RECOMBINANT PROTEINS WITH OX40 ACTIVATING PROPERTIES

Abstract

The disclosure relates to the field of OX40 activating proteins. More specifically, it is disclosed herein recombinant proteins with OX40 agonist antibodies or their antigen-binding fragments fused or linked to OX40 ligand. Also disclosed is the advantageous use of such OX40 activating proteins, in particular for inducing immune responses directed to delivered antigens such as viral or cancer antigens, and/or for treating cancer.

Claims

1. An OX40 activating protein comprising at least the following protein domains: (i) an OX40 agonist antibody or an antigen-binding fragment thereof (αOX40); and, (ii) the OX40 binding domain of OX40L (OX40L).

2. The OX40 activating protein of claim 1, wherein said OX40 agonist antibody or its antigen-binding fragment binds specifically to human OX40 and has at least one or more of the following properties: (i) it induces the proliferation of T cells, as measured in vitro by flow cytometric analysis; or, (ii) it induces the secretion of cytokines from T cells as measured in vitro with a CD4+ T cell activation assay.

3. The OX40 activating protein of claim 1, wherein said OX40 binding domain of OX40L is a fragment of OX40L comprising SEQ ID NO:2.

4. The OX40 activating protein of claim 1, wherein said OX40 binding domain of OX40L is fused to the C-terminus of a light or heavy chain of said OX40 agonist antibody or the antigen-binding fragment thereof.

5. The OX40 activating protein of claim 1, wherein the OX40 agonist antibody comprises a heavy and/or a light chain of an OX40 agonist IgG antibody or an antigen-binding fragment thereof.

6. The OX40 activating protein of claim 5, further comprising a peptide linker between the OX40L and the heavy and/or the light chain of said OX40 agonist IgG antibody or the antigen-binding fragment thereof.

7. The OX40 activating protein of claim 1, wherein said OX40 agonist antibody is selected from the following antibodies: a. an antibody comprising the HCDR1 of SEQ ID NO:3, HCDR2 of SEQ ID NO:4, HCDR3 of SEQ ID NO:5, LCDR1 of SEQ ID NO:6, LCDR2 of SEQ ID NO:7 and LCDR3 of SEQ ID NO:8; b. an antibody comprising VH and VL domains of SEQ ID NO:9 and SEQ ID NO:10 respectively; c. an antibody that competes for binding to OX40 expressing cells with at least one of the antibodies identified in (a) or (b); or, d. an antibody that binds to the same epitope as one of the antibodies identified in (a) or (b).

8. The OX40 activating protein of claim 6, wherein one or more antigens are fused to the heavy and/or light chain of said OX40 agonist antibody or the antigen-binding fragment thereof.

9. The OX40 activating protein of claim 8, wherein the one or more antigens include one or more viral or cancer antigens.

10. The OX40 activating protein of claim 1, comprising a light chain of the formula αOX40Light-PL1-OX40L and a heavy chain of the formula αOX40Heavy-(PL2-Ag)x, wherein αOX40Light is a light chain of said OX40 agonist antibody; αOX40Heavy is a heavy chain of said OX40 agonist antibody; PL1 and PL2 are a bond or a peptide linker, and are identical or different, Ag is one or more viral and/or cancer antigens, which are either identical or different; x is 0, or is an integer from 1 to 20; OX40L is the binding domain of the ligand of OX40 comprising SEQ ID NO:2 and is absent when x is 0 or is a bond.

11. A pharmaceutical composition, comprising the OX40 activating protein of claim 1 and one or more pharmaceutically acceptable excipients.

12. (canceled)

13. (canceled)

14. (canceled)

15. The OX40 activating protein of claim 2, wherein said flow cytometric analysis is an analysis of replicative dilution of CFSE-labelled cells.

16. The OX40 activating protein of claim 2, wherein said T cells are IL5, IL13, IFNγ and/or TNFα cytokines.

17. The OX40 activating protein of claim 6, wherein the peptide linker is the flexible linker FlexV1 of SEQ ID NO:13.

18. The OX40 activating protein of claim 10 wherein said PL1 and/or said PL2 is/are FlexV1 of SEQ ID NO:13.

19. The OX40 activating protein of claim 10 wherein x is 1, 2, 3, 4, or 5.

20. A method for treating or preventing cancer in a subject in need thereof comprising administering to the subject a therapeutically effective amount of the OX40 activating protein of claim 1.

