Dataset Information

TGM4: an immunogenic prostate-restricted antigen.

ABSTRACT:

Background

Prostate cancer is the second leading cause of cancer-related death in men in the USA; death occurs when patients progress to metastatic castration-resistant prostate cancer (CRPC). Although immunotherapy with the Food and Drug Administration-approved vaccine sipuleucel-T, which targets prostatic acid phosphatase (PAP), extends survival for 2-4 months, the identification of new immunogenic tumor-associated antigens (TAAs) continues to be an unmet need.

Methods

We evaluated the differential expression profile of castration-resistant prostate epithelial cells that give rise to CRPC from mice following an androgen deprivation/repletion cycle. The expression levels of a set of androgen-responsive genes were further evaluated in prostate, brain, colon, liver, lung, skin, kidney, and salivary gland from murine and human databases. The expression of a novel prostate-restricted TAA was then validated by immunostaining of mouse tissues and analyzed in primary tumors across all human cancer types in The Cancer Genome Atlas. Finally, the immunogenicity of this TAA was evaluated in vitro and in vivo using autologous coculture assays with cells from healthy donors as well as by measuring antigen-specific antibodies in sera from patients with prostate cancer (PCa) from a neoadjuvant clinical trial.

Results

We identified a set of androgen-responsive genes that could serve as potential TAAs for PCa. In particular, we found transglutaminase 4 (Tgm4) to be highly expressed in prostate tumors that originate from luminal epithelial cells and only expressed at low levels in most extraprostatic tissues evaluated. Furthermore, elevated levels of TGM4 expression in primary PCa tumors correlated with unfavorable prognosis in patients. In vitro and in vivo assays confirmed the immunogenicity of TGM4. We found that activated proinflammatory effector memory CD8 and CD4 T cells were expanded by monocyte-derived dendritic cell (moDCs) pulsed with TGM4 to a greater extent than moDCs pulsed with either PAP or prostate-specific antigen (PSA), and T cells primed with TGM4-pulsed moDCs produce functional cytokines following a prime/boost regiment or in vitro stimulation. An IgG antibody response to TGM4 was detected in 30% of vaccinated patients, while fewer than 8% of vaccinated patients developed antibody responses to PSA or prostate-specific membrane antigen (PSMA).

Conclusions

These results suggest that TGM4 is an immunogenic, prostate-restricted antigen with the potential for further development as an immunotherapy target.

SUBMITTER: Lopez-Bujanda ZA

PROVIDER: S-EPMC8246381 | biostudies-literature |

REPOSITORIES: biostudies-literature

ACCESS DATA

Similar Datasets

Project description:The present study aimed to identify, express and purify an immunogenic fragment in the ectodomain of prostate-specific membrane antigen (PSMA) within a fusion protein. The PSMA amino acid sequence published in National Center for Biotechnology Information GenBank was used to determine sequence homology and immunogenic index analyses, additionally using BLASTN, Protean and ExPASy software to predict the polypeptide sequences of immunogenic epitopes. The gene sequence encoding the ectodomain of the polypeptide immunogenic fragments, containing the identified immunogenic epitopes, was generated using whole-gene synthesis. Prokaryotic expression vector pET-32a-r-ectodomain-PSMA was constructed and the recombinant plasmids were transformed into competent BL21 (DE3) Escherichia coli, which was followed by induction of recombinant protein expression using isopropyl-β-D-thiogalactopyranoside. Fusion proteins were isolated and purified using affinity chromatography and their immune activity was subsequently investigated using western blot analysis. Purified protein was used to immunize BALB/c mice in order to generate polyclonal antibodies, and the binding of polyclonal antibodies to prostate cancer cell lines in vitro was evaluated using flow cytometry. A total of 3 polypeptide fragments with high specificity were identified following analysis using numerous software packages, and the gene sequences encoding regions containing the 2 most immunogenic fragments were synthesized and successfully inserted into the prokaryotic expression vector pET-32a-r-ectodomain-PSMA. The recombinant PSMA protein fragment had a molecular weight of ~50 kDa and 95% purity. Western blot analysis revealed that the r-ectodomain-PSMA fusion protein specifically bound to the anti-PSMA ectodomain monoclonal antibody. Flow cytometry demonstrated that polyclonal antibodies raised against these recombinant proteins could specifically bind to PSMA-positive LNCaP cells, but not to PSMA-negative PC-3 cells. An immunogenic fragment in the ectodomain of PSMA was successfully expressed and purified. The present study, therefore, provides a basis for the preparation of an anti-PSMA small humanized monoclonal antibody.