Control of T(H)17/T(reg) balance by hypoxia-inducible factor 1.

T cell differentiation into distinct functional effector and inhibitory subsets is regulated, in part, by the cytokine environment present at the time of antigen recognition. Here, we show that hypoxia-inducible factor 1 (HIF-1), a key metabolic sensor, regulates the balance between regulatory T cell (T(reg)) and T(H)17 differentiation. HIF-1 enhances T(H)17 development through direct transcriptional activation of RORγt and via tertiary complex formation with RORγt and p300 recruitment to the IL-17 promoter, thereby regulating T(H)17 signature genes. Concurrently, HIF-1 attenuates T(reg) development by binding Foxp3 and targeting it for proteasomal degradation. Importantly, this regulation occurs under both normoxic and hypoxic conditions. Mice with HIF-1α-deficient T cells are resistant to induction of T(H)17-dependent experimental autoimmune encephalitis associated with diminished T(H)17 and increased T(reg) cells. These findings highlight the importance of metabolic cues in T cell fate determination and suggest that metabolic modulation could ameliorate certain T cell-based immune pathologies.

Nivolumab with or without ipilimumab in patients with recurrent or metastatic cervical cancer (CheckMate 358): a phase 1-2, open-label, multicohort trial.

BACKGROUND: In preliminary findings from the recurrent or metastatic cervical cancer cohort of CheckMate 358, nivolumab showed durable anti-tumour responses, and the combination of nivolumab plus ipilimumab showed promising clinical activity. Here, we report long-term outcomes from this cohort. METHODS: CheckMate 358 was a phase 1-2, open-label, multicohort trial. The metastatic cervical cancer cohort enrolled patients from 30 hospitals and cancer centres across ten countries. Female patients aged 18 years or older with a histologically confirmed diagnosis of squamous cell carcinoma of the cervix with recurrent or metastatic disease, an Eastern Cooperative Oncology Group performance status of 0 or 1, and up to two previous systemic therapies were enrolled into the nivolumab 240 mg every 2 weeks group, the randomised groups (nivolumab 3 mg/kg every 2 weeks plus ipilimumab 1 mg/kg every 6 weeks [NIVO3 plus IPI1] or nivolumab 1 mg/kg every 3 weeks plus ipilimumab 3 mg/kg every 3 weeks for four cycles then nivolumab 240 mg every 2 weeks [NIVO1 plus IPI3]), or the NIVO1 plus IPI3 expansion group. All doses were given intravenously. Patients were randomly assigned (1:1) to NIVO3 plus IPI1 or NIVO1 plus IPI3 via an interactive voice response system. Treatment continued until disease progression, unacceptable toxicity, or consent withdrawal, or for up to 24 months. The primary endpoint was investigator-assessed objective response rate. Anti-tumour activity and safety were analysed in all treated patients. This study is registered with ClinicalTrials.gov (NCT02488759) and is now completed. FINDINGS: Between October, 2015, and March, 2020, 193 patients were recruited in the recurrent or metastatic cervical cancer cohort of CheckMate 358, of whom 176 were treated. 19 patients received nivolumab monotherapy, 45 received NIVO3 plus IPI1, and 112 received NIVO1 plus IPI3 (45 in the randomised group and 67 in the expansion group). Median follow-up times were 19·9 months (IQR 8·2-44·8) with nivolumab, 12·6 months (7·8-37·1) with NIVO3 plus IPI1, and 16·7 months (7·2-27·5) with pooled NIVO1 plus IPI3. Objective response rates were 26% (95% CI 9-51; five of 19 patients) with nivolumab, 31% (18-47; 14 of 45 patients) with NIVO3 plus IPI1, 40% (26-56; 18 of 45 patients) with randomised NIVO1 plus IPI3, and 38% (29-48; 43 of 112 patients) with pooled NIVO1 plus IPI3. The most common grade 3-4 treatment-related adverse events were diarrhoea, hepatic cytolysis, hyponatraemia, pneumonitis, and syncope (one [5%] patient each; nivolumab group), diarrhoea, increased gamma-glutamyl transferase, increased lipase, and vomiting (two [4%] patients each; NIVO3 plus IPI1 group), and increased lipase (nine [8%] patients) and anaemia (seven [6%] patients; pooled NIVO1 plus IPI3 group). Serious treatment-related adverse events were reported in three (16%) patients in the nivolumab group, 12 (27%) patients in the NIVO3 plus IPI1 group, and 47 (42%) patients in the pooled NIVO1 plus IPI3 group. There was one treatment-related death due to immune-mediated colitis in the NIVO1 plus IPI3 group. INTERPRETATION: Nivolumab monotherapy and nivolumab plus ipilimumab combination therapy showed promise in the CheckMate 358 study as potential treatment options for recurrent or metastatic cervical cancer. Future randomised controlled trials of nivolumab plus ipilimumab or other dual immunotherapy regimens are warranted to confirm treatment benefit in this patient population. FUNDING: Bristol Myers Squibb and Ono Pharmaceutical.

