Tyr, substitution of the 618.sup.th residue with Ser, substitution of the 624.sup.th residue with Asp or Cys, substitution of the 630.sup.th residue with Leu, substitution of the 635.sup.th residue with Val, substitution of the 643.sup.rd residue with Phe, substitution of the 650.sup.th residue with His, substitution of the 651.sup.st residue with Asp, substitution of the 654.sup.th residue with Gly, substitution of the 655.sup.th residue with Val, substitution of the 656.sup.th residue with Arg or His, substitution of the 658.sup.th residue with His, substitution of the 664.sup.th residue with Asn, and substitution of the 672.sup.nd residue with Val.

4. A fusion protein comprising: (a) the variant protein of ADAMTS13 or a functional fragment thereof of claim 1; and (b) an Fc region of the IgG4 immunoglobulin conjugated to (a).

5. The fusion protein of claim 4, wherein the Fc region comprises substitutions of one or more amino acid residues selected from the group consisting of the 22.sup.nd, 24.sup.th, and 26.sup.th residues of SEQ ID NO: 2.

6. The fusion protein of claim 5, wherein the 22.sup.nd residue is substituted with Tyr, the 24.sup.th residue with Thr, and the 26.sup.th residue with Glu, respectively.

7. The fusion protein of claim 5, further comprising a hinge region of an IgG1 immunoglobulin between said (a) and (b).

8. A nucleotide encoding the ADAMTS13 variant protein or a functional fragment thereof according to claim 1; or a fusion protein comprising (a) the variant protein of ADAMTS13 or a functional fragment thereof of claim 1 and (b) an Fc region of the IgG4 immunoglobulin conjugated to (a).

9. A composition comprising, as an active ingredient: (i) the ADAMTS13 variant protein or a functional fragment thereof according to claim 1; (ii) a fusion protein comprising (a) the variant protein of ADAMTS13 or a functional fragment thereof of claim 1; and (b) an Fc region of the IgG4 immunoglobulin conjugated to (a); (iii) a nucleotide encoding the ADAMTS13 variant protein or a functional fragment thereof of (i); (iv) a nucleotide encoding the fusion protein of (ii).

10.-11. (canceled)

12. A method for screening ADAMTS13 variant with an improved escaping rate for autoantibodies, comprising the following steps of: (a) preparing an ADAMTS13 variant, in which one or more amino acid residues selected from the group consisting of the 85.sup.th, 93.sup.rd, 126.sup.th, 135.sup.th, 278.sup.th, 282.sup.nd, 308.sup.th, 314.sup.th, 317.sup.th, 334.sup.th, 364.sup.th, 376.sup.th, 413.sup.rd, 427.sup.th, 452.sup.nd, 465.sup.th, 567.sup.th, 578.sup.th, 585.sup.th, 589.sup.th, 607.sup.th, 608.sup.th, 609.sup.th, 612.sup.th, 618.sup.th, 624.sup.th, 630.sup.th, 635.sup.th, 643.sup.rd, 650.sup.th, 651.sup.st, 654.sup.th, 655.sup.th, 656.sup.th, 658.sup.th, 664.sup.th, and 672.sup.nd residues of SEQ ID NO: 1 are substituted or deleted; (b) contacting an autoantibody for ADAMTS13 with the ADAMTS13 variant prepared in step (a) above; and (c) selecting ADAMTS13 variant to which the autoantibody shows a binding affinity lower than that for wild-type ADAMTS13.

13. The method of claim 12, wherein step (a) above is performed by substituting one or more amino acid residues selected from the group consisting of the 85.sup.th, 93.sup.rd, 126.sup.th, 135.sup.th, 278.sup.th, 282.sup.nd, 308.sup.th, 314.sup.th, 317.sup.th, 334.sup.th, 376.sup.th, 413.sup.rd, 427.sup.th, 465.sup.th, 567.sup.th, 578.sup.th,

Description

BRIEF DESCRIPTION OF DRAWINGS

[0066] FIGS. 1a and 1b show diagrams illustrating the construction of a human ADAMTS13 variant library using random mutagenesis and the results of confirming the variation rate. FIG. 1a is a schematic diagram of a library construction process by random mutagenesis. Random variations were induced in the MDTCS region or S domain through error-prone PCR (EP PCR), inserted into the acceptor vector, cloned, and then transformed into E. coli to secure colonies. DNA was extracted from the colonies and the mutated nucleotide sequences were confirmed through Sanger sequencing analysis. FIG. 1B shows graphs illustrating the results of sequencing analysis. The total variation rate and variation form in the library constructed by targeting the MDTCS or S domain were confirmed, respectively.

[0067] FIGS. 2a-2c diagrams illustrating the results of confirming the binding site of each domain of ADAMTS13 that binds to the neutralizing antibody. FIG. 2a shows a schematic diagram of six kinds of ADAMTS13 fragments (or wild-type full-length ADAMTS13), in which various domains are combined, prepared to confirm the expression rate of ADAMTS13 or the binding site of a neutralizing antibody. FIG. 2b shows the results of performing western blotting with an anti-V5 antibody after immunoprecipitation by mixing Protein A-Sepharose and each neutralizing antibody using proteins expressed in each ADAMTS13 domain. Input shows the expression level of the protein for each domain. FIG. 2c is a schematic diagram of the binding site of the ADAMTS13 neutralizing antibody illustrating that Ab 4-16 antibody specifically binds to the S domain, the Ab 67 antibody to the D domain, and the Ab 66 antibody to the T2-T8 domain, respectively.

[0068] FIGS. 3a and 3b show diagrams illustrating the screening results of variants that escape anti-ADAMTS13 antibodies or have superior activity to wild-type ADAMTS13. The escaping rate and ADAMTS13 activity for Ab 4-16 antibody or Ab 67 antibody were measured in the wild-type clones and the ADAMTS13 variants. Relative activity was calculated by confirming the specific titers of the wild-type clones and the variants and substituting them in the following equation: Relative activity (%)=specific titer of variants/specific titer of wild-type ADAMTS13100.

[0069] FIGS. 4a and 4b show the results confirming the escaping rate of 12 kinds of full-length ADAMTS13 variants for single neutralizing antibodies. Antibody escaping rates for 7 kinds of neutralizing antibodies (Ab4-16, Ab4-20, Ab60, Ab61, Ab64, Ab65, and Ab67) were measured in the 12 kinds of full-length ADAMTS13 variants. The neutralizing antibody escaping rate was calculated by comparing the relative binding level with the WT ADAMTS13 value, and substituting the relative escaping rate into the following equation using the same: Escaping rate (%)=(1binding ability of variants/binding ability of WT ADAMTS13)100.

[0070] FIGS. 5a and 5b show the results confirming the ability to escape single neutralizing antibodies in a culture medium expressing an Fc-attached MDTCS fragment variant. The selected 12 kinds of variants were subjected to MDTCS fragmentation, and then the Fc-attached variants were combined with the selected mutated amino acid residues, and the neutralizing antibody escaping rate and the relative activity of ADAMTS13 of eight kinds of neutralizing antibodies (Ab3-01, Ab4-16, Ab4-20, Ab60, Ab61, Ab64, Ab65, and Ab67) were measured using a culture medium in which a total of 14 kinds of variants including DM1 and DM2 variants having two amino acid variations were expressed. Escaping rate (%)=(1binding ability of variants/binding ability of WT ADAMTS13)100; relative activity (%)=specific titer of variants/specific titer of WT ADAMTS13100

[0071] FIG. 6 shows the results confirming the ability of escaping mixed neutralizing antibodies under a condition of a culture medium expressing an Fc-attached MDTCS fragment variant. Nine kinds of neutralizing antibodies (Ab3-01, Ab4-16, Ab4-20, Ab60, Ab61, Ab64, Ab65, Ab66, and Ab67), which were mixed at an equal ratio, were mixed into a 4 nM culture medium expressing MDTCS-Fc (i.e., a control group) and 12 kinds of candidate variants, and allowed to react. Then, the residual activity of each candidate variant was calculated by substituting into the following equation: Residual activity (%)=AB100 (A: activity under the condition of mixed neutralizing antibodies, B: activity under the condition of untreated with neutralizing antibodies).

