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Despite the success of programmed cell death-1 (PD-1) and PD-1 ligand (PD-L1) inhibitors in treating solid tumors, only a proportion of patients respond. Here, we describe a first-in-class bifunctional therapeutic molecule, STAR0602, that comprises an antibody targeting germline Vβ6 and Vβ10 T cell receptors (TCRs) fused to human interleukin-2 (IL-2) and simultaneously engages a nonclonal mode of TCR activation with costimulation to promote activation and expansion of αβ T cell subsets expressing distinct variable β (Vβ) TCR chains. In solution, STAR0602 binds IL-2 receptors in cis with Vβ6/Vβ10 TCRs on the same T cell, promoting expansion of human Vβ6 and Vβ10 CD4 + and CD8 + T cells that acquire an atypical central memory phenotype. Monotherapy with a mouse surrogate molecule induced durable tumor regression across six murine solid tumor models, including several refractory to anti-PD-1. Analysis of murine tumor-infiltrating lymphocyte (TIL) transcriptomes revealed that expanded Vβ T cells acquired a distinct effector memory phenotype with suppression of genes associated with T cell exhaustion and TCR signaling repression. Sequencing of TIL TCRs also revealed an increased T cell repertoire diversity within targeted Vβ T cell subsets, suggesting clonal revival of tumor T cell responses. These immunological and antitumor effects in mice were recapitulated in studies of STAR0602 in nonhuman primates and human ex vivo models, wherein STAR0602 boosted human antigen-specific T cell responses and killing of tumor organoids. Thus, STAR0602 represents a distinct class of T cell-activating molecules with the potential to deliver enhanced antitumor activity in checkpoint inhibitor-refractory settings.

Tumor biopsy is often not available or difficult to obtain in patients with solid tumors. Investigation of the peripheral immune system allows for in-depth and dynamic profiling of patient immune response prior to and over the course of treatment and disease. Phosphoflow cytometry is a flow cytometry‒based method to detect levels of phosphorylated proteins in single cells. This method can be applied to peripheral immune cells to determine responsiveness of signaling pathways in specific immune subsets to cytokine stimulation, improving on simply defining numbers of populations of cells based on cell surface markers. Here, we review studies using phosphoflow cytometry to (a) investigate signaling pathways in cancer patients' peripheral immune cells compared with healthy donors, (b) compare immune cell function in peripheral immune cells with the tumor microenvironment, (c) determine the effects of agents on the immune system, and (d) predict cancer patient response to treatment and outcome. In addition, we explore the use and potential of phosphoflow cytometry in preclinical cancer models. We believe this review is the first to provide a comprehensive summary of how phosphoflow cytometry can be applied in the field of cancer immunology, and demonstrates that this approach holds promise in exploring the mechanisms of response or resistance to immunotherapy both prior to and during the course of treatment. Additionally, it can help identify potential therapeutic avenues that can restore normal immune cell function and improve cancer patient outcome., (© 2023. Italian National Cancer Institute ‘Regina Elena’.)

With the rapid adoption of immunotherapy into clinical practice for HPV-associated malignancies, assessing tumor burden using "liquid biopsies" would further our understanding of clinical outcomes mediated by immunotherapy and allow for tailoring of treatment based on real-time tumor dynamics. In this review, we examine translational studies on peripheral surrogates of tumor burden derived from peripheral blood in HPV-associated malignancies, including levels and methylation of circulating tumor DNA (ctDNA), miRNA derived from extracellular vesicles, circulating tumor cells (CTCs), and HPV-specific antibodies and T cell responses. We review their utility as prognostic and predictive biomarkers of response to chemotherapy and radiation, with a focus on how they may inform and guide immunotherapies to treat locally advanced and metastatic HPV-associated malignancies. We also highlight unanswered questions that must be addressed to translate and integrate these peripheral tumor biomarkers into the clinic.

Background: Before 2018, there was no standard of care for non-metastatic (M0) castration resistant prostate cancer nmCRPC. Androgen receptor antagonists (ARAs) were commonly used sequentially nmCRPC., Methods: This was a multicenter, randomized clinical trial comparing the ARA flutamide+/-PROSTVAC, a pox viral vaccine targeting PSA that includes T-cell co-stimulatory molecules. Eligible men had negative CT and Tc99 bone scans, and rising PSA on ADT. Previous treatment with ARA was a stratification factor. Patients were also evaluated for antigen-specific immune responses using intracellular cytokine staining., Results: Thirty-three patients randomized to flutamide and 31 to flutamide+vaccine. The median age was 71.8 and 69.8 years, respectively. The median time to treatment failure after a median potential follow-up of 46.7 months was, 4.5 months (range 2-70) for flutamide alone vs. 6.9 months (2.5-40; P = .38) with flutamide+vaccine. Seven patients in each arm had a >50% PSA response. Antigen-specific responses were similar in both arms (58% of patients in flutamide alone and 56% in flutamide+vaccine). The treatments were well tolerated. The most common side effect > grade 2 was injection site reaction seen in 29/31 vaccine patients which were self-limiting., Conclusion: The combination of flutamide+PROSTVAC did not improve outcomes in men with nmCRPC compared with flutamide alone. (ClinicalTrials.gov Identifier: NCT00450463)., (Published by Oxford University Press 2023. This work is written by (a) US Government employee(s) and is in the public domain in the US.)

