Immune recognition of somatic mutations leading to complete durable regression in metastatic breast cancer

Abstract

Immunotherapy using either checkpoint blockade or the adoptive transfer of antitumor lymphocytes has shown effectiveness in treating cancers with high levels of somatic mutations-such as melanoma, smoking-induced lung cancers and bladder cancer-with little effect in other common epithelial cancers that have lower mutation rates, such as those arising in the gastrointestinal tract, breast and ovary^1-7. Adoptive transfer of autologous lymphocytes that specifically target proteins encoded by somatically mutated genes has mediated substantial objective clinical regressions in patients with metastatic bile duct, colon and cervical cancers^8-11. We present a patient with chemorefractory hormone receptor (HR)-positive metastatic breast cancer who was treated with tumor-infiltrating lymphocytes (TILs) reactive against mutant versions of four proteins-SLC3A2, KIAA0368, CADPS2 and CTSB. Adoptive transfer of these mutant-protein-specific TILs in conjunction with interleukin (IL)-2 and checkpoint blockade mediated the complete durable regression of metastatic breast cancer, which is now ongoing for >22 months, and it represents a new immunotherapy approach for the treatment of these patients.

Fig. 1 |. TIL populations from patient 4136 recognize autologous mutant SLC3A2 and KIAA0368 antigens.

a, Interferon (IFN)-γ production, as determined by ELISPOT assay, following coculture with autologous B cells pulsed with the respective peptide pools, showing that TIL fragments F8 and F12 recognize a component of peptide pool PP1 and that F13 recognizes a component of peptide pool PP6. b, IFN-γ production following coculture with autologous B cells that were electroporated with TMG RNA, showing that fragment F13 also recognizes one of the TMGs within TMG6. c, IFN-γ production following coculture with autologous B cells that were pulsed with the individual peptides within peptide pool PP1, showing that TIL fragments F8 and F12 recognize the mutSLC3A2 (p.Lys94Thr, p.Lys63Thr) peptides. d, IFN-γ production following coculture with autologous B cells that were pulsed with the individual peptides within peptide pool PP6, showing that F13 specifically recognizes the mutKIAA0368 (p.Ser186Phe). e, Flow cytometry analysis of F12 T cells after coculture with B cells that were pulsed with mutSLC3A2 peptides, showing that 4–1BB expression was upregulated. No upregulation was observed when mutant transmembrane protein 53 (TMEM53) or other mutant peptides from PP1 (not shown) were tested. f, Flow cytometry analysis of F13-reactive T cells after coculture with B cells that were pulsed with mutKIAA0368 (p.Ser186Phe) peptide, showing that 4–1BB expression was upregulated. No upregulation was observed when mutant zinc finger CCCH-type containing 3 (ZC3H3) or other mutant peptides from PP6 (not shown) were tested. In e and f, cells were gated on CD3, and data are representative of at least three independent experiments. g, Flow cytometry analysis of allogeneic T cells that were transduced with the sequences encoding TCR K, which specifically recognized the 18-mer mutSLC3A2 peptide (amino acid sequence LLASSDPPALASTNAEVT), as shown by upregulation of 4–1BB. These results are representative of all seven TCRs that were found to specifically recognize the mutSLC3A2 peptide. h, Flow cytometry analysis of allogeneic T cells that were transduced with sequences encoding TCR R, which specifically recognized the 25-mer mutKIAA0368 (p.Ser186Phe) peptide, as shown by upregulation of 4–1BB. In g and h, cells were gated on CD3, and data are representative of two independent experiments. WT, wild type.

Fig. 2 |. Adoptive transfer of autologous TILs targeting immunogenic tumor mutations mediated tumor regression.

a, Treatment schema, with characteristics of the infusion product. Initial gating for flow cytometry analysis was done on live CD3⁺ cells. b, Interferon (IFN)-γ production, as determined by ELISPOT assay, showing that the infusion product, consisting of TILs expanded from fragments 8, 12 and 13 maintained their reactivity to mutSLC3A2 and mutKIAA0368. c, Top, response curves of target lesions (tumor size measurements). All lesions resolved 1 year after TIL transfer, and the patient continued to demonstrate complete response 22 months after cell infusion and 20 months after the last dose of pembrolizumab. Bottom, cross-sectional imaging was obtained 1 week before cell infusion (pre-treatment) and 22 months after infusion (22 months post-treatment). Arrows indicate target lesions (from top to bottom: retrosternal mediastinum, left axilla with clinical brachial plexopathy and compressed axillary vein, and multiple liver segments). SubQ, subcutaneous. d, Top, images showing pre-treatment tumor biopsy of a left chest wall mass (present at time of treatment), demonstrating ductal breast adenocarcinoma with scattered peripheral PD-1⁺ lymphocytes and few intratumoral lymphocytes (magnification: 40× ). Bottom, images showing the subcutaneous tumor that served as the source of TILs, with intratumoral PD-1+ lymphocytes and PD-L1⁺ stroma. Tumor cells were negative for PD-L1 expression (magnification: H&E-stained images, 10×; CD3-, PD-1- or PD-L1-stained images, 20× ).