[0072] FIGS. 7a and 7b show the results confirming the ability of escaping single neutralizing antibodies under the conditions of an Fc-attached MDTCS fragment variant culture medium and a purifying solution. The escaping rate and relative activity of neutralizing antibodies for eight kinds of single neutralizing antibodies (Ab3-01, Ab4-16, Ab4-20, Ab60, Ab61, Ab64, Ab65, and Ab67) were measured using the culture medium in which 12 kinds of variants (1C03, 2B01, 2B02, 3B05, 4E11, 4H07, 5G08, 7A02, 8D01, 8D05, DM1, and DM2) were expressed or a purifying solution purified using the Phytip system (FIGS. 7a and 7b). The concentration of the purifying solution was confirmed through Fc ELISA. Escaping rate (%)=(1binding ability of variants/binding ability of MDTCS-Fc)100, Relative activity (%)=specific titer of variants/specific titer of MDTCS-Fc100. Black bars indicate purified variant proteins and white bars indicate the median values of the expressed variant proteins, respectively.

[0073] FIG. 8 shows the results confirming the ability of escaping mixed neutralizing antibodies under the conditions of an Fc-attached MDTCS fragment variant culture medium and a purifying solution. Eight kinds of neutralizing antibodies (Ab3-01, Ab4-16, Ab4-20, Ab60, Ab61, Ab64, Ab65, and Ab67), which were mixed at an equal ratio, were mixed into a culture medium expressing MDTCS-Fc (i.e., a control group) and 12 kinds of candidate variants or a 4 nM purifying solution, and allowed to react. Then, the residual activity of each candidate variant was measured. The residual activity was calculated by substituting into the following equation: residual activity (%)=AB100 (A: activity under the condition of mixed neutralizing antibodies, B: activity under the condition of untreated with neutralizing antibodies).

[0074] FIG. 9 shows the graphs illustrating the pharmacokinetic results of Fc-attached MDTCS fragment variants. After administration of MDTCS or Fc (IgG1-YTE)-attached MDTCS and the four final candidate variants (1C03, 5C09, 7A02, and DM2) fragment proteins to the tail vein of mice, and plasma was obtained every hour. The protein was administered so that the specific titer of each material can be 160 IU/kg, and the activity of the materials remaining in the plasma obtained every hour was measured through the activity assay.

[0075] FIGS. 10a and 10b show the results confirming the ability of escaping single neutralizing antibodies under the condition of an IgG1-YTE-attached MDTCS fragment variant culture medium. The selected five kinds of variants were subjected to MDTCS fragmentation, and the rate of escaping neutralizing antibodies and relative activity of the eight kinds of single neutralizing antibodies (Ab3-01, Ab4-16, Ab4-20, Ab60, Ab61, Ab64, Ab65, and Ab67) were measured using the culture medium in which the IgG1-YTE-attached variants were expressed (FIGS. 10a and 10b). Escaping rate (%)=(1binding ability of variants/binding ability of MDTCS-IgG1-YTE)100; Relative activity (%)=specific titer of variants/specific titer of MDTCS-IgG1-YTE)100.

[0076] FIG. 11 shows the results confirming the ability of escaping mixed neutralizing antibodies under a condition of a culture medium expressing an IgG1-YTE-attached MDTCS fragment variant. Nine kinds of neutralizing antibodies (Ab3-01, Ab4-16, Ab4-20, Ab60, Ab61, Ab64, Ab65, Ab66, and Ab67), which were mixed at an equal ratio, were mixed into a 4 nM culture medium expressing MDTCS-IgG1-YTE (i.e., a control group) and five kinds of candidate variants, and allowed to react. Then, the residual activity of each candidate variant was calculated by the following equation: Residual activity (%)=AB100 (A: activity under the condition of mixed neutralizing antibodies, B: activity under the condition of untreated with neutralizing antibodies).

[0077] FIG. 12 shows the results confirming the ability of escaping mixed neutralizing antibodies under the condition of an IgG1-YTE-attached MDTCS fragment variant culture medium. Nine kinds of neutralizing antibodies (Ab3-01, Ab4-16, Ab4-20, Ab60, Ab61, Ab64, Ab65, Ab66, and Ab67), which were mixed at an equal ratio, were mixed into a 4 nM purifying solution with MDTCS-IgG1-YTE (i.e., a control group) and five kinds of candidate variants, and allowed to react. Then, the residual activity of each candidate variant was calculated by the following equation: Residual activity (%)=AB100 (A: activity under the condition of mixed neutralizing antibodies, B: activity under the condition of untreated with neutralizing antibodies).

[0078] FIGS. 13a and 13b show a schematic diagram illustrating a procedure for testing the rate of escaping neutralizing antibodies using an aTTP-mimic mouse model (FIG. 13a) and a diagram illustrating the residual ADAMTS13 activity according to the dose of variant candidates (FIG. 13b), respectively.

[0079] FIGS. 14a-14c show a schematic diagram illustrating a test procedure for maintaining residual activity of ADAMTS13 and clinical symptoms according to the concentration of DM2-IgG1-YTE, which showed the most excellent neutralizing antibody escaping rate using an aTTP-mimic mouse model (FIG. 14a), a schematic diagram illustrating an improvement in the platelet and LDH levels and ADAMTS13 activity (FIG. 14b), and the observation results of clinical symptoms (FIG. 14c), respectively.

[0080] FIGS. 15a and 15b show a schematic diagram illustrating a test procedure for the presence/absence of an improvement in hematological and clinical symptoms and the degree of recovery of human ADAMTS13 activity by administering DM2-IgG1-YTE to a cTTP mouse model (FIG. 15a), and an improvement of platelet and LDH levels and ADAMTS13 activity, and the observation results of the recovery of ADAMTS13 activity (FIG. 15b), respectively.

MODE FOR THE INVENTION

[0081] Hereinafter, the present invention will be described in more detail through examples. These examples are only for illustrating the present invention in more detail, and it will be apparent to those of ordinary skill in the art that the scope of the present invention is not limited by these examples according to the gist of the present invention.

EXAMPLES

Experimental Method

Construction of Human ADAMTS13 Variant Library Through Random Mutagenesis

[0082] An expression vector containing human wild-type ADAMTS13 and two receptor vectors for cloning of mutated MDTCS (Metalloprotease, Disintegrin-like, Thrombospondin type 1 (TSP1) repeat, Cys-rich, and Spacer) or Spacer (S) domain regions were prepared. Error-prone PCR (GeneMorph II Random Mutagenesis Kit, Agilent Technologies) was performed for the MDTCS or S domain region of ADAMTS13 using the oligonucleotides of Table 1 below, and the PCR product for the amplified MDTCS or S domain was obtained by elution after agarose gel electrophoresis eluted by electrophoresis. After ligation of the eluted PCR product and the acceptor vectors using the Golden Gate cloning method, the resultant was transformed into Escherichia coli (E. coli) to prepare a library of variants produced by random mutagenesis. Plasmid DNA of a transfection grade was extracted from 384 E. coli colonies per library using the Plasmid Plus 96 Miniprep kit (QIAGEN) to obtain a total of 768 mutated ADAMTS13 variants. The type and position of amino acid variations were confirmed through nucleotide sequence analysis of the corresponding DNA.

TABLE-US-00001 TABLE1 Nucleotidesequencesofprimersforerror-prone PCR pPrimer Sequence(5.fwdarw.3) MDTCS-Forward CACCGGTCTCNTAGAGCTGCTGGCGGCAT MDTCS-Forward CACCGGTCTCNTAGAGCTGCTGGCGGCAT MDTCS-Reverse GGCTGGTCTCNCCCAAGCCTGCCGAGGCTTA S-forward CACCGGTCTCATAGAAGAGAATACGTGACCTTCC TGAC S-reverse GGCTGGTCTCACCCAGTATTCCTCGCCGTATCTT CTGTA

Transient Expression of Human ADAMTS13 Variants

[0083] For the expression of the proteins of ADAMTS13 variants, Expi293F cells (Thermo Fisher, Cat. No. A14527) were prepared to a concentration of 310.sup.6 cells/mL by diluting in the Expi293 Expression Medium. In the first tube, 2.5 s of plasmid DNA was diluted in the opti-MEM I Reduced Serum Medium (Thermo Fisher, Cat. No. 31985-070) to a final volume of 152.5 L, and in the other tube, 8 L of ExpiFectamine 293 and 140 L of opti-MEM I Reduced Serum Medium were mixed and allowed to stand for 5 minutes. 152.5 L of the diluted plasmid DNA was added to the mixed solution in the second tube and allowed to stand for 20 minutes. 2.5 mL of the prepared cells was aliquoted into each well of the 24 deep well plates. The mixture of ExpiFectamine 293 and plasmid DNA prepared in advance was dispensed into the cells and cultured with shaking at 225 rpm in an incubator at 37 C. and CO.sub.2 conditions. After 24 hours, 15 L of ExpiFectamine transfection enhancer 1 and 150 L of enhancer 2 were added thereto and cultured for 5 days. On the 6.sup.th day, the culture was spun down at 300 ref for 5 minutes to collect the supernatant and the resultant was used for analysis for selection of variants.

