Canine IL4-10 fusion protein provides disease modifying activity in a canine model of OA; an exploratory study

Abstract

Objective: An ideal disease modifying osteoarthritis drug (DMOAD) has chondroprotective, anti-inflammatory, and analgesic effects. This study describes the production and characterization of a canine IL4-10 fusion protein (IL4-10 FP) and evaluates its in vivo DMOAD activity in a canine model of osteoarthritis (OA).

Design: The canine Groove model was used as an in vivo model of degenerative knee OA. Six weeks after OA induction dogs were intra-articularly injected weekly, for ten weeks, with either IL4-10 FP or phosphate buffered saline (PBS). In addition to the use of human IL4-10 FP, canine IL4-10 FP was developed and characterized in vitro, and tested in vivo. Force plate analysis (FPA) was performed to analyze joint loading as a proxy measure for pain. After ten weeks dogs were euthanized and cartilage and synovial tissue samples were analyzed by histochemistry (OARSI scores) and biochemistry (cartilage proteoglycan turnover).

Results: Repetitive intra-articular injections with human IL4-10 FP led to antibody formation, that blocked its functional activity. Therefore, a canine IL4-10 FP was developed, which completely inhibited LPS-induced TNFα production by canine blood cells, and increased proteoglycan synthesis of canine cartilage in vitro (p = 0.043). In vivo, canine IL4-10 FP restored the, by OA impaired, joint loading (p = 0.002) and increased cartilage proteoglycan content (p = 0.029).

Conclusions: This first study on the potential DMOAD activity upon prolonged repeated treatment with IL4-10 FP demonstrates that a species-specific variant has anti-inflammatory and chondroprotective effects in vitro and chondroprotective and analgesic effects in vivo. These data warrant further research on the DMOAD potential of the IL4-10 FP.

Fig 1. Experimental set-up of the canine in vivo experiment.

The first part of the canine in vivo experiment was performed treating four dogs with i.a. human IL4-10 FP and four dogs with i.a. PBS as a control group. Because of IL4-10 FP neutralizing antibody formation the second part was performed treating four dogs with i.a. canine IL4-10 FP and four dogs with i.a. PBS.

Fig 2. Immune response against hIL4-10 FP.

A. Macroscopic pictures of synovial fatpad of control, PBS injected and hIL4-10 FP injected knees. B. IgG titers in serum of PBS injected dogs (dotted line) and hIL4-10FP injected dogs (continuous line). C. TNFα inhibition of hIL4-10 FP after adding serum taken before i.a. injections (above) and after i.a. injections with hIL4-10 FP (below). TNFα inhibition is expressed as percentage of inhibition in serum of non-injected dogs.

Fig 3. Molecular characterization of cIL4-10 FP.

A. Schematic overview of the cIL4-10 FP. B. Coomassie Brilliant Blue stained SDS gel in Ni-NTA protein purification steps; M: Marker, L: Load, FT: Flow-through, W: Wash, E: Elution. C. Western blot analysis of purified cIL4-10 FP (untreated and deglycosylated; DG). D. HP-SEC profile showing; A: Aggregate, D: Dimer, M: Monomer.

Fig 4. In vitro activity of cIL4-10 FP.

A. Anti-inflammatory activity of cIL4-10 FP. Whole canine blood was stimulated with LPS with or without IL4-10 FP, IL10, or IL4 for 18 hours. Canine TNFα was measured using ELISA. B. Chondroprotective activity of cIL4-10 FP. Healthy canine cartilage explants were cultured in TNFα containing medium with or without cIL4-10 FP for four days. Proteoglycan synthesis rate was measured using liquid scintillation of ³⁵SO₄^2—labelled GAGs. Values are expressed per dog and are the mean of eight tissue samples per dog. Mean proteoglycan synthesis rate was 2.0 and 4.0 for controls and cIL4-10 FP treated conditions, respectively.

Fig 5. Immunogenicity of cIL4-10 FP in the canine Groove model of OA after injections with PBS or cIL4-10 FP.