21. A method for eliciting cell proliferation and/or inducing cytokine proliferation of T cells in a subject in need thereof comprising administering to the subject a therapeutically effective amount of the OX40 activating protein of claim 1.

22. A method for boosting immunity against cancer cells in a subject in need thereof suffering from cancer comprising administering to the subject a therapeutically effective amount of the OX40 activating protein of claim 1.

Description

DESCRIPTION OF THE FIGURES

[0227] FIG. 1: A cartoon illustrating the anti-Receptor antibody-Ligand fusion concept exemplified by anti-OX40 mAb24 OX40 IgG1. Shown are relevant domains: V.sub.L are the light chain variable regions; V.sub.H are the heavy chain variable regions; C.sub.H are the H chain constant regions 1-3; C.sub.L are the light chain constant regions; linker sequences and key disulfide bonds are represented by grey lines. The disclosure anticipate alternate form of fusion including Ligand fused to H chain C-termini and diverse antibody forms and isotypes.

[0228] FIG. 2: Binding of anti-OX40 antibodies to human OX40-transfected CHO cells. CHO cells stably transfected with a construct for expressing human OX40 ectodomain fused to FAS transmembrane and intracellular residues were incubated at 4° C. for 30 min with a titration series of anti-OX40 IgG1, anti-OX40-OX40L IgG1, and control hIgG4. The cells were washed in PBS and probed with anti-human IgG-PE reagent at 4° C. for 30 min, washed with PBS, then 500K cells per point were analyzed by flow cytometry on a FACS Array Bioanalyzer (BD Biosciences) scoring percentage of cells brighter than the gate set for cells probed only with the detecting reagent (% P 1 of Parent).

[0229] FIG. 3. SPR analysis of soluble OX40 ectodomain binding to solid phase anti-OX40 antibodies with or without directly linked human OX40L. Cohesin-human OX40 ectodomain protein (400 nM injected for 6 min plus 5 min dissociation time at 25 μL/min) over a protein A coated surface with bound anti-OX40 IgG1 mAbs with (B) and without (A) human OX40L linked to the L chain C-termini. The cartoons show the OX40L configuration and F indicates attachment via a Flex V1 linker. Kinetic parameters are shown in Table 4. Control human IgG1 and IgG1-Flex v1-OX40L proteins run in this assay gave no or low (<6 RU) binding to OX40 in this assay.

[0230] FIG. 4. Potentiation via OX40 binding of proliferation and cytokine production by CD4+ T cells. CD4+ T cells from PBMCs of two normal donors (left panels are Donor 1; right panels are Donor 2) were primed for two days with PHA, IL-2, and anti-CD3/CD28 beads, then labeled with CFSE and cultured an additional 5 days with a titration series of the test antibodies and controls. Cells were analyzed for proliferation by flow cytometry scoring CFSE dilution (A). Culture supernatants were analyzed for IL-13 (B) and TNFα (C).

EXAMPLES

[0231] 1. Methods

[0232] Surface plasmon resonance assay. Surface plasmon resonance (SPR) assay binding measurements were performed on a SensiQ Pioneer instrument (SensiQ Technologies, Inc., Oklahoma City, Okla., USA). Protein A or Protein G (100 μg/mL in 10 mM NaAc pH4.5) were immobilized using amine coupling chemistry on COOH2 or COOH5 sensor chips at 25° C. following the manufacturer's recommended protocols. Running buffer was 10 mM HEPES, 3.4 mM EDTA, 0.005% Tween 20, 8.8 g/L NaCl, pH 7.5. Subsequently, Channel 1-2 were used to inject a dilution series of cohesin-human CD40 ectodomain (Genbank AAO43990.1 residues 22-193) or human OX40 (GenBank AAB33944.1 residues 26-211) protein (25, 12.5, 6.25, 3.125, 1.6, 0.8 nM at 25 uL/min for 2 min); finally, surfaces were regenerated through injection of 20 mM NaOH for 1 min (25 μL). The binding data were analyzed with Qdat software (SensiQ Technologies).

