Targeting vesicular GABA Transporter (vGAT)-Expressing Cells with a Polyclonal Antibody to the Lumenal Domain ofvGAT: Results with a Saporin Conjugate. Chelsea Friedman, Brian Russell, Matt Kohls, Leonardo Ancheta, Patrick Shramm, *Douglas Lappi. Advanced Targeting Systems, San Diego, CA

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IntroductionThe vesicular GABA transporter (vGAT) mediates both accumulation and release of GABA from synaptic vesicles. vGAT is expressed in nerve endingsof GABAergic neurons throughout the CNS. The GABAergic system is crucialfor the development and functional maturation of the nervous system, aswell as the maintenance of balance between excitation and inhibition required for normal neural circuit function. A panel of research tools hasbeen created that target the lumenal domain of vGAT. The main objectiveswere to create an antibody against the vesicular GABA transporter and to characterize that antibody in order to create an effective targeted toxin forGABAergic neurons. This investigation was extended to in vitro researchusing a cell line transfected with rat vGAT to demonstrate the internalizationof the antibody coupled to saporin.

Anti-vGAT-SAP could be an important tool in studying diseases involving dysfunction of GABAergic neurons. GABAergic neuron dysfunction isthought to be an underlying factor in Epilepsy, Down Syndrome, Fragile XSyndrome, Schizophrenia and Autism. In vivo, elimination of vGAT-expressing cells in a particular area (rather than knocking out vGAT systemically) makes it possible to study the functions of those regional cells.Animals can then be tested behaviorally before and after injections of Anti-vGAT-SAP to demonstrate the effects of loss of cells in a particular re-gion of interest.

Figure 1. vGAT Schematic.

Figures 3, 4 & 5.

MethodsAntiserum was raised against a peptide from the C-terminus of rat vGAT andresulted in an affinity-purified antibody and an immunotoxin specific forvGAT-expressing cells. The antigen sequence is identical among human, rat,mouse, pig and guinea pig. A stably-transfected clone of HEK-293 cells (HEK-293vGAT) that expresses vGAT on the cell surface shows excellent results for western blot, ICC and flow cytometry using both the antiserumand affinity-purified antibody.

The affinity-purified antibody was used to create an immunotoxin by conjugating it to the ribosome-inactivating protein, saporin. Saporin irreversibly inactivates ribosomes, blocking protein synthesis, after it is escorted into a cell. Saporin cannot enter a cell on its own, but when escorted by something that binds to a cell surface marker, it is internalizedalong with the binding moiety and causes cell death. The immunotoxin(Anti-vGAT-SAP) is 1000-fold more cytotoxic to HEK-293vGAT cells than non-conjugated saporin, based on the EC50 in a cytotoxicity assay. The affinity-purified vGAT antibody binds specifically to cells that express vGAT,and delivers a payload to the interior of these cells.

Figure 2. Flow Cytometry Data.HEK-293 cells (orange line) and HEK-293vGAT transfectantsclone 2E11 (red line) were incubated with 10 μg of Anti-vGAT for 1 hour. PE* was usedas the secondary and was ap-plied at 2 μg per sample for 30 minutes. The controls (greenand blue lines) are HEK-293 cellsand HEK-293vGAT cells alone,respectively. Each sample contained 2 x 106 cells. Data analysis was done with CellQuest Software.

*Biotinylated Anti-vGAT mixed with Streptavidin-PE.

Conclusions

We made a rabbit polyclonal antibody to the lumenal domain of rat vGAT.Analysis by flow cytometry and immunocytochemistry showed that thisantibody binds to cells expressing rat vGAT. When conjugated to the

ribosome-inactivating protein Saporin, this antibody selectively kills cellsthat express vGAT. Anti-vGAT and Anti-vGAT-SAP are useful reagents for thestudy of rat vGAT and possibly other species. The lumenal domain sequenceof rat vGAT is identical to those of mouse and human, and more testingneeds to be done to determine if this antibody will bind in those species.

The level of expression of vGAT on the cell surface as determined by flow•cytometry correlates with the cytotoxicity of Anti-vGAT-SAP.

Anti-vGAT-SAP was 14 billion-fold more toxic to HEK-293vGAT cells than to•the parental cells.

Anti-vGAT-SAP was 1000-fold amount more toxic to vGAT cells than saporin•without a targeting moiety, based on the EC50.

Figures 6 & 7.

Figure 8. Cytotoxicity Assay.

Figure 3 Figure 4

Figure 5

Figure 3. Stained HEK293vGAT cells showing CellMask plasma membranecounterstain (GFP filter).

Figure 4. Stained HEK-293vGAT cells showing Anti-vGAT-Cy3 staining (Y3 filter).

Figure 5. Merged image of HEK-293vGAT cells showing CellMask plasmamembrane counterstain and Anti-vGAT-Cy3 staining.

Immunocytochemistry. Analysis of Anti-vGAT-Cy3, DAPI and CellMask onHEK-293vGAT cells demonstrated antibody binding on the cell membrane(Figures 3-7). HEK-293vGAT cells were fixed with 4% paraformaldehyde andthen treated with 10 ug/ml Anti-vGAT-Cy3 overnight. 5 μg/ml of DAPI nu-clear counterstain was added to the nuclei, and 1.5X CellMask was addedfor plasma membrane staining. Images captured on Leica DMIL Inverted Fluorescent Microscope at 40X magnification outfitted with Spot Basic Imaging Software and camera.

Figure 6. Stained HEK-293vGAT cells showing DAPI nuclear counterstain (A4 filter).

Figure 7. Merged image of HEK-293vGAT cells showing DAPI nuclear counterstain, CellMask plasma membrane stain and Anti-vGAT-Cy3 staining.

Figure 6 Figure 7

Flow Cytometry. Analysis of HEK-293 cells and HEK-293vGAT cells with Anti-vGAT and PE demonstrated differential expression of vGAT (Figure 2).

HEK-293 is a human embryonic kidney cell line, which does not endogenously express vGAT. Cells transfected with vGAT were tested alongside the parental cell line in flow cytometry to analyze surface vGAT expression. HEK-293vGAT cells showed a 98% shift, while the parental cellsshowed a 22% shift. There was a 76% shift difference between the two celllines treated with Anti-vGAT and PE. All cells were incubated with 10 μg of Anti-vGAT for 1 hour and washed. 2 μg of PE was added as secondary for 30 minutes. After washing, labeled cells were run on a BD FACScan flow cytometer and analyzed by CellQuest software).

Flow cytometry service provided by CytoLogistics LLC, San Diego.

Cytotoxicity. Assay of vGAT-SAP on HEK-293 cells correlates with vGAT levelof expression.

Anti-vGAT-SAP was tested on HEK-293vGAT cells and the parental HEK-293 cells. Anti-vGAT-SAP was 14 billion-fold more toxic to HEK-293vGAT cells than to the parental cells. Anti-vGAT-SAP was 1000-foldmore toxic to cells expressing vGAT than saporin without a targeting moiety,based on the EC50. Both cell lines were plated at 1000 cells/well in a 96-wellplate. Cells were incubated overnight before adding Anti-vGAT-SAP. Cellswere then incubated for 72 hours, followed by development with Sulforho-damine B. The plates were read on Spectramax 24 (Molecular Devices) andthe data were analyzed by PRISM (GraphPad).

Design. The c-terminus of vGAT is located on the lumenal domain and is exposed to the extracellular milieu when the vesicle opens. This is important, because in order to create a targeted toxin, the target must belocated on the cell surface and be endocytosed as a result of binding.

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