Nucleic Acid Molecule and Uses Thereof

Abstract

The present invention relates to a nucleic acid molecule encoding a) a modified tyrosylprotein sulfotransferase of a wildtype tyrosylprotein sulfotransferase, wherein the cytoplasmic transmembrane stem (CTS) region of the wild-type tyrosylprotein sulfotransferase is replaced by a heterologous CTS region, or b) a fusion protein comprising a catalytically active fragment of a tyrosylprotein sulfotransferase fused to a heterologous CTS region.

Claims

1. A nucleic acid molecule encoding a) a modified tyrosylprotein sulfotransferase of a wildtype tyrosylprotein sulfotransferase, wherein the cytoplasmic transmembrane stem (CTS) region of the wild-type tyrosylprotein sulfotransferase is replaced by a heterologous CTS region, or b) a fusion protein comprising a catalytically active fragment of a tyrosylprotein sulfotransferase fused to a heterologous CTS region.

2. Nucleic acid molecule according to claim 1, wherein the heterologous CTS region is a plant or animal CTS region.

3. Nucleic acid molecule according to claim 1 or 2, wherein the heterologous CTS region is a CTS region of a glycosyltransferase or a tyrosylprotein sulfotransferase.

4. Nucleic acid molecule according to claim 3, wherein the glycosyltransferase is selected from the group consisting of a fucosyltransferase, preferably an 1,3-fucosyltransferase, more preferably an 1,3-Fucosyltransferase 11, and a sialytransferase, preferably an 2,6-sialytransferase.

5. Nucleic acid molecule according to any one of claims 1 to 4, wherein the tyrosylprotein sulfotransferase is a plant or animal tyrosylprotein sulfotransferase.

6. Nucleic acid molecule according to any one of claims 1 to 5, wherein the heterologous CTS region of said fusion protein is N- or C-terminally, preferably N-terminally, fused to the tyrosylprotein sulfotransferase or the catalytically active fragment thereof.

7. Polypeptide encoded by a nucleic acid molecule according to any one of claims 1 to 6.

8. A vector comprising a nucleic acid molecule according to any one of claims 1 to 6.

9. A plant or plant cell capable of transferring a sulfate moiety to a tyrosine residue of a polypeptide heterologously produced in said plant or plant cell comprising a nucleic acid molecule according to any one of claims 1 to 6 or a vector according to claim 8.

10. Plant or plant cell according to claim 9, wherein said transgenic plant or plant cell comprises further a nucleic acid molecule encoding a heterologous polypeptide operably linked to a promoter region.

11. Plant or plant cell according to claim 10, wherein the heterologous polypeptide of animal origin is a mammalian, more preferably human, polypeptide.

12. Plant or plant cell according to claim 11, wherein the heterologous animal polypeptide is an antibody.

13. Plant or plant cell according to claim 12, wherein the antibody is an antibody binding to an HIV surface protein.

14. Plant or plant cell according to claim 12 or 13, wherein the antibody is an antibody selected from the group consisting of PG9, PG16, PGT141-145, 47e, 412d, Sb1, C12, E51, CM51 and a variant thereof.

15. Plant or plant cell according to claim 14, wherein the antibody variant is a PG9 antibody comprising modifications at R.sup.L94SH.sup.L95A.

16. A method of recombinantly producing a polypeptide of animal origin carrying an animal-type sulfation comprising the step of cultivating a plant or plant cell according to any one of claims 9 to 15.

Description

[0093] The present invention is further illustrated by the following figures and examples without being restricted thereto.

[0094] FIG. 1 shows the PG9 and TPST1 constructs used in the examples. PG9HC, PG9LC and PG9LC-RSH were cloned into Magnlcon vectors. Three versions of hsTPST1 containing different CTS regions were cloned into pPT2 giving rise to p.sup.FullhsTPST1, p.sup.RSThsTPST1 and p.sup.FullhsTPST1.

[0095] FIG. 2 shows the purification of plant-produced PG9 and RSH and comparison to .sup.CHOPG9. Coomassie staining (top panels) and immunoblotting (middle and bottom panels) of purified PG9 and RSH after separation by SDS-PAGE under reducing (right) and non-reducing (left) conditions. 1: .sup.XFPG9; 2: .sup.XFPG9.sub.sulf; 3: .sup.XFPG9.sub.sulfsia; 4: .sup.XFRSH; 5: .sup.XFRSH.sub.Sulf; 6: .sup.XFRSH.sub.sulfsia.

[0096] FIG. 3 shows .sup.XFPG9 and .sup.CHOPG9 are singly and doubly sulfated on tyrosine residues Y.sup.100E, Y.sup.100G or Y.sup.100H. The sulfation sites of .sup.XFPG9 and .sup.CHOPG9 were mapped by LC-ESI-MS to the tryptic PG9 peptide N.sup.100CGYNYYDFYDGYYNYHYMDVWGK.sup.105 (SEQ ID No. 27) (panels A and D, respectively). Further digestion with AspN revealed nonsulfated, singly and doubly sulfated variants of the peptide N.sup.100CGYNYY.sup.100H (SEQ ID No. 28) (panels B and E for .sup.XFPG9 and .sup.CHOPG9). No sulfated residues were found on the other AspN fragment, D.sup.1001FYDGYYNYHYM.sup.100T (SEQ ID No. 29) (panels C and F for .sup.XFPG9 and .sup.CHOPG9).

