Identification of IgG1 isotype phosphorylcholine antibodies for the treatment of inflammatory cardiovascular diseases.

BACKGROUND: Phosphorylcholine (PC) is an important pro-inflammatory damage-associated molecular pattern. Previous data have shown that natural IgM anti-PC protects against cardiovascular disease. We aimed to develop a monoclonal PC IgG antibody with anti-inflammatory and anti-atherosclerotic properties. METHODS: Using various techniques PC antibodies were validated and optimized. In vivo testing was performed in a femoral artery cuff model in ApoE3*Leiden mice. Safety studies are performed in rats and cynomolgus monkeys. RESULTS: A chimeric anti-PC (PC-mAb(T15), consisting of a human IgG1 Fc and a mouse T15/E06 Fab) was produced, and this was shown to bind specifically to epitopes in human atherosclerotic tissues. The cuff model results in rapid induction of inflammatory genes and altered expression of genes associated with ER stress and choline metabolism in the lesions. Treatment with PC-mAb(T15) reduced accelerated atherosclerosis via reduced expression of endoplasmic reticulum stress markers and CCL2 production. Recombinant anti-PC Fab fragments were identified by phage display and cloned into fully human IgG1 backbones creating a human monoclonal IgG1 anti-PC (PC-mAbs) that specifically bind PC, apoptotic cells and oxLDL. Based on preventing macrophage oxLDL uptake and CCL2 production, four monoclonal PC-mAbs were selected, which to various extent reduced vascular inflammation and lesion development. Additional optimization and validation of two PC-mAb antibodies resulted in selection of PC-mAb X19-A05, which inhibited accelerated atherosclerosis. Clinical grade production of this antibody (ATH3G10) significantly attenuated vascular inflammation and accelerated atherosclerosis and was tolerated in safety studies in rats and cynomolgus monkeys. CONCLUSIONS: Chimeric anti-PCs can prevent accelerated atherosclerosis by inhibiting vascular inflammation directly and through reduced macrophage oxLDL uptake resulting in decreased lesions. PC-mAb represents a novel strategy for cardiovascular disease prevention.

C57BL/6 NK cell gene complex is crucially involved in vascular remodeling.

OBJECTIVE: NK cells are known to be involved in cardiovascular disease processes. One of these processes, vascular remodeling, may strongly differ between individuals and mouse strains such as the C57BL/6 and BALB/c. Moreover, C57BL/6 and BALB/c mice vary in immune responses and in the composition of their Natural Killer gene Complex (NKC). Here we study the role of NK cells, and in particular the C57BL/6 NKC in vascular remodeling and intimal hyperplasia formation. METHODS AND RESULTS: C57BL/6, BALB/c and CMV1(r) mice, a BALB/c strain congenic for the C57BL/6 NKC, were used in an injury induced cuff model and a vein graft model. NK cell depleted C57BL/6 mice demonstrated a 43% reduction in intimal hyperplasia after femoral artery cuff placement compared to control C57BL/6 mice (p<0.05). Cuff placement and vein grafting resulted in profound intimal hyperplasia in C57BL/6 mice, but also in CMV1(r) mice, whereas this was significantly less in BALB/c mice. Significant more leukocyte infiltrations and IFN-γ staining were seen in both C57BL/6 and CMV1(r) vein grafts compared to BALB/c vein grafts. CONCLUSIONS: These data demonstrate an important role for NK cells in intimal hyperplasia and vascular remodeling. Furthermore, the C57BL/6 NKC in CMV1(r) mice stimulates vascular remodeling most likely through the activation of (IFN-γ-secreting) NK-cells that modulate the outcome of vascular remodeling.

T-cell co-stimulation by CD28-CD80/86 and its negative regulator CTLA-4 strongly influence accelerated atherosclerosis development.

OBJECTIVE: T-cells are central to the immune response responsible for native atherosclerosis. The objective of this study is to investigate T-cell contribution to post-interventional accelerated atherosclerosis development, as well as the role of the CD28-CD80/86 co-stimulatory and Cytotoxic T-Lymphocyte Antigen (CTLA)-4 co-inhibitory pathways controlling T-cell activation status in this process. METHODS AND RESULTS: The role of T-cells and the CD28-CD80/86 co-stimulatory and CTLA-4 co-inhibitory pathways were investigated in a femoral artery cuff mouse model for post-interventional remodeling, with notable intravascular CTLA-4+ T-cell infiltration. Reduced intimal lesions developed in CD4(-/-) and CD80(-/-)CD86(-/-) mice compared to normal C57Bl/6J controls. Systemic abatacept-treatment, a soluble CTLA-4Ig fusion protein that prevents CD28-CD80/86 co-stimulatory T-cell activation, prevented intimal thickening by 58.5% (p=0.029). Next, hypercholesterolemic ApoE3*Leiden mice received abatacept-treatment which reduced accelerated atherosclerosis development by 78.1% (p=0.040) and prevented CD4 T-cell activation, indicated by reduced splenic fractions of activated KLRG1+, PD1+, CD69+ and CTLA-4+ T-cells. This correlated with reduced plasma interferon-γ and elevated interleukin-10 levels. The role of CTLA-4 was confirmed using CTLA-4 blocking antibodies, which strongly increased vascular lesion size by 66.7% (p=0.008), compared to isotype-treated controls. CONCLUSIONS: T-cell CD28-CD80/86 co-stimulation is vital for post-interventional accelerated atherosclerosis development and is regulated by CTLA-4 co-inhibition, indicating promising clinical potential for prevention of post-interventional remodeling by abatacept.