Nav1.9 target polypeptide, antibody and antibody fragment combined with same, and related pharmaceutical composition

Abstract

Provided is an antibody or a fragment thereof, which can be specifically bound to an S3-4 ring of a voltage sensor paddle of a domain III of voltage-gated sodium channel Nav1.9 α sub-unit, and is able to inactivate a voltage sensor valve to keep sodium ions from entering nerve cells normally. Also provided is an epitope polypeptide specifically bound to the antibody or the fragment thereof, a pharmaceutical composition comprising the antibody or the fragment thereof, and a use of the antibody or the fragment thereof in preparing a drug for treating and diseases related to pains.

Claims

1. An antibody or an antigen binding fragment thereof that specifically binds to a cytomembrane voltage-gated sodium ion channel a subunit Nav1.9, wherein the heavy chain variable region of the antibody or antigen binding fragment thereof comprises CDR sequences of CDRH1 as shown in SEQ ID NO:1, CDRH2 as shown in SEQ ID NO:2 and CDRH3 as shown in SEQ ID NO:3; and the light chain variable region of the antibody or antigen binding fragment thereof comprises CDR sequences of CDRL1 as shown in SEQ ID NO:4, CDRL2 as shown in SEQ ID NO:5, and CDRL3 as shown in SEQ ID NO:6.

2. The antibody or antigen binding fragment thereof according to claim 1, wherein the antibody or antigen binding fragment comprises a heavy chain variable region as shown in SEQ ID NO:7 and/or a light chain variable region as shown in SEQ ID NO:8.

3. The antibody or antigen binding fragment thereof according to claim 1, wherein the antibody further comprises an antibody constant region.

4. The antibody or antigen binding fragment thereof according to claim 1, wherein the antibody or antigen binding fragment is in a structural form selected from the group consisting of a full antibody, Fab, F(ab′)2, dsFv, scFv, a diabody, a minibody, a bispecific antibody, a multispecific antibody, a chimeric antibody, and a CDR-grafted antibody.

5. The antibody or antigen binding fragment thereof according to claim 1, wherein the antibody is a monoclonal antibody.

6. The antibody or antigen binding fragment thereof according to claim 5, wherein the antibody is a humanized antibody.

7. The antibody or antigen binding fragment thereof according to claim 1, wherein the heavy chain variable region of the antibody or antigen binding fragment thereof comprises an antigen binding region consisting of the CDR sequences of CDRH1 as shown in SEQ ID NO:1, CDRH2 as shown in SEQ ID NO:2 and CDRH3 as shown in SEQ ID NO:3; and the light chain variable region of the antibody or antigen binding fragment thereof comprises an antigen binding region consisting of CDR sequences of CDRL1 as shown in SEQ ID NO:4, CDRL2 as shown in SEQ ID NO:5, and CDRL3 as shown in SEQ ID NO:6.

8. A pharmaceutical composition comprising the antibody or antigen binding fragment thereof of claim 1, and a pharmaceutically acceptable carrier.

Description

DESCRIPTION OF THE INVENTION

(1) FIG. 1: Structure diagram of the sodium ion channel Nav1.9

(2) FIG. 2: Target design diagram of the sodium ion channel Nav1.9

(3) FIG. 3: Analysis of specificity of monoclonal antibodies to human neural tissues

(4) A. 42H10; B. 51H10; C. 52F11; D. 55E9

(5) FIG. 4: Immunogenicity analysis of monoclonal antibodies by Western blotting

(6) FIG. 5: Analgesic effect of antibody 51H10D12 on 5% Formalin-induced acute inflammatory pain model in wild type mice.

DETAILED DESCRIPTION OF THE INVENTION

(7) The invention is illustrated, but not limited by the following detailed description of the preferred embodiments of the invention.

(8) Material Sources:

(9) The materials and reagents used below are commercially available unless otherwise stated.

Example 1 [Synthesis of Antigen]

(10) According to the amino acid sequence (GenBank No. NP_001274152) and the crystal structural model of Nav1.9, the hydrophilicity and antigenicity analyses were performed to screen the sequence DVEFSGEDNAQRIT, the hydrophilicity and antigenicity of which met the requirements of the antigen. The DVEFSGEDNAQRIT (SEQ ID NO.9) polypeptide was synthesized using a fully automated synthesizer.