EXAMPLES

Materials and Methods

[0097] 1. Cloning of Neutralizing Anti-HIV Antibody PG9 and its Variant RSH

[0098] The signal peptide of barley a-amylase (amino acid residues 1 to 24 of the amino acid sequence of acc. no. CAX51374.1) was cloned into Magnlcon vectors pICH26033 and pICH31160 (Niemer, M., et al. Biotechn J 9(2014):493-500) to give rise to pICH26033 and pICH31160. cDNA codon-optimized for Nicotiana benthamiana and encoding a Bsal site followed by the PG9 (McLellan J S et al. Nature 480(2011):336-343; Protein Data Bank accession nos. 3U36_A, 3U36_B, 3U36_C, 3U36_D, 3U36_E, 3U36_F) variable heavy and C.sub.H1 domain without signal peptide was PCR-amplified.

[0099] PG9LC (SEQ ID No. 30; PDB-database: 3U2S, 3U36, 3U4E, 3MUH) and PG9LC-RSH (SEQ ID No. 32) cDNAs (both encoding an N23Q mutation) were synthesized with codons optimized for Nicotiana benthamiana, without signal peptide but with 5- and 3 Bsal restriction sites.

[0100] PG9LC consists of amino acid sequence SEQ ID No. 30, whereby the signal peptide of barley a-amylase is marked in bold and italic:

TABLE-US-00021 QSALTQPASVSGSPGQSITISCQGT SNDVGGYESVSWYQQHPGKAPKVVIYDVSKRPSGVSNRFSGSKSGNTAS LTISGLQAEDEGDYYCKSLTSTRRRVFGTGTKLTVLGQPKAAPSVTLFP PSSEELQANKATLVCLISDFYPGAVTVAWKADSSPVKAGVETTTPSKQS NNKYAASSYLSLTPEQWKSHKSYSCQVTHEGSTVEKTVAPTECS

[0101] PG9LC is encoded by nucleotide sequence SEQ ID No. 31 (including stop codon), whereby the nucleic acid stretch encoding the signal peptide of barley a-amylase is marked in bold and italic:

TABLE-US-00022 CAGAGTGCTCTTACTCAGCCTGCTTC TGTTTCTGGTTCTCCTGGTCAGAGCATCACCATTTCTTGCCAGGGAACC TCTAACGATGTGGGAGGTTACGAGTCCGTGTCTTGGTATCAACAGCATC CTGGTAAGGCTCCTAAGGTGGTGATCTACGATGTGAGCAAGAGGCCTTC TGGTGTGAGCAATAGGTTCAGCGGTAGCAAGTCTGGTAACACCGCTTCT CTTACCATCTCTGGACTTCAGGCTGAGGATGAGGGAGATTACTACTGCA AGTCTCTGACCTCCACTAGAAGAAGGGTGTTCGGAACCGGTACTAAGCT TACTGTTCTGGGTCAACCTAAGGCTGCTCCTTCTGTGACTTTGTTCCCT CCATCTTCTGAGGAACTGCAGGCTAACAAGGCTACCCTTGTGTGCCTGA TCAGCGATTTTTACCCTGGTGCTGTTACCGTGGCTTGGAAGGCTGATTC TTCACCTGTTAAGGCTGGTGTGGAAACCACCACTCCTAGCAAGCAGAGC AACAACAAGTACGCTGCTAGCTCCTACCTTAGCCTTACTCCTGAACAGT GGAAGTCCCACAAGAGCTACTCATGCCAGGTTACCCATGAGGGTTCTAC CGTGGAAAAGACTGTTGCTCCTACTGAGTGCAGCTAG

[0102] PG9LC-RSH consists of amino acid sequence SEQ ID No. 32, whereby the signal peptide of barley -amylase is marked in bold and italic:

TABLE-US-00023 QSALTQPASVSGSPGQSITISCQGT SNDVGGYESVSWYQQHPGKAPKVVIYDVSKRPSGVSNRFSGSKSGNTAS LTISGLQAEDEGDYYCKSLTSRSHRVFGTGTKLTVLGQPKAAPSVTLFP PSSEELQANKATLVCLISDFYPGAVTVAWKADSSPVKAGVETTTPSKQS NNKYAASSYLSLTPEQWKSHKSYSCQVTHEGSTVEKTVAPTECS

[0103] PG9LC-RSH is encoded by nucleotide sequence SEQ ID No. 33 (including stop codon), whereby the nucleic acid stretch encoding the signal peptide of barley a-amylase is marked in bold and italic:

TABLE-US-00024 CAGAGTGCTCTTACTCAGCCTGCTTC TGTTTCTGGTTCTCCTGGTCAGAGCATCACCATTTCTTGCCAGGGAACC TCTAACGATGTGGGAGGTTACGAGTCCGTGTCTTGGTATCAACAGCATC CTGGTAAGGCTCCTAAGGTGGTGATCTACGATGTGAGCAAGAGGCCTTC TGGTGTGAGCAATAGGTTCAGCGGTAGCAAGTCTGGTAACACCGCTTCT CTTACCATCTCTGGACTTCAGGCTGAGGATGAGGGAGATTACTACTGCA AGTCTCTGACCTCCAGAAGTCACAGGGTGTTCGGAACCGGTACTAAGCT TACTGTTCTGGGTCAACCTAAGGCTGCTCCTTCTGTGACTTTGTTCCCT CCATCTTCTGAGGAACTGCAGGCTAACAAGGCTACCCTTGTGTGCCTGA TCAGCGATTTTTACCCTGGTGCTGTTACCGTGGCTTGGAAGGCTGATTC TTCACCTGTTAAGGCTGGTGTGGAAACCACCACTCCTAGCAAGCAGAGC AACAACAAGTACGCTGCTAGCTCCTACCTTAGCCTTACTCCTGAACAGT GGAAGTCCCACAAGAGCTACTCATGCCAGGTTACCCATGAGGGTTCTAC CGTGGAAAAGACTGTTGCTCCTACTGAGTGCAGCTAG

[0104] PG9LC and PG9LC-RSH were inserted into pICHa26033 in frame after the barley a-amylase signal peptide. All vectors were transformed into E. coli by electroporation and upon sequence confirmation into the Agrobacterium tumefaciens strain GV3101pMP90.