A. Macroscopic pictures of synovial fat pad of control, PBS injected and cIL4-10 FP injected knees. B. Differences in macroscopic synovial inflammation (OARSI score) between experimental and contralateral joint, representing a change from baseline to end of the study for the experimental joint. Values are expressed per dog (dots) and as median ± IQR (bars). Mean macroscopic OARSI scores in contralateral joints were 0.5 (95%CI: -0.2–1.2) and 0.4 (95%CI: -0.4–1.1) in PBS group and cIL4-10 FP group, respectively. Mean macroscopic OARSI scores in experimental joints were 2.8 (95%CI: 0.9–4.6) and 2.0 (95%CI: 1.4–2.7) in PBS group and cIL4-10 FP group, respectively. C. IgG titers in serum of cIL4-10 FP injected dogs (continuous line) and, as a positive control, a hIL4-10FP injected dog (dotted line).

Fig 6. Histologic synovial inflammation in the canine Groove model of OA after injections with PBS or cIL4-10 FP.

A. Representative images of Haematoxylin-eosin stained synovial tissue sections of control (without Groove surgery), PBS injected and cIL4-10 FP injected knees. Samples were collected at the end of the experiment; 16 weeks after Groove surgery and after ten weekly i.a. injections. B. Differences in histologic synovial inflammation (OARSI score) between experimental and contralateral joint, representing a change from baseline to end of the study for the experimental joint. Values are expressed per dog (dots) and as median ± IQR (bars). Mean histologic OARSI scores in contralateral joints were 3.0 (95%CI: 1.7–4.3) and 1.3 (95%CI: -0.1–2.6) in PBS group and cIL4-10 FP group, respectively. Mean histologic OARSI scores in experimental joints were 1.8 (95%CI: -0.8–4.5) and 1.5 (95%CI: -0.8–3.8) in PBS group and cIL4-10 FP group, respectively.

Fig 7. Analgesic effects of cIL4-10 FP in the canine Groove model of OA.

Six weeks after Groove surgery dogs received weekly i.a. injections with cIL4-10 FP or PBS for ten weeks. Objective force plate analysis was used to determine joint loading as a proxy for pain. After OA induction, Fz RH/LH was clearly reduced. After treatment the Fz RH/LH statistically significant increased in the cIL4-10 FP treated group (continuous line, n = 4) compared to the PBS injected group (dotted line, n = 4); p = 0.002 over the whole time span. Values are expressed as a mean of four dogs ± SEM.

Fig 8. Cartilage damage in the canine Groove model of OA after injections with PBS or cIL4-10 FP.

A. Representative images of Safranin-O stained tibial cartilage section of control (without Groove surgery), PBS injected and cIL4-10 FP injected knees. Samples were collected at the end of the experiment; 16 weeks after Groove surgery and after ten weekly i.a. injections. B. Differences in tibial cartilage structure (part of the OARSI score) between experimental and contralateral joint, representing a change from baseline to end of the study for the experimental joint. Values are expressed per dog (dots) and as median ± IQR (bars). Mean scores in contralateral joints were 1.3 (95%CI: -0.4–2.5) and 1.4 (95%CI: 0.0–2.8) in PBS group and cIL4-10 FP group, respectively. Mean histologic OARSI scores in experimental joints were 3.8 (95%CI: 3.0–4.5) and 2.1 (95%CI: 0.4–3.8) in PBS group and cIL4-10 FP group, respectively. C. Differences in proteoglycan content in tibia of injected knees. Values are expressed per dog (dots) and as median ± IQR (bars). Mean proteoglycan content in contralateral joints were 37.8 (95%CI: 20.4–55.2) and 30.7 (95%CI: 27.3–34.1) in PBS group and cIL4-10 FP group, respectively. Mean proteoglycan content in experimental joint were 27.8 (95%CI: 24.6–31.0) and 34.1 (95%CI: 28.3–39.9) in PBS group and cIL4-10 FP group, respectively.