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Introduction: Costimulatory members of the tumor necrosis factor receptor family, such as OX40 (CD134), provide essential survival and differentiation signals that enhance T cell function. Specifically, OX40 (CD134) agonists stimulate potent anti-tumor immunity in a variety of preclinical models but their therapeutic impact in patients with advanced malignancies has been limited thus far., Areas Covered: In this review, we discuss the current state of combination immunotherapy with OX40 agonists including preclinical studies and recent clinical trials. We also discuss the strengths and limitations of these approaches and provide insight into alternatives that may help enhance the efficacy of combination OX40 agonist immunotherapy., Expert Opinion: OX40 agonist immunotherapy has not yet demonstrated significant clinical activity as a monotherapy or in combination with immune checkpoint blockade (ICB), likely due to several factors including the timing of administration, drug potency, and selection of agents for combination therapy clinical trials. We believe that careful consideration of the biological mechanisms regulating OX40 expression and function may help inform new approaches, particularly in combination with novel agents, capable of increasing the therapeutic efficacy of this approach.

Chemoimmunotherapy with anti-programmed cell death 1/ligand 1 and cytotoxic chemotherapy is a promising therapeutic modality for women with triple-negative breast cancer, but questions remain regarding optimal chemotherapy backbone and biomarkers for patient selection. We report final outcomes from a phase Ib trial evaluating pembrolizumab (200 mg IV every 3 weeks) with either weekly paclitaxel (80 mg/m 2 weekly) or flat-dose capecitabine (2000 mg orally twice daily for 7 days of every 14-day cycle) in the 1st/2nd line setting. The primary endpoint is safety (receipt of 2 cycles without grade III/IV toxicities requiring discontinuation or ≥21-day delays). The secondary endpoint is efficacy (week 12 objective response). Exploratory aims are to characterize immunologic effects of treatment over time, and to evaluate novel biomarkers. The trial demonstrates that both regimens meet the pre-specified safety endpoint (paclitaxel: 87%; capecitabine: 100%). Objective response rate is 29% for pembrolizumab/paclitaxel (n = 4/13, 95% CI: 10-61%) and 43% for pembrolizumab/capecitabine (n = 6/14, 95% CI: 18-71%). Partial responses are observed in two subjects with chemo-refractory metaplastic carcinoma (both in capecitabine arm). Both regimens are associated with significant peripheral leukocyte contraction over time. Response is associated with clinical PD-L1 score, non-receipt of prior chemotherapy, and the H&E stromal tumor-infiltrating lymphocyte score, but also by a novel 27 gene IO score and spatial biomarkers (lymphocyte spatial skewness). In conclusion, pembrolizumab with paclitaxel or capecitabine is safe and clinically active. Both regimens are lymphodepleting, highlighting the competing immunostimulatory versus lymphotoxic effects of cytotoxic chemotherapy. Further exploration of the IO score and spatial TIL biomarkers is warranted. The clinical trial registration is NCT02734290., (© 2023. The Author(s).)

Previously designed flow cytometry panels have provided a framework to analyze T-cell activation; however, few provide an extensive view of lymphocyte populations, and none are optimized for murine models. This article describes a panel designed specifically to assess the expression of activation and exhaustion markers in expanding lymphocyte populations in tumor-bearing mice across two distinct genetic backgrounds: BALB/c and C57BL/6. This comprehensive panel enables the assessment of multiple functional states and immune checkpoint markers across cytotoxic CD8 + T cells, helper and regulatory CD4 + T cells and natural killer cells in murine whole blood, lymph nodes and tumor.

Cancer immunotherapy such as anti-PD-1/anti-PD-L1 immune checkpoint blockade (ICB) can provide significant clinical benefit in patients with advanced malignancies. However, most patients eventually develop progressive disease, thus necessitating additional therapeutic options. We have developed a novel agent, a-TEA-LS, that selectively induces tumor cell death while sparing healthy tissues, leading to increased activation of tumor-reactive T cells and tumor regression. In the current study, we explored the impact of combined a-TEA-LS + ICB in orthotopic and spontaneously arising murine models of mammary carcinoma. We found that a-TEA-LS + ICB led to increased production of pro-inflammatory cytokines that were associated with a reduction in tumor growth and prolonged survival. Together, these data demonstrate the potential utility of a-TEA-LS + ICB for the treatment of breast cancer and provide the rationale for clinical translation of this novel approach., Competing Interests: Author EA was employed by and has ownership interest in Veana Therapeutics. WR: Research support from Bristol-Myers Squibb, GlaxoSmithKline, MiNA Therapeutics, Inhibrx, Veana Therapeutics, Shimadzu, OncoSec, Galecto, Turn Bio, CanWell Pharma, and Calibr. Patents/Licensing fees: Galectin Therapeutics. Scientific Advisory Boards: Vesselon, Medicenna, Veana Therapeutics. The remaining author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Redmond, Kasiewicz and Akporiaye.)

While immune checkpoint blockade has revolutionized cancer treatment, unfortunately most patients do not benefit from this treatment. Many pharmacodynamic (PD) studies have revealed essential requirements for successful cancer immunotherapy that may provide insight into how we can improve these agents. Despite enormous efforts focused on interrogating the immune system using different biospecimens (e.g. blood, primary tumor, metastatic tumor, microbiome samples), a variety of technologies (e.g. flow cytometry, bulk and single-cell RNA-sequencing, immunohistochemistry), and wide-ranging disciplines (e.g. pathology, genomics, bioinformatics, immunology, cancer biology, metabolomics, bacteriology), discovery of consistent biomarkers of response have remained elusive. Pharmacokinetics (PK) studies, however, not only provide critical information regarding safe dosing but may also reveal useful biomarkers. For example, recent studies found that trough levels of therapeutic monoclonal antibodies (mAbs) or clearance (CL) of them were associated with clinical outcome, which suggests that trough levels of mAbs may represent a new class of on-treatment cancer immunotherapy biomarker. In this review, we summarize the potential utility of trough levels of mAbs, the mechanism of varying PK, consideration for therapeutic drug monitoring, and assay attributes that will facilitate wider utilization of PK information in conjunction with PD assessments.

Accurate quantitation of antibodies is critical for development of monoclonal antibody therapeutics (mAbs). Therapeutic drug monitoring has been applied to measure levels of mAbs in clinics for dose adjustment for autoimmune disease. Trough levels of mAbs can be a biomarker for cancer immunotherapy. Thus, the deployment of a rapid and universal platform for mAb monitoring may benefit processes ranging from drug development to clinical practice for a wide spectrum of diseases. However, mAb monitoring often requires development and conduct of an individual ligand binding assay such as ELISA, which is impractical to scale. We streamlined quantitation of antibody therapeutics by a nano-surface and molecular-orientation limited (nSMOL) proteolysis assay using LC-MS with a universal reference antibody (refmAb-Q), for accurate multiplexed quantitation of unique signature peptides derived from mAbs. This innovative refmAb-Q nSMOL platform may provide a practical solution for quantitating an ever-increasing number of mAbs from developmental to clinical use settings.

Background: TGF-β is an immunosuppressive cytokine that is upregulated in colorectal cancer. TGF-β blockade improved response to chemoradiotherapy in preclinical models of colorectal adenocarcinoma. We aimed to test the hypothesis that adding the TGF-β type I receptor kinase inhibitor galunisertib to neoadjuvant chemoradiotherapy would improve pathological complete response rates in patients with locally advanced rectal cancer., Methods: This was an investigator-initiated, single-arm, phase 2 study done in two medical centres in Portland (OR, USA). Eligible patients had previously untreated, locally advanced, rectal adenocarcinoma, stage IIA-IIIC or IV as per the American Joint Committee on Cancer; Eastern Cooperative Oncology Group status 0-2; and were aged 18 years or older. Participants completed two 14-day courses of oral galunisertib 150 mg twice daily, before and during fluorouracil-based chemoradiotherapy (intravenous fluorouracil 225 mg/m 2 over 24 h daily 7 days per week during radiotherapy or oral capecitabine 825 mg/m 2 twice per day 5 days per week during radiotherapy; radiotherapy consisted of 50·4-54·0 Gy in 28-30 fractions). 5-9 weeks later, patients underwent response assessment. Patients with a complete response could opt for non-operative management and proceed to modified FOLFOX6 (intravenous leucovorin 400 mg/m 2 on day 1, intravenous fluorouracil 400 mg/m 2 on day 1 then 2400 mg/m 2 over 46 h, and intravenous oxaliplatin 85 mg/m 2 on day 1 delivered every 2 weeks for eight cycles) or CAPEOX (intravenous oxaliplatin 130 mg/m 2 on day 1 and oral capecitabine 1000 mg/m 2 twice daily for 14 days every 3 weeks for four cycles). Patients with less than complete response underwent surgical resection. The primary endpoint was complete response rate, which was a composite of pathological complete response in patients who proceeded to surgery, or clinical complete response maintained at 1 year after last therapy in patients with non-operative management. Safety was a coprimary endpoint. Both endpoints were assessed in the intention-to-treat population. This study is registered with ClinicalTrials.gov, NCT02688712, and is active but not recruiting., Findings: Between Oct 19, 2016, and Aug 31, 2020, 38 participants were enrolled. 25 (71%) of the 35 patients who completed chemoradiotherapy proceeded to total mesorectal excision surgery, five (20%) of whom had pathological complete responses. Ten (29%) patients had non-operative management, three (30%) of whom ultimately chose to have total mesorectal excision. Two (67%) of those three patients had pathological complete responses. Of the remaining seven patients in the non-operative management group, five (71%) had clinical complete responses at 1 year after their last modified FOLFOX6 infusion. In total, 12 (32% [one-sided 95% CI ≥19%]) of 38 patients had a complete response. Common grade 3 adverse events during treatment included diarrhoea in six (16%) of 38 patients, and haematological toxicity in seven (18%) patients. Two (5%) patients had grade 4 adverse events, one related to chemoradiotherapy-induced diarrhoea and dehydration, and the other an intraoperative ischaemic event. No treatment-related deaths occurred., Interpretation: The addition of galunisertib to neoadjuvant chemoradiotherapy in patients with locally advanced rectal cancer improved the complete response rate to 32%, was well tolerated, and warrants further assessment in randomised trials., Funding: Eli Lilly via ExIST program, The Providence Foundation., Competing Interests: Declaration of interests TY's spouse has ownership interest in PACT Pharma and Genocea Biosciences; was paid to consult or advise PACT Pharma, Genocea Biosciences, and Turnstone Biologics; and receives royalties from patents held jointly with the National Cancer Institute. AJG's spouse is employed by Thermo Fisher and has a patent held jointly with the Providence Portland Medical Center (Portland, OR, USA). MG is employed by Thermo Fisher and MG's spouse holds a patent jointly with the Providence Portland Medical Center. NF is employed by Thermo Fisher. WLR reports sponsored research agreements with Merck, Tesaro, Nektar Therapeutics, IRX Therapeutics, Galectin Therapeutics, and Bristol Myers Squibb; and licensing or royalties from Galectin Therapeutics. WJU reports consultant or advisory fees from the AstraZeneca data safety monitoring board. MJG reports sponsored research agreements with Bristol Myers Squibb and VIR Biotechnology, and a patent held jointly with Providence Portland Medical Center and their spouse. MRC's spouse has sponsored research agreements with Bristol Myers Squibb and VIR Biotechnology, and a patent held jointly with Providence Portland Medical Center and spouse. KHY reports ExIST trial funding of less than 50% of the trial costs from Eli Lilly (the remainder of the study was funded by The Providence Foundation); sponsored research agreements with Bristol Myers Squibb; grant funding held through Susan G Komen Foundation and Providence Foundation H&N Developmental Research Program; and has a patent held jointly with the Providence Portland Medical Center. All other authors declare no competing interests., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

