Plant-based production of highly potent anti-HIV antibodies with engineered posttranslational modifications

Broadly neutralising antibodies (bNAbs) against human immunodeficiency virus type 1 (HIV-1), such as CAP256-VRC26 are being developed for HIV prevention and treatment. These Abs carry a unique but crucial post-translational modification (PTM), namely O-sulfated tyrosine in the heavy chain complementarity determining region (CDR) H3 loop. Several studies have demonstrated that plants are suitable hosts for the generation of highly active anti-HIV-1 antibodies with the potential to engineer PTMs. Here we report the expression and characterisation of CAP256-VRC26 bNAbs with posttranslational modifications (PTM). Two variants, CAP256-VRC26 (08 and 09) were expressed in glycoengineered Nicotiana benthamiana plants. By in planta co-expression of tyrosyl protein sulfotransferase 1, we installed O-sulfated tyrosine in CDR H3 of both bNAbs. These exhibited similar structural folding to the mammalian cell produced bNAbs, but non-sulfated versions showed loss of neutralisation breadth and potency. In contrast, tyrosine sulfated versions displayed equivalent neutralising activity to mammalian produced antibodies retaining exceptional potency against some subtype C viruses. Together, the data demonstrate the enormous potential of plant-based systems for multiple posttranslational engineering and production of fully active bNAbs for application in passive immunisation or as an alternative for current HIV/AIDS antiretroviral therapy regimens.

www.nature.com/scientificreports www.nature.com/scientificreports/ this emulate the gp120 affinity for the sulfated CCR5 5 . We know that this posttranslational modification (PTM) is crucial for the Ab's functional activities 6 . The absence of tyrosine sulfation leads to a significant decrease in antigen binding and subsequent loss of function 7 . This typical human type PTM is catalysed by tyrosyl protein sulfotransferases (TPSTs) 8,9 , which makes the expression of such an antibody version restricted to mammalian cell systems which are generally costly and cumbersome 10 . However, alternative and potentially more cost-effective and scalable platforms are available for mass-production of antibodies 10 .
Plants, and in particular Nicotiana benthamiana, are well suited for the production of efficacious monoclonal HIV antibodies, such as 2G12, VRC01 and PG9 [11][12][13][14] . The efficacy of plant-produced versions of some of these antibodies has been tested in animal trials 7 . A remarkable achievement is the generation of Abs with engineered Fc glycosylation resulting in similar or increased Fc receptor binding activities compared to the non-glycoengineered mammalian cell-derived variants 15-17 . However, the in planta generation of bNAbs that need mammalian type PTMs, like tyrosine sulfation, is hampered by the lack of the respective plant enzymatic repertoire and generated recombinant mAbs remain functionally inactive 7 . Notwithstanding, extensive in planta engineering approaches allowed for the generation of IgGs (and other proteins) with engineered PTMs 18 . Concurrently, in planta CDR H3 tyrosine O-sulfation of PG9 and PG16 bNAbs that, like CAP256-VRC26, require this PTM for antigen binding, was reported. This was achieved by the overexpression of human TPST1 (hTPST1) in N. benthamiana 14 . Also, the presence of engineered Fc glycans allowed for the production of PG9 and PG16 versions with increased effector functions compared to the mammalian cell-derived variants 14 .
In this report, we demonstrate the expression and characterisation of CAP256-VRC26 bNAbs with engineered PTMs to maintain Ab function. Two versions of CAP256-VRC26 (08, 09) were produced in glycoengineered N. benthamiana (ΔXTFT), exhibiting a glycoprofile previously shown in vitro to positively impact HIV effector functions of some antibodies 16 . Higher ADCC and ADCVI has been observed in vitro only for some HIV bNAbs and no in vivo impact has been observed for glycoengineered b12 16 . We further show that by the coexpression of hTPST1, CDR H3 tyrosine sulfation was installed. Our data reveal that PTM engineered CAP256-VRC26 bNAbs exhibited similar structural and functional features compared to HEK293-produced variants, and suggest that plants could be used to mass-produce this antibody for human use.

