Angiopoietin-like 3-derivative LNA043 for cartilage regeneration in osteoarthritis: a randomized phase 1 trial

Figures

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  Fig. 1. LNA043 induces chondrogenesis and DKK1, PRG4 and collagen type II secretion, but not angiogenesis. a, Left, PRG4 production by 3D hMSCs. PRG4 IHC staining after 28 d of LNA043 treatment. Green, PRG4; blue, DAPI. Comparable effects of LNA043 were detected in hMSCs from four (out of four) donors. Scale bar, 100 μm. Right, PRG4 secretion, as determined by ELISA, generated from one donor in quadruplicate (one representative experiment shown out of eight). b, DKK1 secretion by 3D hMSCs. The accumulation of DKK1 between days 8 and 11, 15 and 18 and 22 and 25 was quantified by ELISA (one representative experiment shown out of three; n = 3 biological replicates). Comparable effects of LNA043 were detected in hMSCs from two (out of four) donors. c, Osteogenic and adipogenic marker expression in 3D hMSCs. ALPL (left) and LEP expression (middle) were determined by quantitative reverse transcription PCR (one representative experiment shown out of six; n = 3 biological replicates). Right, LEP secretion as determined by ELISA (one representative experiment shown out of four; n = 6 biological replicates) following 28 d of LNA043 treatment. Comparable LNA043 effects on ALPL, LEP and LEP levels…

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  Fig. 2. Integrin α5β1 is required for LNA043-induced DKK1 secretion by C-28/I2 chondrocytes and binds LNA043. a, LNA043-induced DKK1 secretion is integrin α5-dependent. Integrins α5 and αv were knocked down in C-28/I2 cells by siRNA (siITGA5 and siITGAV, respectively; siITGA5+V for both combined). Left, DKK1 secretion induced by LNA043 (200 μg ml−1), as analyzed by ELISA. The data represent means ± s.e.m. of 3–8 independent experiments (from left to right, ***P = 0.0003, *P = 0.0391, *P = 0.0178 and ****P ≤ 0.0001). Statistical significance was determined by one-way ANOVA with mixed-effect analysis and Šídák’s multiple comparison test. Middle, efficiency of the knockdown, as monitored by immunoblotting. GAPDH was used as a loading control. Right, quantification of the immune bands, as determined by densitometry. The data are presented as a percentage decrease ± s.e.m. versus control siRNA (siCtrl) and represent six independent experiments (****P ≤ 0.0001). Statistical significance was determined by one-way ANOVA with Tukey’s multiple comparison test. b, LNA043 interacts with α5β1 integrin in vitro, as shown by co-IP experimentation. Recombinant human α5β1 integrin (rhα5β1) or recombinant…

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  Fig. 3. LNA043 treatment regenerates cartilage in rat OA and minipig cartilage injury models. a, Short-term rat OA model. Top left, schematic of the experimental design. i.a., intra-articular. Bottom left, representative images of knee joint sections from rats treated with vehicle or 20 μg LNA043 and stained with Safranin-O (red) or combined collagen type II (green) and X (red) IHC and counterstained with DAPI at 4 weeks post-RMT surgery. Scale bar, 200 μm. Right, cartilage damage, as determined by modified OARSI scoring (Supplementary Table 4) for naive (no surgery; n = 5), vehicle-treated (n = 9) and LNA043-treated rats (for 2 μg, n = 8; for 20 μg, n = 8; for 200 μg, n = 10). The data represent means ± s.d. Each data point represents the results from an individual rat. The percentage cartilage repair in LNA043-treated rats was calculated as the improvement in the score in vehicle-treated (0%) compared with naive rats (100%). Statistical significance was determined by Kruskal–Wallis non-parametric test with Dunn’s multiple comparison (*P = 0.279; **P = 0.0041). b, Therapeutic rat OA model. Top left, schematic of the experimental design. Bottom left, representative images of knee joint…

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  Fig. 4. LNA043 FIH study design and baseline data. a, CLNA043X2101 study design summarizing the study periods and treatment arms. The end of study visit was at day 36 for cohorts 1–4 and 7, at day 29 for cohort 5 and at day 50 for cohort 6. b, CONSORT diagram showing the flow of patients at each stage of the FIH trial. c, Baseline data and demographics. BMI, body mass index.