Construction of Human and Mouse ADAMTS13 Neutralizing Antibodies

[0084] In order to construct ADAMTS13 neutralizing antibodies that bind to different domains, 16 kinds of Fabs having excellent binding ability to ADAMTS13 were selected using a full-length recombinant human ADAMTS13 (R&D Systems, Cat. No. 6156-AD) as an antigen and PLATINUM phage library of Human Combinatorial Antibody Library (HuCAL, Bio-Rad), and among them, six kinds of antibodies (Ab60, Ab61, Ab64, Ab65, Ab66, and Ab67) that bind to each of the MDTCS part and the C-terminal part were prepared as full-length antibodies. Ab3-01, Ab 4-16, and Ab 4-20, which are known to bind specifically to the S domain in the plasma of an aTTP patient, were also constructed as full-length antibodies and used for selection analysis of variants.

Measurement of Expression Concentration of Human ADAMTS13 Variant

[0085] In order to confirm the expression level of the ADAMTS13 variant protein present in a culture medium obtained through transient expression, ELISA was performed using an anti-ADAMTS13 antibody (Ab 66, GC Green Cross antibody). 100 L of the anti-ADAMTS13 antibody diluted to 2 g/mL in PBS (Lonza, 17-516Q) was dispensed per well in a 96-well EIA/RIA high bind microplate (Corning, Cat. No. 3590) and allowed to react at room temperature for 2 hours. Then, the microplate was washed three times using wash buffer (0.1 v/v % tween 20 in PBS). Blocking buffer (1 w/v % bovine serum albumin in PBS) was dispensed at 300 L per well and reacted at room temperature for 2 hours, followed by washing three times with wash buffer. Recombinant human ADAMTS13 (R&D Systems, 6156-AD) was used as a standard sample, and the culture to be measured was diluted 16-fold or 32-fold with blocking buffer, and dispensed at 100 L per well and reacted by stirring at 500 rpm for one hour at room temperature. After washing three times, an anti-6His tag antibody (Abcam, Cat. No. Ab1187) was diluted 1:10,000 and dispensed at 100 L per well, followed by reaction by stirring at 500 rpm at room temperature for 1 hour. After washing 6 times, TMB (3,3,5,5-tetramethylbenzidine) was dispensed at 100 L per well and allowed to react at room temperature for 10 minutes. Thereafter, stop buffer (H.sub.2SO.sub.4) was dispensed at 100 L per well, and the absorbance was measured at 450 nm using a microplate reader. After substituting the measured absorbance into the standard sample response curve and calculating the concentration, the dilution factor was calibrated to determine the final concentration.

Measurement of Expression Concentrations of Fc Fusion MDTCS Fragments and Variants

[0086] Goat anti-human IgG Fc (Abeam, Cat. No. A3803) diluted to 2 g/mL using PBS (Lonza, Cat. No. 17-516Q) was dispensed at 100 L per well into a 96 well EIA/RIA high bind microplate (Corning, Cat. No. 3590) and allowed to react at room temperature for two hours. Then, the microplate was washed three times using wash buffer (0.1 v/v % tween 20 in PBS). Blocking buffer (1 w/v % bovine serum albumin in PBS) was dispensed at 300 L per well and allowed to react at room temperature for two hours, followed by washing three times with wash buffer. As a standard sample, the Fc fusion MDTCS fragment protein whose concentration was secured was used, and the sample to be measured was diluted with blocking buffer to be included in the standard range. Thereafter, the resultant was dispensed at 100 L per well and reacted by stirring at 400 rpm at room temperature for one hour. After washing three times, an anti-human IgG (Fc specific) peroxidase antibody (Sigma-aldrich, Cat. No. A0170) was diluted 1:10,000 and dispensed at 100 L per well and reacted by stirring at 400 rpm at room temperature for one hour. After washing six times, TMB was dispensed at 100 L per well and allowed to react at room temperature for 30 minutes. Thereafter, stop buffer (0.5 M H.sub.2SO.sub.4) was dispensed at 100 L per well, and the absorbance was measured at 450 nm with a microplate reader.

Measurement of Activity of Human ADAMTS13 Variants

[0087] In order to measure the activity of ADAMTS13 variants, an experiment was performed using the manual of the Technozyme ADAMTS13 activity kit (Technoclone, Cat. No. 5450701). A GST-vWF73 substrate was dispensed at 100 L per well, allowed to react at room temperature for one hour, and then washed three times with wash buffer. As a standard sample, the calibrator in the kit was used, and as the culture to be measured, a stock solution or a 3-fold dilution thereof was added at 100 L per well using heat-inactivated plasma and allowed to react at room temperature for 30 minutes. After washing three times with wash buffer, the conjugate was added at 100 L per well, allowed to react at room temperature for one hour, and washed three times with wash buffer again. TMB color reagent was added at 100 L per well and allowed to react for 30 minutes, and then, 100 L of stop buffer was added to stop the reaction, and the absorbance was measured at 450 nm with an ELISA reader. In order to calculate the specific titer (IU/mg), the expression result (m/mL) was applied to the activity value (IU/mL), and the relative activity (%) was calculated by substituting into the following equation using the specific titers of wild-type (WT) ADAMTS13 and variants:

Relative activity (%)=specific titer of variants/specific titer of wild-type ADAMTS13100

Confirmation of Escaping Rate of Human ADAMTS13 Neutralizing Antibody

[0088] In order to confirm the ability of each ADAMTS13 variant for escaping ADAMTS13 neutralizing antibodies, ELISA was performed using eight kinds of anti-ADAMTS13 antibodies (Ab3-01, Ab 4-16, Ab4-20, Ab60, Ab61, Ab64, Ab65, and Ab 67, GC Green Cross). An anti-ADAMTS13 antibody diluted to 1-2 g/mL using PBS (Lonza, Cat. No. 17-516Q) was added at 100 L per well in a 96-well EIA/RIA high bind microplate (Corning, Cat. No. 3590) and allowed to react at room temperature for two hours. Then, the microplate was washed three times with wash buffer (0.1% tween 20 in PBS). Blocking buffer (1% BSA in PBS) was added at 300 L per well and allowed to react at room temperature for two hours, and then washed three times with wash buffer. As a standard sample, the recombinant human ADAMTS13 (R&D systems, Cat. No. 6156-AD) was used, and the culture to be measured was diluted with blocking buffer to 25-50 ng/mL, and dispensed at 100 L per well and reacted by shaking at 500 rpm at room temperature for one hour. After washing three times, an anti-6His tag antibody (Abcam, Cat. No. Ab1187) was diluted 1:10,000 and dispensed at 100 L per well, and reacted by shaking at 500 rpm at room temperature for one hour. After washing six times, TMB was dispensed at 100 L per well and allowed to react at room temperature for 10-20 minutes. Then, stop buffer (H.sub.2SO.sub.4) was dispensed at 100 L per well, and the absorbance was measured at 450 nm using a microplate reader. The neutralizing antibody escaping rate was calculated such that the relative binding level compared to the wild-type ADAMTS13 value, and then, the relative escaping rate was substituted into the following equation using the same:

Escaping rate (%)=(1binding ability of variants/binding ability of wild-type ADAMTS13)100

Construction of MDTCS and Fc (IgG1-YTE)-Conjugated MDTCS Variant and Protein Production

