Saporin as a Commercial Reagent: Its Uses and Unexpected Impacts in the Biological Sciences-Tools from the Plant Kingdom

Abstract

Saporin is a ribosome-inactivating protein that can cause inhibition of protein synthesis and causes cell death when delivered inside a cell. Development of commercial Saporin results in a technology termed 'molecular surgery', with Saporin as the scalpel. Its low toxicity (it has no efficient method of cell entry) and sturdy structure make Saporin a safe and simple molecule for many purposes. The most popular applications use experimental molecules that deliver Saporin via an add-on targeting molecule. These add-ons come in several forms: peptides, protein ligands, antibodies, even DNA fragments that mimic cell-binding ligands. Cells that do not express the targeted cell surface marker will not be affected. This review will highlight some newer efforts and discuss significant and unexpected impacts on science that molecular surgery has yielded over the last almost four decades. There are remarkable changes in fields such as the Neurosciences with models for Alzheimer's Disease and epilepsy, and game-changing effects in the study of pain and itch. Many other uses are also discussed to record the wide-reaching impact of Saporin in research and drug development.

Keywords: Alzheimer’s; Alzheimer’s disease; RIP; Saporin; animal model; cancer; immunotoxins; internalization; lesion; pain.

Figure 1

Cresyl violet staining of the medial septal region after ICV injection of 192-IgG-SAP. (A) normal superior cervical ganglia (SCG) section from a rat 5 days after injection (80 μg). (B) section from a rat 14 days after injection into the lateral ventricle (4 μg). (C) immunoperoxidase (ip) stain for ChAT from same (normal) rat as (A). (D) ip staining for ChAT from same rat as (B). (E) ip stain for p75 receptor from same (normal) rat as (A). (F) ip staining for p75 receptor from same rat as (B,D), showing near complete loss of positively-stained neurons. Bar in F = 100 μm and applies to all panels.

Figure 2

Animal models produced by Saporin conjugates.

Figure 3

Antibody composition of Saporin secondary conjugates. (A) Mab-ZAP uses a bivalent antibody, consisting of both the fragment antigen-binding (Fab) region and the fragment crystallizable (Fc) region of IgG, and is capable of reacting with whole IgG. (B) Fab-ZAP uses a monovalent antibody to prevent capping and recognizes whole IgG. (C) FabFc-ZAP uses a monovalent antibody to prevent capping and recognizes the Fc region.

Figure 4

Cytotoxicity assay of bivalent and monovalent IgG-Saporin secondary conjugates. 7H6 cells, a clone of the rat C6 glioma cell line, were plated at 2500 cells/well. The secondary conjugates Mab-ZAP, Fab-ZAP mouse, and FabFc-ZAP mouse were reacted with monovalent 192-IgG as the targeting agent. A stoichiometric effect was seen when using Fab-ZAP (purple-diamond line) and FabFc-ZAP (orange-circle line) held at a constant concentration (4.5 nM), reacted with the titrated mouse monoclonal 192-IgG.

Figure 5

Cytotoxicity assay of Fc region-specific IgG-Saporin secondary conjugates. Daudi cells were plated at 3000 cells/well. Saporin alone and a direct conjugate of an antibody to CD22 and Saporin were used as controls. Three secondary conjugates were compared: (1) Hum-ZAP alone; whole IgG recognizing human whole IgG, (2) Fab-ZAP Human; monovalent IgG recognizing whole Human IgG, and (3) FabFc-ZAP Human; monovalent IgG recognizing only Human Fc. Data show that the two secondary conjugates that recognize whole IgG caused cell death without a primary antibody as a targeting agent due to endogenous sIg on the cell surface. Only the Fc-specific conjugate displayed cell death similar to the Saporin-negative control.

Figure 6

Substance P–Saporin (SP-SAP): composition and specificity. (A) SP-SAP is a conjugate between the peptide, Substance P, and the ribosome-inactivating protein, Saporin. This conjugate specifically targets neurons that express the substance P receptor (NK1R). (B) Saporin immunofluorescence is in yellow, showing specific targeting and internalization into a neuron that expresses NK1R. Neurons that do not express NK1R are shown in red.

Figure 7

Patients in the study filled out several pain surveys, VAS (Visual Analog Scale) Pain, VAS Bothersome, ODI (Oswestry Disability Index), and BDI (Beck Depression Inventory). Survey scores were tabulated according to standard methods for each survey type and values were reported to sponsor. Surveys were administered first during the screening process, 1 week prior to treatment to initiate a baseline for patient pain levels, then again on day of treatment, followed by weekly surveys during each patient visit with the clinical staff following treatment. Due to the complex pain profile for eligible patients, trend lines were incorporated into data above to visualize the overall pain picture for each of the corresponding doses. Both the 16-mcg (microgram) patient and the 32-mcg patient reported reductions in pain over the 8 weeks following treatment.