Tumor-induced immune suppression of therapeutic cancer vaccines

Therapeutic cancer vaccines offer an attractive strategy for treating cancer. Not only can the immune system specifically target tumor cells, but it also provides long-term memory needed to prevent recurrent disease. Despite the therapeutic appeal of cancer vaccines, they have not been effective clinically. Thus, we set out to determine the mechanisms by which the presence of tumor inhibited vaccine efficacy. For this work, we focused on tolerant preclinical tumor models. Vaccination of tolerant neu-N mice with alphaviral replicon particles expressing rat neu (neuET-VRP) failed to induce regression of established neu-expressing tumors. The inability of VRPs to induce regression in neu-N mice was due to a dominant role of the immunosuppressive tumor environment in these animals, as opposed to central deletion of tumor-specific lymphocytes. Accordingly, transfer of neu-specific lymphocytes into neu-N mice did not inhibit tumor growth. On the other hand, we demonstrated that myeloid-derived suppressor cells (MDSC) were a main mediator of suppression in neu-N mice. Depletion of MDSCs, along with provision of neu-specific lymphocytes and neuET-VRP vaccination, induced tumor regression in the majority of neu-N mice. We also identified other immunosuppressive mechanisms that suppressed anti-tumor immunity in neu-N mice. These included suppression by Treg cells, CD200 and IDO. As MDSCs were the main mediator of suppression following therapeutic vaccination, we evaluated genes important for their function. We found that MDSCs expressed NLRP3, a member of the inflammasome complex that generates IL-1[beta] and IL-18. While the NLRP3 inflammasome is known to enhance immunity in response to both microbial and non-microbial stimuli, its role in anti-tumor immunity is not known. We found that NLRP3 promoted the accumulation of MDSCs in the tumor following therapeutic dendritic cell (DC) vaccination. The decreased accumulation of MDSCs in Nlrp3-/- mice led to a fourfold improvement in survival compared to wild type mice after DC vaccination. These data establish an unexpected role for NLRP3 in impeding anti-tumor immunity and suggest novel approaches to improving cancer vaccines. In summary, our data suggest that current therapeutic vaccines are not effective due to the robust immunosuppressive mechanisms present in the tumor environment

CD8 T Cell Tumor Infiltration Following Tc1 or Tc2 Therapy

Type I (Tc1) CD8+ T cells have been shown to be more effective than type II (Tc2) CD8+ T cells for adoptive cell transfer therapy in several tumor models. Migration differences between Tc1 and Tc2 cells were previously proposed to contribute to this difference in therapeutic efficacy. In order to evaluate Tc1 and Tc2 migration in vivo, we developed a model using transfected EL-4 thymoma tumor cells expressing the p33 peptide antigen from lymphocytic choriomeningitis virus (p33.EL-4). We used P14 mice, which are transgenic for the T cell receptor specific for p33 peptide that is expressed by these p33.EL-4 tumors. We crossed UBI.GFP mice that ubiquitously express GFP with the P14 mice, in order to generate mice that express both GFP and the p33-specific TCR in the CD8+ T cell population. Splenocytes from these mice were cultured to generate Tc1 and Tc2 cells, which were injected intraveinously into tumor-bearing mice. Donor cells were phenotyped before transfer and on days 3 and 7 after transfer. The CD8+ T cells were examined for GFP and adhesion molecule expression by flow cytometry. We examined gene expression of T-bet and enzymes important for selectin ligand glycosylation in Tc1 and Tc2 cultures, as well as the gene expression of cytokines, chemokines, and chemokine receptors in Tc1 and Tc2 treated mice. We found significantly more Tc1 than Tc2 cells in TDLNs and tumors on days 3 and 7 after transfer. Both Tc1 and Tc2 donor cells were found in TDLN and tumor sites of p33 positive-positive tumors compared to sites of p33 antigen-negative tumors. More importantly, all CD8+ T cells isolated from these tumors on days 3 and 7 after therapy, regardless of host or donor origin and Tc1 or Tc2 phenotype, expressed high levels of adhesion molecules important for T cell migration. This suggests that antigen does not alter the adhesion molecule expression of tumor infiltrating CD8+ T cells. These cells expressed high levels of CD44, leukocyte function-associated antigen-1 (LFA-1), and P-selectin glycoprotein ligand 1 (PSGL-1, CD162), suggesting a required “tumor infiltrating phenotype”. Before transfer and 3 days after transfer, Tc1 cells expressed higher levels of this tumor infiltrating phenotype compared to Tc2 cells. Thus, increased Tc1 cell migration to TDLN and infiltration of tumors may be due to higher expression of a tumor infiltrating phenotype compared to Tc2 cells. We found that T-bet expression is higher in cells from Tc1 vs. Tc2 cultures, which may promote the type I phenotype, including higher adhesion molecule expression. However, we did not find a significant difference between the gene expression of selectin ligand glycosylating enzymes or PSGL-1 gene expression in Tc1 vs. Tc2 cells. In addition, when we examined the gene expression of chemokines in the tumors of Tc1 vs. Tc2 treated mice, we found that type I interferon (IFN)−γ inducible protein (IP)-10 is more highly expressed compared to macrophage derived chemokine (MDC) or macrophage inducing protein (MIP)-1α in either treatment group. Also, donor and host CD8+ cells in Tc1 and Tc2 treated mice express chemokine receptor CXCR3 and cytokine IFN−γ, but no interleukin (IL)-4. This data suggest that in addition to surface expression of adhesion molecules CD44, LFA-1, PSGL-1, expression of type I cytokine IFN-γ and chemokine receptor CXCR3 is also a characteristic of a “tumor infiltrating phentoype.

