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Prostate cancer (PCa) is the second most common cancer in men, causing more than 300,000 deaths every year worldwide. Due to their superior cell-killing ability and the relative simplicity of their preparation, immunotoxin molecules have great potential in the clinical treatment of cancer, and several such molecules have been approved for clinical application. In this study, we adopted a relatively simple strategy based on a single-domain antibody (sdAb) and an improved Pseudomonas exotoxin A (PE) toxin (PE24X7) to prepare a safer immunotoxin against prostate-specific membrane antigen (PSMA) for PCa treatment. The designed anti-PSMA immunotoxin, JVM-PE24X7, was conveniently prepared in its soluble form in an Escherichia coli ( E. coli ) system, avoiding the complex renaturation process needed for immunotoxin preparation by the conventional strategy. The product was very stable and showed a very strong ability to bind the PSMA receptor. Cytotoxicity assays showed that this molecule at a very low concentration could kill PSMA-positive PCa cells, with an EC 50 value (concentration at which the cell viability decreased by 50%) of 15.3 pM against PSMA-positive LNCaP cells. Moreover, this molecule showed very good killing selectivity between PSMA-positive and PSMA-negative cells, with a selection ratio of more than 300-fold. Animal studies showed that this molecule at a very low dosage (5 × 0.5 mg/kg once every three days) completely inhibited the growth of PCa tumors, and the maximum tolerable dose (MTD) was more than 15 mg/kg, indicating its very potent tumor-treatment ability and a wide therapeutic window. Use of the new PE toxin, PE24X7, as the effector moiety significantly reduced off-target toxicity and improved the therapeutic window of the immunotoxin. The above results demonstrate that the designed anti-PSMA immunotoxin, JVM-PE24X7, has good application value for the treatment of PCa.

Adoptive cell transfer (ACT) is an emerging immunotherapy technique that restricts tumor growth and invasion in cancer patients. Among the different types of ACT, chimeric antigen receptor (CAR)T‑cell therapy is considered to be the most advanced and a potentially powerful technique for the treatment of cancer in clinical trials. The primary aim of CART‑cell therapy is to destroy cancer cells and therefore, it serves an important role in tumor immunotherapy. CART‑cell therapy has been demonstrated to mainly treat blood cancer by targeting cluster of differentiation (CD)‑19, CD20, CD22, CD33 and CD123. However, the use of CART‑cell therapy for treating solid tumors is currently under extensive investigation. With respect to prostate cancer, prostatic acid phosphatase, prostate‑specific antigen, prostate‑specific membrane antigen (PSMA), prostate stem cell antigen, T‑cell receptor γ alternate reading frame protein, transient receptor potential‑p8 and six‑transmembrane epithelial antigen of the prostate 1 are among the identified target antigens for prostate tumors. However, mesothelin, fibroblast activation protein, epidermal growth factor receptor, carcinoembryonic antigen, disialoganglioside‑2 and human epidermal growth factor 2 are among the main targets of CART‑cell therapy in the case of other types of solid tumors. The main challenges in CART‑cell therapy are the selection of the target antigens and the modulation of the ideal tumor microenvironment for T‑cells to fight against the cancer. The present review focuses on the 1st, 2nd, 3rd and 4th generations of anti‑PSMA CARs and their application for combating prostate carcinoma.

Background: Recently, we described a novel modular platform technology in which T cell-recruitment and tumor-targeting domains of conventional bispecific antibodies are split to independent components, a universal effector module (EM) and replaceable monospecific/monovalent target modules (TMs) that form highly efficient T cell-retargeting complexes. Theoretically, our unique strategy should allow us to simultaneously retarget T cells to different tumor antigens by combining the EM with two or more different monovalent/monospecific TMs or even with bivalent/bispecific TMs, thereby overcoming limitations of a monospecific treatment such as the selection of target-negative tumor escape variants., Methods: In order to advance our recently introduced prostate stem cell antigen (PSCA)-specific modular system for a dual-targeting of prostate cancer cells, two additional TMs were constructed: a monovalent/monospecific TM directed against the prostate-specific membrane antigen (PSMA) and a bivalent/bispecific TM (bsTM) with specificity for PSMA and PSCA. The functionality of the novel dual-targeting strategies was analyzed by performing T cell activation and chromium release assays., Results: Similar to the PSCA-specific modular system, the novel PSMA-specific modular system mediates an efficient target-dependent and -specific tumor cell lysis at low E:T ratios and picomolar Ab concentrations. Moreover, by combination of the EM with either the bispecific TM directed to PSMA and PSCA or both monospecifc TMs directed to either PSCA or PSMA, dual-specific targeting complexes were formed which allowed us to kill potential escape variants expressing only one or the other target antigen., Conclusions: Overall, the novel modular system represents a promising tool for multiple tumor targeting., (© 2014 Wiley Periodicals, Inc.)