Purpose of Review: Despite the efficacy of immune checkpoint blockade (ICB) immunotherapy, most cancer patients still develop progressive disease necessitating additional treatment options. One approach is ligation of the OX40 (CD134) costimulatory receptor which promotes T cell activation, effector function, and the generation of long-lived memory cells., Recent Findings: Numerous preclinical studies have demonstrated that OX40 agonists alone or in combination with ICB (e.g., anti-PD-1, anti-PD-L1, and anti-CTLA-4) augment anti-tumor immunity. In this review, we discuss the impact of OX40 agonists on T cell function and the therapeutic potential of OX40 agonists alone or in conjunction with ICB for patients with advanced malignancies., (© 2022. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Intratumoral delivery of plasmid IL12 via electroporation (IT-tavo-EP) induces localized expression of IL12 leading to regression of treated and distant tumors with durable responses and minimal toxicity. A key driver in amplifying this local therapy into a systemic response is the magnitude and composition of immune infiltrate in the treated tumor. While intratumoral IL12 typically increases the density of CD3+ tumor-infiltrating lymphocytes (TIL), this infiltrate is composed of a broad range of T-cell subsets, including activated tumor-specific T cells, less functional bystander T cells, as well as suppressive T regulatory cells. To encourage a more favorable on-treatment tumor microenvironment (TME), we explored combining this IL12 therapy with an intratumoral polyclonal T-cell stimulator membrane-anchored anti-CD3 to productively engage a diverse subset of lymphocytes including the nonreactive and suppressive T cells. This study highlighted that combined intratumoral electroporation of IL12 and membrane-anchored anti-CD3 plasmids can enhance cytokine production, T-cell cytotoxicity, and proliferation while limiting the suppressive capacity within the TME. These collective antitumor effects not only improve regression of treated tumors but drive systemic immunity with control of nontreated contralateral tumors in vivo. Moreover, combination of IL12 and anti-CD3 restored the function of TIL isolated from a patient with melanoma actively progressing on programmed cell death protein 1 (PD-1) checkpoint inhibitor therapy., Implications: This DNA-encodable polyclonal T-cell stimulator (membrane-anchored anti-CD3 plasmid) may represent a key addition to intratumoral IL12 therapies in the clinic., (©2022 American Association for Cancer Research.)

Background: Tumor cell death caused by radiation therapy (RT) triggers antitumor immunity in part because dying cells release adjuvant factors that amplify and sustain dendritic cell and T cell responses. We previously demonstrated that bempegaldesleukin (BEMPEG: NKTR-214, an immunostimulatory IL-2 cytokine prodrug) significantly enhanced the antitumor efficacy of RT through a T cell-dependent mechanism. Because RT can induce either immunogenic or tolerogenic cell death, depending on various factors (radiation dose, cell cycle phase), we hypothesized that providing a specific immunogenic adjuvant, like intratumoral therapy with a novel toll-like receptor (TLR) 7/8 agonist, NKTR-262, would improve systemic tumor-specific responses through the activation of local innate immunity. Therefore, we evaluated whether intratumoral NKTR-262 combined with systemic BEMPEG treatment would elicit improved tumor-specific immunity and survival compared with RT combined with BEMPEG., Methods: Tumor-bearing mice (CT26; EMT6) received BEMPEG (0.8 mg/kg; intravenously), RT (12 Gy × 1), and/or intratumoral NKTR-262 (0.5 mg/kg). Flow cytometry was used to evaluate CD4 + and CD8 + T cell responses in the blood and tumor 7 days post-treatment. The contribution of specific immune subsets was determined by depletion of CD4 + , CD8 + , or NK cells. CD8 + T cell cytolytic activity was determined by an in vitro CTL assay. Data are representative of 1-2 independent experiments (n=5-14/group) and statistical significance was determined by 1-way analysis of variance (ANOVA) or repeated measures ANOVA (p value cut-off of 0.05)., Results: BEMPEG+NKTR-262 significantly improved survival compared with BEMPEG+RT in a CD8 + T cell-dependent manner. Response to BEMPEG+NKTR-262 was characterized by a significant expansion of activated CD8 + T cells (GzmA + ; Ki-67 + ; ICOS + ; PD-1 + ) in the blood, which correlated with reduced tumor size (p<0.05). In the tumor, BEMPEG+NKTR-262 induced higher frequencies of GzmA + CD8 + T cells exhibiting reduced expression of suppressive molecules (PD-1 + ), compared with BEMPEG+RT (p<0.05). Further, BEMPEG+NKTR-262 treatment induced greater tumor-specific CD8 + T cell cytolytic function than BEMPEG+RT., Conclusions: BEMPEG+NKTR-262 therapy elicited more robust expansion of activated CD8 + T cells compared with BEMPEG+RT, suggesting that intratumoral TLR stimulation provides superior antigen presentation and costimulatory activity compared with RT. A clinical trial of BEMPEG+NKTR-262 for patients with metastatic solid tumors is in progress (NCT03435640)., Competing Interests: Competing interests: Redmond: Research support from Galectin Therapeutics, Bristol Myers Squibb, GlaxoSmithKline, MiNA Therapeutics, Inhibrx, Veana Therapeutics, Aeglea Biotherapeutics, Shimadzu, OncoSec, and Calibr. Patents/Licensing Fees: Galectin Therapeutics. Advisory Boards: Nektar Therapeutics, Vesselon. Rubas: Former employee of Nektar Therapeutics; current employee of Sutra Biopharma. Kïvimäe: Current employee of Nektar Therapeutics. All other authors had no relevant disclosures., (© Author(s) (or their employer(s)) 2022. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.)

Background: Chemoimmunotherapy is a standard treatment for triple-negative breast cancer (TNBC), however, the impacts of different chemotherapies on T-cell populations, which could correlate with clinical activity, are not known. Quantifying T-cell populations with flow cytometry and T-cell receptor (TCR) immunosequencing may improve our understanding of how chemoimmunotherapy affects T-cell subsets, and to what extent clonal shifts occur during treatment. TCR immunosequencing of intratumoral T cells may facilitate the identification and monitoring of putatively tumor-reactive T-cell clones within the blood., Methods: Blood and tumor biopsies were collected from patients with metastatic TNBC enrolled in a phase Ib clinical trial of first or second-line pembrolizumab with paclitaxel or capecitabine. Using identical biospecimen processing protocols, blood samples from a cohort of patients treated for early-stage breast cancer were obtained for comparison. Treatment-related immunological changes in peripheral blood and intratumoral T cells were characterized using flow cytometry and TCR immunosequencing. Clonal proliferation rates of T cells were compared based on intratumoral enrichment., Results: When combined with pembrolizumab, paclitaxel and capecitabine resulted in similar time-dependent lymphodepletions across measured peripheral T-cell subsets. Their effects were more modest than that observed following curative-intent dose-dense anthracycline and cyclophosphamide (ddAC) (average fold-change in CD3 + cells, capecitabine: -0.42, paclitaxel: -0.56, ddAC: -1.21). No differences in T-cell clonality or richness were observed following capecitabine or paclitaxel-based treatments. Regression modeling identified differences in the emergence of novel T-cell clones that were not detected at baseline (odds compared with ddAC, capecitabine: 0.292, paclitaxel: 0.652). Pembrolizumab with paclitaxel or capecitabine expanded T-cell clones within tumors; however, these clones did not always expand within the blood. Proliferation rates within the blood were similar between clones that were enriched and those that were not enriched within tumors., Conclusion: Chemoimmunotherapy for metastatic TNBC with pembrolizumab and capecitabine or paclitaxel resulted in similar peripheral T-cell subset lymphodepletion without altering T-cell clonal diversity. Regression modeling methods are applicable in immune monitoring studies, such as this to identify the odds of novel T-cell clones emerging during treatment, and proliferation rates of tumor-enriched T-cell clones., Competing Interests: Competing interests: YK: Research support from Bristol Myers Squibb (BMS), GlaxoSmithKline, Shimadzu. WLR: Research support from Galectin Therapeutics, BMS, GlaxoSmithKline, Mi. NA Therapeutics, Inhibrx, Veana Therapeutics, Shimadzu, OncoSec Medical, Turn Biotechnologies, CanWell Pharma, Aeglea Biotherapeutics, and Calibr. Patents/Royalties: Galectin Therapeutics. Advisory Boards: Nektar Therapeutics, Vesselon. NP: Financial interest: Adaptive Biotechnologies. PAF: Financial interest: Adaptive Biotechnologies. WJU: Safety advisory board: Astra Zeneca. HLM: Research support: Merck, Lilly, BMS. Advisory/consultancy Merck, Lilly, Spectrum Pharmaceuticals, Amgen, Immunomedics, Pfizer, Genentech, BMS, Genomic Health, ZIOPHARM Oncology; Travel/expenses: Merck, Spectrum Pharmaceuticals, Lilly, Amgen, Immunomedics, Pfizer, Genentech Puma Biotechnology; Speaker Bureau: Lilly. Institutional funding: Merck, Lilly, BMS, ZIOPHARM Oncology DBP: Research support: BMS, Merck, Brooklyn ImmunoTherapeutics. Advisory Boards: BMS, Merck, Syndax, Nektar, Puma, Nanostring, Genetech, Brooklyn Immunotherapeutics, Sanofi, Biotheranostics, NGMBio, Lilly. Speaker Bureau: Genentech, Novartis., (© Author(s) (or their employer(s)) 2022. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.)

