Prostate-specific Membrane Antigen Peptides Research Articles

Theranostic PSMA ligands with optimized backbones for intraoperative multimodal imaging and photodynamic therapy of prostate cancer

IntroductionThe first generation ligands for prostate-specific membrane antigen (PSMA)–targeted radio- and fluorescence-guided surgery followed by adjuvant photodynamic therapy (PDT) have already shown the potential of this approach. Here, we developed three new photosensitizer-based dual-labeled PSMA ligands by crucial modification of existing PSMA ligand backbone structures (PSMA-1007/PSMA-617) for multimodal imaging and targeted PDT of PCa.MethodsVarious new PSMA ligands were synthesized using solid-phase chemistry and provided with a DOTA chelator for 111In labeling and the fluorophore/photosensitizer IRDye700DX. The performance of three new dual-labeled ligands was compared with a previously published first-generation ligand (PSMA-N064) and a control ligand with an incomplete PSMA-binding motif. PSMA specificity, affinity, and PDT efficacy of these ligands were determined in LS174T-PSMA cells and control LS174T wildtype cells. Tumor targeting properties were evaluated in BALB/c nude mice with subcutaneous LS174T-PSMA and LS174T wildtype tumors using µSPECT/CT imaging, fluorescence imaging, and biodistribution studies after dissection.ResultsIn order to synthesize the new dual-labeled ligands, we modified the PSMA peptide linker by substitution of a glutamic acid into a lysine residue, providing a handle for conjugation of multiple functional moieties. Ligand optimization showed that the new backbone structure leads to high-affinity PSMA ligands (all IC50 < 50 nM). Moreover, ligand-mediated PDT led to a PSMA-specific decrease in cell viability in vitro (P < 0.001). Linker modification significantly improved tumor targeting compared to the previously developed PSMA-N064 ligand (≥ 20 ± 3%ID/g vs 14 ± 2%ID/g, P < 0.01) and enabled specific visualization of PMSA-positive tumors using both radionuclide and fluorescence imaging in mice.ConclusionThe new high-affinity dual-labeled PSMA-targeting ligands with optimized backbone compositions showed increased tumor targeting and enabled multimodal image-guided PCa surgery combined with targeted photodynamic therapy.

Bispecific radioligands targeting prostate-specific membrane antigen and gastrin-releasing peptide receptors on the surface of prostate cancer cells.

Metastases of prostate cancer usually show highly heterogeneous or partly lost prostate-specific membrane antigen (PSMA) expression. In order to image and treat both PSMA positive and negative tissues, the extension of PSMA tracer specificity to other receptors, also highly expressed on the surface of prostate cancer cells, has been suggested. Prostate cancer cells usually express both PSMA and gastrin-releasing peptide (GRP) receptors; thus, bispecific heterodimeric molecules, addressing both targets at the same time, may significantly improve prostate cancer imaging and therapy. This article summarizes preclinical data regarding low-molecular weight molecules targeting PSMA and GRP receptors.

The Effect of Total Tumor Volume on the Biologically Effective Dose to Tumor and Kidneys for 177Lu-Labeled PSMA Peptides.

The aim of this work was to simulate the effect of prostate-specific membrane antigen (PSMA)-positive total tumor volume (TTV) on the biologically effective doses (BEDs) to tumors and organs at risk in patients with metastatic castration-resistant prostate cancer who are undergoing 177Lu-PSMA radioligand therapy. Methods: A physiologically based pharmacokinetic model was fitted to the data of 13 patients treated with 177Lu-PSMA I&T (a PSMA inhibitor for imaging and therapy). The tumor, kidney, and salivary gland BEDs were simulated for TTVs of 0.1-10 L. The activity and peptide amounts leading to an optimal tumor-to-kidneys BED ratio were also investigated. Results: When the TTV was increased from 0.3 to 3 L, the simulated BEDs to tumors, kidneys, parotid glands, and submandibular glands decreased from 22 ± 15 to 11.0 ± 6.0 Gy1.49, 6.5 ± 2.3 to 3.7 ± 1.4 Gy2.5, 11.0 ± 2.7 to 6.4 ± 1.9 Gy4.5, and 10.9 ± 2.7 to 6.3 ± 1.9 Gy4.5, respectively (where the subscripts denote that an α/β of 1.49, 2.5, or 4.5 Gy was used to calculate the BED). The BED to the red marrow increased from 0.17 ± 0.05 to 0.32 ± 0.11 Gy15 For patients with a TTV of more than 0.3 L, the optimal amount of peptide was 273 ± 136 nmol and the optimal activity was 10.4 ± 4.4 GBq. Conclusion: This simulation study suggests that in patients with large PSMA-positive tumor volumes, higher activities and peptide amounts can be safely administered to maximize tumor BEDs without exceeding the tolerable BED to the organs at risk.

