In vitro and in vivo studies of plant-produced Atezolizumab as a potential immunotherapeutic antibody

Immune checkpoint inhibitors are a well-known class of immunotherapeutic drugs that have been used for effective treatment of several cancers. Atezolizumab (Tecentriq) was the first antibody to target immune checkpoint PD-L1 and is now among the most commonly used anticancer therapies. However, this anti-PD-L1 antibody is produced in mammalian cells with high manufacturing costs, limiting cancer patients’ access to the antibody treatment. Plant expression system is another platform that can be utilized, as they can synthesize complex glycoproteins, are rapidly scalable, and relatively cost-efficient. Herein, Atezolizumab was transiently produced in Nicotiana benthamiana and demonstrated high expression level within 4–6 days post-infiltration. After purification by affinity chromatography, the purified plant-produced Atezolizumab was compared to Tecentriq and showed the absence of glycosylation. Furthermore, the plant-produced Atezolizumab could bind to PD-L1 with comparable affinity to Tecentriq in ELISA. The tumor growth inhibitory activity of plant-produced Atezolizumab in mice was also found to be similar to that of Tecentriq. These findings confirm the plant’s capability to serve as an efficient production platform for immunotherapeutic antibodies and suggest that it could be used to alleviate the cost of existing anticancer products.

In this study, the plant platform was used to produce anti-PD-L1 mAb and determine its activity.The purified plant-produced Atezolizumab was characterized using SDS-PAGE and western blot and its activity was compared with the commercial anti-PD-L1 mAb (Tecentriq).Results showed that the plant-produced Atezolizumab was slightly larger in size than Tecentriq.In terms of functional analysis, the plant-produced Atezolizumab demonstrated similar results in binding to huPD-L1 in vitro and reducing tumor weight and volume in mice in vivo.Our data confirms that the plant system can produce biologically active proteins with functions similar to those of other well-established platforms.More importantly, this platform has the potential to reduce associated costs in the upstream process of drug production, thereby increasing patient access to biological treatments.

Results
Expression of recombinant atezolizumab in N. benthamiana.To generate a non-glycan version of Atezolizumab, three amino acids (N298A, D359E, and L361M) of heavy chain were mutated using overlap PCR.The gene was inserted into a geminiviral vector and transformed into A. tumefaciens.The transformed bacterial cells containing anti-PD-L1 non-glycan heavy chain or light chain were co-infiltrated into N. benthamiana leaves.The level of protein expression was determined using day optimization.Accordingly, the infiltrated leaves were harvested at various days post infiltration (1, 3, 4, 5, 6 and 7 dpi) and the expression levels of Atezolizumab were measured by quantitative sandwich ELISA.The presence of symptoms on the infiltrated leaf area confirms the expression of mAb.However, when necrosis occurred on the later days, Atezolizumab expression decreased.The highest expression level of plant-produced Atezolizumab yielded approximately 1.8 mg/g fresh weight within 5 dpi (Fig. 1).SDS-PAGE and western blot were used to compare infiltrated crude extract to non-infiltrated crude extract (Supplementary Figs. 1 and 2).Under reducing and non-reducing conditions, the crude proteins were stained by InstantBlue dye (Supplementary Fig. 1a) and the expression of Atezolizumab in infiltrated N. benthamiana extract revealed bands at 50 and 150 kDa using anti-human IgG (Supplementary Fig. 1b, lane 2) and at 25 and 150 kDa using anti-human Kappa (Supplementary Fig. 1c, lane 2), respectively.As expected, these bands were absent in non-infiltrated N. benthamiana extract (Supplementary Figs.1b,c, lane 1).

Purification of plant-produced atezolizumab from N. benthamiana proteins. Plant-produced
Atezolizumab was purified from infiltrated N. benthamiana crude extract by protein A affinity chromatography.The characteristics of purified plant-produced Atezolizumab was examined by SDS-PAGE and western blot (Fig. 2 and Supplementary Fig. 3).Under non-reducing condition, the plant-produced Atezolizumab was detected at 150 kDa with InstantBlue (Fig. 2a), anti-human IgG (Fig. 2c), and anti-human Kappa (Fig. 2e).Under reducing condition, the plant-produced Atezolizumab was observed at 50 kDa of heavy chain and 25 kDa of light chain with InstantBlue (Fig. 2b), anti-human IgG (Fig. 2d), and anti-human Kappa (Fig. 2f).On the other hand, the plant-produced Atezolizumab was slightly larger, with bands of higher molecular weight than Tecentriq (Fig. 2, lanes 1 and 2).The results of western blots probed with anti-human-IgG and anti-human Kappa confirm the co-expression of heavy and light chains, resulting in fully assembled mAb.