Comparing anti-tumor and anti-self immunity in a patient with melanoma receiving immune checkpoint blockade.

BACKGROUND: Tumor regression following immune checkpoint blockade (ICB) is often associated with immune-related adverse events (irAEs), marked by inflammation in non-cancerous tissues. This study was undertaken to investigate the functional relationship between anti-tumor and anti-self immunity, to facilitate irAE management while promoting anti-tumor immunity. METHODS: Multiple biopsies from tumor and inflamed tissues were collected from a patient with melanoma experiencing both tumor regression and irAEs on ICB, who underwent rapid autopsy. Immune cells infiltrating melanoma lesions and inflamed normal tissues were subjected to gene expression profiling with multiplex qRT-PCR for 122 candidate genes. Subsequently, immunohistochemistry was conducted to assess the expression of 14 candidate markers of immune cell subsets and checkpoints. TCR-beta sequencing was used to explore T cell clonal repertoires across specimens. RESULTS: While genes involved in MHC I/II antigen presentation, IFN signaling, innate immunity and immunosuppression were abundantly expressed across specimens, irAE tissues over-expressed certain genes associated with immunosuppression (CSF1R, IL10RA, IL27/EBI3, FOXP3, KLRG1, SOCS1, TGFB1), including those in the COX-2/PGE2 pathway (IL1B, PTGER1/EP1 and PTGER4/EP4). Immunohistochemistry revealed similar proportions of immunosuppressive cell subsets and checkpoint molecules across samples. TCRseq did not indicate common TCR repertoires across tumor and inflammation sites, arguing against shared antigen recognition between anti-tumor and anti-self immunity in this patient. CONCLUSIONS: This comprehensive study of a single patient with melanoma experiencing both tumor regression and irAEs on ICB explores the immune landscape across these tissues, revealing similarities between anti-tumor and anti-self immunity. Further, it highlights expression of the COX-2/PGE2 pathway, which is known to be immunosuppressive and potentially mediates ICB resistance. Ongoing clinical trials of COX-2/PGE2 pathway inhibitors targeting the major COX-2 inducer IL-1B, COX-2 itself, or the PGE2 receptors EP2 and EP4 present new opportunities to promote anti-tumor activity, but may also have the potential to enhance the severity of ICB-induced irAEs.

Balance and imbalance in the immune system: life on the edge.

CTLA-4 is a key immune checkpoint in maintaining self-tolerance, which can be co-opted by cancer to evade immune attack. In Science, Kuehn et al. (2014) describe clinical manifestations from inherited heterozygous CTLA4 mutations, and some are reminiscent of immune-related consequences from anti-CTLA-4 cancer therapy.

Neoadjuvant PD-1 Blockade in Resectable Lung Cancer.