Lutetium 177 PSMA radionuclide therapy for men with prostate cancer: a review of the current literature and discussion of practical aspects of therapy.

Prostate‐specific membrane antigen (PSMA) is a receptor on the surface of prostate cancer cells that is revolutionising the way we image and treat men with prostate cancer. New small molecule peptides with high‐binding affinity for the PSMA receptor have allowed high quality, highly specific PET imaging, in addition to the development of targeted radionuclide therapy for men with prostate cancer. This targeted therapy for prostate cancer has, to date, predominately used Lutetium 177 (Lu) labelled PSMA peptides. Early clinical studies evaluating the safety and efficacy of Lu PSMA therapy have demonstrated promising results with a significant proportion of men with metastatic prostate cancer, who have already failed other therapies, responding clinically to Lu PSMA. This review discusses the practical issues of administering Lu PSMA, and gives an overview of the findings from currently published trials in regards to treatment response rates, expected toxicities and safety.

Investigating the Effect of Ligand Amount and Injected Therapeutic Activity: A Simulation Study for 177Lu-Labeled PSMA-Targeting Peptides.

In molecular radiotherapy with 177Lu-labeled prostate specific membrane antigen (PSMA) peptides, kidney and/or salivary glands doses limit the activity which can be administered. The aim of this work was to investigate the effect of the ligand amount and injected activity on the tumor-to-normal tissue biologically effective dose (BED) ratio for 177Lu-labeled PSMA peptides. For this retrospective study, a recently developed physiologically based pharmacokinetic model was adapted for PSMA targeting peptides. General physiological parameters were taken from the literature. Individual parameters were fitted to planar gamma camera measurements (177Lu-PSMA I&T) of five patients with metastasizing prostate cancer. Based on the estimated parameters, the pharmacokinetics of tumor, salivary glands, kidneys, total body and red marrow was simulated and time-integrated activity coefficients were calculated for different peptide amounts. Based on these simulations, the absorbed doses and BEDs for normal tissue and tumor were calculated for all activities leading to a maximal tolerable kidney BED of 10 Gy2.5/cycle, a maximal salivary gland absorbed dose of 7.5 Gy/cycle and a maximal red marrow BED of 0.25 Gy15/cycle. The fits yielded coefficients of determination > 0.85, acceptable relative standard errors and low parameter correlations. All estimated parameters were in a physiologically reasonable range. The amounts (for 25−29 nmol) and pertaining activities leading to a maximal tumor dose, considering the defined maximal tolerable doses to organs of risk, were calculated to be 272±253 nmol (452±420 μg) and 7.3±5.1 GBq. Using the actually injected amount (235±155 μg) and the same maximal tolerable doses, the potential improvement for the tumor BED was 1–3 fold. The results suggest that currently given amounts for therapy are in the appropriate order of magnitude for many lesions. However, for lesions with high binding site density or lower perfusion, optimizing the peptide amount and activity might improve the tumor-to-kidney and tumor-to-salivary glands BED ratio considerably.

