Internalization of PSMA along microtubules.

<p>(A) COS-1 cells were transiently transfected with cDNA encoding YFP-PSMA. Two days after transfection cells were fixed and α-tubulin was detected by indirect immunofluorescence. In these cells α-tubulin did not colocalize with YFP-PSMA. (B) COS-1 cells were transiently transfected with cDNA encoding YFP-PSMA. Two days after transfection cells were activated by antibody-induced cross-linking of PSMA, fixed and α-tubulin was detected by indirect immunofluorescence. The figure shows internalization of PSMA upon activation and several endosomes containing YFP-PSMA colocalize with α-tubulin.</p

PSMA associates with Lubrol WX-DRMs as a dimeric protein.

<p>(A) LNCaP cells were solubilized with either 1% Lubrol WX or 1% Triton X-100 (w/v) in PBS. Lysates were subjected to centrifugation at 100.000×g for 90 min in order to separate the insoluble (P) DRM-fraction and the soluble (S) material. After centrifugation the insoluble fraction (P) was solubilized with a lysis-buffer containing 0.5% (w/v) Triton X-100 and 0.5% (w/v) sodium-deoxycholate and equal amounts of total protein from both fractions were loaded for each lane on SDS gels and blotted. The figure shows that PSMA is completely soluble in Triton X-100 and is partially insoluble in Lubrol WX. To verify these DRMs flotillin-2 and caveolin-1 were used as raft marker proteins and the early endosomal antigen 1 (EEA1) was utilized as a non-raft marker. P pellet, S supernatant (B) After centrifugation the insoluble fraction (P) was solubilized with 1% (w/v) Triton X-100 and 1 ml of both insoluble (P) and soluble (S) fraction was loaded on top of a 5–25% continuous sucrose gradient. After 18 h of centrifugation at 100.000×g, 24 fractions of 500 µl each were collected from bottom to top. Half of each fraction was precipitated using ethanol and PSMA was detected by western blot analysis. The figure shows monomeric PSMA peaking in fractions 16–19 where the mannose-rich glycosylated form (PSMA_M) can be distinguished from its complex glycosylated counterpart (PSMA_C). Dimers of PSMA are located in fractions 13–15 that exclusively contain complex glycosylated PSMA (PSMA_C). PSMA in Lubrol WX-DRMs migrates like the dimeric form of PSMA whereas detergent-soluble PSMA is distributed in later fractions (16 and 17) that contain monomeric PSMA.</p

Internalization of PSMA is followed by its association with Triton X-100-DRMs.

<p>(A) COS-1 cells were transiently transfected with cDNA encoding YFP-PSMA. Two days after transfection cells were either activated by antibody-induced cross-linking of PSMA or directly visualized using a Leica TCS SP5 confocal laser microscope. The figure shows that PSMA is internalized and appears in vesicular structures after activation with antibody cross-linking. (B) LNCaP cells were cell-surface biotinylated for 30 min on ice. Cells were washed and quenched twice with 0.1% bovine serum albumin. Afterwards, internalization of PSMA was induced by antibody cross-linking. Remaining biotin on surface proteins was then cleaved by glutathione. Cells were washed again, solubilized with Triton X-100 and DRMs were extracted. After centrifugation the insoluble fraction (P) was solubilized with a lysis-buffer containing 0.5% (w/v) Triton X-100 and 0.5% (w/v) sodium-deoxycholate and PSMA was immunoprecipitated from both insoluble (P) and soluble (S) fractions. Immunoprecipitates were subjected to SDS/PAGE and western blot analysis. The chemiluminescent detection of biotinylated PSMA was carried out using streptavidin horseradish peroxidase. The blots confirm substantial internalization of PSMA upon antibody-induced cross-linking and the internalized PSMA in the antibody-activated samples was localized in Triton X-100-DRMs in contrast to PSMA in the non-activated cells. P pellet, S supernatant * PSMA_C ∧ PSMA_M.</p

PSMA associates with Triton X-100-DRMs upon activation.

