Covalent Immune Proximity-Induction Strategy Using SuFEx-Engineered Bifunctional Viral Peptides.

[ES] La apoptosis es el principal mecanismo regulado de muerte celular, que tiene\n\t\t\t\t un papel crucial no sólo en condiciones de daño celular o estrés, sino también\n\t\t\t\t durante el desarrollo normal, la morfogénesis, el mantenimiento de la homeostasis de\n\t\t\t\t los tejidos y para un sistema inmune efectivo. Por ello, su alteración está implicada\n\t\t\t\t en numerosas patologías, como enfermedades neurodegenerativas, daño isquémico,\n\t\t\t\t enfermedades autoinmunes y cáncer.\n\t\t\t\t La maquinaria apoptótica intracelular está controlada por una familia de\n\t\t\t\t proteasas que presentan cisteína en el sitio activo y procesan a sus proteínas diana a\n\t\t\t\t través de residuos específicos de aspartato, denominadas caspasas, que son\n\t\t\t\t necesarias para la apoptosis y la respuesta inflamatoria. Las caspasas están reguladas\n\t\t\t\t a nivel posttraduccional por eventos de fosforilación/desfosforilación que afectan a\n\t\t\t\t la conformación de la proteína diana y/o a su interacción con otras proteínas, así\n\t\t\t\t como a su actividad, función, vida media, estabilidad, afinidad de sustrato o\n\t\t\t\t localización subcelular. Este mecanismo está controlado por dos grandes familias de\n\t\t\t\t proteínas: las proteín kinasas y las proteín fosfatasas.\n\t\t\t\t En este trabajo hemos abordado el estudio de las interacciones entre Caspasa-\n\t\t\t\t 9 y las fosfatasas PP1 y PP2A, así como la interacción entre Caspasa-9 y el inhibidor\n\t\t\t\t 2 de PP2A, SET. Además, hemos estudiado la abundancia relativa de las variantes de\n\t\t\t\t empalme alternativo aberrante de Caspasa-9 y PP2Acα en pacientes de CLL y hemos\n\t\t\t\t podido aplicar el conocimiento de la interacción Caspasa-9 – PP2Acα para\n\t\t\t\t desarrollar un péptido capaz de inducir apoptosis específicamente en células B de\n\t\t\t\t CLL y otro tipo de células tumorales, así como en modelos de xenotransplantes de\n\t\t\t\t cáncer de mama. Con el objetivo de llevar a cabo ensayos preclínicos para\n\t\t\t\t desarrollar este péptido como fármaco, hemos evaluado el uso de nanopartículas\n\t\t\t\t como vehículos para transportar este péptido a las céulas diana.

5018 Background: The abundant expression of prostate specific membrane antigen (PSMA) on prostate cancer cells provides a rationale for antibody therapy. PSMA ADC is a fully human antibody to PSMA linked to the microtubule disrupting agent monomethyl auristatin E (MMAE). It binds PSMA and is internalized within the cancer cell where cleavage by lysosomal enzymes release free MMAE, causing cell cycle arrest and apoptosis. A phase 1 dose escalation study of PSMA ADC in taxane-refractory mCRPC has been completed. Methods: Patients with progressive mCRPC following taxane-containing chemotherapy and ECOG status of 0 or 1 were eligible. PSMA ADC was administered by IV infusion Q3W for up to 4 cycles. Safety, pharmacokinetics, PSA, circulating tumor cells (CTC), immunogenicity and clinical progression were assessed. Serum PSMA ADC and total anti-PSMA ADC antibodies were measured by ELISA, and free MMAE was measured by LC/MS/MS.The dosing cohorts ranged from 0.4 mg/kg to 2.8 mg/kg. Subjects who benefitted from PSMA ADC were eligible for treatment in an extension study. Results: 52 subjects were dosed in 9 dose levels. All subjects received prior docetaxel, 6 also received cabazitaxel and 3 subjects also received paclitaxel. PSMA ADC was generally well tolerated with the most commonly seen adverse events being anorexia and fatigue. 16 patients reported peripheral neuropathies, including 3 with grade 3. Dose limiting toxicities (DLT) seen at 2.8 mg/kg were neutropenia (one death) and reversible elevations in liver function tests (LFTs). Antitumor activity was manifested as reductions either in PSA or in CTCs in approximately 50% of patients at ≥ 1.8 mg/kg PSMA ADC. Exposure to PSMA ADC increased with dose and was ~1,000-fold greater than MMAE exposure. There was no accumulation. Conclusion: PSMA ADC in this study was generally well tolerated in subjects with progressive mCRPC, previously treated with taxane. Antitumor activity was seen at doses ≥ 1.8 mg/kg. DLTs were neutropenia and reversible LFT abnormalities. The maximum tolerated dose was determined to be 2.5 mg/kg. A phase 2 trial of PSMA ADC in taxane refractory mCRPC has been initiated at 2.5 mg/kg. Clinical trial information: NCT01414283.