(11) Specific steps were as follows:

(12) (1) attaching —COOH of the first AA to Cl-Resin with DIEA, and then blocking the unreacted functional groups on the resin with MeOH;

(13) (2) washing with DMF;

(14) (3) removing the protecting group Fmoc of —NH.sub.2 in the first AA with Pip to expose the —NH.sub.2;

(15) (4) washing with DMF;

(16) (5) activating —COOH of the second AA with DIC+HOBT, and then condensing it with —NH.sub.2 in the first AA to form an amide bond;

(17) (6) washing with DMF;

(18) (7) removing the protecting group Fmoc of —NH.sub.2 in the second AA with Pip to expose the —NH.sub.2;

(19) (8) washing with DMF;

(20) (9) . . . repeating the steps 5-8 until exposing the —NH.sub.2 of the last AA;

(21) (10) cutting the polypeptide from the resin and removing the side chain protecting groups of all amino acids, with the cleavage reagent as: trifluoroacetic acid+ethanedithiol+phenol+thioanisole+water;

(22) (11) adding the cleavage solution into diethyl ether to precipitate the polypeptide, and centrifuging to obtain the crude peptide (C2363BB030-1);

(23) (12) purifying with a peptide HPLC C18 preparative/analytical column, designated as C2363BB030-1, to obtain the purified polypeptide for immunizing animals.

(24) Note: To facilitate peptide coupling, an additional cysteine may be added to the end of the polypeptide.

Example 2 [Preparation of Monoclonal Cell Lines]

(25) 2.1 Animal Immunization

(26) Freund's complete adjuvant (Sigma, F5881) and Freund's incomplete adjuvant (Sigma, F5506) were prepared. The polypeptide was coupled to the carrier protein KLH by the terminal —SH of polypeptide C2363BB030-1 and used as an immunogen.

(27) Five 8-week-old female BALB/c mice (animal numbers: #1939, #1940, #1941, #1942, #1943) were selected and immunized intraperitoneally three times to stimulate the body to produce an immune response and then to produce antibodies. Primary immunization: 50 μg/mouse; the secondary immunization was performed after three weeks, at a dose of 50 μg/mouse; the third immunization was carried out 2 weeks after the secondary immunization at a dose of 50 μg/mouse; 1 week after the third immunization, blood was collected for antibody test.

(28) 2.2 ELISA Test of Animal Serum

(29) 2.2.1 Instruments and Equipments:

(30) Washing machine: Beijing Nanhua ZDMX

(31) Microtiter-plate reader: Thermo MultiskanAscent

(32) 2.2.2 Reagents:

(33) Coating antigen: polypeptide C2363BB030-1; coating solution: 1*PBS (pH 7.4); washing buffer: 1*PBS (pH 7.4), 0.05% PBS; primary antibody: anti-serum after the third immunization; enzyme-labeled secondary antibody: Peroxidase-AffiniPure Goat Anti-Mouse IgG, Fcγ Fragment Specific (min X Hu, Bov, HrsSr Prot); TMB chromogenic solution; stop solution: 1 M hydrochloric acid.

(34) The specific method was as follows:

(35) (1) Coating: The antigen was diluted to 1 μg/ml with the coating solution, mixed and then added to the plate at 100 μl per well, covered with the cover film, and placed at 4° C. overnight.

(36) (2) Blocking: The microtiter-plate was taken out to discard the coating solution, added with the blocking solution, covered with the cover film, and incubated at 37° C. for 0.5 h.

(37) (3) Addition of primary antibody: The anti-serum after the third immunization was first diluted 1000-fold, and then subjected to doubling dilution for 9 gradients, covered with the cover film, and incubated at 37° C. for 1 h.

(38) (4) Addition of secondary antibody: The enzyme-labeled microtiter-plate was taken out to discard the solution inside, added with the diluted secondary antibody at a concentration of 0.033 μg/ml, covered with the cover film, and incubated at 37° C. for half an hour.

(39) (5) Color development: The enzyme-labeled microtiter-plate was taken out to discard the solution inside, added with the chromogenic solution to develop the color at 25° C. for 13 minutes.

(40) (6) Stop of reaction: The stop solution was added to stop the reaction.

(41) (7) The value was read at 450 nm on a microtiter-plate reader immediately after the addition of the stop solution. The maximum dilution corresponding to the well having an OD value of more than 2.1 times the OD value of the set negative control was determined as the titer of the sample, and the test results are shown in Table 2. NC is a negative control of unimmunized serum, and the initial dilution factor is 1:1,000. The anti-serum after the third immunization was tested. The anti-serum titer of animal No. #1940 was 1:128,000; the anti-serum titer of animal No. #1939 was 1:512,000; the anti-serum titers of the remaining 3 animals (#1941, #1942, #1943) were 1:256,000.

