Cancer immunotherapy based on mutation-specific CD4+ T cells in a patient with epithelial cancer

Abstract

Limited evidence exists that humans mount a mutation-specific T cell response to epithelial cancers. We used a whole-exomic-sequencing-based approach to demonstrate that tumor-infiltrating lymphocytes (TIL) from a patient with metastatic cholangiocarcinoma contained CD4+ T helper 1 (T(H)1) cells recognizing a mutation in erbb2 interacting protein (ERBB2IP) expressed by the cancer. After adoptive transfer of TIL containing about 25% mutation-specific polyfunctional T(H)1 cells, the patient achieved a decrease in target lesions with prolonged stabilization of disease. Upon disease progression, the patient was retreated with a >95% pure population of mutation-reactive T(H)1 cells and again experienced tumor regression. These results provide evidence that a CD4+ T cell response against a mutated antigen can be harnessed to mediate regression of a metastatic epithelial cancer.

Trial registration: ClinicalTrials.gov NCT01174121.

Fig. 1.. Patient 3737–TIL harbor ERBB2IP mutation–specific CD4+ T cells.

(A to C) Patient 3737–TIL were cocultured with dendritic cells (DCs) transfected with nothing (Mock), green fluorescent protein (GFP) RNA, or the indicated TMG construct encoding the various mutations identified by whole-exomic sequencing.(A) IFN-γ enzyme-linked immunosorbent spot assay at 20 hours. “>“ denotes greater than 500 spots per 1e3 cells. (B and C) Flow-cytometric analysis at 24 hours. Data are gated on live CD3+ cells and, for (C), further gated on CD4+ cells. (D) A CD4+ Vβ22+ clone was co-cultured for 6 hours with autologous B cells pulsed overnight with wild-type (wt) ERBB2IP, mutated (mut) anaplastic lymphoma receptor tyrosine kinase (ALK), or mut ERBB2IP 25-amino acid- long peptides. Flow cytometry was used to detect intracellular IFN-γ production in the CD4+ population. Data are representative of two clones sharing the same TCR-Vβ sequence. (E) Autologous open-repertoire peripheral blood T cells were transduced (Td) with nothing (Mock) or the TCR derived from the Vβ22+ clone and then assessed for reactivity as described in (D). The endogenous Vβ22+ TCR constant regions were swapped with mouse constant regions, allowing for the detection of the introduced TCR using antibodies against the mouse TCRβ constant region (mTCRβ). Plate-bound OKT3 was used as a control in all assays. All data are representative of at least two independent experiments. Error bars are SD.

Fig. 2.. Adoptive transfer ofTILcontainingERBB2IP mutation–reactive T cells.

(A) Flow-cytometric analysis of the TCR-Vβ repertoire of 3737-TIL, gated on live CD4+ or CD8+ T cells. (B) Patient 3737–TIL were cocultured with DCs transfected with TMG-1 or TMG-1 encoding the wt ERBB2IP reversion, and flow cytometry was used to assess OX40 and Vβ22 expression on CD4+ T cells at 24 hours post-stimulation. Plate-bound OKT3 stimulation was used as a positive control. (A) and (B) are representative of at least two independent experiments. (C) IFN-γ enzyme-linked immunosorbent assay on patient 3737 serum samples pre- and post-adoptive cell transfer of 3737-TIL. Error bars are SEM. (D) Tumor growth curves [Response Evaluation Criteria in Solid Tumors (RECIST), sum of maximum diameters] before and after infusion of 3737-TIL. Data are expressed as a change in percent from pretreatment baseline and stratified on lung, liver, and total tumors.

Fig. 3.. Functional phenotype and persistence of ERBB2IP mutation–specific CD4+ T cells.

(A) Patient 3737–TIL were cocultured for 6 hours with autologous B cells pulsed overnight with wt ERBB2IP, mut ALK, or mut ERBB2IP 25–amino acid–long peptides. Flow cytometry was used to assess expression of Vβ22 and to detect intracellular production of IL-2, TNF, and IFN-γ in the CD4+ population. Pie charts display the percentage of Vβ22+ cells that expressed the indicated number of cytokines. Data are representative of at least three independent experiments. (B) TCR-Vβ deep sequencing of 3737-TIL and blood and tumors of patient 3737 at various times pre- and post-adoptive cell transfer with 3737-TIL. Data show the frequency of the two ERBB2IP mutation–specific TCRβ-CDR3 clonotypes. ⊗, not detected.

Fig. 4.. Evidence of tumor regression after treatment with a highly pure population of Vβ22+ ERBB2IP mutation–reactive CD4+ T cells.

(A) Flow-cytometric analysis of the TIL product used for retreatment. Data are gated on live CD3+ T cells (left) and further gated on CD4+ cells (right).(B) Retreatment TIL were cocultured for 6 hours with autologous B cells pulsed overnight with wt or mut ERBB2IP 25–amino acid–long peptides. Flow cytometry was used to detect intracellular TNF production in the CD4+ population. (A) and (B) are representative of at least two independent experiments. (C) Tumor growth curves (sum of maximum diameters) of patient 3737 before and after first and second adoptive cell transfers. The first infusion product consisted of 42.4 billion T cells containing about 25% (10 billion) Vβ22+ ERBB2IP mutation–reactive T_H1 cells. The second infusion product consisted of 126 billion T cells containing over 95% (120 billion) Vβ22+ ERBB2IP mutation–reactive T_H1 cells. Data are expressed as a change from baseline and stratified on lung, liver, and total tumors. Some target lesions selected in (C) differ from Fig. 2D because only lesions that were present throughout both treatments were selected for measurement.