Abstract B38: Pilot study of combination PSMA peptide and TARP peptide vaccine with poly-IC:LC adjuvant in HLA-A2 positive patients with elevated PSA after initial definitive treatment of prostate cancer

Abstract Background: For men with elevation of their prostate specific antigen (PSA) as the only sign of progression after initial definitive treatment, immune therapies could impact further disease progression. Epitopes from prostate-specific membrane antigen (PSMA) and T-cell receptor gamma alternate reading frame protein (TARP) capable of inducing in-vitro T-cell responses against prostatic tumors were used to synthesize the vaccine peptide. Each has a class II epitope and an HLA-A2 specific class I epitope. Working from prior studies showing responses of both cytotoxic T lymphocytes and T helper cells are more immunogenic than either alone, this prospective pilot study set to estimate the frequency that this peptide vaccine and poly-IC:LC adjuvant would induce ELISPOT identifiable improved titers. Secondary objectives included evaluation of the safety and toxicity of vaccination and impact on PSA change. Methods: Estimation of immunologic efficacy was accomplished using an ELISPOT assay of peripheral blood which assessed interferon-gamma secretion by activated T-cells in response to vaccination with PSMA-derived and TARP-derived peptides. Patients were vaccinated every 2 weeks for 8 total injections. Twenty-one patients had full ELISPOT data for analysis, which included pre-vaccination, on-treatment, and follow-up titers of PBMS alone, PSMA CD4, PSMA CD8, TARP CD4, and TARP CD8. Twenty-nine patients had PSA data, which were normalized by both date and PSA measurement. A table recorded the frequencies of adverse effects and dose-limiting toxicities (DLT). Results: Quarter-root power means were found to be the best for normalization of the ELISPOT results. The averaged PSMA CD4, PSMA CD8, TARP CD4, and TARP CD8 titers were all increased from baseline PBMC at time points 6 and 8. PSMA and TARP CD4 ELISPOT titers were also increased from PBMC baseline at time points 4 and 10. When restricted to time points 4, 6, and 8, the mean expression of PSMA CD4 and TARP CD4 compared to PBMC alone were significantly different, with a power mean fold increase of 2.70 (p=0.0001) and 2.45 (p=0.0006) respectively. The PSMA CD8 and TARP CD8 compared to PBMC were not significantly up during time points 4, 6, and 8. There were no DLT observed. Ten of the 29 patients had excellent stabilization of the PSA and did not have a PSA doubling before their last study visit; the last study date for this subset was a mean of 458 days from baseline PSA (55-613 days). Nineteen of the 29 patients had either a doubling of the PSA or proceeded to another therapy, occurring at a mean of 248 days (23-526 days). In these retrospectively divided subsets, the group with the longer PSA-doubling time appeared to have higher ELISPOT titer increases than the shorter PSA-doubling time group. Conclusions: A continued need for less intensive, nontoxic therapy such as vaccines exists in the treatment of prostate cancer patients, including those with biochemical failure. This pilot study showed feasibility of the class I + class II + poly-IC: LC combination protocol. Interestingly, significant increases in PMSA CD4 and TARP CD4 ELISPOT titers, as opposed to CD8 titer changes, were observed. A third of the subjects did not have PSA-doubling during the study period. The higher ELISPOT titers in the longer PSA-doubling time group also suggest an association, but not proof of causation, for better vaccine response and longer disease progression timelines. This pilot experience supports further clinical testing using peptide and poly-IC:LC vaccination combination therapies. Citation Format: Matthew Whitehurst, Xiuhua Zhao, Michael Schell, Esteban Celis, Andres Salazar, Mayer Fishman. Pilot study of combination PSMA peptide and TARP peptide vaccine with poly-IC:LC adjuvant in HLA-A2 positive patients with elevated PSA after initial definitive treatment of prostate cancer. [abstract]. In: Proceedings of the AACR Special Conference: Tumor Immunology and Immunotherapy: A New Chapter; December 1-4, 2014; Orlando, FL. Philadelphia (PA): AACR; Cancer Immunol Res 2015;3(10 Suppl):Abstract nr B38.