Background: In preclinical studies, combining M9241 (a novel immunocytokine containing interleukin (IL)-12 heterodimers) with avelumab (anti-programmed death ligand 1 antibody) resulted in additive or synergistic antitumor effects. We report dose-escalation and dose-expansion results from the phase Ib JAVELIN IL-12 trial investigating M9241 plus avelumab., Methods: In the dose-escalation part of JAVELIN IL-12 (NCT02994953), eligible patients had locally advanced or metastatic solid tumors; in the dose-expansion part, eligible patients had locally advanced or metastatic urothelial carcinoma (UC) that had progressed with first-line therapy. Patients received M9241 at 4, 8, 12, or 16.8 µg/kg every 4 weeks (Q4W) plus avelumab 10 mg/kg every 2 weeks (Q2W, dose levels (DLs) 1-4) or M9241 16.8 µg/kg Q4W plus avelumab 800 mg once a week for 12 weeks followed by Q2W (DL5/dose expansion). Primary endpoints for the dose-escalation part were adverse events (AEs) and dose-limiting toxicities (DLTs), and those for the dose-expansion part were confirmed best overall response (BOR) per investigator (Response Evaluation Criteria in Solid Tumors V.1.1) and safety. The dose-expansion part followed a two-stage design; 16 patients were enrolled and treated in stage 1 (single-arm part). A futility analysis based on BOR was planned to determine whether stage 2 (randomized controlled part) would be initiated., Results: At data cut-off, 36 patients had received M9241 plus avelumab in the dose-escalation part. All DLs were well tolerated; one DLT occurred at DL3 (grade 3 autoimmune hepatitis). The maximum-tolerated dose was not reached, and DL5 was declared the recommended phase II dose, considering an observed drug-drug interaction at DL4. Two patients with advanced bladder cancer (DL2 and DL4) had prolonged complete responses. In the dose-expansion part, no objective responses were recorded in the 16 patients with advanced UC; the study failed to meet the criterion (≥3 confirmed objective responses) to initiate stage 2. Any-grade treatment-related AEs occurred in 15 patients (93.8%), including grade ≥3 in 8 (50.0%); no treatment-related deaths occurred. Exposures for avelumab and M9241 concentrations were within expected ranges., Conclusions: M9241 plus avelumab was well tolerated at all DLs, including the dose-expansion part, with no new safety signals. However, the dose-expansion part did not meet the predefined efficacy criterion to proceed to stage 2., Competing Interests: Competing interests: JSt received institutional research funding from Bavarian Nordic, ImmunityBio, Merck, PDS Biotechnology, and Precigen. J-LD received honoraria from Bristol Myers Squibb, Janssen Oncology, and MSD; served as a consultant or advisor for Bristol Myers Squibb, Janssen Oncology, and Sanofi; and was a member of a speakers’ bureau for Astellas Pharma. MS served as a consultant or advisor for AbbVie, Adaptimmune, Agenus, Alligator Bioscience, Almac Diagnostics, Anaeropharma, Array BioPharma, AstraZeneca/MedImmune, BioNTech, Boehringer Ingelheim, Boston Pharmaceuticals, Bristol Myers Squibb, Chugai/Roche, Dragonfly Therapeutics, Genentech/Roche, Genmab, Genocea Biosciences, GI Innovation, Immunocore, Innate Pharma, Lilly, Modulate Pharma, Molecular Partners, Nektar, Newlink Genetics, Numab, Pieris Pharmaceuticals, Pierre Fabre, Seattle Genetics, SERVIER, Symphogen, Torque, Verastem, and Zelluna; owns stocks in Actym Therapeutics, Adaptive Biotechnologies, Amphivena, EvolveImmune Therapeutics, Intensity Therapeutics, Nextcure, and Torque; and had other relationships with CEC Oncology, Dava Oncology, Haymarket Media, and Physican’s Education Resource. AR received institutional research funding from Ipsen, Merck, and Pfizer; served as a consultant or advisor for AstraZeneca, Bristol Myers Squibb, Eisai, Ipsen, Merck, MSD, Novartis, Pfizer, and Roche; and received reimbursement for travel and accommodation expenses from AstraZeneca, Bristol Myers Squibb, Ipsen, Merck, MSD, Novartis, Pfizer, and Roche. MMar served as a consultant or advisor for Bristol Myers Squibb, Janssen Cilag, Ipsen, Merck, MSD, and Pfizer. RKP received research funding from Exelixis, Janssen Oncology, and Pharmacyclics; served as a consultant or advisor for AstraZeneca, Bayer, Blue Earth Diagnostics, Bristol Myers Squibb, Dendreon, Genomic Health, Jounce Therapeutics, Merck, Pfizer, and Sanofi; was a member of a speakers’ bureau for AstraZeneca, Dendreon, Genentech/Roche, Genomic Health, MSD, and Sanofi; and received reimbursement for travel and accommodation expenses from Genentech/Roche. TSG received research funding from Ferring and served as a consultant or advisor for Exelixis, Pfizer, and Merck. MMai received honoraria from Alfasigma, Amgen, AstraZeneca, Bristol Myers Squibb, Merck, MSD, Pierre Fabre, and Roche; served as a consultant or advisor for Alfasigma, AstraZeneca, Bristol Myers Squibb, Merck, MSD, Pierre Fabre, and Roche; owned patents, royalties, and other intellectual properties for ‘DNA hypomethylating agents for cancer therapy’; received reimbursement for travel and accommodation expenses from Alfasigma, Amgen, AstraZeneca, Bristol Myers Squibb, Merck, MSD, Pierre Fabre, and Roche; and owned stock in Theravance. LD had no competing interests. JSc received institutional research funding from Bavarian Nordic, ImmunityBio, Incyte, Merck, NextCure, PDS Biotechnology, and Precigen. RND received institutional research funding from Bavarian Nordic, ImmunityBio, Incyte, Merck, NextCure, PDS Biotechnology, and Precigen. Y-TT received institutional research funding from Bavarian Nordic, ImmunityBio, Incyte, Merck, NextCure, PDS Biotechnology, and Precigen. XW and YV are employed by EMD Serono Research & Development Institute, Inc., Billerica, MA, USA, an affiliate of Merck KGaA. FB, JSe, and AS are employed by Merck. SC was employed by Merck at the time of study. JLG received institutional research funding from Bavarian Nordic, ImmunityBio, Incyte, Janssen Oncology, and Merck., (© Author(s) (or their employer(s)) 2023. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.)

Background: There is an unmet clinical need for interventions to prevent disease progression in patients with localized prostate cancer on active surveillance (AS)., Objective: To determine the immunologic response to the PROSTVAC vaccine and the clinical indicators of disease progression in patients with localized prostate cancer on AS., Design, Setting, and Participants: This was a phase 2, double-blind, randomized controlled trial in 154 men with low- or intermediate-risk prostate cancer on AS., Intervention: Participants were randomized (2:1) to receive seven doses of subcutaneous PROSTVAC, a vaccinia/fowlpox viral vector-based immunotherapy containing a prostate-specific antigen (PSA) transgene and three T-cell co-stimulatory molecules, or an empty fowlpox vector (EV) over 140 d., Outcome Measurements and Statistical Analysis: The primary outcome was the change from baseline in CD4 and CD8 T-cell infiltration in biopsy tumor tissue. Key secondary outcomes were safety and changes in prostate biopsy tumor pathology, peripheral antigen-specific T cells, and serum PSA. Continuous variables were compared using nonparametric tests. Categorical variables were compared using Fisher's exact test., Results and Limitations: The PROSTVAC/EV vaccination was well tolerated. All except one participant completed the vaccination series. Changes in CD4 or CD8 density in biopsy tumor tissue did not differ between the PROSTVAC and EV arms. The proportions of patients with Gleason upgrading to grade group 3 after treatment was similar between the arms. There were no differences in postvaccination peripheral T-cell responses or the PSA change from baseline to 6-mo post-treatment follow-up between the groups., Conclusions: In this first-of-kind trial of immunotherapy in patients on AS for prostate cancer, PROSTVAC did not elicit more favorable prostate tissue or peripheral T-cell responses than the EV. There was no difference between the arms in clinicopathologic effects. Despite the null findings, this is the first study reporting the feasibility and acceptability of an immunotherapy intervention in the AS setting., Patient Summary: We looked at responses after an experimental prostate cancer vaccine in patients with prostate cancer on active surveillance (AS). Participants who received the vaccine did not show more favorable outcomes than those receiving the control. Despite these findings, this is the first report showing the feasibility and acceptability of immunotherapy for prostate cancer in patients on AS., (Copyright © 2022 European Association of Urology. All rights reserved.)

Background: NHS-IL12 is a first-in-class, recombinant fusion protein composed of the human monoclonal antibody NHS76 (binds exposed DNA/histones at sites of intratumoral necrosis) fused to 2 IL-12 heterodimers. The maximum tolerated dose (MTD) and recommended phase II dose (RP2D) of NHS-IL12 monotherapy given subcutaneously (SC) every 4 weeks was previously reported. The study was expanded to include a high-exposure cohort with NHS-IL12 SC every 2 weeks (q2w)., Methods: This single-arm, phase I trial evaluated NHS-IL12 12 µg/kg SC q2w or 16.8µg/kg SC q2w in patients with metastatic solid tumors. The primary endpoint was safety., Results: Using a 3+3 design, 13 patients with advanced cancer were enrolled and 12 were dose-limiting toxicity (DLT) evaluable. There was 1 DLT (Grade 3 aspartate transaminase/alanine transaminase [AST/ALT] elevation). Other grade 3 toxicities included: flu-like symptoms 1/13 (8%), decreased absolute lymphocyte count (ALC) 1/13 (8%), decreased white blood cell count (WBC) 1/13 (8%), but most adverse events reported were low grade and self-limiting grade. Fifty percent of evaluable patients (6/12) experienced stable disease (SD) with 42% (5/12) developing progressive disease (PD) at the first restaging., Conclusion: Biweekly NHS-IL12 was well tolerated in this small phase I study. Additional studies incorporating NHS-IL12 with other immunomodulating agents are underway. (ClinicalTrials.gov Identifier: NCT01417546)., (Published by Oxford University Press 2023.)

CV301, a poxviral-based vaccine, has been evaluated in a phase 1 clinical trial (NCT02840994) and shown to be safe and immunologically active (phase 1a). Preclinical data support a combination of CV301 with programmed death-1 inhibitors, which has been evaluated in the phase 1b part of this trial and is reported here. Patients with advanced nonsquamous non-small cell lung cancer (NSCLC) without actionable genomic alterations received two priming doses of modified vaccinia Ankara-BN-CV301 (MVA) 4 weeks apart, followed by boosting doses of fowlpox-CV301 (FPV) at increasing time intervals for a maximum of 17 doses in combination with nivolumab for cohort 1 (C1) and 15 doses in combination with pembrolizumab for cohort 2 (C2). The primary objective was evaluation of safety and tolerability. Between October 2017 and September 14, 2018, patients were enrolled (C1: 4; median age: 64 years). Mean treatment duration was 332 days in C1 and 289 days in C2. CTCAE ≥grade 3 adverse events (AEs) were observed in four (100%) patients in C1 and three (37.5%) patients in C2. There was one death on trial. Immune-related AEs (irAEs) fulfilling criteria for a dose-limiting toxicity included 1 case of pneumonitis. Among 11 evaluable patients, 1 (9%) had a complete response, 1 (9%) had a partial response and 9 (82%) had stable disease. We conclude that CV301 administered with PD-1 inhibitors is safe and clinically active in patients with advanced NSCLC. The frequency or severity of AEs is not increased, including irAEs for each component of the combination., (© 2022 UICC. This article has been contributed to by U.S. Government employees and their work is in the public domain in the USA.)