276. Evaluation of an Oncolytic Adenovirus: Efficacy Studies on Primary Human Tumor Cell Cultures and Safety Studies in Non-Human Primates

Plants are the most efficient bioreactors for the production of complex proteins like antibodies, and are the only viable transgenic system capable of producing secretory antibodies. Plant-based monoclonal antibody production allows the generation of virtually unlimited quantities of antibodies at prices that are 25 to 100 times less expensive than mammalian cell culture, while reducing manufacturing capital by 90%. The human sequences encoding anti-HSV (Herpes Simplex Virus) antibodies as an IgG, IgA, dIgA, or sIgA were introduced into rice cells. Transgenic cells were selected and allowed to regenerate into plants. More than half of the independent rice lines assayed produced assembled antibody. The rice-produced antibodies performed at least as well as the original CHO-cell expressed anti-HSV antibody in standard in vitro neutralization assays of HSV-2. Furthermore, the rice-produced anti-HSV antibodies were able to prevent vaginal HSV-2 infection in mice. We are interested in developing a topical gel that contains an anti-HSV antibody for protection against vaginal transmission of HSV-2. For large over-the-counter applications, plants may offer the only method for producing protective antibodies cost effectively and in sufficient quantities to be employed worldwide.

In the present work, we have attempted a comprehensive analysis of cytosolic and microsomal proteomes to elucidate the signaling pathways impaired in human hepatoma (Huh7) cells upon herpes simplex virus type 1 (HSV-1; Cgal(+)) infection. Using a combination of differential in-gel electrophoresis and nano liquid chromatography/tandem mass spectrometry, 18 spots corresponding to 16 unique deregulated cellular proteins were unambiguously identified, which were involved in the regulation of essential processes such as apoptosis, mRNA processing, cellular structure and integrity, signal transduction, and endoplasmic-reticulum-associated degradation pathway. Based on our proteomic data and additional functional studies target proteins were identified indicating a late activation of apoptotic pathways in Huh7 cells upon HSV-1 Cgal(+) infection. Additionally to changes on RuvB-like 2 and Bif-1, down-regulation of Erlin-2 suggests stimulation of Ca(2+)-dependent apoptosis. Moreover, activation of the mitochondrial apoptotic pathway results from a time-dependent multi-factorial impairment as inferred from the stepwise characterization of constitutive pro- and anti-apoptotic factors. Activation of serine-threonine protein phosphatase 2A (PP2A) was also found in Huh7 cells upon HSV-1 Cgal(+) infection. In addition, PP2A activation paralleled dephosphorylation and inactivation of downstream mitogen-activated protein (MAP) kinase pathway (MEK(1/2), ERK(1/2)) critical to cell survival and activation of proapoptotic Bad by dephosphorylation of Ser-112. Taken together, our results provide novel molecular information that contributes to define in detail the apoptotic mechanisms triggered by HSV-1 Cgal(+) in the host cell and lead to the implication of PP2A in the transduction of cell death signals and cell survival pathway arrest.

A Theranostic “SMART” Aptamer for Targeted Therapy of Prostate Cancer

Prostate specific membrane antigen (PSMA) is a suggested target for antibody-based therapy of prostate cancer potentially involved in the regulation of cell migration. This study was undertaken, to gain more insight on the role of PSMA in early prostate cancer and its distribution in various normal tissues. A total of 1,700 different prostate cancers treated by radical prostatectomy and 612 samples from 76 different normal tissue types were successfully analyzed by immunohistochemistry (IHC) in a tissue microarray (TMA) format. PSMA immunostaining in cancers was also compared with clinical follow-up, preexisting HER2 expression and Ki67 labeling index data. PSMA staining was only found in prostate epithelium and expression was higher in cancer cells than in benign tissue. PSMA staining was found in 94.1% of cancers and was significantly associated with tumor stage, high Gleason grade, preoperative PSA, and HER2 expression (P < 0.0001 each). Tumors with strong PSMA expression had a higher risk of biochemical recurrence than cancers with only weak PSMA staining intensity (P = 0.0483). There was no significant association between PSMA expression and Ki67 labeling index (P = 0.442). Based on the high frequency of PSMA overexpression in all stages and grades of prostate cancer and the high prevalence of PSMA overexpression, it can be speculated that increased PSMA expression may be related with prostate cancer development rather than progression. The known function of PSMA activating cell migration would be in line with the suggested role in cancer progression and the missing association between PSMA overexpression and tumor cell proliferation.