BACKGROUND: Antibodies that block programmed death 1 (PD-1) protein improve survival in patients with advanced non-small-cell lung cancer (NSCLC) but have not been tested in resectable NSCLC, a condition in which little progress has been made during the past decade. METHODS: In this pilot study, we administered two preoperative doses of PD-1 inhibitor nivolumab in adults with untreated, surgically resectable early (stage I, II, or IIIA) NSCLC. Nivolumab (at a dose of 3 mg per kilogram of body weight) was administered intravenously every 2 weeks, with surgery planned approximately 4 weeks after the first dose. The primary end points of the study were safety and feasibility. We also evaluated the tumor pathological response, expression of programmed death ligand 1 (PD-L1), mutational burden, and mutation-associated, neoantigen-specific T-cell responses. RESULTS: Neoadjuvant nivolumab had an acceptable side-effect profile and was not associated with delays in surgery. Of the 21 tumors that were removed, 20 were completely resected. A major pathological response occurred in 9 of 20 resected tumors (45%). Responses occurred in both PD-L1-positive and PD-L1-negative tumors. There was a significant correlation between the pathological response and the pretreatment tumor mutational burden. The number of T-cell clones that were found in both the tumor and peripheral blood increased systemically after PD-1 blockade in eight of nine patients who were evaluated. Mutation-associated, neoantigen-specific T-cell clones from a primary tumor with a complete response on pathological assessment rapidly expanded in peripheral blood at 2 to 4 weeks after treatment; some of these clones were not detected before the administration of nivolumab. CONCLUSIONS: Neoadjuvant nivolumab was associated with few side effects, did not delay surgery, and induced a major pathological response in 45% of resected tumors. The tumor mutational burden was predictive of the pathological response to PD-1 blockade. Treatment induced expansion of mutation-associated, neoantigen-specific T-cell clones in peripheral blood. (Funded by Cancer Research Institute-Stand Up 2 Cancer and others; ClinicalTrials.gov number, NCT02259621 .).

Characterization of the tumor immune microenvironment in human papillomavirus-positive and -negative head and neck squamous cell carcinomas.

Approximately 15% of advanced head and neck squamous cell carcinomas (HNSCC) respond to anti-PD-(L)1 monotherapies. Tumor PD-L1 expression and human papillomavirus (HPV) status have been proposed as biomarkers to identify patients likely to benefit from these treatments. We aimed to understand the potential immune effects of HPV in HNSCC and to characterize additional potentially targetable immune-regulatory pathways in primary, treatment-naïve tumors. CD3, CD4, CD8, CD20, CD68, FoxP3, PD-1, PD-L2, LAG-3, IDO-1, and GITR cell densities were determined in 27 HNSCC specimens. IHC for PD-L1 assessed percentage of positive tumor cells and immune cells separately or as a combined positive score (CPS), and whether PD-L1 was expressed in an adaptive or constitutive pattern (i.e., PD-L1+ tumor cells juxtaposed to TILs or in the absence of TILs, respectively). HPV testing with p16 IHC was confirmed by HPV genotyping. When compared to HPV(-) tumors (n = 14), HPV+ tumors (n = 13) contained significantly higher densities of CD3+, CD4+, CD8+, CD20+, and PD-1+ cells (P < 0.02), and there was a trend towards increased density of FoxP3 + cells. PD-L1 expression patterns did not vary by tumor viral status, suggesting possible heterogeneous mechanisms driving constitutive vs adaptive PD-L1 expression patterns in HNSCC. IDO-1 expression was abundant (> 500 IDO-1+ cells/mm2 in 17/27 specimens) and was found on tumor cells as well as immune cells in 12/27 (44%) cases (range 5-80% tumor cells+). Notably, the studied markers varied on a per-patient basis and were not always related to the degree of T cell infiltration. These findings may inform therapeutic co-targeting strategies and raise consideration for a personalized treatment approach.

PD-1 Blockade with Pembrolizumab in Advanced Merkel-Cell Carcinoma.

