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. 2015 Oct 1;21(19):4431-9.
doi: 10.1158/1078-0432.CCR-14-3341.

Targeting of HPV-16+ Epithelial Cancer Cells by TCR Gene Engineered T Cells Directed against E6

Lindsey M Draper  1 , Mei Li M Kwong  1 , Alena Gros  1 , Sanja Stevanović  1 , Eric Tran  1 , Sid Kerkar  2 , Mark Raffeld  2 , Steven A Rosenberg  1 , Christian S Hinrichs  3
Affiliations

Targeting of HPV-16+ Epithelial Cancer Cells by TCR Gene Engineered T Cells Directed against E6

Lindsey M Draper et al. Clin Cancer Res. .

Abstract

Purpose: The E6 and E7 oncoproteins of HPV-associated epithelial cancers are in principle ideal immunotherapeutic targets, but evidence that T cells specific for these antigens can recognize and kill HPV(+) tumor cells is limited. We sought to determine whether TCR gene engineered T cells directed against an HPV oncoprotein can successfully target HPV(+) tumor cells.

Experimental design: T-cell responses against the HPV-16 oncoproteins were investigated in a patient with an ongoing 22-month disease-free interval after her second resection of distant metastatic anal cancer. T cells genetically engineered to express an oncoprotein-specific TCR from this patient's tumor-infiltrating T cells were tested for specific reactivity against HPV(+) epithelial tumor cells.

Results: We identified, from an excised metastatic anal cancer tumor, T cells that recognized an HLA-A*02:01-restricted epitope of HPV-16 E6. The frequency of the dominant T-cell clonotype from these cells was approximately 400-fold greater in the patient's tumor than in her peripheral blood. T cells genetically engineered to express the TCR from this clonotype displayed high avidity for an HLA-A*02:01-restricted epitope of HPV-16, and they showed specific recognition and killing of HPV-16(+) cervical, and head and neck cancer cell lines.

Conclusions: These findings demonstrate that HPV-16(+) tumors can be targeted by E6-specific TCR gene engineered T cells, and they provide the foundation for a novel cellular therapy directed against HPV-16(+) malignancies, including cervical, oropharyngeal, anal, vulvar, vaginal, and penile cancers.

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Conflict of interest statement

Conflict of interest statement: Drs. Hinrichs and Rosenberg are inventors on an NIH patent related to this work. This research was funded in part by a cooperative research and development agreement with Kite Pharma.