Olaparib combined with abiraterone in patients with metastatic castration-resistant prostate cancer: a randomised, double-blind, placebo-controlled, phase 2 trial

Background Patients with metastatic castration-resistant prostate cancer and homologous recombination repair (HRR) mutations have a better response to treatment with the poly(ADP-ribose) polymerase inhibitor olaparib than patients without HRR mutations. Preclinical data suggest synergy between olaparib and androgen pathway inhibitors. We aimed to assess the efficacy of olaparib plus the androgen pathway inhibitor abiraterone in patients with metastatic castration-resistant prostate cancer regardless of HRR mutation status. Methods We carried out this double-blind, randomised, placebo-controlled phase 2 trial at 41 urological oncology sites in 11 countries across Europe and North America. Eligible male patients were aged 18 years or older with metastatic castration-resistant prostate cancer who had previously received docetaxel and were candidates for abiraterone treatment. Patients were excluded if they had received more than two previous lines of chemotherapy, or had previous exposure to second-generation antihormonal drugs. Patients were randomly assigned (1:1) using an interactive voice or web response system, without stratification, to receive oral olaparib 300 mg twice daily or placebo. All patients received oral abiraterone 1000 mg once daily and prednisone or prednisolone 5 mg twice daily. Patients and investigators were masked to treatment allocation. The primary endpoint was investigator-assessed radiographic progression-free survival (rPFS; based on Response Evaluation Criteria in Solid Tumors version 1.1 and Prostate Cancer Clinical Trials Working Group 2 criteria). Efficacy analyses were done in the intention-to-treat population, which included all randomly assigned patients, and safety analyses included all patients who received at least one dose of olaparib or placebo. This trial is registered with ClinicalTrials.gov, number NCT01972217, and is no longer recruiting patients. Findings Between Nov 25, 2014, and July 14, 2015, 171 patients were assessed for eligibility. Of those, 142 patients were randomly assigned to receive olaparib and abiraterone (n=71) or placebo and abiraterone (n=71). The clinical cutoff date for the final analysis was Sept 22, 2017. Median rPFS was 13·8 months (95% CI 10·8–20·4) with olaparib and abiraterone and 8·2 months (5·5–9·7) with placebo and abiraterone (hazard ratio [HR] 0·65, 95% CI 0·44–0·97, p=0·034). The most common grade 1–2 adverse events were nausea (26 [37%] patients in the olaparib group vs 13 [18%] patients in the placebo group), constipation (18 [25%] vs eight [11%]), and back pain (17 [24%] vs 13 [18%]). 38 (54%) of 71 patients in the olaparib and abiraterone group and 20 (28%) of 71 patients in the placebo and abiraterone group had grade 3 or worse adverse events, including anaemia (in 15 [21%] of 71 patients vs none of 71), pneumonia (four [6%] vs three [4%]), and myocardial infarction (four [6%] vs none). Serious adverse events were reported by 24 (34%) of 71 patients receiving olaparib and abiraterone (seven of which were related to treatment) and 13 (18%) of 71 patients receiving placebo and abiraterone (one of which was related to treatment). One treatment-related death (pneumonitis) occurred in the olaparib and abiraterone group. Interpretation Olaparib in combination with abiraterone provided clinical efficacy benefit for patients with metastatic castration-resistant prostate cancer compared with abiraterone alone. More serious adverse events were observed in patients who received olaparib and abiraterone than abiraterone alone. Our data suggest that the combination of olaparib and abiraterone might provide an additional clinical benefit to a broad population of patients with metastatic castration-resistant prostate cancer