BACKGROUND: Merkel-cell carcinoma is an aggressive skin cancer that is linked to exposure to ultraviolet light and the Merkel-cell polyomavirus (MCPyV). Advanced Merkel-cell carcinoma often responds to chemotherapy, but responses are transient. Blocking the programmed death 1 (PD-1) immune inhibitory pathway is of interest, because these tumors often express PD-L1, and MCPyV-specific T cells express PD-1. METHODS: In this multicenter, phase 2, noncontrolled study, we assigned adults with advanced Merkel-cell carcinoma who had received no previous systemic therapy to receive pembrolizumab (anti-PD-1) at a dose of 2 mg per kilogram of body weight every 3 weeks. The primary end point was the objective response rate according to Response Evaluation Criteria in Solid Tumors, version 1.1. Efficacy was correlated with tumor viral status, as assessed by serologic and immunohistochemical testing. RESULTS: A total of 26 patients received at least one dose of pembrolizumab. The objective response rate among the 25 patients with at least one evaluation during treatment was 56% (95% confidence interval [CI], 35 to 76); 4 patients had a complete response, and 10 had a partial response. With a median follow-up of 33 weeks (range, 7 to 53), relapses occurred in 2 of the 14 patients who had had a response (14%). The response duration ranged from at least 2.2 months to at least 9.7 months. The rate of progression-free survival at 6 months was 67% (95% CI, 49 to 86). A total of 17 of the 26 patients (65%) had virus-positive tumors. The response rate was 62% among patients with MCPyV-positive tumors (10 of 16 patients) and 44% among those with virus-negative tumors (4 of 9 patients). Drug-related grade 3 or 4 adverse events occurred in 15% of the patients. CONCLUSIONS: In this study, first-line therapy with pembrolizumab in patients with advanced Merkel-cell carcinoma was associated with an objective response rate of 56%. Responses were observed in patients with virus-positive tumors and those with virus-negative tumors. (Funded by the National Cancer Institute and Merck; ClinicalTrials.gov number, NCT02267603.).

Identification and Characterization of Complex Glycosylated Peptides Presented by the MHC Class II Processing Pathway in Melanoma.

The MHC class II (MHCII) processing pathway presents peptides derived from exogenous or membrane-bound proteins to CD4+ T cells. Several studies have shown that glycopeptides are necessary to modulate CD4+ T cell recognition, though glycopeptide structures in these cases are generally unknown. Here, we present a total of 93 glycopeptides from three melanoma cell lines and one matched EBV-transformed line with most found only in the melanoma cell lines. The glycosylation we detected was diverse and comprised 17 different glycoforms. We then used molecular modeling to demonstrate that complex glycopeptides are capable of binding the MHC and may interact with complementarity determining regions. Finally, we present the first evidence of disulfide-bonded peptides presented by MHCII. This is the first large scale study to sequence glyco- and disulfide bonded MHCII peptides from the surface of cancer cells and could represent a novel avenue of tumor activation and/or immunoevasion.

Safety and activity of anti-PD-L1 antibody in patients with advanced cancer.

BACKGROUND: Programmed death 1 (PD-1) protein, a T-cell coinhibitory receptor, and one of its ligands, PD-L1, play a pivotal role in the ability of tumor cells to evade the host's immune system. Blockade of interactions between PD-1 and PD-L1 enhances immune function in vitro and mediates antitumor activity in preclinical models. METHODS: In this multicenter phase 1 trial, we administered intravenous anti-PD-L1 antibody (at escalating doses ranging from 0.3 to 10 mg per kilogram of body weight) to patients with selected advanced cancers. Anti-PD-L1 antibody was administered every 14 days in 6-week cycles for up to 16 cycles or until the patient had a complete response or confirmed disease progression. RESULTS: As of February 24, 2012, a total of 207 patients--75 with non-small-cell lung cancer, 55 with melanoma, 18 with colorectal cancer, 17 with renal-cell cancer, 17 with ovarian cancer, 14 with pancreatic cancer, 7 with gastric cancer, and 4 with breast cancer--had received anti-PD-L1 antibody. The median duration of therapy was 12 weeks (range, 2 to 111). Grade 3 or 4 toxic effects that investigators considered to be related to treatment occurred in 9% of patients. Among patients with a response that could be evaluated, an objective response (a complete or partial response) was observed in 9 of 52 patients with melanoma, 2 of 17 with renal-cell cancer, 5 of 49 with non-small-cell lung cancer, and 1 of 17 with ovarian cancer. Responses lasted for 1 year or more in 8 of 16 patients with at least 1 year of follow-up. CONCLUSIONS: Antibody-mediated blockade of PD-L1 induced durable tumor regression (objective response rate of 6 to 17%) and prolonged stabilization of disease (rates of 12 to 41% at 24 weeks) in patients with advanced cancers, including non-small-cell lung cancer, melanoma, and renal-cell cancer. (Funded by Bristol-Myers Squibb and others; ClinicalTrials.gov number, NCT00729664.).

