Investigating the Therapeutic Potential of Anti-Human PDGFR-beta mAb In the Treatment of Rheumatoid Arthritis (RA)

During my visit, we will be investigating the therapeutic efficacy of anti-human PDGFR-beta in inhibiting PDGF-BB-driven synovitis and follicular dendritic cell (FDC) differentiation in Rheumatoid Arthritis (RA).

In this study, I will contribute to the immunohistochemistry (IHC) part where quantitative morphometry using markers for the endothelium; FDC perivascular stromal precursors and FDCs, as well as cellular distribution and synovial pathotypes under the effect of different treatments will be identified.

For this study, we hypothesize that PDGFR-beta blockade in RA could simultaneously interfere with development and progression of the FDC-dependent lymphoid and the fibroblast-dominated fibroid synovial pathotypes.

Several lines of evidence, support the role of PDGFR-beta in the pathogenic mechanisms of RA and other inflammatory conditions associated with fibrosis.

For example: * PDGFR-beta displays enhanced expression and activation in the synovium of RA patients, * PDGFR-beta blockade ameliorates disease manifestations in refractory cases of RA, prevents and treats arthritis induced by type II collagen Ab in mice, prevents and reduces synovitis, pannus, and erosion in CIA and inhibits liver and pulmonary fibrosis in different pre-clinical animal models.

Our preliminary studies indicate that RA synovial PDGFR-beta/PDGF-BB: •Directly induce the fibroblastic markers on synovial stromal cells; and strongly correlate with fibroblast-dominated (fibroid) RA synovial pathotype, •Directly induce TNF-alpha-R expression in RA synovial fibroblasts and positively correlate with TNF-alpha-R and LT-beta-R expression in the RA synovium.

Subsequent engagement of TNF-alpha-R and LT-beta-R with TNF-alpha and LT-beta promotes ectopic lymphoid neogenesis and inhibits fibroid synovitis, and •Induces FDC differentiation from synovial NG2+/alphaSMA+ type 1 pericytes.

Experimental Approach in Brief: •We will use a Recombinant Human PDGF R beta Fc Chimera Protein as a decoy PDGFR-beta (R and D 385-PR). • The ED50 of this decoy receptor in inhibiting the biological activity of PDGF-BB using NR6R-3T3 mouse fibroblast cells is 1-3 μg/mL in the presence of 50 ng/mL Recombinant Human PDGF-BB. • RA Synovial organ cultures will be set up in 300 ul RPMI, and treated with the following and compared with PBS injected controls; i.

PDGF-BB alone ii. TNF-alpha+ LT-beta alone iii. PDGF-BB followed by TNF-alpha+ LT-beta iv. Soluble human PDGFR-B followed by PDGF-BB v. Soluble human PDGFR-B followed by TNF-alpha+ LT-beta vi.

Soluble human PDGFR-B followed by PDGF-BB then TNF-alpha+ LT-beta . •Biopsies will be recovered after culture; each biopsy will be divided into 2 halves, •One half will be embedded in OCT for IHC •One half will be stored in RNA later for RNA extraction •qPCR will assess the expression of the genes encoding the proteins assessed by IHC.

Expected outcomes of the study: a.

PDGF-BB alone would induce early CNA42+ FDC differentiation, stromal cell proliferation and activation, but not mature FDC reticular formation. b. TNF-alpha/LT-beta alone would activate the endothelium but not mature FDC+ follicular structures. c. PDGF-BB would prime the stroma for the effect of TNF-alpha/LT-beta which would induce mature FDC+ differentiation d.

Pre-treatment with the soluble decoy PDGFR-B would inhibit the priming effect in a and c.