[0233] Assay for human OX40 binding. CHO-S cells stably transfected with a plasmid construct for expressing human OX40 ectodomain (GenBank AAB33944.1residues 26-211) fused to FAS transmembrane and intracellular domains (GenPept XP_011538069.1 residues 187-350) were incubated at 4° C. for 30 min with a titration series of anti-OX40 IgG1, anti-OX40-OX40L IgG1, and control hIgG4. The cells were washed in PBS and probed with anti-human IgG-PE reagent at 4° C. for 30 min, washed with PBS, then 500K cells per point were analyzed by flow cytometry on a FACSArray Bioanalyzer (BD Biosciences) scoring the percentage of cells brighter than the gate set for cells probed only with the detecting reagent (defined as % P1 of Parent).

[0234] Assay for OX40 agonistic activity. Blood from 2 different donors was collected in Acid-Citrate-Dextrose (ACD) tubes was processed by Ficoll-Paque® separation in SepMate™ (Stemcell Technologies, Cambridge, Mass.), using 3 ACD tubes for each SepMate™ according to the manufacturers protocol. Recovery of viable cells was in the range 1.1E8, 98.5%; and 1.13E8, 99.3%. The PBMCs were processed with the EasySep™ CD4.sup.+ T cell Negative Isolation Kit (Stemcell Technologies, 17952). Cells were first stimulated with PHA/IL-2/anti-CD3/CD28 beads. PHA stock (Remel purified phytohaemagglutinin, Thermo Fisher, R30852801) was in PBS, 2% FCS at 1 mg/ml and was used at 2 μg/ml. Anti-CD3/CD28 beads for polyclonal stimulation (Gibco Dynabeads Human T activator, reference 11161D, 40,000 bead per 1l) were used at 5 T cells to 1 bead. Interlekin-2 (IL-2, Pharmaceutical Grade Proleukin®) at 10E6 Units per ml was used at 20 U/ml. The isolated CD4.sup.+ T cells from both donors were individually stimulated at 1E6/ml in 50 ml conical tubes with 10% FCS, cRPMI and kept at 37° C. at 7% CO.sub.2 for 48 hours. A small subset was tested by flow cytometry at 24 and 48 hours to validate increased expression of OX40 (data not shown). After 48 hours, anti-CD3/CD28 beads were removed using a magnet (Stem Cell) and cells were labeled with CFSE to enable monitoring the proliferation status using the Cell Trace Labeling Kit (Thermo Fisher, 34554) reconstituted with 18 μl DMSO to make a 5 mM stock. Cells were washed in twice in PBS warmed to 37° C. following the bead depletion. Cells were then resuspended in 1.25 μM CFSE in the warm PBS at a concentration of 1E6/ml at RT for 10 min, then quenched with 10 volumes of ice cold 10% FCS cRPMI and kept at 4° C. for an additional 5 min. Cells were washed twice in media or 2% FCS with PBS and then resuspended in media and added at 100K cells per well (50 l) into V-bottom culture plates for stimulation. Test proteins were added as a dilution series from 2 μM to 0.05 nM. Cell controls without test vehicles were included as controls and some were also stimulate with anti-CD3/CD28 (100K T cells to 40K beads, ratio of 2.5 T cells to 1 bead) as positive signal controls. Cells were then cultured an additional 5 days and culture supernatants were analyzed by Luminex® for IL-5, IL-13, IFNγ and TNFα cytokine levels (Millipore). The remaining cells were analyzed by flow cytometry (FACS CANTO, BD Biosciences) for CFSE proliferation after staining with Live/Dead™ Fixable Aqua Dead Cell Stain (Thermo Fisher) and anti-CD4 mAb using FlowJo® software (Ashland, Or).