Immune checkpoint inhibitors (ICIs) have revolutionized the treatment of advanced cancers. However, activation of the immune system can occasionally cause life-threatening toxicity involving critical organs. Induction of immune-mediated toxicity is a significant concern for patients with thymic epithelial tumors (TETs) due to defects in immune tolerance. An increased risk of skeletal and cardiac muscle inflammation following treatment with ICIs is well recognized in patients with advanced TETs. However, uncommon musculoskeletal and rheumatic complications can also occur. The cases presented in this report highlight the spectrum of presentation of immune-mediated, joint-predominant musculoskeletal adverse events in patients with advanced TETs treated with ICIs, including polymyalgia rheumatica-like illness and inflammatory arthritis., (Published by Oxford University Press 2022.)

Purpose: Bintrafusp alfa is a bifunctional agent consisting of an anti-human PD-L1 antibody linked to two TGFβRII. It is designed to act both as a checkpoint inhibitor and to 'trap' TGFβ in the tumor microenvironment. Phase I and II clinical studies demonstrated clinical activity in patients with a range of human papillomavirus (HPV)-associated cancers. The purpose of the studies reported here was the interrogation of various aspects of the peripheral immunome in patients with HPV-associated cancers, both prior to and early in the treatment regimen of bintrafusp alfa to better understand the mode of action of the agent and to help define which patients are more likely to benefit from bintrafusp alfa treatment., Patients and Methods: The peripheral immunome of patients (n=65) with HPV + malignancies was analyzed both prior to treatment with bintrafusp alfa and day 14 post-treatment for levels and changes in (1) 158 different immune cell subsets, (2) multiple plasma soluble factors including analytes reflecting immune stimulatory and inhibitory status, (3) complete blood counts, and in a subset of patients (4) TCR diversity and (5) HPV-specific T-cell responses., Results: Interrogation of the peripheral immunome prior to bintrafusp alfa treatment revealed several factors that associated with clinical response, including (1) higher levels of sCD27:sCD40L ratios, (2) lower levels of TGFβ1 and 12 additional factors associated with tumor mesenchymalization, and (3) higher CD8 + T cell:MDSC ratios. Analysis at 2 weeks post bintrafusp alfa revealed that eventual clinical responders had fewer increases in IL-8 levels and the neutrophil to lymphocyte ratio, and higher levels of HPV-16 specific CD8 + T cells. This study also provided information concerning differences in the peripheral immunome for patients who were naïve versus refractory to prior checkpoint inhibition therapy. While preliminary, two multivariate models developed predicted clinical benefit with 76%-91% accuracy., Conclusions: These studies add insight into the mechanism of action of bintrafusp alfa and provide evidence that the interrogation of both cellular and soluble components of the peripheral immunome of patients with HPV-associated malignancies, either prior to or early in the therapeutic regimen, can provide information as to which patients are more likely to benefit with bintrafusp alfa therapy., Competing Interests: Competing interests: None declared., (© 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: Metformin may have anticancer effects that are independent of its hypoglycemic effects. Retrospective studies have shown that metformin use is associated with decreased incidence of prostate cancer and prostate cancer-specific mortality. Preclinical studies suggesting additive anticancer effects of combining metformin and bicalutamide prompted this clinical trial (NCT02614859)., Methods: This open-label, randomized, phase 2 trial enrolled non-diabetic patients with biochemically recurrent prostate cancer, a PSADT of 3-9 months, BMI > 25 and normal testosterone. Patients were randomized 1:2 to observation for an initial 8 weeks (Arm A) or metformin 1000 mg twice daily (Arm B). Bicalutamide 50 mg/day was added after 8 weeks to both arms. The primary objective was to evaluate the number of patients with undetectable PSA ( < 0.2 ng/mL) at the end of 32 weeks. Immune correlatives were assessed as exploratory endpoints., Results: A total of 29 patients were enrolled from March 2015 to January 2020. No difference was seen between the 2 arms in the proportion of patients with undetectable PSA. Modest PSA decrease ranging from 4% to 24% were seen in 40.0% (95% CI: 19.1-64.0%) of patients with metformin monotherapy, compared to 11.1% (95% CI: 0.3-48.3%) in the observation arm. Metformin monotherapy reduced PD-1 + NK cells, and increased NKG2D + NK cells. The combination of metformin and bicalutamide led to greater reductions in PD-1 expressing NK, CD4 + T, and CD8 + T-cell subsets compared to bicalutamide alone. The trial was stopped early due to predicted inability to achieve its primary endpoint., Conclusions: Although metformin plus bicalutamide was well tolerated, there was no improvement in rates of achieving undetectable PSA at 32 weeks. Metformin monotherapy induced modest PSA declines in 40% of patients after 8 weeks. Metformin, given alone and in combination with bicalutamide, displayed immune modifying effects, primarily within NK and T cells subsets., Trial Registration: Trial Registration Number: NCT02614859., (© 2022. This is a U.S. government work and not under copyright protection in the U.S.; foreign copyright protection may apply.)

Background: FOLFOX plus bevacizumab is a standard of care (SOC) for first-line treatment of microsatellite-stable metastatic colorectal cancer (MSS mCRC). This study randomized patients to SOC or SOC plus avelumab (anti-PD-L1) plus CEA-targeted vaccine., Methods: Patients with untreated MSS mCRC enrolled to a lead-in arm assessing safety of SOC + immuno-oncology agents (IO). Next, patients were randomized to SOC or SOC + IO. The primary endpoint was progression-free survival (PFS). Multiple immune parameters were analyzed., Results: Six patients enrolled to safety lead-in, 10 randomized to SOC, and 10 to SOC + IO. There was no difference in median PFS comparing SOC versus SOC + IO (8.8 months (95% CI: 3.3-17.0 months) versus 10.1 months (95% CI: 3.6-16.1 months), respectively; hazard ratio 1.061 [P = .91; 95% CI: 0.380-2.966]). The objective response rate was 50% in both arms. Of patients analyzed, most (8/11) who received SOC + IO developed multifunctional CD4+/CD8+ T-cell responses to cascade antigens MUC1 and/or brachyury, compared to 1/8 who received SOC alone (P = .020). We detected post-treatment changes in immune parameters that were distinct to the SOC and SOC + IO treatment arms. Accrual closed after an unplanned analysis predicted a low likelihood of meeting the primary endpoint., Conclusions: SOC + IO generated multifunctional MUC1- and brachyury-specific CD4+/CD8+ T cells despite concurrent chemotherapy. Although a tumor-directed immune response is necessary for T-cell-mediated antitumor activity, it was not sufficient to improve PFS. Adding agents that increase the number and function of effector cells may be required for clinical benefit., (Published by Oxford University Press 2022. This work is written by (a) US Government employee(s) and is in the public domain in the US.)