[0089] An expression vector in which IgG1-YTE (hinge-Fc) was conjugated to MDTCS or an MDTCS variant fragment was constructed and gene synthesis (Thermo Fisher Scientific, GeneArt Gene Synthesis) was performed as shown in Table 2 below. Expression vectors were constructed by inserting each of the synthetic genes in Table 2 into the pMSID2 vector (constructed by GC Green Cross). CHO DG44(S) cells were subcultured according to the expected amount using CDM4CHO (GE, Cat. No. SH30557.02) medium at 310 5 cells/mL. On the day of transfection, live 310.sup.7 cells were collected, suspended in OptiPro SFM (Gibco, Cat. No. 12309019), diluted to a final volume of 110.sup.7 cells/mL (3 mL) and added into a 125 mL shake flask, and cultured in the CO.sub.2 shaking incubator (37 C., 5% CO.sub.2) at 140 rpm. 30 g of the expression vector was mixed to a total volume of 1.5 mL using OptiPro SFM (tube 1). 90 L of the transporter 5 PEI (Polyscience, Cat. No. 26008-5) and 1,410 L of the OptiPro SFM were mixed (tube 2). After adding the previously prepared tube 2 into the tube 1 and gently mixing, the mixture was allowed to react at room temperature for 20-30 minutes. 3 mL of the DNA:PEI complex was added dropwise to 3 mL of previously prepared CHO DG44(S) cells. The resultant was cultured at 140 rpm in a CO.sub.2 shaking incubator (37 C., 5% CO.sub.2), and 4 hours thereafter, 24 mL of CDM4CHO medium was added to 6 mL of the CHO DG44(S)/DNA:PEI complex and cultured under the same conditions. After 48 hours of incubation, the cell number and cell viability were measured, and the resultant was added into a centrifuge tube to a volume of 1510.sup.5 cells, centrifuged at 1,200 rpm for 5 minutes, and the supernatant was removed, and suspended in 30 mL of CDM4CHO medium containing 20 nM MTX, and then inoculated into a shake flask and cultured. Subculture was performed every 3 to 4 days until the cell viability was recovered to 90% or more. The final overexpressing cell line was cultured on a large scale and used for protein purification. For MDTCS protein purification, the prepared culture was centrifuged to remove the cells and then filtered using a sterile filter made of cellulose acetate (Sartobran P, 5235307H7-OO, Sartorius) after centrifuging the prepared culture solution. The filtered culture was concentrated and then purified using anion exchange chromatography (Q Sepharose Fast Flow, GE), multi-mode chromatography (hydroxyapatite, Bio-Rad), and affinity chromatography (Blue Sepharose, GE), and the MDTCS variant IgG1-YTE-conjugated protein was filtered such that the culture of the overexpressing cell line was centrifuged to remove the cells and then purified using an antibacterial filter made of cellulose acetate (Sartobran P, 5235307H7-00, Sartorius). Purification was performed using the filtered culture using affinity chromatography (Protein A, MabSelect SuRe, GE Healthcare) and size exclusion chromatography. It was confirmed that the purified protein had a purity of 95% or more.

TABLE-US-00002 TABLE2 AminoacidsequenceofMDTCS-IgG1-YTEfusion Hinge IgG1 Fc(CH2-CH3) IgG4(YTE) AminoAcid MHQRHPRARCPPLCVAGILACGFLLGCWGPSHFQQSCLQALEP Sequence QAVSSYLSPGAPLKGRPPSPGFQRQRQRQRRAAGGILHLELLV AVGPDVFQAHQEDTERYVLTNLNIGAELLRDPSLGAQFRVHL VKMVILTEPEGAPNITANLTSSLLSVCGWSQTINPEDDTDPGHA DLVLYITRFDLELPDGNRQVRGVTQLGGACSPTWSCLITEDTG FDLGVTIAHEIGHSFGLEHDGAPGSGCGPSGHVMASDGAAPRA GLAWSPCSRROLLSLLSAGRARCVWDPPRPQPGSAGHPPDAQ PGLYYSANEQCRVAFGPKAVACTFAREHLDMCQALSCHTDPL DQSSCSRLLVPLLDGTECGVEKWCSKGRCRSLVELTPIAAVHG RWSSWGPRSPCSRSCGGGVVTRRRQCNNPRPAFGGRACVGAD LQAEMCNTQACEKTQLEFMSQQCARTDGQPLRSSPGGASFYH WGAAVPHSQGDALCRHMCRAIGESFIMKRGDSFLDGTRCMPS GPREDGTLSLCVSGSCRTFGCDGRMDSQQVWDRCQVCGGDN STCSPRKGSFTAGRAREYVTFLTVTPNLTSVYIANHRPLFTHLA VRIGGRYVVAGKMSISPNTTYPSLLEDGRVEYRVALTEDRLPR LEEIRIWGPLQEDADIQVYRRYGEEYGNLTRPDITFTYFQPKPR QAEPKSCDKTHTCPPCPAPEFLGGPSVFLFPPKPKDTLYITREPE VTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRV VSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQV YTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTT PPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQK SLSLSLGK *Black: MDTCS/Underline: Hinge/Italic: Fc

Confirmation of Pharmacokinetics of MDTCS-Hinge-Fc Fusion

[0090] In order to compare the duration in the blood and pharmacokinetic parameters of the MDTCS-IgG1-YTE (hinge-Fc) fusion, 44 or 52 C57BL/6 mice (4 mice/blood collection time) were used in each group. After injecting 160 IU/kg of MDTCS (control group) and 3 kinds of materials, in which IgG1-YTE is fused to MDTCS variants (test group), into the tail vein, the blood samples were collected at 0.083, 0.25, 0.5, 1, 2, 4, 8, 12, 24, 48, 72, 96, and 168 hours. 500 L of blood was collected from the heart through a syringe containing 10% sodium tri-citrate (i.e., an anticoagulant), and centrifuged at 2,000g at 4 C. for 20 minutes to separate the supernatant, and thereby the plasma was obtained. The activity of the MDTCS variant-hinge-Fc present in the plasma was measured according to the manual of Technozyme ADAMTS13 activity kit (Technoclone, Cat. No. 5450701). In addition, an activity assay was performed for the plasma obtained from the 44 mice to obtain an average value (0.0772 IU/mL) of the murine ADAMTS13 activity in the mouse plasma. By removing the corresponding value from the measured value of activity of the PK study sample, the murine ADAMTS13 activity inherent in the mouse plasma could be excluded and only the result of activity of the administered material was obtained. The data was analyzed through non-compartmental analysis using a native pooled method and WINNONLIN. t.sub.1/2 indicates the blood half-life of the drug, AUC1 indicates the area under the activity graph from 0 to infinity time, and mean residence time (MRT) indicates the average residence time of the drug molecule in the body.

Confirmation of ADAMTS13 Binding Site of Neutralizing Antibodies

[0091] Nucleic acid sequences of six kinds of ADAMTS13 in which full-length wild-type or C-terminal domains were sequentially deleted were cloned into pcDNA3.1 V5-His B vector (Invitrogen, Cat. No. V81020). The constructed DNA was immunoprecipitated by adding Protein A-Sepharose 4B using the culture obtained through the above-mentioned transient expression method, followed by Western blotting. Specifically, three kinds of 30 nM ADAMTS13 neutralizing antibodies (Ab4-16, Ab66, and Ab67) were added to normal control plasma (HemosIL, Cat. No. 00020003110). After adding 10 mL of plasma containing neutralizing antibodies and a culture containing 50 ng of each fragment protein of ADAMTS13 together, 500 L of binding buffer (50 mM Tris-HCl pH 8.0, 150 mM NaCl, 1% Triton X-100, and 1% BSA) was added thereto, and allowed to react at 4 C. for 24 hours. After completion of the reaction, 20 L of Protein A-Sepharose 4B resin (Thermo Fisher Scientific, Cat. No. 101041) was added thereto as a 50% slurry and allowed to react at 4 C. for 4 hours. The resin was collected by centrifugation at 845 rcf for 1 minute, and then washed three times with wash buffer (50 mM Tris-HCl pH 8.0, 150 mM NaCl, 1% Triton X-100). The proteins bound to the resin was eluted through an SDS-sample buffer (Novex, Cat. No. NP0007) containing 10% -mercaptoethanol and heated at 95 C. for 5 minutes, followed by SDS-PAGE and anti-immunoblotting with the V5 antibody (Invitrogen, Cat. No. R960-25).

PoC Evaluation of Variants in Mouse Model with aTTP Mimic Disease

[0092] The mouse model with aTTP mimic disease was established by treating ADAMTS13 KO mice with a human ADAMTS13 neutralizing antibody. ADAMTS13 KO mice were prepared by Macrogen by way of inducing large deletions and frameshift variations between exons 1 and 6 of the ADAMTS13 gene using the CRISPR/Cas9 system. Nine kinds of ADAMTS13 neutralizing antibodies (Ab3-01, Ab4-16, Ab4-20, Ab60, Ab61, Ab64, Ab65, Ab66, and Ab67), which were mixed at an equal ratio, were administered intravenously to 12-25 week old ADAMTS13 KO mice at a dose of 0.54 mg/kg. After 15 minutes, the control material (MDTCS-Fc) or five kinds of variants (1C03, 2B02, 5C09, 7A02, and DM2) were administered at a dose of 5,000 IU/kg or 7,000 IU/kg. The blood sample was collected 6 hours after the administration to measure ADAMTS13 activity.