Fig. 3 |. Persistence of known mutant-reactive TCR clonotypes at time of infusion and identification of new dominant clonotypes of unknown reactivity present in an apheresis product obtained 6 weeks after treatment.

a, TRBV diversity of the infused TILs with eight unique CDR3s (antigen, TCR, %, rank) associated with mutant-protein-specific reactive TCRs that account for 23.1% of the infused product. Unlabeled sections represent different TCRBV families (TCRBV01–30) without known reactivity. b, TRBV diversity of peripheral blood sampled at 6 weeks after infusion (post treatment). Known reactive TCR clonotypes accounted for 1.73% of circulating lymphocytes. The TCRBV20 and TCRVB28 populations were FACS-sorted from post-treatment peripheral blood for additional screening of mutant-peptide reactivity. c, The TRBV28 population was screened against peptide pools and TMGs as previously described, and it demonstrated concordant reactivity to PP6 and TMG6 by an IFN-γ ELISPOT assay. Data are representative of a single experiment and were confirmed by individual peptide experiments. d, The TCRBV28 population demonstrated reactivity against peptides encoded by two nonsynonymous mutations within PP6—CADPS2 (p.Arg1266His) and CTSB (p.Asp159His). TCRs were isolated from CD3⁺4–1BB⁺ peripheral blood lymphocytes (PBLs) following coculture with autologous B cells pulsed with either mutCADPS2 or mutCTSB. e, Flow cytometry analysis showing that allogeneic peripheral blood T cells that were transduced with constructs encoding TCR J (TRBV28–01–TRAV26–02) specifically recognized the 25-mer mutCADPS2 peptide and not the WT peptide, as shown by upregulation of 4–1BB. f, Flow cytometry analysis showing that allogeneic peripheral blood T cells that were transduced with constructs encoding TCR M (left) or TCR O (right), both of which are TRBV28–01–TRAV12–01, specifically recognized the 25-mer mutCTSB peptide, and minimal recognition was observed for the WT CTSB peptide, as shown by upregulation of 4–1BB. Data in e and f are representative of two independent experiments.

Fig. 4 |. Persistence of the 11 mutant-reactive TCR clonotypes from cell infusion to 17 months after cell transfer.

a, Of the 11 clonotypes, only 2 were detected in pre-treatment peripheral blood (denoted by the asterisk) and comprised 0.005% of all CDR3 sequences. The total percentage of reactive clonotypes identified in the peripheral blood was highest at the earliest time point (3.29%), but eight of these clonotypes persisted 17 months after treatment, comprising 0.81% of all CD3⁺ cells. Clonotypes (of the TRBV20 and TRBV28 families; see Fig. 3b) of unknown reactivity that dominated the peripheral blood at 6 weeks after transfer persisted as 3.6% of productive CDR3 sequences at 17 months after transfer (data not shown). Sequences corresponding to TCR J were detected only with ultra-deep sequencing of the infusion bag, but TCR J was a dominant intratumoral clonotype that expanded after treatment. b, The pattern of persistence in peripheral blood for each of the eight mutant-reactive clonotypes that persisted at 17 months after transfer. Bars indicate the percentage of infusion product. Rank (in parentheses) indicates relative position of the unique CDR3 sequence when all productive CDR3 sequences are sorted in decreasing frequency. Crossed circles indicate ‘not detected’ (n.d.) with a cut-off frequency of 0.0005. For the +1-week and +3-week samples, the lower limits of detection were 0.0044 and 0.0016, respectively. Rx1, treatment 1. FrTu, fresh tumor. c, Clonal architecture of the resected tumor, with recognized neoantigens identified. The top panel provides a histogram of the density of the nonsynonymous mutations depicted in the bottom panel. a.u., arbitrary units.