L-Selectin Is Dispensable for T Regulatory Cell Function Postallogeneic Bone Marrow Transplantation: CD62L−/− Tregs Inhibit Acute GvHD

In murine models, the adoptive transfer of CD4+/CD25+ regulatory T cells (Tregs) inhibited graft-versus-host disease (GvHD). Previous work has indicated a critical role for the adhesion molecule L-selectin (CD62L) in the function of Tregs in preventing GvHD. Here we examined the capacity of naive wild-type (WT), CD62L−/− and ex vivo expanded CD62LLo Tregs to inhibit acute GvHD. Surprisingly, we found that CD62L−/− Tregs were potent suppressors of GvHD, whereas CD62LLo Tregs were unable to inhibit disease despite being functionally competent to suppress allo T cell responses in vitro. Concomitant with improved outcomes, WT and CD62L−/− Tregs significantly reduced liver pathology and systemic pro-inflammatory cytokine production, although CD62L−/− Tregs were less effective in reducing lung pathology. While accumulation of CD62L−/− Tregs in GvHD target organs was equivalent to WT Tregs, CD62L−/− Tregs did not migrate as well as WT Tregs to peripheral lymph nodes (PLNs) over the first 2 weeks posttransplantation. This work demonstrated that CD62L was dispensable for Treg-mediated protection from GvHD

The Inflammasome Component Nlrp3 Impairs Antitumor Vaccine by Enhancing the Accumulation of Tumor-Associated Myeloid-Derived Suppressor Cells

The inflammasome is a proteolysis complex that generates the active forms of the pro-inflammatory cytokines IL-1β and IL-18. Inflammasome activtation is mediated by NLR proteins that respond to microbial and nonmicrobial stimuli. Among NLRs, NLRP3 senses the widest array of stimuli and enhances adaptive immunity. However, its role in antitumor immunity is unknown. Therefore, we evaluated the function of the NLRP3 inflammasome in the immune response using dendritic cell vaccination against the poorly immunogenic melanoma cell line B16-F10. Vaccination of Nlrp3−/− mice led to a relative 4-fold improvement in survival relative to control animals. Immunity depended upon CD8+ T cells and exhibited immune specificity and memory. Increased vaccine efficacy in Nlrp3−/− hosts did not reflect differences in dendritic cells but rather differences in myeloid-derived suppressor cells (MDSCs). Although Nlrp3 was expressed in MDSCs, the absence of Nlrp3 did not alter either their functional capacity to inhibit T cells or their presence in peripheral lymphoid tissues. Instead, the absence of Nlrp3 caused a 5-fold reduction in the number of tumor-associated MDSCs found in host mice. Adoptive transfer experiments also showed that Nlrp3−/− MDSCs were less efficient in reaching the tumor site. Depleting MDSCs with an anti-Gr-1 antibody increased the survival of tumor-bearing wild-type mice but not Nlrp3−/− mice. We concluded that Nlrp3 was critical for accumulation of MDSCs in tumors and for inhibition of antitumor T cell immunity after dendritic cell vaccination. Our findings establish an unexpected role for Nlrp3 in impeding antitumor immune responses, suggesting novel approaches to improve the response to antitumor vaccines by limiting Nlrp3 signaling