Prostate-specific membrane antigen (PSMA) is a membrane protein that is overexpressed manifold in prostate cancer and provides an attractive target for therapy. PSMA ADC is an antibody-drug conjugate (ADC) that consists of a fully human anti-PSMA monoclonal antibody conjugated to monomethylauristatin E through a valine-citrulline linker. In this study, the antitumor activity of PSMA ADC was evaluated against a panel of prostate cancer cell lines in vitro and in a novel in vivo model of taxane-refractory human prostate cancer. In vitro cell killing was efficient for cells with abundant PSMA expression (>10(5) molecules/cell; IC(50) ≤ 0.022 nmol/L) and 1,000-fold less efficient for cells with undetectable PSMA (IC(50) > 30 nmol/L). Intermediate potency (IC(50) = 0.80 nmol/L) was observed for cells with approximately 10(4) molecules of PSMA per cell, indicating a threshold PSMA level for selective cell killing. Similar in vitro activity was observed against androgen-dependent and -independent cells that had abundant PSMA expression. In vitro activity of PSMA ADC was also dependent on internalization and proper N-glycosylation/folding of PSMA. In contrast, less potent and nonselective cytotoxic activity was observed for a control ADC, free monomethylauristatin E, and other microtubule inhibitors. PSMA ADC showed high in vivo activity in treating xenograft tumors that had progressed following an initial course of docetaxel therapy, including tumors that were large (>700 mm(3)) before treatment with PSMA ADC. This study defines determinants of antitumor activity of a novel ADC. The findings here support the clinical evaluation of this agent in advanced prostate cancer.

Preferentially expressed antigen in melanoma (PRAME) and prostate-specific membrane antigen (PSMA) are tumor-associated antigens implicated in cellular differentiation, genetic stability, and angiogenesis. MKC1106-PP is an immunotherapeutic regimen cotargeting PRAME and PSMA, comprised of a recombinant plasmid (pPRA-PSM encoding fragments derived from both antigens) and 2 peptides (E-PRA and E-PSM derived from PRAME and PSMA, respectively). This multicenter study evaluated MKC1106-PP with a fixed plasmid dose and 2 different peptide doses, administered by intralymph node injection in a prime-boost sequence in human leukocyte antigen-A*0201 and tumor-antigen-positive patients with progressing metastatic solid tumors who had failed standard therapy. Immune monitoring was done by tetramer and enzymatic-linked immune spot analysis. The treatment was well tolerated, with no significant differences in safety, immune response, and clinical outcome relative to peptide doses. Fifteen of 24 evaluable patients showed an immune response, as defined by the expansion of PRAME-specific or PSMA-specific T cells in the blood. There were no partial or complete responses by the Response Evaluation Criteria in Solid Tumors. Seven patients showed stable disease (SD) for 6 months or longer, or prostate specific antigen decline: 4 of 10 with prostate carcinoma, 2 of 2 with renal clear cell carcinoma, and 1 of 10 with metastatic melanoma. In addition, there was an association between the induction and persistence of antigen-specific T cells in blood above baseline levels and disease control, defined as SD for 6 months or longer. These results support further development of MKC1106-PP in specific clinical indications.

Prostate cancer remains a leading cause of death for men in Western civilization. Despite the effectiveness of surgical prostatectomy, radiotherapy and hormonal therapy, a significant proportion of patients progress to advanced metastatic disease for which there are currently no curative treatment options. Therefore, new therapeutic approaches need to be considered. The prostate-specific membrane antigen is a cell-surface glycoprotein that is highly and specifically expressed on prostate epithelial cells and strongly upregulated in prostate cancer at all stages. These characteristics make it an attractive target for antibody-based imaging and therapies and the first anti-prostate-specific membrane antigen agents have already entered clinical trials. The proposed strategies include targeted toxins and radiotherapeutics as well as immunotherapeutic agents and vaccines.

Purpose: Based on prostate-specific membrane antigen (PSMA) expression on the vasculature of solid tumors, we performed a phase I trial of antibody J591, targeting the extracellular domain of PSMA, in patients with advanced solid tumor malignancies. This was a proof-of-principle evaluation of PSMA as a potential neovascular target. The primary end points were targeting,toxicity, maximum-tolerated dose, pharmacokinetics (PK), and human antihuman antibody (HAHA) response., Patients and Methods: Patients had advanced solid tumors previously shown to express PSMA on the neovasculature. They received 111Indium (111ln)-J591 for scintigraphy and PK, followed 2 weeks later by J591 with a reduced amount of 111In for additional PK measurements. J591 dose levels were 5, 10, 20, 40, and 80 mg. The protocol was amended for six weekly administrations of unchelated J591. Patients with a response or stable disease were eligible for re-treatment. Immunohistochemistry assessed PSMA expression in tumor tissues., Results: Twenty-seven patients received monoclonal antibody (mAb) J591. Treatment was well tolerated. Twenty (74%) of 27 patients had at least one area of known metastatic disease targeted by 111In-J591, with positive imaging seen in patients with kidney, bladder, lung, breast, colorectal, and pancreatic cancers, and melanoma. Seven of 10 patient specimens available for immunohistochemical assessment of PSMA expression in tumor-associated vasculature demonstrated PSMA staining. No HAHA response was seen. Three patients of 27 with stable disease received re-treatment., Conclusion: Acceptable toxicity and excellent targeting of known sites of metastases were demonstrated in patients with multiple solid tumor types, highlighting a potential role for the anti-PSMA antibody J591 as a vascular-targeting agent.