Safety, activity, and immune correlates of anti-PD-1 antibody in cancer.

BACKGROUND: Blockade of programmed death 1 (PD-1), an inhibitory receptor expressed by T cells, can overcome immune resistance. We assessed the antitumor activity and safety of BMS-936558, an antibody that specifically blocks PD-1. METHODS: We enrolled patients with advanced melanoma, non-small-cell lung cancer, castration-resistant prostate cancer, or renal-cell or colorectal cancer to receive anti-PD-1 antibody at a dose of 0.1 to 10.0 mg per kilogram of body weight every 2 weeks. Response was assessed after each 8-week treatment cycle. Patients received up to 12 cycles until disease progression or a complete response occurred. RESULTS: A total of 296 patients received treatment through February 24, 2012. Grade 3 or 4 drug-related adverse events occurred in 14% of patients; there were three deaths from pulmonary toxicity. No maximum tolerated dose was defined. Adverse events consistent with immune-related causes were observed. Among 236 patients in whom response could be evaluated, objective responses (complete or partial responses) were observed in those with non-small-cell lung cancer, melanoma, or renal-cell cancer. Cumulative response rates (all doses) were 18% among patients with non-small-cell lung cancer (14 of 76 patients), 28% among patients with melanoma (26 of 94 patients), and 27% among patients with renal-cell cancer (9 of 33 patients). Responses were durable; 20 of 31 responses lasted 1 year or more in patients with 1 year or more of follow-up. To assess the role of intratumoral PD-1 ligand (PD-L1) expression in the modulation of the PD-1-PD-L1 pathway, immunohistochemical analysis was performed on pretreatment tumor specimens obtained from 42 patients. Of 17 patients with PD-L1-negative tumors, none had an objective response; 9 of 25 patients (36%) with PD-L1-positive tumors had an objective response (P=0.006). CONCLUSIONS: Anti-PD-1 antibody produced objective responses in approximately one in four to one in five patients with non-small-cell lung cancer, melanoma, or renal-cell cancer; the adverse-event profile does not appear to preclude its use. Preliminary data suggest a relationship between PD-L1 expression on tumor cells and objective response. (Funded by Bristol-Myers Squibb and others; ClinicalTrials.gov number, NCT00730639.).

Safety and immunologic correlates of Melanoma GVAX, a GM-CSF secreting allogeneic melanoma cell vaccine administered in the adjuvant setting.