(42) TABLE-US-00001 TABLE 2 Test results of serum ELISA after the third immunization: Animal No. Dilution factor No.1939 No.1940 No.1941 No.1942 No.1943 Negative 1:1,000 0.087 0.090 0.093 0.143 0.101 control Dilution 1 1:1,000 2.798 2.350 2.404 2.546 2.706 Dilution 2 1:2,000 2.607 1.980 2.036 2.147 2.339 Dilution 3 1:4,000 2.557 1.750 1.970 1.830 1.986 Dilution 4 1:8,000 2.264 1.473 1.522 1.381 1.463 Dilution 5 1:16,000 1.908 0.956 1.105 0.953 1.024 Dilution 6 1:32,000 1.453 0.754 0.725 0.595 0.659 Dilution 7 1:64,000 1.071 0.427 0.513 0.366 0.407 Dilution 8 1:128,000 0.625 0.274 0.278 0.216 0.261 Dilution 9 1:256,000 0.368 0.138 0.194 0.164 0.175 Dilution 10 1:512,000 0.229 0.106 0.128 0.109 0.146 Dilution 11 Blank control 0.077 0.077 0.077 0.077 0.077 Titer 1:512,000 1:128,000 1:256,000 1:256,000 1:256,000

(43) 2.3 Cell Fusion and Screening of Hybridoma Cell Lines

(44) 2.3.1 Cell Fusion:

(45) According to the ELISA test results of Example 2.2, combined with the tissue specificity results, two animals #1942, #1943 were selected for final immunization, and three days later the spleen cells of the two animals were fused with tumor cells. The mouse myeloma cells (SP2/0) and spleen cells were electrofused in a 1:3 ratio and the fused cells were plated into 15 feeder cell plates using HAT medium, and cultured in a CO.sub.2 incubator.

(46) 2.3.2 Screening of Hybridoma Cell Lines:

(47) After the fused cells were cultured for 7-10 days, the whole medium was replaced and ELISA test was carried out after 4 hours of the medium replacement.

(48) The specific materials and procedures of ELISA were the same as that of ELISA test of animal serum in 2.2.

(49) First ELISA Screening:

(50) There is a total of fifteen 96-well microtiter-plates (from plate No. 41 to plate No. 55). Well 55H12 (well H12 of plate No. 55) was set as positive control, a 1000-fold diluted solution of fused animal serum was added, and the OD value was determined to be 2.215; Well 55G12 (well G12 of plate No. 55) was set as negative control, a blank medium was added, and the OD value was determined to be 0.088. The clones with OD>0.5 were selected.

(51) Second ELISA Screening:

(52) The 68 clones with OD>0.5 of a single-well cell from the first screening were subjected to a second test (same as the above test method). The results are shown in Table 3. 10 clones with an OD of more than 1.0 were selected, namely 41C12, 47A11, 42A3, 42H10, 48D6, 51H10, 52F11, 52H5, 55E9, 55H7.

(53) TABLE-US-00002 TABLE 3 Second ELISA screening of hybridoma cells 1 2 3 4 5 6 7 8 9 10 11 12 41C12 41F4 42A3 42D8 42H10 43B1 43B7 44D1 46A6 47A11 48B12 48D6 1.942 0.748 0.966 0.660 1.254 0.524 0.638 0.540 0.629 1.102 0.791 1.362 50C6 51D10 51H9 51H10 51H11 52B4 52C5 52F6 52F11 52H5 52H6 53D9 0.564 0.459 0.834 1.962 0.084 0.588 0.713 0.864 1.118 1.777 0.109 0.808 53E2 55E9 55G5 55H2 55H7 NC PC 0.558 1.294 0.757 0.716 1.148 0.084 1.985

(54) Third ELISA Screening:

(55) The 10 lines from the second screening were subjected to positive confirmation test (same as the above test method). The experiment results are shown in Table 4. The above 10 cell lines were expanded into a 24-well microtiter-plate, and 2 ml of supernatant of each line was collected for next confirmation step.

(56) TABLE-US-00003 TABLE 4 Third ELISA screening of hybridoma cells 1 2 3 4 5 6 7 8 9 10 11 12 41C12 47A11 42A3 42H10 48D6 51H10 52F11 52H5 55E9 55H7 PC NC 2.259 1.454 1.379 1.562 1.574 1.833 1.326 1.748 1.385 1.325 2.025 0.072

(57) 2.3.3. Cytological Specificity Confirmation

(58) In order to confirm whether these cell lines have specificity to nerve tissues, the supernatants of the third ELISA screening of the 2.3.2 hybridoma cell lines were subjected to immunohistochemical examination. The specific experimental methods were as follows:

(59) 2.3.3.1 Tissue Dehydration Treatment:

(60) The human nerve tissues were taken for dehydration treatment, and the dehydration treatment was carried out by Leica ASP300S. The specific process was as follows:

(61) The tissues was dehydrated with 70%, 85%, 90%, anhydrous ethanol for 30 minutes, respectively; then dehydrated twice with anhydrous ethanol for 60 minutes each time; then treated with clearing agent for 30 minutes, then treated with clearing agent twice for 60 minutes each time; and then treated 3 times with paraffin, for 60 minutes, 120 minutes and 180 minutes, respectively, and then subjected to embedding operation using a Leica EG1150 embedding machine, to prepare a wax block, which was cut into sections with a thickness of 4 μm.

