Using a recombinant single chain antibody for targeting liver myofibroblasts with anti-fibrogenic therapeutics

Introduction 

Fibrosis is a progressive disease characterised by the accumulation of scarring extracellular matrix proteins which disrupt normal tissue architecture. As fibrosis worsens, tissue function is impaired and in vital organs such as the liver – in the absence of transplantation – morbidity and mortality result [1]. A pragmatic approach to the treatment of chronic disease is the alleviation of fibrosis. Previous work has demonstrated that the liver myofibroblast (derived from hepatic stellate cells or other sources) is primarily responsible for fibrogenesis in the liver and that experimental therapies that stimulate liver myofibroblast apoptosis reverse fibrosis and improve liver function in animal models [2], [3], [4], [5].

However, directing cell death as a mode of treatment for a disease comes with risk because most therapeutics are unlikely to act specifically on the target cells alone. The risk is therefore the stimulation of cell death in cells other than the target cells, with potential adverse effects.

To increase the specificity of action of a potential therapeutic to stimulate liver myofibroblast death, a human recombinant single chain antibody (scAb) was developed (termed C1-3 [6]) to an extracellularly-exposed region of synaptophysin, a plasma membrane protein expressed in the liver specifically in human and rodent myofibroblasts [7]. Synaptophysin is primarily associated with neural tissue and is thought to be involved in synaptic vesicle exo-endocytosis. However, synaptophysin knock out mice show no phenotype or detectable effects on synaptic transmission [8]. A potential advantage of using synaptophysin as a target to deliver therapeutics is that this protein – at least in neural tissue – forms part of an endocytosing vesicle, which may increase chances of scAb and conjugated therapeutic uptake.

Phage display 

Phage display is a powerful technique for selecting and cloning sequences that encode proteins with a native affinity for a particular ligand. In the case of C1-3, a phage library was employed in which potentially the entire human repertoire of variable heavy and variable light chains (which encode antibody antigen binding sites) were cloned as a single fused domain into the gIII M13 bacteriophage coat protein (Fig. 1). The phage “display” different variable domains on their surface, thus generating a phage display library. Variable domains which interact with a particular ligand can be selected by a process of “panning”, whereby phage are exposed to an immobilized target sequence. Unbound phage are removed by washing and bound phage eluted and amplified through re-infection of Escherichia coli (Fig. 1) The process of panning is repeated several times allowing for isolation of specific high-affinity antigen binding phage. Single clones are then isolated and analysed prior to the sub-cloning the scAb-encoding region into an expression vector. Using established procedures such as protein tagging, high levels of pure monoclonal scAb can be generated with relative ease.

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Fig. 1. A, schematic diagram of recombinant M13 bacteriophage incorporating a scAb within its coat protein and the procedure of phage display.

C1-3 scAb 

A range of human recombinant scAbs were generated to synaptophysin by phage display. One of these – C1-3 – was selected and its ability to act as a targeting agent for liver myofibroblasts examined. Fluorescently-labelled C1-3 scAb avidly bound to myofibroblasts (see Fig. 2) but not hepatocytes [6]. The C1-3 was taken up into myofibroblasts by pinocytosis [6]. The C1-3 scAb alone was not toxic to myofibroblasts in vitro but when conjugated with tributyl tin, the scAb directed the toxin to myofibroblasts (with toxin activity retained) [6].

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Fig. 2. Mouse liver myofibroblasts bind C1-3 scAb. Cells were fixed and co-stained for C1-3 using FITC-labelled anti-human Cκ light chain antibody (green); the myofibroblast marker α-smooth muscle actin (red) and DNA using DAPI (blue).