Overall Survival with Adjuvant Pembrolizumab in Renal-Cell Carcinoma

BackgroundAdjuvant pembrolizumab therapy after surgery for renal-cell carcinoma was approved on the basis of a significant improvement in disease-free survival in the KEYNOTE-564 trial. Whether the results regarding overall survival from the third prespecified interim analysis of the trial would also favor pembrolizumab was uncertain.MethodsIn this phase 3, double-blind, placebo-controlled trial, we randomly assigned (in a 1:1 ratio) participants with clear-cell renal-cell carcinoma who had an increased risk of recurrence after surgery to receive pembrolizumab (at a dose of 200 mg) or placebo every 3 weeks for up to 17 cycles (approximately 1 year) or until recurrence, the occurrence of unacceptable toxic effects, or withdrawal of consent. A significant improvement in disease-free survival according to investigator assessment (the primary end point) was shown previously. Overall survival was the key secondary end point. Safety was a secondary end point.Download a PDF of the Research Summary.ResultsA total of 496 participants were assigned to receive pembrolizumab and 498 to receive placebo. As of September 15, 2023, the median follow-up was 57.2 months. The disease-free survival benefit was consistent with that in previous analyses (hazard ratio for recurrence or death, 0.72; 95% confidence interval [CI], 0.59 to 0.87). A significant improvement in overall survival was observed with pembrolizumab as compared with placebo (hazard ratio for death, 0.62; 95% CI, 0.44 to 0.87; P=0.005). The estimated overall survival at 48 months was 91.2% in the pembrolizumab group, as compared with 86.0% in the placebo group; the benefit was consistent across key subgroups. Pembrolizumab was associated with a higher incidence of serious adverse events of any cause (20.7%, vs. 11.5% with placebo) and of grade 3 or 4 adverse events related to pembrolizumab or placebo (18.6% vs. 1.2%). No deaths were attributed to pembrolizumab therapy.ConclusionsAdjuvant pembrolizumab was associated with a significant and clinically meaningful improvement in overall survival, as compared with placebo, among participants with clear-cell renal-cell carcinoma at increased risk for recurrence after surgery. (Funded by Merck Sharp and Dohme, a subsidiary of Merck; KEYNOTE-564 ClinicalTrials.gov number, NCT03142334.

Subsequent therapy following pembrolizumab + axitinib or sunitinib treatment for advanced renal cell carcinoma (RCC) in the phase III KEYNOTE-426 study

Background: In the phase III KEYNOTE-426 study, pembrolizumab + axitinib showed significant improvement in OS, PFS, and ORR vs sunitinib in patients with RCC. This analysis assessed subsequent treatment in patients enrolled in KEYNOTE-426. Methods: Treatment-naive patients with clear cell RCC, KPS score �70%, and measurable disease (RECIST v1.1) were randomly assigned 1:1 to receive pembrolizumab 200 mg IV every 3 weeks for up to 35 doses + axitinib 5 mg orally twice daily or sunitinib 50 mg once daily (4 weeks on/2 weeks off) until progression, toxicity, or withdrawal. Type of and time to subsequent therapy were assessed. Results: Of patients in the pembrolizumab + axitinib arm and in the sunitinib arm, 81.4% (349/432) and 90.6% of patients (385/429), respectively, discontinued treatment; radiologic or clinical PD was the most common reason for discontinuation in both (pembrolizumab + axitinib: 65.0% [227/349]; sunitinib: 68.1% [262/385]). Of patients who discontinued, 58.5% of patients (204/349) in the pembrolizumab + axitinib arm and 73.0% (281/385) in the sunitinib arm received subsequent therapy (Table). Although a similar proportion of patients in both arms received subsequent therapy with a VEGF/VEGFR inhibitor (pembrolizumab + axitinib: 88.2% [180/204]; sunitinib: 68.7% [193/281]), a greater proportion of patients in the sunitinib arm (74.4% [209/281]) received subsequent PD-1/PD-L1 inhibitor therapy than in the pembrolizumab + axitinib arm (21.6% [44/204]). Of patients in the pembrolizumab + axitinib arm and the sunitinib arm, 32.4% (66/204) and 22.8% (64/281), respectively, received other therapies

Pembrolizumab (pembro) plus axitinib (axi) versus sunitinib as first-line therapy for advanced clear cell renal cell carcinoma (ccRCC): Results from 42-month follow-up of KEYNOTE-426

https://ascopubs.org/doi/abs/10.1200/JCO.2021.39.15_suppl.450

Phase 3 CLEAR study in patients with advanced renal cell carcinoma: outcomes in subgroups for the lenvatinib-plus-pembrolizumab and sunitinib arms

