Challenges and opportunities in the development of combination immunotherapy with OX40 agonists.

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.

A phase Ib trial of pembrolizumab plus paclitaxel or flat-dose capecitabine in 1st/2nd line metastatic triple-negative breast cancer.

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/m2 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.

Characterization of murine lymphocyte activation and exhaustion markers by a 14-color flow cytometry panel.

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.

Flow cytometry is a technique that allows researchers to analyze protein expression on single cells through the detection of fluorescence markers that is widely used to assess immune cell phenotypes. The selection of target proteins expressed on specific cell subsets and accompanying fluorophores is known as 'panel building' and is critically important for accurate flow cytometry results. However, there is a lack of optimized and reproducible panels for mouse models of cancer. This article describes the development and performance of a robust panel that characterizes immune cell diversity and activation, while leaving room for customization. This flow cytometry panel provides a starting point for exploring the spectrum of mouse lymphocyte activation and is adaptable for subsequent studies.

Enhancement of anti-tumor efficacy of immune checkpoint blockade by alpha-TEA.

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.

A Novel Class of On-Treatment Cancer Immunotherapy Biomarker: Trough Levels of Antibody Therapeutics in Peripheral Blood.

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.

Galunisertib plus neoadjuvant chemoradiotherapy in patients with locally advanced rectal cancer: a single-arm, phase 2 trial.

BACKGROUND: TGF-ß is an immunosuppressive cytokine that is upregulated in colorectal cancer. TGF-ß blockade improved response to chemoradiotherapy in preclinical models of colorectal adenocarcinoma. We aimed to test the hypothesis that adding the TGF-ß 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/m2 over 24 h daily 7 days per week during radiotherapy or oral capecitabine 825 mg/m2 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/m2 on day 1, intravenous fluorouracil 400 mg/m2 on day 1 then 2400 mg/m2 over 46 h, and intravenous oxaliplatin 85 mg/m2 on day 1 delivered every 2 weeks for eight cycles) or CAPEOX (intravenous oxaliplatin 130 mg/m2 on day 1 and oral capecitabine 1000 mg/m2 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.

A rapid and universal liquid chromatograph-mass spectrometry-based platform, refmAb-Q nSMOL, for monitoring monoclonal antibody therapeutics.

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.

Combining bempegaldesleukin (CD122-preferential IL-2 pathway agonist) and NKTR-262 (TLR7/8 agonist) improves systemic antitumor CD8+ T cell cytotoxicity over BEMPEG+RT.

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).

Current Clinical Trial Landscape of OX40 Agonists.

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.

Intratumoral Electroporation of Plasmid Encoded IL12 and Membrane-Anchored Anti-CD3 Increases Systemic Tumor Immunity.

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.

Changes in T-cell subsets and clonal repertoire during chemoimmunotherapy with pembrolizumab and paclitaxel or capecitabine for metastatic triple-negative breast cancer.

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.

Trough levels of ipilimumab in serum as a potential biomarker of clinical outcomes for patients with advanced melanoma after treatment with ipilimumab.

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.

A Case Series of Metastatic Metaplastic Breast Carcinoma Treated With Anti-PD-1 Therapy.

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.

Enhancing clinical and immunological effects of anti-PD-1 with belapectin, a galectin-3 inhibitor.

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.

Galectin-3 inhibition with belapectin combined with anti-OX40 therapy reprograms the tumor microenvironment to favor anti-tumor immunity.

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-IIhi 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.

Developing an enhanced 7-color multiplex IHC protocol to dissect immune infiltration in human cancers.

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.

Enhancing the Generation of Eomeshi CD8+ T Cells Augments the Efficacy of OX40- and CTLA-4-Targeted Immunotherapy.

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 EomeshiCD8+ 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 EomeshiCD8+ 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 Eomeslo 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.

Arginase Therapy Combines Effectively with Immune Checkpoint Blockade or Agonist Anti-OX40 Immunotherapy to Control Tumor Growth.

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.

Multiplex immunofluorescence to measure dynamic changes in tumor-infiltrating lymphocytes and PD-L1 in early-stage breast cancer.

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 .

Intracellular Galectin-3 Is Essential for OX40-Mediated Memory CD8+ T Cell Development.

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 ß-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.