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.

Prostate cancer (PCa) has a unique tropism to bone. Indeed, bone is the most frequent site of distant metastasis and cause of morbidity due to skeletal complications. 99mTc-Methylene diphosphonate (MDP) bone scintigraphy/ scan (BS) is the current standard imaging due to increase adsorption of the tracer at osteoblastic sites. However, it has limited specificity due to false positives in degenerative changes, benign causes and false negatives in bone marrow metastasis and lytic lesions. Another drawback of BS is flare response. Prostate Specific Membrane Antigen (PSMA) has been the most studies target in prostate cancer imaging in recent time due to 100-1000 time overexpression in cancer cells. 68Ga-PSMA-11, a small molecule with PSMA enzyme inhibition activity has been found promising in recurrence and lymph-node staging. In our experience of 97 staging prostate cancer patients, PSMA PET-CT showed 57.41% with pure sclerotic metastasis. Mixed (33.33%), marrow (7.14%) and lytic (2.3%) types of lesions constitute the rest and thus BS alone in these patients may leads to underestimation of bony disease burden. PSMA has not been found positive in degenerative changes however its role in response to anti-androgen needs caution due to know synergistic effect on PSMA expression. We concluded, PSMA PET-CT would have better sensitivity and specificity due to unique distinction for detecting non-sclerotic metastases. We presumed if PSMA has been performed for staging workup then there is limited role of BS except in clinical trial patient. Overall PSMA PET may become one-stop-shop for PCa workup.

Anti-PSMA monoclonal antibody increases the toxicity of paclitaxel carried by carbon nanotubes.

&lt;div&gt;Abstract&lt;p&gt;Prostate-specific membrane antigen (PSMA) expression increases with prostate cancer grade and progression; however, the role of PSMA in prostate cancer progression remains poorly understood. Telomere stability is essential for the survival and genome stability of cancer cells. We found massive telomere DNA damage in PSMA-negative prostate cancer cells (PC-3 and DU145) compared with PSMA-positive prostate cancer (LNCaP) cells. The ectopic expression of PSMA suppressed telomere DNA damage in PC3 cells. PSMA inhibitor, 2-PMPA, and PSMA knockdown induced telomere DNA damage in PSMA-positive LNCaP cells but not in PSMA-negative PC-3 cells, suggesting that PSMA plays a critical role in telomere stability in prostate cancer cells. In addition, we observed that inhibition of PSMA or inhibition of glutamate receptor, which mediates PSMA-dependent activation of AKT, suppressed AKT phosphorylation, and caused telomere DNA damage. Furthermore, 2-PMPA-induced telomere DNA damage in LNCaP cells was associated with telomere aberrations, such as telomere–telomere fusions, sister–chromatid telomere fusions, and telomere breakages. AKT is reported to promote cell growth by stabilizing telomere association with telomere-binding proteins TRF1 and TPP1. We observed that TRF1 and TPP1 transfection of LNCaP cells attenuated the inhibitory effect of 2-PMPA on cell growth and telomere DNA damage. Together, these observations indicate that PSMA role in maintaining telomere stability in prostate cancer cells is mediated by AKT. Thus, these studies reveal an important role of PSMA in maintaining telomere stability that can promote cell survival and, thereby, prostate cancer progression.&lt;/p&gt;Implications:&lt;p&gt;Role of PSMA in telomere stability suggests a strong correlation between PSMA expression and prostate cancer progression.&lt;/p&gt;&lt;/div&gt;

