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

Abstract

Infections of humans and livestock with African trypanosomes are treated with drugs introduced decades ago that are not always fully effective and often have severe side effects. Here, the trypanosome haptoglobin-haemoglobin receptor (HpHbR) has been exploited as a route of uptake for an antibody-drug conjugate (ADC) that is completely effective against Trypanosoma brucei in the standard mouse model of infection. Recombinant human anti-HpHbR monoclonal antibodies were isolated and shown to be internalised in a receptor-dependent manner. Antibodies were conjugated to a pyrrolobenzodiazepine (PBD) toxin and killed T. brucei in vitro at picomolar concentrations. A single therapeutic dose (0.25 mg/kg) of a HpHbR antibody-PBD conjugate completely cured a T. brucei mouse infection within 2 days with no re-emergence of infection over a subsequent time course of 77 days. These experiments provide a demonstration of how ADCs can be exploited to treat protozoal diseases that desperately require new therapeutics.

Fig 1. The generation of ADCs that target the T. brucei HpHbR.

(A) Workflow for the generation of anti-trypanosomal ADCs. (B) Structures of the two PBD toxins (SG3199 and SG3552) and their corresponding toxins plus linker derivatives (SG3249 and SG3376) used in this study. Note that the linker of SG3249 contains a cleavable dipeptide motif whereas the linker of SG3376 does not.

Fig 2. Receptor mediated endocytosis of humanised anti-HpHbR IgG1s.

Uptake of Alexa594-labelled antibodies into T. b. brucei Lister 427 HpHbR wild type and -/- cells was monitored by microscopy. Uptake of five of the seven selected antibodies was detected at 10 nM in wild-type (indicated by arrows in upper panel) but not in HpHbR -/- cells (lower panel). No specific uptake of the remaining antibody (Tb086) or a control antibody (NIP228) was detected. Scale bar represents 10 μm.

Fig 3. HpHbR antibody-PBD conjugates result in T. brucei cell death at low picomolar concentrations in vitro in a HpHbR-dependent manner.

(A) Toxin SG3199 kills T. b. brucei wild type cells at sub-picomolar concentrations (IC₅₀ 0.86 pM), killing activity is reduced by the addition of a linker (SG3249 IC₅₀ 236.0 pM). Conjugation of SG3249 to a non-specific control antibody (NIP228) further reduces trypanosome killing activity to low nanomolar concentrations (IC₅₀ 2.1 nM) whereas conjugation of SG3249 to antibodies that target the HpHbR increased killing activity to low picomolar concentrations (IC₅₀ values range from 86 pM for Tb073-SG3249 to 9.4 pM for Tb085-SG3249). All assays were carried out in triplicate over 48 hours. Lines represents nonlinear regression lines of best fit on Log₁₀ transformed data. Error bars represent standard error of the mean (s.e.m.), n = 3 biological replicates (carried out in parallel). (B) Toxin SG3552 kills T. b. brucei wild type and HpHbR -/- cells with sub-picomolar IC₅₀ concentrations The IC₅₀ is increased by orders of magnitude by the addition of a linker (Table 1). Conjugation of SG3376 to a non-specific control antibody (NIP228) further increases the IC₅₀ to nanomolar concentrations in both trypanosome cell lines. HpHbR antibody SG3376 conjugates have an IC₅₀ in the low/sub picomolar range for wild type T. b. brucei. In contrast, IC₅₀ values with T. b. brucei HpHbR -/- cells remained similar to the control ADC. All assays were carried out in triplicate over 48 hours. Lines represents nonlinear regression lines of best fit on Log₁₀ transformed data. See Table 1 for corresponding IC₅₀ values. Error bars represent s.e.m., n = 3 biological replicates (carried out in parallel).

Fig 4. A single low dose of Tb085-SG3376 was able to cure infection in a mouse model of trypanosomiasis.

Three groups of 5 mice were infected with pleomorphic T. b. brucei GVR35-VSL2 cells [32, 33], which allow for parasite burden in live mice to be assessed over a time course by bioluminescent imaging (BLI). BLI was performed prior to any treatment at 3 dpi and then at regular time points following treatment on 3 dpi with a single intravenous dose of (1) 0.25 mg/kg Tb085-SG3376 (n = 5), (2) 0.25 mg/kg NIP228-SG3376 (n = 5) or (3) PBS alone (n = 5). Unlike the control-treated mice, Tb085-SG3376 treatment caused a decrease in the luminescent signal to that obtained from uninfected control animals within 2 days and this remained the case for the duration of the infection, including following the immunosuppression of Tb085-SG3376 treated mice at 66 dpi. Mice treated with NIP228-SG3376 or PBS were culled at a humane endpoint on day 14. (A) Quantification shown is the combined (dorsal + ventral) luminescence over the whole mouse in photons per second (p/s). The corresponding quantification data from the 18 individual mice are shown in S5 Fig. Error bars represent standard deviation. Downward error bars are missing from 4 data points due to scale constraints. (B) For each group of mice selected ventral images for the BLI are shown. Corresponding dorsal images of the same mice are shown in S4 Fig. The scale bar represents the photons emitted at any given point on the image. Exposure times range from 0.5 seconds (for heavily burdened mice) to 5 minutes (for uninfected animals). One mouse in the PBS control group had a lower BLI signal than all other infected mice at 3 dpi (S5 Fig). In the image shown here this mouse appears negative, however, this is due to the low exposure time required for adjacent mice.