[0235] Flow cytometry analysis. Cells were transferred in a V bottom plate, washed twice in PBS and incubated for 20 minutes at 4° C. with Live/Dead™ Fixable Aqua Dead Cell Stain Kit (Thermo Fisher Scientific, Cat. L34965) at 1:50 in a volume of 50 μL or with L/D-ef780 (Thermo Fisher, Cat. 65-0865-14) added to the antibody mix. Cells were wash twice and incubated for 30 minutes on ice with the mix of antibodies in a volume of 50 μL. Finally, cells were washed and resuspended in 200 μL BD™ stabilizing fixative (BD Biosciences, Cat. 338036) diluted 1:3. All analysis plots were pre-gated on live (using Live/Dead stain) and singlet events. Cells were analyzed with a FACS Canto II, FACSArray Bioanalyzer or a LSR Fortessa (BD Biosciences). Data was analyzed with FlowJo Software. The following antibodies were used for analysis of human DC activation: hCD80-PE, Mouse clone L307.4, ref 340294 (BD), hCD83-APC, Mouse clone HB15e, ref 551073 (BD); hCD86-FITC, Mouse clone 2331 (FUN-1), ref 555657 (BD); hCD11c-PEpCyanine7, Mouse clone 3.9, ref 25011642, (eBioscience); hHLA-DR-V450, Mouse clone G46-6, ref561359 (BD); hCD40-PE-Cyanine5, Mouse clone 5C3, ref 555590 (BD). hCD19-APC, mouse clone HIB19, ref 555415 (BD) and hCD3-PerCP, Mouse clone SK7, ref 347344 (BD) were used in the human B cell proliferation panel. The following antibodies were used for the mouse ex vivo cell analyses: mB220-AF488, clone RA3-6B2, ref 103225 (BD); mOX40L-PerCPCy5.5, clone RM134L, ref 65-5905-U025 (Tonbo Biosciences); mCD69-PE, clone H1.2F3, ref 104508 (Biolegend); mCD11c-PECy7, clone N418, ref 117318, (Biolegend); mLangerin-APC, clone 4C7, ref144206 (Biolegend); mMHCII-AF700, clone M5/114.15.2, ref 107622 (Biolegend); mCD11b-PacificBlue, clone M1/70, ref 101224 (Biolegend); mLy6G-BV510, clone 1A8, ref 127633 (Biolegend); mCD86-BV605, clone GL1, ref 105037 (Biolegend); mIgD-BV711, clone 11-26c.2a, ref 405731 (Biolegend); FC bloc, clone 2.4G2, ref 70-0161-U500 (Tonbo Biosciences).

[0236] Statistical analyses. Data are presented as means (±SEM). Statistical significance was determined by Student's t test with or without Welch's correction. A P value <0.05 was considered as statistically significant. GraphPad Prism software was used for statistical calculations.

[0237] 2. Results

[0238] Fusing OX40 Ligand to an Agonistic OX40 Antibody Improves Agonist Potency and Efficacy

[0239] We linked human OX40L to the light chain C-terminus of an agonistic anti-OX40 IgG1 antibody in clinical development (humanized OX40mAb24, U.S. Pat. No. 9,738,723).

[0240] FIG. 2 shows that both the humanized anti OX40 Ab24 IgG1 and anti OX40 Ab24-OX40L fusion IgG1 proteins bound similarly to CHO cells expressing the human OX40 ectodomain. However, SPR analysis detected an ˜2-fold enhancement in off-rate associated with the addition of OX40L to the anti-OX40 mAb L chain (FIG. 3 and Table 4).

TABLE-US-00004 TABLE 4 Kinetic parameters and affinity constants for the interaction between immobilized anti-human OX40 IgG1 mAbs and liquid phase soluble human OX40 ectodomain as shown in FIG. 3. mAh Ka (M.sup.−1s.sup.−1) kd (s.sup.−1) KD (nM) Anti-OX40 IgG1 1.4E+04 [1.0] 1.9E−03 [1.0] 131 [1] Anti-OX40-FlexV1- 1.6E+04 [1.1] 8.7E−04 [2.2] 55 [0.42] OX40L IgG1

[0241] Anti-OX40 hIgG1, anti-OX40-OX40L hIgG1 and hIgG1-OX40L proteins were tested for their ability to evoke proliferation and cytokine production of primed human CD4.sup.+ T cells in vitro. In the absence of any Fc cross-linking agent, picomolar levels of anti-OX40-OX40L were sufficient to drive both proliferation and cytokine production in this assay. In contrast, nanomolar amounts of hIgG1-OX40L were required to drive proliferation and cytokine production, while anti-OX40 was inactive in this assay.

[0242] These data (FIG. 4) show that fusing OX40L to agonistic OX40 antibody profoundly changes the characteristics of the separate entities: i) the anti-OX40-OX40L IgG1 fusion increases potency of the OX40L IgG1 by >2 logs; ii) the efficacy (maximum response) is increased; and iii) the activity is independent of Fc cross-linking.

[0243] In conclusion, we selected a clinical candidate anti-X40 mAb agonist and fused OX4L to this mAb. This improved in vitro potency compared to the OX40L IgG1 by ≥2 logs; the anti-X40 mAb activity became independent of Fc cross-linking; and the efficacy (maximum response) increased.

