Construction and in vitro characterization of the PSMA-DMAb plasmid

Human PSMA is a type II integral membrane glycoprotein that is highly expressed in prostate secretory-acinar epithelium as well as in several extra-prostatic tissues, and it possesses 86% identity and 91% similarity to mouse PSMA [20]. A plasmid capable of directing in vivo antibody production was designed by (1) creating a cassette consisting of the full-length coding sequences for the variable heavy (V H ) and light (V L ) immunoglobulin (Ig) chains from the published sequence of an anti-PSMA mAb driven off a CMV promoter; (2) optimizing the cassette sequence to improve its expression; and (3) cloning the cassette into a pVax1plasmid (Fig. 1a). Antibodies targeting PSMA produced from this optimized DNA plasmid will henceforth be referred to as PSMA-DMAb.

Fig. 1 Construct design for human PSMA-DMAb and in vitro expression. a The schematic of the PSMA-DMAb plasmid construction. b–d Confirmation of in vitro expression and specificity of PSMA-DMAb produced antibodies in transfected 293T cells; b ELISA to quantitate human IgG levels in supernatants collected at 48 h post-transfection. c Binding specificity of supernatant IgG (1:50 dilution) to recombinant human PSMA (rPSMA) by ELISA. d Western blot analysis of rPSMA or recombinant HIV envelope protein (rHIV-Env) blotted with tissue culture supernatants from transfected 293T cells. Error bars in b and c are SDs Full size image

To confirm that the plasmid directs production of fully assembled IgG, human embryonic kidney 293T cells were transfected with either empty pVax1 or PSMA-DMAb plasmid. Supernatants collected from cells at 48 h post-transfection were assayed by ELISA to quantify total human IgG levels. A concentration of nearly 800 ng/ml of human IgG was measured in supernatants of PSMA-DMAb plasmid-transfected cells (Fig. 1b). A binding ELISA performed on the same supernatants indicated that the IgG produced from PSMA-DMAb plasmid-transfected cells bound to recombinant human PSMA with high affinity (Fig. 1c). Western blot analysis further confirmed the specificity of PSMA-DMAb plasmid-derived antibodies for binding to recombinant human PSMA protein (Fig. 1d). The results indicate that the PSMA-DMAb plasmid can direct the production of anti-PSMA-specific antibodies in vitro.

PSMA-DMAb plasmid administration generates PSMA-specific antibodies in vivo

The ability of the PSMA-DMAb plasmid to direct antibody production in vivo was evaluated in both immune-deficient B6.Cg-Foxn1 nu/J (C57BL/6 nude) and immune-competent C57BL/6J mice. Groups of five mice received a single 100 μg injection of PSMA-DMAb plasmid intramuscularly in their quadriceps muscle followed by EP for enhanced delivery [16]. Injected mice were bled at various time points post-injection to obtain sera that was evaluated by ELISA to quantitate human IgG levels. Human IgG became detectable in sera of injected mice beginning on day 5 post-injection, with peak levels achieved at day 14 post-injection in both C57BL/6 nude (1.17 ± 0.41 μg/ml, Fig. 2a) and C57BL/6 (0.82 ± 0.11 μg/ml, Fig. 2b) mice. While elevated human IgG levels persisted in C57BL/6 nude mice beyond 50 days, the levels in C57BL/6 mice dropped to baseline values by day 35 post-injection likely due to the mouse anti-human antibody response [21, 22]. Serum collected at day 14 post-injection from PSMA-DMAb plasmid-injected C57BL/6 nude mice was evaluated by ELISA (Fig. 2c) and Western blot (Fig. 2d) to evaluate the affinity and specificity of serum IgG for recombinant human PSMA. Both assays show that the IgG in day 14 sera recognized human PSMA, but not irrelevant HIV envelope protein with high affinity and specificity, suggesting that the IgG are properly folded and functional PSMA-DMAb.

Fig. 2 Confirmation of in vivo expression and specificity of the PSMA-DMAb in mice. Measurement of human IgG in sera from a immunodeficient B6.Cg-Foxn1 nu/J (C57BL/6 nude) mice (n = 5) and b immune-competent (C57BL/6) mice. Mice were injected with PSMA-DMAb plasmid as described in “Materials and methods” and sera levels of human IgG were measured at various time points post-injection. c Binding specificity, as a function of dilution, measured by ELISA in sera from PSMA-DMAb plasmid-injected nude mice collected at day 14 post-DNA administration. rPSMA and rHIV-1 Env proteins (negative control) were used as the binding antigen. d Binding specificity of sera from PSMA-DMAb plasmid-administered nude mice to rPSMA by Western blot analysis. rHIV-Env is used as a negative control. e, f Measurement of anti-PSMA levels in prostate tissue of PSMA-DMAb injected mice. e Quantification of human IgG in prostate tissue of PSMA-DMAb-plasmid and pVax1 injected mice at day 14 by ELISA. Individual IgG concentrations and mean values are shown. f Immunohistochemical (IHC) staining for human IgG of prostate tissues from PSMA-DMAb plasmid and pVax1 injected mice at day 14. Samples were evaluated at magnification 20×. Scale bar represents 100 µm Full size image

