A nanobody with high affinity to human dr3 ectodomain

Abstract

The present application relates to the technical field of biopharmaceuticals, and particularly relates to a kind of nanobody with high affinity to human DR3 extracellular domain.The present application immunizes alpaca with the extracellular domain of DR3 of insect expression system, separates peripheral blood lymphocyte to carry out total RNA extraction, reverses transcription and amplifies nanobody sequence, finally separates and obtains nanobody, and is named H10.SPR result shows that the nanobody shows high affinity binding to human DR3 extracellular domain, and the nanobody provided by the present application has the potential to provide experimental fact basis for the treatment idea of DR3 related diseases.

Description

[0001] This application is a divisional application of application number 202310649138.3, application date June 2, 2023, entitled "A nanobody targeting the extracellular domain of DR3 and its application". Technical Field

[0002] This invention relates to the field of biopharmaceutical technology, and in particular to a nanobody with high affinity for the extracellular domain of human DR3. Technical Background

[0003] The tumor necrosis factor receptor superfamily (TNFRSF) is one of the most important families in the immune system, broadly regulating various immune responses such as the development of immune organs, co-stimulation of lymphocytes, and determination of lymphocyte fate. Within the TNFRSF family, death receptor 3 (DR3) shares the highest amino acid sequence homology with TNFR1. DR3 is highly expressed in thymic and splenic lymphocytes, as well as peripheral blood lymphocytes. It plays a role in promoting apoptosis during thymocyte development, while in lymphocytes, it primarily exerts a co-stimulatory effect with its unique ligand TL1A (TNF-like ligand 1A, TNFSF15). When TL1A activates the signaling pathway, the intracellular DR3 can recruit its downstream aptamer molecule TRADD (TNFR-associated death domain), ultimately activating NF-κB signaling. The most prevalent chronic autoimmune diseases associated with TL1A-DR3 are inflammatory bowel disease (IBD) and rheumatoid arthritis (RA). Studies have shown that the DR3-TL1A signaling pathway can play a pathogenic role in inflammatory bowel disease by promoting Th1 and Th17 immune responses. For example, TL1A / DR3 can exert an inflammatory effect in the gut by activating ILC3 (Activation of DR3 signaling causes loss of ILC3s and exacerbates intestinal inflammation).

[0004] Given the crucial roles of the TL1A and DR3 signaling pathways in autoimmune and inflammatory diseases, studies have shown that neutralizing TL1A with soluble DR3 protein can effectively alleviate α-CD40 and DSS-induced colitis. This suggests that inhibiting TL1A-DR3 interaction may be an effective therapeutic strategy for improving autoimmune diseases and local inflammation in target organs. In recent years, the incidence and prevalence of inflammatory bowel diseases such as ulcerative colitis and Crohn's disease have been on the rise. However, the efficacy of traditional drug treatments, such as 5-aminosalicylic acid ester, is uncertain, and the relapse rate is high; furthermore, they are ineffective in patients with moderate to severe IBD. Targeting ligands and receptors of related signaling pathways has become the most promising target for anti-IBD drug development. Developing an antibody that targets the extracellular domain of DR3 and prevents it from activating the signaling pathway with its ligand TL1A will have significant clinical implications. Summary of the Invention

[0005] To address the problems in the prior art, one objective of this invention is to provide a nanobody targeting the extracellular domain of DR3, wherein the nanobody is any one of A2, A6, and H10, and each of A2, A6, and H10 contains three antigen complementarity-determining regions CDR1, CDR2, and CDR3, wherein:

[0006] The amino acid sequences of the three antigen complementarity-determining regions of A2 have a homology of greater than or equal to 80% with the amino acid sequences shown in SEQ ID NO:1, SEQ ID NO:2 and SEQ ID NO:3, and preferably at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% of the amino acid sequences.

[0007] The amino acid sequences of the three antigen complementarity-determining regions of A6 have a homology of greater than or equal to 80% with the amino acid sequences shown in SEQ ID NO:4, SEQ ID NO:5 and SEQ ID NO:6, preferably at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% of the amino acid sequences.

[0008] The amino acid sequences of the three antigen complementarity-determining regions of the H10 have a homology of 80% or more with the amino acid sequences shown in SEQ ID NO:7, SEQ ID NO:8 and SEQ ID NO:9, preferably at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%.