[0093] In order to confirm whether the recovery of ADAMTS13 activity and hematological properties shown in aTTP model mice are improved by the administration of the DM2 variant, nine kinds of ADAMTS13 neutralizing antibodies (Ab3-01, Ab4-16, Ab4-20, Ab60, Ab61, Ab64, Ab65, Ab66, and Ab67) which were mixed at an equal ratio, were administered intravenously to 12-25 week old ADAMTS13 KO mice at a dose of 0.54 mg/kg. After 15 minutes, the control material (MDTCS-Fc) or variant DM2 was administered at a dose of 5,000 IU/kg, 7,000 IU/kg, or 14,000 IU/kg. After administration of 2,000 IU/kg of recombinant human vWF (VEYVONDI) 30 minutes after the administration of neutralizing antibodies, 700 L of blood was collected 6 hours thereafter, and 300 L of the blood was added into an EDTA tube (BD Medical, REF365974) and the other 300 L was added into an SST tube (BD Medical, REF365967), placed at room temperature for 30 minutes, and then centrifuged at 4 C. at 3,000 rpm for 15 minutes to separate the serum. The remaining blood was mixed with sodium citrate in a 9:1 ratio (blood:sodium citrate), and the plasma was separated and used to analyze the residual activity of human ADAMTS13 in plasma. While the whole blood in the EDTA tube was subjected to general blood tests such as platelets (PLT) using a hemocytometer (ADVIA 2120i, Siemens), the serum separated in the SST tube was used to measure lactate dehydrogenase (LDH) using a blood biochemical analyzer (Hitachi, 7180). Statistical analysis used one-way ANOVA test with the Tukey test.

PoC Evaluation of Variants in Mouse Model with cTTP Disease

[0094] The mouse model with cTTP disease was established using ADAMTS13 KO mice. 12-25 week old ADAMTS13 KO mice were administered intravenously with a vehicle or DM2 variant at a dose of 20 IU/kg, 60 IU/kg, 180 IU/kg, and 360 IU/kg, and 15 minutes thereafter, recombinant human vWF (VEYVONDI) was administered at a dose of 2,000 IU/kg, and the blood sample was collected 6 hours thereafter. While the whole blood was used for general blood tests including the measurement of the platelet level, the serum was used for the measurement of the LDH level, and the plasma was used for the analysis of human ADAMTS13 activity.

Experiment Results

Construction of Human ADAMTS13 Variants Using Random Mutagenesis

[0095] In the case of ADAMTS13 neutralizing antibodies possessed by aTTP patients, it has been reported that the proportion of binding to MDTCS among various domains of ADAMTS13 is relatively very high being more than 97%, and that the S domain is the most prevalent binding site (Tersteeg et al, 2016; Klaus et al., 2004; Leken et al., 2005). Based on the above, each library was prepared by performing error-prone PCR so that variations could occur targeting the MDTCS part or the S domain (FIG. 1a). As a result of obtaining DNA for 384 ADAMTS13 variants from each library and analyzing the nucleotide sequences, the total variation rate in the library of the MDTCS part was 72.9%, and specifically, silent mutation or non-mutation showed a rate of 23.7%, empty vectors or poor sequences 3.1%, nonsense mutation 8.9%, frameshift mutation 4.9%, and missense variation 59.1% (FIG. 1b). In the case of the S domain library, the overall mutation rate was 50.8%, and silent mutation or non-mutation accounted for 44.5%, empty vectors or poor sequences 4.7%, nonsense mutation 2.3%, frameshift mutation 3.1%, and missense mutation 45.3% (FIG. 1b). A selection experiment was performed using about 500 variants, excluding those variants which showed silent or non-mutation, empty vectors or poor sequences, and nonsense mutation among the above mutations.

Preparation of ADAMTS13 Neutralizing Antibody and Confirmation of Binding Site

[0096] To confirm whether a variant having a mutation in the MDTCS region or the S domain can escape the neutralizing antibodies of ADAMTS13, with regard to the antibodies that recognize different epitopes for human ADAMTS13, Fab was constructed using the HuCAL system (Bio-Rad), and 16 kinds having excellent binding ability to human ADAMTS13 were selected. Ab 66 and Ab 67, which were confirmed to specifically bind to different domains among the 16 kinds, were produced in the form of a full-length antibody. Since Ab 4-16 was known to specifically bind to the S domain in the plasma of the aTTP patient, it was produced in the form of a full-length antibody (Casina et al., 2015), and the binding region of each antibody was, as shown in FIG. 2c, such that Ab 4-16 specifically binds to the S domain, Ab 67 to the MDTCS region, and Ab 66 to the C-terminal end (TSP-2 to TSP-8 domains).

[0097] Setting Selection Criteria for Variants

[0098] The evaluation of the variants was conducted by analyzing the relative results with regard to the non-mutation or silent mutation variants (hereinafter, wild-type clones) having the same amino acid sequence as the wild-type ADAMTS13, among the variants constructed through random mutagenesis. A total of 59 wild-type clones were analyzed for activity and binding affinities for Ab 4-16 and Ab 67, and the analysis results of each wild-type clone were indicated relative to the results of the wild-type ADAMTS13 construct, and are shown in FIGS. 3a and 3b. As can be seen in FIGS. 3a and 3b, the escaping rates and relative activity with respect to wild-type ADAMTS13 (59 kinds), mutated ADAMTS13 (304 kinds), and selected ADAMTS13 (26 kinds) containing a conservative amino acid substitution for Ab 4-16 and Ab 67 antibodies are shown. The wild-type clone showed a relative difference in result with the wild-type ADAMTS13 construct despite no variation, and among the mutated ADAMTS13 (304 kinds), 26 kinds of ADAMTS13 mutants having a specific value of escaping rate or a value greater than or equal to relative activity were selected as shown in Table 3 below.

[0099] Specifically, as for the mutated ADAMTS13, the escaping rate for Ab 4-16 was in the range of 29.5% to 33.4%, the escaping rate for Ab 67 was in the range of 24.4% to 30.5%, and the relative activity was shown to be in the range of 62.7% to 128.9% (Table 3). As a result of applying the normal distribution using the three-sigma rule, the escaping rate for Ab 4-16 was 34.1% to 38.5%, the escaping rate for Ab 67 was 38.5% to 42.3%, and the relative activity was in the range of 47.0% to 145.2%, and thus it was predicted that almost all WT clones would fall within this distribution. Therefore, in the selection of variants, the selection criteria were applied as follows: the escaping rate of exceeding 38.5% for Ab 4-16, the escaping rate of exceeding 42.3% for Ab 67, or relative activity of 47.0% or more based on the maximum of three-sigma.

TABLE-US-00003 TABLE 3 Range of escaping rate and relative activity for neutralizing antibody Ab4-16 or Ab67 Escaping Escaping Rate for Rate for Relative Category Ab 4-16 Ab 67 Activity Minimum 29.5% 24.4% 62.7% Maximum 33.4% 30.5% 128.9% Average 2.2% 1.9% 96.1% Standard Deviation 12.1% 13.5% 16.4% Mean 3 Standard 34.1% 38.5% 47.0% Deviation (Mean 3) Mean + 3 Standard 38.5% 42.3% 145.2% Deviation (Mean + 3)
Selection of Variants which Evade ADAMTS13 Neutralizing Antibody and have Activity Higher than or Equal to the Level of Wild-Type ADAMTS13

[0100] After transfection of WT clones and variants with amino acid variations into cells, the amount of proteins present in the culture was measured, and the binding affinity for neutralizing antibodies and activity assays were performed for 304 variants having an expression concentration of 50 ng/mL or more. The variants having amino acid variations were shown to have various distributions in binding affinity for neutralizing antibodies or relative activity compared to WT clones, and among them, 26 kinds of variants satisfying the selection criteria were finally selected (FIGS. 3a and 3b). Among the 26 variants, 18 kinds of variants were shown to have S domain variations, and in particular, 13 kinds of the variants having S domain variations (1C03, 1G07, 2B01, 2B02, 3B05, 3G04, 5C09, 5G08, 6B12, 7A02, 8C04, 8D01, and 8F01) were shown to have a high escaping rate for Ab 4-16, and 5 kinds of the variants (7E01, 7G08, 8C02, 8D01, and 8D05) were shown to have the characteristic of high relative activity (Table 4). Six kinds of variants were shown to have variations in the D domain, and five kinds among them (46, 3A06, 4C07, 4E11, and 4H07) showed the characteristic of a high escaping rate for Ab 67. In addition, 6 kinds of variants having a variation for the M domain, 2 kinds of variants having a variation for the C domain, and 2 kinds of variants having a variation for the T domain were identified. The repeated reproducibility of the results was confirmed through three repeated experiments for the 26 selected variants, and 12 kinds of variants that continuously maintained superiority compared to the WT clones even in repeated tests were selected as the subject for the in vitro potency test. Among the 12 kinds of variants, 1C03, 2B01, 2B02, 3B05, 5C09, 7A02, and 8D01 were selected for their excellent Ab4-16 antibody escaping rate, and 4C07, 4E11 and 4H07 were selected for their excellent escaping rate for Ab67 antibody. Finally, 8D05 was selected for its excellent relative activity.

