Cancer immunotherapy can induce long lasting responses in patients with metastatic cancers of a wide range of histologies. Broadening the clinical applicability of these treatments requires an improved understanding of the mechanisms limiting cancer immunotherapy. The interactions between the immune system and cancer cells are continuous, dynamic, and evolving from the initial establishment of a cancer cell to the development of metastatic disease, which is dependent on immune evasion. As the molecular mechanisms of resistance to immunotherapy are elucidated, actionable strategies to prevent or treat them may be derived to improve clinical outcomes for patients.

Purpose

CTLA-4 was the first inhibitory immune checkpoint to be identified. Two mAbs, ipilimumab (IgG1) and tremelimumab (IgG2), which block the function of CTLA-4, have demonstrated durable clinical activity in a subset of patients with advanced solid malignancies by augmenting effector T-cell-mediated immune responses. Studies in mice suggest that anti-CTLA-4 mAbs may also selectively deplete intratumoral FOXP3⁺ regulatory T cells via an Fc-dependent mechanism. However, it is unclear whether the depletion of FOXP3⁺ cells occurs in patients with cancer treated with anti-CTLA-4 therapies.

Experimental design

Quantitative IHC was used to evaluate the densities of intratumoral CD4⁺, CD8⁺, and FOXP3⁺ cells in stage-matched melanoma (n = 19), prostate cancer (n = 17), and bladder cancer (n = 9) samples treated with ipilimumab and in paired melanoma tumors (n = 18) treated with tremelimumab. These findings were corroborated with multiparametric mass cytometry analysis of tumor-infiltrating cells from paired fresh melanoma tumors (n = 5) treated with ipilimumab.

Results

Both ipilimumab and tremelimumab increase infiltration of intratumoral CD4⁺ and CD8⁺ cells without significantly changing or depleting FOXP3⁺ cells within the tumor microenvironment.

Conclusions

Anti-CTLA-4 immunotherapy does not deplete FOXP3⁺ cells in human tumors, which suggests that their efficacy could be enhanced by modifying the Fc portions of the mAbs to enhance Fc-mediated depletion of intratumoral regulatory T cells.See related commentary by Quezada and Peggs, p. 1130.

Background

Sipuleucel-T is a US Food and Drug Administration-approved autologous cellular immunotherapy that improves survival in patients with metastatic castration-resistant prostate cancer (mCRPC). We examined whether administering ipilimumab after sipuleucel-T could modify immune and/or clinical responses to this treatment.

Methods

A total of 50 patients with mCRPC were enrolled into a clinical trial (NCT01804465, ClinicalTrials.gov) where they received ipilimumab either immediately or delayed 3 weeks following completion of sipuleucel-T treatment. Blood was collected at various timepoints of the study. Luminex assay for anti-prostatic acid phosphatase (PAP) and anti-PA2024-specific serum immunoglobulin G (IgG) and ELISpot for interferon-γ (IFN-γ) production against PAP and PA2024 were used to assess antigen-specific B and T cell responses, respectively. Clinical response was defined as >30% reduction in serum prostate-specific antigen levels compared with pretreatment levels. The frequency and state of circulating immune cells were determined by mass cytometry by time-of-flight and statistical scaffold analysis.

Results

We found the combination to be well tolerated with no unexpected adverse events occurring. The timing of ipilimumab did not significantly alter the rates of antigen-specific B and T cell responses, the primary endpoint of the clinical trial. Clinical responses were observed in 6 of 50 patients, with 3 having responses lasting longer than 3 months. The timing of ipilimumab did not significantly associate with clinical response or toxicity. The combination treatment did induce CD4 and CD8 T cell activation that was most pronounced with the immediate schedule. Lower frequencies of CTLA-4 positive circulating T cells, even prior to treatment, were associated with better clinical outcomes. Interestingly, these differences in CTLA-4 expression were associated with prior localized radiation therapy (RT) to the prostate or prostatic fossa. Prior radiation treatment was also associated with improved radiographic progression-free survival.

Conclusion

Combining CTLA-4 blockade with sipuleucel-T resulted in modest clinical activity. The timing of CTLA-4 blockade following sipuleucel-T did not alter antigen-specific responses. Clinical responses were associated with both lower baseline frequencies of CTLA-4 expressing T cells and a history of RT. Prior cancer therapy may therefore result in long-lasting immune changes that influence responsiveness to immunotherapy with sipuleucel-T and anti-CTLA-4.

With the recent success of checkpoint inhibitors and other immunomodulating agents, there has been renewed interest in the combination of such agents with radiation. The biologic premise behind such a strategy is that the tumor-antigen release achieved by localized radiation will promote specific tumor targeting by the adaptive immune system, which can be augmented further by systemic immune-stimulating agents. In this manner, clinicians hope to induce a phenomenon known as the abscopal effect, whereby localized radiation results in immune-mediated tumor regression in disease sites well outside of the radiation field. Herein, we present a comprehensive overview of the early clinical and preclinical evidence behind this approach.

