Description and preparation of venom antigens (toxins)

D. polylepis venom was fractionated using RP-HPLC11, resolving the key dendrotoxins in four venom fractions (Dp5, Dp6, Dp7, and Dp8) that cannot be further resolved in quantitative yields with standardized techniques. While Dp8 contains almost pure dendrotoxin-1 (P00979 (https://www.uniprot.org/uniprot/P00979)), the venom fractions Dp5, Dp6, and Dp7 are ‘mixed fractions’ that contain similar amounts of at least one dendrotoxin and at least one type II α-neurotoxins. Previous proteomic studies have identified the toxin components of Dp5, Dp6, and Dp7 to contain the same dendrotoxin (a homolog of dendrotoxin-δ, P00982 (https://www.uniprot.org/uniprot/P00982), from the Eastern green mamba, D. angusticeps) and a type II α-neurotoxin (α-elapitoxin Dpp2c, P01397 (https://www.uniprot.org/uniprot/P01397))11.

Phage display selection and screening of scFv binders

Following three rounds of panning against selected venom fractions containing dendrotoxins, polyclonal ELISAs revealed that antibody binders had been enriched (Fig. 1). Antibody genes (in scFv format) were isolated from both the second and third panning rounds, sub-cloned into an scFv bacterial expression vector19, and 188 clones were picked and their antibody expressed20. Recombinant monoclonal antibodies were tested for binding to respective target antigens (see example with Dp8 as antigen Fig. 2). Using a cut-off score of 5000 fluorescence units (25 times above the background binding signal), the top binders (up to 94) were picked for each antigen for further characterization (DNA sequencing and affinity ranking). An Expression-Normalized Capture (ENC) assay was used to rank the antibody clones by affinity. In this assay, limiting amounts of anti-FLAG antibody were used to capture FLAG-tagged scFvs in the expression culture supernatant. Since the scFv expression level for each clone in culture supernatant is well above the capture capacity of the anti-FLAG antibody coated in each well, differences in antibody expression are normalized so that the binding signal better reflects differences in affinity between scFv and antigen.

Fig. 1 Polyclonal phage ELISA signals for the output phages for each selection. A significant increase in binding signal is observed from round 2 to 3 for all selections, and for selections against Dp5, Dp6, and Dp7, a high degree of cross-reactive binding exists for the phages against all three antigens (Dp5, Dp6, and Dp7). Negative control antigens: Cbtx α-cobratoxin, b-Gal β-galactosidase, Strep Streptavidin. All experiments were performed in triplicates on distinct samples. Error bars represent standard deviations Full size image

Fig. 2 Schematic overview of selected results from the employed discovery process. Here, demonstrated for human IgGs against the antigen, Dp8. a Polyclonal phage ELISA signals against different venom fractions and negative control antigens (Cbtx α-cobratoxin, b-Gal β-galactosidase, Strep Streptavidin). b Monoclonal scFv ELISA signals against Dp8. c Summary of DNA sequencing results. Sequences are defined as unique based on V H and V L CDR3 sequences. d Monoclonal IgG ELISA signals for converted clones demonstrating retained binding for the majority of the clones upon conversion from the scFv format Full size image

Conversion of scFvs to IgG format and characterization

A panel of unique scFv-formatted antibodies that yielded the highest binding signals in the ENC assay were selected for conversion to IgG format. Following expression by transient transfection in Expi293TM cells, ELISA was used to confirm retention of target binding.

Twenty-five IgG-formatted antibodies were produced and purified by protein A chromatography. Using a fluid-phase technique based on protein G-beads pull-down of antigen-antibody complexes, followed by acid dissociation and MALDI-TOF MS analysis (see example in Fig. 3), binding to a dendrotoxin homologous to dendrotoxin-δ11 in the venom fractions was confirmed for 4/4 IgGs against Dp5, 3/4 IgGs against Dp6, and 1/8 IgGs against Dp7 (Table 1). The Dp8 fraction was known to consist predominantly of dendrotoxin-1, with only minor traces of other toxins11. A pull-down experiment was performed for clone 367_01_H01, which confirmed that dendrotoxin-1 was indeed the target for this clone (Table 1).

Fig. 3 Example of MALDI-TOF MS analysis of antigen-antibody complex pull-down experiments. a IgG 361_01_F07 pull-down from venom fraction Dp5. b IgG 361_01_F07 pull-down from whole venom. c IgG 363_01_F07 pull-down from venom fraction Dp6. d IgG 363_01_F07 pull-down from whole venom. Dtx Dendrotoxin, 3FTx Three-finger toxin Full size image

Table 1 Overview of all MALDI-TOF MS pull-down experiments Full size table

In vivo neutralization of dendrotoxins

In total, 24 out of 25 recombinant human IgGs targeting black mamba neurotoxins were tested in vivo. All IgGs were evaluated for neutralization of lethality by the intracerebroventricular (i.c.v.) route, where nine showed full (100%) protection against the venom fraction they were raised against (Tables 2 and 3). Even at the highest dose tested, seven IgGs failed to provide survival in the 24 h assay, although most of these IgGs showed prolonged survival time, as compared to controls, during the assay. Eight IgGs provided partial survival in the 24 h assay at one or more dose regimes (Tables 2 and 3).