We developed a theranostic radiopharmaceutical that engages two key cell surface proteases, fibroblast activation protein alpha (FAP) and prostate-specific membrane antigen (PSMA), each frequently overexpressed within the tumor microenvironment (TME). The latter is also expressed in most prostate tumor epithelium. To engage a broader spectrum of cancers for imaging and therapy, we conjugated small-molecule FAP and PSMA-targeting moieties using an optimized linker to provide 64Cu-labeled compounds. We synthesized FP-L1 and FP-L2 using two linker constructs attaching the FAP and PSMA-binding pharmacophores. We determined in vitro inhibition constants (Ki) for FAP and PSMA. Cell uptake assays and flow cytometry were conducted in human glioma (U87), melanoma (SK-MEL-24), prostate cancer (PSMA + PC3 PIP and PSMA - PC3 flu), and clear cell renal cell carcinoma lines (PSMA + /PSMA - 786-O). Quantitative positron emission tomography/computed tomography (PET/CT) and tissue biodistribution studies were performed using U87, SK-MEL-24, PSMA + PC3 PIP, and PSMA + 786-O experimental xenograft models and the KPC genetically engineered mouse model of pancreatic cancer. 64Cu-FP-L1 and 64Cu-FP-L2 were produced in high radiochemical yields (> 98%) and molar activities (> 19MBq/nmol). Ki values were in the nanomolar range for both FAP and PSMA. PET imaging and biodistribution studies revealed high and specific targeting of 64Cu-FP-L1 and 64Cu-FP-L2 for FAP and PSMA. 64Cu-FP-L1 displayed more favorable pharmacokinetics than 64Cu-FP-L2. In the U87 tumor model at 2h post-injection, tumor uptake of 64Cu-FP-L1 (10.83 ± 1.02%ID/g) was comparable to 64Cu-FAPI-04 (9.53 ± 2.55%ID/g). 64Cu-FP-L1 demonstrated high retention 5.34 ± 0.29%ID/g at 48h in U87 tumor. Additionally, 64Cu-FP-L1 showed high retention in PSMA + PC3 PIP tumor (12.06 ± 0.78%ID/g at 2h and 10.51 ± 1.82%ID/g at 24h). 64Cu-FP-L1 demonstrated high and specific tumor targeting of FAP and PSMA. This compound should enable imaging of lesions expressing FAP, PSMA, or both on the tumor cell surface or within the TME. FP-L1 can readily be converted into a theranostic for the management of heterogeneous tumors.

The objective of this article is to rectify tumor shrinkage as a valid diagnosis of cancer therapy. Cancer incidence and cancer mortality keep on increasing, which are an indication that cancer has not been handled right. Perpetual proliferation of cancer cells (CCs) is the most outstanding feature of cancer. Elimination of CCs to shrink tumor became the commanding principle, which was wrong, because cancer evolved due to wound unhealing. Killing to create wound definitely is contra-indication of cancer therapy. The mistake was committed at a time when we did not have the full knowledge of cancer, which was excusable. Now we have better knowledge of cancer, but the mistake is carrying on to result in 10 million annual mortality worldwide in 2019 with an anticipated annual increment of 5%, which is not excusable. Cytotoxic therapies can only benefit a small minority of cancer patients in the early stage whose chem-surveillance have not yet fatally damaged, relying on the restoration of chemo-surveillance to subdue cancer stem cells (CSCs), which are resistant to cytotoxic agents. Under this circumstance, tumor shrinkage is a promising diagnosis toward remission. Cytotoxic therapies cause the death of a majority of cancer patients in the advanced stage whose chemo-surveillance have been fatally damaged. Under this circumstance, tumor shrinkage is an ominous diagnosis toward fatality. Tumor shrinkage should be used with discretion. Cancer is a disease created by multiple factors. The collapse of chemo-surveillance or immuno-surveillance, the evolution of cancer stem cells (CSCs) from progenitor stem cells (PSCs) due to wound unhealing, and the progression of CSCs to faster replicating CCs by the activation of oncogenes or the inactivation of suppressor genes all contribute significantly to the development of cancer. An effective cancer therapy must be able to rectify all these contributing factors. Focusing on a specific factor is insufficient. The obsession to eliminate CCs to achieve tumor shrinkage is a grave mistake of cancer establishments to result in ever-increasing cancer mortality. Actually, elimination of CSCs is more important than the elimination of CCs, because CSCs contribute most fatal effects of cancer such as metastasis, drug resistance, anti-apoptosis, angiogenesis and recurrence. Induction of terminal differentiation by cell differentiation agent (CDA) formulations is the only option to eliminate CSCs which are critically linked to wound unhealing at the primary site. Induction of terminal differentiation can also result in the elimination of CCs, which are not as tightly linked to wound unhealing as CSCs. Terminal differentiation of CSCs and CCs cannot make the tumor to disappear. Thus, CDAs violated the commanding principle of tumor shrinkage put up by the cancer establishments, and were blocked as acceptable cancer drugs. CDA formulations are obviously the only drugs that can save the lives of advanced cancer patients whose chemo-surveillance have been fatally damaged. Oncologists and advanced cancer patients must unite to push for the approval of CDA formulations to save advanced cancer patients.

Oncolytic virotherapy: Molecular targets in tumor-selective replication and carrier cell-mediated delivery of oncolytic viruses

B7-H4 as a potential target for immunotherapy for gynecologic cancers: A closer look