In vivo distribution of PSMA-DMAb in prostate tissue was studied in mice by harvesting tissues 7 days post-plasmid injection and performing ELISA and immunohistochemistry for IgG quantification. Prostate tissue from mice administered the PSMA-DMAb plasmid exhibited higher levels of human IgG compared to prostate tissue from empty pVax1 plasmid-injected mice as measured by ELISA of tissue homogenates (Fig. 2e). Further, prostate tissues were evaluated by immunohistochemistry staining for anti-human-Fc expression. A strong immunostaining signal was detected on the cell membranes and within the prostate for the PSMA-DMAb plasmid-injected mice, but not pVax1-treated controls (Fig. 2f). Together, these findings demonstrated that the PSMA-DMAb plasmid can direct the production of robust levels of PSMA-specific human IgG in vivo.

In vivo generated PSMA-DMAbs bind to PSMA on prostate cancer cells

We next evaluated the ability of PSMA-DMAb in mouse sera to bind PSMA on tumor cells and tissues. Two PSMA-expressing prostate cancer cell lines were chosen for the initial studies: (1) LNCaP cells, derived from human prostate adenocarcinoma cells; and (2) transgenic adenocarcinoma mouse prostate (TRAMP)-C2 cells derived from a heterogeneous 32-week tumor grown in the TRAMP mouse model. Both cell lines were incubated sequentially with day 14 sera from pVax1 or PSMA-DMAb plasmid-injected C57BL/6 nude mice followed by a fluorescently labeled anti-human IgG secondary antibody. Histograms (Fig. 3a) and mean fluorescent intensity MFI (Fig. 3b) obtained from flow cytometry analysis of stained cells show that in vivo produced PSMA-DMAbs bind to both PSMA-positive tumor cell lines. No staining was observed on PSMA-negative PC3 cells (data not shown).

Fig. 3 Flow cytometry analysis of PSMA-expressing LNCaP and TRAMP-C2 positive cells. a Overlaid histogram of PSMA expression on LNCaP (upper panel) and TRAMP-C2 (lower panel) cells. Histograms show live LNCaP or TRAMP-C2 cells stained with either day 14 sera from mice injected with pVax1 plasmid (pink) or day 14 sera from mice injected with PSMA-DMAb plasmid (blue). Representative flow cytometry of LNCaP and TRAMP-C2 cells stained with a commercial anti-PSMA antibody as a control. b Quantitation of MFI of PSMA binding for all mouse sera to LNCaP and TRAMP-C2 based on values measured in the five mice presented in a. Error bars indicated in b are SD Full size image

In addition to normal and cancerous prostate cells, several studies have reported PSMA expression on a wide variety of tumors, especially on tumor neovasculature [23, 24]. Immunofluorescence assays were used to evaluate the ability of PSMA-DMAb to bind to PSMA expressed on tissue sections of human bladder and kidney tumors (Fig. 4). The results show that PSMA-DMAb was able to stain cells in the bladder and kidney tumor tissue sections, but not cells in normal ovarian tissues, confirming previous reports of PSMA expression in these tumors [25]. Furthermore, the staining shows that PSMA distribution is homogeneous throughout the bladder and kidney tumor sections. This data confirms that a PSMA-DMAb retains specificity for PSMA and specifically binds PSMA on the surface of human tumor cells.

Fig. 4 PSMA-DMAb generated antibodies bind to human bladder and kidney carcinoma tissue sections. Tissue sections were stained with pooled sera from mice collected 14 days after the administration of PSMA-DMAb plasmid. Results of the staining of the bladder and kidney carcinoma tissue sections are indicated in the top and middle rows of the panels, respectively, and normal ovarian tissues in the bottom. The a panels show DAPI staining of cell nuclei. The b panels show staining with anti-human IgG Alexa Fluor 488 following incubation with PSMA-DMAb sera. Panel c shows composite staining (DAPI +Alexa Fluor 488), while panel d is a magnified in photo of the composite panels Full size image

PSMA-DMAbs possess antibody-dependent cell-mediated cytotoxicity activity

The biological activity of PSMA-DMAb was next evaluated by using an antibody-dependent cell-mediated cytotoxicity (ADCC) mechanism of action assay [26, 27]. The assay involves incubating PSMA-expressing LNCaP cells with effector cells for 6 h in the presence of different concentrations of serum from pVax1 or PSMA-DMAb plasmid-injected mice. The effector cells are Jurkat cells that stably express high-affinity V158 FcγRIIIa and a gene for firefly luciferase driven off a nuclear factor of activated T cell (NFAT) response element [28]. The assay readout is based on activation of gene transcription in effector cells as measured by firefly luciferase production. As indicated in Fig. 5a, day 14 serum from PSMA-DMAb plasmid-injected mice mediates an ADCC effect.