[0009] The aforementioned homology sequences also include amino acid sequences that have one or more (preferably 1, 2 or 3) conserved amino acid mutations (preferably substitutions, insertions or deletions) compared to the sequences shown in the sequence listing.

[0010] Preferably, the amino acid sequences of A2, A6, and H10 are as follows:

[0011] The amino acid sequence of A2:

[0012] QVQLVESGGGLVQAGGSLRLSCAAS GDTICISAM GWYRQAPGKERE LVAGITSS TYYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYC NADLGSCPGVYE YEYWGQGTQVTVSS(SEQ ID NO:10)

[0013] The amino acid sequence of A6:

[0014] QVQLVESGGGLVQPGGSLRLSCAAS GDTICISGM GWYRQAPGKERE LVAAITSGGS TYYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYC NADGSRCPGE YDYWGQGTQVTVSS(SEQ ID NO:11)

[0015] The amino acid sequence of H10:

[0016] QVQLVESGGGLVQPGGSLRLSCTAS GLTSDDYTM GWFRQAPGKERE GVSCISRVDGN TYYADSVKGRFTISRGNAKNTVYLQMNSLKPEDTAVYYC ATDCRLTPNSDWNGPLRFGS WGQGTQVTVSS(SEQ ID NO:12)

[0017] The amino acid sequences of the three antigen complementarity-determining regions (CDR1, CDR2, and CDR3) of the above-mentioned nanobody are shown in bold underlined parts, namely:

[0018] The amino acid sequence of the antigen complementarity-determining region of A2:

[0019] CDR1:GDTICISAM(SEQ ID NO:1)

[0020] CDR2:LVAGITSS(SEQ ID NO:2)

[0021] CDR3:NADLGSPGVYE(SEQ ID NO:3)

[0022] The amino acid sequences of the three antigen complementarity-determining regions of A6 are as follows:

[0023] CDR1:GDTICISGM(SEQ ID NO:4)

[0024] CDR2:LVAAITSGGS(SEQ ID NO:5)

[0025] CDR3:NADGSRCPGE(SEQ ID NO:6)

[0026] The amino acid sequences of the three antigen complementarity-determining regions of H10 are as follows:

[0027] CDR1:GLTSDDYTM(SEQ ID NO:7)

[0028] CDR2:GVSCISRVDGN(SEQ ID NO:8)

[0029] CDR3:ATDCRLTPNSDWNGPLRFGS(SEQ ID NO:9)

[0030] The present invention also provides an antibody targeting the extracellular domain of DR3, which has any one of the nanobodies of A2, A6, and H10 as described above and an Fc domain.

[0031] Preferably, the Fc domain is the human IgG1 Fc domain, and the amino acid sequence is shown in SEQ ID NO:13.

[0032] The present invention also provides a polynucleotide encoding the nanobody A2, A6 or H10 as described above, or encoding an antibody having an Fc domain as described above; wherein the nucleotide sequence encoding A2 is shown in SEQ ID NO:14, the nucleotide sequence encoding A6 is shown in SEQ ID NO:15, and the nucleotide sequence encoding H10 is shown in SEQ ID NO:16.

[0033] The present invention also provides an expression vector comprising the polynucleotides described above, and a host cell comprising the expression vector as described above. Preferably, the host cell is a host cell for expressing exogenous proteins, such as bacteria, yeast, insect cells, or mammalian cells.

[0034] The present invention provides a pharmaceutical composition comprising nanobodies A2, A6 or H10 as described above, or comprising antibodies having an Fc domain as described above.

[0035] Preferably, the pharmaceutical composition is in a form suitable for administration by subcutaneous injection, intradermal injection, intravenous injection, intramuscular injection, or intralesional injection.

[0036] The nanobodies / antibodies provided by this invention can be used to prepare treatments and / or diagnoses of DR3-related diseases, such as Crohn's disease, ulcerative colitis, and rheumatoid arthritis.

[0037] The present invention also provides the use of the above-mentioned nanobodies A2, A6 or H10, or antibodies containing the Fc domain as described above, in the preparation of kits for the treatment and / or diagnosis of diseases related to DR3, such as Crohn's disease, ulcerative colitis or rheumatoid arthritis.