Results
transient in planta coexpression of CAP256-VRC26 bNAbs and hTPST1. Here we used N. benthamiana (ΔXTFT), a glycoengineered mutant host that lacks N-glycan residues with a core β1,2-xylose and α1,3-fucose moieties 19 for transient expression of CAP256-VRC26 bNAbs. Several Potato virus X (PVX) and Tobacco mosaic virus (TMV) vector combinations carrying light and heavy chains, were delivered into plant leaves. Expression levels were measured eight days post-infiltration (d.p.i) by ELISA (Table 1), with the highest production being achieved using the murine IgG heavy chain signal peptide and PVX-mHC + TMV-mLC vector combinations. Expression levels of assembled Abs were 489 and 487 mg.kg −1 , respectively. MagReSyn ® Protein A microsphere-based approach for the one-step protein A purification of CAP256-VRC26 bNAbs. Magnetic Protein A microspheres were used as a one-step protein A purification method for IgG purification from centrifugally clarified N. benthamiana (ΔXTFT) leaf extract which was then analysed on SDS-PAGE (Fig. 1). Under non-reducing conditions IgG1s typically display a single band pattern, ~150 kDa -assembled IgG, whereas, under reducing conditions IgG1s typically display a two-band pattern, ~50 kDa -heavy chain (HC) and ~25 kDa -light chain (LC). The use of MagReSyn ® Protein A microspheres resulted in successful purification of CAP256-VRC26 bNAbs from clarified samples. Nicotiana benthamiana (ΔXTFT)-produced CAP256-VRC26 bNAb eluents display a similar protein banding pattern to their HEK293-produced counterparts. A prominent signal at position 150 kDa was obtained under non-reducing conditions, corresponding to the size of an assembled IgG. Under reducing conditions, two prominent signals at position 55 kDa and 25 kDa were obtained, corresponding to the size of IgG HC and LC, respectively (Fig. 1). In addition, under reducing condition, there were additional bands at position ~10 kDa and ~40 kDa (Fig. 1, lane 5, 6 and 11, 12) N. benthamiana (ΔXTFT)-produced CAP256-VRC26 bNAb eluents; these correspond to proteolytic degradation fragments of the IgGs heavy chain as determined by liquid chromatography-tandem mass spectrometry (LC-MS/MS) and liquid chromatography mass spectrometry (LC-MS) (Supplementary Fig. S4 and S8). Heavy chain proteolytic degradation fragments were also observed between 45-48 kDa, with the lighter fragment being undetected.

Plant and HEK293 cell produced bNAbs exhibit similar structural features. Preliminary structural
analysis was done between the HEK293 and one-step protein A purified N. benthamiana (ΔXTFT)-produced CAP256-VRC26 bNAbs, revealing no detectable secondary structural difference between the respective bNAbs. The characteristic minima of the circular dichroism (CD) spectra of the HEK293 and N. benthamiana (ΔXTFT)-produced CAP256-VRC26 bNAbs (Fig. 2(a)) is typical of a protein structure with a dominant β-sheet content and low levels of α-helices 24 . The folds of HEK293 and N. benthamiana (ΔXTFT)-produced CAP256-VRC26 bNAbs were probed by intrinsic fluorescence (Fig. 2(b,c)). The HC species of CAP256-VRC26.08 and CAP256-VRC26.09 have 11 Trp residues and 17 Tyr residues. The LC species of CAP256-VRC26.08 has 4 Trp residues and 9 Tyr residues and LC species CAP256-VRC26.09 has 5 Trp residues and 7 Tyr residues. All Trp and Tyr residues were distributed similarly throughout CAP256-VRC26.08 and CAP256-VRC26.09. The fluorescence spectral data of both the HEK293 and N. benthamiana (ΔXTFT) depict a λ emm max of 337.4 nm. The same λ emm max was exhibited by HEK293 and N. benthamiana (ΔXTFT) suggesting that the Trp and Tyr residues are in similar structural environments. This is indicative of similar folds of the CAP256-VRC26 bNAbs from both expression hosts.