Background: MVA-BN-brachyury-TRICOM is a recombinant vector-based therapeutic cancer vaccine designed to induce an immune response against brachyury. Brachyury, a transcription factor overexpressed in advanced cancers, has been associated with treatment resistance, epithelial-to-mesenchymal transition, and metastatic potential. MVA-BN-brachyury-TRICOM has demonstrated immunogenicity and safety in previous clinical trials of subcutaneously administered vaccine. Preclinical studies have suggested that intravenous administration of therapeutic vaccines can induce superior CD8 + T cell responses, higher levels of systemic cytokine release, and stronger natural killer cell activation and proliferation. This is the first-in-human study of the intravenous administration of MVA-BN-brachyury-TRICOM., Methods: Between January 2020 and March 2021, 13 patients were treated on a phase 1, open-label, 3+3 design, dose-escalation study at the National Institutes of Health Clinical Center. The study population was adults with advanced solid tumors and was enriched for chordoma, a rare sarcoma of the notochord that overexpresses brachyury. Vaccine was administered intravenously at three DLs on days 1, 22, and 43. Blood samples were taken to assess drug pharmacokinetics and immune activation. Imaging was conducted at baseline, 1 month, and 3 months post-treatment. The primary endpoint was safety and tolerability as determined by the frequency of dose-limiting toxicities; a secondary endpoint was determination of the recommended phase 2 dose., Results: No dose-limiting toxicities were observed and no serious adverse events were attributed to the vaccine. Vaccine-related toxicities were consistent with class profile (ie, influenza-like symptoms). Cytokine release syndrome up to grade 2 was observed with no adverse outcomes. Dose-effect trend was observed for fever, chills/rigor, and hypotension. Efficacy analysis of objective response rate per RECIST 1.1 at the end of study showed one patient with a partial response, four with stable disease, and eight with progressive disease. Three patients with stable disease experienced clinical benefit in the form of improvement in pain. Immune correlatives showed T cell activation against brachyury and other tumor-associated cascade antigens., Conclusions: Intravenous administration of MVA-BN-brachyury-TRICOM vaccine was safe and tolerable. Maximum tolerated dose was not reached. The maximum administered dose was 10 9 infectious units every 3 weeks for three doses. This dose was selected as the recommended phase 2 dose., Trial Registration Number: NCT04134312., Competing Interests: Competing interests: BK: Bavarian Nordic (E). TA: Bavarian Nordic (E). CP-N: Bavarian Nordic (E). CH: Arcellx (E) (previously employed by Bavarian Nordic). The other authors indicate no financial relationships. (E) Employment., (© 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.)

Background: Recurrent respiratory papillomatosis (RRP) is a human papillomavirus (HPV) driven neoplastic disorder of the upper aerodigestive tract that causes significant morbidity and can lead to fatal airway obstruction. Prior clinical study demonstrated clinical benefit with the programmed death-ligand 1 (PD-L1) monoclonal antibody avelumab. Bintrafusp alpha is a bifunctional inhibitor of PD-L1 and transforming growth factor-beta (TGF-b) that has shown clinical activity in several cancer types., Methods: We conducted a phase II clinical trial evaluating bintrafusp alpha in adults with RRP. Papilloma samples before and after treatment with bintrafusp alpha were assessed for correlates of response with multiplex immunofluorescence as well as immunological and genomic analyses. Post hoc analyses of papilloma samples before and after treatment with avelumab were assessed for comparison., Results: Dual PD-L1/TGF-b inhibition failed to abrogate papilloma growth in most subjects and increased the frequency of clinically indicated interventions after treatment in four of eight subjects based on each subject's own historical control. TGF-b neutralization consistently decreased pSMAD3 and p21 and increased Ki67 expression within the basal layers of papillomas, indicating that TGF-b restrained proliferation. These alterations were not observed in papillomas treated with PD-L1 blockade alone. Dual PD-L1/TGF-b inhibition did not enhance anti-HPV immunity within papillomas beyond that observed with PD-L1 blockade. Genomic alterations in TGF-b superfamily genes were infrequent in papillomas and normal mucosa but present in a significant fraction of head and neck carcinomas., Conclusions: Intact TGF-b signaling restrains proliferation within papillomas, and the use of clinical agents that abrogate this pathway should be avoided in patients with RRP., Trial Registration Numbers: NCT03707587 and NCT02859454., Competing Interests: Competing interests: None., (© 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.)

Background: Brachyury is a transcription factor overexpressed in chordoma and is associated with chemotherapy resistance and epithelial-to-mesenchymal transition. GI-6301 is a recombinant, heat-killed Saccharomyces cerevisiae yeast-based vaccine targeting brachyury. A previous phase I trial of GI-6301 demonstrated a signal of clinical activity in chordomas. This trial evaluated synergistic effects of GI-6301 vaccine plus radiation., Materials and Methods: Adults with locally advanced, unresectable chordoma were treated on a randomized, placebo-controlled trial. Patients received three doses of GI-6301 (80 × 10 7 yeast cells) or placebo followed by radiation, followed by continued vaccine or placebo until progression. Primary endpoint was overall response rate, defined as a complete response (CR) or partial response (PR) in the irradiated tumor site at 24 months. Immune assays were conducted to evaluate immunogenicity., Results: Between May 2015 and September 2019, 24 patients enrolled on the first randomized phase II study in chordoma. There was one PR in each arm; no CRs were observed. Median progressive-free survival for vaccine and placebo arms was 20.6 months (95% confidence interval [CI], 5.7-37.5 months) and 25.9 months (95% CI, 9.2-30.8 months), respectively. Hazard ratio was 1.02 (95% CI, 0.38-2.71). Vaccine was well tolerated with no vaccine-related serious adverse events. Preexisting brachyury-specific T cells were detected in most patients in both arms. Most patients developed T-cell responses during therapy, with no difference between arms in frequency or magnitude of response., Conclusion: No difference in overall response rate was observed, leading to early discontinuation of this trial due to low conditional power to detect statistical difference at the planned end of accrual., Implications for Practice: Chordoma is a rare neoplasm lacking effective systemic therapies for advanced, unresectable disease. Lack of clinically actionable somatic mutations in chordoma makes development of targeted therapy quite challenging. While the combination of yeast-brachyury vaccine (GI-6301) and standard radiation therapy did not demonstrate synergistic antitumor effects, brachyury still remains a good target for developmental therapeutics in chordoma. Patients and their oncologists should consider early referral to centers with expertise in chordoma (or sarcoma) and encourage participation in clinical trials., (Published 2021. This article is a U.S. Government work and is in the public domain in the USA.)

Breast tumors commonly harbor low mutational burden, low PD-L1 expression, defective antigen processing/presentation, and an immunosuppressive tumor microenvironment (TME). In a malignancy mostly refractory to checkpoint blockade, there is an unmet clinical need for novel combination approaches that increase tumor immune infiltration and tumor control. Preclinical data have guided the development of this clinical trial combining 1) BN-Brachyury (a poxvirus vaccine platform encoding the tumor associated antigen brachyury), 2) bintrafusp alfa (a bifunctional protein composed of the extracellular domain of the TGF-βRII receptor (TGFβ "trap") fused to a human IgG1 anti-PD-L1), 3), entinostat (a class I histone deacetylase inhibitor), and 4) T-DM1 (ado-trastuzumab emtansine, a standard of care antibody-drug conjugate targeting HER2). We hypothesize that this tetratherapy will induce a robust immune response against HER2 + breast cancer with improved response rates through 1) expanding tumor antigen-specific effector T cells, natural killer cells, and immunostimulatory dendritic cells, 2) improving antigen presentation, and 3) decreasing inhibitory cytokines, regulatory T cells, and myeloid-derived suppressor cells. In an orthotopic HER2 + murine breast cancer model, tetratherapy induced high levels of antigen-specific T cell responses, tumor CD8 + T cell/Treg ratio, and augmented the presence of IFNγ- or TNFα-producing CD8 + T cells and IFNγ/TNFα bifunctional CD8 + T cells with increased cytokine production. Similar effects were observed in tumor CD4 + effector T cells. Based on this data, a phase 1b clinical trial evaluating the stepwise addition of BN-Brachyury, bintrafusp alfa, T-DM1 and entinostat in advanced breast cancer was designed. Arm 1 (TNBC) receives BN-Brachyury + bintrafusp alfa. Arm 2 (HER2 + ) receives T-DM1 + BN-Brachyury + bintrafusp alfa. After safety is established in Arm 2, Arm 3 (HER2 + ) will receive T-DM1 + BN-Brachyury + bintrafusp alfa + entinostat. Reimaging will occur every 2 cycles (1 cycle = 21 days). Arms 2 and 3 undergo research biopsies at baseline and after 2 cycles to evaluate changes within the TME. Peripheral immune responses will be evaluated. Co-primary objectives are response rate and safety. All arms employ a safety assessment in the initial six patients and a 2-stage Simon design for clinical efficacy (Arm 1 if ≥ three responses of eight then expand to 13 patients; Arms 2 and 3 if ≥ four responses of 14 then expand to 19 patients per arm). Secondary objectives include progression-free survival and changes in tumor infiltrating lymphocytes. Exploratory analyses include changes in peripheral immune cells and cytokines. To our knowledge, the combination of a vaccine, an anti-PD-L1 antibody, entinostat, and T-DM1 has not been previously evaluated in the preclinical or clinical setting. This trial (NCT04296942) is open at the National Cancer Institute (Bethesda, MD)., Competing Interests: CP is an inventor in NIH patents related to brachyury. 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 Gatti-Mays, Gameiro, Ozawa, Knudson, Hicks, Palena, Cordes, Steinberg, Francis, Karzai, Lipkowitz, Donahue, Jochems, Schlom and Gulley.)