[0244] Tables 5 and 6: Useful Sequences for Practicing the Invention

TABLE-US-00005 SEQ ID Type Brief description 1 aa Full amino acid sequence of OX40 2 aa Amino acid sequence of OX40 binding domain of human OX40L 3 aa HCDR1 amino acid sequence of mAb24 OX40 agonist antibody 4 aa HCDR2 amino acid sequence of mAb24 OX40 agonist antibody 5 aa HCDR3 amino acid sequence of mAb24 OX40 agonist antibody 6 aa LCDR1 amino acid sequence of mAb24 OX40 agonist antibody 7 aa LCDR2 amino acid sequence of mAb24 OX40 agonist antibody 8 aa LCDR3 amino acid sequence of mAb24 OX40 agonist antibody 9 aa VH amino acid sequence of mAb24 OX40 agonist antibody 10 aa VL amino acid sequence of mAb24 OX40 agonist antibody 11 aa Amino acid sequence of anti-OX40-OX40L IgG1 heavy chain 12 aa Amino acid sequence of anti-OX40-OX40L light chain 13 aa Amino acid sequence of FlexV1 peptidic linker 14 nt VH nucleotide sequence of mAb24 OX40 agonist antibody 15 nt VL nucleotide sequence of mAb24 OX40 agonist antibody 16 nt Nucleotide sequence of anti-OX40-OX40L IgG1 heavy chain 17 nt Nucleotide sequence of anti-OX40-OX40L light chain 18 aa Ectodomain of OX40