<p>(A) LNCaP cells were activated by antibody-induced cross-linking of PSMA at 37°C or 4°C. Subsequently non activated and activated cells were solubilized with either 1% Lubrol WX or 1% Triton X-100 (w/v) in PBS. DRMs (P) and soluble (S) fractions were isolated as described in Fig. 1A. Equal amounts of total protein from all fractions were loaded for each lane on SDS gels and blotted. The distribution of PSMA activated with 7e11c mAb, canine PSMA activated with D2B mAb as well as LPH activated with 909 mAb were analyzed as negative controls. Western blots were developed using D2B mAb for PSMA and anti-GFP mAb for canine PSMA and LPH. The activation of PSMA by antibody-induced cross-linking results in redistribution of PSMA to Triton X-100-DRMs whereas the negative controls show no changes in their detergent solubility properties. P pellet, S supernatant (B) Statistical analysis of results from five independent experiments was performed using GraphPad Prism5 applying the parametric paired t test (two-tailed). Significance was accepted when p<0.05. P nAct - pellet of non activated cells, S nAct - supernatant of non activated cells, P Act - pellet of activated cells, S Act - supernatant of activated cells.</p

Schematic presentation of DRM-associated internalization of PSMA.

<p>PSMA is transported to the Golgi where it associates with Lubrol WX-DRMs. Likewise, homodimeric forms as well as cell surface forms of PSMA associate with similar type of DRMs at the cell surface. Antibody-induced cross-linking of PSMA homodimers results in a partitioning of PSMA and α-tubulin into Triton X-100-DRMs. Concomitantly internalization of PSMA occurs along α-tubulin filaments.</p

Identification of proteins potentially interacting with PSMA.

<p>(A) LNCaP cells were either activated by antibody-induced cross-linking of PSMA or subsequently solubilized with 1% Triton X-100 and DRMs were extracted. After centrifugation the insoluble fraction (P) was solubilized with a lysis-buffer containing 0.5% (w/v) Triton X-100 and 0.5% (w/v) sodium-deoxycholate and equal amounts of protein (400 µg) were precipitated with ethanol over night. Pellets were resuspended in rehydration buffer and 2-dimensional gel electrophoresis was performed. Gels were stained with colloidal coomassie G-250 and protein spots were quantified by using ImageJ. The 2-D gels show variations in the expression levels of different proteins in Triton X-100-DRMs due to antibody-induced activation of PSMA. One of these proteins is tubulin that increased substantially in Triton X-100-DRMs isolated from activated cells. (B) LNCaP cells were either activated by antibody-induced cross-linking of PSMA or subsequently solubilized with 1% Triton X-100 and DRMs were extracted. After centrifugation the insoluble fraction (P) was solubilized with a lysis-buffer containing 0.5% (w/v) Triton X-100 and 0.5% (w/v) sodium-deoxycholate. Equal amounts of total protein from all fractions were loaded for each lane on SDS gels and α- as well as β-tubulin concentrations were detected by western blot analysis. The blots confirm the redistribution of α-tubulin as well as β-tubulin to Triton X-100-DRMs upon activation of PSMA. P pellet, S supernatant (C) Statistical analysis of results from three independent experiments was performed using GraphPad Prism5 applying the parametric paired t test (two-tailed). Significance was considered at p<0.05. P nAct - pellet of non activated cells, S nAct - supernatant of non activated cells, P Act - pellet of activated cells, S Act - supernatant of activated cells.</p

Time course of association of PSMA with Triton X-100-DRMs.

<p>(A) LNCaP cells were activated by antibody-induced cross-linking of PSMA for different time periods. Subsequently non activated and activated cells were solubilized with 1% (w/v) Triton X-100 in PBS. Lysates were subjected to centrifugation at 100.000×g for 90 min in order to separate the insoluble (P) DRM-fractions. Equal amounts of total protein from each fraction were further analyzed by western blotting. The figure shows that redistribution of PSMA to Triton X-100-DRMs occurs in a time-dependent manner. (B) Statistical analysis of results from three independent experiments was performed using GraphPad Prism5 applying the parametric paired t test. Significance was accepted when p<0.05.</p

