Anti–programmed cell death protein 1 (PD-1) therapy provides long-term clinical benefits to patients with advanced melanoma. The composition of the gut microbiota correlates with anti–PD-1 efficacy in preclinical models and cancer patients. To investigate whether resistance to anti–PD-1 can be overcome by changing the gut microbiota, this clinical trial evaluated the safety and efficacy of responder-derived fecal microbiota transplantation (FMT) together with anti–PD-1 in patients with PD-1–refractory melanoma. This combination was well tolerated, provided clinical benefit in 6 of 15 patients, and induced rapid and durable microbiota perturbation. Responders exhibited increased abundance of taxa that were previously shown to be associated with response to anti–PD-1, increased CD8+ T cell activation, and decreased frequency of interleukin-8–expressing myeloid cells. Responders had distinct proteomic and metabolomic signatures, and transkingdom network analyses confirmed that the gut microbiome regulated these changes. Collectively, our findings show that FMT and anti–PD-1 changed the gut microbiome and reprogrammed the tumor microenvironment to overcome resistance to anti–PD-1 in a subset of PD-1 advanced melanoma.

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◥ Purpose: Natural killer (NK) cells play a critical role in tumor immunosurveillance. Multiple activating and inhibitory receptors (IR) regulate NK-cell-mediated tumor control. The IR T-cell immunoglobulin and ITIM domain (TIGIT) and its counterreceptor CD226 exert opposite effects on NK-cell-mediated tumor reactivity.Experimental Design: We evaluated the frequency, phenotype, and functions of NK cells freshly isolated from healthy donors and patients with melanoma with multiparameter flow cytometry. We assessed TIGIT and CD226 cell surface expression and internalization upon binding to CD155. We evaluated the role of IL15 and TIGIT blockade in increasing NK-cell-mediated cytotoxicity in vitro and in two mouse models.Results: NK cells are present at low frequencies in metastatic melanoma, are dysfunctional, and downregulate both TIGIT and CD226 expression. As compared with TIGIT À NK cells, TIGIT þ NK cells exhibit higher cytotoxic capacity and maturation, but paradoxically lower cytotoxicity against CD155 þ MHC class Ideficient melanoma cells. Membrane bound CD155 triggers CD226 internalization and degradation, resulting in decreased NK-cell-mediated tumor reactivity. IL15 increases TIGIT and CD226 gene expression by tumor-infiltrating NK cells (TiNKs) and, together with TIGIT blockade, increases NK-cell-mediated melanoma cytotoxicity in vitro and decreases tumor metastasis in two mouse melanoma models. Specific deletion of TIGIT on transferred NK cells enhances the antimetastatic activity of IL15, while CD226 blockade decreases the effects of IL15 and TIGIT blockade. Conclusions: Our findings support the development of novel combinatorial immunotherapy with IL15 and TIGIT blockade to promote NK-cell-mediated destruction of MHC class I-deficient melanoma, which are refractory to CD8 þ T-cell-mediated immunity.

was captured on anti-mouse Fc tips. Mouse Tim-3-human Fc fusion protein was captured on anti-human Fc tips. Captured Tim-3 was first saturated with an antibody (aTim-3.18, aTim-3.22, RMT3-23, or nonblocking anti-mouse aTim-3 control mAb), and then binding of PS liposomes was tested. The observed PS binding signal was normalized to the highest binding response in the assay.