Preclinical evaluation of a bispecific low‐molecular heterodimer targeting both PSMA and GRPR for improved PET imaging and therapy of prostate cancer

It has recently been reported that metastases of prostate cancer usually show highly heterogeneous or partly lost prostate-specific membrane antigen (PSMA) expression. In order to image and treat both PSMA positive and negative tissues PSMA targeting probes need to be extended by a further specificity. Since prostate cancer cells usually express both PSMA and gastrin-releasing peptide receptor (GRPR) a bispecific low-molecular heterodimeric molecule, addressing both targets at the same time, may significantly improve prostate cancer imaging and therapy. The nonapeptide BZH3 representing the GRPR binding part was combined with the urea-based PSMA inhibitor Glu-urea-Lys(Ahx)-HBED-CC. The syntheses of the compounds were performed according to standard Fmoc-solid phase peptide synthesis. The binding properties were analyzed by competitive cell binding and internalization experiments. The in vivo targeting properties were investigated by means of biodistribution studies. Cell binding experiments revealed high binding affinities to both GRPR and PSMA expressing cell lines. The heterodimer bound with IC50 -values essentially matching the IC50 values of the respective monomers (25.0 ± 5.4 nM for PSMA and 9.0 ± 1.8 nM for GRPR, respectively). In vivo, the heterodimer showed dual targeting of PSMA (5.4%ID/g for PSMA-positive tumors) and GRPR receptors (3.3% ID/g for GRPR-positive tumors) while exhibiting fast pharmacokinetic properties. The clearance from background was comparable to the monomeric PSMA-targeting reference. The heterodimeric molecule is a promising agent for PET imaging of primary and recurrent prostate cancer covering two receptor entities which might lead to an improved diagnostic sensitivity and therapeutic efficiency.

DNA fusion gene vaccines induce cytotoxic T‐cell attack on naturally processed peptides of human prostate‐specific membrane antigen

For long-term attack on tumor cells in patients with prostate cancer, induction of cytolytic T cells is desirable. Several lineage-specific target proteins are known and algorithms have identified candidate MHC class I-binding peptides, particularly for HLA-A*0201. We have designed tolerance-breaking DNA fusion vaccines incorporating a domain of tetanus toxin fused to candidate tumor-derived peptide sequences. Using three separate peptide sequences from prostate-specific membrane antigen (PSMA) (peptides PSMA(27) , PSMA(663) , and PSMA(711) ), this vaccine design induced high levels of CD8(+) T cells against each peptide in a HLA-A(*) 0201 preclinical model. In contrast, the full-length PSMA sequence containing all three epitopes was poorly immunogenic. Induced T cells were cytotoxic against peptide-loaded tumor cells, but only those against PSMA(27) or PSMA(663) peptides, and not PSMA(711) , were able to kill tumor cells expressing endogenous PSMA. Cytotoxicity was also evident in vivo. The preclinical model provides a powerful tool for generating CD8(+) T cells able to predict whether target cells can process and present peptides, essential for planning peptide vaccine-based clinical trials.

Binding of prostate-specific membrane antigen to dendritic cells: a critical step in vaccine preparation

Dendritic cell (DC)-based vaccines hold promise as a safe therapy for prostate cancer (PCa), and prostate-specific membrane antigen (PSMA) fulfils the requirements for a tumor-associated antigen (TAA) to be clinically effective. We evaluated the actual binding of selected HLA-A2-restricted PSMA peptides to HLA class I molecules on ex vivo-generated mature (m) DC. mDC were generated from peripheral monocytes of HLA-A2 normal donors. The PSMA peptides PSMA(711) (ALFDIESKV), PSMA(27) (VLAGGFFLL) and PSMA(663) (MMNDQLMFL) were selected based on computer-assisted prediction programs, documented CTL epitope activity or previous use in clinical trials. The model cell line T2 and the clinical grade (CD83+ CCR7+) mDC were pulsed with fluorescein (FL)-conjugated peptides and an anti-HLA-A2 monoclonal antibody (MAb) and analyzed. Flow cytometry analysis showed best binding efficiency to be by PSMA(27.) Confocal microscopy confirmed coincident fluorescence emission of HLA-A2 MAb and FL-PSMA(27). Virtual co-localization of PSMA(27) and HLA class I molecules was supported further by fluorescence resonance energy transfer (FRET) analysis. The clinical relevance of our findings has to be validated in vivo. The present report is the first to score selected PSMA peptides based on their detectable binding to mDC. It identifies PSMA(27) as the choice candidate among other PSMA peptides and it should be included in developing DC vaccine protocols for HLA-A2 PCa patients.

