Affimer reagents enable targeted delivery of therapeutic agents and RNA via virus-like particles

Abstract

Monoclonal antibodies have revolutionized therapies, but non-immunoglobulin scaffolds are becoming compelling alternatives owing to their adaptability. Their ability to be labeled with imaging or cytotoxic compounds and to create multimeric proteins is an attractive strategy for therapeutics. Focusing on HER2, a frequently overexpressed receptor in breast cancer, this study addresses some limitations of conventional targeting moieties by harnessing the potential of these scaffolds. HER2-binding Affimers were isolated and characterized, demonstrating potency as binding reagents and efficient internalization by HER2-overexpressing cells. Affimers conjugated with cytotoxic agent achieved dose-dependent reductions in cell viability within HER2-overexpressing cell lines. Bispecific Affimers, targeting HER2 and virus-like particles, facilitated efficient internalization of virus-like particles carrying enhanced green fluorescent protein (eGFP)-encoding RNA, leading to protein expression. Anti-HER2 affibody or designed ankyrin repeat protein (DARPin) fusion constructs with the anti-VLP Affimer further underscore the adaptability of this approach. This study demonstrates the versatility of scaffolds for precise delivery of cargos into cells, advancing biotechnology and therapeutic research.

Keywords: Molecular biology; Virology.

Graphical abstract

Figure 1

Identification of Affimers that bind cellularly expressed HER2 (A) Phage ELISA of 32 Affimer clones identified as binding the ECD of HER2. Identical clones are shown with the same shading. (B) Phage ELISA of 32 Affimer clones identified as binding the ECD of HER2 in the presence of HER2. Identical clones are shown with the same shading. (C) Affinity precipitation of endogenous HER2 from HER2-overexpressing cell lines (AU-565 and BT-474) and the HER2-negative cell line (MDA-MB-231) using HER2-binding Affimers identified by phage screening against the ECD of HER2. (n = 3 independent experiments with representative blots shown; white lines delineate individual membranes; all blots were run concurrently). (D) Phage ELISA of 48 Affimer-clones identified as preferential binding fixed HER2-expressing cells (MDA-MB-453) compared to HER2-negative cells (MCF7). An arbitrary cutoff of 3 was used to determine which clones would be taken forward for DNA sequence analysis identifying the two unique binding Affimers D11 and H7. Identical clones are shown with the same shading. ECD, extracellular domain (amino acids 1-652); WCL, whole-cell lysates; Cont., control Affimer (Variable regions AAAA; AAE).

Figure 2

Affimers D11 and H7 colocalize with HER2 and show internalization into HER2-positive cells AlexaFluor 488-conjugated Affimers (green) D11 (A) and H7 (B) were incubated with HER2-positive (AU-565 and BT-474) and -negative (MDA-MB-231) cell lines for 1 h before fixation. Cells were stained for HER2 (pink) and with Hoechst (blue). The internalization of the Affimers D11 and H7 was assessed as mean AlexaFluor 488 intensity per cell (C). The mechanism of internalization was assessed with costaining for EEA1 [pink, (D) and (E)] and LAMP2 [pink, (F) and (G)]. Colocalization of Affimers with HER2/EEA1/LAMP2 is shown by arrows. Images were taken on a Zeiss confocal microscope with a 40x magnification under the same exposure for AlexaFluor 488-conjugated Affimers. Images were analyzed using ZenLite and ImageJ software. Data are mean ± SEM, n = 3 independent experiments, with representative images shown, unpaired t test. ∗p < 0.05 (C).

Figure 3

Impact of Affimers on the metabolic activity of breast cancer cell lines A range of breast cancer cell lines AU-565, SKBR3, BT-474, MDA-MB-453, MCF-7, MDA-MB-231, and ZR-75-1 were seeded one day prior to the addition of HER2-binding Affimers or HER2-binding Affimer-MMAE conjugates. Cells were incubated for a further 72 h, and metabolic activity was analyzed by AlamarBlue measurement. HER-2-binding Affimers did not affect metabolic activity in either HER2-positive cell lines AU-565 and BT474 or the HER-2-negative cell line MDA-MB-231 [(A) and (B)]. HER2-binding Affimers were conjugated with the cytotoxin MMAE via a cathepsin cleavable group and a PABC spacer (C). Seven breast cancer cell lines with varied HER2 expression level as measured by immunoblotting (D); representative blot shown; dotted line indicates removal of a lane containing lysates from a non-breast cancer cell line were treated with the Affimer-MMAE conjugates and showed dose-dependent inhibition of metabolic activity [(E) and (F)], the efficacy of which varied with HER2 expression level. All values were normalized to cells incubated with media alone. Data are mean ± SEM; dose-response curves were fitted using GraphPad Prism v 9.0, [Inhibitor] vs. response -- Variable slope (four parameters); n = 3 independent experiments for all panels. MMAE, monomethyl auristatin E.

Figure 4

HER2-binding Affimers mediate targeted delivery of VLPs Affimer dimer containing HER2-binding Affimer D11 and CPMV-VLP-binding Affimer were used to target CPMV VLPs containing eGFP RNA to HER2-positive cells (A). Affimer dimer increased cellular uptake of eGFP RNA-containing CPMV VLPs to HER2-positive cell lines (AU-565 and SKBR3) in a dose-dependent manner (Kruskal-Wallis test; p = 0.0109 and p = 0.0099, respectively) while uptake was minimal in HER2-negative cell line MDA-MB-231 [Kruskal-Wallis test; p = 0.3133; (B) and (C)]. Affimer dimer:VLP conjugates were non-toxic at doses used (0.07 μM VLP and increasing ratios of Affimer dimer). Two-way ANOVA; p = 0.5687 (D). Data are mean ± SEM; n = 3 independent experiments. (A) Created with BioRender.com (2023).

Figure 5

HER2-binding Affimer D11 bind different epitopes to other HER2-binding SBPs The Alphafold2-predicted model shows HER2-binding Affimer D11 level binds subdomains II and III of the HER2 ECD. The prediction is color coded based on pLDDT values that indicate the confidence level of the prediction (A) while Affibody Z_HER2:342 (yellow; PDB code: 3MZW) binds subdomain III and DARPinG3 (gray; PDB code:4HRN) binds subdomain IV. Affimer D11 is shown in magenta (B). Replacement of HER2-binding Affimer D11 with either Affibody Z_HER2:342 or DARPinG3 in Affimer dimer still results in an increased percentage of eGFP-positive cells over VLP alone (C) but not to the same magnitude as the Affimer dimer (D). Data are mean ± SEM; n = 3 independent experiments, with representative images shown in (C); scale bar is 50 μm. One-way ANOVA with Dunnett’s post hoc test ∗∗∗∗p < 0.0001 (D). Image created with Pymol v2.5.4 (Schrodinger LLC) (B).