While vaccines directed against the SARS-CoV-2 spike protein will have varying degrees of effectiveness in preventing SARS-CoV-2 infections, the severity of infection will be determined by multiple host factors including the ability of immune cells to lyse virus-infected cells. This review will discuss the complexity of both adaptive and innate immunomes and how a flow-based assay can detect up to 158 distinct cell subsets in the periphery. This assay has been employed to show the effect of age on differences in specific immune cell subsets, and the differences in the immunome between healthy donors and age-matched cancer patients. Also reviewed are the numerous soluble factors, in addition to cytokines, that may vary in the pathogenesis of SARS-CoV-2 infections and may also be employed to help define the effectiveness of a given vaccine or other antiviral agents. Various steroids have been employed in the management of autoimmune adverse events in cancer patients receiving immunotherapeutics and may be employed in the management of SARS-CoV-2 infections. The influence of steroids on multiple immune cells subsets will also be discussed., Competing Interests: Competing interests: None declared., (© 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.)

Background: Antitumor vaccines targeting tumor-associated antigens (TAAs) can generate antitumor immune response. A novel vaccine platform using adenovirus 5 (Ad5) vectors [E1-, E2b-] targeting three TAAs-prostate-specific antigen (PSA), brachyury, and MUC-1-has been developed. Both brachyury and the C-terminus of MUC-1 are overexpressed in metastatic castration-resistant prostate cancer (mCRPC) and have been shown to play an important role in resistance to chemotherapy, epithelial-mesenchymal transition, and metastasis. The transgenes for PSA, brachyury, and MUC-1 all contain epitope modifications for the expression of CD8+ T-cell enhancer agonist epitopes. We report here the first-in-human trial of this vaccine platform., Methods: Patients with mCRPC were given concurrently three vaccines targeting PSA, brachyury, and MUC-1 at 5×10 11 viral particles (VP) each, subcutaneously every 3 weeks for a maximum of three doses (dose de-escalation cohort), followed by a booster vaccine every 8 weeks for 1 year (dose-expansion cohort only). The primary objective was to determine the safety and the recommended phase II dose. Immune assays and clinical responses were evaluated., Results: Eighteen patients with mCRPC were enrolled between July 2018 and September 2019 and received at least one vaccination. Median PSA was 25.58 ng/mL (range, 0.65-1006 ng/mL). The vaccine was tolerable and safe, and no grade >3 treatment-related adverse events or dose-limiting toxicities (DLTs) were observed. One patient had a partial response, while five patients had confirmed PSA decline and five had stable disease for >6 months. Median progression-free survival was 22 weeks (95% CI: 19.1 to 34). Seventeen (100%) of 17 patients mounted T-cell responses to at least one TAA, whereras 8 (47%) of 17 patients mounted immune responses to all three TAAs. Multifunctional T-cell responses to PSA, MUC-1, and brachyury were also detected after vaccination in the majority of the patients., Conclusions: Ad5 PSA/MUC-1/brachyury vaccine is well tolerated. The primary end points were met and there were no DLTs. The recommended phase II dose is 5×10 11  VP. The vaccine demonstrated clinical activity, including one partial response and confirmed PSA responses in five patients. Three patients with prolonged PSA responses received palliative radiation therapy. Further research is needed to evaluate the clinical benefit and immunogenicity of this vaccine in combination with other immuno-oncology agents and/or palliative radiation therapy., Trial Registration Number: NCT03481816., Competing Interests: Competing interests: ImmunityBio authors are employees of ImmunityBio, Inc., (© 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.)

Background: The standard treatment for non-metastatic castration sensitive prostate cancer (nmCSPC) is androgen deprivation therapy (ADT) or surveillance. This study evaluated the potential synergy of immunotherapy and enzalutamide (without ADT) in nmCSPC. In addition, the immunologic impact of enzalutamide was also evaluated in men with normal testosterone., Methods: Patients with rising prostate-specific antigen (PSA) after definitive therapy, normal testosterone and no radiographic metastasis were randomized to enzalutamide for 3 months with/without PROSTVAC for 6 months. Thereafter, patients could be retreated with another 3 month course of enzalutamide when PSA returned to baseline. Immune profiles were evaluated in these patients., Results: Thirty-eight patients were randomized with a median PSA=4.38 ng/dL and PSA doubling time=4.1 months. No difference was observed between the two groups for PSA growth kinetics, but PSA responses to enzalutamide were noteworthy regardless of PROSTVAC. The median PSA decline after short-course enzalutamide without ADT/testosterone lowering therapy was 99% in both courses. The median time to PSA recovery to baseline after each 84-day course of enzalutamide was also noteworthy because of the duration of response after enzalutamide was discontinued. After the first and second 3 month cycle of enzalutamide, PSA recovery to baseline took a median 224 (range 84-1246) and 189 days (78-400), respectively. The most common adverse events related to the enzalutamide were grade 1 fatigue (71%) and grade 1 breast pain/nipple tenderness (81%). The only grade 3 toxicity was aspartate aminotransferase (AST)/alanine aminotransferase (ALT) elevation in two patients. Enzalutamide was independently associated with immune changes, increasing natural killer cells, naïve-T cells, and decreasing myeloid-derived suppressor cells., Conclusions: Three months of enzalutamide without ADT induced substantial PSA control beyond the treatment period and was repeatable, perhaps representing an alternative to intermittent ADT in nmCSPC. In addition, enzalutamide was associated with immune changes that could be relevant as future immune combinations are developed. TRAIL REGISTRATION NUMBER: clinicaltrials.gov (NCT01875250)., Competing Interests: Competing interests: No, there are no competing interests., (© 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.)

Mesenchymal stem cell (MSC) therapy has been widely tested in clinical trials to promote healing post-myocardial infarction. However, low cell retention and the need for a large donor cell number in human studies remain a key challenge for clinical translation. Natural biomaterials such as gelatin are ideally suited as scaffolds to deliver and enhance cell engraftment after transplantation. A potential drawback of MSC encapsulation in the hydrogel is that the bulky matrix may limit their biological function and interaction with the surrounding tissue microenvironment that conveys important injury signals. To overcome this limitation, we adopted a gelatin methacrylate (gelMA) cell-coating technique that photocross-links gelatin on the individual cell surface at the nanoscale. The present study investigated the cardiac protection of gelMA coated, hypoxia preconditioned MSCs (gelMA-MSCs) in a murine myocardial infarction (MI) model. We demonstrate that the direct injection of gelMA-MSC results in significantly higher myocardial engraftment 7 days after MI compared to uncoated MSCs. GelMA-MSC further amplified MSC benefits resulting in enhanced cardioprotection as measured by cardiac function, scar size, and angiogenesis. Improved MSC cardiac retention also led to a greater cardiac immunomodulatory function after injury. Taken together, this study demonstrated the efficacy of gelMA-MSCs in treating cardiac injury with a promising potential to reduce the need for donor MSCs through enhanced myocardial engraftment.

