CXorf61 is a target for T cell based immunotherapy of triple-negative breast cancer

Abstract

Triple-negative breast cancer (TNBC) is a high medical need disease with limited treatment options. CD8+ T cell-mediated immunotherapy may represent an attractive approach to address TNBC. The objectives of this study were to assess the expression of CXorf61 in TNBCs and healthy tissues and to evaluate its capability to induce T cell responses. We show by transcriptional profiling of a broad comprehensive set of normal human tissue that CXorf61 expression is strictly restricted to testis. 53% of TNBC patients express this antigen in at least 30% of their tumor cells. In CXorf61-negative breast cancer cell lines CXorf61 expression is activated by treatment with the hypomethylating agent 5-aza-2'-deoxycytidine. By vaccination of HLA-A*02-transgenic mice with CXorf61 encoding RNA we obtained high frequencies of CXorf61-specific T cells. Cloning and characterization of T cell receptors (TCRs) from responding T cells resulted in the identification of the two HLA-A*0201-restricted T cell epitopes CXorf6166-74 and CXorf6179-87. Furthermore, by in vitro priming of human CD8+ T cells derived from a healthy donor recognizing CXorf6166-74 we were able to induce a strong antigen-specific immune response and clone a human TCR recognizing this epitope. In summary, our data confirms this antigen as promising target for T cell based therapies.

Keywords: CD8+ T cell epitope; CXorf61; T-cell receptor cloning; TNBC; immunotherapy.

Figure 1. Frequent expression of CXorf61 mRNA in TNBC samples and absence from the vast majority of normal human tissue types

CXorf61 expression was analysed by qRT-PCR using the BioMark™ HD system on 62 normal tissue types A. and 53 TNBC samples B, C. Expression of CXorf61 in human breast cancer cell lines by qRT-PCR without (−) or after addition of 5-aza-dC. After normalization to the housekeeping gene HPRT1, the relative quantification value was expressed as 2^−ΔΔCt. Expression analysis was done in triplicates. Standard deviation is indicated.

Figure 2. Robust expression of CXorf61 protein in primary TNBC, TNBC cell lines and normal testis

A. CXorf61 protein expression was analyzed with antibody anti-CXorf61-B in the lysates of 2 TNBC samples (patients # 40 and 19, Supplementary Table S1). GAPDH was used as loading control. Positive control: lysate of HEK 293T transfected with a plasmid coding for CXorf61. Negative control: HEK 293T transfected with empty vector. B. Nuclei and cytosol isolated from the MDA-MB-468 cell line were analysed by Western Blot with the CXorf61 specific antibody anti-CXorf61-B or antibodies against different cellular compartments (Histon, Lamin B, GAPDH). C. Staining of tissue sections by immunohistochemistry with antibody anti-CXorf61-A. Tissues were obtained by xenografting HEK 293T-CXorf61 (a) or HEK 293T-mock (b), MDA-MB-468 cells (c) and MDA-MB-231 cells (d) in mice. Human testis (e) and 4 TNBC tissues (g-j) were stained. Negative control: staining of the testis without any primary antibody (f). Tissues g-j correspond to patients # 40, 56, 48 and 62 (Supplementary table S1). Scale bar = 20 μM.

Figure 3. Cloning of murine TCRs from CXorf61-specific T cells induced by immunization of HLA-transgenic mice and identification of HLA-A*02-restricted epitopes

A. Detection of CXorf61-specific T cells after 5 rounds of immunization of HLA-A*0201-transgenic mice with CXorf61 encoding IVT RNA. Spleen cells from three mice were analyzed for reactivity against CXorf61 overlapping peptide pool (CXorf61 pool) or predicted HLA-A*0201-binding CXorf61-derived peptides by IFNy-ELISPOT assay. B. FACS sorting of CXorf61-specific murine CD8+ T cells from spleen cells of immunized HLA-A*0201-transgenic mice after in vitro restimulation with overlapping peptide pools. Cells were gated on CD3+/CD8+ lymphocytes. Single CD8+/CD137+ T cells were isolated C. Specificity testing of murine TCRs isolated from CD8+ T cells of CXorf61-immunized mice. CD8+ T cells of a HLA-A*0201-positive healthy donor were transfected with TCR-α/β chain RNAs and tested for recognition of K562-A*0201 transfected with CXorf61 IVT RNA or pulsed with CXorf61 peptide pool or HLA-A*02 binding peptides by IFNγ-ELISPOT. D. OKT3-activated CD8+ T cells from a healthy donor were transfected with the murine TCR#4 IVT RNA and co-cultured with autologous immature DCs transfected with CXorf61 IVT RNA or loaded with CXorf61_66–74. Specific killing was analyzed by luciferase cytotoxicity assay. Positive controls: Phorbol-12-myristate-13-acetate treated spleen cells (PMA); Staphylococcus Enterotoxin B treated cells (SEB); negative controls: spleen cells without stimulus (medium); irrelevant HLA-A*0201-restricted peptide PLAC1_31–39 (control peptide); pool of overlapping 15mer peptides representing the irrelevant antigen HIV-gag (control pool); IVT RNA encoding the irrelevant antigen TPTE (control RNA), T cells transfected without TCR RNA (CD8 mock).

Figure 4. Induction of human CXorf61-specific T cells in vitro and cloning of a CXorf61-specific human TCR

A. CD8+ T cells of a healthy HLA-A*0201 expressing donor were primed using autologous mDC transfected with CXorf61 RNA. After three rounds of stimulation CXorf61-specific T cells were detected by CXorf61_66–74/A*0201 dextramer staining and isolated by flow cytometry for TCR cloning. B. Specificity testing of a TCR isolated from human CD8+ T cells sensitized against CXorf61. CD8+ T cells transfected with the cloned TCR chains were tested by IFNγ-ELISPOT for recognition of K562-A*0201 cells either transfected with CXorf61 IVT RNA or loaded with CXorf61_66–74; negative controls: irrelevant RNA CLDN6 (control RNA); irrelevant peptide PLAC1_31–39 (control peptide); positive control: SEB treated cells.