Table 2 In vivo neutralization results for monoclonal IgG antibodies raised against Dp5, Dp6, and Dp7 Full size table

The i.c.v. assay is particularly useful for assaying toxicity of dendrotoxins, as these neurotoxins are highly potent when administered i.c.v., but display lower toxicity by intravenous (i.v.) administration, requiring relatively high doses to induce lethality11. In contrast, α-neurotoxins are less potent when administered i.c.v. and are better assayed using the i.v. route of administration. Three of the four investigated venom fractions contain a mixture of dendrotoxins and type II α-neurotoxins11, and it was observed that only dendrotoxin-targeting IgGs were able to provide full survival in the i.c.v. assay (compare Table 1 with Tables 2 and 3). Clones 364_01_A01, 364_01_B01, 364_01_D03, and 364_01_D04 target the type II α-neurotoxin present in Dp7 and were therefore also assayed using the i.v. route. Unfortunately, these IgGs failed in providing full protection, although clone 364_01_B01 provided a low survival rate (1/4) when mice were challenged with 10.6 µg toxin pre-incubated with the IgG at a molar ratio of 3:1 (IgG:toxin). Similarly, clone 364_01_A01 provided significantly prolonged survival, although all challenged mice died between 12–18 h. The present findings thus demonstrate that effective dendrotoxin-targeting human IgGs were discovered, but more work (such as affinity maturation) may be needed to improve the discovered IgGs that target type II α-neurotoxins. Moreover, this work also demonstrates that the discovered human IgGs could neutralize the lethal effect of the target dendrotoxins present in more than one venom fraction (e.g., 363_01_F07 which completely neutralizes the dendrotoxins in Dp5 and Dp6 and provides some protection against Dp7) (Table 2). The mass spectrometry data (Table 1) suggests that, in this case, the observed neutralization ability is due to the presence of a key dendrotoxin (or very similar dendrotoxins) in more than one venom fraction.

To explore whether the dendrotoxin-mediated neurotoxicity of the whole venom could be completely abrogated using the discovered human IgGs, antibody cocktails were designed (Table 4) and evaluated against whole venom by the i.c.v. route. In all cases, the combination of antibodies to dendrotoxin-1 and the dendrotoxin-δ homolog was superior to anti-dendrotoxin-1 (367_01_H01) alone. Anti-dendrotoxin-1 (367_01_H01) was used along with different combinations of antibodies to dendrotoxin-δ. For example, Cocktail 2 included 363_01_F07 and 365_01-G06 whereas Cocktail 1 included these, as well as an additional antibody (361_01_F07) (Tables 3 and 4). Both cocktails successfully provided full protection against whole venom when injected via the i.c.v. route at a challenge dose of 1.5 µg of whole venom pre-incubated with the IgG cocktails at IgG:toxin molar ratios of 4:1 and 3:1 (Fig. 4). Omission of one or other of the anti-dendrotoxin-δ-homolog antibodies from Cocktail 2 yielded reduced protection. For example, neutralization tests against whole venom were performed i.c.v. using Cocktail 3 (where 365_01_G06 had been substituted with additional 363_01_F07), Cocktail 4 (where 363_01_F07 had been substituted with additional 365_01_G06) (Table 4), and 367_01_H01 alone. As seen in Fig. 5, none of these cocktails were able to provide equivalent protection at equimolar doses compared to Cocktail 2. Thus, Cocktail 2 represented the minimum cocktail providing full protection in this experiment.

Table 3 In vivo neutralization results for monoclonal IgG antibodies raised against Dp8 and oligoclonal IgG cocktails Full size table

Table 4 Composition of the IgG cocktails Full size table

Fig. 4 Kaplan-Meier survival curves for antibody cocktails. Here, shown for a Cocktail 1 and b Cocktail 2 at different molar ratios against black mamba whole venom administered i.c.v., demonstrating full protection at an IgG:toxin molar ratio of 4:1 for Cocktail 1 and 3:1 for Cocktail 2. Cocktail 1 contains the IgGs: 361_01_F07 (anti-Dp5), 363_01_F07 (anti-Dp6), 365_01_G06 (anti-Dp7), 367_01_H01 (anti-Dp8). Cocktail 2 contains the IgGs: 363_01_F07 (anti-Dp6), 365_01_G06 (anti-Dp7), 367_01_H01 (anti-Dp8). Each individual curve represents survival of a cohort of 4 animals Full size image

Fig. 5 Kaplan–Meier survival curves for antibody cocktails. Here, shown for a Cocktail 3, b Cocktail 4, and c clone 367_01_H01 against black mamba whole venom administered i.c.v. at an IgG:toxin molar ratio of 3:1. Cocktail 3 contains the IgGs: 363_01_F07 (anti-Dp6) and 367_01_H01 (anti-Dp8). Cocktail 4 contains the IgGs: 365_01_G06 (anti-Dp7) and 367_01_H01 (anti-Dp8). Each individual curve represents survival of a cohort of 4 animals Full size image

Finally, Cocktail 1 and 2 were tested against whole venom by the i.v. route to demonstrate that these cocktails were truly dendrotoxin-specific and that whole venom cannot be neutralized, when using the i.v. route, unless both dendrotoxins and α-neurotoxins in the venom are neutralized21. As expected, similar survival curves (rapid death upon injection) were observed for mice challenged with 25.8 µg of whole venom alone and mice challenged with a similar dose of whole venom pre-incubated with Cocktail 1 and 2 at molar ratios of 3:1 and 4:1 (IgG:toxin), respectively.