Glycan analysis of plant-produced atezolizumab.
The N-glycan composition of plant-produced Atezolizumab heavy chain was analyzed using liquid chromatography-electrospray ionization-mass spectrometry (LC-ESI-MS) analysis and compared to Tecentriq.Three sites in the mAb Fc region were mutated in an attempt to inhibit N-glycosylation of our plant-produced mAb.The findings confirmed that the mutant plant-produced Atezolizumab lacks glycan structures and follows the same pattern as Tecentriq (Fig. 3).
Plant-produced atezolizumab binds to human PD-L1.The specific antigen recognition of plantproduced Atezolizumab was determined and compared to that of Tecentriq and plant-produced Nivolumab 23 by functional ELISA.Both the plant-produced Atezolizumab and Tecentriq demonstrated specific and comparable binding to the huPD-L1 protein, whereas plant-produced Nivolumab showed no antigen binding (Fig. 4).This    www.nature.com/scientificreports/treatment administration.On day 21, the plant-produced Atezolizumab (TGI TV = 41.90%) and Tecentriq (TGI TV = 24.59%)significantly reduced the growth of CT26-hPD-L1 tumors in mice when compared to the negative control (Supplementary tables 1 and 2) (P<0.05).Furthermore, tumor volume reduction did not differ between anti-PD-L1 mAb-treated groups (Fig. 5c) (P > 0.05).At the end of the study, all mice were terminated, and tumors were collected and weighed (tumor weight; TW).The tumor sizes in groups treated with plant-produced Atezolizumab (TGI TW = 29.03%)were significantly smaller than those in the PBS control group (P < 0.05), as depicted in Fig. 5d and Supplementary table 3. Most importantly, the antitumor efficacy of plant-produced Atezolizumab was not significantly different from that of its mammalian cell-produced mAb counterpart in this syngeneic murine colorectal cancer model (P > 0.05).

