Povetacicept (ALPN-303; TACI vTD-Fc), an enhanced, potent dual inhibitor of BAFF and APRIL, ameliorates experimental autoimmune myasthenia gravis in C57BL/6N mice

Abstract

Background and objectives: Myasthenia gravis (MG) is a T cell-dependent, B cell-mediated autoimmune disease targeting the acetylcholine receptor (AChR) and other proteins of the neuromuscular junction postsynaptic membrane. Production of pathogenic autoantibodies results from B cell activation and expansion of antibody-secreting cells, including plasma cells, whose differentiation and survival are reliant on the TNF family cytokines APRIL and BAFF. Povetacicept (ALPN-303; TACI vTD-Fc) is an Fc fusion protein of an engineered TACI domain with significantly more potent dual inhibition of APRIL and BAFF than wild-type (WT) TACI-Fc (e.g., telitacicept).

Methods: In this study, the activity of povetacicept was evaluated in the mouse experimental autoimmune MG (EAMG) model, compared to (i) telitacicept, (ii) a depleting anti-CD20 antibody, (iii) neonatal Fc receptor blocker efgartigimod, (iv) a matched Fc control protein, and (v) PBS as vehicle.

Results: Therapeutic administration of povetacicept ameliorated clinical manifestations in EAMG mice and was associated with significantly lower levels of immunoglobulin subclasses and anti-AChR antibody titers in serum, along with increased muscle AChR content - superior to the evaluated comparators. Povetacicept treatment also reduced the number of total B220⁺ and Ki67⁺ proliferating cells in draining lymph node follicles and resulted in modifications of splenic T and B cell subset frequencies, compared to controls.

Discussion: The potent, dual BAFF/APRIL inhibitor povetacicept significantly improves clinical disease activity in EAMG, associated with reductions in pathogenic anti-AChR autoantibodies and superior to comparator therapeutic interventions based on WT TACI-Fc, CD20 depletion, or FcRn inhibition. Povetacicept may therefore confer beneficial clinical outcomes in the treatment of MG and other autoantibody-related neurological diseases.

Keywords: APRIL (TNFSF13); B cell; BAFF - B-cell activating factor; TACI (TNFRSF13B); animal model; experimental autoimmune myasthenia gravis; myasthenia gravis.

Figure 1

Administration of povetacicept reduces clinical signs of EAMG. (A) EAMG pilot experiment. Povetacicept was administered i.p. twice weekly from Week (wk) 7 (grey squares, n=5 mice, 10 doses total) or from Week 9 (black squares, n=5 mice, 6 doses total). EAMG control mice received vehicle (PBS) only, from Week 7 (white circles, n=5 mice, 10 doses total) (PBS-EAMG). TAChR immunization and boosts are indicated with upward arrows and i.p. treatments with downward arrows on the x-axis. (B) EAMG therapeutic experiment. EAMG mice received povetacicept (grey squares, n=12 mice, 7 doses total), telitacicept (light grey squares, n=12 mice, 7 doses total), an Fc control (light grey circles, n=12 mice, 7 doses total), or vehicle (PBS-EAMG) (white circles, n=12 mice, 7 doses total) via twice weekly i.p. injections. TAChR immunization and boosts are indicated with upward arrows and i.p. treatments with downward arrows on the x-axis. Statistical significance of clinical scores between groups in A and B was calculated with the Kruskal-Wallis non-parametric test with Dunn’s multiple comparisons test; data are presented as mean EAMG scores ± the standard error of the mean (SEM), with mice being scored for disease as described in the Methods section. (C) Anti-mouse AChR antibody titers in individual mice were measured by a radioimmunoprecipitation assay detailed in the Methods and expressed as pmoles of immunoprecipitated ¹²⁵I-αBuTx binding sites per ml of serum; horizontal and vertical bars represent the mean and standard deviation (SD), respectively. (D) AChR muscle content in individual mice expressed as fmol of immunoprecipitated ¹²⁵I-αBuTx binding sites per gram of muscle is shown, with horizontal and vertical bars representing the mean and SD, respectively. Statistical analysis in (C, D) was performed using one-way ANOVA, corrected for multiple comparisons, and assays carried out as described in the Methods section. ns, not significant.

Figure 2

Povetacicept treatment reduces concentrations of total serum immunoglobulins. Total IgM, IgG1, IgG2b, IgG3, and IgA concentrations in serum samples collected just prior to the start of dosing (Week 9; “baseline”) and at termination (Week 13) from PBS-EAMG, povetacicept, telitacicept, or Fc control mice were measured using a magnetic bead multiplex kit as described in the Methods. Data from individual mice for terminal concentrations (A) and the difference in concentrations (terminal minus baseline) (B) are plotted, with horizontal and vertical bars representing the median and interquartile range, respectively. Statistical analysis was performed using the Kruskal-Wallis test with Dunn’s multiple comparisons test.

Figure 3

Serum exposure and anti-drug antibody levels. (A) Concentrations (ng/ml) of povetacicept and telitacicept in serum collected over time, just prior to administration of test articles at Week 9.6, 10.6, and 11.6, and at Week 12.6 and 13 (termination), measured using an ELISA as described in the Methods. Data are shown as the mean ± SD. (B) Anti-drug antibody (ADA) titers in individual mice from terminal serum, analyzed as described in the Methods. Titers <1:100 were considered undetectable. (C) Optical density (OD) values at 450 nm in the ADA ELISA for terminal serum samples from mice treated with povetacicept or telitacicept across all dilutions, or from naïve female C57BL/6 mice (n = 6) diluted 1:100 to obtain the cut-point OD. Data from individual mice are shown.