N. benthamiana (ΔXTFT) derived bNAbs can neutralise HIV-1 with equivalent efficacy to their HEK293 counterparts in vitro.
The CAP256-VRC26 lineage is highly potent against HIV-1 subtype A and C strains, with reduced activity against subtype B viruses 3 . Both the one-step protein A purified N. benthamiana (ΔXTFT) and HEK293-produced CAP256-VRC26 bNAbs was assessed using the TZM-bl neutralisation assay against a multi-subtype panel of 17 pseudoviruses (Table 4). Representative neutralisation curves of the most sensitive viruses from each subtype are shown in Supplementary Fig. S9. The IC 50 values of HEK293-produced CAP256-VRC26 bNAbs were similar to those obtained for N. benthamiana (ΔXTFT) antibodies co-expressed with hTPST1 neutralising 8 of the 9 subtype C viruses and 3 of the 4 subtype B viruses. A similar pattern was observed for the neutralization of subtype A viruses, where CAP256-VRC26.08 and CAP256-VRC26.09 from   www.nature.com/scientificreports www.nature.com/scientificreports/ both expression systems neutralized 3 and 4 viruses, respectively. Importantly, the exceptional potency against some subtype C and A viruses were maintained by the plant-derived mAbs. However, the non-sulfated N. benthamiana (ΔXTFT) antibodies showed loss of both breadth and potency against the panel as indicated by the higher IC 50 values.

Discussion
This study demonstrates the efficient production of functional anti-HIV bNAbs, CAP256-VRC26 (08 and 09) in N. benthamiana (ΔXTFT). At the moment, determining the best signal peptide/vector backbone is more empirical rather than rationally designed. Four different signal peptide/vector backbone combinations were screened to eliminate any low producing combinations. All combinations signal peptide and vector backbone combinations had high (330-490 mg.kg −1 ) absolute levels of recovered mAb.
The presence and functional impact of O-sulfated tyrosine in the antigen-binding domains of Abs have so far only been reported for anti-HIV bNAbs that target the V1V2 region of the HIV envelope trimer. Notably, most plants, including N. benthamiana, are installed with all the necessary repertoire for carrying out most PTMs, such as transferases for glycosylation, however, previous attempts to find a TPST candidate in the N. benthamiana draft genome has been unsuccessful 14 . Indeed Abs that need this modification, remain inactive, as shown in this study and others 7,14 . In the current study, the engineering of tyrosine sulfation within the CDR H3 of the bNAbs was done through co-expression with hTPST1 engineered for post-Golgi targeting 14 . Sulfation was observed in both HEK293 and N. benthamiana (ΔXTFT)-produced bNAbs based on a characteristic mass shift observed for tryptic CDR H3 fragments and intact HCs. A mass shift of 79.96 Da per sulfate moiety was observed in both HEK293 and N. benthamiana (ΔXTFT)-produced bNAbs, corresponding to reports by Parker and co-workers 25 . Tryptic peptide fragments of CAP256-VRC26 (08 and 09) bNAbs revealed sulfation at two tyrosine residues, Tyr112 and Tyr 113, of which Tyr 112 is essential for the efficacy of the bNAbs 3 . LC-MS revealed mono-and di-sulfated CAP256-VRC26 species in both HEK293 and plant-produced bNAbs. It was, however, observed that there were higher levels of these mono-and di-sulfated CAP256-VRC26 species in the HEK293-produced bNAbs relative to the plant-produced bNAbs. Levels of ~60% sulfation was achieved, similar to what was achieved with PG9 14 . This suggests that the ability of the transiently coexpressed hTPST1 to sulfate tyrosines in the CDR H3 domain might not be as efficient as the native machinery of the HEK293 cells.