[0105] 2. Cloning of Tyrosylprotein Sulfotransferase (TPST) Constructs

[0106] For expression in plants, hsTPST1 (accession number AK313098.1, open reading frame from start to stop codon) was cloned into vector pPT2 (Strasser R et al. Biochem J 387(2005):385-391). Three different constructs containing different CTS regions were constructed (FIG. 1). p.sup.FullhsTPST1 contains the authentic CTS region (SEQ ID No. 3), in p.sup.Fut11hsTPST1 Met.sup.1-Ser.sup.39 of hsTPST1 is replaced by the CTS of A. thaliana Fut11 (Met.sup.1-Val.sup.68) (SEQ ID No. 5), and in p.sup.RSThsTPST it is replaced by the CTS region of rat sialyltransferase (Met.sup.1-Gly.sup.54) (SEQ ID No. 7). The nucleic acid sequences encoding the respective polypeptides having SEQ ID No. 1, 11 and 12 consist of SEQ ID No. 2, 9 and 10, respectively. After transformation into E. coli and sequence confirmation, all constructs were transformed into Agrobacterium tumefaciens strain UIA143pMP90.

[0107] 3. In Planta Expression of PG9 and RSH

[0108] N. benthamiana XT/FT plants (age: 4-5 weeks) were used for co-infiltration with agrobacteria as described previously (Strasser R, et al. Plant Biotech J 6(2008):392-402). Briefly, liquid cultures of agrobacterial strains carrying pPG9HC, pPG9LC, pPG9LC-RSH, p.sup.FullhsTPST1 , p.sup.Fut11hsTPST1 and p.sup.RSThsTPST1 were grown overnight, pelleted and resuspended in infiltration buffer (25 mM MES (pH 5.5), 25 mM MgSO.sub.4, 0.1 mM acetosyringone). Mixtures of bacteria containing pPG9HC and pPG9LC (or pPG9LC-RSH) were infiltrated with or without different TPST constructs into N. benthamiana XT/FT leaves. For in planta galactosylation and sialylation, an additional 6 genes had to be infiltrated (Castilho A et al. J Biol Chem 285(2010):15923-15930). OD.sub.600 for infiltration was 0.01 for each of the IgG vectors, up to 0.8 for the TPST constructs and 0.05 for the vectors required for galactosylation/sialylation. Plants were harvested 3-6 days post infiltration.

[0109] 4. Cloning, Expression and Purification of gp120.sup.ZM109

[0110] The codon-optimized coding sequence (SEQ ID No. 34) for gp120 of HIV strain ZM109 (gp120.sup.109) (SEQ ID No. 35) was appended with a C-terminal hexahistidine tag (bold and underlined in SEQ ID No. 34 and 35) and inserted into the HindIII/NotI sites of pCEP4 (Life Technologies).

[0111] gp120.sup.ZM109 (including a C-terminal histidine tag) is encoded by the nucleic acid sequence SEQ ID No. 34:

TABLE-US-00025 ATGCCTATGGGCAGCCTGCAGCCCCTGGCCACACTGTATCTGCTGGGAA TGCTGGTGGCCAGCTGCCTGGGCGTGTGGAAAGAGGCCAAGACCACCCT GTTCTGCGCCAGCGACGCCAAGAGCTACGAGCGCGAGGTGCACAATGTG TGGGCCACCCATGCCTGCGTGCCCACCGATCCTGATCCCCAGGAACTCG TGATGGCCAACGTGACCGAGAACTTCAACATGTGGAAGAACGACATGGT GGACCAGATGCACGAGGACATCATCAGCCTGTGGGACCAGAGCCTGAAG CCCTGCGTGAAGCTGACCCCTCTGTGCGTGACCCTGAACTGCACATCTC CTGCCGCCCACAACGAGAGCGAGACAAGAGTGAAGCACTGCAGCTTCAA CATCACCACCGACGTGAAGGACCGGAAGCAGAAAGTGAACGCCACCTTC TACGACCTGGACATCGTGCCCCTGAGCAGCAGCGACAACAGCAGCAACA GCTCCCTGTACAGACTGATCAGCTGCAACACCAGCACCATCACCCAGGC CTGCCCCAAGGTGTCCTTCGACCCCATCCCCATCCACTACTGTGCCCCT GCCGGCTACGCCATCCTGAAGTGCAACAACAAGACCTTCAGCGGCAAGG GCCCCTGCAGCAACGTGTCCACCGTGCAGTGTACCCACGGCATCAGACC CGTGGTGTCCACCCAGCTGCTGCTGAATGGCAGCCTGGCCGAAGAGGAA ATCGTGATCAGAAGCGAGAACCTGACCGACAACGCCAAGACAATCATTG TGCATCTGAACAAGAGCGTGGAAATCGAGTGCATCAGGCCCGGCAACAA CACCAGAAAGAGCATCAGACTGGGCCCTGGCCAGACCTTTTACGCCACC GGGGATGTGATCGGCGACATCCGGAAGGCCTACTGCAAGATCAACGGCA GCGAGTGGAACGAGACACTGACAAAGGTGTCCGAGAAGCTGAAAGAGTA CTTTAACAAGACCATTCGCTTCGCCCAGCACTCTGGCGGCGACCTGGAA GTGACCACCCACAGCTTCAATTGCAGAGGCGAGTTCTTCTACTGCAATA CCAGCGAGCTGTTCAACAGCAACGCCACCGAGAGCAATATCACCCTGCC CTGCCGGATCAAGCAGATCATCAATATGTGGCAGGGCGTGGGCAGAGCT ATGTACGCCCCTCCCATCCGGGGCGAGATCAAGTGCACCTCTAACATCA CCGGCCTGCTGCTGACCAGGGACGGCGGAAACAACAACAATAGCACCGA GGAAATCTTCCGGCCCGAGGGCGGCAACATGAGAGACAATTGGAGATCC GAGCTGTACAAGTACAAGGTGGTGGAAATCAAGGGCCTGCGGGGCAGCC ACCACCATCATCACCATTGA

