A single dose of antibody-drug conjugate cures a stage 1 model of African trypanosomiasis

Abstract

22Infections of humans and livestock with African trypanosomes are treated with drugs 23 introduced decades ago that are not always fully effective and often have severe 24 side effects. Here, the trypanosome haptoglobin-haemoglobin receptor (HpHbR) has 25 been exploited as a route of uptake for an antibody-drug conjugate (ADC) that is 26 completely effective against Trypanosoma brucei in the standard mouse model of 27 infection. Recombinant human anti-HpHbR monoclonal antibodies were isolated and 28 shown to be internalised in a receptor-dependent manner. Antibodies were 29 conjugated to a pyrrolobenzodiazepine (PBD) toxin and killed T. brucei in vitro at 30 picomolar concentrations. A single therapeutic dose (0.25 mg/kg) of a HpHbR 31 antibody-PBD conjugate completely cured a T. brucei mouse infection within 2 days 32 with no re-emergence of infection over a subsequent time course of 77 days. These 33 experiments provide a demonstration of how ADCs can be exploited to treat 34 protozoal diseases that desperately require new therapeutics. 35 36 Author Summary 37Here we show that antibody-drug conjugates (ADCs) can be re-purposed from 38 cancer immunotherapeutics to anti-protozoals by changing the specificity of the 39 immunoglobulin to target a trypanosome cell surface receptor. Trypanosomes were 40 used as a model system due to the availability of receptor null cell lines that allowed 41 the unambiguous demonstration that ADCs targeted to a parasite surface receptor 42 could be specifically internalised via receptor-mediated endocytosis. A single low 43 dose of the resulting ADC was able to cure a stage 1 mouse model of trypanosome 44 infection. We have used toxins and conjugation chemistry that are identical to anti-45 3 cancer ADCs demonstrating the ability to piggy-back onto the huge research efforts 46 and resources that are being invested in the development of such ADCs. 47The potential for development of ADCs against a wide range of human pathogens is 48 vast, where only epitope binding sites need vary in order to provide selectivity. This 49 provides a far-reaching opportunity for the rapid development of novel anti-50 protozoals for the targeted killing of a wide range of pathogens that cause disease 51 worldwide, especially in developing countries.

A receptor for the complement regulator factor H increases transmission of trypanosomes to tsetse flies

“…Live-cell-binding assays with FH and moAb. PCFs were incubated in culture with 100 nM of fluorescently labelled FH purified from bovine serum (see above) or moAb raised against the FHR by phage display (AstraZeneca) 68,69 . In brief, phage display selection strategy involved panning selections with biotinylated FHR on streptavidin-coated plates or with unlabelled FHR on MaxiSorp plates, and soluble selections with biotinylated FHR on magnetic streptavidin-coated Dynabeads.…”

“…When a GST-tagged protein was used, a GST deselection step was performed as a control. In all selections, 1 × 10 12 phage particles were incubated with immobilized FHR, followed by extensive wash steps (PBS or with 0.1% Tween-20), de-selections (e.g., removal of non-antigen-binding phage-scFv by pre-incubation with beads) and elution of bound phage with trypsin 69 .…”

“…Nine purified anti-FHR scFv were selected, which bound unique epitopes and converted into full-length human IgG1 using standard techniques. Secreted antibodies were purified using protein A affinity chromatography and labelled using an AlexaFluor 594 NHS ester labelling kit (ThermoFisher) 69 . After 2 h of incubation, live cells were fixed in 1% formaldehyde for 10 min.…”

How can monoclonal antibodies be harnessed against neglected tropical diseases and other infectious diseases?

“…However, in the most recent study on antibody-based therapeutics against trypanosomiasis, researchers circumvented this challenge. Here, the development was reported of an antibody-drug conjugate (ADC) targeting the trypanosome haptoglobin-haemoglobin receptor, which was shown to be efficacious against Trypanosoma brucei in a murine infection model [9] (Figure 3). In comparison, the malaria parasite avoids the human immune system by infecting red blood cells and hiding intracellularly.…”

“…To a large extent, this trend is driven by financial incentives and the reality that enterprises operating in the pharmaceutical industry typically must deliver a monetary return on investment to their shareholders. With the traditional business and reimbursement models for pharmaceuticals, achieving financial success may be challenging for antibody-based therapies against many neglected tropical diseases and other infectious diseases, despite the existence of a huge unmet humanitarian need for better therapies in areas such as infectious and parasitic diseases [1,[7][8][9][10][11], envenomings by animal bites and stings [12][13][14], and mushroom poisonings [15]. In these areas, existing therapeutic options are either suboptimal, or resistance towards therapy is developing.…”