(62) 2.3.3.2 In Situ Hybridization:

(63) The nerve tissue sections were baked at 85° C. for 20 min; treated 3 times with a dewaxing agent for 1 minute each time; dewaxed 3 times with anhydrous alcohol for 1 minute each time; washed 3 times with water for 1 minute each time; thermal repaired with ER2 (pH=9 buffer solution) for 20 minutes, cooled for 12 minutes, then washed 3 times with water for 1 minute each time; then blocked for 30 minutes; washed 3 times with water for 1 minute each time; added with the supernatant of the cell line and incubated for 30 minutes, washed 3 times with water for 1 minute each time; incubated for 8 minutes with an enhancing agent, washed 3 times with water for 2 minutes each time, added with the secondary antibody and incubated for 8 minutes; washed 3 times with water for 2 minutes each time; developed color with DAB for 8 minutes; washed 3 times with water for 1 minute each time, stained with hematoxylin for 10 minutes; washed 3 times with water for 1 minute each time, dehydrated with alcohol, air-dried and sealed. Observations were performed using an Olympus optical microscope.

(64) It was observed by optical microscopy that the specificity and hybridization signals of the antibodies secreted by cell lines 42H10, 51H10, 52F11, 55E9 in neural tissues met the requirements.

(65) 2.3.4 Subcloning

(66) According to the results of the cytology test of Example 2.3.3, 42H10, 51H10, 52F11, 55E9 were selected for subcloning. The four cell lines were subcloned by limiting dilution method. The four cell lines were plated into a 96-well feeder cell plate. After 7-10 days of culture, 12 monoclones were selected from each line for ELISA test (same as above test method). The results showed that 16 clones such as 42H10E3, 42H10E4, 42H10F3, 42H10G4, 51H10D9, 51H10D12, 51H10E11, 51H10F8, 52F11B2, 52F11H3, 52F11H6, 52F11H11, 55E9A4, 55E9C6, 55E9D9, and 55E9E3 were positive and the others were negative. These positive clones were selected for a second confirmation and the OD values were confirmed to be relatively higher (see Table 5). These positive monoclones were then expanded into a 24-well microtiter-plate and 2 ml of supernatant of each clone culture was collected for cytological confirmation. It was confirmed by tissue immunochemistry that the clone 51H10 was immunohistochemically positive (FIG. 3).

(67) TABLE-US-00004 TABLE 5 Confirmation of second ELISA test of positive subclones 42H10E3 42H10E4 42H10F3 42H10G4 51H10D9 51H10D12 51H10E11 51H10F8 52F11B2 1.566 1.579 1.544 1.592 2.188 2.254 2.154 1.601 1.616 52F11H3 52F11H6 52F11H11 55E9A4 55E9C6 55E9D9 55E9E3 NC PC 2.158 1.488 1.504 1.485 1.348 1.439 1.542 0.073 2.034

(68) 51H10D9, 51H10D12, and 51H10F8 were selected for the second subcloning (subcloning method was the same as above). After 7-10 days of culture, 12 monoclonal wells were selected for ELISA test. The results showed that they were all positive, indicating that these were homozygous monoclones. Five sub-clones were randomly selected, and the supernatants were subjected to titer assay and subtype identification (Southern Biotech kit, Cat. No. ST17). The experimental results showed that the OD values of the five clones were identical, the titer was 1:2400 and the subtypes were IgG2b, K. Finally, 51H10D12 was selected for cell cryopreservation.

Example 3 [Antibody Sequencing]

(69) In order to determine the monoclonal antibody sequence, one monoclone 51H10D12 was selected for sequencing. Total RNA was isolated from hybridoma cells according to the technical manual of TRIzol reagent. The total RNA was then reverse transcribed into cDNA using isotype-specific antisense primers or universal primers, according to the PrimeScript™ First Strand cDNA Synthesis Kit Technical Manual. Antibody fragments of VH and VL were amplified according to the standard operating procedure (SOP) method of rapid amplification of cDNA ends (RACE) of GenScript. The amplified antibody fragments were cloned into standard cloning vectors, respectively. Colony PCR was performed to screen for clones containing inserts of the correct size. At least 5 colonies with the correct size inserts were sequenced. The sequences of the different clones were aligned to determine the consensus sequence of these clones.

