Immunogenicity Risk Profile of Nanobodies

Abstract

Nanobodies (Nbs), the variable domains of camelid heavy chain-only antibodies, are a promising class of therapeutics or in vivo imaging reagents entering the clinic. They possess unique characteristics, including a minimal size, providing fast pharmacokinetics, high-target specificity, and an affinity in the (sub-)nanomolar range in conjunction with an easy selection and production, which allow them to outperform conventional antibodies for imaging and radiotherapeutic purposes. As for all protein theranostics, extended safety assessment and investigation of their possible immunogenicity in particular are required. In this study, we assessed the immunogenicity risk profile of two Nbs that are in phase II clinical trials: a first Nb against Human Epidermal growth factor Receptor 2 (HER2) for PET imaging of breast cancer and a second Nb with specificity to the Macrophage Mannose Receptor (MMR) for PET imaging of tumor-associated macrophages. For the anti-HER2 Nb, we show that only one out of 20 patients had a low amount of pre-existing anti-drug antibodies (ADAs), which only marginally increased 3 months after administering the Nb, and without negative effects of safety and pharmacokinetics. Further in vitro immunogenicity assessment assays showed that both non-humanized Nbs were taken up by human dendritic cells but exhibited no or only a marginal capacity to activate dendritic cells or to induce T cell proliferation. From our data, we conclude that monomeric Nbs present a low immunogenicity risk profile, which is encouraging for their future development toward potential clinical applications.

One sentence summary: Nanobodies, the recombinant single domain affinity reagents derived from heavy chain-only antibodies in camelids, are proven to possess a low immunogenicity risk profile, which will facilitate a growing number of Nanobodies to enter the clinic for therapeutic or in vivo diagnostic applications.

Keywords: DC activation; T cell—DC interactions; anti drug antibodies; dendritic cells; immunogenicity; nanobody.

Figure 1

ECL measurement of ADA in samples from healthy volunteers and from patients. Anti-drug antibody (ADA) analysis by Electrochemiluminescence (ECL) for 50 serum samples of healthy donors for cut-off point determination (A,B) and for the 20 samples of patients enrolled in the phase I PET study, both prior to and 3 months after Nb-tracer injection (C). Four patients out of 20, showed a positive ECL result (i.e., a signal above the screening cut-off, indicated by a green horizontal line) of which according to the confirmation assay, one patient (#13) possesses ADA in the serum both prior to and 3 months after Nb-injection.

Figure 2

DLS measurement of both Nanobodies. Dynamic Light Scattering (DLS). Black dots and line represent the experimental data and fit, respectively. The residuals (Res.) are shown. The percentage of mass as a function of the R_h distribution is given in the inset.

Figure 3

Uptake of pHrodo-labeled Nanobodies by DCs. Both moDCs and cDCs of the same donor were exposed to pHrodo-labeled Nbs for 1 h or left uninduced (A). Results are expressed as MFI (*P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001). The fluorescence signal of the different pHrodo labeled Nbs and their NOTA conjugates was measured after 1 h incubation in PBS at different pH (*P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001) (B).

Figure 4

Gating strategy for moDCs and cDCs generated or isolated from human buffy coats. MoDCs were gated based on singlets and CD11c⁺, CD3⁻, CD14⁻, CD19⁻ live cells (A) and cDCs were gated based on CD3⁻, CD14⁻, CD19⁻ live cells, and subdivided in cDC1 type (CD141^high) and cDC2 type (CD1c⁺) cells (B).

Figure 5

Surface marker expression on human moDCs and cDCs after stimulation with different antigens. In vitro generated moDCs (A) as well as directly isolated primary cDCs (B,C) from healthy donors were exposed to control antigens or to Nbs for 24 h or left uninduced. The moDCs, cDC1, and cDC2 cells were gated as shown in Figure 4. Five different surface markers (CD80, CD83, CD86, CD40, and HLA-DR) were analyzed by flow cytometry. Results are expressed as median fluorescence intensity (MFI) of 25 (moDCs) or nine (cDCs) independent experiments (*P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001, Mann–Whitney test).

Figure 6

Cytokine secretion by human moDCs and cDCs after stimulation with different antigens. In vitro generated moDCs (A) as well as directly isolated primary cDCs (B) of healthy donors were exposed to control antigens or to Nbs for 24 h or were left uninduced. The supernatant of each cell type and condition was analyzed for presence of IL-12, IL-6, TNF-α, and IL-10 by ELISA. For moDCs, 22 donors were analyzed. For cDCs, nine donors were analyzed. For cDCs, 9 donors were analyzed (* P < 0.05, **P < 0.01, ***P < 0.001 and ****P < 0.0001, Mann-Whitney test).

Figure 7

T cell proliferation after co-culture with autologous moDCs stimulated with different antigens. In vitro generated moDCs were exposed to control antigens or to Nbs and the cells were stimulated overnight with a maturation cocktail (A) or left untreated (B). The maturation cocktail was washed away before adding autologous T cells. After 6 days of co-culture, cell proliferation was monitored by ³H-thymidine incorporation. Results are expressed as counts per minute (cpm), for fifteen donors in co-culture with maturation cocktail and nine donors in co-culture without maturation (*P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001).