Background: Immune checkpoint blockade (ICB) using anti-CTLA-4 and anti-PD-1/PD-L1 has revolutionized the treatment of advanced cancer. However, ICB is effective for only a small fraction of patients, and biomarkers such as expression of PD-L1 in tumor or serum levels of CXCL11 have suboptimal sensitivity and specificity. Exposure-response (E-R) relationships have been observed with other therapeutic monoclonal antibodies. There are many factors influencing E-R relationships, yet several studies have shown that trough levels of anti-PD-1/PD-L1 correlated with clinical outcomes. However, the potential utility of anti-CTLA-4 levels as a biomarker remains unknown., Methods: Serum was obtained at trough levels at weeks 7 and 12 (after doses 2 and 4) from patients with advanced melanoma who received ipilimumab alone (3 mg/kg every 3 weeks for four treatments) via an expanded access program (NCT00495066). We have successfully established a proteomics assay to measure the concentration of ipilimumab in serum using an liquid chromatography with tandem mass spectrometry-based nanosurface and molecular-orientation limited proteolysis (nSMOL) approach. Serum samples from 38 patients were assessed for trough levels of ipilimumab by the nSMOL assay., Results: We found that trough levels of ipilimumab were higher in patients who developed immune-related adverse events but did not differ based on the presence or absence of disease progression. We found that patients with higher trough levels of ipilimumab had better overall survival when grouped based on ipilimumab trough levels. Trough levels of ipilimumab were inversely associated with pretreatment serum levels of CXCL11, a predictive biomarker we previously identified, and soluble CD25 (sCD25), a prognostic biomarker for advanced melanoma, as well as C reactive protein (CRP) and interleukin (IL)-6 levels at week 7., Conclusions: Our results suggest that trough levels of ipilimumab may be a useful biomarker for the long-term survival of patients with advanced melanoma treated with ipilimumab. The association of ipilimumab trough levels with pretreatment serum levels of CXCL11 and sCD25 is suggestive of a baseline-driven E-R relationship, and the association of ipilimumab trough levels with on-treatment levels of CRP and IL-6 is suggestive of response-driven E-R relationship. Our findings highlight the potential utility of trough levels of ipilimumab as a biomarker., Trial Registration Number: NCT00495066., Competing Interests: Competing interests: YK: research support from Bristol Myers Squibb (BMS), GlaxoSmithKline, and Shimadzu. NI and TS: employees of Shimadzu Scientific Instruments. BDC: research support from AstraZeneca, Clinigen, and Galectin Therapeutics; patents/royalties: none; advisory boards: Clinigen, Nektar, and Merck. WJU: research support from BMS; royalties: data safety monitoring board for AstraZeneca. BDP: research support from Heat Biologics and Shimadzu; advisory boards: Bayer and UnitedHealthcare. WLR: research support from Galectin Therapeutics, BMS, GlaxoSmithKline, MiNA Therapeutics, Inhibrx, Veana Therapeutics, Aeglea Biotherapeutics, Shimadzu, OncoSec, and Calibr; patents/licensing fees: Galectin Therapeutics; advisory boards: Nektar Therapeutics and Vesselon., (© Author(s) (or their employer(s)) 2021. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.)

Metaplastic breast cancer is a rare and often chemo-refractory subtype of breast cancer with poor prognosis and limited treatment options. Recent studies have reported overexpression of programmed death ligand 1 (PD-L1) in metaplastic breast cancers, and there are several reports of anti-PD-1/L1 being potentially active in this disease. In this case series, we present 5 patients with metastatic metaplastic breast cancer treated with anti-PD-1-based therapy at a single center, with 3 of 5 cases demonstrating a response to therapy, and one of the responding cases being a metaplastic lobular carcinoma with low-level hormone receptor expression. Cases were evaluated for PD-L1 expression, tumor infiltrating lymphocytes (TILs), DNA mutations, RNA sequencing, and T-cell receptor sequencing. Duration of the response in these cases was limited, in contrast to the more durable responses noted in other recently published reports., Competing Interests: WR: Research support from Galectin Therapeutics, Bristol Myers Squibb (BMS), Merck, Tesaro/GlaxoSmithKline, MiNA Therapeutics, Inhibrx, Veana Therapeutics, Aeglea Biotherapeutics, Shimadzu, OncoSec, and Calibr. Patents/Royalties: Galectin Therapeutics. Advisory Boards: Nektar Therapeutics, Vesselon. DP: Received advisory board honoraria and institutional research funding support from Brooklyn ImmunoTherapeutics, Bristol-Myers Squibb, and Merck Laboratories. DP receives speaker bureau honoraria from Genentech and Novartis. Unrelated to this work, DP has received additional advisory board honoraria from other entities. RB: Consulting fees: Genomic Health, Astra Zeneca, Genentech; Institutional research funding: Seattle Genetics, Ichnos Biosciences, Merck; Honoraria for speakers bureau: Genentech, Seattle Genetics. HM: Consulting or Advisory Role - AstraZeneca; Bristol-Myers Squibb; Daiichi Sankyo; Genentech; Genomic Health; Immunomedics; Lilly; Merck; Pfizer; Puma Biotechnology; Puma Biotechnology; Seattle Genetics, Research Funding - Bristol-Myers Squibb (Inst); Lilly (Inst); Merck, (Inst); ZIOPHARM Oncology (Inst), Travel, Accommodations, Expenses - Amgen; AstraZeneca; Bristol-Myers Squibb; DAVA Pharmaceuticals; Genentech; Immunomedics; Lilly; Merck; Pfizer; Puma Biotechnology; Spectrum Pharmaceuticals, Other Relationship - Genomic Health; Lilly. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Kim, Rajamanickam, Bernard, Chun, Wu, Martel, Sun, Redmond, Sanchez, Basho, McArthur and Page.)

CTLA-4 blockade in combination with an agonist OX40-specific monoclonal antibody synergizes to augment antitumor immunity through enhanced T-cell effector function, leading to increased survival in preclinical cancer models. We have shown previously that anti-OX40/anti-CTLA-4 combination therapy synergistically enhances the expression of Eomesodermin (Eomes) in CD8 + T cells. Eomes is a critical transcription factor for the differentiation and memory function of CD8 + T cells. We hypothesized that Eomes hi CD8 + T cells were necessary for anti-OX40/anti-CTLA-4 immunotherapy efficacy and that further enhancement of this population would improve tumor-free survival. Indeed, CD8 + T cell-specific deletion of Eomes abrogated the efficacy of anti-OX40/anti-CTLA-4 therapy. We also found that anti-OX40/anti-CTLA-4-induced Eomes hi CD8 + T cells expressed lower levels of checkpoint receptors (PD1, Tim-3, and Lag-3) and higher levels of effector cytokines (IFNγ and TNFα) than their Eomes lo counterparts. Eomes expression is negatively regulated in T cells through interleukin-2-inducible T-cell kinase (ITK) signaling. We investigated the impact of modulating ITK signaling with ibrutinib, an FDA-approved tyrosine kinase inhibitor, and found that anti-OX40/anti-CTLA-4/ibrutinib therapy further enhanced CD8 + T cell-specific Eomes expression, leading to enhanced tumor regression and improved survival, both of which were associated with increased T-cell effector function across multiple tumor models. Taken together, these data demonstrate the potential of anti-OX40/anti-CTLA-4/ibrutinib as a triple therapy to improve the efficacy of immunotherapy., (©2021 American Association for Cancer Research.)

Metabolic dysregulation is a hallmark of cancer. Many tumors exhibit auxotrophy for various amino acids, such as arginine, because they are unable to meet the demand for these amino acids through endogenous production. This vulnerability can be exploited by employing therapeutic strategies that deplete systemic arginine in order to limit the growth and survival of arginine auxotrophic tumors. Pegzilarginase, a human arginase-1 enzyme engineered to have superior stability and enzymatic activity relative to the native human arginase-1 enzyme, depletes systemic arginine by converting it to ornithine and urea. Therapeutic administration of pegzilarginase in the setting of arginine auxotrophic tumors exerts direct antitumor activity by starving the tumor of exogenous arginine. We hypothesized that in addition to this direct effect, pegzilarginase treatment indirectly augments antitumor immunity through increased antigen presentation, thus making pegzilarginase a prime candidate for combination therapy with immuno-oncology (I-O) agents. Tumor-bearing mice (CT26, MC38, and MCA-205) receiving pegzilarginase in combination with anti-PD-L1 or agonist anti-OX40 experienced significantly increased survival relative to animals receiving I-O monotherapy. Combination pegzilarginase/immunotherapy induced robust antitumor immunity characterized by increased intratumoral effector CD8 + T cells and M1 polarization of tumor-associated macrophages. Our data suggest potential mechanisms of synergy between pegzilarginase and I-O agents that include increased intratumoral MHC expression on both antigen-presenting cells and tumor cells, and increased presence of M1-like antitumor macrophages. These data support the clinical evaluation of I-O agents in conjunction with pegzilarginase for the treatment of patients with cancer., (©2021 American Association for Cancer Research.)

