Rapid T cell–based identification of human tumor tissue antigens by automated two-dimensional protein fractionation

Abstract

Identifying the antigens that have the potential to trigger endogenous antitumor responses in an individualcancer patient is likely to enhance the efficacy of cancer immunotherapy, but current methodologies do not efficiently identify such antigens. This study describes what we believe to be a new method of comprehensively identifying candidate tissue antigens that spontaneously cause T cell responses in disease situations. We used the newly developed automated, two-dimensional chromatography system PF2D to fractionate the proteome of human tumor tissues and tested protein fractions for recognition by preexisting tumor-specific CD4 + Th cells and CTLs. Applying this method using mice transgenic for a TCR that recognizes an OVA peptide presented by MHC class I, we demonstrated efficient separation, processing, and cross-presentation to CD8 + T cells by DCs of OVA expressed by the OVA-transfected mouse lymphoma RMA-OVA. Applying this method to human tumor tissues, we identified MUC1 and EGFR as tumor-associated antigens selectively recognized by T cells in patients with head and neck cancer. Finally, in an exemplary patient with a malignant brain tumor, we detected CD4 + and CD8 + T cell responses against two novel antigens, transthyretin and calgranulin B/S100A9, which were expressed in tumor and endothelial cells. The immunogenicity of these antigens was confirmed in 4 of 10 other brain tumor patients. This fast and inexpensive method therefore appears suitable for identifying candidate T cell antigens in various disease situations, such as autoimmune and malignant diseases, without being restricted to expression by a certain cell type or HLA allele.

Proteomics for development of vaccine

“…Beckhove et al [183] used the newly developed automated, two-dimensional chromatography system PF2D to fractionate the proteome of human tumor tissues and subsequently tested protein fractions for recognition of preexisting tumorspecific CD4 + Th cells and CTLs. Fractioning by the PF2D system is based on the isoelectric point (pI) and hydrophobicity of processed proteins.…”

“…The PF2D system is suitable to identify "classic" TAAs that are not recognized by preexisting memory cells [183]. Response to such TAAs can be potentially developed de novo by vaccination.…”

Immunotherapy for head and neck cancer

“…To increase the effectiveness of immunotherapeutic strategies, efforts are underway to improve the identification of candidate tumor antigens that cause T cell responses. One such effort involves the use of a newly developed, automated, two-dimensional chromatography system PF2D that fractionates the proteome of human tumor tissues [4]. Advances in this area have the potential to reveal more immunogenic proteins to target, leading to stronger and more enduring responses to therapy.…”

“…Increased intraepithelial CD8 + tumor-infiltrating lymphocytes (TILs) in HNSCC metastases, as well as increased numbers of CD20 + B cells in involved lymph nodes, are associated with a better prognosis [8]. Also, both CD4 + and CD8 + T cells isolated from patients with HNSCC can be activated in response to tumor antigens [4]. Antigen processing and cross-presentation by dendritic cells to CD8 + T cells can stimulate their reactivity to tumor antigens.…”

Non-invasive glioblastoma immunoprofiling by printed peptide arrays

“…As a consequence, there is a need for meaningful immune assays to profile and monitor the immune status of tumor patients. As for GBMs, antitumor T-cell responses were described, [2][3][4] but are still challenging to quantify in a timely and highthroughput manner. On the contrary, little is known about antitumor B-cell response in GBM, but serum antibodies could be observed 5 and are comparatively easy to quantify.…”