Figure 4

Secondary lymphoid organ weights are reduced in povetacicept-treated EAMG mice. Representative images of spleens (A) and draining (inguinal) lymph nodes (B) collected at termination (Week 13) are shown. Length of representative spleens are: PBS-EAMG controls = 19 mm, povetacicept = 15 mm, telitacicept = 14 mm, Fc control = 16.5 mm, healthy donor (HD) = 14.5 mm. Mean weights (expressed as grams) of spleens and LNs are presented in graphs (C, D), respectively, for each treatment group (n=12 mice each) and for HD mice (n=4). (E) Total cellularity of spleens, processed for flow cytometry as described in the Methods. Individual mice are plotted, with horizontal and vertical bars as the mean and SD, respectively. Statistical analysis was performed using one-way ANOVA, corrected for multiple comparisons.

Figure 5

Povetacicept treatment results in significantly lower B cells and proliferating (Ki67⁺) cells within draining LNs (drLN). (A) Representative images of immunofluorescence on drLN, with maximum intensity projection of z-stack images (objective 20X). The drLNs were stained and imaged for Ki67+ cells (green) and B220+ cells (red) as described in Methods. The regions of interest (ROI) are delimited by white dotted lines and shown graphically in (B-E). HD = healthy donor [mice]. Integrated densities of B220⁺ and Ki67⁺ cells in whole immunofluorescence microscopy images (B, C, respectively) and in the region of interest (D, E, respectively) were determined as described in the Methods section. Individual mice are plotted, with horizontal and vertical bars as the mean and SD, respectively. Statistical analysis was performed using one-way ANOVA, corrected for multiple comparisons. ns, not significant.

Figure 6

Povetacicept treatment modifies B and T cell subset compositions in spleens. (A) Flow cytometry analysis of CD19⁺B220⁺ (% of viable cells), B_GC CD95⁺GL7⁺ (% of CD19⁺B220⁺ cells), PC CD138⁺TACI⁺ (% of viable cells), PC_LL CD138⁺TACI⁺CD19-B220- (% of viable cells). (B) CD4+ T cell and (C) CD8+ T cell compartments were analyzed to detect changes in CD4_EM (CD44⁺CD62L^-), CD4_naive (CD44^-CD62L⁺), CD4_CM (CD44⁺CD62L⁺) and CD8_EM (CD44⁺CD62L^-), CD8_naive (CD44^-CD62L⁺), CD8_CM (CD44⁺CD62L⁺). n=12 spleens/treatment group collected at termination (Week 13); healthy donor (HD) = 6 spleens. Individual mice are plotted, with horizontal and vertical bars as the mean and SD, respectively. Statistical analysis was performed using one-way ANOVA, corrected for multiple comparisons.

Figure 7

Povetacicept efficacy in ameliorating clinical signs of EAMG. (A) EAMG mice received povetacicept (black squares, n=12 mice, twice weekly for 8 doses total), efgartigimod (light grey circles, n=12 mice, 8 doses total), anti-CD20 (dark grey circles, n=12, once weekly for 4 doses total), Fc control protein (light grey squares, n=12 mice, 8 doses total), or PBS for the PBS-EAMG group (white circles, n=12 mice, 8 doses). TAChR immunization and boosts are indicated with the larger upward arrows and i.p. treatments with smaller upward arrows along the x-axis. Statistical significance of clinical scores was calculated using the Kruskal-Wallis test with Dunn’s multiple comparisons test; data are presented as mean EAMG scores (± SEM). Insert: Anti-mouse AChR antibody titers in individual mice (pmoles of immunoprecipitated ¹²⁵I-αBuTx binding sites per ml of serum). (B) Total cellularity of spleens collected at termination (Week 13) and processed for flow cytometry as described in the Methods. Anti-mouse AChR Ab and spleen cellularity data are presented as scattered dot plot graphs of individual mice, with horizontal and vertical bars representing the mean and SD, respectively. Statistical analysis was performed using one-way ANOVA, corrected for multiple comparisons.

Figure 8

Povetacicept reduces serum concentrations of total immunoglobulins and impacts splenic T and B cell frequencies. (A) Total IgM, IgG1, IgG2b, IgG3, and IgA concentrations were measured in serum samples collected just prior to the start of dosing (Week 9; “baseline”) and at termination (Week 13) from EAMG mice treated with PBS (EAMG controls), povetacicept, efgartigimod, anti-CD20, or Fc control. Concentrations from individual mice for terminal concentrations (A) and the difference in concentrations (terminal minus baseline) (B) are plotted, with horizontal and vertical bars representing the median and interquartile range, respectively. Statistical analysis was performed using the Kruskal-Wallis test with Dunn’s multiple comparisons test; p values for significant comparisons (p<0.05) are shown. (C) Flow cytometry analysis of T cell and B cell subsets in splenocytes (n=8 mice/group) collected at termination (Week 13). Data for individual mice are plotted, with horizontal and vertical bars representing the mean and SD, respectively. Statistical analysis was performed using one-way ANOVA, corrected for multiple comparisons.