The Pentraxins 1975-2018: Serendipity, Diagnostics and Drugs

Abstract

The phylogenetically ancient, pentraxin family of plasma proteins, comprises C-reactive protein (CRP) and serum amyloid P component (SAP) in humans and the homologous proteins in other species. They are composed of five, identical, non-covalently associated protomers arranged with cyclic pentameric symmetry in a disc-like configuration. Each protomer has a calcium dependent site that mediates the particular specific ligand binding responsible for all the rigorously established functional properties of these proteins. No genetic deficiency of either human CRP or SAP has been reported, nor even any sequence polymorphism in the proteins themselves. Although their actual functions in humans are therefore unknown, gene deletion studies in mice demonstrate that both proteins can contribute to innate immunity. CRP is the classical human acute phase protein, routinely measured in clinical practice worldwide to monitor disease activity. Human SAP, which is not an acute phase protein, is a universal constituent of all human amyloid deposits as a result of its avid specific binding to amyloid fibrils of all types. SAP thereby contributes to amyloid formation and persistence in vivo. Whole body radiolabelled SAP scintigraphy safely and non-invasively localizes and quantifies systemic amyloid deposits, and has transformed understanding of the natural history of amyloidosis and its response to treatment. Human SAP is also a therapeutic target, both in amyloidosis and Alzheimer's disease. Our drug, miridesap, depletes SAP from the blood and the brain and is currently being tested in the DESPIAD clinical trial in Alzheimer's disease. Meanwhile, the obligate therapeutic partnership of miridesap, to deplete circulating SAP, and dezamizumab, a humanized monoclonal anti-SAP antibody that targets residual SAP in amyloid deposits, produces unprecedented removal of amyloid from the tissues and improves organ function. Human CRP binds to dead and damaged cells in vivo and activates complement and this can exacerbate pre-existing tissue damage. The adverse effects of CRP are completely abrogated by compounds that block its binding to autologous ligands and we are developing CRP inhibitor drugs. The present personal and critical perspective on the pentraxins reports, for the first time, the key role of serendipity in our work since 1975. (345 words).

Keywords: C-reactive protein; amyloidosis; complement; dezamizumab; drugs; miridesap; pentraxin; serum amyloid P component.

Figure 1

Molecular appearance of the pentraxins. (A) Negatively stained electron microscopic image of human CRP with the characteristic symmetrical pentameric ring viewed face on. Inset shows disc-like appearance of single molecules side on. (B) Negatively stained electron microscopic image of human SAP with the characteristic symmetrical pentameric ring viewed face on. Inset shows typical face to face double pentamer forming the decameric assembly present in calcium free conditions. This was thought to be the normal assembly of human SAP until the actual physiological native pentameric structure was demonstrated (15). (C) Cryoelectron microscope image of our preparations of human CRP (mass 115,135) and of (D) human SAP (mass 127,310) showing the actual native pentraxin structure with no staining or artefactual enhancement (courtesy of Dr Richard Henderson).

Figure 2

Structure of human SAP. (A) Electron micrograph of negatively stained human SAP molecules. (B) Ribbon diagram of the 3D X-ray crystal structure of human SAP face on (“B” face uppermost). (C) Ribbon diagram of the 3D X-ray crystal structure of human SAP side on. Calcium atoms are represented as yellow spheres located on the binding, “B” face; the single small α-helix of each protomer is shown in red, located on the “A” face (29); β-sheets are in pale blue and loops in dark blue.

Figure 3

Structure of human CRP with bound phosphocholine and bis(phosphocholine)-hexane. (A) Space filling model of “B” face of human CRP with phosphocholine bound in each of the protomer binding sites. (B) 3D X-ray crystal structure of phosphocholine in the binding pocket of a single CRP protomer within the native molecule, showing the ligand interactions with calcium and the CRP residues responsible for binding. (C) The structure of bis(phosphocholine)-hexane (above) and the structure of the complex formed by two CRP molecules cross linked by five bis(phosphocholine)-hexane molecules; face on (left) and side on (right) [From reference (32) with permission of Macmillan Publishers Ltd].

