T Cells that Recognize HPV Protein Can Target Virus-Infected Cells

The T cell clone from which the E6 TCR was isolated was approximately 400 times more prevalent in the tumor than in the peripheral blood. The complementarity-determining regions (CDRs) sequence of the E6 TCR was determined. TCR clone analysis was performed by ImmunoSeq. Clones occurring at greater than 1 percent frequency are shown. The E6 T cell is in red. The frequency and rank (by frequency) of the E6 T cell in the tumor and in peripheral blood is indicated in text.

Adoptive T-cell transfer (ACT) is a promising form of cancer immunotherapy. Treating patients with T cells isolated from a tumor and subsequently expanded in the lab can cause the complete regression of some melanomas and cervical cancers, but the treatment is currently restricted to a few cancer types. An approach that may be applied to a wider array of cancers involves modifying peripheral blood T cells with chimeric antigen receptors or T-cell receptors (TCR) that target specific tumor antigens. Unfortunately, epithelial cancers, which are the vast majority of cancers diagnosed, have proven difficult to treat this way because most identified antigens are shared with healthy tissues and targeting them leads to toxic side effects. However, cancers caused by persistent human papillomavirus (HPV) infection, including cervical, head and neck, anal, vaginal, vulvar, and penile cancers, may be particularly amenable to the latter form of ACT since the E6 and E7 viral proteins are essential for cancer formation but are not produced in normal tissues. To test this idea, Christian Hinrichs, M.D., and his colleagues examined tumor infiltrating lymphocytes (TILs) from a patient who experienced a prolonged disease-free period after her second surgical removal of metastatic anal cancer in the hopes of identifying a TCR against one of the HPV oncoproteins.

The patient’s metastasis was removed from a portal lymph node. Real-time reverse-transcription polymerase chain reaction (RT-PCR) of tumor tissue demonstrated expression of E6 and E7 genes from the HPV-16 strain, which causes the majority of HPV-related cancers. Immunohistochemistry revealed the presence of human leukocyte antigen (HLA) class I and the infiltration of CD8-positive immune cells. Given this, the researchers grew lymphocyte cultures from a number of tumor fragments and found that nine of the cultures reacted to HPV-16 E6 but not E7 or the control protein gp100.

The investigators next isolated a population of the T cells with enriched E6 reactivity, dubbed E6 TILs. They tested the cells’ ability to recognize full-length E6 expressed by a cell line with stable expression of HLA-A*02:01, the most common HLA allele in Caucasian patients. The E6 TILs recognized the cells expressing E6 but not similar cells expressing only green fluorescent protein, suggesting the cells targeted a naturally processed region of E6 and that recognition might be HLA-A*02:01 restricted. To test this possibility, the scientists analyzed the binding of E6 TILs to a proposed HLA-A*02:01-restricted region of E6, E29-38, with flow cytometry. Thirty-two percent of the E6 TILs bound the HLA-A*02:01/E29-38 peptides.

The researchers then asked whether the dominant E6-reactive clone from the E6 TIL localized to the patient’s tumor. They performed TCR clone analysis using 5’ rapid analysis of cDNA ends (5’ RACE) and sequenced the TCR α and β chains. The analysis revealed a dominant β-chain clone, named E6 TCR, which was the eighth most common T-cell clone in the tumor with a frequency of 1.6 percent. In peripheral blood, however, the clone had a frequency of 0.004 percent, suggesting it preferentially accumulated in the tumor likely in response to the presence of its target antigen.

To generate T cells that could recognize HPV-16 E6, the investigators designed a retroviral vector encoding the α- and β-chains of the E6 TCR and transduced T cells isolated from peripheral blood mononuclear cells with the retrovirus. Both CD8-positive and -negative engineered T cells could bind to HLA-A*02:01/E29-38tetramers. However, the CD8-positive T cells displayed higher functional activity, suggesting that the CD8 co-receptor contributed to target recognition.

The engineered T cells recognized target cells, including cervical and head and neck cancer cell lines, as well as cell lines transfected with full-length E6. The binding of the engineered cells to the cancer cell lines was blocked by anti-HLA class I, but not class II, antibodies, while binding to the transfected cells required co-expression of HLA-A*02:01. Recognition of target cells led to the production of interferon-γ and tumor necrosis factor α by the T cells and lysis of target cells. Encouragingly, the T cells expressing E6 TCR did not react to human peptides that resembled the HPV-16 E6 target sequence.

These results are the first to describe high-avidity T cells targeting E6 that can recognize HPV-positive tumor cells. Using the associated TCR to engineer T cells for ACT may permit the treatment of a family of otherwise incurable epithelial tumors. Future studies will need to test the activity of these T cells in vivo, and a clinical trial (NCT02280811) of E6 TCR gene therapy is currently underway.

Summary Posted: Sun, 11/01/2015


Draper LM, Kwong MLM, Gros A, Stevanovic S, Tran E, Kerkar S, Raffeld M, Rosenberg SA, and Hinrichs CS. Targeting of HPV-16+ Epithelial Cancer Cells by TCR Gene Engineered T Cells Directed Against E6. Clinical Cancer Research. October 1, 2015. PubMed Link