Bi-specific autoantigen-T cell engagers as targeted immunotherapy for autoreactive B cell depletion in autoimmune diseases

Abstract

Introduction: In autoimmune diseases, autoreactive B cells comprise only the 0.1-0.5% of total circulating B cells. However, current first-line treatments rely on non-specific and general suppression of the immune system, exposing patients to severe side effects. For this reason, identification of targeted therapies for autoimmune diseases is an unmet clinical need.

Methods: Here, we designed a novel class of immunotherapeutic molecules, Bi-specific AutoAntigen-T cell Engagers (BiAATEs), as a potential approach for targeting the small subset of autoreactive B cells. To test this approach, we focused on a prototype autoimmune disease of the kidney, membranous nephropathy (MN), in which phospholipase A₂ receptor (PLA₂R) serves as primary nephritogenic antigen. Specifically, we developed a BiAATE consisting of the immunodominant Cysteine-Rich (CysR) domain of PLA₂R and the single-chain variable fragment (scFv) of an antibody against the T cell antigen CD3, connected by a small flexible linker.

Results: BiAATE creates an immunological synapse between autoreactive B cells bearing an CysR-specific surface Ig⁺ and T cells. Ex vivo, the BiAATE successfully induced T cell-dependent depletion of PLA₂R-specific B cells isolated form MN patients, sparing normal B cells. Systemic administration of BiAATE to mice transgenic for human CD3 reduced anti-PLA₂R antibody levels following active immunization with PLA₂R.

Discussion: Should this approach be confirmed for other autoimmune diseases, BiAATEs could represent a promising off-the-shelf therapy for precision medicine in virtually all antibody-mediated autoimmune diseases for which the pathogenic autoantigen is known, leading to a paradigm shift in the treatment of these diseases.

Keywords: anti-PLA2R antibodies; autoimmune diseases; autoreactive B cell; bi-specific autoantigen-T cell engagers; membranous nephropathy; targeted immunotherapies.

Figure 1

Design and characterization of the BiAATE for MN. (A, B) Schematic representation of the structure (A) and the proposed mechanism of action (B) of the BiAATE, a bi-specific molecule that expresses the autoantigen involved in a specific autoimmune disease linked to the single-chain variable fragment (scFv) of an anti-CD3 by a small flexible linker. As such, the BiAATE redirects the lytic activity of T cells against the surface immunoglobulin (sIg)⁺ included in the B cell receptor (BCR) of autoreactive B cells. Activation of T cells leads to the production of perforin (PFN), granzyme B (GzmB), interferon γ (IFNγ), and tumor necrosis factor α (TNFα) that induce the selective removal of autoreactive B cells. All drawings were created by using BioRender.com. (C) Representative ponceau red staining of 1 μg BiAATE loaded under reducing (R) and non-reducing (NR) condition. Molecular weights (MW) are reported for each Western Blot and expressed in kilo Dalton (kDa). The BiAATE is indicated by arrows and exhibited a MW of approximatively 50 kDa. (D) Schematic representation of the Western Blot performed to test the immunogenic properties of the CysR antigen included in the BiAATE. (E) Representative Western Blot of 1 μg BiAATE incubated with sera from healthy controls (CTRL sera) or MN patients (MN sera). Data derive from n=6 experiments from three independent batches of BiAATE. (F) Schematic representation of the Western Blot performed to test the ability of the BiAATE to simultaneously bind CD3 in T cells and the pathogenic antibodies in the MN sera. (G) Representative Western Blot of total peripheral blood mononuclear cell (PBMC) extracts loaded under R and NR condition and incubated with 5 μg/mL BiAATE followed by exposure to CTRL sera (BiAATE + CTRL sera) or MN sera (BiAATE + MN sera), or incubated with MN sera without prior exposure to 5 μg/mL BiAATE (MN sera). Actin was used as sample loading control. Representative western blot of CD3 expression in PBMC extracts to confirm CD3 specificity (anti-human CD3). Data derive from n=6 experiments with PBMCs isolated from 6 healthy subjects and exposed to sera from n=6 CTRL or MN patients. (H) Representative dot plots of anti-human IgG₄ FITC antibody binding and 7AAD staining after gating singlet lymphocytes. PBMCs were incubated with 5 µg/mL BiAATEs in the presence of CTRL or MN sera, then with mouse anti-human IgG₄ FITC antibody and 7AAD. (I) Percentage of lymphocytes bound by anti-human IgG₄ FITC antibody after incubation with 5 µg/mL BiAATEs in the presence of CTRL or MN sera (n=6 per group). Data represent mean ± SEM and were analyzed using unpaired t-test. **p-value<0.01 vs CTRL sera.