Background: Bintrafusp alfa is a first-in-class bifunctional fusion protein composed of the extracellular domain of transforming growth factor (TGF)-βRII (a TGF-β 'trap') fused to a human IgG1 mAb blocking programmed cell death ligand 1. This is the largest analysis of patients with advanced, pretreated human papillomavirus (HPV)-associated malignancies treated with bintrafusp alfa., Methods: In these phase 1 (NCT02517398) and phase 2 trials (NCT03427411), 59 patients with advanced, pretreated, checkpoint inhibitor-naive HPV-associated cancers received bintrafusp alfa intravenously every 2 weeks until progressive disease, unacceptable toxicity, or withdrawal. Primary endpoint was best overall response per Response Evaluation Criteria in Solid Tumors (RECIST) V.1.1; other endpoints included safety., Results: As of April 17, 2019 (phase 1), and October 4, 2019 (phase 2), the confirmed objective response rate per RECIST V.1.1 in the checkpoint inhibitor-naive, full-analysis population was 30.5% (95% CI, 19.2% to 43.9%; five complete responses); eight patients had stable disease (disease control rate, 44.1% (95% CI, 31.2% to 57.6%)). In addition, three patients experienced a delayed partial response after initial disease progression, for a total clinical response rate of 35.6% (95% CI, 23.6% to 49.1%). An additional patient with vulvar cancer had an unconfirmed response. Forty-nine patients (83.1%) experienced treatment-related adverse events, which were grade 3/4 in 16 patients (27.1%). No treatment-related deaths occurred., Conclusion: Bintrafusp alfa showed clinical activity and manageable safety and is a promising treatment in HPV-associated cancers. These findings support further investigation of bintrafusp alfa in patients with advanced, pretreated HPV-associated cancers., Competing Interests: Competing interests: JStrauss, CSH, and JLG disclose inventorship on a National Institutes of Health patent related to the work. JSchlom and JLG disclose that the National Cancer Institute has a Cooperative Research and Development Agreement (CRADA) with EMD Serono Research & Development Institute; an affiliate of Merck KGaA, Darmstadt, Germany. BCC discloses research funding from Novartis, Bayer, AstraZeneca, MOGAM Institute, Dong-A ST, Champions Oncology, Janssen, Yuhan, Ono, Dizal Pharma, and Merck Sharp & Dohme; discloses consultancy with Novartis, AstraZeneca, Boehringer-Ingelheim, Roche, Bristol Myers Squibb, Ono, Yuhan, Pfizer, Eli Lilly, Janssen, Takeda, and Merck Sharp & Dohme; discloses stock ownership with TheraCanVac; and discloses royalty from Champions Oncology. CSH is an employee of Merck KGaA. LSO and PAR are employees of EMD Serono Research and Development Institute. CSH discloses CRADA with Kite Pharma., (© 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: The risk-benefit calculation for corticosteroid administration in the management of COVID-19 is complex and urgently requires data to inform the decision. The neutrophil-to-lymphocyte ratio (NLR) is a marker of systemic inflammation associated with poor prognosis in both COVID-19 and cancer. Investigating NLR as an inflammatory marker and lymphocyte levels as a critical component of antiviral immunity may inform the dilemma of reducing toxic hyperinflammation while still maintaining effective antiviral responses., Methods: We performed a retrospective analysis of NLR, absolute neutrophil counts (ANCs) and absolute lymphocyte counts (ALCs) in patients with cancer enrolled in immunotherapy trials who received moderate-dose to high-dose corticosteroids. We compared paired presteroid and available poststeroid initiation values daily during week 1 and again on day 14 using the Wilcoxon signed-rank test. Associated immune subsets by flow cytometry were included where available., Results: Patients (n=48) with a variety of solid tumors received prednisone, methylprednisolone, or dexamethasone alone or in combination in doses ranging from 20 to 190 mg/24 hours (prednisone equivalent). The median NLR prior to steroid administration was elevated at 5.0 (range: 0.9-61.2). The corresponding median ANC was 5.1 K/µL (range: 2.03-22.31 K/µL) and ALC was 1.03 K/µL (0.15-2.57 K/µL). One day after steroid administration, there was a significant transient drop in median ALC to 0.54 K/µL (p=0.0243), driving an increase in NLR (median 10.8, p=0.0306). Relative lymphopenia persisted through day 14 but was no longer statistically significant. ANC increased steadily over time, becoming significant at day 4 (median: 7.31 K/µL, p=0.0171) and remaining significantly elevated through day 14. NLR was consistently elevated after steroid initiation, significantly at days 1, 7 (median: 8.2, p=0.0272), and 14 (median: 15.0, p=0.0018). Flow cytometry data from 11 patients showed significant decreases in activated CD4 cells and effector memory CD8 cells., Conclusions: The early drop in ALC with persistent lymphopenia as well as the prolonged ANC elevation seen in response to corticosteroid administration are similar to trends associated with increased mortality in several coronavirus studies to include the current SARS-CoV-2 pandemic. The affected subsets are essential for effective antiviral immunity. This may have implications for glucocorticoid therapy for COVID-19., Competing Interests: Competing interests: The NCI has Collaborative Research and Development Agreements with a number of different companies, including EMD Serono, Bavarian Nordic, and Immunity Bio. These agreements provide resources for codevelopment of compounds., (© Author(s) (or their employer(s)) 2020. Re-use permitted under CC BY. Published by BMJ.)

It is important to develop vaccines that can also mediate T-cell responses to SARS-CoV-2 to limit severity of infections, and to analyze the cellular immunome in the use of anti-SARS-CoV-2 therapeutics., (Published by Oxford University Press for the Infectious Diseases Society of America 2020.)

Background: Although immune checkpoint inhibitors have revolutionized cancer treatment, clinical benefit with this class of agents has been limited to a subset of patients. Hence, more effective means to target tumor cells that express immune checkpoint molecules should be developed. For the first time, we report a novel natural killer (NK) cell line, programmed death-ligand 1 (PD-L1) targeting high-affinity natural killer (t-haNK), which was derived from NK-92 and was engineered to express high-affinity CD16, endoplasmic reticulum-retained interleukin (IL)-2, and a PD-L1-specific chimeric antigen receptor (CAR). We show that PD-L1 t-haNK cells also retained the expression of native NK receptors and carried a high content of granzyme and perforin granules., Methods: NanoString, flow cytometry, and immunofluorescence analyses were performed to characterize the phenotype of irradiated PD-L1 t-haNK cells. In vitro PD-L1 t-haNK cell activity against cancer cell lines and human peripheral blood mononuclear cells (PBMCs) was determined via flow-based and 111 In-release killing assays. The antitumor effect of PD-L1 t-haNK cells in vivo was investigated using MDA-MB-231, H460, and HTB1 xenograft models in NOD-scid IL2Rgamma null (NSG) mice. Additionally, the antitumor effect of PD-L1 t-haNK cells, in combination with anti-PD-1 and N-803, an IL-15 superagonist, was evaluated using mouse oral cancer 1 syngeneic model in C57BL/6 mice., Results: We show that PD-L1 t-haNK cells expressed PD-L1-targeting CAR and CD16, retained the expression of native NK receptors, and carried a high content of granzyme and perforin granules. In vitro, we demonstrate the ability of irradiated PD-L1 t-haNK cells to lyse 20 of the 20 human cancer cell lines tested, including triple negative breast cancer (TNBC) and lung, urogenital, and gastric cancer cells. The cytotoxicity of PD-L1 t-haNK cells was correlated to the PD-L1 expression of the tumor targets and can be improved by pretreating the targets with interferon (IFN)-γ. In vivo, irradiated PD-L1 t-haNK cells inhibited the growth of engrafted TNBC and lung and bladder tumors in NSG mice. The combination of PD-L1 t-haNK cells with N-803 and anti-PD-1 antibody resulted in superior tumor growth control of engrafted oral cavity squamous carcinoma tumors in C57BL/6 mice. In addition, when cocultured with human PBMCs, PD-L1 t-haNK cells preferentially lysed the myeloid-derived suppressor cell population but not other immune cell types., Conclusion: These studies demonstrate the antitumor efficacy of PD-L1 t-haNK cells and provide a rationale for the potential use of these cells in clinical studies., Competing Interests: Competing interests: JHL is an employee of NantKwest and ImmunityBio; RS is an employee of NantOmics and ImmunityBio; PS-S is a founder of NantKwest, NantOmics and ImmunityBio., (© 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: Clinical trials have shown the ability of therapeutic vaccines to generate immune responses to tumor-associated antigens (TAAs). What is relatively less known is if this translates into immune-cell (IC) infiltration into the tumor microenvironment. This study examined whether neoadjuvant prostate-specific antigen (PSA)-targeted vaccination with PROSTVAC could induce T-cell immunity, particularly at the tumor site., Methods: An open-label, phase II study of neoadjuvant PROSTVAC vaccine enrolled 27 patients with localized prostate cancer awaiting radical prostatectomy (RP). We evaluated increases in CD4 and CD8 T-cell infiltrates (RP tissue vs baseline biopsies) using a six-color multiplex immunofluorescence Opal method. Antigen-specific responses were assessed by intracellular cytokine staining after in vitro stimulation of peripheral blood mononuclear cells with overlapping 15-mer peptide pools encoding the TAAs PSA, brachyury and MUC-1., Results: Of 27 vaccinated patients, 26 had matched prevaccination (biopsy) and postvaccination (RP) prostate samples available for non-compartmentalized analysis (NCA) and compartmentalized analysis (CA). Tumor CD4 T-cell infiltrates were significantly increased in postvaccination RP specimens compared with baseline biopsies by NCA (median 176/mm² vs 152/mm²; IQR 136-317/mm² vs 69-284/mm²; p=0.0249; median ratio 1.20; IQR 0.64-2.25). By CA, an increase in both CD4 T-cell infiltrates at the tumor infiltrative margin (median 198/mm² vs 151/mm²; IQR 123-500/mm² vs 85-256/mm²; p=0.042; median ratio 1.44; IQR 0.59-4.17) and in CD8 T-cell infiltrates at the tumor core (median 140/mm² vs 105/mm²; IQR 91-175/mm² vs 83-163/mm²; p=0.036; median ratio 1.25; IQR 0.88-2.09) were noted in postvaccination RP specimens compared with baseline biopsies. A total of 13/25 patients (52%) developed peripheral T-cell responses to any of the three tested TAAs (non-neoantigens); five of these had responses to more than one antigen of the three evaluated., Conclusion: Neoadjuvant PROSTVAC can induce both tumor immune response and peripheral immune response., Trial Registration Number: NCT02153918., Competing Interests: Competing interests: The NCI has a collaborative research and development agreement (CRADA) with Bavarian Nordic, the manufacturer of the vaccine used in this study. Under this CRADA, resources to develop agents are provided, including the vaccine., (© 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.)