(70) The DNA sequence of VH is thus determined as shown in SEQ ID NO:10; the DNA sequence of VL is determined as shown in SEQ ID NO:11.

(71) TABLE-US-00005 Leader sequence-FR1-CDR1-FR2-CDR2-FR3- CDR3-FR4-constant region-stop codon Leader sequence TGCAGCAGCCTGGGGCTGAGCTGGTGAGGCCTGGAGCTTCAGTGAAGCTG TCCTGCAAGGCTTCTGG CTACTCCTTCACCAGTTACTGGATGAACTGGGTGAAGCAGAGGCCTGGAC AAGGCCTTGAGTGGATT CDR1 GGCATGATTCATCCTTCCGATAGTGAAACTAGGTTAAATCAGAAGTTCAA GGACAAGGCCACATTGA CDR2 CTGTAGACAAATCCTCCAGCACAGCCTACATGCAACTCAGCAGCCCGACA TCTGAGGACTCTGCGGT CDR3 Constant region (SEQ ID NO: 10) 0 TGA Leader sequence-FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4- constant region-stop codon Leader sequence AGATGAACCAGTCTCCATCCAGTCTGTCTGCATCCCTTGGAGACACAATT ACCATCACTTGCCATGC CAGTCAGAACATTAATGTTTGGTTAAGCTGGTACCAGCAGAAACCAGGAA ATATTCCTAAACTATTG CDR 1 ATCTATAAGGCTTCCAACTTGCGCACAGGCGTCCCATCAAGGTTTAGTGG CAGTGGATCTGGAACAG CDR2 GTTTCACATTAACCATCAGCAGCCTGCAGCCTGAAGACATTGCCACTTAC TACTGTCACCAGGGTCA CDR3 0 Constant region (SEQ ID NO: 11)

(72) The amino acid sequences of VH and VL can be deduced from their DNA sequences. The amino acid sequence of VH is as shown in SEQ ID NO:7, and the amino acid sequence of VL is as shown in SEQ ID NO:8.

(73) TABLE-US-00006 Leader sequence-FRl-CDRl-FR2-CDR2-FR3-CDR3-FR4- constant region (SEQ ID NO: 7) Leader sequence CDR1 CDR2 CDR3 Constant region 0 Leader sequence-FRl-CDRl-FR2-CDR2-FR3-CDR3-FR4- constant region (SEQ ID NO: 8) Leader sequence CDR1 CDR2 CDR3 Constant region

(74) It can be deduced that the RNA sequence encoding VH is as shown in SEQ ID NO:12, and the RNA sequence encoding VL is as shown in SEQ ID NO:13.

Example 4 [Specificity Test of Antibody]

(75) 4.1 Preparation of hNav1.9 Antigen

(76) The human Nav1.9-HA plasmid (7-8 μg) was transfected into ND7/23 cells (10 cm cell dishes), cultured at 37° C. for 10 hours, and then cultured in a cell dish at 29° C. for 20 hours to collect the cells.

(77) 4.2 Western Blotting Analysis

(78) 1. The cell culture medium was aspirated, 2 ml of sterilized PBS was added and the cell dish was gently rotated to wash the cells twice.

(79) 2. 1 ml of Western and IP cell lysate were added, and the cells were collected by cell scraper and lysed on ice for half an hour.

(80) 3. A sonicator was used for ultrasonically disruption twice for 3s each time.

(81) 4. The cell lysate was centrifuged at 12000 rpm for 10 minutes at 4° C. to collect the cell lysis supernatant.

(82) 5. 40 μl of the supernatant of the cell lysate was taken and electrophoresed on an 8% SDS-polyacrylamide gel.

(83) 6. After transformation, the cells were incubated with HA tag (1:2000) antibody and 51H10D12 (1:300) antibody for 1 hour at room temperature.

(84) 7. The cells were washed three times with PBS for 5 minutes each time;

(85) 8. A digoxigenin-labeled anti-mouse antibody (1:20000) was added and incubated for 1 hour at room temperature;

(86) 9. The cells were washed three times with PBS for 5 minutes each time;

(87) 10. A digoxin substrate was added and developed for 5-10 minutes.

(88) 11. Western blot hybridization was performed.

(89) 4.3 Western Hybridization Results

(90) As shown in FIG. 4, the antibody 51H10D12 and the HA tag can produce a hybridization signal between 130-250 KD and no corresponding signal was detected in the negative control, the hybridization signal was close to the molecular weight of hNav1.9 of about 210 kDa, indicating that the antibody can recognize the protein of hNav1.9.