Image_1_PSMA-Specific CAR-Engineered T Cells for Prostate Cancer: CD28 Outperforms Combined CD28-4-1BB “Super-Stimulation”.jpeg

Prostate cancer (PCa) is the second leading cause of malignancy-related mortality in males in the Western world. Although treatment like prostatectomy and radiotherapy for localized cancer have good results, similar positive outcomes are not achieved in metastatic PCa. Consequently, these aggressive and metastatic forms of PCa urgently need new methods of treatment. We already described an efficient and specific second-generation (2G) Chimeric Antigen Receptor (CAR) against Prostate Specific Membrane Antigen (PSMA), a glycoprotein overexpressed in prostate cancer and also present on neovasculature of several tumor entities. In an attempt to improve efficacy and in vivo survival of anti-PSMA 2G CAR-T cells, we developed a third generation (3G) CAR containing two costimulatory elements, namely CD28 and 4-1BB co-signaling domains, in addition to CD3ζ. Differently from what described for other 3G receptors, our third generation CAR disclosed an antitumor activity in vitro similar to the related 2G CAR that comprises the CD28 co-signaling domain only. Moreover, the additional costimulatory domain produced detrimental effects, which could be attributed to an increased activation-induced cell death (AICD). Indeed, such “superstimulation” resulted in an exhausted phenotype of CAR-T cells, after prolonged in vitro restimulation, a higher frequency of cell death, and an impairment in yielding sufficient numbers of transgenic T lymphocytes. Thus, the optimal combination of costimulatory domains for CAR development should be assessed cautiously and evaluated case-by-case.</p

Assessment of T-body <i>in vivo</i> systemic therapeutic efficacy against disseminated prostate carcinoma.

<p>Rag2^−/−/γc^−/− mice (n = 6) and NOD/SCID mice (n = 6) bearing established bioluminescent PC3-PIP tumors were injected i.v. with 2×10⁷ T-body-hPSMA/eGFP for 3 times at a two day interval. Untreated animals (n = 6 for both mouse strains) were used as controls. (A) Pictures show two representative Rag2^−/−/γc^−/− and NOD/SCID mice (<i>left</i> and <i>right panels</i>, respectively) imaged by BLI at different time points, whereas (B) and (C) graphs report cumulative results of the regions of interest (ROI) in lungs and in total body, respectively. Tumor growth was monitored as photon flux and quantified as photon * sec⁻¹ * cm⁻² * sr⁻¹. Graphs show mean ± SD of three independent experiments. *: P<0.05. The t-Test was used for statistical analysis. (D) Cumulative Kaplan-Meier survival curves of Rag2^−/−/γc^−/− (<i>left panel</i>; untreated mice, black line; median survival = 54 days; treated mice, red line; median survival = 74 days; P = 0.046) and NOD/SCID mice (<i>right panel</i>; untreated mice, black line; median survival = 60 days; treated mice, red line; median survival = not evaluable; P<0.001).</p

Assessment of T-body <i>in vivo</i> loco-regional therapeutic efficacy.

<p>(A) Winn Assay. PC3-PIP (<i>left panel</i>) and PC3 (<i>right panel</i>) tumor cells were inoculated s.c. in SCID mice, alone or mixed 1∶1 with T-body-hPSMA/eGFP at opposite flanks of the same animal. Tumor growth was monitored over time by caliper measurement. Number of mice per group, n = 6. (B) Loco-regional therapy. T-body-hPSMA/eGFP at 72 hours post transduction were administered intralesionally and perilesionally in SCID mice 4 days after s.c. injection of PC3-PIP tumor cells (n = 6); untreated animals served as control group (n = 6). <i>Left panel</i> shows tumor volumes, while <i>right panel</i> reports Kaplan-Meyer survival curves of treated and untreated mice. (C) Expression of hPSMA antigen in prostate tumors. PC3-PIP tumor cells from <i>in vitro</i> cultures (first quadrant) or isolated <i>ex-vivo</i> from control or treated mice (second and third quadrant, respectively; dark line) were evaluated for hPSMA expression by flow cytometry. The grey plot corresponds to the isotype control.</p