[0101] In order to confirm the ability to escape additional neutralizing antibodies in the 12 kinds of selected variants, the present inventors attempted to confirm whether it is also possible to escape even the neutralizing antibodies Ab4-20, Ab60, Ab61, Ab64, and Ab65 in addition to the Ab4-16 and Ab67 antibodies which were used for screening. Among the 12 kinds of variants, eight kinds of variants of the nine kinds of variants with amino acid variations in the S domain, except for 8D05, 1C03, 2B01, 2B02, 3B05, 5G08, 7A02, and 8D01 showed an excellent escaping rate for Ab4-16 and Ab4-20, which were prepared based on the sequences of ADAMTS13 autoantibodies possessed by aTTP patients, and these variants also showed excellent escaping ability for Ab60 and Ab61. In contrast, in the case of variants 4C07, 4E11, and 4H07 having amino acid variations in the D domain, the escaping rate for Ab67 was excellent (FIGS. 4a and 4b, Table 5).

TABLE-US-00004 TABLE 4 Selection of 26 kinds of variants with excellent escaping ability or relative activity for Ab4-16 and Ab67 neutralizing antibodies Variant Escaping Escaping Mutation Amino Rate for Rate for Applied Acid Ab4-16 Ab67 Relative Variant ID Domain Residues Antibody Antibody Activity 46 M, D L85F 46.30% 53.90% 66.80% P317H 1C03 S S612Y 48.00% 10.40% 104.60% 1G07 M, C, S V282A 41.40% 57.90% 63.80% A465D D672V 2B01 S D635V 87.40% 5.40% 62.20% 2B02 C, S R452I 69.80% 15.30% 88.80% S612Y 3A06 M, D, T R278I 42.60% 58.50% 62.90% A334T D427N 3B05 S P618S 66.00% 48.40% 71.20% 3E01 M T135I 52.30% 36.00% 60.00% 3G04 M, S V126M 42.00% 29.40% 103.50% A567S E651D 3H12 T N413D 69.00% 104.60% 162.40% 4C07 D A334V 6.60% 72.30% 153.10% 4E11 D A314T 10.30% 96.40% 71.90% 4H03 M, D F93V 40.30% 37.60% 65.70% K364R E376D 4H07 D N308K 30.20% 64.60% 106.40% 5C09 S S612F 60.90% 10.50% 88.50% 5G08 S Q656H 41.40% 26.00% 91.90% 6B12 S G607R 43.90% 101.60% 93.80% 7A02 S S612F 57.00% 80.50% 128.50% G624D 7E01 S R589Q 36.60% 89.70% 149.90% 7G08 S Q650H 25.20% 47.60% 160.70% Q656R 8C02 S I643F 19.20% 50.90% 168.10% 8C04 S I585N 51.30% 34.80% 69.00% Y658H 8C12 S V630L 12.00% 73.20% 48.10% D654G E664N 8D01 S R589Q 85.10% 78.30% 193.60% K608M M609L G624C I655V 8D05 S P578L 1.20% 5.70% 172.30% 8F01 S I585M 45.20% 34.40% 99.40%

TABLE-US-00005 TABLE 5 Confirmation of escaping ability and relative activity for single neutralizing antibodies in 12 kinds of selected variants Variant Position of Neutralizing Antibody Escaping Rate (%) Relative ID Variation Ab4-16 Ab4-20 Ab60 Ab61 Ab64 Ab65 Ab67 Activity 1C03 S612Y 57.5% 59.4% 97.2% 94.7% 5.2% 11.0% 15.1% 103.7% 2B01 D635V 82.9% 88.6% 92.4% 97.4% 96.3% 8.1% 7.9% 66.8% 2B02 R452I 65.4% 61.1% 97.3% 95.5% 0.9% 15.0% 15.1% 112.6% S612Y 3B05 P618S 51.3% 6.5% 24.9% 39.4% 19.4% 63.8% 37.4% 77.3% 4C07 A334V 4.2% 5.0% 3.3% 6.9% 2.2% 4.7% 78.5% 152.2% 4E11 A314T 16.6% 1.3% 2.7% 3.2% 5.4% 0.8% 98.9% 78.3% 4H07 N308K 27.9% 1.5% 11.0% 16.4% 17.0% 12.8% 61.3% 94.7% 5C09 S612F 64.6% 54.7% 97.3% 95.2% 7.8% 1.6% 11.8% 104.8% 5G08 Q656H 47.0% 7.2% 32.1% 46.2% 27.4% 51.4% 30.3% 95.9% 7A02 S612F 57.1% 55.4% 97.6% 95.5% 1.9% 26.2% 12.9% 125.3% G624D 8D01 R589Q 90.1% 98.5% 95.2% 94.1% 20.0% 95.9% 7.3% 127.0% K608M M609L G624C I655V 8D05 P578L 9.3% 43.1% 3.2% 15.9% 0.9% 116.4% 11.5% 154.7%

Confirmation of Neutralizing Antibody Escaping Ability of Fc-Attached MDTCS Fragment Variants

[0102] In a structure-function study, it was reported that among a total of 14 domains of ADAMTS13, the MDTCS domain, in which the CUB2 domain was removed from the C-terminal TSP-2, was shown to have a metalloprotease function similar to ADAMTS13 and is thus capable of cleaving VWF (Shelat et al., 2005). This suggests that even only a MDTCS fragment, instead of a full-length ADAMTS13, can function as a therapeutic agent for TTP disease, and that it can escape neutralizing antibodies present in the patient's plasma in a truncated form that bind to the C-terminal portion.

[0103] In addition, the present inventors attempted to further improve the structural stability of the MDTCS fragment and extend the half-life by attaching an Fc. The selected 12 kinds of variants were fragmented with MDTCS, and thereby Fc-attached variants were prepared, and DM1 and DM2 variants having two amino acid variations were additionally prepared by combining the selected variant amino acid residues. The final candidate materials were selected by measuring the escaping rate and relative activity of single or mixed neutralizing antibodies using the culture expressed in the cells by the above-mentioned method and a purification solution. As a result of measuring the escaping rates of binding to and relative activity for eight kinds of single neutralizing antibodies using the culture, 2B01, 3B05, 4H07, 5G08, 8D05, and DM1 were shown to escape all neutralizing antibodies at a similar level (FIGS. 5a and 5b, Table 6). 1C03, 2B02, 5C09, 7A02, 8D01, and DM2 showed excellent escaping rates of binding for 3-01 and Ab60. All of the six kinds of candidates were shown to have an amino acid variation in the S domain, and except for 8D01, have a variation in the 612.sup.th amino acid in common. In the cases of 4E11 and DM2, they were shown to have excellent escaping rates of binding to Ab67, and both were shown to have a variation in the 314.sup.th amino acid of the D domain. In the case of relative activity, DM1 showed the lowest of 57.9% and 1C03 showed the highest of 133.1%. All candidates except for the above two kinds showed relative activity of 78.2% to 125.1%, which was similar to that of the MDTCS-Fc control group (Table 6). In order to confirm the escaping ability of the candidate variants excluding 4C07 and 5C09, compared to the control group MDTCS-Fc, under the condition in which nine kinds of neutralizing antibodies (Ab3-01, Ab4-16, Ab4-20, Ab60, Ab61, Ab64, Ab65, Ab66, and Ab67) were mixed in the same ratio, 7.5 nM of the mixed neutralizing antibodies and 4 nM of each variant expressing culture were mixed, and allowed to react at room temperature for one hour to measure the residual activity. MDTCS-Fc was shown to maintain a residual activity of 3.5%, and 3B05, 4E11, 4H07, 5G08, and 8D05 were confirmed to have a residual activity of 1.24% to 2.42%, thus showing that the residual activity was lower than that of MDTCS-Fc (FIG. 6). In contrast, in the cases of 1C03, 2B01, 2B02, 7A02, 8D01, DM1, and DM2, the residual activity was maintained at 7.69% to 18.81%, thus confirming that the escaping ability by the mixed neutralizing antibodies was excellent compared to MDTCS-Fc (FIG. 6).