Figures

Fig. 1
Fig. 1
The excised tumor of a patient experiencing a prolonged disease free interval harbored T cells with HPV-16 E6 reactivity. (A) Timeline for the clinical course of Patient 3809. Gray segments indicate progressively longer disease-free intervals including the current, ongoing 22-month period. (B) Contrast-enhanced computed tomography showing the portal lymph node metastasis (yellow arrow) that was resected and used to generate TIL. (C) Microscopic examination of the resected tumor including hematoxylin and eosin stain (H&E), as well as p16, HLA class I, and CD8 immunohistochemistry. (D) Testing of TIL cultures for HPV-16 E6 and E7 reactivity. The tumor fragment number is indicated on the x-axis. Target cells were autologous immature DCs loaded with pools of peptides spanning the antigen indicated in the legend. The W6/32 anti-MHC class I blocking antibody was added as indicated in the legend. TIL1 and TIL2 were TIL cultures generated from melanoma tumors. The concentration of IFN-γ in the supernatants following overnight coculture is shown.
Fig. 2
Fig. 2
TIL enriched for E6 reactivity displayed recognition of a naturally processed epitope of E6 and exhibited binding to HLA-A*02:01/E629-38 tetramer. (A) TIL were enriched for E6 reactivity as described in the Materials and Methods section and tested for recognition of a 293-A2 line transfected with a plasmid encoding full length E6 (293-A2+E6 FL) or GFP (293-A2+GFP FL). Other targets in this assay were an autologous EBV LCL line with (3809 LCL+E6) or without (3809 LCL) the E6 peptide pool. The OKT3 condition was a non-specific stimulation with plate-bound anti-CD3 antibody. The quantity of IFN-γ produced following overnight coculture with each target is displayed. (B) Flow cytometric analysis of E629-38 tetramer binding by E6-reactivity-enriched TIL from Patient 3809 or Patient 3775 (negative control). HLA-A2/E711-19 was a control tetramer composed of an HLA-A*02:01-restricted epitope of HPV-16 E7. The frequency of events in the right upper quadrant is indicated on each dot plot. The data are graphed on a logarithmic scale. (C) The frequency of T cell clonotypes from the tumor or blood. The β-chain complementary determining regions for clonotypes present at greater than one percent frequency are shown in color and labeled in the legend. The dominant TCR from E6-reactive 3809 TIL is indicated with arrows, and its frequency and clonotypic rank in tumor and peripheral blood is shown in the adjacent text.
Fig. 3
Fig. 3
Genetically engineered T cells expressing the E6 TCR displayed high avidity for cognate antigen. (A) Schematic of the E6 TCR cassette that was cloned into the MSGV1 retroviral vector. Human TCR α- and β-chain constant regions were replaced with their murine equivalents (mTRAC and mTRBC1) and α- (TRAV35*02/TRAJ41) and β- (TRBV7-6/TRBJ2-3/TRBD1) chains were linked with a Furin P2A connector. (B) Flow cytometric analysis of CD8- and CD8+ fractions of E6 TCR-transduced and un-transduced T cells. Mouse TCR β-chain constant region (mTRBC) expression and HLA-A2*02:01/E629-38 tetramer binding is shown. Dot plots are displayed on a logarithmic scale. (C) Functional avidity assay testing recognition by T cells transduced to express the E6 TCR (E6 TCR) of T2 cells pulsed with titrated concentrations of E629-38 peptide. Control effector cells were not transduced (UT). The concentration of IFN-γ in supernatants after overnight coculture is shown. Error bars represent the standard deviation of duplicate measurements. Data are representative of three separate experiments.
Fig. 4
Fig. 4
Recognition of cell lines by E6 TCR gene engineered T cells was HLA-A*2:01-restricted. The quantity of IFN-γ in supernatants following overnight coculture of E6 TCR transduced T cells with the targets indicated on the x-axis is shown. Target cells were 293 or 293-A2 lines transfected with a vector encoding full length E6 (E6 FL), or the cell lines SCC90, CaSki, or 624. SCC90 and CaSki are HLA-A*02:01+ and HPV-16+. The 624 line is HLA-A*02:01+ and HPV-16−. HLA class I (W6/32 clone) or class II (HB145 clone) blocking antibodies were added as indicated in the legend. The HLA class I blocking control consisted of T cells expressing an HLA-A*02:01-restricted MART-1 specific TCR that were cocultured with the 624 cell line. The HLA class II blocking control consisted of T cells expressing an HLA-DR4-restricted E7 specific TCR that were cocultured with dendritic cells loaded with the E7 peptide pool. Error bars represent the standard deviation of duplicate measurements, and data are representative of two experiments.
Fig. 5
Fig. 5
E6 TCR gene engineered T cells specifically recognized and killed HLA-A2+ HPV-16+ tumor cells. (A) IFN-γ and (B) TNF-α production by E6 TCR gene engineered T cells cocultured with a panel of targets possessing or lacking the HPV-16 E6 antigen and/or HLA-A*02:01 restriction element. Target expression of HLA-A*02:01 and HPV-16 E6 is indicated below the graph in panel B. Error bars represent the standard deviation of duplicate measurements, and data are representative of two independent experiments. In some experiments recognition of SCC90 and SCC152 was weaker. (C) Specific cytolysis of tumor cells by E6 TCR gene engineered (E6 TCR) or untransduced (UT) T cells as measured by Cr51 release assay. The target cell line is indicated above each graph. Error bars represent the standard error of the mean of triplicate measurements.
Fig. 6
Fig. 6
E6 TCR gene engineered T cells did not display cross-reactivity against human peptides. E6 TCR transduced T cells were cocultured with T2 cells pulsed with human peptides identified as sharing six (or five plus a conservative substitution) or more amino acids with the E629-38 epitope (Table S1). The concentration of IFN-γ in the supernatants following overnight coculture is shown. The MART (MART127-35) and gp100 (gp100154-162) peptides are HLA-A*02:01-restricted. The OKT3 condition was stimulation with plate-bound OKT3 antibody. Error bars represent the standard error of the mean of duplicate measurements. Asterisks indicate peptides derived from the same protein.
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