[0038] Preferably, the nanobody / antibody further includes a second antibody, which includes a detectable label, such as a radioactive isotope, a luminescent substance, a colored substance, an enzyme, or polyethylene glycol.

[0039] The beneficial effects of this invention are as follows:

[0040] 1. Alpacas were immunized three times using the extracellular domain of the DR3 expression system of an insect. Peripheral blood lymphocytes (PBMCs) were then isolated from the blood. Total RNA was extracted from the isolated PBMCs and immediately reverse transcribed into cDNA. The cDNA was used as a template to amplify nanobody sequences. Finally, three nanobodies were isolated and named A2, A6 and H10, respectively.

[0041] This nanobody (VHH) is derived from a natural camel heavy chain antibody and has the following advantages: 1) simple structure and small molecular weight, which are conducive to expression and use; 2) convenient for efficient and large-scale expression in E. coli and various eukaryotic systems; 3) due to having only one binding site, it is a single-domain antibody, which has better permeability, specificity and detection linearity as a diagnostic reagent; 4) easy to couple with various fusion proteins or more easily labeled by various markers; 5) easier to prepare bifunctional antibodies, which is more conducive to targeted drug development and cellular target-directed delivery; 6) as a drug development agent, it has the advantages of low immunogenicity in humans and is less likely to cause immune rejection.

[0042] 2. The three nanobodies provided in this invention have different antigen complementarity-determining regions. Antibodies binding to the extracellular domain of human DR3 were expressed and secreted in mammalian cells (HEK293F). The antibodies were fused with human IgG1 Fc and cloned into the mammalian expression vector pTT5. The vector was transfected into mammalian cells 293F, and the supernatant was collected after 4 days of culture. The fusion protein in the supernatant was purified using a Protein A column. The yields of all three nanobodies were greater than 50 mg / L.

[0043] ELISA results showed that all three nanobodies exhibited high affinity binding to the extracellular domain of human DR3, with the A2-IgG1 Fc nanobody showing the highest affinity binding. 50 EC50 of A6-IgG1 Fc at 0.072±0.021 nM50 At 0.146±0.041 nM, the EC of H10-IgG1 Fc 50 At 0.063±0.017 nM. The nanobody provided by this invention holds promise for providing experimental evidence for treatment strategies related to DR3-related diseases. Attached Figure Description

[0044] Figure 1 ELISA results for monoclonal phages that specifically bind to the extracellular domain of human DR3.

[0045] Figure 2 Figure 1 shows the results of antigen and antibody purification and SDS-PAGE analysis. Figure A shows the 24 mL molecular sieve result and SDS-PAGE result of the extracellular region of human DR3 antigen; Figure B shows the elution diagram and SDS-PAGE result of nanobody A2 purified by Protein A column; Figure C shows the elution diagram and SDS-PAGE result of nanobody A6 purified by Protein A column; Figure D shows the elution diagram and SDS-PAGE result of nanobody H10 purified by Protein A column.

[0046] Figure 3 Results of competitive ELISA analysis of the binding of nanobodies to the extracellular region of human DR3; ECGs of three nanobodies 50 The values ​​are A2 and EC respectively. 50 =0.072±0.021nM; A6: EC 50 =0.146±0.041nM; G10: EC 50 =0.063±0.017nM. Detailed Implementation

[0047] To facilitate understanding, the technical solution of the present invention will be described in more detail below with reference to embodiments:

[0048] Example 1

[0049] Purified extracellular domains of human DR3 were used for immunization and screening of alpacas.

[0050] 1) An expression plasmid was constructed and transformed into DH10Bac competent cells containing a Bacmid and a helper plasmid. The target gene was transposable onto the Bacmid by transposase. Clones containing recombinant transposons were obtained through kanamycin, gentamicin, and tetracycline resistance selection and blue-white screening. After further identification by PCR, high-purity, high-concentration recombinant Bacmid was purified and transfected into corresponding insect cells to obtain recombinant baculovirus containing the target gene. The recombinant baculovirus was amplified three times in SF9 cells, and the virus in the supernatant was collected and added to Hi5 cells at a ratio of 1:1000 for viral infection and expression of the target protein. Four days later, the cell supernatant was collected, and residues 26-199 of the extracellular region of the human DR3 protein were purified by affinity chromatography and size exclusion chromatography. The sequence is shown in SEQ ID NO:17.

