A resource of high-quality and versatile nanobodies for drug delivery

Abstract

Therapeutic and diagnostic efficacies of small biomolecules and chemical compounds are hampered by suboptimal pharmacokinetics. Here, we developed a repertoire of robust and high-affinity antihuman serum albumin nanobodies (Nb_HSA) that can be readily fused to small biologics for half-life extension. We characterized the thermostability, binding kinetics, and cross-species reactivity of Nb_HSAs, mapped their epitopes, and structurally resolved a tetrameric HSA-Nb complex. We parallelly determined the half-lives of a cohort of selected Nb_HSAs in an HSA mouse model by quantitative proteomics. Compared to short-lived control nanobodies, the half-lives of Nb_HSAs were drastically prolonged by 771-fold. Nb_HSAs have distinct and diverse pharmacokinetics, positively correlating with their albumin binding affinities at the endosomal pH. We then generated stable and highly bioactive Nb_HSA-cytokine fusion constructs "Duraleukin" and demonstrated Duraleukin's high preclinical efficacy for cancer treatment in a melanoma model. This high-quality and versatile Nb toolkit will help tailor drug half-life to specific medical needs.

Keywords: Biochemical Engineering; Biotechnology; Drug Delivery System; Proteomics; Structural Biology.

Graphical abstract

Figure 1

Identification and characterization of Nb_HSA (A) Schematic structure and amino acid composition of Nb_HSA. 71 different Nbs were analyzed by Yvis. The amino acid frequency at each CDR position was calculated. Amino acids were color-coded and classified based on the physicochemical properties. (B) Circos and sequence logo plots showing the diversity of CDR3. (C) ELISA heat map of albumin cross-species binding of 71 different Nbs. (D) Binding kinetics of 6 Nbs by SPR (surface plasmon resonance). (E) Correlation between ELISA O.D. (optical density) and SPR K_D affinity. (F) Immunoprecipitation of the HSA-Nb₁₃ complex at different concentrations of Nb_13.. (G) Validation of Nb cross-species reactivity by immunoprecipitation. Three Nbs (Nb₁₄₃, Nb_85, and Nb₁₃₂) were immunoprecipitated by affinity resins coupled with albumin of different species, including human, monkey, mouse, bovine, and llama. (H) The plot of the thermostability of 20 Nb_HSA selected for measurement by differential scanning fluorimetry.

Figure 2

Cross-link models of the HSA-Nb_HSA complexes (A) The epitope clusters of HSA based on converged cross-link models. HSA Epitope cluster A: residues 29–37, 81–93, 118–124, 232, 236, 252–290, 300–310, 345–350; cluster B: residues 35–44, 179–212, 282–286, 298–318, 460–465; cluster C: residues 56–62, 101–108, 1234–169; cluster D: residues 426, 566–582, 595, 598–606; cluster F: 440–446, 488–525. Cluster E has two subtypes that share the same helices region and vary on other binding residues. E_type1: residues 321–331, 334–335, 358, 360–365, 395–413, 465–470; E_type2: residues 371–387, 396–410, 494–519. (B) Percentage of epitope-specific Nb_HSA based on converged cross-link models. (C) The RMSD (root-mean-square deviation, Å) distribution of the cross-link models. (D) Integrative structural models of HSA-Nb_HSA complexes. Only the top-scored Nb_HSA models were shown. Different molecules were presented in different colors. Gray (surface presentation): HSA (with three domains DI, DII, and DIII). Nb_HSA corresponding to different epitopes were presented as cartoons. Epitope Cluster (A): blue; (B): purple; (C): salmon; (D): light pink; (E): green; (F): yellow.

