Phase 1 trial of CV301 in combination with anti-PD-1 therapy in nonsquamous non-small cell lung cancer.

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.

A Phase I Single-Arm Study of Biweekly NHS-IL12 in Patients With Metastatic Solid Tumors.

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).

Flutamide With or Without PROSTVAC in Non-metastatic Castration Resistant (M0) Prostate Cancer.

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).

Joint-Predominant Rheumatic Complications of Immune Checkpoint Inhibitor Therapy in Patients with Thymic Epithelial Tumors.

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.

A Randomized Phase II Trial of mFOLFOX6 + Bevacizumab Alone or with AdCEA Vaccine + Avelumab Immunotherapy for Untreated Metastatic Colorectal Cancer.

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.

Randomized, Double-Blind, Placebo-Controlled Phase II Study of Yeast-Brachyury Vaccine (GI-6301) in Combination with Standard-of-Care Radiotherapy in Locally Advanced, Unresectable Chordoma.

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 × 107 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.

The Importance of Cellular Immunity in the Development of Vaccines and Therapeutics for COVID-19.

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.

A Phase I Trial Using a Multitargeted Recombinant Adenovirus 5 (CEA/MUC1/Brachyury)-Based Immunotherapy Vaccine Regimen in Patients with Advanced Cancer.

LESSONS LEARNED: Concurrent ETBX-011, ETBX-051, and ETBX-061 can be safely administered to patients with advanced cancer. All patients developed CD4+ and/or CD8+ T-cell responses after vaccination to at least one tumor-associated antigen (TAA) encoded by the vaccine; 5/6 patients (83%) developed MUC1-specific T cells, 4/6 (67%) developed CEA-specific T cells, and 3/6 (50%) developed brachyury-specific T cells. The presence of adenovirus 5-neutralizing antibodies did not prevent the generation of TAA-specific T cells. BACKGROUND: A novel adenovirus-based vaccine targeting three human tumor-associated antigens-CEA, MUC1, and brachyury-has demonstrated antitumor cytolytic T-cell responses in preclinical animal models of cancer. METHODS: This open-label, phase I trial evaluated concurrent administration of three therapeutic vaccines (ETBX-011 = CEA, ETBX-061 = MUC1 and ETBX-051 = brachyury). All three vaccines used the same modified adenovirus 5 (Ad5) vector backbone and were administered at a single dose level (DL) of 5 × 1011 viral particles (VP) per vector. The vaccine regimen consisting of all three vaccines was given every 3 weeks for three doses then every 8 weeks for up to 1 year. Clinical and immune responses were evaluated. RESULTS: Ten patients enrolled on trial (DL1 = 6 with 4 in the DL1 expansion cohort). All treatment-related adverse events were temporary, self-limiting, grade 1/2 and included injection site reactions and flu-like symptoms. Antigen-specific T cells to MUC1, CEA, and/or brachyury were generated in all patients. There was no evidence of antigenic competition. The administration of the vaccine regimen produced stable disease as the best clinical response. CONCLUSION: Concurrent ETBX-011, ETBX-051, and ETBX-061 can be safely administered to patients with advanced cancer. Further studies of the vaccine regimen in combination with other agents, including immune checkpoint blockade, are planned.

Phase I Trial of a Modified Vaccinia Ankara Priming Vaccine Followed by a Fowlpox Virus Boosting Vaccine Modified to Express Brachyury and Costimulatory Molecules in Advanced Solid Tumors.

LESSONS LEARNED: Modified vaccinia Ankara-Bavarian Nordic (MVA-BN)-Brachyury followed by fowlpox virus-BN-Brachyury was well tolerated upon administration to patients with advanced cancer. Sixty-three percent of patients developed CD4+ and/or CD8+ T-cell responses to brachyury after vaccination. BN-Brachyury vaccine also induced T-cell responses against CEA and MUC1, which are cascade antigens, that is, antigens not encoded in the vaccines. BACKGROUND: Brachyury, a transcription factor, plays an integral role in the epithelial-mesenchymal transition, metastasis, and tumor resistance to chemotherapy. It is expressed in many tumor types, and rarely in normal tissues, making it an ideal immunologic target. Bavarian Nordic (BN)-Brachyury consists of vaccination with modified vaccinia Ankara (MVA) priming followed by fowlpox virus (FPV) boosting, each encoding transgenes for brachyury and costimulatory molecules. METHODS: Patients with metastatic solid tumors were treated with two monthly doses of MVA-brachyury s.c., 8 × 108 infectious units (IU), followed by FPV-brachyury s.c., 1 × 109 IU, for six monthly doses and then every 3 months for up to 2 years. The primary objective was to determine safety and tolerability. RESULTS: Eleven patients were enrolled from March 2018 to July 2018 (one patient was nonevaluable). No dose-limiting toxicities were observed. The most common treatment-related adverse event was grade 1/2 injection-site reaction observed in all patients. Best overall response was stable disease in six patients, and the 6-month progression-free survival rate was 50%. T cells against brachyury and cascade antigens CEA and MUC1 were detected in the majority of patients. CONCLUSION: BN-Brachyury vaccine is well tolerated and induces immune responses to brachyury and cascade antigens and demonstrates some evidence of clinical benefit.