Example 5 [Analgesic Efficacy of 51H10D12 Antibody in Wild-Type Mice]

(91) In order to test whether the antibody 51H10D12 has an analgesic effect, we used Formalin inflammatory pain model to evaluate the efficacy of the antibody. After intravenous injection of the antibody, 2 μl of 5% formalin was injected to the mice hind paw after 30 minutes injection of the antibody. The time of paw licking and withdrawal was recorded every 5 minutes, to evaluate the analgesic efficacy of different treatments on Formalin-induced spontaneous inflammatory pain.

(92) 5.1 Experimental Steps

(93) According to the method of Lee et al. (2014, Cell 157, 1393-1404), 12 wild-type mice were tested after 2 days of adaptation. They were randomly divided into 2 groups, one served as control group, injected with PBS by tail vein, and the other served as experimental group, injected with 10 mg/kg of antibody 51H10D12 by tail vein. After half an hour, 20 μL of 5% Formalin was injected subcutaneously into the hind paw to produce pain caused by acute inflammation, and the time of paw licking and withdrawal was recorded within every 5 minutes for a total of 45 minutes. Phase I (0-10 minutes) and phase II (10-45 minutes) were statistically analyzed, respectively. Phase I represented acute pain, and phase II represented spontaneous persistent pain. After the experiment, the two phases as well as the differences between the drug injection group and the control group during various phases in the wild-type mice were statistically analyzed.

(94) 5.2 Experimental Results

(95) As shown in FIG. 5, the wild-type mice were injected with antibody 51H10D12 by tail vein, and given 5% Formalin to induce acute inflammatory pain. The antibody can reduce the total time of paw licking within 10-15 minutes and 30-35 minutes after subcutaneous injection of 5% Formalin by hind paw, namely, the total time of paw licking in Phase II, which was significantly different compared to that of the negative control. The results showed that antibody 51H10D12 can alleviate phase II inflammatory pain induced by 5% Formalin in wild type mice, which is equivalent to the effect reported in the literature.