BACKGROUND: Limited adjuvant treatment options exist for patients with high-risk surgically resected melanoma. This first-in-human study investigated the safety, tolerability and immunologic correlates of Melanoma GVAX, a lethally irradiated granulocyte-macrophage colony stimulating factor (GM-CSF)-secreting allogeneic whole-cell melanoma vaccine, administered in the adjuvant setting. METHODS: Patients with stage IIB-IV melanoma were enrolled following complete surgical resection. Melanoma GVAX was administered intradermally once every 28 days for four cycles, at 5E7 cells/cycle (n = 3), 2E8 cells/cycle (n = 9), or 2E8 cells/cycle preceded by cyclophosphamide 200 mg/m(2) to deplete T regulatory cells (Tregs; n = 8). Blood was collected before each vaccination and at 4 and 6 months after treatment initiation for immunologic studies. Vaccine injection site biopsies and additional blood samples were obtained 2 days after the 1st and 4th vaccines. RESULTS: Among 20 treated patients, 18 completed 4 vaccinations. Minimal treatment-related toxicity was observed. One patient developed vitiligo and patches of white hair during the treatment and follow-up period. Vaccine site biopsies demonstrated complex inflammatory infiltrates, including significant increases in eosinophils and PD-1+ lymphocytes from cycle 1 to cycle 4 (P < 0.05). Serum GM-CSF concentrations increased significantly in a dose-dependent manner 48 h after vaccination (P = 0.0086), accompanied by increased numbers of activated circulating monocytes (P < 0.0001) and decreased percentages of myeloid-derived suppressor cells among monocytes (CD14+ , CD11b+ , HLA-DR low or negative; P = 0.002). Cyclophosphamide did not affect numbers of circulating Tregs. No significant changes in anti-melanoma immunity were observed in peripheral T cells by interferon-gamma ELIPSOT, or immunoglobulins by serum Western blotting. CONCLUSION: Melanoma GVAX was safe and tolerable in the adjuvant setting. Pharmacodynamic testing revealed complex vaccine site immune infiltrates and an immune-reactive profile in circulating monocytic cell subsets. These findings support the optimization of Melanoma GVAX with additional monocyte and dendritic cell activators, and the potential development of combinatorial treatment regimens with synergistic agents. TRIAL REGISTRATION: NCT01435499.

Structure-based design of altered MHC class II-restricted peptide ligands with heterogeneous immunogenicity.

Insights gained from characterizing MHC-peptide-TCR interactions have held the promise that directed structural modifications can have predictable functional consequences. The ability to manipulate T cell reactivity synthetically or through genetic engineering might thus be translated into new therapies for common diseases such as cancer and autoimmune disorders. In the current study, we determined the crystal structure of HLA-DR4 in complex with the nonmutated dominant gp100 epitope gp10044-59, associated with many melanomas. Altered peptide ligands (APLs) were designed to enhance MHC binding and hence T cell recognition of gp100 in HLA-DR4(+) melanoma patients. Increased MHC binding of several APLs was observed, validating this approach biochemically. Nevertheless, heterogeneous preferences of CD4(+) T cells from several HLA-DR4(+) melanoma patients for different gp100 APLs suggested highly variable TCR usage, even among six patients who had been vaccinated against the wild-type gp100 peptide. This heterogeneity prevented the selection of an APL candidate for developing an improved generic gp100 vaccine in melanoma. Our results are consistent with the idea that even conservative changes in MHC anchor residues may result in subtle, yet crucial, effects on peptide contacts with the TCR or on peptide dynamics, such that alterations intended to enhance immunogenicity may be unpredictable or counterproductive. They also underscore a critical knowledge gap that needs to be filled before structural and in vitro observations can be used reliably to devise new immunotherapies for cancer and other disorders.

Structural insights into the editing of germ-line-encoded interactions between T-cell receptor and MHC class II by Vα CDR3.