Discussion
Immune checkpoint inhibitors (ICIs) are a type of immunotherapy that is increasingly relevant in cancer treatment.These anti-cancer agents have shown robust and long-term clinical benefits, but they are also expensive 14,26 .
The lengthy timeline of research and development, financial obligations of clinical trials, and cost of production process are just few of the many factors that lead to the prohibitive pricing of biopharmaceutical medicines.Until now, the affordability of cancer care and biologics has always been a major concern.Drug prices should be reduced so that more patients, particularly those in underdeveloped nations, can receive adequate healthcare and cancer treatment.In doing so, the manufacturing cost of drugs can be lowered via maximizing production technologies.Plants represent a promising platform for the production of biopharmaceutical products at relatively low cost 17,27,28 .They have the potential to rapidly produce plant-made therapeutics en masse with low contamination risk 19 .The production and economic advantages of plants fueled its promise as a competent pharmaceutical factory.Plant cells, tissues, or whole plants are among the primary systems used in the manufacturing of therapeutic recombinant proteins for commercial, industrial, or pharmaceutical applications 29 .To investigate the plant platform, commercial Atezolizumab (Tecentriq) produced in mammalian cells, which is one of seven commercial ICIs approved by the FDA 12 , was used as a control in comparison to Atezolizumab produced in N. benthamiana.
In previous studies, geminiviral vectors have set the stage for transient expression of effective recombinant proteins and mAbs derived from plants 23,30,31 .Efforts to improve the protein expression levels in plants by exploiting targeting sequences have long been considered.The endoplasmic reticulum (ER) retention motif, SEKDEL (Ser-Glu-Lys-Asp-Glu-Leu), for example, demonstrated more efficient targeting, leading to enhanced accumulation of proteins in plants 32,33 .In addition, using cell secretion signals such as murine signal peptide has shown increased levels of plant-produced mAbs against rabies virus, whereby rabies mAb constructs carrying this murine signal sequence were more highly expressed than those carrying a plant signal peptide 34 .As demonstrated by our previous study, high level of glycosylated Atezolizumab expression was also found in N. benthamiana plants containing the heavy chain and light chain genes with a murine signal peptide on the N-terminus along with a SEKDEL sequence on the C-terminus 24 .Similarly, we have utilized these transgenes into this study and introduced mutations (N298A, D359E, and L361M) into the heavy chain, in order to alter the normal glycosylation of the mAb.Our results revealed that the mutated Atezolizumab was successfully expressed in plants, with highest amounts reaching up to 1.8 mg/g fresh weight in crude extracts within 5 days post-infiltration, which was consistent with prior report 24 .Generally, geminiviral-based vectors achieve transient expression of plant-made biopharmaceutical proteins in 2-6 days after agroinfiltration, depending on the plant leaf hypersensitive response 25,35,36 .
The plant-produced mAb was further purified and analyzed for physicochemical characteristics and molecular structures.Our findings show that heavy chain and light chain bands migrated closely to their expected molecular weights (50 and 25 kDa) under reducing SDS-PAGE and western blot.Likewise, non-reducing SDS-PAGE and immunoblotting analyses revealed that the plant-produced mutated Atezolizumab was completely and correctly assembled (150 kDa).However, the apparent molecular weights of the heavy chain, light chain, and assembled plant-synthesized mAb were slightly higher when compared with Tecentriq.This small increase in size may be attributed to the addition of SEKDEL [37][38][39] and the 19-amino acid murine signal peptide 40 .Another possible explanation could be due to the unsuccessful cleavage of signal peptide 39 , which we have not confirmed.Asparagine (N)-linked glycosylation is a common post-translational modification involved in several biological functions such as protein folding, stability, biological activity, interaction, and others 41,42 .The early stages of plant N-glycosylation appear to be conserved and similar to that of mammalian cells 43 , which usually occurs in the ER 44 .mAb N-glycan processing has become a crucial aspect in biologic manufacturing.In the case of commercial Atezolizumab (Tecentriq), glyco-modification via amino acid substitution at position 298 (asparagine to alanine; N298A) of the heavy chain results in a non-glycosylated mAb.This removal of N-glycans reduces or abrogates the binding affinity of anti-PD-L1 mAb for Fcγ receptors 45,46 .Herein, using our plant expression platform, we generated a non-glycosylated Atezolizumab by incorporating glycan-deleting mutations in its Fc region and compared it to Tecentriq.Site-specific glycosylation was analysed by LC-ESI-MS and no glycan structures were identified in our plant-produced Atezolizumab.These findings confirm the successful removal of N-glycans following mutations on the conserved glycosylation site and are consistent with the data from non-glycosylated reference mAb.Furthermore, the murine signal peptide and SEKDEL motif fused to plantproduced mAb only act as leading and targeting sequences for protein expression.However, it is well-known that aglycosylation of mAbs yields to aggregation 42 , which in turn may induce anti-drug antibodies 47 .More recently, several studies attempted to produce glycosylated variants of Atezolizumab in the hopes of improving antibody stability and activity 48,49 .Furthermore, two amino acid substitutions were introduced at positions 359 (glutamic acid to aspartic acid; D359E) and 361 (leucine to methionine; L361M) to follow the protein sequence Vol:.(1234567890 The plant-produced Atezolizumab binds specifically to human PD-L1 protein in functional ELISA.It also exhibited comparable binding with Tecentriq, whereas plant-produced Nivolumab did not bind to the target as predicted.These findings indicate that Fc modifications had no effect on the antigen-binding capacity of our plant-produced mAb, in agreement with previous studies 24,48 .Overall, the results presented here illustrate that a functional non-glycosylated anti-PD-L1 mAb can be produced in plants with specific binding activity to PD-L1.Atezolizumab immunotherapy has been considered as a first-line treatment option for patients with metastatic lung cancer 51 and some patients with advanced urothelial cancer 52 .It has also been used in combination to treat certain types of cancer, including liver, breast, and colon cancer [53][54][55] .In this study, we evaluated the efficiency our plant-produced Atezolizumab in syngeneic mouse model subcutaneously grafted with murine CT26-hPD-L1 tumors.At a dose of 3 mg/kg, our findings revealed significant inhibition of tumor growth from plant-derived mAb treatment.Notably, the plant-produced Atezolizumab demonstrated similar regression of tumor volumes and weights as compared with Tecentriq.In addition, the antitumor responses in anti-PD-L1 mAb-treated groups differed significantly from those in PBS control group.Prior research has shown that high doses of Atezolizumab (10 mg/kg) increased antitumor activity in vivo 48 .Having said that, dose-escalation experiments for our plantproduced Atezolizumab may be investigated in the future to optimize tumor growth inhibitory response.On the other hand, tumor-bearing mice displayed no significant body weight changes in any of the treatment groups, indicating that plant-produced Atezolizumab treatment is safe and tolerable 56 .
In conclusion, we have successfully generated an anti-PD-L1 Atezolizumab in N. bethamiana plants with no glycosylation in its heavy chain.The glyco-modified plant-produced Atezolizumab had the same N-glycan pattern as the aglycosylated commercial Atezolizumab (Tecentriq).Moreover, both the plant-based mAb and Tecentriq had comparable antigen binding affinities to human PD-L1 in vitro.Most importantly, our plant-derived Atezolizumab is as effective as Tecentriq in inhibiting tumor growth in vivo.These findings confirm the feasibility of plant platforms both for biopharmaceutical production as well as for immunotherapy.If desired, plants could be considered as another production system of choice, with the goal of lowering the cost of biopharmaceutical drugs in developing countries.

