Induction of autoantibodies to syngeneic prostate-specific membrane antigen by xenogeneic vaccination

Abstract

Prostate-specific membrane antigen (PSMA) is a prototypical differentiation antigen expressed on normal and neoplastic prostate epithelial cells, and on the neovasculature of many solid tumors. Monoclonal antibodies specific for PSMA are in development as therapeutic agents. Methodologies to actively immunize against PSMA may be limited by immunologic ignorance and/or tolerance that restrict the response to self-antigens. Our studies have previously shown that xenogeneic immunization with DNA vaccines encoding melanosomal differentiation antigens induces immunity in a mouse melanoma model. Here we apply this approach to PSMA to establish proof of principle in a mouse model. Immunization with xenogeneic human PSMA protein or DNA induced antibodies to both human and mouse PSMA in mice. Monoclonal antibodies specific for mouse PSMA were generated to analyze antibody isotypes and specificity for native and denatured PSMA at the clonal level. Most antibodies recognized denatured PSMA, but C57BL/6 mice immunized with xenogeneic PSMA DNA followed by a final boost with xenogeneic PSMA protein yielded autoantibodies that reacted with native folded mouse PSMA. Monoclonal antibodies were used to confirm the expression of PSMA protein in normal mouse kidney. These results establish the basis for clinical trials to test PSMA DNA vaccines in patients with solid tumors that either express PSMA directly or that depend on normal endothelial cells expressing PSMA for their continued growth.

Figure 1

Xenogeneic immunization induces autoantibodies against recombinant mouse PSMA. (a) Sera from mice immunized with immune complexes containing hPSMA, or with DNA vaccines containing hPSMA, mPSMA or empty vector were assayed on ELISA plates coated with recombinant mPSMA protein produced in baculovirus. Data represent the average absorbance for groups of 9–10 mice on wells coated with mPSMA less the absorbance of control wells. Sera were diluted 1/200 for mice that received immune complexes and 1/100 for all other groups. (b) Immunoblot analysis. Three recombinant proteins hPSMA (H), mPSMA (M) and human tyrosinase (Ty) were stained with the MAb anti-TAG (TAG) and with a 1/400 dilution of sera from a mouse immunized with hPSMA DNA (HuPSMA) that recognizes hPSMA and mPSMA but not Tyrosinase. The molecular weight of the recombinant mPSMA protein is less than that of hPSMA because this construct contained only the extracellular domain.

Figure 2

Xenogeneic immunization with hPSMA induces autoantibodies against native mouse PSMA. Sera from representative mice were used to stain NIH 3T3 cells transduced with empty SFV vector (solid purple), hPSMA (green line) or mPSMA (pink line) by flow cytometry. Mice were immunized with hPSMA protein in an immune complex, mPSMA, or hPSMA DNA vaccines.

Figure 3

Cross-reactive monoclonal antibodies were obtained from a mouse immunized with hPSMA protein. Whole cell lysates were prepared from NIH 3T3 cells transduced with empty SFV vector (vec), hPSMA (Hu) or mPSMA (Mo). After separation by SDS-PAGE, PSMA was visualized by incubation with cross-reactive (13D6, 3E2) or hPSMA-specific CYT-356 (CYT) monoclonal antibodies.

Figure 4

Monoclonal antibodies 5H12 and 7C12 are specific for native hPSMA. Flow cytometry data is presented for representative hybridoma supernatants isolated from a mouse immunized with hPSMA protein. Representative MAb supernatants were used to stain NIH 3T3 cells transduced with empty SFV vector (solid purple), hPSMA (green line) or mPSMA (pink line).

Figure 5

mPSMA is expressed in normal mouse kidney sections. Antibodies to mPSMA were used to stain paraffin-embedded sections of normal mouse kidney. Primary antibodies were naive rabbit sera (a), immune sera from a rabbit immunized with peptide 483 QSP (b), or mouse MAbs 3E2, 13D6 or 7E11 (c,d,e, respectively).

Figure 6

Cross-reactive MAbs recognize a 100 kDa protein in mouse kidney lysates. Thirty micrograms total protein from normal mouse kidney (K) or from cultured human LNCaP cells (L) were separated by SDS-PAGE and PSMA proteins were visualized by Western analysis. The MAb supernatants 3E2 and 13D6 recognize both hPSMA and mPSMA, while 5H12 and CYT-356 recognize only the hPSMA protein in LNCaP cell lysates.

Figure 7

Flow cytometry of cross-reactive hybridomas. Two individual hybridomas, 9C1 and 11C8, were identified by flow cytometry with 3T3mPSMA cells. MAb supernatants were used to stain NIH 3T3 cells transduced with empty SFV vector (solid purple), hPSMA (green line) or mPSMA (pink line) by flow cytometry.