The conserved diagonal docking mode observed in structures of T-cell receptors (TCRs) bound to peptide-MHC ligands is believed to reflect coevolution of TCR and MHC genes. This coevolution is supported by the conservation of certain interactions between the germ-line-encoded complementarity-determining region (CDR)1 and CDR2 loops of TCR and MHC. However, the rules governing these interactions are not straightforward, even when the same variable (V) region recognizes the same MHC molecule. Here, we demonstrate that the somatically generated CDR3 loops can markedly alter evolutionarily selected contacts between TCR and MHC ("CDR3 editing"). To understand CDR3 editing at the atomic level, we determined the structure of a human melanoma-specific TCR (G4) bound to the MHC class II molecule HLA-DR1 and an epitope from mutant triose phosphate isomerase (mutTPI). A comparison of the G4-mutTPI-DR1 complex with a complex involving a TCR (E8) that uses the same Vα region to recognize the same mutTPI-DR1 ligand as G4 revealed that CDR1α adopts markedly different conformations in the two TCRs, resulting in an almost entirely different set of contacts with MHC. Based on the structures of unbound G4 and E8, the distinct conformations of CDR1α in these TCRs are not induced by binding to mutTPI-DR1 but result from differences in the length and sequence of CDR3α that are transmitted to CDR1α. The editing of germ-line-encoded TCR-MHC interactions by CDR3 demonstrates that these interactions possess sufficient intrinsic flexibility to accommodate large structural variations in CDR3 and, consequently, in the TCR-binding site.

T antigen-specific CD8+ T cells associate with PD-1 blockade response in virus-positive Merkel cell carcinoma.

Merkel cell carcinoma (MCC) is a highly immunogenic skin cancer primarily induced by Merkel cell polyomavirus, which is driven by the expression of the oncogenic T antigens (T-Ags). Blockade of the programmed cell death protein-1 (PD-1) pathway has shown remarkable response rates, but evidence for therapy-associated T-Ag-specific immune response and therapeutic strategies for the nonresponding fraction are both limited. We tracked T-Ag-reactive CD8+ T cells in peripheral blood of 26 MCC patients under anti-PD1 therapy, using DNA-barcoded pMHC multimers, displaying all peptides from the predicted HLA ligandome of the oncoproteins, covering 33 class I haplotypes. We observed a broad T cell recognition of T-Ags, including identification of 20 T-Ag-derived epitopes we believe to be novel. Broadening of the T-Ag recognition profile and increased T cell frequencies during therapy were strongly associated with clinical response and prolonged progression-free survival. T-Ag-specific T cells could be further boosted and expanded directly from peripheral blood using artificial antigen-presenting scaffolds, even in patients with no detectable T-Ag-specific T cells. These T cells provided strong tumor-rejection capacity while retaining a favorable phenotype for adoptive cell transfer. These findings demonstrate that T-Ag-specific T cells are associated with the clinical outcome to PD-1 blockade and that Ag-presenting scaffolds can be used to boost such responses.

Circulating cancer-specific CD8 T cell frequency is associated with response to PD-1 blockade in Merkel cell carcinoma.

Understanding cancer immunobiology has been hampered by difficulty identifying cancer-specific T cells. Merkel cell polyomavirus (MCPyV) causes most Merkel cell carcinomas (MCCs). All patients with virus-driven MCC express MCPyV oncoproteins, facilitating identification of virus (cancer)-specific T cells. We studied MCPyV-specific T cells from 27 patients with MCC using MCPyV peptide-HLA-I multimers, 26-color flow cytometry, single-cell transcriptomics, and T cell receptor (TCR) sequencing. In a prospective clinical trial, higher circulating MCPyV-specific CD8 T cell frequency before anti-PD-1 treatment was strongly associated with 2-year recurrence-free survival (75% if detectable, 0% if undetectable, p = 0.0018; ClinicalTrial.gov: NCT02488759). Intratumorally, such T cells were typically present, but their frequency did not significantly associate with response. Circulating MCPyV-specific CD8 T cells had increased stem/memory and decreased exhaustion signatures relative to their intratumoral counterparts. These results suggest that cancer-specific CD8 T cells in the blood may play a role in anti-PD-1 responses. Thus, strategies that augment their number or mobilize them into tumors could improve outcomes.

Nivolumab + Tacrolimus + Prednisone ± Ipilimumab for Kidney Transplant Recipients With Advanced Cutaneous Cancers.