Here, we used N. benthamiana (ΔXTFT) as an expression platform which allowed the generation of bNAbs carrying virtually exclusively GnGn structures, lacking core xylose and fucose. In contrast, HEK293 cell-derived variants showed four prominent glycan species, the majority decorated with core fucose. Various studies have demonstrated that the lack of fucose on IgG antibodies have superior anti-viral activities due an increased binding to the respective Fcγ-receptor 26,27 . While non-fucosylated anti-HIV mAbs have enhanced FcγR-mediated antiviral activity in vitro [14][15][16] and show increased ADCC 14 , non-human primate studies are not entirely conclusive. www.nature.com/scientificreports www.nature.com/scientificreports/ For example fucose free anti-HIV-1 bNAb b12 does not improve protection against SHIV challenge in macaques despite enhanced in vitro activities 16 . Further experiments are needed to fully explore the potential of using glycoengineered anti-HIV antibodies in vivo.
An important issue with the plant-made CAP256-VRC26 Abs is the incomplete glycosylation of the Fc domain. While Fc domain of HEK293 derived variants are virtually fully occupied by N-glycans only about 50% of plant-produced versions are glycosylated. Incomplete glycosylation of transiently plant-produced Abs has been reported earlier 13,14,19,28 ; however, glycosylation levels of 74.3% of G0 glycans were achieved with plant-produced VRC01 13 . It seems that the Fc N-glycosylation site (N297) is inefficiently recognized in some instances by the plant oligosaccharyltransferase complex (OST complex), resulting in under glycosylation of the recombinant glycoproteins. Notwithstanding, in planta overexpression of foreign OST subunits is a viable approach to increase N-glycosylation efficiency in plants 12 . Glycosylation of the light chain has been shown to influence the clearance of Ab from blood in pharmacokinetics studies 7 . In contrast to the sialylated glycosylating glycan of the HEK293-made Ab light chain, the glycosylating glycan of the plant-made Ab light chain terminates in a GlcNAc, which has been shown to induce rapid receptor-mediated removal 7 .
Functional activity of CAP256-VRC26 bNAbs was assessed by HIV-1 neutralisation assays in vitro. The results demonstrate similar activity of HEK293 cell-derived and sulfated plant-produced Abs. Anti-viral activity was successfully tested against HIV-1 strains (encompassing subtype A, B, and C), confirming maintenance of the broad anti-viral activities of the Abs. In contrast, non-sulfated Ab versions showed a loss of both breadth and potency, emphasizing the importance of O-sulfated tyrosine in the CDR H3 domain for functional activity. Interestingly, although the levels of sulfation between HEK293 and plant-expressed antibodies were different (~90% and ~60%, respectively) their neutralization potency was similar. A major problem which is encountered with the plant-based production of protein in Nicotiana species, is the proteolytic degradation of some recombinantly produced proteins in planta 29,30 . Despite the presence of protease degradation products in the plant produced Abs, similar neutralization potency was seen between the HEK293 cell-derived and sulfated plant-produced Abs samples which contained proteolytic degradation products. This suggests that these degradation products were still functionally active, with cleavage having occurred in a region of the Abs that does not compromise neutralising potency detectable by in vitro assay.
Taken together, we demonstrate the efficient in planta expression of functionally active CAP256-VRC26 bNAbs with engineered PTMs to optimise efficacy. This study paves the way for further in vivo studies to determine the potential of these Abs for treatment of, or passive immunization against HIV/AIDS.
Secondary structure analysis. Far-UV Circular dichroism (CD) spectrum (260-180 nm) measurements of mAb samples in 10 mM Tris-Acetate, pH 7.4 were taken using a 1 mm path length on an Applied Photophysics Chirascan CD spectrometer (Surrey, UK) at 20 °C. Averaged ellipticity values were were converted to mean residue ellipticity (MRE) and corrected for the buffer blank baseline.
Threefold dilution series of the bNAbs were prepared in Dulbecco's Modified Eagle's medium (DMEM)