(96) TABLE-US-00007 Sequence listing: (SEQ ID NO: 1) SYWMN (SEQ ID NO: 2) MIHPSDSETRLNQKFKD (SEQ ID NO: 3) QGFAY (SEQ ID NO: 4) HASQNINVWLS (SEQ ID NO: 5) KASNLRT (SEQ ID NO: 6) HQGQSYPWT (SEQ ID NO: 7) MGWSSIILFLVATATGVHSQVQLQQPGAELVRPGASVKLSCKASGYSF TSYWMNWVKQRPGQGLEWIGMIHPSDSETRLNQKFKDKATLTVDKSSS TAYMQLSSPTSEDSAVYYCARQGFAYWGQGTLVTVSTAKTTPPSVYPL APGCGDTTGSSVTLGCLVKGYFPESVTVTWNSGSLSSSVHTFPALLQS GLYTMSSSVTVPSSTWPSQTVTCSVAHPASSTTVDKKLEPSGPISTIN PCPPCKECHKCPAPNLEGGPSVFIFPPNIKDVLMISLTPKVTCVVVDV SEDDPDVQISWFVNNVEVHTAQTQTHREDYNSTIRVVSTLPIQHQDWM SGKEFKCKVNNKDLPSPIERTISKIKGLVRAPQVYILPPPAEQLSRKD VSLTCLVVGFNPGDISVEWTSNGHTEENYKDTAPVLDSDGSYFIYSKL NMKTSKWEKTDSFSCNVRHEGLKNYYLKKTISRSPGK (SEQ ID NO: 8) MRVLAELLGLLLFCFLGVRCDIQMNQSPSSLSASLGDTITITCHASQN INVWLSWYQQKPGNIPKLLIYKASNLRTGVPSRFSGSGSGTGFTLTIS SLQPEDIATYYCHQGQSYPWTFGGGTKLEIKRADAAPTVSIFPPSSEQ LTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDST YSMSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNEC (SEQ ID NO: 9) DVEFSGEDNAQRIT (SEQ ID NO: 10) ATGGGATGGAGCTCTATCATCCTCTTCTTGGTAGCAACAGCTACAGGT GTCCACTCCCAGGTCCAACTGCAGCAGCCTGGGGCTGAGCTGGTGAGG CCTGGAGCTTCAGTGAAGCTGTCCTGCAAGGCTTCTGGCTACTCCTTC ACCAGTTACTGGATGAACTGGGTGAAGCAGAGGCCTGGACAAGGCCTT GAGTGGATTGGCATGATTCATCCTTCCGATAGTGAAACTAGGTTAAAT CAGAAGTTCAAGGACAAGGCCACATTGACTGTAGACAAATCCTCCAGC ACAGCCTACATGCAACTCAGCAGCCCGACATCTGAGGACTCTGCGGTC TATTACTGTGCAAGACAAGGGTTTGCTTACTGGGGCCAAGGGACTCTG GTCACTGTCTCTACAGCCAAAACAACACCCCCATCAGTCTATCCACTG GCCCCTGGGTGTGGAGATACAACTGGTTCCTCCGTGACTCTGGGATGC CTGGTCAAGGGCTACTTCCCTGAGTCAGTGACTGTGACTTGGAACTCT GGATCCCTGTCCAGCAGTGTGCACACCTTCCCAGCTCTCCTGCAGTCT GGACTCTACACTATGAGCAGCTCAGTGACTGTCCCCTCCAGCACCTGG CCAAGTCAGACCGTCACCTGCAGCGTTGCTCACCCAGCCAGCAGCACC ACGGTGGACAAAAAACTTGAGCCCAGCGGGCCCATTTCAACAATCAAC CCCTGTCCTCCATGCAAGGAGTGTCACAAATGCCCAGCTCCTAACCTC GAGGGTGGACCATCCGTCTTCATCTTCCCTCCAAATATCAAGGATGTA CTCATGATCTCCCTGACACCCAAGGTCACGTGTGTGGTGGTGGATGTG AGCGAGGATGACCCAGACGTCCAGATCAGCTGGTTTGTGAACAACGTG GAAGTACACACAGCTCAGACACAAACCCATAGAGAGGATTACAACAGT ACTATCCGGGTGGTCAGCACCCTCCCCATCCAGCACCAGGACTGGATG AGTGGCAAGGAGTTCAAATGCAAGGTCAACAACAAAGACCTCCCATCA CCCATCGAGAGAACCATCTCAAAAATTAAAGGGCTAGTCAGAGCTCCA CAAGTATACATCTTGCCGCCACCAGCAGAGCAGTTGTCCAGGAAAGAT GTCAGTCTCACTTGCCTGGTCGTGGGCTTCAACCCTGGAGACATCAGT GTGGAGTGGACCAGCAATGGGCATACAGAGGAGAACTACAAGGACACC GCACCAGTCCTGGACTCTGACGGTTCTTACTTCATATATAGCAAGCTC AATATGAAAACAAGCAAGTGGGAGAAAACAGATTCCTTCTCATGCAAC GTGAGACACGAGGGTCTGAAAAATTACTACCTGAAGAAGACCATCTCC CGGTCTCCGGGTAAATGA (SEQ ID NO: 11) ATGAGGGTCCTTGCTGAGCTCCTGGGGCTGCTGCTGTTCTGCTTTTTA GGTGTGAGATGTGACATCCAGATGAACCAGTCTCCATCCAGTCTGTCT GCATCCCTTGGAGACACAATTACCATCACTTGCCATGCCAGTCAGAAC ATTAATGTTTGGTTAAGCTGGTACCAGCAGAAACCAGGAAATATTCCT AAACTATTGATCTATAAGGCTTCCAACTTGCGCACAGGCGTCCCATCA