Immunotherapy resistance driven by loss of NY-ESO-1 expression in response to transgenic adoptive cellular therapy with PD-1 blockade

Abstract

Background: The tumor antigen NY-ESO-1 has been shown to be an effective target for transgenic adoptive cell therapy (ACT) for the treatment of sarcoma and melanoma. However, despite frequent early clinical responses, many patients ultimately develop progressive disease. Understanding the mechanisms underlying treatment resistance is crucial to improve future ACT protocols. Here, we describe a novel mechanism of treatment resistance in sarcoma involving loss of expression of NY-ESO-1 in response to transgenic ACT with dendritic cell (DC) vaccination and programmed cell death protein-1 (PD-1) blockade.

Methods: A HLA-A*02:01-positive patient with an NY-ESO-1-positive undifferentiated pleomorphic sarcoma was treated with autologous NY-ESO-1-specific T-cell receptor (TCR) transgenic lymphocytes, NY-ESO-1 peptide-pulsed DC vaccination, and nivolumab-mediated PD-1 blockade.

Results: Peripheral blood reconstitution with NY-ESO-1-specific T cells peaked within 2 weeks of ACT, indicating rapid in vivo expansion. There was initial tumor regression, and immunophenotyping of the peripheral transgenic T cells showed a predominantly effector memory phenotype over time. Tracking of transgenic T cells to the tumor sites was demonstrated in on-treatment biopsy via both TCR sequencing-based and RNA sequencing-based immune reconstitution, and nivolumab binding to PD-1 on transgenic T cells was confirmed at the tumor site. At the time of disease progression, the promoter region of NY-ESO-1 was found to be extensively methylated, and tumor NY-ESO-1 expression was completely lost as measured by RNA sequencing and immunohistochemistry.

Conclusions: ACT of NY-ESO-1 transgenic T cells given with DC vaccination and anti-PD-1 therapy resulted in transient antitumor activity. NY-ESO-1 expression was lost in the post-treatment sample in the setting of extensive methylation of the NY-ESO-1 promoter region.

Biological/clinical insight: Antigen loss represents a novel mechanism of immune escape in sarcoma and a new point of improvement in cellular therapy approaches.

Trial registration number: NCT02775292.

Keywords: Immunotherapy, Adoptive; Nivolumab; Sarcoma; Therapies, Investigational; Tumor Escape.

Figure 1

ACT of NY-ESO-1 transgenic T cells and given with DC vaccination and concurrent anti-PD-1 therapy resulted in transient antitumor activity. (A) Overview of the clinical and sample collection timeline for the analysis performed in this study. Dosing schedule for nivolumab was every 2 weeks and is represented by red arrows. (B) Pretreatment and (C) post-treatment PET-CT images from the patient showing evidence of initial antitumor activity (decreased 18F-FDG uptake). Arrows point to the pulmonary nodule. (D–G) CT scans of the inguinal mass (arrows point to the mass) are shown at (D) baseline before ACT, (E, F) during tumor regression on days +22 and +61 of ACT, and (G) during tumor progression on day +112 of ACT. ACT, adoptive cell therapy; Cy, cytarabine; DC, dendritic cell; Flu, fludarabine; IL, interleukin; PET, positron emission tomography; TCR, T-cell receptor; 18F-fluorodeoxyglucose, 18F-FDG.

Figure 2

Immunophenotyping of peripheral T cells shows a predominantly EM phenotype over time and confirms nivolumab-mediated PD-1 blockade. (A) Postinfusion peripheral blood levels of NY-ESO-1 TCR transgenic CD3⁺ cells over time. (B, C) Immunophenotyping of adoptively transferred TCRs overtime the CD8⁺ population. T-cell compartments analyzed include naïve, CM, EM, and EMRA. (C) analysis of T cell exhaustion among total CD8⁺ and NY-ESO-1 TCR transgenic CD8⁺ populations. exhaustion determined by CD39/PD1 positivity. (D) PD-1-receptor occupancy analysis. IgG4 positivity serves as a measure of nivolumab binding. PD1 positivity represents residual PD1 not bound by nivolumab. (E) same as in (D) excluding PD1−/IgG4− population. CM, central memory; EM, effector memory; EMRA, effector memory cells re-expressing CD45RA; TCR, T-cell receptor.

Figure 3

Nivolumab and NY-ESO-1 transgenic TCR track to the site of the tumor. (A) IHC of the patient’s tumor at the time of biopsy for CD8, IgG4, and 4′,6-diamidino-2-phenylindole (DAPI). (B) Same as in (A) but for sarcoma marker TLE1, PD-L1, and DAPI. (C) Same as in (A) but for sarcoma marker PD-1 alone, a combination of CD8, IgG4, TLE1, PD-L1, PD-1, or the same combination with DAPI. (D) Post-therapy TCRβ repertoire as determined by deep sequencing of TCRβ alleles using genomic DNA from post-therapy tumor biopsy. Blue represents the frequency of NY-ESO-1 TCRβ alleles; beige represents the frequency of all other sequenced TCRβ alleles. (E) Post-therapy TCRα repertoire as determined by repertoire reconstruction using bulk RNA-sequencing from post-therapy tumor biopsy. Blue represents the frequency of NY-ESO-1 TCRα alleles; beige represents the frequency of all other sequenced TCRα alleles. (F) Same as in (E) except for TCRβ repertoire. IHC, immunohistochemistry; TCR, T-cell receptor.

Figure 4

NY-ESO-1 antigen expression is lost following ACT treatment. (A) Histogram of mapped reads corresponding to NY-ESO-1 expression in the pretreatment biopsy sample. (B) Same as in (A) but for two post-treatment biological replicates. Scale is normalized to pretreatment maximum. (C) NY-ESO-1 expression (TPM) from the pretreatment biopsy and both post-treatment biological replicates. (D) IHC for NY-ESO-1 using pretreatment tumor biopsy. (E) Same as in (D) but for post-treatment tissue. (F) Genomic structure of the NY-ESO-1 promoter region and transcriptional start site. Bent arrow represents the +1 TSS; blue arrow indicates CDS; blue arrowheads represent bisulfite primer sites; upright lines represent CpG dinucleotides. (G) Analysis of methylation status at CpG dinucleotides in the NY-ESO-1 promoter region. CpG sites assayed for methylation are depicted (top) and numbered relative to the TSS. Methylation level for the aggregate experiments quantified for each site (middle). Schematic depiction of the aggregate methylation level depicted (bottom). ACT, adoptive cell therapy; CDS, coding sequence; CpG, cytosine-phosphate-guanine; IHC, immunohistochemistry; TPM, transcript per million; TSS, transcription start site.