Methods
Research involving plants.The study was permitted to be carried out by the internal CU-IBC (Chulalongkorn University-Institutional Biosafety Committee) following all the Biosafety guidelines for modern biotechnology.All methods were performed in accordance with the relevant guidelines/regulations/legistration.The seeds of N. benthamiana used in the present study were kindly gifted by Dr. Supaart Sirikantaramas, Faculty of Science, Chulalongkorn University.

Approval for animal experiments.
This study (Animal Protocol No. GPTAP20220128-4) was reviewed and approved by the Institutional Animal Care and Use Committee (IACUC) of GemPharmatech Co., Ltd.(Nanjing, China).The care and use of animals for experiments were performed in accordance with the Animal welfare act and the Association for Assessment and Accreditation of Laboratory Animal Care (AAALAC).The study adheres to the recommendations in the ARRIVE guidelines.

Gene cloning in expression vector.
The heavy chain (HC) and light chain (LC) genes of Atezolizumab carrying an N-terminal murine signal peptide and a C-terminal SEKDEL peptide had previously been obtained 24 .Briefly, the gene encoding sequences for the HC and LC were optimized in silico with N. benthamiana codons by Invitrogen GeneArt Gene Synthesis (Thermo Scientifc, USA).The variable domains of Atezolizumab HC and LC were commercially synthesized and fused separately to the constant domains of human IgG1 gamma chain or kappa chain, respectively.Here, multiple nucleotide substitutions were incorporated into the coding sequence of HC by overlapping PCR method (N298A, D359E, and L361M) to generate a non-glycosylated Atezolizumab (NGAte) with desired mutations.The primers used to introduce specific point mutations into Atezolizumab HC are listed in Table 1.The resulting mutated HC product was confirmed by sequencing.Both the Atezolizumab HC and LC constructs were digested with XbaI and SacI restriction enzymes (BioLabs, Massachusetts, USA) and subcloned into the pBY2eK geminiviral vector (provided by Professor Hugh Mason 30 ).The Agrobacterium tumefaciens strain GV3101 was used for bacterial transformation.
For purification experiments performed here, infiltrated plants were collected at the optimal harvest time of peak mAb production and homogenized.The plant-produced Atezolizumab was purified by protein A affinity chromatography, as described previously 23 .