The HLA‐A2‐restricted PSMA peptide LLHETDSAV is poorly immunogenic in patients with metastatic prostate cancer

Increasing evidence suggests that prostate cancer is visible to the immune system and is potentially responsive to immunotherapeutic interventions. Previous work has identified prostate-specific membrane antigen (PSMA) as a potential antigen for T-cell recognition, and specific PSMA epitopes presented by HLA-A2 have been described. One vaccination strategy that is being pursued in the clinic utilizes peptide-pulsed peripheral blood mononuclear cells (PBMC) + IL-12. HLA-A2(+) patients with castrate-resistant prostate cancer and normal organ function were considered. Vaccines were prepared using autologous PBMC loaded with a PSMA peptide previously reported to be immunogenic (LLHETDSAV) administered subcutaneously every 3 weeks. T-cell responses and tumor activity were assessed. Although the vaccine was well tolerated, no clinical responses were observed in 12 enrolled patients and no immune responses were detected based on an ex vivo IFN-gamma ELISPOT assay. To examine immunogenicity of this PSMA peptide in more detail, an in vitro priming assay was utilized with normal donor PBMC, and no detectable reactivity was observed. Our results suggest that the PSMA peptide LLHETDSAV is poorly immunogenic in humans and that alternative prostate cancer antigens should be pursued.

A Novel Alphavirus Vaccine Encoding Prostate-Specific Membrane Antigen Elicits Potent Cellular and Humoral Immune Responses

Prostate-specific membrane antigen (PSMA) is an attractive target for active immunotherapy. Alphavirus vaccines have shown promise in eliciting immunity to tumor antigens. This study investigated the immunogenicity of alphavirus vaccine replicon particles (VRP) that encode PSMA (PSMA-VRP). Cells were infected with PSMA-VRP and evaluated for PSMA expression and folate hydrolase activity. Mice were immunized s.c. with PSMA-VRP or purified PSMA protein. Sera, splenocytes, and purified T cells were evaluated for the magnitude, durability, and epitope specificity of the anti-PSMA response. Antibodies were measured by flow cytometry, and cellular responses were measured by IFN-gamma enzyme-linked immunospot and chromium release assays. Cellular responses in BALB/c and C57BL/6 mice were mapped using overlapping 15-mer PSMA peptides. A Good Laboratory Practice-compliant toxicology study was conducted in rabbits. PSMA-VRP directed high-level expression of active PSMA. Robust T-cell and B-cell responses were elicited by a single injection of 2 x 10(5) infectious units, and responses were boosted following repeat immunizations. Anti-PSMA responses were detected following three immunizations with 10(2) infectious units and increased with increasing dose. PSMA-VRP was more immunogenic than adjuvanted PSMA protein. Responses to PSMA-VRP were characterized by Th-1 cytokines, potent CTL activity, and IgG2a/IgG2b antibodies. T-cell responses in BALB/c and C57BL/6 mice were directed toward different PSMA peptides. Immunogenic doses of PSMA-VRP were well tolerated in mice and rabbits. PSMA-VRP elicited potent cellular and humoral immunity in mice, and specific anti-PSMA responses were boosted on repeat dosing. PSMA-VRP represents a promising approach for immunotherapy of prostate cancer.

A xenogeneic PSMA DNA vaccine for patients (pts) with non-castrate metastatic (NCMPC) and castrate metastatic prostate cancer (CMPC)—A phase I trial of proof of principle