Background: PD-1/PD-L1 engagement and overexpression of galectin-3 (Gal-3) are critical mechanisms of tumor-induced immune suppression that contribute to immunotherapy resistance. We hypothesized that Gal-3 blockade with belapectin (GR-MD-02) plus anti-PD-1 (pembrolizumab) would enhance tumor response in patients with metastatic melanoma (MM) and head and neck squamous cell carcinoma (HNSCC)., Methods: We performed a phase I dose escalation study of belapectin+pembrolizumab in patients with advanced MM or HNSCC (NCT02575404). Belapectin was administered at 2, 4, or 8 mg/kg IV 60 min before pembrolizumab (200 mg IV every 3 weeks for five cycles). Responding patients continued pembrolizumab monotherapy for up to 17 cycles. Main eligibility requirements were a functional Eastern Cooperative Oncology Group status of 0-2, measurable or assessable disease, and no active autoimmune disease. Prior T-cell checkpoint antibody therapy was permitted., Results: Objective response was observed in 50% of MM (7/14) and and 33% of HNSCC (2/6) patients. Belapectin+pembrolizumab was associated with fewer immune-mediated adverse events than anticipated with pembrolizumab monotherapy. There were no dose-limiting toxicities for belapectin within the dose range investigated. Significantly increased effector memory T-cell activation and reduced monocytic myeloid-derived suppressor cells (M-MDSCs) were observed in responders compared with non-responders. Increased baseline expression of Gal-3+ tumor cells and PD-1+CD8+ T cells in the periphery correlated with response as did higher serum trough levels of pembrolizumab., Conclusions: Belapectin+pembrolizumab therapy has activity in MM and HNSCC. Increased Gal-3 expression, expansion of effector memory T cells, and decreased M-MDSCs correlated with clinical response. Further investigation is planned., Competing Interests: Competing interests: WLR: research support from Galectin Therapeutics, Bristol Myers Squibb (BMS), Merck, Tesaro/GlaxoSmithKline, MiNA Therapeutics, Inhibrx, Veana Therapeutics, Aeglea Biotherapeutics, Shimadzu, OncoSec, and Calibr. Patents/licensing fees: Galectin Therapeutics. Advisory boards: Nektar Therapeutics, Vesselon. BC: research support from AstraZeneca, Clinigen, and Galectin Therapeutics. Advisory Boards: Replimmune, Clinigen, BMS, and Merck. Leidner: research support from BMS. Advisory boards: Merck, Oncolys, Sanofi, and Regeneron. YW: advisory boards: Roche, Amgen, Alexion, and AstraZeneca. NI and TS: employees of Shimadzu Scientific Instruments. Traber: stockholder of Galectin Therapeutics; employee and stockholder for Selecta Biosciences. EZ, JRH, and HS: employees and stockholders of Galectin Therapeutics. All other authors had no relevant disclosures., (© Author(s) (or their employer(s)) 2021. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.)

Treatment with an agonist anti-OX40 antibody (aOX40) boosts anti-tumor immunity by providing costimulation and driving effector T cell responses. However, tumor-induced immune suppression contributes significantly to poor response rates to aOX40 therapy, thus combining aOX40 with other agents that relieve tumor-mediated immune suppression may significantly improve outcomes. Once such target is galectin-3 (Gal-3), which drives tumor-induced immunosuppression by increasing macrophage infiltration and M2 polarization, restricting TCR signaling, and inducing T cell apoptosis. A wide-variety of tumors also upregulate Gal-3, which is associated with poor prognosis. Tumor-bearing (MCA-205 sarcoma, 4T1 mammary carcinoma, TRAMP-C1 prostate adenocarcinoma) mice were treated with a Gal-3 inhibitor (belapectin; GR-MD-02), aOX40, or combination therapy and the extent of tumor growth was determined. The phenotype and function of tumor-infiltrating lymphocytes was determined by flow cytometry, multiplex cytokine assay, and multiplex immunohistochemistry. Gal-3 inhibition synergized with aOX40 to promote tumor regression and increase survival. Specifically, aOX40/belapectin therapy significantly improved survival of tumor-bearing mice through a CD8 + T cell-dependent mechanism. Combination aOX40/belapectin therapy enhanced CD8 + T cell density within the tumor and reduced the frequency and proliferation of regulatory Foxp3 + CD4 + T cells. Further, aOX40/belapectin therapy significantly reduced monocytic MDSC (M-MDSCs) and MHC-II hi macrophage populations, both of which displayed reduced arginase 1 and increased iNOS. Combination aOX40/belapectin therapy alleviated M-MDSC-specific functional suppression compared to M-MDSCs isolated from untreated tumors. Our data suggests that Gal-3 inhibition plus aOX40 therapy reduces M-MDSC-meditated immune suppression thereby increasing CD8 + T cell recruitment leading to increased tumor regression and survival., (© 2021 The Author(s). Published with license by Taylor & Francis Group, LLC.)

The TSA Opal multiplex immunohistochemistry (mIHC) protocol (PerkinElmer) has been used to characterize immune infiltration in human cancers. This technique allows multiple biomarkers to be simultaneously stained in a single tissue section, which helps to elucidate the spatial relationship among individual cell types. We developed and optimized two improved mIHC protocols for a 7-color panel containing 6 biomarkers (CD3, CD8, CD163, PD-L1, FoxP3, and cytokeratin (CK)) and DAPI. The only difference between these two protocols was the staining sequence of those 6 biomarkers as the first sequence is PD-L1/CD163/CD8/CK/CD3/FoxP3/DAPI and the second sequence is FoxP3/CD163/CD8/CK/CD3/PD-L1/DAPI. By comparing PD-L1/FoxP3 staining in mIHC and singleplex PD-L1/FoxP3 staining on the adjacent slide, we demonstrated that the staining sequence does not affect the staining intensity of individual biomarkers as long as a proper antigen retrieval method was used. Our study suggests that use of an antigen retrieval buffer with higher pH value (such as Tris-EDTA pH9.0) than that of the stripping buffers (such as citrate buffer pH6.0) is helpful when using this advanced mIHC method to develop panels with multiple biomarkers. Otherwise, individual biomarkers may exhibit different intensities when the staining sequence is changed. By using this protocol, we characterized immune infiltration and PD-L1 expression in head and neck squamous cell carcinoma (HNSCC), breast cancer (BCa), and non-small cell lung cancer (NSCLC) specimens. We observed a statistically significant increase in CD3+ cell populations within the stroma of NSCLC as compared to BCa and increased PD-L1+ tumor cells in HNSCC as opposed to BCa., Competing Interests: WR has received research grants from Galectin Therapeutics, Bristol-Myers Squibb, Merck, GlaxoSmithKline, MiNA Therapeutics, Nektar Therapeutics, Inhibrx, Veana Therapeutics, Aeglea Biotherapeutics, Shimadzu, OncoSec, and Calibr; patents and licensing fees from Galectin Therapeutics; and has served on Scientific Advisory Boards for Nektar Therapeutics and Vesselon. ZS, RN, YW, and BB have no conflicts of interest to disclose. This does not alter our adherence to PLOS ONE policies on sharing data and materials.

Background: The H&E stromal tumor-infiltrating lymphocyte (sTIL) score and programmed death ligand 1 (PD-L1) SP142 immunohistochemistry assay are prognostic and predictive in early-stage breast cancer, but are operator-dependent and may have insufficient precision to characterize dynamic changes in sTILs/PD-L1 in the context of clinical research. We illustrate how multiplex immunofluorescence (mIF) combined with statistical modeling can be used to precisely estimate dynamic changes in sTIL score, PD-L1 expression, and other immune variables from a single paraffin-embedded slide, thus enabling comprehensive characterization of activity of novel immunotherapy agents., Methods: Serial tissue was obtained from a recent clinical trial evaluating loco-regional cytokine delivery as a strategy to promote immune cell infiltration and activation in breast tumors. Pre-treatment biopsies and post-treatment tumor resections were analyzed by mIF (PerkinElmer Vectra) using an antibody panel that characterized tumor cells (cytokeratin-positive), immune cells (CD3, CD8, CD163, FoxP3), and PD-L1 expression. mIF estimates of sTIL score and PD-L1 expression were compared to the H&E/SP142 clinical assays. Hierarchical linear modeling was utilized to compare pre- and post-treatment immune cell expression, account for correlation of time-dependent measurement, variation across high-powered magnification views within each subject, and variation between subjects. Simulation methods (Monte Carlo, bootstrapping) were used to evaluate the impact of model and tissue sample size on statistical power., Results: mIF estimates of sTIL and PD-L1 expression were strongly correlated with their respective clinical assays (p < .001). Hierarchical linear modeling resulted in more precise estimates of treatment-related increases in sTIL, PD-L1, and other metrics such as CD8+ tumor nest infiltration. Statistical precision was dependent on adequate tissue sampling, with at least 15 high-powered fields recommended per specimen. Compared to conventional t-testing of means, hierarchical linear modeling was associated with substantial reductions in enrollment size required (n = 25➔n = 13) to detect the observed increases in sTIL/PD-L1., Conclusion: mIF is useful for quantifying treatment-related dynamic changes in sTILs/PD-L1 and is concordant with clinical assays, but with greater precision. Hierarchical linear modeling can mitigate the effects of intratumoral heterogeneity on immune cell count estimations, allowing for more efficient detection of treatment-related pharmocodynamic effects in the context of clinical trials., Trial Registration: NCT02950259 .

