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. 2024 May;22(5):1224-1237.
doi: 10.1111/pbi.14260. Epub 2023 Dec 4.

Plant-derived Durvalumab variants show efficient PD-1/PD-L1 blockade and therapeutically favourable FcR binding

Shiva Izadi  1 , Simon Gumpelmair  2 , Pedro Coelho  3   4 , Henrique O Duarte  3   4 , Joana Gomes  3   4 , Judith Leitner  2 , Vinny Kunnummel  1 , Lukas Mach  1 , Celso A Reis  3   4   5   6 , Peter Steinberger  2 , Alexandra Castilho  1
Affiliations

Plant-derived Durvalumab variants show efficient PD-1/PD-L1 blockade and therapeutically favourable FcR binding

Shiva Izadi et al. Plant Biotechnol J. 2024 May.

Abstract

Immune checkpoint blocking therapy targeting the PD-1/PD-L1 inhibitory signalling pathway has produced encouraging results in the treatment of a variety of cancers. Durvalumab (Imfinzi®) targeting PD-L1 is currently used for immunotherapy of several tumour malignancies. The Fc region of this IgG1 antibody has been engineered to reduce FcγR interactions with the aim of enhancing blockade of PD-1/PD-L1 interactions without the depletion of PD-L1-expressing immune cells. Here, we used Nicotiana benthamiana to produce four variants of Durvalumab (DL): wild-type IgG1 and its 'Fc-effector-silent' variant (LALAPG) carrying further modifications to increase antibody half-life (YTE); IgG4S228P and its variant (PVA) with Fc mutations to decrease binding to FcγRI. In addition, DL variants were produced with two distinct glycosylation profiles: afucosylated and decorated with α1,6-core fucose. Plant-derived DL variants were compared to the therapeutic antibody regarding their ability to (i) bind to PD-L1, (ii) block PD-1/PD-L1 inhibitory signalling and (iii) engage with the neonatal Fc receptor (FcRn) and various Fcγ receptors. It was found that plant-derived DL variants bind to recombinant PD-L1 and to PD-L1 expressed in gastrointestinal cancer cells and are able to effectively block its interaction with PD-1 on T cells, thereby enhancing their activation. Furthermore, we show a positive impact of Fc amino acid mutations and core fucosylation on DL's therapeutic potential. Compared to Imfinzi®, DL-IgG1 (LALAPG) and DL-IgG4 (PVA)S228P show lower affinity to CD32B inhibitory receptor which can be therapeutically favourable. Importantly, DL-IgG1 (LALAPG) also shows enhanced binding to FcRn, a key determinant of serum half-life of IgGs.

Keywords: Nicotiana benthamiana; Durvalumab; FcRn; FcγR; PD‐1/PD‐L1; checkpoint inhibitors.

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Conflict of interest statement

