Preclinical studies of a new anti-psma (prostate specific membrane antigen) antibody for diagnosis and immunotargeting

Prostate cancer (PCa) is the second leading cause of cancer death in men in western countries. Whereas primary organ-confined disease can be properly treated and cured by surgery and/or radiation therapy, there are limited therapeutic options for the advanced forms of this disease (metastatic, hormone-refractory). Since the conventional therapeutic approaches are poorly effective in the advanced stages of PCa, several investigations, prompted by the discovery of antigens expressed in organ-specific patterns, have proposed and studied different immunological strategies for immunotherapy and immunodiagnosis. Prostate Specific Membrane Antigen (PSMA) has recently emerged as one of the most promising biomarkers in the diagnosis and treatment of prostate cancer and its clinical relevance is presently being evaluated in several immunotherapy trials. Aims of my work were: produce and characterize a new mAb anti-PSMA and its scFv format and study their immunotherapeutical and diagnostic applications. Antibody, scFv fragment and Immunotoxins Our anti-PSMA mAb D2/B as whole molecule or in scFv format recognizes PSMA expressed on LNCaP cells with good specificity and affinity; D2/B shows either much affinity than J591 Ab which is currently under phase II clinical trials; at the same time the scFv format of D2/B shows only 20-fold lower affinity in respect to whole Ab. Moreover we have demonstrated that our Ab is able to recognize the PSMA antigen in the native and denaturated form (western Blot, IHC). When compared to the 7E11c, D2/B mAb shows, in IHC analysis, the same pattern of recognition both on criopreserved and on formalin fixed prostate cancer tissues; no labeling was demonstrated on antigen negative normal tissues. D2/B mAb was chemically linked to ricin A chain toxin obtaining a powerful immunodelivered drug (immunotoxin, IT) with specific cytotoxic activity on PSMA+ cells; in a cytotoxic assay on LNCaP cells D2B-RTA IT shows an IC50=10-10 M/L only 35 times higher than the IC50 of J591- RTA IT (3.5*10-11). In order to reduce the molecular mass of this IT and subsequently improve the penetration in the tumor mass, we decided to clone the single-chain (scFv) variable region of PSMA-specific antibody into a prokaryotic expression vector containing the catalytic subunit of Pseudomonas aeruginosa exotoxin A (PE40). In an 3H-Tdr incorporation assay the PSMA-specific fusion immunotoxin (IT) inhibit half the proliferation of the PSMA-positive population tested LNCaP at an estimated concentration of approximately 2*10-11 M/L. No IC50 can be defined on PSMA-negative PC3 cell line, neither once tested at a concentration of 10-7 M/L. Similarly our IT shows a great cytotoxicity against hPSMA-transfected MCF-7 cell line (IC50 below 10-11 M/L) but not against mock-transfected one (IC50 3*10-8 M/L). As a further proof of specificity we observed that the cytotoxic activity of 10-9 M/L of the abovementioned IT, on LNCaP cells, is fully prevented by addition of whole molecule PSMA-specific antibody at a concentration of 0,7*10-5 M/L. Gold Nanoparticles Specific targeted delivery and control drug release are desirable properties of a drug for tumor therapy. Nanomedicine, the science which studies the application of nanotechnology to disease treatment, might be of help. Targeted Nanoparticles (NP) for their size and structure are able to enhance the accumulation in the tumor of encapsulated of linked/adsorbed molecules (gene, drug). We have therefore investigated the binding properties of gold-NPs (20 um Æ) charged with a reporter solution (Texas red) and conjugated to mAb D2/B. D2/B-NP binding to LNCaP (PSMA+) cells has been assessed by cytometry. We have measured a MFI (mean fluorescence value) of 1,383 for D2/B-NP whereas the MFI of the negative control (CTRL-) was 68. The binding specificity was confirmed on PSMA– Jurkat cells (MFI of 121 and 101 for D2/B-NP and CTRL-, respectively). Binding and internalization of D2/B-NP in LNCaP cells were assayed by confocal microscopy; detection of D2/B-NP by surface-enhanced Raman scattering also revealed the selective binding of the NPs to Ag+ cells. Our results of specific delivery and internalization support our idea to use mAb anti-PSMA targeted NPs for imaging of tumor sites and to carry toxic drugs inside the tumor. Syngeneic mouse model: A syngeneic mouse models has been created transfecting B16 murine melanoma cells with phCMV3-hPSMA vector. In FACS analysis B16 hPSMA MFI value was 11,578 respect a negative control value of 188; cytotoxic assays performed on this clone with scFvD2B-PE40 IT show an IC50= 4*10-9 M/L; the whole toxin PE shows in the same assays an IC50= 6*10-9 M/L. When B16 WT cells have been used the IC50 values were >3*10-7 M/L and 6*10-9 M/L for scFvD2B-PE40 and PE respectively. To verify the tumorigenicity of our B16-hPSMA in C57BL/6 mice, different amount of transfected cells 0.5, 1 and 2 millions has been inoculated iv; after 21 days we have measured the number of metastasis at the lung level. There are about 140 metastasis/lung in mice inoculated with 1 and 2 millions of cells and 60 metastasis in mice inoculated with 0.5 millions of cells. Immunoimaging The first series of experiments to conjugate Ab D2B with quantum dot, don’t have showed good results. So we have decided to use the fluorocrome Cy5.5; after conjugation with the fluorocrome D2/B mAb Ab preserves its ability to recognize LNCaP cells without altering the specificity and affinity. Therefore we have plan analyze if it was possible to capture “in vivo” the fluorescence signal of labelled LNCaP cells; we have labeled LNCaP cells “in vitro” with Ab D2B-Cy5.5 and than we have inoculated SCID mice sc with 1*106 and 7.5*106 cells. With this experiment we have demonstrated that Explore Optix instrument is able “in vivo” to show the fluorescence signal derived from the tumor labeled cells. At the same time we are able to identify “in vivo” the tumor mass, stable tumor obtained inoculating sc LNCaP cells, injecting iv Ab D2/B labeled with Cy5.5.