Figure 2

The BiAATE selectively depletes PLA₂R-specific autoreactive B cells isolated from MN patients. (A, B) Schematic representation of the experimental procedure (A) and quantification overtime of IgG anti-PLA₂R in the supernatants (B) during the expansion of B cells isolated from CTRL and MN PBMCs treated or not with 3 μg/mL BiAATE for 15 hours (n=4 per group). Data represent mean ± SEM and were analyzed using 2-way ANOVA corrected with Tukey post hoc test. *p-value<0.05, and **p-value<0.01 vs MN at 14 days; °°p-value<0.01 vs CTRL at the corresponding time; ^## p-value<0.01 vs CTRL + BiAATE at the corresponding time; and ^$p-value<0.05 vs MN+ BiAATE at the corresponding time. (C, D) Schematic representation of the experimental procedure (C) and quantification overtime of IgG anti-PLA₂R antibody in the supernatants (D) during the expansion of B cells isolated from PBMCs of MN patients (n=6 per group). Data represent mean ± SEM and were analyzed using 1-way ANOVA corrected with Tukey post hoc test. *p-value<0.05 vs 14 days; °°p-value<0.01 vs 10 days. (E) Quantification of IgG anti-PLA₂R in the supernatants of expanded B cells from MN patients treated with autologous T cells alone or in the presence of 3 μg/mL BiAATEs or 3 μg/mL Blinatumomab (BLINA) for 3 days (n=6 per group). Data represent mean ± SEM and were analyzed using 1-way ANOVA corrected with Tukey post hoc test. *p-value<0.05, **p-value<0.01, and ***p-value<0.001 vs T cells alone. (F) Percentages on singlets of viable (7AAD negative) CD3^-CD19⁺ B cells co-cultured with autologous T cells alone or in the presence of 3 μg/mL BiAATE or 3 μg/mL BLINA for 3 days (n=6 per group). The percentage of viable B cells in co-culture with autologous T cells alone were taken as 100%. Data represent mean ± SEM and were analyzed using 1-way ANOVA corrected with Tukey post hoc test. ***p-value<0.001 vs T cells alone and °°°p-value<0.001 vs BIAATE.

Figure 3

Human CD3 is expressed and functional on Pan hCD3 mice, and the BiAATE reduces PLA₂R antibody titer in these mice following active immunization with PLA₂R. (A) Expression of mouse CD3 in splenocytes from Pan hCD3 and WT mice (n=3) determined flow cytometry. Data represent mean ± SEM and were analyzed using unpaired t-test. ***p-value<0.001 vs WT mice. (B) Binding of different antibody clones of human anti-CD3 (SP34.2, OKT3, HIT3α, MEM-57, UCHT-1) to CD4⁺ T cells from Pan hCD3 mice (n=3) determined by flow cytometry. (C, D) Percentage of dividing CD8⁺ T cells (C) and IFN-γ release (D) determined by flow cytometry and ELISA, respectively, in unstimulated (NA) T cells isolated from Pan hCD3 or in response to coated human anti-CD3 (OKT3) activation for 3 days at different concentration (n=6 per group). Human anti-CD3 antibody clone SP34.2 (5 µg/mL) was used as a positive control. Data represent mean ± SEM and were analyzed using 1-way ANOVA corrected with Tukey post hoc test. **p-value<0.01, and ***p-value<0.001 vs NA; °°°p-value<0.001 vs OKT3 1 μg/mL. (E) Representative Western Blot of MN patients’ IgG₄ binding to CD3 in total splenocyte extracts from hCD3 mice (n=6) incubated with (upper panel on the left) or without (lower panel on the left) 5 μg/mL BiAATE followed by MN sera. The right panel shows representative Western Blot of IgG₄ binding to CD3 in total splenocyte extracts from hCD3 mice (n=6) incubated with 5 μg/mL BiAATE followed by incubation with CTRL sera. Actin was used as sample loading control. MW are reported in each Western Blot and expressed in kDa. (F) Quantification of IgG anti-PLA₂R (mg/mL) in the sera of pan hCD3 mice (n=18) immunized with two doses of 50 μg PLA₂R antigen. Data represent mean ± SEM and were analyzed with unpaired t-test. ***p-value<0.001 vs Baseline. (G) Quantification overtime of IgG anti-PLA₂R expressed as % changes over D28 in the sera of immunized pan hCD3 mice (n=9 per group) treated at D28 and D35 (black arrows) with vehicle or 1 mg/kg BiAATE. Data represent mean ± SEM and were analyzed with 2-way ANOVA corrected with Šídák’s multiple comparisons test. *p-value<0.05 vs BiAATE at the corresponding time; °°p-value<0.01, and °°°p-value<0.001 vs BiAATE at D28; and ^#p-value<0.05 vs BiAATE at D42.