TABLE-US-00006 SEQ ID Type SEQUENCE 1 aa RCCHECRPGNGMVSRCSRSQNTVCRPCGPGFYNDVVSSKP CKPCTWCNLRSGSERKQLCTATQDTVCRCRAGTQPLDSYK PGVDCAPCPPGHFSPGDNQACKPWTNCTLAGKHTLQPASN SSDAICEDRDPPATQPQETQGPPARPITVQPTEAWPRTSQ GPSTRPVEVPGGRAVAAILGLGLVLGLLGPLAILLALYLL RRDQRLPPDAHKPPGGGSFRTPIQEEQADAHSTLAKI 2 aa ASHFSALQVSHRYPRIQSIKVQFTEYKKEKGFILTSQKED EIMKVQNNSVIINCDGFYLISLKGYFSQEVNISLHYQKDE EPLFQLKKVRSVNSLMVASLTYKDKVYLNVTTDNTSLDDF HVNGGELILIHQNPGEFCVLAS 3 aa GGSFSSGYWN 4 aa YISYNGITYHNPSLKS 5 aa YKYDYDGGHAMDY 6 aa RASQDISNYLN 7 aa YTSKLHS 8 aa QQGSALPWT 9 aa QVQLQESGPGLVKPSQTLSLTCAVYGGSFSSGYWNWIRKH PGKGLEYIGYISYNGITYHNPSLKSRITINRDTSKNQYSL QLNSVTPEDTAVYYCARYKYDYDGGHAMDYWGQGTLVTVS SASTKGPSVFPLAPCSRSTSGGTAALGCLVKDYFPEPVTV SWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQ TYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLG GPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFN WYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNG KEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRD ELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPP VLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHY TQKSLSLSPGKAS 10 aa DIQMTQSPSSLSASVGDRVTITCRASQDISNYLNWYQQKP GKAPKLLIYYTSKLHSGVPSRFSGSGSGTDYTLTISSLQP EDFATYYCQQGSALPWTFGQGTKVEIKRTVAAPSVFIFPP SDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQ ESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQG LSSPVTKSFNRGECAS 11 aa QVQLQESGPGLVKPSQTLSLTCAVYGGSFSSGYWNWIRKH PGKGLEYIGYISYNGITYHNPSLKSRITINRDTSKNQYSL QLNSVTPEDTAVYYCARYKYDYDGGHAMDYWGQGTLVTVS SASTKGPSVFPLAPCSRSTSGGTAALGCLVKDYFPEPVTV SWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQ TYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLG GPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFN WYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNG KEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRD ELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPP VLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHY TQKSLSLSPGKAS 12 aa DIQMTQSPSSLSASVGDRVTITCRASQDISNYLNWYQQKP GKAPKLLIYYTSKLHSGVPSRFSGSGSGTDYTLTISSLQP EDFATYYCQQGSALPWTFGQGTKVEIKRTVAAPSVFIFPP SDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQ ESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQG LSSPVTKSFNRGECASQTPTNTISVTPTNNSTPTNNSNPK PNPASHFSALQVSHRYPRIQSIKVQFTEYKKEKGFILTSQ KEDEIMKVQNNSVIINCDGFYLISLKGYFSQEVNISLHYQ KDEEPLFQLKKVRSVNSLMVASLTYKDKVYLNVTTDNTSL DDFHVNGGELILIHQNPGEFCVLAS 13 aa ASQTPTNTISVTPTNNSTPTNNSNPKPNPAS 14 nt ATGGATCCTAAGGGCTCTCTGTCTTGGAGAATCCTGCTGT TTCTGTCTCTGGCCTTTGAGCTGTCTTACGGCCAGGTGCA GCTGCAGGAGTCTGGACCTGGCCTGGTGAAGCCTTCTCAG ACCCTGTCTCTGACATGTGCCGTGTACGGAGGCTCTTTTT CTTCTGGCTACTGGAACTGGATTAGAAAGCACCCTGGAAA GGGCCTGGAGTACATTGGCTACATCTCTTACAACGGCATC ACATATCATAACCCTTCTCTGAAGTCTAGAATCACAATTA ACAGAGATACATCTAAGAACCAGTACTCTCTGCAGCTGAA CTCTGTGACACCTGAGGATACAGCCGTGTACTACTGTGCC AGATACAAGTACGATTATGATGGCGGCCACGCTATGGATT ACTGGGGACAGGGAACACTGGTGACCGTGTCTTCTGCTTC TACAAAGGGCCCATCGGTCTTCCCCCTGGCGCCCTGCTCC AGGAGCACCTCTGGGGGCACAGCGGCCCTGGGCTGCCTGG TCAAGGACTACTTCCCCGAACCGGTGACGGTGTCGTGGAA CTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCT GTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGG TGACCGTGCCCTCCAGCAGCTTGGGCACCCAGACCTACAT CTGCAACGTGAATCACAAGCCCAGCAACACCAAGGTGGAC AAGAGAGTTGAGCCCAAATCTTGTGACAAAACTCACACAT GCCCACCGTGCCCAGCACCTGAACTCCTGGGGGGACCGTC AGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATG ATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACG TGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGT GGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGG GAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCC TCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTA CAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCATC GAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAAC CACAGGTGTACACCCTGCCCCCATCCCGGGATGAGCTGAC CAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTC TATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGC AGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGA CTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTG GACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCT CCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAA GAGCCTCTCCCTGTCTCCGGGTAAAGCTAGCTGA 