[0112] gp120.sup.ZM109 (including a C-terminal histidine tag) consists of the amino acid sequence SEQ ID No. 35:

TABLE-US-00026 MPMGSLQPLATLYLLGMLVASCLGVWKEAKTTLFCASDAKSYEREVHNV WATHACVPTDPDPQELVMANVTENFNMWKNDMVDQMHEDIISLWDQSLK PCVKLTPLCVTLNCTSPAAHNESETRVKHCSFNITTDVKDRKQKVNATF YDLDIVPLSSSDNSSNSSLYRLISCNTSTITQACPKVSFDPIPIHYCAP AGYAILKCNNKTFSGKGPCSNVSTVQCTHGIRPVVSTQLLLNGSLAEEE IVIRSENLTDNAKTIIVHLNKSVEIECIRPGNNTRKSIRLGPGQTFYAT GDVIGDIRKAYCKINGSEWNETLTKVSEKLKEYFNKTIRFAQHSGGDLE VTTHSFNCRGEFFYCNTSELFNSNATESNITLPCRIKQIINMWQGVGRA MYAPPIRGEIKCTSNITGLLLTRDGGNNNNSTEEIFRPEGGNMRDNWRS ELYKYKVVEIKGLRGSHHHHHH

[0113] Transient expression in FreeStyle293F cells (Life Technologies) was performed following the instructions of the manufacturer. Culture supernatants were subjected to affinity chromatography using Ni.sup.2+-charged Chelating Sepharose (GE Healthcare), omitting the addition of phosphatase and protease inhibitors. Fractions eluted with 250 mM imidazole were dialyzed against PBS containing 0.02% (v/v) NaN.sub.3 and then concentrated by ultrafiltration.

[0114] 5. Monoclonal Antibody (mAb) Purification

[0115] Leaf material (see item 3.) infiltrated with the transformed Agrobacterium tumefaciens strains described under item 2 was crushed under liquid nitrogen, extracted twice for 20 min on ice with 45 mM Tris/HC1 (pH 7.4) containing 1.5 M NaCl, 40 mM ascorbic acid and 1 mM EDTA (2 ml per g leaf material) and cleared by centrifugation (4 C., 30 min, 27.500 g). Upon vacuum filtration through 10-m cellulose filters (Roth, AP27.1) and centrifugation (4 C., 30 min, 27.500 g), the extract was filtered through a series of filters with pore sizes ranging from 10 m to 0.2 m (Roth, AP27.1, Roth, AP51.1, Roth, CT92.1, Roth, KH54.1) before being applied to a 1.5 ml Protein A Sepharose 4FF column (GE Healthcare, 17-1279-01) at 1 ml/min. Upon washing with PBS, bound mAbs were eluted with 100 mM glycine/HCl (pH 2.5). The eluate was immediately neutralized (1 M Tris/HCl (pH 8.0), 1.5 M NaCl), mAb-containing fractions were identified by their absorbance at 280 nm, pooled and the buffer was exchanged to PBS by dialysis.

[0116] 6. SDS-PAGE and Western Blotting

[0117] Samples were separated on 12% polyacrylamide gels under reducing conditions and on 8% polyacrylamide gels under non-reducing conditions, followed by either staining with Coomassie Brilliant Blue or blotting onto a nitrocellulose membrane (GE Healthcare). mAb heavy and light chains were detected with anti-human IgG gamma chain-peroxidase conjugate (Sigma A8775) or anti-human lambda light chain-peroxidase conjugate (Sigma A5175) and visualized with a chemiluminescence detection kit (Bio-Rad).

[0118] 7. Glycosylation Analysis of mAbs and gp120

[0119] The N-glycosylation profiles of mAbs (Asn.sup.297) and gp120.sup.ZM109 (Asn.sup.160, Asn.sup.173) were determined by LC-ESI-MS as published by Stadlmann et al. (Proteomics 8(2008):2858-2871) and Pabst et al. (Biol Chem 393(2012):719-730), respectively. Briefly, purified mAbs or gp120 were separated by reducing SDS-PAGE, stained with Coomassie Brilliant Blue and the relevant bands were excised from the gel. Upon S-alkylation with iodoacetamide and tryptic or tryptic/chymotryptic digestion (gp120.sup.ZM109 Asn.sup.173), fragments were eluted from the gel with 50% acetonitrile and separated on a reversed phase column (1500.32 mm BioBasic-18, Thermo Scientific) with a gradient of 1-80% acetonitrile. Glycopeptides were analysed with a Q-TOF Ultima Global mass spectrometer (Waters). Spectra were summed and deconvoluted for identification of glycoforms. Annotation of glycoforms was done according to the proglycan nomenclature (Stadlmann J. et al. (Proteomics 8(2008), 2858-2871).

