Mutated PPP1R3B is recognized by T cells used to treat a melanoma patient who experienced a durable complete tumor regression

Abstract

Adoptive cell therapy with tumor-infiltrating lymphocytes (TILs) represents an effective treatment for patients with metastatic melanoma. However, most of the Ag targets recognized by effective melanoma-reactive TILs remain elusive. In this study, patient 2369 experienced a complete response, including regressions of bulky liver tumor masses, ongoing beyond 7 y following adoptive TIL transfer. The screening of a cDNA library generated from the autologous melanoma cell line resulted in the isolation of a mutated protein phosphatase 1, regulatory (inhibitor) subunit 3B (PPP1R3B) gene product. The mutated PPP1R3B peptide represents the immunodominant epitope recognized by tumor-reactive T cells in TIL 2369. Five years following adoptive transfer, peripheral blood T lymphocytes obtained from patient 2369 recognized the mutated PPP1R3B epitope. These results demonstrate that adoptive T cell therapy targeting a tumor-specific Ag can mediate long-term survival for a patient with metastatic melanoma. This study also provides an impetus to develop personalized immunotherapy targeting tumor-specific, mutated Ags.

FIGURE 1

Response of patient 2369 after adoptive cell therapy. Computed tomography (CT) and magnetic resonance imaging (MRI) scans showed the disease status after the hepatic resection for TIL harvest (A), prior to the treatment (B) and seven years after the adoptive cell therapy (C). White arrows indicate the lesions.

FIGURE 2

Identification of dominant HLA-restriction element of TIL 2369 T cells. (A) Mel 2369 cells were pre-incubated with HLA-B,C (B1.23.1) or HLA-A,B,C (W6/32) blocking antibodies for 3 hr, followed by co-cultured with TIL 2369 T cells, Mage-A3 TCR-transduced or Mage-A12 TCR-transduced T cells overnight. (B) Mel 2369 cells were transfected with HLA-A*01 siRNA and cDNA, and the expression of HLA-A1 and A26 was determined by flow cytometry (NT, non-targeting). (C) Mel 2369 cells were transfected with HLA-A*01 siRNA and cDNA, and then co-cultured with autologous TIL 2369 T cells. The secretion of IFN-γ by T cells was determined by ELISA.

FIGURE 3

Identification of mutated PPP1R3B as the potential antigen. (A) cDNA library screening of the potential antigen. A single colony containing a mutated PPP1R3B cDNA fragment was isolated from a pool of 50 colonies. (B) Gene structure of PPP1R3B. Both transcripts encode the same protein. (C) DNA sequence chromatogram results for PPP1R3B genomic DNA obtained from autologous PBMC and Mel 2369 cells. (D) DNA sequence chromatogram results for PPP1R3B cDNA obtained from autologous Mel 2369 cells.

FIGURE 4

TIL 2369 T cells recognize mutated, but not wild-type PPP1R3B gene product. (A) PPP1R3B mRNA expression in melanoma cell lines after the transfection of Mel 2369 cells with PPP1R3B siRNAs. The copy numbers of PPP1R3B transcript variant 1 and variant 2 from Mel 2369 cells after siRNA transfection were determined by quantitative PCR (NT, non-targeting). (B) Mel 2369 cells were transfected with PPP1R3B siRNAs, and then co-cultured with TIL 2369 T cells or HLA-A*01-restricted Mage-A3 TCR-transduced T cells. (C) COS-7 cells were transfected with HLA cDNA constructs, together with wild-type or mutated PPP1R3B cDNA construct. These transfected cells were co-culture with TIL 2369 T cells overnight. The secretion of IFN-γ was determined by ELISA.

FIGURE 5

Identification of the PPP1R3B epitope. (A) Constructs encoding the truncations of the carboxy terminus of the mutated PPP1R3B (ORF, open reading frame). (B) 293-A1 cells were transfected with these constructs, followed by co-cultured with TIL 2369 T cells. The secretion of IFN-γ was determined by ELISA. (C) Multiple peptides covered the region of 175 ~ 186 a.a. were synthesized. (D) 293-A1 cells were pulsed with these peptides, followed by co-cultured with TIL 2369 T cells. (E) Target cells, including melanoma cells or 293-A1 cells pulsed with 172wt or 172mut peptide, were incubated with TIL 2369 T cells in a ⁵¹Cr-release assay. HLA-A*01⁺ Mel 2556 cells lack the PPP1R3B mutation.

FIGURE 6

Mutated PPP1R3B-reactive T cells are responsible for tumor recognition. Intracellular IFN-γ staining (A) and TCR Vβ14 co-staining (B) of TIL 2369 T cells after co-culture with autologous Mel 2369 cells or 293-A1 cells pulsed with PPP1R3B 172wt or 172mut peptide. (C) Healthy donor T cells were transduced with mut PPP1R3B TCR. These T cells were co-cultured with Mel 2369, Mel 2556 (HLA-A*01⁺) cells or 293-A1 cells pulsed with PPP1R3B 172wt or 172mut peptide. (D) In a ⁵¹Cr-release assay, health donor T cells transduced with mut PPP1R3B TCR or un-transduced T cells were incubated with autologous Mel 2369 cells or Mel 2556 cells.

FIGURE 7

The long-term persistence of mutated PPP1R3B-reactive T cells. Patient 2369 PBMC samples were thawed and incubated overnight in medium with IL-2, followed by stimulation with PPP1R3B 172wt or 172mut peptides. The secretion of IFN-γ was determined by ELISA after overnight culture.