BackgroundNeoadjuvant PD-1 blockade produces major pathological responses (MPR) in ~30% of patients (pts) with high-risk resectable melanoma (MEL) with durable relapse-free benefit, and increased circulating activated CD8+ T cells.1 2 CMP-001 is a type A CpG packaged within a virus-like particle that activates tumor-associated plasmacytoid dendritic cells (pDC) via TLR9 inducing type I interferons and anti-tumor CD8+ T cells. CMP-001/pembrolizumab produces durable anti-tumor responses in PD-1 refractory melanoma.3 We previously reported preliminary evidence of efficacy of neoadjuvant IT CMP/Nivo in high-risk resectable MEL; and herein present final results on 30 evaluable patients.Methods30 pts with stage III B/C/D MEL were enrolled. Pre-operatively, CMP-001 was dosed at 5 mg subcutaneous (SC, 1st), then 10 mg IT (2nd-7th) weekly; Nivo was dosed 240 mg q2 weeks for 3 doses – both agents given for 7 weeks. Post-operatively, Nivo was dosed 480 mg q4 weeks with CMP-001 5 mg q4 weeks SC for 48 weeks. Primary endpoints included major pathologic response rate (MPR), and incidence of dose-limiting toxicities (DLT). Secondary endpoints were radiographic response, relapse-free survival (RFS) and overall survival (OS). Pathological response was scored blinded by pathologists based on residual volume of tumor (RVT) using prior specified cutoffs:4 60% (complete response, pCR); 0%<rvt<rvt50% (non-response, pNR). Radiographic response was assessed using RECIST v1.1. Sequential blood draws and tumor biopsies were collected and analyzed for CD8+ T cell infiltrate (TIL), multiparameter flow cytometry (MFC) and multiplex immunofluorescence (mIF).Results30 pts with regionally advanced MEL were enrolled, of stages IIIB (57%), IIIC (37%), IIID (7%). 29/30 (97%) of pts completed 7 weeks of neoadjuvant Nivo/CMP; while 1 pt had a delay in surgery related to a pre-operative infection unrelated to therapy. No DLTs were reported; grade 3/4 irAE were reported in 3 pts (11%) leading to CMP-001 discontinuation in 2 pts (7%). Radiographic responses were seen in 13 pts (43%), while 9 pts (30%) had stable disease and 8 pts (27%) had progressive disease. Pathological responses (RVT <50%) were seen in 70% of pts: pCR 15 (50%), pMR 3 (10%), 3 pPR (10%); only 9 (30%) had pNR. Pathological responders (pCR/pMR) had increased CD8+ TIL and CD303+ pDC intra-tumorally by mIF; and peripherally activated PD1+/Ki67+ CD8+ T cells by MFC.ConclusionsNeoadjuvant CMP/Nivo has acceptable toxicity and promising efficacy. MPR is 60% in 30 pts. 1-year RFS was 82% (all pts) and 89% (among those with pCR/pMR); median RFS is 9 months (among pNR/pPR) and not reached (among pCR/pMR). Response is associated with evidence of immune activation intra-tumorally and peripherally. IT CMP001 increases clinical efficacy of PD-1 blockade with minimal additional toxicity in pts with regionally advanced MEL. Further study of this combination in high-risk resectable MEL is planned.AcknowledgementsWe thank Dr. Jagjit Singh and the pathology grossing room staff for their assistance and Checkmate Pharmaceuticals for funding and CMP-001.Trial RegistrationClinical trial information: NCT03618641Ethics ApprovalThe study was approved by University of Pittsburgh’s Institutional Review Board, approval number MOD19040237-002.ConsentWritten informed consent was obtained from the patient for publication of this abstract and any accompanying images. A copy of the written consent is available for review by the Editor of this journal.ReferencesAmaria RN, Reddy SM, Tawbi HA, et al. Neoadjuvant immune checkpoint blockade in high-risk resectable melanoma. Nat Med 2018. Nov;24(11):1649–1654.Huang AC, Orlowski RJ, Xu X, et al. A single dose of neoadjuvant PD-1 blockade predicts clinical outcomes in resectable melanoma. Nat Med 2019. Mar;25(3):454–461. doi: 10.1038/s41591-019-0357-y.Milhem M, Gonzales R, Medina T, et al. Abstract CT144: Intratumoral toll-like receptor 9 (TLR9) agonist, CMP-001, in combination with pembrolizumab can reverse resistance to PD-1 inhibition in a phase Ib trial in subjects with advanced melanoma. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14–18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract CT144.Tetzlaff MT, Messina JL, Stein JE, et al. Pathological assessment of resection specimens after neoadjuvant therapy for metastatic melanoma. Ann Oncol 2018. Aug 1;29(8):1861–1868.Cottrell TR, Thompson ED, Forde PM, et al. Pathologic features of response to neoadjuvant anti-PD-1 in resected non-small-cell lung carcinoma: a proposal for quantitative immune-related pathologic response criteria (irPRC). Ann Oncol 2018 Aug 1;29(8):1853–1860. doi: 10.1093/annonc/mdy218.Stein JE, Soni A, Danilova L, et al. Major pathologic response on biopsy (MPRbx) in patients with advanced melanoma treated with anti-PD-1: evidence for an early, on-therapy biomarker of response. Ann Oncol 2019 Apr 1;30(4):589–596. doi: 10.1093/annonc/mdz019.