To determine whether the C1-3 scAb could be effective as a drug targeting agent in vivo, mice with liver fibrosis (via carbon tetrachloride administration) were injected i.p. with C1-3 scAb. Initial studies used fluorescently-labelled C1-3 to examine its distribution throughout the body up to 24h after injection. The results of these studies showed that the C1-3 scAb appeared in the serum within 20min and was eliminated with an approximately half-life of 2h [9]. The C1-3 scAb was detectable in liver homogenate but undetectable in brain, muscle or spleen [9]. Immunohistochemical analysis indicated that the C1-3 scAb localised to the liver, in regions where scars were present. There was minimal immunohistochemical evidence for the presence of C1-3 scAb in non-fibrotic liver [9]. Co-staining of fibrotic liver sections showed that the C1-3 scAb co-localised with myofibroblast α-smooth muscle actin, but not the monocyte and macrophage marker F4/80 [9]. These data indicate that the C1-3 scAb readily and selectively targeted liver myofibroblasts in an animal model of liver fibrosis.

To determine whether C1-3 could deliver a functional experimental anti-fibrogenic therapeutic to myofibroblasts, C1-3 protein was chemically conjugated with gliotoxin. Free gliotoxin has been shown in previous work to stimulate the apoptosis of liver myofibroblasts in vitro [10] and in vivo [11] and to enhance recovery from liver fibrosis in vivo [2], [11], [5]. However, gliotoxin also causes – although to a lesser degree – the apoptosis of hepatocytes and stimulates Kupffer cell death [4], [12].

Gliotoxin was chemically conjugated to either a C1-3 scAb or a control CSBD9 scAb (selected for its ability to bind to an irrelevant ligand) using N-(p-maleimidophenyl) isocyanate and S-acetyl thioglycolic acid N-hydroxysuccinimide [9]. Conjugation did not significantly alter scAb affinities for their respective antigens (as determined using antigen ELISA and BIAcore [9]) or ablate gliotoxin’s ability to cause liver myofibroblasts apoptosis (see Fig. 3).

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Fig. 3. Time course for the effects of free gliotoxin or C1-3-GT on sub-stratum adherence in vitro. Mouse myofibroblasts (culture-activated hepatic stellate cells) were sub-cultured into 24 well plates in 300μl medium and treated with either free gliotoxin added from a 1000-fold molar concentrated stock in DMSO vehicle (total 2.25nmol/well); 22.5μg C1-3/well or 22.5μg C1-3-GT scAb (to give approximately 450pmol gliotoxin/well for conjugated scAb). Data are the mean of three separate wells, typical of at least four separate experiments.

Mice were administered carbon tetrachloride twice weekly for eight weeks to generate liver fibrosis. During the last week prior to the final injection of carbon tetrachloride, mice were treated with a single dose of C1-3 scAb or C1-3-gliotoxin scAb. The mice were therefore treated with a potential therapeutic and injury was sustained by further treatment with carbon tetrachloride. Fibrotic mice were also treated with an equivalent dose of free gliotoxin or gliotoxin conjugated to CSBD9 (i.e., CSBD9-GT).

The results are summarised in Table 1 and show that free gliotoxin reduced the number of myofibroblasts but did not reduce fibrosis severity in the liver. C1-3-gliotoxin more potently reduced the number of myofibroblasts present compared to the same dose of free gliotoxin and additionally significantly reduced the severity of fibrosis in the liver. C1-3 scAb, CSBD9 scAb and CSBD9-gliotoxin had no effect on the number of liver myofibroblasts or fibrosis severity. Interestingly, free gliotoxin reduced the number of F4/80 positive cells (i.e., monocytes and macrophages/Kupffer cells) and MMP-13 levels in the liver whereas C1-3-gliotoxin did not. Macrophage depletion abrogates recovery from liver fibrosis [13]. Retaining macrophages in C1-3-gliotoxin treated animals may account for the improved anti-fibrogenic effect of gliotoxin when targeted to liver myofibroblasts with C1-3.

Table 1. The effects of gliotoxin and C1-3-gliotoxin on parameters of liver fibrosis in a sustained carbon tetrachloride model of liver fibrosis.