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Figures

Figure 1
Figure 1
Expression and purification of plant‐derived Durvalumab variants. (a) Schematic representation of an IgG highlighting the differences in amino acid sequence within the hinge and CH2 domain of Durvalumab (Imfinzi®) and plant‐derived DL variants. (b) DL variants expressed in N. benthamiana ΔXF plants and purified by affinity chromatography were analysed in reducing conditions. SDS‐PAGE gels were either stained with Coomassie brilliant blue (CBB) or used for immunoblotting with anti‐gamma chain (heavy chain) and anti‐kappa chain (light chain) antibodies conjugated to HRP. The apparent molecular mass of marker proteins is shown in kilo Dalton (kDa). (c) Size exclusion chromatography profiles and CBB of SDS‐PAGE gels run under non‐reducing conditions.
Figure 2
Figure 2
Glycosylation profiles of Durvalumab variants. Glycosylation profiles of Durvalumab (Imfinzi®) and DL variants expressed in N. benthamiana ΔXF plants without or with co‐expression of core α1,6‐fucosyltransferase (FUT8) are shown. MS spectra refer to the peptide carrying the N297 N‐glycan: EEQFNSTYR for DL‐IgG4(PVA)S288P and EEQYNSTYR for the other variants. The assigned N‐glycan structures were labelled according to the ProGlycAn nomenclature. A cartoon illustration highlights the main glycan structures detected for each peptide. For details see http://www.functionalglycomics.org/.
Figure 3
Figure 3
Binding of Durvalumab variants to PD‐L1‐expressing gastrointestinal cancer cells. (a,b) In vitro IFNγ stimulation induces the cell surface expression of PD‐L1 and allows the binding of clinical‐grade DL to the NCI‐N87 and SW48 gastrointestinal cancer cell lines. (c,d) The binding of plant‐derived DL variants to cell surface PD‐L1 was assessed via flow cytometry in NCI‐N87 and SW48 cells. The variants DL‐IgG1 (GnGn), DL‐IgG1 (GnGnF6) and DL‐IgG1 (LALAPG) (GnGnF6) showed similar binding as Durvalumab (Imfinzi®), while the variants DL‐IgG4 (PVA)S288P (GnGn), DL‐IgG1 (LALAPG) (GnGn) and DL‐IgG4 (PVA)S288P (GnGnF6) showed a 20% decrease in the binding to cell surface PD‐L1 expressed by cancer cells.
Figure 4
Figure 4
Blocking PD‐1/PD‐L1 interaction by Durvalumab. (a) Simplified schematic representation of the use of a transcriptional PD‐1+NF‐κB::eGFP reporter T‐cell line to evaluate the impact of plant‐derived DL on T‐cell activation. Cross‐linking of the TCR‐CD3 complex with CD3‐antibody fragments expressed on K562‐based stimulator cells (K562S‐PD‐L1) results in a strong expression of the eGFP reporter gene. Negative co‐stimulatory signals induced by engagement of PD‐1 with K562S expressing PD‐L1 lead to the inhibition of TCR/CD3 signalling and reduced eGFP expression. Antibodies targeting PD‐L1 block PD‐1 engagement and restore eGFP expression. (b) Example of flow cytometric measurement of eGFP expression in stimulated PD‐1 reporter cells in the absence or presence of Durvalumab. (c) PD‐1 expressing NF‐κB::eGFP reporter cells were stimulated for 24 h with K562S‐PD‐L1 in presence of Durvalumab variants (DL‐IgG1, DL‐IgG1 (LALAPG) and DL‐IgG4 (PVA)S288P) and glyco‐variants (GnGn and GnGnF6) at indicated concentrations (1000 to 1 ng/mL). Untreated PD‐1 reporter cells stimulated with K562S‐PD‐L1 used as controls. Data are derived from two independent experiments performed in triplicates (n = 6). Data were normalized to the eGFP expression of PD‐1 reporter cells stimulated under conditions where PD‐L1 was fully blocked (1 μg/mL of PD‐L1 antibody). Inhibition curves (Figure S2) and half maximum effective concentrations (EC50) were calculated from normalized data using a 4‐parameter logistic function.
Figure 5
Figure 5
Binding of Durvalumab variants to different FcγRs. (a) Plant‐derived Durvalumab variants (DL‐IgG1, DL‐IgG1 (LALAPG) and DL‐IgG4 (PVA)S288P) and glyco‐variants (GnGn and GnGnF6) were compared to DL (Imfinzi®) regarding their ability to bind to cells expressing CD16, CD16V, CD32A, CD32B and CD64 Fcγ receptors. Binding was determined at indicated antibody concentrations (0.3–10 μg/mL) and bound antibodies were detected using an Allophycocyanin‐conjugated AffiniPure F(ab)2 Fragment of Donkey Anti‐Human IgG (H + L specific). Cells not expressing human FcγR served as control to normalize data. Two independent experiments were performed in duplicates. The table summarizes the reduction of binding to FcγR upon core fucosylation. Statistical analysis was performed with multiple comparisons two‐way ANOVA (n = 2). (b) Comparison of the binding of DL and plant‐derived core‐fucosylated (GnGnF6) DL variants (used at 10 μg/mL) to different FcγRs.
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