[0051] For alpaca immunization, Freund's complete adjuvant was used for the initial immunization, and Freund's incomplete adjuvant was used for the subsequent two immunizations. Each immunization used a dose of 200 μg of antigen, mixed at a 1:1 volume ratio with the adjuvant. The first two injections were administered subcutaneously, and the final injection was given intramuscularly. Alpacas (Vicugna pacos) were immunized a total of three times, with each injection spaced three weeks apart.

[0052] 2) At week 11, blood was drawn from the vein and peripheral blood lymphocytes were isolated using Solarbio's Ficoll.

[0053] 3) Total RNA was extracted using an Omega Biotek RNA extraction kit, and genomic DNA was removed. Takara's PrimeScript was used. TM II. First-strand cDNA Synthesis Kit: Reverse transcription of RNA into cDNA.

[0054] 4) Construction of the nanobody phage display library: Using the above cDNA as a template, the coding sequence of the nanobody was obtained by PCR amplification using specific alpaca VHH primers. The amplified nanobody sequence was then inserted into the NcoI and NotI sites of the phage particle pR2 using Gibson assembly. The resulting Gibson assembly product was the initial nanobody phage library, and the product was recovered.

[0055] 5) *E. coli* T1 (MRC Laboratory of Molecular Biology) competent cells were prepared using the 10% glycerol washing method. Activated TG1 cells were cultured in 250 mL of 2×YT medium (formulation 1L: 16 g tryptone, 10 g yeast extract, 5 g NaCl) until OD500. 600After centrifuging at 5000g for 15 min at approximately 0.6-0.8, wash three times with 250mL, 250mL, and 100mL of pre-cooled 10% glycerol, and finally resuspend in 1mL of 10% glycerol and dispense into 500μL tubes.

[0056] 6) Transform *E. coli* TG1 competent cells using a BTX ECM 399 electroporator. The product recovered from Gibson assembly was mixed with 500 μL of TG1 competent cells and transferred to a 0.1 cm electroporation cuvette, followed by electroporation at 2.5 kV. The electroporated product was resuspended in 20 mL of 2× medium and incubated at 37°C and 220 rpm for 1 h. 10 μL of the bacterial culture was diluted to 990 μL of 2×YT, and then another 40 μL of the bacterial culture was diluted to 160 μL of 2×YT. A 100 μL plate was then plated and incubated overnight at 37°C. The next day, the library size was calculated by counting the cells (library size = count × 10⁻⁶). 5 Bacteria were spread onto five 150 mm 2×YT plates supplemented with 100 μg / ml ampicillin and 2% glucose to amplify the phage library, and incubated overnight at 37°C. Next, transformed colonies were scraped from the plates, vortexed thoroughly with a final concentration of 25% glycerol, flash-frozen in 1 mL aliquots with liquid nitrogen, and stored at -80°C. The size of the phage library was then calculated.

[0057] 7) Amplification of the nanobody phage display library: To amplify the nanobody phage library, 0.2 ml of the frozen library was thawed on ice and diluted in 200 mL of 2×YT medium supplemented with 100 μg / mL ampicillin and 2% glucose, and incubated at 37°C and 220 rpm. Next, 1×10⁻⁶ phage display libraries were added to the library. 12 PFU-modified KM13 helper phage (MRC Laboratory of Molecular Biology) was added to the culture and incubated at 37°C (water bath) for 45 minutes. The cell pellet was separated by high-speed centrifugation and resuspended in 200 mL of 2×YT medium supplemented with 0.1% glucose, 50 μg / mL kanamycin, and 100 μg / mL ampicillin. Cells were incubated at 25°C and 220 rpm for 20 hours to amplify the phage library. After centrifugation, polyethylene glycol (PEG) was added to the culture supernatant to precipitate the phage particles. The precipitated phage particles were dissolved in 1×PBS (formulation: 10 mmol / L Na₂HPO₄; 1.75 mmol / L KH₂PO₄; 137 mmol / L NaCl; 2.65 mmol / L KCl; pH 7.2–7.6) and stored in 1 mL aliquots at -80°C in the presence of 25% glycerol.