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  Fig. 5. LNA043 is safe in the FIH study. a, Table summarizing the adverse events (AEs) that occurred at least twice, reported per treatment arm and not showing clear trends in safety. b, Table reporting all of the serious adverse events (SAEs), also not showing safety trends, and with none of them suspected to be related to LNA043. Cohorts 1 and 4–6 are not included as there were no SAEs in these cohorts; however, the final column includes these participants in the total.

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  Fig. 6. LNA043 penetrates damaged cartilage and reverts the OA transcriptional signature in FIH study patients. a, Representative images of LNA043 detection by IHC in TKR cartilage from patients with OA in the FIH trial 2 h post-LNA043 injection (left) or after placebo treatment (right). LNA043 (brown stain) showed deeper penetration into damaged (red bracket) than intact cartilage (green bracket). Cartilage from placebo-injected patients showed no LNA043-specific staining. Unspecific staining was seen in all specimens at the tidemark (arrows). The arrowheads indicate the superficial tear characteristic of damaged cartilage. These images are representative of 21 (LNA043 injection) and 49 (placebo treatment) IHC-stained cartilage samples. Scale bar, 600 μm. b, Transcriptomics study design. Damaged and undamaged articular cartilage samples were profiled from patients with OA injected with placebo (n = 7) or 20 mg LNA043 at 2 h, 7 d or 21 d (n = 3 each) before TKR. c, Left, heatmap showing the expression changes expressed as log2[fold change] (log2[FC]) of the 28 genes statistically significantly regulated by LNA043. OA damage refers to damaged versus undamaged cartilage in the placebo…

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  Extended Data Fig. 1. Viability of C-28/I2 cells upon LNA043 treatment and absence of a proliferative effect of LNA043 during chondrogenic rescue in UE7T-13 cells. a, Viability of C-28/I2 cells upon LNA043 treatment. Absence of cytotoxicity of LNA043 in C-28/I2 cells treated for 24 h in C-28/I2 test medium supplemented with LNA043 was determined by CellTiter-Glo® (Promega). Levels are expressed in relative light units (RLU). Values are mean ± s.d. of measurements from a representative experiment with biological triplicates. b, Absence of a proliferative effect of LNA043 during the chondrogenic rescue in UE7T-13 cells. Proliferation was assessed in UE7T-13 cells cultivated for 3, 7, 11 and 14 d in UE7T-13 chondrogenic medium supplemented or not with 0.5 μg ml-1 Poly I:C (PIC), 100 or 200 μg ml-1 LNA043 by CellTiter-Glo® (Promega). Levels are expressed in relative light units (RLU). Values are mean ± s.d. of measurements from biological triplicates. In a and b statistical significance was determined by one-way ANOVA. No significant differences were observed between any of the conditions.

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  Extended Data Fig. 2. LNA043 partially preserves chondrogenic activity in immortalized hMSC and primary human OA chondrocytes in inflammatory conditions. a-d, UE7T-13 cells cultivated in chondrogenic medium supplemented with Poly I:C (PIC) and/or LNA043 (100 or 200 μg ml-1) as indicated. a, ACAN, SOX9 and COMP gene expression after 3, 7, 11 and 14 d culture. COMP secretion (ELISA) following 14 d treatment; one representative experiment is shown out of three, with n=3 biological replicates. b, Phase-contrast microscopic appearance of representative UE7T-13 cells cultured for 11 d in chondrogenic medium (A) or supplemented with 0.5 μg ml-1 Poly I:C (B) or 0.5 μg ml-1 Poly I:C and 100 μg ml-1 LNA043 (C) or 0.5 μg ml-1 Poly I:C and 200 μg ml-1 LNA043 (D). Areas of cartilage nodule formation are encircled by lines; scale bar = 100 μm. Middle: Alcian blue staining at 10 d in a 96-well plate in chondrogenic medium supplemented as indicated in A, B, C and D; one representative experiment shown out of three. c, IL6 gene and protein expression after 3 (gene only), 7, 11 and 14 d culture; one representative experiment shown out of three, with n=3 biological replicates. d, PRG4 ELISA of supernatant from 3D…