[0120] 8. Sulfation Analysis of PG9 and RSH

[0121] Tryptic peptides were prepared as above (see item 7), digested with AspN where appropriate and then separated using a Dionex Ultimate 3000 HPLC system using a Thermo BioBasic C18 separation column (5 m particle size, 1500.32 mm) with a gradient from 95% solvent A (65 mM ammonium formate) and 5% solvent B (acetonitrile) to 75% B in 50 min at a flow rate of 6 L/min. Peptides were analysed on a maXis 4G ETD QTOF mass spectrometer (Bruker Daltonik) equipped with the standard ESI source in the positive ion, DDA mode (=switching to MSMS mode for eluting peaks). MS2 scans of dominant precursor peaks were acquired and manually analysed with DataAnalysis software version 4.0 (Bruker Daltonik).

[0122] 9. Quantification of mAb Content and gp120 Binding by ELISA

[0123] For determination of PG9 and RSH content, wells were coated with 100 l of 2 g/ml anti-human gamma chain (Sigma-Aldrich 13391) in 0.1 N NaHCO.sub.3 buffer (pH 9.6) overnight at 4 C. After washing with PBS containing 0.05% Tween-20 (PBST), samples and .sup.CHOPG9 standards (100 l) appropriately diluted (1 - 100 ng/ml) in PBST containing 1% BSA were added, the plate was incubated for 60 min, washed and incubated for 60 min with 100 l of a 1:20.000 dilution of anti-human lambda light chain-peroxidase conjugate (Sigma A5175). After washing with PBST, the wells were incubated for 20 min with 100 l 3,3,5,5-tetramethylbenzidine (TMB) substrate solution (Sigma-Aldrich T0440). The reaction was then stopped with 100 l 30% H.sub.2SO.sub.4 prior to spectrophotometry at 450 nm.

[0124] To test binding to gp120, the ELISA setup was adopted as follows: 1 g/ml gp120.sup.ZM109 was coated, and the mAb sample concentrations ranged from 10 ng/ml to 4 g/ml. As detection antibody a 1:2.000 dilution of anti-human IgG (Fcgamma specific)peroxidase conjugate (Invitrogen 62-8420) was used.

[0125] 10. Biolayer Interferometry

[0126] PG9 was bound at 20 g/ml to Dip and Read Protein A Biosensor sticks (fortBio) and antigen binding kinetics were determined with gp120.sup.ZM109 solutions ranging from 50 g/ml to 6.25 g/ml (1:2 dilutions). Blanks were run without PG9 and/or without gp120.sup.ZM109. All measurements were conducted at 30 C. Results were analysed with Octet Data Analysis Software 6.4 with single reference-well subtractions. The kinetic constants were computed for each curve separately assuming that dissociation does not reach the pre-association baseline. All estimates with a coefficient of determination (R.sup.2) above 0.85 were considered for calculation of the dissociation constant K.sub.d.

[0127] 11. Virus Neutralization Assays

[0128] Pseudotyped virions were generated as described previously (Gach J S, et al. PLoS One 8(2013):e72054). In brief, 510.sup.5 human embryonic kidney 293T cells (ATCC, # CRL-3216) were cotransfected with 4 g of the HIV Env-deleted backbone plasmid pSG3Env and 2 g of the respective Env complementation plasmid using polyethyleneimine (18 g) as a transfection reagent. Cell culture supernatants were harvested 48 h after transfection, cleared by centrifugation at 4.000 g for 10 min, and then used for single-round infectivity assays as described elsewhere (Gach J S, et al. PLoS One 9(2014):e85371). Briefly, pseudotyped virus was added at a 1:1 volume ratio to serially diluted (1:3) mAbs (starting at 40 g/ml) and incubated at 37 C. After 1 h TZM-bl reporter cells (NIH AIDS Reagent Program, # 8129) were added (1:1 by volume) at 110.sup.4 cells/well, supplemented with 10 g/ml DEAE-dextran and then incubated for a further 48 h at 37 C. Next, the cells were washed, lysed, and developed with luciferase assay reagent according to the manufacturer's instructions (Promega). Relative light units were then measured using a microplate luminometer (BioTek, Synergy 2 luminescence microplate reader). All experiments were performed at least in duplicate. The extent of virus neutralization in the presence of antibody was determined as the 50% or 90% inhibitory concentration (IC.sub.50, IC.sub.90) as compared to samples treated without mAb.

[0129] 12. Protein Quantification

[0130] The total protein content of gp120 and mAb samples was quantified with the BCA Protein Assay Kit (Pierce) using BSA as standard according to the protocol provided by the manufacturer.

Example 1

Natural .SUP.XF.PG9 Binds gp120 Less Efficiently than .SUP.CHO.PG9

[0131] Previously the in planta production of PG9 in XT/FT Nicotiana benthamiana plants that have been glycoengineered to remove the plant-typical N-glycan residues 1,2-xylose and core 1,3-fucose has been reported (Niemer M, et al. Biotech J 9(2014):493-500). Another recent study has revealed that changing three consecutive amino acids of the PG9 light chain into the corresponding PG16 residues (T.sup.L94RR.sup.L95A to R.sup.L94SH.sup.L95A) leads to improved antigen-binding characteristics and higher neutralization efficiency (Pancera M, et al. Nat Struct Mol Biol 20(2013):804-+). Therefore, a .sup.XFPG9-R.sup.L94SH.sup.L95A variant termed .sup.XFRSH was constructed. Using protein A affinity chromatography, .sup.XFPG9 and .sup.XFRSH could be purified in good yields from leaf extracts. When analyzed by SDS-PAGE under reducing conditions, the .sup.XFPG9 and .sup.XFRSH heavy and light chains showed the expected migration pattern, with the light chains displaying higher electrophoretic mobilities than their CHO-derived counterpart (.sup.CHOPG.sub.9) due to the removal of a functionally unnecessary N-glycosylation site. Under non-reducing conditions, .sup.XFPG9 and .sup.XFRSH yielded single major bands co-migrating with .sup.CHOPG9 (FIG. 2).