Fig. 5 PSMA-DMAb mediates targeted death of LNCaP cells. a ADCC activity of PSMA-DMAb-generated antibodies. Target LNCaP cells were incubated for 6 h with the engineered Jurkat effector cells along with various dilutions of day 14 PSMA-DMAb sera samples. Negative controls such as absence of target cells (LNCaP) and no antibody, and a rPSMA-mAb as a positive control were used. Luciferase activity was measured. Results are representative data from two independent experiments. b Flow cytometric analysis of the effects of sera collected from PSMA-DMAb plasmid-administered mice on LNCaP cell death. Day 14 sera were incubated with LNCaP cells in the absence or presence of human PBMCs. Following washing, cells were stained with Annexin-V and propidium iodide (PI), according to the manufacturer’s assay specifications. Gated FACS scan panels are shown for the various treatments: pVax1 control (10 μg), 1 or 10 μg PSMA-DMAb and non-treated cell control. Figure illustrates a representative experiment out of two performed independently Full size image

As a second demonstration of the biological activity of PSMA-DMAb, flow cytometry was used to measure apoptosis and necrosis of LNCaP cells that were co-cultured with human PBMCs in the presence of sera from pVax1 or PSMA-DMAb plasmid-injected mice. The results (Fig. 5b) show that there was a statistically significant increase in apoptosis (Q3 section of the histogram) as well as necrosis (Q2 section in the histogram) for LNCaP cells co-cultured with human PBMCs in the presence of PSMA-DMAb in comparison to control pVax1 sera. Combined, these findings show that the synthetic PSMA-DMAb can bind Fc receptors and mediate an ADCC effect on tumor cells [29].

PSMA-DMAb represses tumor growth in a TRAMP-C2 tumor challenge mouse model

In vivo functional activity of PSMA-DMAb was assessed using a TRAMP-C2 tumor challenge mouse model [30]. For this assay, C57BL/6 mice were subcutaneously implanted with 1 × 106 TRAMP-C2 tumor cells and then injected 1 week later with 100 μg of either pVax1 or PSMA-DMAb plasmid by intramuscular injection with enhanced EP [30]. Mice were followed for up to 56 days with regular measurements of tumor size made on each mouse during this period (Fig. 6a). Tumors in the pVax1-treated mice began to grow at day 7–10 post-implantation, while tumors were not detectable in PSMA-DMAb-treated mice until days 15–17. Rapid tumor growth was noted for the control groups (pVax1), but the PSMA-DMAb-treated group exhibited an obvious suppression of tumor growth due to the antibody-mediated tumor-protective immunity. Over the course of the 56-day observation period, there was a statistically significant reduction in average tumor volumes (p = 0.0201) (Fig. 6b) and a significant improvement in survival (p = 0.0280) in mice receiving the PSMA-DMAb construct compared to the control mice (Fig. 6c). It is likely that this effect might be further enhanced in the absence of the mouse anti-human antibody response. Visual inspection of tumors (Fig. 6d) that developed in each group revealed that the tumors in the PSMA-DMAb group were impacted early and remained small and subdermal, while tumors in the pVax1 control group protruded out of the skin and became ulcerated.

Fig. 6 PSMA-DMAb administration induces anti-tumor immunity in a TRAMP-C2 tumor cell mouse challenge model. a Schema of TRAMP-C2 tumor cell administration and plasmid administration into C57BL/6 mice. Mice were administered subcutaneously 1 × 106 TRAMP-C2 cells followed 1 week later by intramuscular injection of 100 μg of the DNA. b–d Assessment of tumor development in pVax1 and PSMA-DMAb plasmid-injected mice. b Tumor volumes (mm3) were measured weekly, in mice for up to 56 days post-tumor administration. c Kaplan–Meier curves (n = 10) showed the tumor survival time of mice in the pVax1 and PSMA-DMAb groups. d Representative mice with TRAMP-C2 tumors from pVax1 and PSMA-DMAb plasmid-injected groups at day 56 post-tumor administration. e Kaplan–Meier curves (n = 10) show the effect of NK cell depletion on PSMA-DMAb-mediated tumor survival time Full size image

The anti-tumor activity of many therapeutic antibodies including ADCC and antibody-dependent cellular phagocytosis (ADCP) is dependent on the interaction of the IgG-Fc domain with Fc gamma receptors (FcγRs) on effector cells. Natural killer cells express high levels of FcγRs, therefore we also examined the contribution of NK cells to the observed effects of PSMA-DMAb on tumor growth. Previous studies have reported that human IgG can bind to all activating mouse FcγRs and can induce ADCC/ADCP with mouse NK cells and mouse macrophages [29]. Groups of mice were treated with either control IgG or the NK cell-depleting anti-AGM1 IgG antibody and then implanted with TRAMP-C2 cells. One week later, mice were given a single injection of either pVax1 or PSMA-DMAb plasmid and were subsequently evaluated for tumor growth up to 56 days. There was a rapid onset of tumor development, accelerated tumor growth, and decreased survival in PSMA-DMAb-immunized, NK cell-depleted mice (p = 0.0019, Fig. 6e), but not in those pretreated with the control IgG. Taken together, these data demonstrate that PSMA-DMAb can exert a profound therapeutic effect on a PSMA-expressing tumor in vivo, supporting the possible application of this therapy for the treatment of prostate cancer.