[0058] 8) Screening: The purified extracellular fragment of human DR3 was diluted with PBS to a final concentration of 0.1 mg / ml and coated into one well of a 96-well immunoassay plate (Nunc Maxsorp plate), reserving one well as a negative control. After washing three times with 1×PBS, 300 μL of MPBS (1×PBS containing 5% skim milk) was added to each well of the ELISA plate, and the plate was incubated at room temperature for 2 hours to block unbound sites. Next, the plate was washed three times with 1×PBS, and 1×10⁻⁶ mg / ml of the PBS containing the extracellular fragment targeting human DR3 was added to each well. 11 A PFU (diluted in 100 μL MPBS) phage library was added to each well. After incubation at room temperature for one hour, the cells were washed three times with PBST (1×PBS containing 0.1% Tween 20). Phages displaying extracellular nanobodies specifically targeting human DR3 were eluted by incubation at room temperature for one hour with trypsin at a final concentration of 0.5 mg / mL. 10 μL of the eluted phage was added to 1 mL of *E. coli* TG1 competent cells and incubated at 37°C (water bath) for 45 minutes for infection. The bacterial culture was then plated on 2×YT supplemented with 100 μg / mL ampicillin and 2% glucose and incubated overnight at 37°C.

[0059] 9) Preparation of monoclonal phages: After one round of panning, 48 individual colonies were picked into a 96-well round-bottom culture dish containing 100 μL of 2×YT medium supplemented with 100 μg / mL ampicillin and 2% glucose (w / v). The culture was incubated at 37°C and 180 rpm for 12 hours. Next, 5 μL of this culture was inoculated into a new 96-well round-bottom culture dish containing 200 μL of 2×YT medium supplemented with 100 μg / mL ampicillin and 2% glucose (w / v). The freshly inoculated plate was incubated at 37°C and 250 rpm for 1.5 hours until the OD260 was approximately 0.5. 50 μL of the culture containing 4×10⁴ phages was then inoculated into the phage. 8 PFU KM13 helper phage 2×YT medium was added to each well of the plate and incubated at 37°C for 45 minutes without shaking for infection. After infection, 150 μL of supernatant was discarded, and the remaining volume was centrifuged at 3500g for 15 minutes, the supernatant was discarded, and the bacterial pellet was resuspended in 200 mL of 2×YT medium supplemented with 100 μg / mL ampicillin, 50 μg / mL kanamycin, and 0.1% glucose (w / v). The culture was incubated overnight at 25°C and 250 rpm for approximately 14-16 hours. The next day, the culture was centrifuged at 3500g for 30 minutes, and 150 μL of supernatant was transferred to a new 96-well plate and stored at 4°C for screening nanobodies.

[0060] 10) Phage ELISA Assay. A 96-well round-bottom immunoassay ELISA plate was coated with 100 μL of human DR3 extracellular fragment to a final concentration of 0.2 μg / mL using 1×PBS dilution and incubated at 4°C for 16 hours. Next, the ELISA plate was washed three times with 1×PBS. The plate was then blocked with MPBS (1×PBS containing 5% skim milk) at room temperature for 2 hours. After blocking, the wells were washed four times with PBS-0.1% Tween 20. Finally, 100 μL of 1×10⁻⁶ phage ELISA solution was added. 11 PFU phage was added to each well and incubated at room temperature for 1 hour. The ELISA plate was then washed 5 times with PBS-0.1% Tween 20. Next, 100 μL of HRP-KM13 diluted 1:8000 with MPBS was added to each well and incubated at room temperature for 1 hour. The wells were then washed 4 times with PBS-0.1% Tween 20, and 100 μL of 3,3',5,5'-tetramethylbenzidine (TMB) was added to each well. The plates were incubated in the dark for 7 minutes for color development, and then immediately 50 μL of 1M H2SO4 was added to terminate the reaction. The OD was measured using a microplate reader. 450nm Value, result as Figure 1 As shown.