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  Extended Data Fig. 3. Tables summarizing the key pharmacokinetic parameters. a, LNA043 pharmacokinetics, demonstrating fast clearance of LNA043 from circulation, and b, ANGPTL3 pharmacokinetics, not showing any impact of exposure to LNA043.

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  Extended Data Fig. 4. LNA043 cartilage penetration in the FIH study. Data are analyzed using a linear mixed-effect model with repeated measures. The two-sided P-value was obtained by t-statistic. a, LNA043 staining detected only in cohort 5 samples from the CLNA043X2101 study, when LNA043 was injected 2 h before TKR. Individual data points and boxplot of the LNA043 staining score of osteochondral samples from all cohorts are shown. Positive staining was observed in cohort 5 (*P = 0.0002). Boxplot data is expressed as median and lower/upper quartiles and whiskers at 1.5 times the interquartile range. b, LNA043 diffuses deeper into damaged compared to undamaged cartilage. Scatter plot of LNA043 penetration depth in cartilage samples from cohort 5, expressed as % of cartilage thickness, which is positively stained. Measurements were performed by an expert pathologist (KAW) on n=11 samples, with 4 samples harvested from different areas of the knee (osteochondral biopsies from femoral condyle, patella, trochlea, tibia) for each of the 3 patients (1 sample missing, because there was no cartilage left). Dotted lines separate data from individual patients. P = 0.000000475 for LNA043 penetration depth in…

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  Extended Data Fig. 5. Genes significantly regulated by LNA043 treatment and RAAK OA signature counter-regulated by LNA043. a, Log2[FC] (log2FC) of genes significantly regulated at either 2 h, 7 or 21 d after LNA043 treatment in damaged OA cartilage compared to placebo (Limma moderated t-test, Bejamini and Hochberg, P ≤ 0.05). Most-significantly regulated genes are shown in bold; fold upregulation is visualized by red color intensity, fold downregulation by blue color intensity, adjusted P-values (adj. P) ≤ 0.05 are shaded in yellow b, Temporal counter-regulation of RAAK OA signatures by LNA043. Signatures were generated from the RAAK study published by Ramos et al.38. The enrichment significance for each geneset, defined as the negative log10 of the False Discovery Rate (-log10(FDR)), is plotted over time. Signs depict the direction of enrichment (positive for geneset upregulation and negative for geneset downregulation). Heatmap of the expression profiles of individual genes in the RAAK signatures. OA-associated changes in damaged versus undamaged OA cartilage are reverted by LNA043 treatment across all three timepoints in the FIH study. Downregulated genes, besides FN1 and OPN, encode TNFRSF11B/OPG…

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  Extended Data Fig. 6. LNA043 MoA hypothesis. LNA043 binding to the FN1 receptor, integrin α5β1, and intracellular signaling e.g. via FAK and AKT phosphorylation induces transcriptional counter-regulation of OA-relevant gene expression. Specifically, a, up-regulation of essential cartilage matrix components PRG4, COL2A1, ACAN, COMP, MATN4, COL9A1 results in cartilage matrix synthesis; b, regulation of genes DKK1, FRZB, WNT16 inhibits WNT signaling, which induces chondrogenesis and cartilage regeneration; c, regulation of genes CHRDL2, GREM1, BMP6 inhibits BMP signaling, which prevents detrimental chondrocyte hypertrophy and mineralization in OA; d, downregulation of OA progression mediators OPN, DNER, OPG as well as FN1 splice variants and FN1, which in the OA environment is cleaved into cartilage degradation-promoting fragments, reduces cartilage degeneration and progression of OA. Transparent symbols depict transcriptomics results, colored symbols in vitro assay-confirmed data.