An engineered substance P variant for receptor-mediated delivery of synthetic antibodies into tumor cells

Abstract

We have developed and tested a robust delivery method for the transport of proteins to the cytoplasm of mammalian cells without compromising the integrity of the cell membrane. This receptor-mediated delivery (RMD) technology utilizes a variant of substance P (SP), a neuropeptide that is rapidly internalized upon interaction with the neurokinin-1 receptor (NK1R). Cargos in the form of synthetic antibody fragments (sABs) were conjugated to the engineered SP variant (SPv) and efficiently internalized by NK1R-expressing cells. The sABs used here were generated to bind specific conformational forms of actin. The internalized proteins appear to escape the endosome and retain their binding activity within the cells as demonstrated by co-localization with the actin cytoskeleton. Further, since the NK1R is over-expressed in many cancers, SPv-mediated delivery provides a highly specific method for therapeutic utilization of affinity reagents targeting intracellular processes in diseased tissue.

Fig. 1.

Receptor-mediated delivery. A fluorescently-labeled cargo is attached to a natural ligand. Upon binding to the receptor, the cargo is internalized where it can carry out the desired function.

Fig. 2.

Specific delivery of cargo to tumor cells by SPv. (A) U87 glioblastoma incubated with Alexa555-labeled SP for 2 h followed by fixing and nuclear staining (blue) show internalization of the peptide (red). (B) Normal human astrocytes show no detectable internalization of the peptide. (C) Conjugation of SPv to protein cargo. SPv is synthesized to contain the amino acid sequence of SP (boxed) in addition to an N-terminal maleimide for attachment of protein cargo via a surface cysteine residue. An amine reactive fluorophore (star) is attached to the cargo via surface lysines.

Fig. 3.

Delivery of actin-binding sABs to cells expressing the NK1 receptor. (A) Electron micrographs of the bundling effect of sAB-27; left panel: actin only; right panel: actin in the presence of 6.4 μM sAB-27. (B) Severing of TMR-labeled actin filaments by sAB-4 viewed by TIRF microscopy. Numbers indicate time in seconds following addition of 0.5 μM sAB-4. (C) TIRF microscopy imaging of TMR-labeled actin filaments (green) in the presence of 0.25 μM cy5-labeled sAB-19 (red). (D) Phalloidin staining of untreated U87 glioblastoma cell. (E) The actin-bundling effect in U87 cells treated with 20 nM cy5-labeled sAB-27-SPv conjugate (red) followed by fixing and staining with TMR-phallodin (green). (F) U87 cells treated with 20 nM Alexa488-labeled sAB-4-SPv conjugate (red), followed by fixing and staining with TMR-phalloidin (green), inset: enlarged box showing severed actin filaments. (G) U87 cells treated with 20 nM Alexa488-labeled sAB-19-SPv conjugate (red) followed by fixing and staining with TMR-phalloidin (green); arrow in inset indicates an example of sAB-19 localizing to the base of filopodia. (H) U87 cells treated with 200 nM sAB-4 only exhibiting normal actin filaments. (I–K) HeLa cells transiently transfected with the NK1 receptor. (I) HeLa cells were treated with 100 nM SP-Alexa555 (red), then fixed and stained with FITC-phalloidin (green). (J) HeLa cells treated with 20 nM cy5-labeled sAB-27-SPv conjugate (red), followed by fixing and staining with TMR-phalloidin (green). (K) HeLa cells treated with 20 nM cy5-labeled sAB-4-SP conjugate (red) followed by staining with TMR-phallodin (green). (Scale bar, 10 μm.)

Fig. 4.

Cell viability assay of U87 glioblastoma cells. (A) Forty-eight hour treatment with sAB27, SP or sAB-27-SPv conjugate; left panel, 10 nM; right panel, 20 nM. (B) Time course of inhibition of cell viability by the sAB27-SPv conjugate; open circles, 10 nM; closed circles, 20 nM.

Fig. 5.

Quantification of sAB-SPv conjugate internalization by U87 cells. Cells plated on a 96-well plate were treated with increasing amounts of sAB-27-SPv conjugate fluorescently labeled with cy5. After 24 h incubation, the cells were extensively washed, and the amount of the internalized conjugate was calculated from fluorescence values using a standard curve.