[0132] To investigate the antigen-binding properties of .sup.XFPG9 in comparison to .sup.CHOPG9, suitable ligand was required. PG9 has been described to bind with high affinity to trimeric envelope glycoproteins of a wide variety of HIV isolates and also to gp120 monomers of selected HIV strains including ZM109. Therefore gp120.sup.ZM109 containing a C-terminal hexahistidine tag was expressed in FreeStyle 293 (FS293) cells and purified by metalchelate affinity chromatography to apparent homogeneity. SDSPAGE revealed a diffuse band as expected for a heavily glycosylated protein. N-glycosylation of two gp120 asparagines (Asn.sup.160 and Asn.sup.173) has been shown to be important for PG9 binding. Glycosylation analysis by mass spectrometry revealed mainly Man5 structures on either of these N-glycosylation sites. Importantly, PG9 is known to prefer such N-glycans on Asn.sup.160 while tolerating them on Asn.sup.173. Only minor amounts of other N-glycans were detected on either site, indicating that FS293-derived gp120.sup.ZM109 meets the prerequisites for a high-affinity PG9 ligand. When tested by ELISA, binding of .sup.XFPG9 to gp120.sup.ZM109 was found to be considerably weaker than observed for .sup.CHOPG9 (Table A).

TABLE-US-00027 TABLE A Sulfation enhances binding of PG9 and RSH to gp120/140. Binding of PG9 and RSH antibodies to immobilized antigen was measured by ELISA in triplicates (monomeric gp120.sup.ZM109) or duplicates (trimeric gp140.sup.BG505.SOSIP.664). Data are presented as means SD. EC.sub.50 [ng/ml] mAb gp120 gp140 .sup.CHOPG9 89 3 290 120 .sup.XFPG9 452 72 4870 1560 .sup.XFPG9.sub.Sulf 92 16 490 240 .sup.XFPG9.sub.SulfSia 101 12 310 50 .sup.XFRSH 179 85 2230 170 .sup.XFRSH.sub.Sulf 82 33 180 10 .sup.XFRSH.sub.SulfSia 73 22 180 40

Example 2

Co-expression of TPST1 Enables the in Planta Production of Sulfated PG9

[0133] Sulfation of one or two tyrosine residues in the CDR H3 domain of PG9 and other V1/V2-directed bnAbs has been described to enhance antigen binding. However, a functional tyrosylprotein sulfotransferase is not contained in the N. benthamiana genome. Therefore, it is obvious that .sup.XFPG9 was not properly sulfated. Analysis of CDR H3 peptides by mass spectrometry revealed a high degree (82%) of sulfation in the case of .sup.CHOPG9, whereas this post-translational modification was not detected for .sup.XFPG9 (Table B).

TABLE-US-00028 TABLE B Tyrosine sulfation of plant-produced PG9 and RSH. The sulfation status of .sup.CHOPG9 is shown for comparison. .sup.CHOPG9 PG9 PG9.sub.Sulf PG9.sub.SulfSia RSH RSH.sub.Sulf RSH.sub.SulfSia 0S [%] 18.5 >98 49.2 42.9 >98 52.7 48.9 1S [%] 60.4 <1 33.2 33.8 <1 30.5 34.3 2S [%] 21.1 <1 17.6 23.3 <1 16.8 16.8 1 + 2S [%] 81.5 <2 50.8 57.1 <2 47.3 51.1

[0134] Therefore, .sup.XFPG9 was co-expressed with human TPST1 (hsTPST1) in N. benthamiana. To mediate proper targeting to sub-Golgi compartments, three constructs carrying different cytoplasmic tail, transmembrane domain and stem (CTS) regions were tested. Expression of hsTPST1 in combination with its authentic CTS region (p.sup.FullhsTPST1) led to 15-20% sulfated .sup.XFPG9 (Table C). Interestingly, replacement of the natural hsTPST1 CTS region with the corresponding parts of glycosylation enzymes known to be targeted to the trans-region of the plant Golgi (p.sup.Fut11hsTPST1 and p.sup.RSThsTPST1) led to a substantially higher level of .sup.XFPG9 sulfation, almost reaching the extent of tyrosine sulfation observed for .sup.CHOPG9. Using p.sup.RSThsTPST1, up to 57% of .sup.XFPG9 could be mono- or disulfated (Table B; Table C).

TABLE-US-00029 TABLE C Tyrosine sulfation of PG9 and RSH. Relative amounts of unsulfated (0S), singly (1S) and doubly (2S) sulfated PG9 and RSH when coexpressed with different hsTPST1 constructs in plants. The sulfation status of .sup.CHOPG9 is shown for comparison. Data are presented as means SD of 2-9 analyses. mAb TPST1 OD.sub.600 0S 1S 2S .sup.XFPG9.sub.Sulf p.sup.FullhsTPST1 0.01 91.5 7.0 8.2 6.4 0.3 0.7 .sup.XFPG9.sub.Sulf p.sup.FullhsTPST1 0.05 83.4 13.9 16 13.1 0.6 0.9 .sup.XFPG9.sub.Sulf p.sup.FullhsTPST1 0.2 83.7 8.8 15.6 8.2 0.7 0.9 .sup.XFPG9.sub.Sulf p.sup.FullhsTPST1 0.8 82.8 11.7 16.8 11 0.4 0.7 .sup.XFPG9.sub.Sulf p.sup.Fut11hsTPST1 0.01 73.7 5.8 17.8 3.3 8.6 2.6 .sup.XFPG9.sub.Sulf p.sup.Fut11hsTPST1 0.05 57.4 0.8 32.1 2.3 10.6 3.1 .sup.XFPG9.sub.Sulf p.sup.Fut11hsTPST1 0.2 59.6 5.8 28.1 5.4 12.3 5.4 .sup.XFPG9.sub.Sulf p.sup.Fut11hsTPST1 0.8 58.7 8.7 32.7 10.8 8.6 2.1 .sup.XFPG9.sub.Sulf p.sup.RSThsTPST1 0.01 69.5 8.9 20.6 6.9 9.9 2.3 .sup.XFPG9.sub.Sulf p.sup.RSThsTPST1 0.05 53.9 6.1 30.8 2.3 15.3 3.8 .sup.XFPG9.sub.Sulf p.sup.RSThsTPST1 0.2 57.2 10.4 29.3 7.4 13.5 5.1 .sup.XFPG9.sub.Sulf p.sup.RSThsTPST1 0.8 53.7 11.1 36.8 11.8 9.5 2.7 .sup.XFRSH.sub.Sulf p.sup.RSThsTPST1 0.2 57.4 7.6 29.6 5.2 13.1 3.6 .sup.CHOPG9 18.5 1.5 60.4 3.9 21.1 5.4

