Rapid T cell–based identification of human tumor tissue antigens by automated two-dimensional protein fractionation
Philipp Beckhove, Rolf Warta, Britt Lemke, Diana Stoycheva, Frank Momburg, Martina Schnölzer, Uwe Warnken, Hubertus Schmitz-Winnenthal, Rezvan Ahmadi, Gerhard Dyckhoff, Mariana Bucur, Simone Jünger, Thomas Schueler, Volker Lennerz, Thomas Woelfel, Andreas Unterberg, Christel Herold-Mende
Philipp Beckhove, Rolf Warta, Britt Lemke, Diana Stoycheva, Frank Momburg, Martina Schnölzer, Uwe Warnken, Hubertus Schmitz-Winnenthal, Rezvan Ahmadi, Gerhard Dyckhoff, Mariana Bucur, Simone Jünger, Thomas Schueler, Volker Lennerz, Thomas Woelfel, Andreas Unterberg, Christel Herold-Mende
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Technical Advance Oncology

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 individual cancer 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.

Authors

Philipp Beckhove, Rolf Warta, Britt Lemke, Diana Stoycheva, Frank Momburg, Martina Schnölzer, Uwe Warnken, Hubertus Schmitz-Winnenthal, Rezvan Ahmadi, Gerhard Dyckhoff, Mariana Bucur, Simone Jünger, Thomas Schueler, Volker Lennerz, Thomas Woelfel, Andreas Unterberg, Christel Herold-Mende

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Figure 5

TTR as target antigen of autologous T cells from NCH550.

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TTR as target antigen of autologous T cells from NCH550. (A) Molecular s...
(A) Molecular structure and amino acid sequence (AAS) of TTR and TTR precursor (protein ID NP_000362). Black lines indicate synthetic polypeptides used for T cell stimulation. (B) Recognition of synthetic polypeptides by NCH550 T cells in IFN-γ ELISPOT assay. Autologous PB-L, human IgG, T cells, and DCs served as negative controls. DCs pulsed by TTR3 resulted in significantly increased IFN-γ spot numbers. (C) Significant reactivity of total CD3, CD8+, and CD4+ T cells against DCs pulsed with either total autologous tumor lysate or with synthetic TTR3 as compared with PB-L control. (D) AAS of the immunogenic region of TTR as revealed by IFN-γ ELISPOT assays using synthetic polypeptides. (E) AAS of predicted epitopes potentially presented by different HLA-I molecules of NCH550 (HLA-A*0101–restricted epitopes, black; HLA-A*0201–restricted epitopes, blue; HLA-B*4101, red; HLA-B*5101, green). Colored asterisks indicate epitopes that may be also presented by other HLA-I molecules. (F) Recognition of HLA-I–restricted peptides (E). DCs pulsed with autologous PB-L, human IgG, and HLA-A0201–restricted peptide from HIVgag served as negative control antigens. White bars indicate unstimulated or PB-L–stimulated subpopulations of CD3+, CD8+, or CD4+ T cells; black bars show significantly increased IFN-γ spot numbers as compared with PB-L control; gray bars show nonsignificant T cell responses. (G) AAS of identified immunogenic target epitopes of TTR restricted to HLA-A*0101, HLA-A*0201, and HLA-B*4101 as recognized by T cells from patient NCH550 are indicated by black lines. Small indices correspond to peptide numbers as shown in E and F.