Avelumab for metastatic or locally advanced previously treated solid tumours (JAVELIN Solid Tumor): a phase 1a, multicohort, dose-escalation trial.

BACKGROUND: Avelumab (MSB0010718C) is a human IgG1 monoclonal antibody that binds to PD-L1, inhibiting its binding to PD-1, which inactivates T cells. We aimed to establish the safety and pharmacokinetics of avelumab in patients with solid tumours while assessing biological correlatives for future development. METHODS: This open-label, single-centre, phase 1a, dose-escalation trial (part of the JAVELIN Solid Tumor trial) assessed four doses of avelumab (1 mg/kg, 3 mg/kg, 10 mg/kg, and 20 mg/kg), with dose-level cohort expansions to provide additional safety, pharmacokinetics, and target occupancy data. This study used a standard 3 + 3 cohort design and assigned patients sequentially at trial entry according to the 3 + 3 dose-escalation algorithm and depending on the number of dose-limiting toxicities during the first 3-week assessment period (the primary endpoint). Patient eligibility criteria included age 18 years or older, Eastern Cooperative Oncology Group performance status 0-1, metastatic or locally advanced previously treated solid tumours, and adequate end-organ function. Avelumab was given as a 1-h intravenous infusion every 2 weeks. Patients in the dose-limiting toxicity analysis set were assessed for the primary endpoint of dose-limiting toxicity, and all patients enrolled in the dose-escalation part were assessed for the secondary endpoints of safety (treatment-emergent and treatment-related adverse events according to National Cancer Institute Common Terminology Criteria for Adverse Events version 4.0), pharmacokinetic and pharmacodynamic profiles (immunological effects), best overall response by Response Evaluation Criteria, and antidrug antibody formation. The population for the pharmacokinetic analysis included a subset of patients with rich pharmacokinetic samples from two selected disease-specific expansion cohorts at the same study site who had serum samples obtained at multiple early timepoints. This trial is registered with ClinicalTrials.gov, number NCT01772004. Patient recruitment to the dose-escalation part reported here is closed. FINDINGS: Between Jan 31, 2013, and Oct 8, 2014, 53 patients were enrolled (four patients at 1 mg/kg, 13 at 3 mg/kg, 15 at 10 mg/kg, and 21 at 20 mg/kg). 18 patients were analysed in the dose-limiting toxicity analysis set: three at dose level 1 (1 mg/kg), three at dose level 2 (3 mg/kg), six at dose level 3 (10 mg/kg), and six at dose level 4 (20 mg/kg). Only one dose-limiting toxicity occurred, at the 20 mg/kg dose, and thus the maximum tolerated dose was not reached. In all 53 enrolled patients (the safety analysis set), common treatment-related adverse events (occurring in >10% of patients) included fatigue (21 patients [40%]), influenza-like symptoms (11 [21%]), fever (8 [15%]), and chills (6 [11%]). Grade 3-4 treatment-related adverse events occurred in nine (17%) of 53 patients, with autoimmune disorder (n=3), increased blood creatine phosphokinase (n=2), and increased aspartate aminotransferase (n=2) each occurring in more than one patient (autoimmune disorder in two patients at 10 mg/kg and one patient at 20 mg/kg, increased blood creatine phosphokinase in two patients at 20 mg/kg, and increased aspartate aminotransferase in one patient at 1 mg/kg, and one patient at 10 mg/kg). Six (11%) of 53 patients had a serious treatment-related adverse event: autoimmune disorder (two [13%]), lower abdominal pain (one [7%]), fatigue (one [7%]), and influenza-like illness (one [7%]) in three patients treated at 10 mg/kg dose level, and autoimmune disorder (one [5%]), increased amylase (one [5%]), myositis (one [5%]), and dysphonia (one [5%]) in three patients who received the 20 mg/kg dose. We recorded some evidence of clinical activity in various solid tumours, with partial confirmed or unconfirmed responses in four (8%) of 53 patients; 30 (57%) additional patients had stable disease. Pharmacokinetic analysis (n=86) showed a dose-proportional exposure between doses of 3 mg/kg and 20 mg/kg and a half-life of 95-99 h (3·9-4·1 days) at the 10 mg/kg and 20 mg/kg doses. Target occupancy was greater than 90% at doses of 3 mg/kg and 10 mg/kg. Antidrug antibodies were detected in two (4%) of 53 patients. No substantial differences were found in absolute lymphocyte count or multiple immune cell subsets, including those expressing PD-L1, after treatment with avelumab. 31 (58%) of 53 patients in the overall safety population died; no deaths were related to treatment on study. INTERPRETATION: Avelumab has an acceptable toxicity profile up to 20 mg/kg and the maximum tolerated dose was not reached. Based on pharmacokinetics, target occupancy, and immunological analysis, we chose 10 mg/kg every 2 weeks as the dose for further development and phase 3 trials are ongoing. FUNDING: National Cancer Institute and Merck KGaA.