CD8 + T cells are critical mediators of adaptive immunity, and enhancing their function can promote robust responses against invading pathogens and neoplastic cells. In addition to TCR stimulation, the provision of costimulation through ligation of TNFR family members, such as OX40 (CD134), provides essential signals driving T cell differentiation, survival, and memory in part through enhanced IL-2/IL-2R signaling. Interestingly, TCR stimulation in the presence of IL-2 upregulates intracellular expression of the β-galactoside binding protein, Galectin-3 (Gal-3). Gal-3 has been shown to regulate Th1/Th2 polarization of CD4 + T cells; however, the extent to which Gal-3 regulates the OX40/IL-2 signaling axis and CD8 + T cell proliferation, effector function, and/or survival is unknown. In this study, we demonstrate that murine Gal-3-deficient CD8 + T cells exhibited no defects in early (36 h) activation or proliferation following TCR stimulation. In contrast, Gal-3 -/- CD8 + T cells exhibited decreased survival and a reduced capacity to develop into memory cells following stimulation with cognate Ag plus agonist anti-OX40 mAb or IL-2 in vivo. Decreased survival of Gal-3 -/- T cells was associated with increased apoptosis and occurred in a cell-intrinsic manner. Together, these data implicate intracellular Gal-3 as a critical mediator of OX40-mediated CD8 + T cell survival and memory formation following Ag exposure., (Copyright © 2020 by The American Association of Immunologists, Inc.)

Background: Checkpoint inhibitors can induce profound anticancer responses, but programmed cell death protein-1 (PD-1) inhibition monotherapy has shown minimal activity in prostate cancer. A published report showed that men with prostate cancer who were resistant to the second-generation androgen receptor inhibitor enzalutamide had increased programmed death-ligand 1 (PD-L1) expression on circulating antigen-presenting cells. We hypothesized that the addition of PD-1 inhibition in these patients could induce a meaningful cancer response., Methods: We evaluated enzalutamide plus the PD-1 inhibitor pembrolizumab in a single-arm phase II study of 28 men with metastatic castration-resistant prostate cancer (mprogressing on enzalutamide alone. Pembrolizumab 200 mg intravenous was given every 3 weeks for four doses with enzalutamide. The primary endpoint was prostate-specific antigen (PSA) decline of ≥50%. Secondary endpoints were objective response, PSA progression-free survival (PFS), time to subsequent treatment, and time to death. Baseline tumor biopsies were obtained when feasible, and samples were sequenced and evaluated for the expression of PD-L1, microsatellite instability (MSI), mutational and neoepitope burdens., Results: Five (18%) of 28 patients had a PSA decline of ≥50%. Three (25%) of 12 patients with measurable disease at baseline achieved an objective response. Of the five responders, two continue with PSA and radiographic response after 39.3 and 37.8 months. For the entire cohort, median follow-up was 37 months, and median PSA PFS time was 3.8 months (95% CI: 2.8 to 9.9 months). Time to subsequent treatment was 7.21 months (95% CI: 5.1 to 11.1 months). Median overall survival for all patients was 21.9 months (95% CI: 14.7 to 28 .4 months), versus 41.7 months (95% CI: 22.16 to not reached (NR)) in the responders. Of the three responders with baseline biopsies, one had MSI high disease with mutations consistent with DNA-repair defects. None had detectable PD-L1 expression., Conclusions: Pembrolizumab has activity in mCRPC when added to enzalutamide. Responses were deep and durable and did not require tumor PD-L1 expression or DNA-repair defects., Trial Registration Number: clinicaltrials.gov (NCT02312557)., Competing Interests: Competing interests: JG has received research funding from Astellas/Medivation, Merck, Sanofi, Janssen Biotech, and travel support from Sanofi. CD has received research funding from Bristol Myers Squibb (BMS). He has received consulting fees from BMS, Merck, AstraZeneca (AZ) and Medimmune. He has patents licensed to AZ, BMS and Medimmune. WLR has received research grants, consulting fees, and/or royalties from Bristol-Myers Squibb, Merck, Galectin Therapeutics, and Nektar Therapeutics. TMB has research funding from Astellas/Medivation, Alliance Foundation Trials, Boehringer Ingelheim, Corcept Therapeutics, Endocyte Inc, Janssen Research & Development, OncoGenex, Sotio, and Theraclone Sciences/OncoResponse. TMB receives consulting fees from AbbVie, AstraZeneca, Astellas Pharma, Bayer, Boehringer Ingelheim, Clovis Oncology, GlaxoSmithKline, Janssen Biotech, Janssen Japan, Merck, and Pfizer and has stock ownership in Salarius Pharmaceuticals, and Arvinas Inc. JJA, GVT, and RS have no conflicts. RCB is a co-owner of Third Coast Therapeutics Inc, which has an option to license patents of which he is an owner. AEM has a sponsored research agreement with AstraZeneca and has received reagents from Genetech. JJA has received consulting fees from Merck., (© Author(s) (or their employer(s)) 2020. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.)

Background: High-dose radiotherapy (RT) is known to be immunogenic, but is rarely capable of driving clinically relevant abscopal antitumor immunity as monotherapy. RT is known to increase antigen presentation, type I/II interferon responses, and immune cell trafficking to irradiated tumors. Bempegaldesleukin (NKTR-214) is a CD122-preferential interleukin 2 (IL-2) pathway agonist that has been shown to increase tumor-infiltrating lymphocytes, T cell clonality, and increase PD-1 expression. NKTR-214 has increased drug half-life, decreased toxicity, and increased CD8 + T cell and natural killer cell stimulation compared with IL-2., Methods: Animals bearing bilateral subcutaneous MCA-205 fibrosarcoma or CT26 colorectal tumors were treated with NKTR-214, RT, or combination therapy, and tumor growth of irradiated and abscopal lesions was assessed. Focal RT was delivered using a small animal radiation research platform. Peripheral and tumor-infiltrating immune phenotype and functional analyses were performed by flow cytometry. RNA expression profiling from both irradiated and abscopal lesions was performed using microarray., Results: We demonstrate synergy between RT of a single tumor and NKTR-214 systemic therapy resulting in dramatically increased cure rates of mice bearing bilateral tumors compared with RT or NKTR-214 therapy alone. Combination therapy resulted in increased magnitude and effector function of tumor-specific CD8 + T cell responses and increased trafficking of these T cells to both irradiated and distant, unirradiated, tumors., Conclusions: Given the increasing role of hypofractionated and stereotactic body RT as standard of care treatments in the management of locally advanced and metastatic cancer, these data have important implications for future clinical trial development. The combination of RT and NKTR-214 therapy potently stimulates systemic antitumor immunity and should be evaluated for the treatment of patients with locally advanced and metastatic solid tumors., Competing Interests: Competing interests: DCR is a former employee and current shareholder of Nektar therapeutics, a consultant to Venrock Ventures and Third Rock Ventures, a shareholder of Maze Therapeutics, and on the scientific advisory board of Akrevia Inc. UH is an employee and shareholder of Nektar therapeutics. WLR has received research funding from Galectin Therapeutics, Bristol-Myers Squibb, AstraZeneca, Merck, Nektar Therapeutics, Tesaro/GSK, Aeglea BioTherapeutics, Shimadzu, Inhibrx, Veana Therapeutics, and MiNA Therapeutics, is on the advisory board of Vesselon, and receives royalties from Galectin Therapeutics. JMW is a consultant to Jounce Therapeutics and Novocure., (© Author(s) (or their employer(s)) 2020. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.)

Purpose: To evaluate the safety and feasibility of preoperative locoregional cytokine therapy (IRX-2 regimen) in early-stage breast cancer, and to evaluate for intratumoral and peripheral immunomodulatory activity., Patients and Methods: Sixteen patients with stage I-III early-stage breast cancer (any histology type) indicated for surgical lumpectomy or mastectomy were enrolled to receive preoperative locoregional immunotherapy with the IRX-2 cytokine biological (2 mL subcutaneous × 10 days to periareolar skin). The regimen also included single-dose cyclophosphamide (300 mg/m 2 ) on day 1 to deplete T-regulatory cells and oral indomethacin to modulate suppressive myeloid subpopulations. The primary objective was to evaluate feasibility (i.e., receipt of therapy without surgical delays or grade 3/4 treatment-related adverse events). The secondary objective was to evaluate changes in stromal tumor-infiltrating lymphocyte score. The exploratory objective was to identify candidate pharmacodynamic changes for future study using a variety of assays, including flow cytometry, RNA and T-cell receptor DNA sequencing, and multispectral immunofluorescence., Results: Preoperative locoregional cytokine administration was feasible in 100% ( n = 16/16) of subjects and associated with increases in stromal tumor-infiltrating lymphocytes ( P < 0.001). Programmed death ligand 1 (CD274) was upregulated at the RNA ( P < 0.01) and protein level [by Ventana PD-L1 (SP142) and immunofluorescence]. Other immunomodulatory effects included upregulation of RNA signatures of T-cell activation and recruitment and cyclophosphamide-related peripheral T-regulatory cell depletion., Conclusions: IRX-2 is safe in early-stage breast cancer. Potentially favorable immunomodulatory changes were observed, supporting further study of IRX-2 in early-stage breast cancer and other malignancies., (©2019 American Association for Cancer Research.)