15 nt ATGGATCCTAAGGGCTCTCTGTCTTGGAGAATCCTGCTGT TCCTGTCTCTGGCTTTTGAGCTGTCTTACGGCGATATCCA GATGACACAGTCTCCTTCTTCTCTGTCTGCCTCTGTGGGC GATAGAGTGACCATCACATGTAGAGCCTCTCAGGATATCT CTAACTACCTGAACTGGTACCAGCAGAAGCCTGGCAAGGC CCCAAAGCTGCTGATCTATTACACATCTAAGCTGCACTCT GGCGTGCCTTCTAGATTTTCTGGCTCTGGATCTGGCACAG ATTATACACTGACCATCTCTTCTCTGCAGCCTGAAGATTT TGCTACATACTACTGTCAGCAGGGCTCTGCTCTGCCTTGG ACCTTTGGACAGGGCACAAAGGTGGAGATCAAGCGAACTG TGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGA GCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTG AATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGG TGGATAACGCCCTCCAATCGGGTAACTCCCAGGAGAGTGT CACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGC AGGACCCTGACGCTGAGCAAAGCAGACTAGGAGAAACACA AAGTCTATGCCTGCGAAGTCACCCATCAGGGCCTGAGCTC GCCCGTCACAAAGAGCTTCAACAGGGGAGAGTGTGCTAGC TGA 16 nt ATGGATCCTAAGGGCTCTCTGTCTTGGAGAATCCTGCTGT TTCTGTCTCTGGCCTTTGAGCTGTCTTACGGCCAGGTGCA GCTGCAGGAGTCTGGACCTGGCCTGGTGAAGCCTTCTCAG ACCCTGTCTCTGACATGTGCCGTGTACGGAGGCTCTTTTT CTTCTGGCTACTGGAACTGGATTAGAAAGCACCCTGGAAA GGGCCTGGAGTACATTGGCTACATCTCTTACAACGGCATC ACATATCATAACCCTTCTCTGAAGTCTAGAATCACAATTA ACAGAGATACATCTAAGAACCAGTACTCTCTGCAGCTGAA CTCTGTGACACCTGAGGATACAGCCGTGTACTACTGTGCC AGATACAAGTACGATTATGATGGCGGCCACGCTATGGATT ACTGGGGACAGGGAACACTGGTGACCGTGTCTTCTGCTTC TACAAAGGGCCCATCGGTCTTCCCCCTGGCGCCCTGCTCC AGGAGCACCTCTGGGGGCACAGCGGCCCTGGGCTGCCTGG TCAAGGACTACTTCCCCGAACCGGTGACGGTGTCGTGGAA CTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCT GTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGG TGACCGTGCCCTCCAGCAGCTTGGGCACCCAGACCTACAT CTGCAACGTGAATCACAAGCCCAGCAACACCAAGGTGGAC AAGAGAGTTGAGCCCAAATCTTGTGACAAAACTCACACAT GCCCACCGTGCCCAGCACCTGAACTCCTGGGGGGACCGTC AGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATG ATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACG TGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGT GGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGG GAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCC TCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTA CAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCATC GAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAAC CACAGGTGTACACCCTGCCCCCATCCCGGGATGAGCTGAC CAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTC TATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGC AGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGA CTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTG GACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCT CCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAA GAGCCTCTCCCTGTCTCCGGGTAAAGCTAGCTGA 17 nt ATGGATCCTAAGGGCTCTCTGTCTTGGAGAATCCTGCTGT TCCTGTCTCTGGCTTTTGAGCTGTCTTACGGCGATATCCA GATGACACAGTCTCCTTCTTCTCTGTCTGCCTCTGTGGGC GATAGAGTGACCATCACATGTAGAGCCTCTCAGGATATCT CTAACTACCTGAACTGGTACCAGCAGAAGCCTGGCAAGGC CCCAAAGCTGCTGATCTATTACACATCTAAGCTGCACTCT GGCGTGCCTTCTAGATTTTCTGGCTCTGGATCTGGCACAG ATTATACACTGACCATCTCTTCTCTGCAGCCTGAAGATTT TGCTACATACTACTGTCAGCAGGGCTCTGCTCTGCCTTGG ACCTTTGGACAGGGCACAAAGGTGGAGATCAAGCGAACTG TGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGA GCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTG AATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGG TGGATAACGCCCTCCAATCGGGTAACTCCCAGGAGAGTGT CACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGC AGCACCCTGACGCTGAGCAAAGCAGACTAGGAGAAACACA AAGTCTATGCCTGCGAAGTCACCCATCAGGGCCTGAGCTC GCCCGTCACAAAGAGCTTCAACAGGGGAGAGTGTGCTAGT CAGACCCCCACCAACACCATCAGCGTGACCCCCACCAACA ACAGCACCCCCACCAACAACAGCAACCCCAAGCCCAACCC CGCTAGTCATTTCTCAGCTTTGCAAGTGTCCCACAGATAC CCAAGGATCCAGTCCATCAAGGTGCAGTTCACAGAGTACA AGAAGGAGAAAGGGTTCATCCTGACATCCCAGAAAGAGGA CGAGATCATGAAGGTCCAGAACAACAGTGTGATTATCAAC TGCGACGGCTTCTACCTCATTAGCCTGAAGGGGTACTTTT CTCAGGAGGTGAATATTTCCCTGCACTACCAGAAGGATGA GGAGCCTCTCTTTCAGTTGAAGAAGGTGCGGAGCGTGAAC AGCCTTATGGTCGCCAGCCTGACATATAAGGACAAGGTGT ACCTGAACGTGACCACTGATAACACCAGCCTCGATGATTT TCACGTCAACGGGGGAGAACTCATTCTGATCCACCAGAAC CCCGGCGAATTTTGTGTCCTGGCTAGC 18 aa HFSALQVSHRYPRIQSIKVQFTEYKKEKGFILTSQKEDEI MKVQNNSVIINCDGFYLISLKGYFSQEVNISLHYQKDEEP LFQLKKVRSVNSLMVASLTYKDKVYLNVTTDNTSLDDFHV NGGELILIHQNPGEFCVL * highlighted AS relates to restriction linker sites

BIBLIOGRAPHY

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