Background: Dendritic cell (DC)-based immunotherapy is a promising approach to augment tumor antigen-specific T cell responses in cancer patients. However, tumor escape with down-regulation or complete loss of target antigens may limit the susceptibility of tumor cells to the immune attack. Concomitant generation of T cell responses against several immunodominant antigens may circumvent this potential drawback. In this trial, we determined the immunostimulatory capacity of autologous DC pulsed with multiple T cell epitopes derived from four different prostate-specific antigens in patients with advanced hormone-refractory prostate cancer., Patients and Methods: Autologous DC of HLA-A*0201(+) patients with hormone-refractory prostate cancer were loaded with antigenic peptides derived from prostate stem cell antigen (PSCA(14-22)), prostatic acid phosphatase (PAP(299-307)), prostate-specific membrane antigen (PSMA(4-12)), and prostate-specific antigen (PSA(154-163)). DC were intradermally applied six times at biweekly intervals followed-in the case of an enhanced immune response-by monthly booster injections. Immune monitoring during the time of ongoing vaccinations (12-59 weeks) included ex vivo ELISPOT measurements, MHC tetramer analysis and in vitro cytotoxicity assays., Results: Of the initial six patients, three qualified for long-term multi-epitope DC vaccination. This regime was tolerated well by all three patients. The vaccination elicited significant cytotoxic T cell responses against all prostate-specific antigens tested. In addition, memory T cell responses against the control peptides derived from influenza matrix protein and tetanus toxoid were efficiently boosted. Clinically, the long-term DC vaccination was associated with an increase in PSA doubling time., Conclusions: DC-based multi-epitope immunotherapy with repeated boosting in men with hormone-refractory prostate carcinoma is feasible and generates efficient cellular antitumor responses.

DNA vaccines provide an attractive technology against cancer because of their safety record in humans and ease of construction, testing and manufacture. In this study, several DNA fragments encoding multiple cytotoxic T lymphocyte (CTL) and T helper cell epitopes were selected from human prostate-specific membrane antigen (hPSM), mouse prostatic acid phosphatase (mPAP), and human prostate-specific antigen (hPSA). These DNA fragments were ligated together to form a novel fusion gene, termed the 3P gene. The secondary lymphoid tissue chemokine (SLC), 3P and human immunoglobulin G Fc genes were inserted into pcDNA3.1 to construct a DNA vaccine, designated pSLC-3P-Fc. After vaccination, the DNA is taken up by cells that produce and secrete the SLC-3P-Fc fusion proteins, termed chemotactic antigen (chemo-antigen). The secreted chemo-antigens, in addition to promoting the co-localization of naive, non-polarized memory T cells and dendritic cells, are efficiently captured and processed by dendritic cells via receptor-mediated endocytosis and then cross-presented to both major histocompatibility complex class I and class II in a cognate manner. The results of this study demonstrate that vaccination with pSLC-3P-Fc by gene gun inoculation induced a strong antitumour response in a mouse tumour model, which significantly inhibited tumour growth and prolonged the survival time of the tumour-bearing mice. In vitro, the secreted SLC-3P-Fc fusion protein can attract lymphocytes from human peripheral blood mononuclear cells (PBMC); when human lymphocytes were stimulated by pSLC-3P-Fc-transfected autologous PBMC, CTLs were induced which could specifically kill hPSM-, hPAP-, or hPSA-expressing tumour cells. These observations provide a new vaccine strategy for cancer therapy through promoting the co-localization of lymphocytes and the concomitant enhancement of antigen-specific CD4+ helper and CD8+ cytotoxic T-cell responses against tumour.

Determination of the immunoreactive fraction (IF) of radiolabeled monoclonal antibodies (MAb) is essential to the understanding of the effects of radiolabeling and subsequent target-specific tumor localization. There has been generally no accepted method of determining the IF of MAbs. The conventional method is based on a radioimmunoassay technique in which the fraction of radiolabeled MAb bound to antigen under conditions of "antigen excess" is determined. Lindmo et al. introduced a modified method in which the IF is determined by extrapolation to conditions representing "infinite antigen excess." Although the Lindmo method, in principle, is insensitive to experimental parameters, it does not always provide a reliable estimate of IF. We, therefore, evaluated an alternate method in which percent cell bound fraction is measured under conditions of fixed antigen concentration and various dilutions of radiolabeled MAb. We developed a mathematical equation to estimate immunoreactivity. J591 MAb specific for prostate-specific membrane antigen was radiolabeled with (111)In, (90)Y and (177)Lu to specific activities of 1-20 mCi/mg. We compared the effect of several experimental conditions on the determination of IF using all three different methods. The Lindmo method requires careful optimization of experimental conditions for each radiolabeled MAb. The alternate method, based on a fixed antigen concentration, appears to be practical and may provide a more reliable measure of immunoreactivity.