Automated reagent preparation, sample processing, and data acquisition have increased the rate at which flow cytometry data can be generated. Furthermore, advances in technology and flow cytometry instrumentation continually increase the complexity and dimensionality of this data. Together, this leads to increased pressure on manual data analysis, which has inherent limitations including subjectivity of the analyst and the length of time needed for data processing. These issues can create bottlenecks in the data processing workflow and potentially compromise data quality. To address these issues, as well as the challenges associated with manual gating in a high-volume human immune profiling laboratory, we sought to implement an automated analysis pipeline. In this report, we discuss considerations for selecting an automated analysis method, the process of implementing an automated pipeline, and detail our successful incorporation of an automated gating strategy with flowDensity into our analysis workflow. This validated pipeline augments our laboratory's ability to provide rapid high-throughput immune profiling for patients participating in cancer immunotherapy clinical trials. © International Society for Advancement of Cytometry., (© International Society for Advancement of Cytometry.)

Prostate cancer is one of the most common cancers in men and a leading cause of cancer-related death. Recent advances in the treatment of advanced prostate cancer, including the use of more potent and selective inhibitors of the androgen signaling pathway, have provided significant clinical benefit for men with metastatic castration-resistant prostate cancer (mCRPC). However, most patients develop progressive lethal disease, highlighting the need for more effective treatments. One such approach is immunotherapy, which harness the power of the patient's immune system to identify and destroy cancer cells through the activation of cytotoxic CD8 T cells specific for tumor antigens. Although immunotherapy, particularly checkpoint blockade, can induce significant clinical responses in patients with solid tumors or hematological malignancies, minimal efficacy has been observed in men with mCRPC. In the current review, we discuss our current understanding of the immunological complexity of the immunosuppressive prostate cancer microenvironment, preclinical models of prostate cancer, and recent advances in immunotherapy clinical trials to improve outcomes for men with mCRPC.

Recent advancements in T-cell biology and antibody engineering have opened doors to significant improvements in cancer immunotherapy. Initial success with monoclonal antibodies targeting key receptors that inhibit T-cell function such as cytotoxic T lymphocyte antigen 4 (CTLA-4) and programmed death-ligand 1 (PD-1) have demonstrated the potency of this new class of therapy, highlighted by long-term complete responses for metastatic cancers once thought incurable. However, only a subset of patients responds to checkpoint blockade because of a multitude of factors, including an immunosuppressive tumor microenvironment and the mutational burden of the cancer. Novel antibodies, as well as ligand-immunoglobulin fusion proteins that target costimulatory immune receptors, are being developed and tested in clinical trials to further enhance the anti-tumor immune response. Many of these costimulatory receptors are in the tumor necrosis factor receptor superfamily (TNFRSF) and are expressed on multiple immune cell types, including inhibitory cells. While TNFRSFs signal through common pathways, the outcome of targeting different receptors depends on the functional status of the cell types expressing the relevant receptors. In this review, we discuss the current state of targeted costimulatory immunotherapy.

Background: Immunotherapy with PD-1 or PD-L1 blockade fails to induce a response in about 80% of patients with unselected non-small cell lung cancer (NSCLC), and many of those who do initially respond then develop resistance to treatment. Agonists that target the shared interleukin-2 (IL-2) and IL-15Rβγ pathway have induced complete and durable responses in some cancers, but no studies have been done to assess the safety or efficacy of these agonists in combination with anti-PD-1 immunotherapy. We aimed to define the safety, tolerability, and activity of this drug combination in patients with NSCLC., Methods: In this non-randomised, open-label, phase 1b trial, we enrolled patients (aged ≥18 years) with previously treated histologically or cytologically confirmed stage IIIB or IV NSCLC from three academic hospitals in the USA. Key eligibility criteria included measurable disease, eligibility to receive anti-PD-1 immunotherapy, and an Eastern Cooperative Oncology Group performance status of 0 or 1. Patients received the anti-PD-1 monoclonal antibody nivolumab intravenously at 3 mg/kg (then 240 mg when US Food and Drug Administration [FDA]-approved dosing changed) every 14 days (either as new treatment or continued treatment at the time of disease progression) and the IL-15 superagonist ALT-803 subcutaneously once per week on weeks 1-5 of four 6-week cycles for 6 months. ALT-803 was administered at one of four escalating dose concentrations: 6, 10, 15, or 20 μg/kg. The primary endpoint was to define safety and tolerability and to establish a recommended phase 2 dose of ALT-803 in combination with nivolumab. Analyses were per-protocol and included any patients who received at least one dose of study treatment. This trial is registered with ClinicalTrials.gov, number NCT02523469; phase 2 enrolment of patients is ongoing., Findings: Between Jan 18, 2016, and June 28, 2017, 23 patients were enrolled and 21 were treated at four dose levels of ALT-803 in combination with nivolumab. Two patients did not receive treatment because of the development of inter-current illness during enrolment, one patient due to leucopenia and one patient due to pulmonary dysfunction. No dose-limiting toxicities were recorded and the maximum tolerated dose was not reached. The most common adverse events were injection-site reactions (in 19 [90%] of 21 patients) and flu-like symptoms (15 [71%]). The most common grade 3 adverse events, occurring in two patients each, were lymphocytopenia and fatigue. A grade 3 myocardial infarction occurred in one patient. No grade 4 or 5 adverse events were recorded. The recommended phase 2 dose of ALT-803 is 20 μg/kg given once per week subcutaneously in combination with 240 mg intravenous nivolumab every 2 weeks., Interpretation: ALT-803 in combination with nivolumab can be safely administered in an outpatient setting. The promising clinical activity observed with the addition of ALT-803 to the regimen of patients with PD-1 monoclonal antibody relapsed and refractory disease shows evidence of anti-tumour activity for a new class of agents in NSCLC., Funding: Altor BioScience (a NantWorks company), National Institutes of Health, and Medical University of South Carolina Hollings Cancer Center., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

The efficacy of cancer immunotherapy is limited, in part, by the multitude of immunosuppressive mechanisms present within the tumor microenvironment (TME). Galectin-3 (Gal-3) is a lectin that contributes to TME immunosuppression and regulates diverse functions including cellular homeostasis and cancer biology. Increased Gal-3 expression during cancer progression augments tumor growth, invasiveness, metastatic potential, and immune suppression, which highlights the potential use of Gal-3 as a therapeutic target capable of modulating anti-tumor immunity. Here, we discuss the mechanisms by which Gal-3 regulates lymphocytes, the role of Gal-3 in lung and prostate tumors, and the contribution of Gal-3 to TME immunosuppression.

Cancer immunotherapy is now established as a powerful way to treat cancer. The recent clinical success of immune checkpoint blockade (antagonists of CTLA-4, PD-1 and PD-L1) highlights both the universal power of treating the immune system across tumour types and the unique features of cancer immunotherapy. Immune-related adverse events, atypical clinical response patterns, durable responses, and clear overall survival benefit distinguish cancer immunotherapy from cytotoxic cancer therapy. Combination immunotherapies that transform non-responders to responders are under rapid development. Current challenges facing the field include incorporating immunotherapy into adjuvant and neoadjuvant cancer therapy, refining dose, schedule and duration of treatment and developing novel surrogate endpoints that accurately capture overall survival benefit early in treatment. As the field rapidly evolves, we must prioritise the development of biomarkers to guide the use of immunotherapies in the most appropriate patients. Immunotherapy is already transforming cancer from a death sentence to a chronic disease for some patients. By making smart, evidence-based decisions in developing next generation immunotherapies, cancer should become an imminently treatable, curable and even preventable disease., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Numerous preclinical studies have demonstrated that combination immunotherapy can significantly reduce tumor growth and improve overall survival as compared to monotherapy. Furthermore, dual CTLA-4/PD-1 checkpoint blockade recently received FDA-approval for patients with metastatic melanoma, becoming the first combination immunotherapy to garner this designation in a rapidly evolving field. Despite this progress, the majority of patients do not respond to treatment, underscoring the critical need for more effective therapies. We have been investigating the mechanisms by which combination immunotherapy with an OX40 agonist plus CTLA-4 checkpoint blockade augments effector T cell responses to elicit anti-tumor immunity. Surprisingly, this approach failed to eradicate well-established tumors, in part due to the induction of anergy in cytotoxic CD8 + T cells. Further work revealed that anergic CD8 + T cells could be rescued by combining a dendritic cell-targeted vaccine with combination immunotherapy. Taken together, these data suggest that novel combinatorial immunotherapeutic strategies incorporating a vaccination strategy may be needed to generate effective anti-tumor responses in the majority of patients with metastatic disease.

While programmed cell death 1 (PD-1) inhibitors have shown clear anti-tumor efficacy in several solid tumors, prior results in men with metastatic castration resistant prostate cancer (mCRPC) showed no evidence of activity. Here we report unexpected antitumor activity seen in mCRPC patients treated with the anti-PD-1 antibody pembrolizumab. Patients with evidence of progression on enzalutamide were treated with pembrolizumab 200 mg IV every 3 weeks for 4 doses; pembrolizumab was added to standard dose enzalutamide. Three of the first ten patients enrolled in this ongoing phase II trial experienced rapid prostate specific antigen (PSA) reductions to ≤ 0.2 ng/ml. Two of these three patients had measurable disease upon study entry; both achieved a partial response. There were three patients with significant immune-related adverse events. One had grade 2 myositis, one had grade 3 hypothyroidism, and one had grade 2 hypothyroidism. None of these patients had a response. Two of the three responders had a baseline tumor biopsy. Immunohistochemistry from those biopsies showed the presence of CD3+, CD8+, and CD163+ leukocyte infiltrates and PD-L1 expression. Genetic analysis of the two responders revealed markers of microsatellite instability in one. The surprising and robust responses seen in this study should lead to re-examination of PD-1 inhibition in prostate cancer., Competing Interests: JNG has received honoraria from Astellas/Medivation. She has received research funding from Astellas/Medivation and Merck. CGD has received research funding from Bristol Myers Squibb (BMS). He has received consulting fees from BMS, Merck, Astra Zeneca (AZ) and Medimmune. He has patents licensed to AZ, BMS and Medimmune. WLR has received research grants, consulting fees, and/or royalties from Bristol-Myers Squibb, Merck, Galectin Therapeutics, and Nektar Therapeutics. TMB Research funding from Astellas and Medivation; consulting fees from Astellas. JJA, GVT, MF, JPC, FSE, RCB and RS have no conflicts. This research was funded by Merck Sharp & Dohme Corporation. Funds from the Bloomberg Kimmel Institute (BKI) supported a portion of the laboratory work. Additional laboratory funding was funded by Merck.