[0061] 11) OD 450nm Twenty positive clones with a value greater than 1 were selected for sequencing and sequence analysis. Finally, three positive phage nanobodies specific to the extracellular region of human DR3 protein were identified and named A2 (amino acid sequence as shown in SEQ ID NO:10), A6 (amino acid sequence as shown in SEQ ID NO:11), and H10 (amino acid sequence as shown in SEQ ID NO:12), respectively. These three nanobodies have specific CDR regions that bind to the extracellular region of human DR3.

[0062] Example 2

[0063] Expression and purification of nanobody-FC fusion protein

[0064] The secretion-guided peptide gene sequence was designed and fused to the N-terminus of the nanobody gene to ensure secretion after expression. Human IgG1 Fc was fused to the C-terminus of the nanobody gene, and the nanobody gene and human IgG1 Fc were recombined. Then, it was cloned into the mammalian expression vector pTT5.

[0065] The construct vector was transfected into HEK293F cells using polyethyleneimine (PEI) at a density of approximately 2.5 x 10⁻⁶ cells / cells. 6 (cells / ml), in Freestyle TMTransfected mammalian cells were cultured in 293 expression medium (purchased from Yonglian Biotechnology) at 5% CO2, 150 rpm, and 37°C for 4 days. Afterward, the cells were centrifuged at 3000 rpm for 10 minutes to culture for 5 days. The supernatant of the mammalian cell culture was then collected by centrifugation at 2000 rpm for 10 minutes. The nanobody-FC fusion protein was purified using a protein A column, eluted with 0.1 M acetic acid, and the eluted protein was analyzed by SDS-PAGE electrophoresis. Figure 2 As shown, high-purity nanobody-Fc fusion protein was obtained from the supernatant.

[0066] Example 3

[0067] Binding affinity analysis of nanobodies to the extracellular domain of human DR3

[0068] Immuno MaxiSorb (Nunc) plates were coated with 2 μg / mL human DR3 extracellular fragment protein and incubated at room temperature for 2 h. After washing the ELISA plates three times with 1×PBS, 240 μL of MPBS was added to each well and incubated at room temperature for 2 h to block unbound sites. Next, the nanobody-Fc was serially diluted with PBST containing 5% milk (starting at 50 nM, three-fold serial dilutions, 12 dilutions). 100 μL was added to each well and incubated at room temperature for 1 h. After 1 h, the ELISA plates were washed three times with 1×PBST, and HRP-anti-human IgG Fc (Beijing Yiqiao Shenzhou) was diluted 1:10000 with PBST containing 5% milk and incubated at room temperature for 1 h. The ELISA plates were then washed three times with 1×PBST, and 100 μL of TMB was added to each well and reacted at room temperature in the dark for 7 min. Finally, 50 μL of 1M H2SO4 was added to stop the reaction, and the absorbance was measured at 450 nm.

[0069] The results of the ELISA analysis are as follows: Figure 3 As shown, the extracellular domain of human DR3 and the EC of nanobody A2-IgG1 Fc 50 At 0.072±0.021 nM, EC with nanobody A6-IgG1 Fc 50 At 0.146±0.041 nM, EC with nanobody G10-IgG1Fc 50 The nanobody prepared at 0.063±0.017 nM showed high affinity for the extracellular domain of human DR3.

[0070] The above embodiments are only used to illustrate the technical solutions of the present invention, and are not intended to limit the present invention. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A Nanobody with high affinity for the extracellular domain of human DR3, characterized in that, The nanobody is named H10, and the H10 comprises three antigenic complementarity determining regions CDR1, CDR2 and CDR3, the amino acid sequences of which are shown in SEQ ID NO: 7, SEQ ID NO: 8 and SEQ ID NO: 9, respectively.

2. The nanobody with high affinity to the extracellular domain of human DR3 according to claim 1, characterized in that, The amino acid sequence of the H10 is shown in SEQ ID NO:

12.

3. A nanobody with high affinity to the extracellular domain of human DR3, which has the nanobody of claim 1 and an Fc domain.

4. The nanobody of claim 3, wherein The Fc domain is a human IgG1 Fc domain.

5. A polynucleotide encoding the nanobody of any one of claims 1-2, or encoding the nanobody of any one of claims 3-4.

6. An expression vector comprising the polynucleotide of claim 5.

7. A host cell comprising the expression vector of claim 6.

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