Antibody quantification.
The heavy chain band was excised, S-alkylated, and trypsin digested.The digested peptide was then analyzed by liquid chromatography-electrospray ionization-mass spectrometry (LC-ESI-MS), as described previously 57 .
Binding assay to PD-L1.The binding ability of plant-produced Atezolizumab to human PD-L1 was investigated in a functional antigen-binding ELISA.A 96-well, half-area microtiter plate (Corning, New York, USA) was coated with recombinant huPD-L1 protein (R&D System, Minneapolis, USA) overnight at 2 µg/ml in 1×PBS.Then, the plate was washed with 1×PBS-T and blocked with 5% (w/v) skim milk in 1×PBS.Plantproduced Atezolizumab, Tecentriq standard, and plant-produced Nivolumab control 23 were serially diluted and incubated on the coated plate for 2 h at 37 °C.Detection was with goat anti-human Kappa-HRP (SouthernBiotech, Alabama, USA) at 1:3,000 in 1×PBS and peroxidase activity was determined by TMB one solution substrate (Promega, Wisconsin, USA).Color development was monitored and stopped with 1M H 2 SO 4 .The absorbance was measured at 450 nm on a NS-100 Nano Scan microplate reader (Hercuvan, Shah Alam.Malaysia).
The CT26 murine colon cancer cell line was procured from ATCC (Virginia, USA).The CT26-hPDL-1 cell line was developed by knocking-out mouse PD-L1 gene using CRISPR/Cas9 technology and inserting a constitutively expressed human PD-L1 gene.The cells were grown in Roswell Park Memorial Institute media (RPMI 1640, Gibco, NY, USA), supplemented with supplemented with 10% fetal bovine serum (ExCell Bio, Shanghai, China), 0.1% Penicillin/Streptomycin (Amresco, Ohio, USA) antibiotics, and 200 μg/mL G418 (Gibco, New York, USA).The mycoplasma-free cells were thawed and cultured at 37ºC and 5% CO 2 .Then, CT26-hPDL-1 cells (Passage: Pn+12) were collected and resuspended in Dulbecco's Phosphate-Buffered Saline (DPBS; Gibco, NY, USA).The cell viability was calculated before and after inoculation and found to be 97.14% and 95.17%, respectively.In this study, each humanized mice at the age of 6-8 weeks were injected subcutaneously with 1.10 6 CT26-hPD-L1 cells into the right flank.All mice (n=18) were randomly separated into groups (n=6 mice/group) when tumor reached a diameter of 100 mm 3 .Treatment groups received either plant-produced Atezolizumab (3 mg/kg) or Tecentriq (3 mg/kg).The regimen consists of intraperitoneal (i.p.) antibody drugs every 3 days for up to 6 doses (Q3D×6).Mice from the vehicle group were injected with 1×PBS.The effect of treatment was determined by twice-weekly monitoring of tumor volume and mouse body weight.All mice were terminated on Day 23.At this www.nature.com/scientificreports/time, tumors were collected for further analysis.Data are expressed as mean ± standard deviation (Mean ± SD) and analyzed using a one-way ANOVA test.

Figure 1 .
Figure 1.Day optimization experiment for plant-produced Atezolizumab.Infiltrated N. benthamiana leaves were harvested on days 1, 3, 4, 5, 6, and 7 post-infiltration (dpi).The antibody expression level at various dpi was calculated by sandwich ELISA.Representative images of leaf necrosis and a graph showing the relative expression of plant-produced Atezolizumab were provided.Data are presented as mean ± SD of triplicate samples.

Figure 2 .
Figure 2. SDS-PAGE and western blot analysis of anti-PD-L1 mAbs.Tecentriq and purified plant-produced Atezolizumab were separated by non-reducing (a, c, and e) and reducing SDS-PAGE (b, d, f).About 2 µg of mAbs were loaded in SDS-PAGE, and 300 ng of mAbs were loaded for western blot analysis.The gels were either stained with InstantBlue dye (a,b) or transferred to nitrocellulose membrane and probed with anti-human IgG (heavy chain-specific) (c,d) and anti-human kappa (light chain-specific) (e,f).Lane M: protein ladder; lane 1: Tecentriq; lane 2: purified plant-produced Atezolizumab.

Figure 3 .
Figure 3. Glycosylation analysis of Tecentriq and purified plant-produced Atezolizumab.Tecentriq and purified plant-produced Atezolizumab were trypsin digested and analyzed with LC-ESI-MS.

Figure 4 .
Figure 4. Binding affinity of purified plant-produced Atezolizumab to huPD-L1 protein at 2 µg/mL.Tecentriq was used as positive control and plant-produced Nivolumab as negative control.Increasing dilution series of mAbs were tested for huPD-L1 binding, which was detected by anti-human kappa-HRP.Data are presented as mean ± SD of triplicate samples.

Figure 5 .
Figure 5. Antitumor efficacy of plant-produced Atezolizumab in CT26-hPD-L1-bearing knocked-in mice.Tecentriq was used as a positive control and PBS as a negative control.Three groups were formed (n = 6 mice/ group) and injected with anti-PD-L1 mAbs (3 mg/kg i.p.) and PBS.The timeline for dose administration and data collection was illustrated in (a).Treatment-related effects were demonstrated on body weight (b), tumor volume (c), and tumor weight (d).Data were analyzed by using one-way ANOVA and presented as mean ± SD. *P < 0.05, **P < 0.01, ***P < 0.001 were considered as statistically significant.