Immune checkpoint inhibitors are transforming the way cancer is treated. However, these therapies do not benefit all patients and frequently cause significant immune-related adverse events. Biomarkers that identify patients with a favorable early response to therapy are essential for guiding treatment decisions and improving patient outcomes. In this report of our study, we present evidence that shortly after administration of dual PD-1/CTLA-4 blockade, the proinflammatory capacity of peripheral lymphocytes is predictive of tumor progression and survival outcomes in multiple murine models. Specifically, we observed that the quantity of interferon-γ (IFNγ) produced by peripheral lymphocytes in response to CD3/CD28 stimulation was robustly correlated with subsequent survival outcomes. In the tumor models and early time points assessed in this study, this relationship was considerably more predictive than a host of other potential biomarkers, several of which have been previously reported. Overall, these findings suggest that measuring the capacity of peripheral lymphocytes to produce IFNγ may help identify which patients are benefitting from combination anti-PD-1/anti-CTLA-4 immunotherapy. Cancer Immunol Res; 4(8); 650-7. ©2016 AACR., (©2016 American Association for Cancer Research.)

Mounting an immune response sufficient to eradicate a tumor is the goal of modern immunotherapy. Single agent therapies with checkpoint inhibitors or costimulatory molecule agonists are effective only for a small portion of all treated patients. Combined therapy, e.g., CTLA-4 and PD-1 checkpoint blockade, is a more effective treatment modality, but in preclinical studies OX40 agonism with CTLA-4 blockade using monoclonal antibodies (aOX40/aCTLA-4) failed to induce tumor regression of larger, more established tumors. We hypothesized that administration of a vaccine with a tumor-associated antigen targeted to the appropriate antigen presenting cell could make combined aOX40/aCTLA-4 therapy more effective. We administered an antibody-based vaccine targeting HER2 to the DEC-205 endocytic receptor on cross-presenting dendritic cells (anti-DEC-205/HER2; aDEC-205/HER2) and a potent adjuvant (poly (I:C)) to assist with maturation, along with aOX40/aCTLA-4 therapy. This therapy induced complete regression of established tumors and a pronounced infiltration of effector CD8 and CD4 T cells, with no effect on regulatory T cell infiltration compared to aOX40/aCTLA-4 alone. To be maximally effective, this therapy required expression of both OX40 and CTLA-4 on CD8 T cells. These data indicate that vaccination targeting cross-presenting dendritic cells with a tumor-associated antigen is a highly effective immunization strategy that can overcome some of the limitations of current systemic immunotherapeutic approaches that lack defined tumor-directed antigenic targets.

Immunotherapy is gathering momentum as a primary therapy for cancer patients. However, monotherapies have limited efficacy in improving outcomes and benefit only a subset of patients. Combination therapies targeting multiple pathways can augment an immune response to improve survival further. Here, we demonstrate that dual aOX40 (anti-CD134)/aCTLA-4 (anti-cytotoxic T-lymphocyte-associated protein 4) immunotherapy generated a potent antigen-specific CD8 T-cell response, enhancing expansion, effector function, and memory T-cell persistence. Importantly, OX40 and CTLA-4 expression on CD8 T cells was critical for promoting their maximal expansion following combination therapy. Animals treated with combination therapy and vaccination using anti-DEC-205 (dendritic and epithelial cells, 205 kDa)-HER2 (human epidermal growth factor receptor 2) had significantly improved survival in a mammary carcinoma model. Vaccination with combination therapy uniquely restricted Th2-cytokine production by CD4 cells, relative to combination therapy alone, and enhanced IFNγ production by CD8 and CD4 cells. We observed an increase in MIP-1α (macrophage inflammatory protein-1α)/CCL3 [chemokine (C-C motif) ligand 3], MIP-1β/CCL4, RANTES (regulated on activation, normal T-cell expressed and excreted)/CCL5, and GM-CSF production by CD8 and CD4 T cells following treatment. Furthermore, this therapy was associated with extensive tumor destruction and T-cell infiltration into the tumor. Notably, in a spontaneous model of prostate adenocarcinoma, vaccination with combination therapy reversed anergy and enhanced the expansion and function of CD8 T cells recognizing a tumor-associated antigen. Collectively, these data demonstrate that the addition of a vaccine with combined aOX40/aCTLA-4 immunotherapy augmented antitumor CD8 T-cell function while limiting Th2 polarization in CD4 cells and improved overall survival.

Recent studies have highlighted the therapeutic efficacy of immunotherapy, a class of cancer treatments that utilize the patient's own immune system to destroy cancerous cells. Within a tumor the presence of a family of negative regulatory molecules, collectively known as "checkpoint inhibitors," can inhibit T cell function to suppress anti-tumor immunity. Checkpoint inhibitors, such as CTLA-4 and PD-1, attenuate T cell proliferation and cytokine production. Targeted blockade of CTLA-4 or PD-1 with antagonist monoclonal antibodies (mAbs) releases the "brakes" on T cells to boost anti-tumor immunity. Generating optimal "killer" CD8 T cell responses also requires T cell receptor activation plus co-stimulation, which can be provided through ligation of tumor necrosis factor receptor family members, including OX40 (CD134) and 4-1BB (CD137). OX40 is of particular interest as treatment with an activating (agonist) anti-OX40 mAb augments T cell differentiation and cytolytic function leading to enhanced anti-tumor immunity against a variety of tumors. When used as single agents, these drugs can induce potent clinical and immunologic responses in patients with metastatic disease. However, each of these agents only benefits a subset of patients, highlighting the critical need for more effective combinatorial therapeutic strategies. In this review, we will discuss our current understanding of the cellular and molecular mechanisms by which OX40 agonists synergize with checkpoint inhibitor blockade to augment T cell-mediated anti-tumor immunity and the potential opportunities for clinical translation of combinatorial immunotherapeutic strategies.

Background: Several members of the common gamma chain (gc) cytokine family are already approved (IL-2) or actively being developed as vaccine adjuvants and cancer immunotherapies. Studies have indicated that co-administration of gc cytokines may enhance the efficacy of immunotherapies that function via direct activation of co-stimulatory T cell receptors. To define the specific influence of gc cytokines on the co-stimulatory capacity of CD8(+) T cells and identify combinations with synergistic potential, we investigated the direct impact of gc cytokines on the differentiation and transcriptional profile of recently antigen-primed CD8(+) T cells., Methods: Naïve CD8(+) T cells were activated with peptide-pulsed APCs. After 48 hours, CD8(+) T cells were harvested and re-cultured in media supplemented with IL-2, IL-4, IL-7, IL-15 or IL-21. After 24 hours, cells were analyzed by cytokine bead array, flow cytometry, and mRNA micro-array. Gene networks responsible for specific CD8(+) T cell functions were constructed through literature-meta review and publicly available annotation databases. Gene expression data from the experimental groups was imported into this network to visualize the impact of each gc cytokine on the functional polarization of recently-activated CD8(+) T cells., Results: Among the gc cytokines, IL-2 induced the greatest increase in the expression of co-stimulatory receptors in recently-activated CD8(+) T cells. IL-2 increased significantly expression of 4-1BB, GITR, ICOS and OX40, at both the transcriptional and protein level. IL-2 also drove the greatest increase in cellular proliferation and the most robust shift towards a pro-survival phenotype, compared with the other gc cytokines. Both IL-4 and IL-21 enhanced expression of cytotoxic effector proteins, but drove distinct phenotypic polarizations, Th2/Tc2 and NK-like, respectively., Conclusions: Overall, these observations suggest that among gc cytokines, IL-2 may be uniquely capable of synergizing with therapeutic strategies that combine immunization with agonists of co-stimulatory T cell receptors. Previous studies have shown that the timing of IL-2 treatment relative to immunization plays a key role in defining the CD8(+) T cell response, and the findings from this study indicate that administration of exogenous IL-2 shortly after the initial antigen-priming event has concluded may augment the receptivity of these cells to subsequent TNFR co-stimulation.

It is becoming clear that combination strategies will be necessary to augment cancer immunotherapy. We report that combination anti-OX40/anti-CTLA-4 mAb immunotherapy improves survival by enhancing effector T cell expansion and function, even while inducing Th2 cytokine production. Furthermore, IL-4 blockade in addition to combination therapy significantly improved anti-tumor efficacy.

Ligation of the TNF receptor family costimulatory molecule OX40 (CD134) with an agonist anti-OX40 monoclonal antibody (mAb) enhances antitumor immunity by augmenting T-cell differentiation as well as turning off the suppressive activity of the FoxP3(+)CD4(+) regulatory T cells (Treg). In addition, antibody-mediated blockade of the checkpoint inhibitor CTLA-4 releases the "brakes" on T cells to augment tumor immunotherapy. However, monotherapy with these agents has limited therapeutic benefit particularly against poorly immunogenic murine tumors. Therefore, we examined whether the administration of agonist anti-OX40 therapy in the presence of CTLA-4 blockade would enhance tumor immunotherapy. Combined anti-OX40/anti-CTLA-4 immunotherapy significantly enhanced tumor regression and the survival of tumor-bearing hosts in a CD4 and CD8 T cell-dependent manner. Mechanistic studies revealed that the combination immunotherapy directed the expansion of effector T-bet(high)/Eomes(high) granzyme B(+) CD8 T cells. Dual immunotherapy also induced distinct populations of Th1 [interleukin (IL)-2, IFN-γ], and, surprisingly, Th2 (IL-4, IL-5, and IL-13) CD4 T cells exhibiting increased T-bet and Gata-3 expression. Furthermore, IL-4 blockade inhibited the Th2 response, while maintaining the Th1 CD4 and effector CD8 T cells that enhanced tumor-free survival. These data demonstrate that refining the global T-cell response during combination immunotherapy can further enhance the therapeutic efficacy of these agents., (©2013 AACR.)