PURPOSE: Cancer-related mortality rates among kidney transplant recipients (KTR) are high, but these patients have largely been excluded from trials of immune checkpoint inhibitors because of immunosuppression and risk of treatment-related allograft loss (TRAL). We conducted a prospective clinical trial testing nivolumab (NIVO) + tacrolimus (TACRO) + prednisone (PRED) ± ipilimumab (IPI) in KTR with advanced cutaneous cancers. METHODS: Adult KTR with advanced melanoma or basal, cutaneous squamous, or Merkel cell carcinomas were eligible. Immunosuppression was standardized to TACRO (serum trough 2-5 ng/mL) + PRED 5 mg once daily. Patients then received NIVO 480 mg IV once every 4 weeks. The primary composite end point was partial or complete (tumor) response (CR) or stable disease per RECIST v1.1 without allograft loss at 16W. Patients with progressive disease (PD) could receive IPI 1 mg/kg IV + NIVO 3 mg/kg once every 3 weeks × 4 followed by NIVO. Donor-derived cell-free DNA (dd-cfDNA) levels were measured approximately once every 2 weeks as a potential predictor of allograft rejection. RESULTS: Among eight evaluable patients, none met the trial's primary end point. All eight patients experienced PD on NIVO + TACRO + PRED; TRAL occurred in one patient. Six patients then received IPI + NIVO + TACRO + PRED. Best overall responses: two CR (one with TRAL) and four PD (one with TRAL). In total, 7 of 8 pre-NIVO tumor biopsies contained a paucity of infiltrating immune cells. In total, 2 of 5 on-NIVO biopsies demonstrated moderate immune infiltrates; both patients later experienced a CR to IPI + NIVO. In 2 of 3 patients with TRAL, dd-cfDNA elevations occurred 10 and 15 days before increases in serum creatinine. CONCLUSION: In most KTR with advanced skin cancer, TACRO + PRED provides insufficient allograft protection and compromises immune-mediated tumor regression after administration of NIVO ± IPI. Elevated dd-cfDNA levels can signal treatment-related allograft rejection earlier than rises in serum creatinine.

Neoadjuvant immune checkpoint blockade: A window of opportunity to advance cancer immunotherapy.

Among new treatment approaches for patients with cancer, few have accelerated as quickly as neoadjuvant immune checkpoint blockade (ICB). Neoadjuvant cancer therapy is administered before curative-intent surgery in treatment-naïve patients. Conventional neoadjuvant chemotherapy and radiotherapy are primarily intended to reduce tumor size, improving surgical resectability. However, recent scientific evidence outlined here suggests that neoadjuvant immunotherapy can expand and transcriptionally modify tumor-specific T cell clones to enhance both intratumoral and systemic anti-tumor immunity. It further offers a unique "window of opportunity" to explore mechanisms and identify novel biomarkers of ICB response and resistance, opening possibilities for refining long-term clinical outcome predictions and developing new, more highly effective ICB combination therapies. Here, we examine advances in clinical and scientific knowledge gleaned from studies in select cancers and describe emerging key principles relevant to neoadjuvant ICB across many cancer types.

Combinatorial biomarker for predicting outcomes to anti-PD-1 therapy in patients with metastatic clear cell renal cell carcinoma.

With a rapidly developing immunotherapeutic landscape for patients with metastatic clear cell renal cell carcinoma, biomarkers of efficacy are highly desirable to guide treatment strategy. Hematoxylin and eosin (H&E)-stained slides are inexpensive and widely available in pathology laboratories, including in resource-poor settings. Here, H&E scoring of tumor-infiltrating immune cells (TILplus) in pre-treatment tumor specimens using light microscopy is associated with improved overall survival (OS) in three independent cohorts of patients receiving immune checkpoint blockade. Necrosis score alone does not associate with OS; however, necrosis modifies the predictive effect of TILplus, a finding that has broad translational relevance for tissue-based biomarker development. PBRM1 mutational status is combined with H&E scores to further refine outcome predictions (OS, p = 0.007, and objective response, p = 0.04). These findings bring H&E assessment to the fore for biomarker development in future prospective, randomized trials, and emerging multi-omics classifiers.