PURPOSE: To assess the safety and tolerability of preoperative cryoablation-mediated tumor antigen presentation and/or ipilimumab-mediated immune modulation in women with operable breast cancer. EXPERIMENTAL DESIGN: In this pilot study, 19 women with breast cancer for whom mastectomy was planned were treated with preoperative tumor cryoablation (n = 7), single-dose ipilimumab at 10 mg/kg (n = 6), or both (n = 6). The primary outcome for this pilot study was safety/tolerability as defined as freedom from delays in pre-planned, curative-intent mastectomy. Exploratory studies of immune activation were performed on peripheral blood and tumor. RESULTS: Preoperative cryoablation and/or ipilimumab were safe and tolerable, with no delays in pre-planned surgery. Grade III toxicity was seen in 1 of 19 (unrelated rash after ipilimumab). Combination therapy was associated with sustained peripheral elevations in: Th1-type cytokines, activated (ICOS+) and proliferating (Ki67+) CD4+ and CD8+ T cells, and posttreatment proliferative T-effector cells relative to T-regulatory cells within tumor. CONCLUSIONS: Preoperative cryoablation and single-dose ipilimumab are safe alone or in combination with no surgical delays incurred. Potentially favorable intratumoral and systemic immunologic effects were observed with the combination, suggesting the possibility for induced and synergistic antitumor immunity with this strategy. Clin Cancer Res; 22(23); 5729-37. ©2016 AACR.

Pancreatic ductal adenocarcinoma (PDAC) is associated with marked fibrosis and stromal myofibroblasts, but their functional contribution remains unknown. Transgenic mice with the ability to delete αSMA(+) myofibroblasts in pancreatic cancer were generated. Depletion starting at either noninvasive precursor (pancreatic intraepithelial neoplasia) or the PDAC stage led to invasive, undifferentiated tumors with enhanced hypoxia, epithelial-to-mesenchymal transition, and cancer stem cells, with diminished animal survival. In PDAC patients, fewer myofibroblasts in their tumors also correlated with reduced survival. Suppressed immune surveillance with increased CD4(+)Foxp3(+) Tregs was observed in myofibroblast-depleted mouse tumors. Although myofibroblast-depleted tumors did not respond to gemcitabine, anti-CTLA4 immunotherapy reversed disease acceleration and prolonged animal survival. This study underscores the need for caution in targeting carcinoma-associated fibroblasts in PDAC.

Immune checkpoint inhibitors (ICI) targeting CTLA4 or PD-1/PD-L1 have transformed cancer therapy but are associated with immune-related adverse events, including myocarditis. Here, we report a robust preclinical mouse model of ICI-associated myocarditis in which monoallelic loss of Ctla4 in the context of complete genetic absence of Pdcd1 leads to premature death in approximately half of mice. Premature death results from myocardial infiltration by T cells and macrophages and severe ECG abnormalities, closely recapitulating the clinical and pathologic hallmarks of ICI-associated myocarditis observed in patients. Using this model, we show that Ctla4 and Pdcd1 functionally interact in a gene dosage-dependent manner, providing a mechanism by which myocarditis arises with increased frequency in the setting of combination ICI therapy. We demonstrate that intervention with CTLA4-Ig (abatacept) is sufficient to ameliorate disease progression and additionally provide a case series of patients in which abatacept mitigates the fulminant course of ICI myocarditis. SIGNIFICANCE: We provide a preclinical model of ICI-associated myocarditis which recapitulates this clinical syndrome. Using this model, we demonstrate that CTLA4 and PD-1 (ICI targets) functionally interact for myocarditis development and that intervention with CTLA4-Ig (abatacept) attenuates myocarditis, providing mechanistic rationale and preclinical support for therapeutic clinical studies.See related commentary by Young and Bluestone, p. 537.This article is highlighted in the In This Issue feature, p. 521.

Identifying pan-tumor biomarkers that predict responses to immune checkpoint inhibitors (ICI) is critically needed. In the AMADEUS clinical trial (NCT03651271), patients with various advanced solid tumors were assessed for changes in intratumoral CD8 percentages and their response to ICI. Patients were grouped based on tumoral CD8 levels: those with CD8 <15% (CD8-low) received nivolumab (anti-PD-1) plus ipilimumab (anti-CTLA4) and those with CD8 ≥15% (CD8-high) received nivolumab monotherapy. 79 patients (72 CD8-low and 7 CD8-high) were treated. The disease control rate was 25.0% (18/72; 95% CI: 15.8-35.2) in CD8-low and 14.3% (1/7; 95% CI: 1.1-43.8) in CD8-high. Tumors from 35.9% (14/39; 95% CI: 21.8-51.4) of patients converted from CD8 <15% pretreatment to ≥15% after treatment. Multiomic analyses showed that CD8-low responders had an inflammatory tumor microenvironment pretreatment, enhanced by an influx of CD8 T cells, CD4 T cells, B cells, and macrophages upon treatment. These findings reveal crucial pan-cancer immunological features for ICI response in patients with metastatic disease.

Treatment with immune checkpoint blockade (ICB) has revolutionized cancer therapy. Until now, predictive biomarkers^1-10 and strategies to augment clinical response have largely focused on the T cell compartment. However, other immune subsets may also contribute to anti-tumour immunity^11-15, although these have been less well-studied in ICB treatment¹⁶. A previously conducted neoadjuvant ICB trial in patients with melanoma showed via targeted expression profiling¹⁷ that B cell signatures were enriched in the tumours of patients who respond to treatment versus non-responding patients. To build on this, here we performed bulk RNA sequencing and found that B cell markers were the most differentially expressed genes in the tumours of responders versus non-responders. Our findings were corroborated using a computational method (MCP-counter¹⁸) to estimate the immune and stromal composition in this and two other ICB-treated cohorts (patients with melanoma and renal cell carcinoma). Histological evaluation highlighted the localization of B cells within tertiary lymphoid structures. We assessed the potential functional contributions of B cells via bulk and single-cell RNA sequencing, which demonstrate clonal expansion and unique functional states of B cells in responders. Mass cytometry showed that switched memory B cells were enriched in the tumours of responders. Together, these data provide insights into the potential role of B cells and tertiary lymphoid structures in the response to ICB treatment, with implications for the development of biomarkers and therapeutic targets.