Figure 4

Whole-body scintigraphy with ¹²³I-labeled serum amyloid P component in systemic amyloidosis. (A) (a) Left: Anterior view of a typical patient with AL amyloidosis showing massive liver and spleen amyloid and the pathognomonic deposits throughout the bone marrow that are not seen in any other type of amyloidosis. Right: Posterior view of a typical patient with AA amyloidosis showing amyloid in the spleen, kidneys, and adrenal. The left adrenal is obscured by the overlying spleen, but the right is clearly visible above the kidney. (b) Posterior scans taken a year apart in a patient with longstanding rheumatoid arthritis who suddenly developed AA amyloidosis. The earlier scan (left) is normal; the later one (right) shows heavy splenic and significant renal amyloidosis. (B) (a) Anterior (left) and posterior (right) views of a patient with AL amyloid who presented with minor proteinuria and no other clinical or investigational evidence of disease. There is substantial renal amyloid but no scintigraphically detectable de- posits elsewhere. (b) Anterior (left) and posterior (right) views of a different patient with AL amyloid who also presented with minor proteinuria and no other clinical or investigational evidence of disease. There is massive amyloid deposition in the liver and spleen. The kidneys are not visualized, probably because the tracer, which distributes according to the amount of amyloid, is all taken up elsewhere. Note that, in contrast to (a), there is no residual tracer in the circulation, indicating a heavy whole-body amyloid load. This patient did not tolerate intensive chemotherapy and developed liver failure. (C) (a) Anterior (left) and posterior (right) views of a patient with AL amyloid who presented with multiple fractures over 4 years. X-ray and bone scan were normal but bone biopsy unexpectedly revealed amyloid. No monoclonal gammopathy was identifiable at that time, but bone amyloid is frequent in AL and may be the main clinical feature. (b) Serial anterior views showing regression of AA amyloidosis in a juvenile rheumatoid arthritis patient treated with chlorambucil, in whom the SAA concentration was suppressed to <10 mg/l. (c) Serial anterior views showing regression of AL amyloidosis in a patient treated with high-dose melphalan and stem cell rescue. [From Pepys (100) with permission of Annual Reviews].

Figure 5

Structure of CPHPC (miridesap) and its complex with human SAP. The palindromic bivalent structure of (R)-1-[6-[(R)-2-carboxy-pyrrolidin-1-yl] -6-oxo-hexanoyl]pyrrolidine-2-carboxylic acid (CPHPC), now known by its WHO INN, miridesap, is shown above. Below is the 3D X-ray crystal structure of the SAP-drug complex which is also the structure of the complex in solution (112). [From Pepys et al. (72) with permission of Macmillan Publishers Ltd].

Figure 6

Depletion of circulating SAP by CPHPC (miridesap) in patients with systemic amyloidosis. (A) Serum concentration of SAP immediately before and 6 weeks after starting daily treatment with CPHPC. (B) Sustained depletion of SAP throughout CPHPC treatment. Each line shows the results of serial measurements in an individual patient. Note different scale for SAP concentration compared to (A). From Gillmore et al. (73) with permission of Blackwell Publishing Ltd). In patients without systemic amyloidosis and the associated massive extracellular load of SAP (98), CPHPC (miridesap) treatment reduces plasma SAP concentration to much lower values, for example, mean (SD) 0.25 (0.16) mg/l, in our 5 patients with Alzheimer's disease (112).

Figure 7

Amyloid clearance mediated by macrophage derived multinucleated giant cells after depletion of circulating SAP followed by treatment with anti-SAP antibody. Thin sections of liver stained with toluidine blue from AA amyloidotic human SAP transgenic mice treated with CPHPC (miridesap) to deplete circulating SAP followed by anti-SAP antibody to target residual SAP in the amyloid deposits. Control mouse, not treated with anti-SAP antibody, show abundant amorphous pink-stained amyloid deposits, with the characteristic absence of any surrounding inflammatory reaction or cellular infiltrate. One day after anti-SAP antibody treatment there is intense, predominantly mononuclear cell infiltration in and around the amyloid. Five days after anti-SAP-antibody treatment there is fusion of macrophages to form multinucleated giant cells surrounding and infiltrating the deposits and containing large masses of ingested amyloid undergoing degradation. At 16 days there is complete elimination of amyloid deposits with no residual cellular infiltrate and restoration of normal tissue architecture. [From Bodin et al. (114) with permission of Macmillan Publishers Ltd].

Figure 8

Whole body scintigraphy with ¹²³I-labeled serum amyloid P component in a patient with systemic amyloidosis before and after depletion of circulating SAP followed by treatment with anti-SAP antibody. (A) Scan immediately before treatment. (B) Scan 42 days after single dose of dezamizumab (fully humanized monoclonal anti-human SAP antibody) infused following depletion of circulating SAP with miridesap. The heavy load of amyloid in the liver has been dramatically reduced. [From Richards et al. (115) with permission of Massachusetts Medical Society].