Example 3

Tyrosine Sulfation of PG9 Produced in Plants and CHO Cells Occurs at the Same Positions

[0135] The tryptic CDR H3 peptide used for analyzing the PG9 sulfation status by LC-ESI-MS (N.sup.100CGYNYYDFYDGYYNYHYMDVWGK.sup.105; SEQ ID No. 27) contains several tyrosine residues that are potential TPST targets. To narrow down which amino acids are sulfated in the case of plant-derived PG9, the tryptic peptide was further digested with AspN to give rise to the shorter peptide N.sup.100CGYNYY.sup.100H (SEQ ID No. 28), containing the tyrosines involved in gp120 binding, as well as D.sup.100IFYDGYYNYHYM.sup.100T (SEQ ID No. 29) and D.sup.101WGK.sup.105. In plant-produced as well as CHO-derived PG9 and RSH, no sulfates were found on D.sup.100IFYDGYYNYHYM.sup.100T (SEQ ID No. XX) (FIG. 3), whereas N.sup.100CGYNYY.sup.100H (SEQ ID No. 28) was found to be singly and doubly sulfated to roughly the same extent as N.sup.100CGYNYYDFYDGYYNYHYMDVWGK.sup.105 (SEQ ID No. 27). This indicates that the sulfate groups are attached to Y.sup.100E, Y.sup.100G and/or Y.sup.100H independent of the expression platform used for PG9 production. It has been shown previously by X-ray crystallography that Y.sup.100G and Y.sup.100H of mammalian cell-produced PG9 can be sulfated. This shows that human TPST1 also modifies the same tyrosine residues in planta.

Example 4

PG9 Carries Human-type N-glycans when Expressed in Glycoengineered Plants

[0136] Mass spectrometric N-glycan analysis of .sup.XFPG9, .sup.XFPG9.sub.Sulf, .sup.XFRSH and .sup.XFRSH.sub.Sulf revealed the presence of a single dominant N-glycan species, GnGn (G0). This glycoform accounted for roughly 45-50% of all N-glycan species. Upon coexpression of PG9 and RSH with mammalian genes necessary for terminal galactosylation and sialylation in planta resulting in the synthesis of .sup.XFPG9.sub.SulfSia and .sub.XFRSH.sub.SulfSia the N-glycosylation profiles shifted to 30-40% galactosylated oligosaccharides and 6-12% sialylated glycans, with G0 reduced to 15-20%. Importantly, core 1,3-fucose and 1,2-xylose residues were barely detectable in all 6 plant-produced variants (below 5%). In the case of .sup.CHOPG9, the vast majority of Asn.sup.297 N-glycans contained 1,6-fucose (more than 95%) and the main N-glycan structure detected (70%) was G0F.sup.6 (GnGnF.sup.6). Roughly 20% of .sup.CHOPG9 was galactosylated and less than 1% sialylated. These results indicate that the N-glycan moieties of .sup.XFPG.sub.SulfSia and .sup.XFRSH.sub.SulfSia are largely devoid of the core fucose residues known to hamper mAb binding to Fc receptors while otherwise being reminiscent of those found on PG9 produced in mammalian cell factories.

Example 5

Antigen Binding by .SUP.XF.PG9 is Enhanced by Tyrosine Sulfation

[0137] Binding of the different PG9 and RSH variants to monomeric gp120.sup.ZM109 or trimeric gp140.sup.BG505.SOSIP.664 was tested by ELISA (Table A), and EC.sub.50 values were calculated. RSH showed up to 3-fold better binding to either antigen than PG9. Sulfation of plant-produced PG9 and RSH increased their affinities 10-16 times for trimeric gp140 and 2-5 times for monomeric gp120.sup.ZM109. As expected, different glycoforms showed very similar EC.sub.50 values. The avidity of the antigen-antibody interaction was also determined by biolayer interferometry (Table D).

TABLE-US-00030 TABLE D Affinities of PG9 and RSH for gp120.sup.ZM109 as determined by biolayer interferometry measurements. Data are presented as means SD of 2 (.sup.XFRSH) or 4-6 individual determinations. The binding of .sup.XFPG9 to gp120.sup.ZM109 was too weak for accurate determination of K.sub.d under the experimental conditions used. K.sub.d [nM] CHO.sub.PG9 756 365 .sup.XFPG9.sub.Sulf 525 167 .sup.XFRSH.sub.Sulf 605 239 .sup.XFRSH 2510 39 .sup.XFPG9 >3000

[0138] .sup.XFPG9.sub.Sulf, .sup.XFRSH.sub.Sulf and .sup.CHOPG9 showed roughly the same affinity to the antigen (K.sub.d of 525, 605 and 756 nM, respectively), whereas unsulfated .sup.XFRSH exhibited a roughly 4-fold lower affinity (K.sub.d of 2.51 M). The results obtained by biolayer interferometry confirmed those from the ELISA experiments, namely that RSH binds stronger to gp120/gp140 than wild-type PG9 and that tyrosine sulfation increases the affinity of both antibodies for either antigen. Furthermore, .sup.XFPG.sup.9.sub.Sulf and .sup.XFPG9.sub.SulfSial displayed essentially the same gp120/140-binding properties as .sup.CHOPG9, demonstrating the suitability of our plant-based expression platform to produce fully active versions of this bnAb.