Figure 3

Integrative structural characterization of a tetrameric HSA-Nb complex (A) Size-exclusion chromatography (SEC) analysis of the reconstituted tetrameric complex, including Nb₁₃, Nb₂₉, Nb_80, and HSA. (B) Overlapping of the cross-link structure model of the tetrameric complex with the negative stain EM structure. Nb_HSA were presented in different colors. Yellow: Nb₁₃; salmon: Nb₂₉; blue: Nb₈₀. (C–E) Close-up views of the interfaces of the tetrameric HSA-Nb complex and the cross-link restraints. (F) A cross-link model of HSA and Nb₈₀ interaction showing the putative salt bridges. Sidechains of two charged residues on HSA (K383 and E400) and the corresponding residues on Nb₈₀ were shown. The HSA sequence (from residue 377 to residue 401) was aligned with camelid albumin. (G) HSA site-directed mutagenesis and immunoprecipitation of the mutant HSA. Nb₈₀-conjugated resins were used to pull down different HSA mutants, including E400R and E400R-K383D.

Figure 4

High-throughput Nb pharmacokinetics in an Alb^−/- FcRn-humanized mouse model (A) Schematics of the MS-based assay for multiplexed pharmacokinetic analysis. Briefly, after consecutive injections of HSA and the Nb mixtures into the mouse model, blood was collected at 21 different time points and processed for proteomics-based pharmacokinetic analysis. (B) Pharmacokinetic analysis of 22 Nbs in an Alb^−/- FcRn-humanized mouse model. Single bolus administration of an equimolar mixture of 22 Nbs, including 20 Nb_HSA and two nonbinder controls were administered to three animals via i.v. injection. Serum samples were collected at different time points and proteolyzed. The resulting peptides were analyzed by LC coupled to MS (PRM). Each data point indicates the median Nb abundance from three different animals. The analysis was repeated three times. The data were then fitted into a two-phase decay model to calculate the Nb half-lives. (C) Heatmap analysis of the distribution and elimination of Nb pharmacokinetics. ∗, See Figure S3A. (D) Correlative analysis between pharmacokinetics and the properties of Nbs. For each correlation analysis, the correlation coefficient Spearman ρ, and the corresponding p-value were calculated using Prism GraphPad.

Figure 5

Development of Duraleukin – a novel class of Nb_HSA-fusion cytokine (A) Schematic design of Duraleukin, including an N-terminal Nb_HSA, a short flexible linker sequence ((GGGGS)₂), and hIL-2 followed by a C-terminal His6 tag. (B) Schematics of large-scale Duraleukin production from E. coli. A representative SDS-PAGE analysis of purified Duraleukin. S: soluble fraction. IB: inclusion body; FT: flow-through; E: elution; PBS: after endotoxin removal and dialysis in PBS. (C) Thermostability of Duraleukins by differential scanning fluorimetry. (D) In vitro CTLL-2 cell proliferation assay of Duraleukin and hIL-2. The X axis is the concentration of hIL-2 (ng/mL). The Y axis is the absorbance at 450 nM subtracted by absorbance at 690 nm (background signal) for the measurement of T cell proliferation. (E) The resistance of three DLs to serum proteases. Purified DLs were incubated with the mouse serum for the indicated periods. After incubation, DLs were detected by western blot using an anti-IL-2 monoclonal antibody (BG5). (F) The SPR kinetic measurement of DL₈₀ for HSA binding. (G) Immunoprecipitation assay (pH-dependent) of DL₈₀ for HSA binding. HSA-conjugated agarose resin was used to pull down DL₈₀ at various concentrations. Immunoprecipitated DL₈₀ at different pH was normalized and quantified by ImageJ.

Figure 6

Evaluation of the in vivo efficacy of Duraleukin in a melanoma mouse model (A) The tumor growth curve. C57BL/6J mice bearing subcutaneous B16-F10 tumors were treated with a combination of TA99 and Duraleukin or hIL-2 (n = 8/group) with different dosage intervals. PBS treatment was used for control. (B) Animal survival curve after treatment. (C) Flow cytometry analysis of tumor-infiltrating immune cells (n = 5/group). The y axis is the respective immune cell count based on a million total cells from the isolated tumor.