3073 Background: Prostate Specific Membrane Antigen (PSMA) represents a target for both primary and metastatic prostate cancer and an anti-angiogenic approach for other solid tumors. Antitumor effects in PC have been observed using autologous dendritic cells pulsed with PSMA peptides. This is a pilot phase I trial of vaccination with a DNA encoding PSMA in patients with PC. The objective was to determine the safety and immunogenicity of vaccination with the genes encoding for mouse and human PSMA in patients with recurrent disease. The hypothesis was that xenogeneic DNA encoding a xenogeneic homologous antigen was a more potent immunogen than self DNA. Methods: Thirty-six HLA-A0201+ pts with either NCMPC or CMPC were randomly assigned to vaccination with either xenogeneic (mouse) or human (self) PSMA DNA delivered i.m. via a high pressure delivery system at 100μg, 1,500μg or 4,000μg every 3 weeks for 3 immunizations. Those pts randomized initially to receive human PSMA DNA received 3 immunizations with mouse PSMA DNA at three week intervals. Sera and peripheral blood mononuclear cells were analyzed for humoral and T cell responses Results: Twenty-nine of 36 pts were evaluable having received all six vaccines. Nine remain active without disease progression or significant change in PSA logslope. One of 6 pts treated at the 4,000μg cohort has been followed by &gt; 92 weeks; at the 1,500μg cohort 1 of 2 pt has been monitored for &gt; 110 weeks; and 1 pt at the 100μg dose has remained on study for &gt; 169 weeks. No high titer antibodies against PSMA were detected by ELISA or FACS against 3T3 fibroblasts transduced to express PSMA. Conclusions: The vaccine was safe at all dose levels without evidence of autoimmune reactivity. While antibodies against PSMA were not detected, studies are ongoing to examine T cell responsiveness. The majority of pts who remain on study were treated at 4,000μg dose level which appears to be the optimal dose. Supported by The Prostate Cancer Foundation and MSKCC Prostate Cancer SPORE No significant financial relationships to disclose.

Identification of a prostate-specific membrane antigen-derived peptide capable of eliciting both cellular and humoral immune responses in HLA-A24+ prostate cancer patients.

We tried to identify prostate-specific membrane antigen (PSMA)-derived peptides capable of eliciting both cellular and humoral immune responses in peripheral blood mononuclear cells (PBMCs) and plasma of HLA-A24(+) prostate cancer patients, respectively. For cellular response, peptide-specific and prostate cancer-reactive responses of in vitro-stimulated PBMCs were examined with regard to interferon (IFN)-gamma production and cytotoxicity against both a parental HLA-A24(-) prostate cancer cell line (PC-93) and an HLA-A24-expressing transfectant cell line (PC93-A24). For humoral response, patients' plasma was tested for reactivity to the peptides by means of an enzyme-linked immunosorbent assay (ELISA). Among 13 PSMA peptides, PSMA 624-632 peptide induced peptide-specific and tumor-reactive cytotoxic T lymphocytes (CTLs) most effectively. The PSMA 624-632 peptide-stimulated PBMCs from either healthy donors or prostate cancer patients produced a significant level of IFN-gamma in response to prostate cancer cells in an HLA-A24-restricted manner, and also showed a higher level of cytotoxicity against PC93-A24 than against PC93. Antibodies to the PSMA 624-632 peptide, but not to any others, were detected in prostate cancer patients. These results demonstrate that the PSMA 624-632 peptide could be an appropriate molecule for use in specific immunotherapy of HLA-A24(+) patients with prostate cancer.

Use of artificial neural networks in evaluating prognostic factors determining the response to dendritic cells pulsed with PSMA peptides in prostate cancer patients.

Our purpose was to compare the importance of over 22 measurements used in evaluating the clinical responses of patients with metastatic or locally recurrent prostate cancer, treated by dendritic cell (DC) infusions with prostate-specific membrane antigen (PSMA) peptides. Artificial neural networks (ANNs) were employed for assessment, as well as the traditional methods of logistic regression. Twenty-six patients with metastatic disease and 37 patients with local recurrence were available for evaluation and comparison. ANN evaluation ranked the collective effects of DC infusion, immune responses (CD3+ cells, CD16+ cells, zeta chain+ cells), and cytokines, e.g., IL-6 and PSMA levels, very highly. Logistic regression identified all of these parameters to some degree, but in a different rank order. Patients with metastases showed a sharp rate of response secondary to the level of DC infusion, in contrast to those patients with local recurrence, in which it was more gradual. ANN analysis emphasizes the importance of level of DC infusion, immune parameters, cytokines, and markers such as PSMA in determining the response to PSMA peptide immunotherapy. The criteria of response were judged to be correct in 86% of metastatic patients and 83% of locally recurrent patients evaluated in this study.