[0104] In order to confirm the neutralizing antibody escaping ability of the candidate material variants using the purifying solution, protein purification was performed in 12 kinds of cultures, excluding 4C07 and 5C09, using the Phytip system (protein A resin). The concentration of the purifying solution was confirmed through Fc ELISA, and it was confirmed that the target protein was eluted through silver staining. As a result of confirming the escaping rate of a single neutralizing antibody using the purifying solution, it was confirmed that most of the variants had similar tendencies to the results evaluated in the culture (FIGS. 7a and 7b). The 2B01 5G08, 8D05, and DM1 variants were shown to escape neutralizing antibodies for all eight kinds of neutralizing antibodies by 24.6% or more, and 1C03, 2B02, 7A02, 8D01, and DM2 showed excellent escaping rates of binding to 3-01 and Ab60 similar to the results of the culture. In the cases of 4E11 and DM2, it was confirmed that they had excellent escaping rates of binding to Ab67 to be the same as the results of the culture. The relative activity of each variant is shown in FIGS. 7a and 7b and Table 7. As a result of measuring the residual activity under the condition of mixed neutralizing antibodies, it was confirmed that MDTCS-Fc maintained the residual activity of 3.76%, and it was confirmed that the 3B05, 4E11, 4H07, and 8D05 variants showed the residual activity of 2.05% to 3.50%, thus showing higher inhibition compared to MDTCS-Fc. In contrast, in the cases of 1C03, 2B01, 2B02, 5G08, 7A02, 8D01, DM1, and DM2, it was confirmed that the residual activity of 6.34% to 12.8% was maintained, thus confirming that the mixed neutralizing antibodies have excellent ability of escaping neutralizing antibodies compared to MDTCS-Fc (FIG. 8).

[0105] Based on the above results, comprehensively considering the relative activity or escaping rates for mixed neutralizing antibodies, 1C03, 2B02, 7A02, DM2, and 5C09, which is predicted to have excellent ability of escaping mixed neutralizing antibodies due to the variation on the 612.sup.th amino acid, were selected as five kinds of candidates for further study.

TABLE-US-00006 TABLE 6 Confirmation of single neutralizing antibody escaping ability and relative activity of MDTCS fragments including 14 variants (culture medium conditions) Variant Position of Neutralizing Antibody Escaping Rate (%) Relative ID Variation 3-01 4-16 4-20 Ab60 Ab61 Ab64 Ab65 Ab67 Activity 1C03 S612Y 100.0% 2.0% 11.0% 91.3% 3.9% 5.9% 16.9% 1.1% 133.1% 2B01 D635V 25.1% 28.2% 38.1% 38.4% 36.2% 26.8% 11.0% 24.9% 78.2% 2B02 R452I 99.0% 1.2% 4.2% 93.5% 2.9% 2.1% 12.9% 3.1% 116.5% S612Y 3B05 P618S 35.3% 18.9% 25.5% 23.6% 19.1% 21.3% 17.9% 17.8% 92.7% 4C07 A334V 5.9% 11.2% 9.6% 11.3% 3.3% 12.4% 14.6% 14.5% 118.2% 4E11 A314T 15.3% 7.3% 11.9% 8.1% 9.0% 9.7% 8.8% 83.5% 89.7% 4H07 N308K 36.1% 20.4% 25.8% 19.5% 22.5% 25.1% 21.0% 53.8% 93.6% 5C09 S612F 100.0% 15.7% 18.7% 100.0% 12.4% 13.0% 0.5% 8.5% 108.2% 5G08 Q656H 48.7% 38.3% 41.2% 44.2% 41.9% 37.2% 27.8% 47.6% 85.4% 7A02 S612F 100.0% 7.5% 17.4% 97.5% 11.2% 7.2% 7.2% 12.3% 125.1% G624D 8D01 R589Q 100.0% 38.9% 49.1% 71.2% 31.6% 36.3% 18.1% 41.8% 78.4% K608M M609L G624C I655V 8D05 P578L 39.6% 29.9% 38.3% 32.6% 32.4% 32.5% 21.5% 43.7% 82.1% DM1 A314T 52.3% 48.2% 56.9% 60.6% 54.7% 51.6% 38.1% 86.8% 57.9% D635V DM2 A314T 100.0% 12.0% 22.0% 95.1% 21.6% 13.0% 7.3% 84.8% 97.7% S612F

TABLE-US-00007 TABLE 7 Confirmation of single neutralizing antibody escaping ability and relative activity of MDTCS fragments including 14 kinds of variants (purifying solution condition) Variant Position of Neutralizing Antibody Escaping Rate (%) Relative ID Variation 3-01 4-16 4-20 Ab60 Ab61 Ab64 Ab65 Ab67 Activity 1C03 S612Y 100.0% 15.6% 21.1% 100.0% 16.3% 7.3% 7.1% 10.9% 81.4% 2B01 D635V 46.0% 37.8% 49.6% 65.5% 43.7% 49.7% 28.5% 28.2% 56.3% 2B02 R452I 100.0% 8.4% 15.0% 100.0% 10.3% 4.4% 9.0% 0.2% 91.1% 2B02 S612Y 100.0% 8.4% 15.0% 100.0% 10.3% 4.4% 9.0% 0.2% 91.1% 3B05 P618S 25.5% 11.9% 13.3% 14.9% 10.5% 13.8% 7.9% 10.2% 83.7% 4E11 A314T 19.6% 6.2% 11.5% 15.7% 1.8% 8.8% 12.6% 84.4% 77.5% 4H07 N308K 32.6% 12.80% 16.8% 29.0% 9.4% 16.9% 17.0% 47.1% 77.5% 5G08 Q656H 89.6% 64.5% 70.2% 81.9% 57.90% 68.9% 59.0% 37.7% 18.3% 7A02 S612F 100.0% 10.4% 12.8% 100.0% 8.5% 3.4% 7.3% 14.4% 97.8% G624D 8D01 R589Q 100.0% 29.5% 48.2% 91.7% 20.5% 17.1% 14.2% 31.5% 65.1% K608M M609L G624C I655V 8D05 P578L 51.0% 27.4% 35.1% 49.5% 27.2% 33.8% 29.9% 46.0% 56.3% DM1 A314T 50.2% 34.0% 54.0% 66.50% 45.3% 52.9% 24.6% 85.1% 50.3% D635V DM2 A314T 100.0% 16.2% 24.2% 100.0% 24.0% 11.5% 11.0% 86.5% 73.1% S612F

[0106] Ability of escaping binding and relative activity of eight kinds of single neutralizing antibodies were measured in the culture expressed in cells by constructing DNA in which IgG1-YTE was conjugated to the finally selected five kinds of MDTCS variant fragments. As a result, 1C03, 2B02, 5C09, and 7A02 showed excellent escaping rates of binding to Ab3-01 and Ab60, and in the case of DM2, it was confirmed that the escaping rates of binding to Ab3-01, Ab60, and Ab67 were excellent (FIG. 10a-10b). The relative activity was 98.6-127.7% being similar to that of MDTCS-IgG1-YTE at (FIGS. 10a-10b). In order to confirm the escaping ability of five kinds of variant materials compared to the control group MDTCS-IgG1-YTE under the condition where nine kinds of neutralizing antibodies were mixed in the same ratio, the mixed neutralizing antibodies (nine kinds) and each variant expressing culture were mixed, allowed to react at room temperature for one hour, and the residual activity was measured. In the case of MDTCS-IgG1-YIE, the residual activity was maintained at 5.5%, and in the case of the five kinds of materials, the residual activity was maintained at 8.18% to 13.42%, thus confirming that the escaping ability by the neutralizing antibodies was excellent compared to the control group (FIG. 11).