Expansion of myeloid-lineage leukocytes in tumor-bearing mice has been proposed as a cause of systemic immunosuppression. We demonstrate that radiation therapy of tumors leads to a decline in myeloid cell numbers in the blood and a decrease in spleen size. The frequency of myeloid cells does not decline to the level seen in tumor-free mice: we demonstrate that metastatic disease can prevent myeloid cell numbers from returning to baseline, and that tumor recurrence from residual disease correlates with re-expansion of myeloid lineage cells. Radiation therapy results in increased proliferation of T cells in the spleen and while T cell responses to foreign antigens are not altered by tumor burden or myeloid cell expansion, responses to tumor-associated antigens are increased after radiation therapy. These data demonstrate that myeloid cell numbers are directly linked to primary tumor burden, that this population contracts following radiation therapy, and that radiation therapy may open a therapeutic window for immunotherapy of residual disease.

The Society for Immunotherapy of Cancer (SITC) 2012 Professional Development Session was held as part of the SITC 27th Annual Meeting, Washington, DC, on October 24, 2012. The session was designed as a new opportunity for early career investigators to learn about relevant career development topics in a didactic setting.

The provision of T cell co-stimulation via members of the TNFR super-family, including OX40 (CD134) and 4-1BB (CD137), provides critical signals that promote T cell survival and differentiation. Recent studies have demonstrated that ligation of OX40 can augment T cell-mediated anti-tumor immunity in pre-clinical models and more importantly, OX40 agonists are under clinical development for cancer immunotherapy. OX40 is of particular interest as a therapeutic target as it is not expressed on naïve T cells but rather, is transiently up-regulated following TCR stimulation. Although TCR engagement is necessary for inducing OX40 expression, the downstream signals that regulate OX40 itself remain unclear. In this study, we demonstrate that OX40 expression is regulated through a TCR and common gamma chain cytokine-dependent signaling cascade that requires JAK3-mediated activation of the downstream transcription factors STAT3 and STAT5. Furthermore, combined treatment with an agonist anti-OX40 mAb and IL-2 augmented tumor immunotherapy against multiple tumor types. Dual therapy was also able to restore the function of anergic tumor-reactive CD8 T cells in mice with long-term well-established (>5 wks) tumors, leading to increased survival of the tumor-bearing hosts. Together, these data reveal the ability of TCR/common gamma chain cytokine signaling to regulate OX40 expression and demonstrate a novel means of augmenting cancer immunotherapy by providing dual anti-OX40/common gamma chain cytokine-directed therapy.

The tumor recurrence from residual local or micrometastatic disease remains a problem in cancer therapy. In patients with soft tissue sarcoma and the patients with inoperable nonsmall cell lung cancer, local recurrence is common and significant mortality is caused by the subsequent emergence of metastatic disease. Thus, although the aim of the primary therapy is curative, the outcome may be improved by additional targeting of residual microscopic disease. We display in a murine model that surgical removal of a large primary sarcoma results in local recurrence in approximately 50% of animals. Depletion of CD8 T cells results in local recurrence in 100% of animals, indicating that these cells are involved in the control of residual disease. We further show that systemic adjuvant administration of αOX40 at surgery eliminates local recurrences. In this model, αOX40 acts to directly enhance tumor antigen-specific CD8 T-cell proliferation in the lymph node draining the surgical site, and results in increased tumor antigen-specific cytotoxicity in vivo. These results are also corroborated in a murine model of hypofractionated radiation therapy of lung cancer. Administration of αOX40 in combination with radiation significantly extended the survival compared with either agent alone, and resulted in a significant proportion of long-term tumor-free survivors. We conclude that αOX40 increases tumor antigen-specific CD8 T-cell cytotoxic activity resulting in improved endogenous immune control of residual microscopic disease, and we propose that adjuvant αOX40 administration may be a valuable addition to surgical and radiation therapy for cancer.

The existence of tumor-specific T cells, as well as their ability to be primed in cancer patients, confirms that the immune response can be deployed to combat cancer. However, there are obstacles that must be overcome to convert the ineffective immune response commonly found in the tumor environment to one that leads to sustained destruction of tumor. Members of the tumor necrosis factor (TNF) superfamily direct diverse immune functions. OX40 and its ligand, OX40L, are key TNF members that augment T-cell expansion, cytokine production, and survival. OX40 signaling also controls regulatory T-cell differentiation and suppressive function. Studies over the past decade have demonstrated that OX40 agonists enhance antitumor immunity in preclinical models using immunogenic tumors; however, treatment of poorly immunogenic tumors has been less successful. Combining strategies that prime tumor-specific T cells together with OX40 signaling could generate and maintain a therapeutic antitumor immune response., (Copyright © 2010 Elsevier Inc. All rights reserved.)

Tumor-specific CD8 T-cell peripheral tolerance occurs through clonal deletion, suppression, and the induction of anergy and can limit the generation of anti-tumor immunity. Several groups have demonstrated that prostate cancer can render tumor-specific CD8 T cells anergic, suggesting reversing tumor-induced anergy may greatly augment anti-tumor immunity. Recent work has demonstrated that signaling through the OX40 (CD134) co-stimulatory receptor, a member of the TNFR super-family, can augment CD4 and CD8 T-cell expansion, differentiation, and the generation of memory cells. However, whether OX40 ligation can reverse CD8 T-cell anergy, and more specifically, tumor-induced CD8 T-cell anergy, remains unclear. In the current study, we demonstrate that OX40 ligation can reverse CD8 T-cell anergy to a prostate-specific self-Ag in non-tumor-bearing hosts. Furthermore, OX40 engagement reversed tumor-specific CD8 T-cell anergy and restored the proliferative capacity of tumor-reactive CD8 T cells, which attenuated tumor growth and enhanced the survival of tumor-bearing hosts. These data demonstrate that OX40 ligation can rescue the function of anergic self- or tumor-reactive CD8 T cells in vivo and suggests that OX40-mediated therapy may provide a novel means of boosting anti-tumor immunity by restoring the responsiveness of previously anergic tumor-specific CD8 T cells.

The extent of T-cell activation, proliferation, and survival that follows T-cell receptor (TCR) ligation is controlled by several factors, including the strength of TCR stimulation, the availability of prosurvival cytokines, and the presence or absence of co-stimulatory signals. In addition to engagement of the CD28 co-stimulatory receptor by its natural ligands, B7.1 (CD80) and B7.2 (CD86), recent work has begun to elucidate the mechanisms by which signaling through the OX40 (CD134) co-stimulatory receptor, a member of the tumor necrosis factor receptor (TNFR) superfamily, affects T-cell responses. Importantly, OX40 ligation has been shown to augment CD4 and CD8 T-cell clonal expansion, effector differentiation, survival, and in some cases, abrogate the suppressive activity of regulatory FoxP3+CD25+CD4+ T cells. In this review, we focus on the mechanisms regulating OX40 expression on activated T cells as well as the role of OX40-mediated co-stimulation in boosting T-cell clonal expansion, effector differentiation, and survival.

Acquisition of full T-cell effector function and memory differentiation requires appropriate costimulatory signals, including ligation of the costimulatory molecule OX40 (TNFRSF4, CD134). Tumors often grow despite the presence of tumor-specific T cells and establish an environment with weak costimulation and immune suppression. Administration of OX40 agonists has been shown to significantly increase the survival of tumor-bearing mice and was dependent on the presence of both CD4 and CD8 T cells during tumor-specific priming. To understand how OX40 agonists work in mice with established tumors, we developed a model to study changes in immune cell populations within the tumor environment. We show here that systemic administration of OX40 agonist antibodies increased the proportion of CD8 T cells at the tumor site in three different tumor models. The function of the CD8 T cells at the tumor site was also increased by administration of OX40 agonist antibody, and we observed an increase in the proportion of antigen-specific CD8 T cells within the tumor. Despite decreases in the proportion of T regulatory cells at the tumor site, T regulatory cell function in the spleen was unaffected by OX40 agonist antibody therapy. Interestingly, administration of OX40 agonist antibody caused significant changes in the tumor stroma, including decreased macrophages, myeloid-derived suppressor cells, and decreased expression of transforming growth factor-beta. Thus, therapies targeting OX40 dramatically changed the tumor environment by enhancing the infiltration and function of CD8 T cells combined with diminished suppressive influences within the tumor.

The maintenance of T cell tolerance in the periphery proceeds through several mechanisms, including anergy, immuno-regulation, and deletion via apoptosis. We examined the mechanism underlying the induction of CD8 T cell peripheral tolerance to a self-Ag expressed on pancreatic islet beta-cells. Following adoptive transfer, Ag-specific clone 4 T cells underwent deletion independently of extrinsic death receptors, including Fas, TNFR1, or TNFR2. Additional experiments revealed that the induction of clone 4 T cell apoptosis during peripheral tolerance occurred via an intrinsic death pathway that could be inhibited by overexpression of Bcl-2 or targeted deletion of the proapoptotic molecule, Bim, thereby resulting in accumulation of activated clone 4 T cells. Over-expression of Bcl-2 in clone 4 T cells promoted the development of effector function and insulitis whereas Bim-/- clone 4 cells were not autoaggressive. Examination of the upstream molecular mechanisms contributing to clone 4 T cell apoptosis revealed that it proceeded in a p53, E2F1, and E2F2-independent manner. Taken together, these data reveal that initiation of clone 4 T cell apoptosis during the induction of peripheral tolerance to a cross-presented self-Ag occurs through a Bcl-2-sensitive and at least partially Bim-dependent mechanism.