	Treatment	Number myofibroblasts	Number F4/80 cells	Fibrosis severity	MMP-13 levels
	DMSO vehicle	+++++	+++	++++	++++
	Free gliotoxin	+++a	++a	++++	+++a
	PBS	+++++	+++	++++	++++
	C1-3
	C1-3-gliotoxin	+b	+++	++b	++++
	CSBD9	+++++	+++	++++	++++
	CSBD9-gliotoxin	+++++	+++	++++	++++

Note: gliotoxin was administered in DMSO at a dose of 0.6mg/kg body weight. scAbs were administered in phosphate buffered saline (PBS) at a dose of 20mg protein/kg body weight. Mice received conjugates at an equivalent dose of 0.6mg gliotoxin/kg body.

a Significantly different (two tailed) from DMSO control using Student’s T-test (P>95%). b Significantly different (two tailed) from PBS control using Student’s T-test (P>95%).

Discussion 

Two major hurdles must be overcome to advance successful treatments for fibrosis. The first is the effective assessment of fibrosis in both experimental and clinical studies. It remains problematic to accurately screen for fibrosis using a battery of serum markers, particularly for those at early and intermediate stages of disease [14]. Biopsy remains the “gold standard” diagnostic tool for liver disease [15] but has significant drawbacks (the procedure is risky; repeated biopsy is inadvisable; and it does not give a globally accurate picture of organ fibrosis). The second hurdle is specificity and efficacy of potential treatments (in the liver – the myofibroblasts lie adjacent to the major drug metabolising cell of the body – the hepatocyte).

This laboratory has examined the potential of an antibody-based approach to tackle both imaging and therapeutic delivery in fibrosis. Although the C1-3 scAb does not directly bind to fibrosis proteins (it binds to the major fibrogenic cell), an image-visible scAb derivative may be effective in providing an indication of fibrosis severity. If successful as an imaging agent, experimental and clinical imaging studies should aid in its development as a targeting agent for anti-fibrogenic therapeutics.

The utility, or therapeutic efficacy of whole antibody, or related antibody structures, in vivo is influenced by a number of competing factors. These include: specificity, affinity (including avidity) and serum half-life. The C1-3 has already been expressed successfully as a monomeric scAb (38kDA). In addition, it could also be used in a slightly smaller but related scFv formulation (25kDa). C1-3 could also be expressed as a dimeric fragment (60kDa) which will typically show one order of magnitude improvement in affinity for antigen through increased avidity. Finally, the C1-3 could be expressed as a full mAb incorporating either a mouse or human Fc region. This construct will have a similar affinity to the C1-3 dimer but an increased serum half-life (150kDa) and the capacity to initiate cell killing upon binding in vivo.

Typically, for imaging agents, a shorter serum half-life is favoured, to maximise signal to noise ratios in the diseased tissue. For therapeutic applications, a whole antibody is often preferred where antibody binding and presentation of the Fc region can recruit components of the mammalian immune system and induce cell killing, etc. However, for certain applications a smaller antibody fragment conjugated to a cell killing agent, may be a more potent therapeutic formulation. This type of agent can provide a powerful alternative to Fc induced cell death, offering greater tissue penetration and rapid clearance of toxic agent from non-target tissues.

The administration of a recombinant antibody specific for the surface of liver myofibroblasts could be used to develop a safer, less invasive and more effective diagnostic for the assessment of fibrosis throughout the entire organ. In the short term, the diagnostic could lead to more refined experimental animal studies; provide for a more effective assessment of potential anti-fibrogenics; and reduce the number of animals required in pre-clinical studies. Experimental animal studies will also contribute to the translation of the diagnostic to the clinic in the long term, as part of in vivo imaging (diagnostic and diseases management). In addition the C1-3 antibody could be used to direct anti-fibrogenics to the myofibroblasts (or analagous pro-fibrogenic cells in other tissues). Directing therapeutics specifically to the target myofibroblast – and away from other cells – will enhance the efficacy of an anti-fibrogenic since it will reduce the amount of therapeutic required and the likelihood of adverse effects.

There are currently no treatments indicated for use as anti-fibrogenics in patients [16]. The major causes of liver fibrosis in the developed world are non-alcoholic steatohepatitis (NASH), hepatitis C infection and alcoholism. NASH has an incidence of at least 2% in the EU and US and the incidence of hepatitis C infection is between 1% and 2% [17], [18]. The incidence of both of these diseases is predicted to rise in the future and further reinforces the urgent need to diagnose, manage and treat this “silent killer”.