AGGTTTAGTGGCAGTGGATCTGGAACAGGTTTCACATTAACCATCAGC AGCCTGCAGCCTGAAGACATTGCCACTTACTACTGTCACCAGGGTCAA AGTTATCCGTGGACGTTCGGTGGAGGCACCAAGCTGGAAATCAAACGG GCTGATGCTGCACCAACTGTATCCATCTTCCCACCATCCAGTGAGCAG TTAACATCTGGAGGTGCCTCAGTCGTGTGCTTCTTGAACAACTTCTAC CCCAAAGACATCAATGTCAAGTGGAAGATTGATGGCAGTGAACGACAA AATGGCGTCCTGAACAGTTGGACTGATCAGGACAGCAAAGACAGCACC TACAGCATGAGCAGCACCCTCACGTTGACCAAGGACGAGTATGAACGA CATAACAGCTATACCTGTGAGGCCACTCACAAGACATCAACTTCACCA TTGTCAAGAGCTTCAACAGGAATGAGTGTTAG (SEQ ID NO: 12) AUGGGAUGGAGCUCUAUCAUCCUCUUCUUGGUAGCAACAGCUACAGGU GUCCACUCCCAGGUCCAACUGCAGCAGCCUGGGGCUGAGCUGGUGAGG CCUGGAGCUUCAGUGAAGCUGUCCUGCAAGGCUUCUGGCUACUCCUUC ACCAGUUACUGGAUGAACUGGGUGAAGCAGAGGCCUGGACAAGGCCUU GAGUGGAUUGGCAUGAUUCAUCCUUCCGAUAGUGAAACUAGGUUAAAU CAGAAGUUCAAGGACAAGGCCACAUUGACUGUAGACAAAUCCUCCAGC ACAGCCUACAUGCAACUCAGCAGCCCGACAUCUGAGGACUCUGCGGUC UAUUACUGUGCAAGACAAGGGUUUGCUUACUGGGGCCAAGGGACUCUG GUCACUGUCUCUACAGCCAAAACAACACCCCCAUCAGUCUAUCCACUG GCCCCUGGGUGUGGAGAUACAACUGGUUCCUCCGUGACUCUGGGAUGC CUGGUCAAGGGCUACUUCCCUGAGUCAGUGACUGUGACUUGGAACUCU GGAUCCCUGUCCAGCAGUGUGCACACCUUCCCAGCUCUCCUGCAGUCU GGACUCUACACUAUGAGCAGCUCAGUGACUGUCCCCUCCAGCACCUGG CCAAGUCAGACCGUCACCUGCAGCGUUGCUCACCCAGCCAGCAGCACC ACGGUGGACAAAAAACUUGAGCCCAGCGGGCCCAUUUCAACAAUCAAC CCCUGUCCUCCAUGCAAGGAGUGUCACAAAUGCCCAGCUCCUAACCUC GAGGGUGGACCAUCCGUCUUCAUCUUCCCUCCAAAUAUCAAGGAUGUA CUCAUGAUCUCCCUGACACCCAAGGUCACGUGUGUGGUGGUGGAUGUG AGCGAGGAUGACCCAGACGUCCAGAUCAGCUGGUUUGUGAACAACGUG GAAGUACACACAGCUCAGACACAAACCCAUAGAGAGGAUUACAACAGU ACUAUCCGGGUGGUCAGCACCCUCCCCAUCCAGCACCAGGACUGGAUG AGUGGCAAGGAGUUCAAAUGCAAGGUCAACAACAAAGACCUCCCAUCA CCCAUCGAGAGAACCAUCUCAAAAAUUAAAGGGCUAGUCAGAGCUCCA CAAGUAUACAUCUUGCCGCCACCAGCAGAGCAGUUGUCCAGGAAAGAU GUCAGUCUCACUUGCCUGGUCGUGGGCUUCAACCCUGGAGACAUCAGU GUGGAGUGGACCAGCAAUGGGCAUACAGAGGAGAACUACAAGGACACC GCACCAGUCCUGGACUCUGACGGUUCUUACUUCAUAUAUAGCAAGCUC AAUAUGAAAACAAGCAAGUGGGAGAAAACAGAUUCCUUCUCAUGCAAC GUGAGACACGAGGGUCUGAAAAAUUACUACCUGAAGAAGACCAUCUCC CGGUCUCCGGGUAAAUGA (SEQ ID NO: 13) AUGAGGGUCCUUGCUGAGCUCCUGGGGCUGCUGCUGUUCUGCUUUUUA GGUGUGAGAUGUGACAUCCAGAUGAACCAGUCUCCAUCCAGUCUGUCU GCAUCCCUUGGAGACACAAUUACCAUCACUUGCCAUGCCAGUCAGAAC AUUAAUGUUUGGUUAAGCUGGUACCAGCAGAAACCAGGAAAUAUUCCU AAACUAUUGAUCUAUAAGGCUUCCAACUUGCGCACAGGCGUCCCAUCA AGGUUUAGUGGCAGUGGAUCUGGAACAGGUUUCACAUUAACCAUCAGC AGCCUGCAGCCUGAAGACAUUGCCACUUACUACUGUCACCAGGGUCAA AGUUAUCCGUGGACGUUCGGUGGAGGCACCAAGCUGGAAAUCAAACGG GCUGAUGCUGCACCAACUGUAUCCAUCUUCCCACCAUCCAGUGAGCAG UUAACAUCUGGAGGUGCCUCAGUCGUGUGCUUCUUGAACAACUUCUAC CCCAAAGACAUCAAUGUCAAGUGGAAGAUUGAUGGCAGUGAACGACAA AAUGGCGUCCUGAACAGUUGGACUGAUCAGGACAGCAAAGACAGCACC UACAGCAUGAGCAGCACCCUCACGUUGACCAAGGACGAGUAUGAACGA CAUAACAGCUAUACCUGUGAGGCCACUCACAAGACAUCAACUUCACCC AUUGUCAAGAGCUUCAACAGGAAUGAGUGUUAG