Example 6

Increased Virus Neutralization by Sulfated PG9 and RSH Variants

[0139] Finally, the neutralization efficiencies of the antibodies on a panel of HIV clade B and clade C pseudoviruses were tested (Table E).

TABLE-US-00031 TABLE E Neutralization efficiencies of PG9 and RSH against a panel of pseudoviruses. IC.sub.50 values (g/ml) are indicated as >50 g/ml; 10-50 g/ml; 1-10 g/ml; <1 g/ml). .sup.CHOPG9 .sup.XFPG9 .sup.XFPG9.sub.Sulf .sup.XFPG9.sub.SulfSia .sup.XFRSH .sup.XFRSH.sub.Sulf .sup.XFRSH.sub.SulfSia JRFL >50 >50 >50 >50 >50 >50 >50 PVO >50 >50 >50 >50 >50 >50 >50 TRO.11 >50 >50 >50 >50 >50 >50 >50 ZM214M >50 >50 >50 >50 >50 >50 >50 YU2 >50 >50 >50 >50 42.47 34.75 26.08 MN >50 >50 >50 >50 43.70 30.77 36.14 ADA 42.81 >50 37.36 40.17 26.15 19.58 20.06 DU422.1 10.93 >50 14.28 6.60 >50 9.72 8.45 ZM109F 0.78 >50 1.66 1.31 >50 1.46 1.38 DU156.12 0.35 >50 1.20 0.90 33.85 0.56 0.56 CAP45 <0.02 0.65 0.03 <0.02 0.25 <0.02 <0.02 JRCSF <0.02 0.19 <0.02 0.03 0.06 <0.02 <0.02

[0140] The viruses included well-neutralized isolates as well as some resistant to PG9 or RSH produced in mammalian cells. As expected, a number of pseudoviruses was not neutralized under the tested conditions (JRFL, ZM214M, PVO, TRO.11), whereas others were neutralized at intermediate (ADA, YU2, MN) to good efficiency (DU156.12, DU422.1, ZM109) and some were neutralized very efficiently (JRCSF, CAP45). Interestingly, the various PG9 variants displayed pronounced differences with respect to their neutralization efficiencies. In accordance with the results of the antigen-binding assays, tyrosine sulfation strongly enhanced neutralization of highly sensitive isolates (50-fold and more; e.g. JRCSF, DU156.12, ZM109, CAP45, DU422.1), whereas only a modest improvement was observed for more resistant strains (1.3-1.5 fold; ADA, YU2 and MN). These data provide unprecedented evidence for the pivotal role of CDR H3 sulfotyrosines in effective HIV neutralization by PG9 as previously proposed based on the tertiary structure of the PG9/gp120 complex. In general, the varying sensitivities of the tested HIV strains to PG9 and RSH were in good agreement with the presence or absence of PG9-interacting residues in their gp120 V2 sequences. Furthermore, the observed differences in neutralization efficiency between PG9 and RSH were comparable to those found in antigen-binding assays. Importantly, glycoengineering of plant-derived PG9 and RSH did not affect virus neutralization, thus demonstrating that fine tuning of Asn.sup.297 N-glycosylation does not compromise the anti-viral potency of these bnAbs.

Conclusion

[0141] The examples provided herein aimed at the establishment of sulfoengineering in plants in order to increase the in vivo efficiency of two HIV-specific mAbs, PG9 and RSH. Although some plants have a tyrosylprotein sulfotransferase and can sulfate phytohormones, PG9 expressed in N. benthamiana did not contain detectable amounts of sulfated peptides, indicating that mammalian-type sulfation does not occur naturally in N. benthamiana leaves. In humans, sulfation of suitable tyrosine residues is carried out by the two tyrosylprotein sulfotransferases hsTPST1 and hsTPST2, and overexpression of hsTPST1 and hsTPST2 has been shown to increase sulfation of recombinantly produced proteins in CHO and HEK293T cells. However, expression of full-length hsTPST1 did not yield high levels of sulfation in N. benthamiana. Replacing the authentic CTS sequence of hsTPST1 with a plant CTS region, for instance, drastically increased the sulfation efficiency and led to mAbs with an increased neutralization efficacy.

[0142] The crystal structure of PG9 in complex with its antigen has revealed that Y.sup.100H and Y.sup.100Gof the PG9 (and RSH) heavy chain can be sulfated and that their sulfation increases antigen binding. By mass spectrometry, the sulfation sites of .sup.CHOPG9 and plant-produced PG9/RSH could be mapped to a short peptide containing 3 tyrosine residues (Y.sup.100E, Y.sup.100H and Y.sup.100G). Sulfation of plant-produced PG9 and RSH enhanced antigen binding and virus neutralization, indicating that also in plants Y.sup.100H and Y.sup.100G are the sulfated residues. While the impact of tyrosine sulfation on neutralization efficiency was so far only assessed for singly and doubly sulfated PG9, sulfated and unmodified PG9/RSH were compared and a far more pronounced difference in anti-viral potency was observed. These results show that singly sulfated PG9 binds and neutralizes HIV better than non-sulfated antibody, and that the doubly sulfated mAb has an even higher efficacy.