Dual PD-L1 and TGF-b blockade in patients with recurrent respiratory papillomatosis

Abstract

Background: Recurrent respiratory papillomatosis (RRP) is a human papillomavirus (HPV) driven neoplastic disorder of the upper aerodigestive tract that causes significant morbidity and can lead to fatal airway obstruction. Prior clinical study demonstrated clinical benefit with the programmed death-ligand 1 (PD-L1) monoclonal antibody avelumab. Bintrafusp alpha is a bifunctional inhibitor of PD-L1 and transforming growth factor-beta (TGF-b) that has shown clinical activity in several cancer types.

Methods: We conducted a phase II clinical trial evaluating bintrafusp alpha in adults with RRP. Papilloma samples before and after treatment with bintrafusp alpha were assessed for correlates of response with multiplex immunofluorescence as well as immunological and genomic analyses. Post hoc analyses of papilloma samples before and after treatment with avelumab were assessed for comparison.

Results: Dual PD-L1/TGF-b inhibition failed to abrogate papilloma growth in most subjects and increased the frequency of clinically indicated interventions after treatment in four of eight subjects based on each subject's own historical control. TGF-b neutralization consistently decreased pSMAD3 and p21 and increased Ki67 expression within the basal layers of papillomas, indicating that TGF-b restrained proliferation. These alterations were not observed in papillomas treated with PD-L1 blockade alone. Dual PD-L1/TGF-b inhibition did not enhance anti-HPV immunity within papillomas beyond that observed with PD-L1 blockade. Genomic alterations in TGF-b superfamily genes were infrequent in papillomas and normal mucosa but present in a significant fraction of head and neck carcinomas.

Conclusions: Intact TGF-b signaling restrains proliferation within papillomas, and the use of clinical agents that abrogate this pathway should be avoided in patients with RRP.

Trial registration numbers: NCT03707587 and NCT02859454.

Keywords: head and neck neoplasms; immunohistochemistry; immunotherapy; investigational; therapies; tumor microenvironment.

Figure 1

Clinical responses following treatment with PD-L1/TGF-b inhibition or PD-L1 blockade alone in patients with recurrent respiratory papillomatosis. (A) Spider plot of change in laryngeal disease burden following treatment with dual PD-L1/TGF-b inhibition (n=9) as measured by anatomic Derkay score are shown. Negative time points on the x-axis indicate disease burden at the time of clinical trial screening. Treatment was initiated at time 0. Per cent change relative to disease burden at time 0. (B) Summary results demonstrating change in laryngeal disease burden following treatment with dual PD-L1/TGF-b inhibition (gray circles) or PD-L1 blockade alone (white circles) as previously reported. P value determined by comparing multiple means over multiple timepoints using the Holm-Sidak method. (C) Representative endoscopic still images from clinic laryngoscopy of patients treated with dual PD-L1/TGF-b inhibition or PD-L1 blockade alone. Black arrows indicate papillomatous disease. (D) Changes in the number of clinically indicated interventions per 12 patient months for patients treated with dual PD-L1/TGF-b inhibition or PD-L1 blockade alone are shown. P value determined by Wilcoxon matched-pairs signed-rank test. PD-L1, programmed death-ligand 1; TGF-b, transforming growth factor-beta.

Figure 2

Assessment of papilloma TGF-b signaling in patients treated with PD-L1/TGF-b inhibition or PD-L1 blockade alone. (A) Representative photomicrographs of papilloma tissue sections and TGF-b signaling pathway protein expression or phosphorylation measured by multiplex immunofluorescence are shown. Changes in quantification of TGF-b signaling pathway protein expression or phosphorylation within the basal layers of papillomas in patients treated with (B) dual PD-L1/TGF-b inhibition (n=9) or (C) PD-L1 blockade alone (n=12) are shown. H-scores were determined by quantifying annotated basal cell fluorescence across entire biopsy sections. P value determined by Wilcoxon matched-pairs signed-rank test. PD-L1, programmed death-ligand 1; TGF-b, transforming growth factor-beta.

Figure 3

Quantification of papilloma infiltrating lymphocytes and HPV specificity. spider plots (left panels) of change in papilloma infiltration of CD8 or CD4 T-lymphocytes following treatment with (A) dual PD-L1/TGF-b inhibition (n=8 evaluable paired samples) or (B) PD-L1 blockade alone (n=12 evaluable paired samples) as measured by immunohistochemistry. Entire papilloma sections were scored for percentage of positive cells for each sample. P value determined by Wilcoxon matched-pairs signed-rank test. Right panels show quantification of the number of IFN spots following stimulation of papilloma infiltrating T-lymphocytes cultured from papillomas biopsied after treatment with dual PD-L1/TGF-b inhibition (A, n=7 evaluable cultures) or PD-L1 blockade alone (B, n=11 evaluable cultures) with autologous B-lymphocytes electroporated with full-length mRNA corresponding to HPV E2, E6 or E7 from HPV 6 or 11 as measured by ELISpot is shown. Also shown are representative ELISpot well photomicrographs from a patient treated with dual PD-L1/TGF-b inhibition (patient 3) or PD-L1 blockade alone (patient 5). HPV, human papillomavirus; PD-L1, programmed death-ligand 1; TGF-b, transforming growth factor-beta.

Figure 4

Assessment of genomic alterations in papillomas and head and neck carcinomas. (A) Boxplot shows the total number of single nucleotide variants (SNVs) and small insertions and deletions (INDELs) called from whole exome sequencing using an ensemble approach was determined for normal laryngeal mucosa (n=8), papillomas (n=21), HPV-negative HNSCC (n=74) and HPV-positive HNSCC (n=314). (B) Barplot shows the fraction of samples from each cohort harboring an SNV or INDEL in one or more of the 43 TGF-b superfamily genes was determined. (C) Barplot shows the mean number of TGF-b superfamily genes amplified or deleted within each cohort was determined. HNSCC, head and neck squamous cell carcinoma; HPV, human papillomavirus; TGF-b, transforming growth factor-beta.