[0107] The protein was purified using the Phytip system (protein A resin) from the expression culture in which IgG1-YTE was conjugated to the five kinds of MDTCS variant fragments above, and the residual activity and specific titer of the mixed neutralizing antibodies were measured in the purifying solution. The concentration of the corresponding purifying solution was confirmed through Fc ELISA, and the elution of the target protein was confirmed through silver staining. As a result of measuring the residual activity under the condition of mixed neutralizing antibodies, it was confirmed that MDTCS-IgG1-YTE was maintained at 0.4%, and the residual activity of 1C03, 2B02, 5C09, 7A02, and DM2 were each maintained at 5.7%, 1.7%, 8.8%, 2.6%, and 7.9%, thus confirming that the mixed antibody escaping ability was superior to that of MDTCS-IgG1-YTE. As a result of measuring specific titers, it was confirmed that the four kinds of variants except for 7A02 (10,288 IU/mg) showed specific titers of 19,091 IU/mg to 22,379 IU/mg similar to the control group (18,030 IU/mg) (FIG. 12). As a result, it was confirmed that the IgG1-YTE-conjugated materials of the five variant fragments had excellent escaping ability in the mixed neutralizing antibodies compared to the control group, and that the four kinds of variants except for 7A02 had specific titers similar to the control group.

Evaluation of Whether Half-Life of MDTCS Fragment Variants has Increased by Fc Attachment

[0108] In order to determine whether Fc attached to MDTCS (IgG1-YTE) will actually increase the half-life, a pharmacokinetic analysis was performed in mice. For this purpose, MDTCS or MDTCS, to which IgG1-YTE is attached, and four final kinds of candidates (1C03, 5C09, 7A02, and DM2) variant fragment proteins were administered to the tail vein of the mice and then plasma was obtained for each set time. These materials were administered to a concentration of 160 IU/kg based on the specific titer of each material and the activity of the materials remaining in the plasma obtained for each set time was measured through an activity assay. The obtained results were summarized by the naive pooled method, and a pharmacokinetic analysis was performed using the noncompartmental analysis method. The half-life of MDTCS was measured to be 2.898 hours, MDTCS-IgG1-YTE was 11.51 hours, and each variant showed a half-life of 5.184 to 9.902 hours. Compared to the control MDTCS, the half-life was extended by 1.79 to 3.97 times by IgG1-YTE conjugation. In addition, the mean residence time (MRT) value indicating the average residence time of the material by IgG1-YTE conjugation was 7.189 to 11.67 hours, thus indicating an increased residence time compared to 3.743 hours (Table 8, FIG. 9). As a result, it was confirmed that the IgG1-YTE fusion was effective in maintaining activity through blood half-life and average duration in vivo.

TABLE-US-00008 TABLE 8 Pharmacokinetic Results Parameters of PK Analysis Candidate t.sub.1/2, AUC.sub.Inf MRT Material Terminal (hr) (IU*hr/mL) (hr) MDTCS 2.898 5.467 3.743 MDTCS-IgG1-YTE 11.51 16.89 11.67 1C03-IgG1-YTE 6.087 12.33 8.695 5C09-IgG1-YTE 5.184 12.06 7.982 7A02-IgG1-YTE 9.902 14.49 10.35 DM2-IgG1-YTE 5.986 11.39 7.189

[0109] As described above, the present inventors have discovered 26 kinds of novel variants that either escape ADAMTS13 neutralizing antibodies binding to the MDTCS region or the S domain or exhibit activity greater than or equal to wild-type ADAMTS13. Among these, 12 kinds having most excellent escaping ability or remarkably excellent comparative activity for the nine kinds of neutralizing antibodies were selected, and then constructed MDTCS fragments essential for vWF cleavage while capable of efficiently escaping the neutralizing antibodies binding to the C-terminus, thereby identifying variants with a significantly improved rate of escaping autoantibodies binding to D, C or S domains. The variants of the present invention can be usefully used as an effective pharmacological ingredient with improved stability and durability of physiological activity by increasing the blood half-life through the IgG1-YTE conjugation.

Confirmation Result of Variant PoC in aTTP Mimic Disease Mouse Model

[0110] In order to select the final candidate materials from the established aTTP-mimic mouse model, the control material (MDTCS-IgG1-YTE) or selected five variant candidate materials (1C03-IgG1-YTE, 2B02-IgG1-YTE, 5C09-IgG1-YTE, 7A02-IgG1-YTE, and DM2-IgG1-YTE) were administered, and the rate of escaping a neutralizing antibody was evaluated (FIG. 13a). Among the five kinds of variant candidate materials, DM2-IgG1-YTE showed the highest human ADAMTS13 residual activity at both doses of 5,000 IU/kg and 7,000 IU/kg (FIG. 13b). The DM2-IgG1-YTE material, which showed the best neutralizing antibody escaping rate, was finally selected and administered at various concentrations to confirm the ability to maintain residual activity of human ADAMTS13 and the degree of alleviation of clinical symptoms (FIG. 14a); as a result, it was confirmed that the degree of alleviation increased as the DM2 dose increased, and it was observed that the improvement of platelet and LDH levels was relatively high by DM2-IgG1-YTE administration compared to MDTCS-IgG1-YTE administration in the 7,000 IU/kg administration group (FIG. 14b). In consistent with the improvement of platelet and LDH levels, as a result of the human ADAMTS13 activity test, it was possible to observe an increase in the residual activity proportional to the administered dose of the control material or candidate material (FIG. 14b). As a result of observation of clinical symptoms, it was confirmed that mortality or hematuria shown in the aTTP-mimic mouse model was reduced by the administration of the control material or candidate material. In particular, in the case of the DM2-IgG1-YTE treatment group, there were no deaths at any dose, and no subjects with hematuria symptoms were observed when administered at a dose of 7,000 IU/kg or more (FIG. 14c). When administered at a dose of 7,000 IU/kg, the average residual activity was 0.32 IU/mL of DM2-IgG1-YTE, which was 9.6 times higher than that of 0.03 IU/mL of MDTCS-IgG1-YTE, and it was determined that the differences in observation results of clinical symptoms was due to such difference in residual activity. As a result of a general blood test, clinical chemical test, and observation of clinical symptoms, it was confirmed the administrations of MDTCS-IgG1-YTE and DM2-IgG1-YTE tended to improve the clinical symptoms of aTTP in the aTTP-mimic mouse model, and through this, it was possible to confirm the in vivo PoC. In particular, it was confirmed that when administered at a dose of 7,000 IU/kg, the DM2-IgG1-YTE administration showed an excellent improvement effect compared to the MDTCS-IgG1-YTE administration.

Result of Confirmation of Variant PoC in cTTP Disease Mouse Model

[0111] The presence of an improvement in hematological and clinical symptoms appearing in TTP disease and the degree of recovery of human ADAMTS13 activity in the cTTP mouse model, was confirmed by using DM2-IgG1-YTE, which showed the most excellent effect among the five kinds of variant candidates in the aTTP minic mouse model (FIG. 15a). It could be confirmed that the improvement of platelet and LDH levels occurred in a concentration-dependent manner according to an increase of the dose of DM2-IgG1-YTE, and a significant increase in platelets was observed in the group administered with DM2-IgG1-YTE 180 at 360 IU/kg compared to the control group, and in particular, in the case of the group administered at 360 IU/kg, a recovery comparable to the normal level was shown (FIG. 15b). In the case of LDH level, it was confirmed that LDH was recovered to a level similar to that of the control group in the group administered at 180 IU/kg (FIG. 15b). In the case of ADAMTS13 activity, starting from the group administered with DM2-IgG1-YTE at a dose of 60 IU/kg group, an average activity of 0.1 IU/mL or more was shown, and in the group administered at a dose of 360 IU/kg, an average activity of 1.08 IU/mL was measured (FIG. 15b). Therefore, it was found that the DM2-IgG1-YTE variant effectively could improve clinical symptoms and restore ADAMTS13 activity in the cTTP disease mice.

[0112] As the specific parts of the present invention have been described in detail above, these specific descriptions are only preferred embodiments to those of ordinary skill in the art, and it is clear that the scope of the present invention is not limited thereto. Accordingly, the substantial scope of the present invention will be defined by the appended claims and equivalents thereof.

REFERENCES

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