Immunosuppressive entities in the tumor microenvironment (TME) remain a major impediment to immunotherapeutic approaches for a majority of patients with cancer. While the immunosuppressive role of transforming growth factor-β (TGF-β) in the TME is well known, clinical studies to date with anti-TGF-β agents have led to limited success. The bifunctional agent bintrafusp alfa (previously designated M7824) has been developed in an attempt to address this issue. Bintrafusp alfa consists of an IgG 1 targeting programmed death ligand 1 (PD-L1) moiety fused via peptide linkers to the extracellular domain of two TGF-β receptor II molecules designed to 'trap' TGF-β in the TME. This agent is able to bring the TGF-β trap to the TME via its anti-PD-L1 component, thus simultaneously attacking both the immunosuppressive PD-L1 and TGF-β entities. A number of preclinical studies have shown bintrafusp alfa capable of (1) preventing or reverting TGF-β-induced epithelial-mesenchymal transition in human carcinoma cells; this alteration in tumor cell plasticity was shown to render human tumor cells more susceptible to immune-mediated attack as well as to several chemotherapeutic agents; (2) altering the phenotype of natural killer and T cells, thus enhancing their cytolytic ability against tumor cells; (3) mediating enhanced lysis of human tumor cells via the antibody-dependent cell-mediated cytotoxicity mechanism; (4) reducing the suppressive activity of T reg cells; (5) mediating antitumor activity in numerous preclinical models and (6) enhancing antitumor activity in combination with radiation, chemotherapy and several other immunotherapeutic agents. A phase I clinical trial demonstrated a safety profile similar to other programmed cell death protein 1 (PD-1)/PD-L1 checkpoint inhibitors, with objective and durable clinical responses. We summarize here preclinical and emerging clinical data in the use of this bispecific and potentially multifunctional agent., Competing Interests: Competing interests: None declared., (© 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.)

The Immunotherapy to Prevent Progression on Active Surveillance Study is the first trial of immunotherapy for localized prostate cancer. We randomized active surveillance patients to PSA-TRICOM (PROSTVAC) or placebo for 5mo. Final results will be available in 2019., (Copyright © 2018 European Association of Urology. All rights reserved.)

Purpose: Despite the strong interest in combining stereotactic ablative radiation therapy (SAR) with immunotherapy, limited data characterizing the systemic immune response after SAR are available. We hypothesized that the systemic immune response to SAR would differ by irradiated site owing to inherent differences in the microenvironment of various organs., Methods and Materials: Patients receiving SAR to any organ underwent prospective blood banking before and 1 to 2 weeks after SAR. Peripheral blood mononuclear cells (PBMCs) and serum were isolated. PBMCs were stained with fluorophore-conjugated antibodies against T and natural killer (NK) cell markers. Cells were interrogated by flow cytometry, and the results were analyzed using FlowJo software. Serum cytokine and chemokine levels were measured using Luminex. We analyzed the changes from before to after therapy using paired t tests or 1-way analysis of variance (ANOVA) with Bonferroni's post-test., Results: A total of 31 patients had evaluable PBMCs for flow cytometry and 37 had evaluable serum samples for Luminex analysis. The total number of NK cells and cytotoxic (CD56 dim CD16 + ) NK cells decreased (P = .02) and T-cell immunoglobulin- and mucin domain-containing molecule-3-positive (TIM3 + ) NK cells increased (P = .04) after SAR to parenchymal sites (lung and liver) but not to bone or brain. The total memory CD4 + T cells, activated inducible co-stimulator-positive and CD25 + CD4 + memory T cells, and activated CD25 + CD8 + memory T cells increased after SAR to parenchymal sites but not bone or brain. The circulating levels of tumor necrosis factor-α (P = .04) and multiple chemokines, including RANTES (P = .04), decreased after SAR to parenchymal sites but not bone or brain., Conclusions: Our data suggest SAR to parenchymal sites induces systemic immune changes, including a decrease in total and cytotoxic NK cells, an increase in TIM3 + NK cells, and an increase in activated memory CD4 + and CD8 + T cells. SAR to nonparenchymal sites did not induce these changes. By comparing the immune response after radiation to different organs, our data suggest SAR induces systemic immunologic changes that are dependent on the irradiated site., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Spinal manipulative therapy, including low-velocity variable-amplitude spinal manipulation (LVVA-SM), relieves chronic low back pain, especially in patients with neuropathic radiating leg pain following peripheral nervous system insult. Understanding the underlying analgesic mechanisms requires animal models. The aim of the current study was to develop an animal model for the analgesic actions of LVVA-SM in the setting of peripheral neuropathic pain. Adult male Sprague-Dawley rat sciatic nerve tibial and common peroneal branches were transected, sparing the sural branch (spared nerve injury, SNI). After 15-18 days, rats were assigned randomly to one of three groups (n=9 each group): LVVA-SM at 0.15-or 0.16-Hz or Control. LVVA-SM (20° flexion at the L5 vertebra with an innovative motorized treatment table) was administered in anesthetized rats for 10 min. Control rats were administered anesthesia and positioned on the treatment table. After 10, 25, and 40 min, the plantar skin of the hindpaw ipsilateral to SNI was tested for mechanical sensitivity (paw withdrawal threshold to a logarithmic series of Semmes-Weinstein monofilaments) and cold sensitivity (duration of paw lifting, shaking, and/or licking to topical acetone application). SNI produced behavioral signs of mechanical and cold allodynia. LVVA-SM reduced mechanical, but not cold, hypersensitivity compared with Control (0.15-Hz: P=0.04 at 10 min; 0.16-Hz: P<0.001 at 10 min, P=0.04 at 25 min). The analgesic effect of LVVA-SM in chronic low back pain patients with neuropathic leg pain can be reverse-translated to a rat model Video abstract: http://links.lww.com/WNR/A453.

Unlabelled: Thiazolidinedione drugs (TZDs) such as pioglitazone are approved by the U.S. Food and Drug Administration for the treatment of insulin resistance in type 2 diabetes. However, whether TZDs reduce painful diabetic neuropathy (PDN) remains unknown. Therefore, we tested the hypothesis that chronic administration of pioglitazone would reduce PDN in Zucker Diabetic Fatty (ZDF(fa/fa) [ZDF]) rats. Compared with Zucker Lean (ZL(fa/+)) controls, ZDF rats developed: (1) increased blood glucose, hemoglobin A1c, methylglyoxal, and insulin levels; (2) mechanical and thermal hyperalgesia in the hind paw; (3) increased avoidance of noxious mechanical probes in a mechanical conflict avoidance behavioral assay, to our knowledge, the first report of a measure of affective-motivational pain-like behavior in ZDF rats; and (4) exaggerated lumbar dorsal horn immunohistochemical expression of pressure-evoked phosphorylated extracellular signal-regulated kinase. Seven weeks of pioglitazone (30 mg/kg/d in food) reduced blood glucose, hemoglobin A1c, hyperalgesia, and phosphorylated extracellular signal-regulated kinase expression in ZDF. To our knowledge, this is the first report to reveal hyperalgesia and spinal sensitization in the same ZDF animals, both evoked by a noxious mechanical stimulus that reflects pressure pain frequently associated with clinical PDN. Because pioglitazone provides the combined benefit of reducing hyperglycemia, hyperalgesia, and central sensitization, we suggest that TZDs represent an attractive pharmacotherapy in patients with type 2 diabetes-associated pain., Perspective: To our knowledge, this is the first preclinical report to show that: (1) ZDF rats exhibit hyperalgesia and affective-motivational pain concurrent with central sensitization; and (2) pioglitazone reduces hyperalgesia and spinal sensitization to noxious mechanical stimulation within the same subjects. Further studies are needed to determine the anti-PDN effect of TZDs in humans., (Copyright © 2016 American Pain Society. Published by Elsevier Inc. All rights reserved.)