IntroductionThe phase 3 CLEAR study demonstrated that lenvatinib plus pembrolizumab significantly improved efficacy versus sunitinib as first-line treatment for patients with advanced renal cell carcinoma (RCC). Prognostic features including presence and/or site of baseline metastases, prior nephrectomy, and sarcomatoid features have been associated with disease and treatment success. This subsequent analysis explores outcomes in patients with or without specific prognostic features.MethodsIn CLEAR, patients with clear cell RCC were randomly assigned (1:1:1) to receive either lenvatinib (20 mg/day) plus pembrolizumab (200 mg every 3 weeks), lenvatinib (18 mg/day) plus everolimus (5 mg/day), or sunitinib alone (50 mg/day, 4 weeks on, 2 weeks off). In this report, progression-free survival (PFS), overall survival (OS), and objective response rate (ORR) were all assessed in the lenvatinib-plus-pembrolizumab and the sunitinib arms, based on baseline features: lung metastases, bone metastases, liver metastases, prior nephrectomy, and sarcomatoid histology.ResultsIn all the assessed subgroups, median PFS was longer with lenvatinib-plus-pembrolizumab than with sunitinib treatment, notably among patients with baseline bone metastases (HR 0.33, 95% CI 0.21–0.52) and patients with sarcomatoid features (HR 0.39, 95% CI 0.18–0.84). Median OS favored lenvatinib plus pembrolizumab over sunitinib irrespective of metastatic lesions at baseline, prior nephrectomy, and sarcomatoid features. Of interest, among patients with baseline bone metastases the HR for survival was 0.50 (95% CI 0.30–0.83) and among patients with sarcomatoid features the HR for survival was 0.91 (95% CI 0.32–2.58); though for many groups, median OS was not reached. ORR also favored lenvatinib plus pembrolizumab over sunitinib across all subgroups; similarly, complete responses also followed this pattern.ConclusionEfficacy outcomes improved following treatment with lenvatinib-plus-pembrolizumab versus sunitinib in patients with RCC—irrespective of the presence or absence of baseline lung metastases, baseline bone metastases, baseline liver metastases, prior nephrectomy, or sarcomatoid features. These findings corroborate those of the primary CLEAR study analysis in the overall population and support lenvatinib plus pembrolizumab as a standard of care in 1L treatment for patients with advanced RCC.Clinical trial registrationClinicalTrials.gov, identifier NCT0281186

Pembrolizumab versus placebo as post-nephrectomy adjuvant therapy for clear cell renal cell carcinoma (KEYNOTE-564): 30-month follow-up analysis of a multicentre, randomised, double-blind, placebo-controlled, phase 3 trial

BACKGROUND: The first interim analysis of the KEYNOTE-564 study showed improved disease-free survival with adjuvant pembrolizumab compared with placebo after surgery in patients with clear cell renal cell carcinoma at an increased risk of recurrence. The analysis reported here, with an additional 6 months of follow-up, was designed to assess longer-term efficacy and safety of pembrolizumab versus placebo, as well as additional secondary and exploratory endpoints. METHODS: In the multicentre, randomised, double-blind, placebo-controlled, phase 3 KEYNOTE-564 trial, adults aged 18 years or older with clear cell renal cell carcinoma with an increased risk of recurrence were enrolled at 213 hospitals and cancer centres in North America, South America, Europe, Asia, and Australia. Eligible participants had an Eastern Cooperative Oncology Group performance status of 0 or 1, had undergone nephrectomy 12 weeks or less before randomisation, and had not received previous systemic therapy for advanced renal cell carcinoma. Participants were randomly assigned (1:1) via central permuted block randomisation (block size of four) to receive pembrolizumab 200 mg or placebo intravenously every 3 weeks for up to 17 cycles. Randomisation was stratified by metastatic disease status (M0 vs M1), and the M0 group was further stratified by ECOG performance status and geographical region. All participants and investigators involved in study treatment administration were masked to the treatment group assignment. The primary endpoint was disease-free survival by investigator assessment in the intention-to-treat population (all participants randomly assigned to a treatment). Safety was assessed in the safety population, comprising all participants who received at least one dose of pembrolizumab or placebo. As the primary endpoint was met at the first interim analysis, updated data are reported without p values. This study is ongoing, but no longer recruiting, and is registered with ClinicalTrials.gov, NCT03142334. FINDINGS: Between June 30, 2017, and Sept 20, 2019, 994 participants were assigned to receive pembrolizumab (n=496) or placebo (n=498). Median follow-up, defined as the time from randomisation to data cutoff (June 14, 2021), was 30·1 months (IQR 25·7-36·7). Disease-free survival was better with pembrolizumab compared with placebo (HR 0·63 [95% CI 0·50-0·80]). Median disease-free survival was not reached in either group. The most common all-cause grade 3-4 adverse events were hypertension (in 14 [3%] of 496 participants) and increased alanine aminotransferase (in 11 [2%]) in the pembrolizumab group, and hypertension (in 13 [3%] of 498 participants) in the placebo group. Serious adverse events attributed to study treatment occurred in 59 (12%) participants in the pembrolizumab group and one (<1%) participant in the placebo group. No deaths were attributed to pembrolizumab. INTERPRETATION: Updated results from KEYNOTE-564 support the use of adjuvant pembrolizumab monotherapy as a standard of care for participants with renal cell carcinoma with an increased risk of recurrence after nephrectomy. FUNDING: Merck Sharp & Dohme LLC, a subsidiary of Merck & Co, Inc, Rahway, NJ, USA