Importance: Previous phase 1 and 2 trials of PANVAC, a poxviral-based cancer vaccine, have suggested clinical efficacy in some patients with breast, ovarian, and colorectal cancer and have shown evidence of immunologic activity. Preclinical data have shown that docetaxel can modify tumor phenotype, making tumor cells more amenable to T cell-mediated killing., Objective: The goal of this study was to determine if the treatment combination of docetaxel and PANVAC improves clinical outcomes in patients with metastatic breast cancer compared with docetaxel treatment alone., Design, Setting, and Participants: Between May 2006 and February 2012, this open-label, phase 2 randomized clinical trial enrolled 48 patients with metastatic breast cancer of all subtypes, without limitation on other lines of previous therapy, to receive treatment with either docetaxel with PANVAC (arm A) or docetaxel alone (arm B). Final clinical data were collected on September 16, 2013. All patients were treated at either the National Cancer Institute or the Department of Breast Medical Oncology, MD Anderson Cancer Center., Main Outcomes and Measures: The primary end point was progression-free survival (PFS), using a phase 2.5 statistical design, with the intent of identifying a trend toward benefit (defined as 1-sided P≤.10) to guide a larger trial design. Secondary end points included safety and immunologic correlative studies., Results: Forty-eight participants were enrolled: 25 were randomized to the combination treatment arm A, and 23 to arm B. No patient remained in the study at the time of the final analysis. Patient and tumor characteristics were well matched. Analysis of adverse events in both treatment arms demonstrated very little difference between the 2 groups. In the combination treatment arm (arm A), statistically significant increases were noted in the frequency of grades 1 and 2 edema (P=.02, likely related to greater median number of docetaxel cycles) and injection-site reactions (P<.001). In the final data analysis, median PFS was 7.9 months in arm A vs 3.9 months in arm B (hazard ratio, 0.65 [95% CI, 0.34-1.14]; P=.09)., Conclusions and Relevance: The results suggest that the combination of PANVAC with docetaxel in metastatic breast cancer may provide a clinical benefit. This study was hypothesis generating and provides both rationale and statistical assumptions for a larger definitive randomized study., Trial Registration: clinicaltrials.gov Identifier: NCT00179309.

Combination therapy for the treatment of cancer is becoming increasingly essential as we gain improved understanding of the complexity of cancer progression and the mechanisms by which cancer cells become resistant to single-agent therapy. Recent studies, both clinical and preclinical, have suggested that immunotherapy is a promising approach to the treatment of cancer; however, strategies to improve its clinical efficacy are still needed. A number of recent studies have indicated that antiangiogenic tyrosine kinase inhibitors (TKIs) target multiple components of the tumor microenvironment and are an ideal class of agents for synergizing with cancer immunotherapy. TKIs are well known to modulate tumor endothelial cells, leading to vascular normalization; however, these agents have also been recently shown to decrease tumor compactness and tight junctions, thereby reducing solid tumor pressure and allowing for improved perfusion of collapsed vessels and increased tumor oxygenation. In addition, some TKIs are capable of inducing immunogenic modulation, whereby tumor cells are sensitized to killing by T lymphocytes. Moreover, a number of TKIs have been shown to be involved in immune subset conditioning, increasing the frequency and function of effector immune elements, while decreasing the number and function of immune suppressor cells. The alteration of the immune landscape, direct modification of tumor cells, and improved vascular perfusion leads to improved antitumor efficacy when antiangiogenic TKIs are combined with immunotherapy. Collectively, the data presented in this review support the clinical combination of multi-targeted antiangiogenic TKIs, including but not limited to cabozantinib, sunitinib, and sorafenib, as well as to other antiangiogenic therapies, such as the anti-VEGF antibody bevacizumab, with cancer vaccines for improved treatment of solid tumors.

Preclinical studies have demonstrated that the combination of systemic subcutaneous (s.c.) vaccination with intratumoral (i.t.) vaccination was superior in the induction of antitumor activity vs. vaccination with either route alone. A subsequent phase I study employing i.t.-s.c. vaccination was carried out in men with locally recurrent or progressive prostate cancer. rF-PSA-TRICOM (PROSTVAC) vaccine was administered intraprostatically and rV-PSA-TRICOM followed by rF-PSA-TRICOM vaccine was administered systemically. In that study no dose limiting toxicities were observed, 19/21 patients had stable or improved prostate-specific antigen (PSA) values and tumor-infiltrating lymphocytes (TILs) increased in post- vs. pre-treatment tumor biopsies, analyzed employing conventional immunohistochemistry (IHC). In the studies reported here, 31 phenotypes of peripheral blood mononuclear cells (PBMCs) were analyzed prevaccination and postvaccination as well as the functions of PBMC regulatory T cells (Tregs) and natural killer cells. A trend was observed in decreases in serum PSA with the reduction of circulating Tregs postvaccination. Digital IHC was employed prevaccination and postvaccination to measure CD4 and CD8 TILs, as well as Treg TILs by conventional IHC. Few correlations were observed with CD4, CD8 or Treg in TILs vs. PBMCs. However, patients with lower levels of CD4 TILs prevaccination showed the greatest increases in CD4 TILs postvaccine, while Treg TILs decreased postvaccine. There was also a strong correlation between decreases in serum PSA and increases in CD8 TILs postvaccine. These studies provide additional rationale for the use of i.t.-s.c. vaccinations and demonstrate a noncoordinate expression of specific immune subsets in PBMCs vs. tumor., (Published 2014. This article is a US Government work and, as such, is in the public domain of the United States of America.)

Recent studies have suggested that pan inhibitors of dipeptidyl peptidase-4 activity and/or structure homologs (DASH), including ARI-4175, can mediate tumor regression by immune-mediated mechanisms. This study assessed the potential of combining ARI-4175 with cancer vaccines. We evaluated ARI-4175's effect on immunogenic modulation, ability to sensitize tumor cells to antigen-specific CTL killing, effect on immune-cell subsets and function, and antitumor activity in 2 tumor models, both as a monotherapy and in combination with a recombinant viral or dendritic cell (DC)-based tumor-cell vaccine. ARI-4175's effects on the growth, surface phenotype, and antigen-specific CTL-mediated lysis of murine and human carcinoma cell lines were assessed in vitro. In vivo, C57BL-6 mice were treated orally with ARI-4175, after which splenocytes were assessed by flow cytometry and functional assays. Antitumor studies were performed in murine models of colon carcinoma (MC38-CEA(+) in CEA-transgenic C57BL-6 mice) and rhabdomyosarcoma (M3-9-M in C57BL-6 mice). Mice received oral ARI-4175 alone or in combination with a vaccine consisting of recombinant vaccinia/fowlpox CEA-TRICOM (colon model) or a DC-based tumor-cell vaccine (rhabdomyosarcoma model). Exposure to ARI-4175 had no effect on the proliferation or viability of carcinoma cells in vitro; however, it did alter tumor phenotype, making murine and human tumor cells more sensitive to antigen-specific CTL killing. Assessment of immune-cell subsets and function indicated that ARI-4175 increased levels of natural killer cells and DCs. Detrimental immune effects, including reduced T effector cells and increased immunosuppressive cells (Tregs, MDSCs), were normalized when treatment stopped, suggesting that scheduling is critical when combining this agent with vaccine. As a monotherapy, ARI-4175 had potent antitumor activity in both tumor models, and had even greater effects when combined with a vaccine (either DC-based or poxviral vector based). These findings provide the rationale for the combined use of cancer immunotherapy with DASH enzyme inhibitors such as ARI-4175., (Published by Elsevier Ltd.)