A phase I study of recombinant (r) vaccinia-CEA(6D)-TRICOM and rFowlpox-CEA(6D)-TRICOM vaccines with GM-CSF and IFN-α-2b in patients with CEA-expressing carcinomas.

Prime-boost vaccination with recombinant (r) vaccinia(V)-CEA(6D)-TRICOM (triad of co-stimulatory molecules B7.1, ICAM-1 and LFA-3) and rFowlpox(F)-CEA(6D)-TRICOM infect antigen-presenting cells and direct expression of co-stimulatory molecules. We hypothesized that co-administration of vaccine with GM-CSF and interferon alpha (IFN-α) would have efficacy in CEA-expressing cancers. Patients with CEA-expressing cancers received the rV-CEA(6D)-TRICOM vaccine subcutaneously (s.c.) on day 1 followed by GM-CSF s.c. to the injection site on days 1-4. In Cycle 1, patients received thrice weekly s.c. injections of IFN-α-2b the week after rV-CEA(6D)-TRICOM. In Cycles 2-4, patients received thrice weekly s.c. injections of IFN-α-2b the same week that rF-CEA(6D)-TRICOM was given. The first cohort received no IFN followed by dose escalation of IFN-α in subsequent cohorts. Thirty-three patients were accrued (mean 59.8 years). Grade 3 toxicities included fatigue and hyperglycemia. Grade 4-5 adverse events (unrelated to treatment) were confusion (1), elevated aspartate transaminase (AST)/alanine transaminase (ALT) (1), and sudden death (1). No patients had a partial response, and eight patients exhibited stable disease of ≥3 months. Median progression-free survival and overall survival (OS) were 1.8 and 6.3 months, respectively. Significantly higher serum CD27 levels were observed after vaccine therapy (p = 0.006 post 1-2 cycles, p = 0.003 post 3 cycles, p = 0.03 post 4-7 cycles) and 42 % of patients assayed developed CEA-specific T cell responses. Pre-treatment levels of myeloid-derived suppressor cells correlated with overall survival (p = 0.04). Administration of IFN-α led to significantly increased OS (p = 0.02) compared to vaccine alone. While the vaccine regimen produced no clinical responses, IFN-α administration was associated with improved survival.

Pan-Bcl-2 inhibitor, GX15-070 (obatoclax), decreases human T regulatory lymphocytes while preserving effector T lymphocytes: a rationale for its use in combination immunotherapy.

Bcl-2 inhibitors are currently being evaluated in clinical studies for treatment of patients with solid tumors and hematopoietic malignancies. In this study we explored the potential for combining the pan-Bcl-2 inhibitor GX15-070 (GX15; obatoclax) with immunotherapeutic modalities. We evaluated the in vitro effects of GX15 on human T cell subsets obtained from PBMCs in terms of activation, memory, and suppressive function. Our results indicated that in healthy-donor PBMCs, mature-activated T cells were more resistant to GX15 than early-activated T cells, and that GX15 preserved memory but not non-memory T cell populations. Furthermore, GX15 increased the apoptosis of regulatory T cells (Tregs), profoundly downregulated FOXP3 and CTLA-4 in a dose-dependent manner, and decreased their suppressive function. Treating PBMCs obtained from ovarian cancer patients with GX15 also resulted in increased CD8(+):Treg and CD4(+):Treg ratios. These results support preclinical studies in which mice vaccinated before treatment with GX15 showed the greatest reduction in metastatic lung tumors as a result of increased apoptotic resistance of mature CD8(+) T cells and decreased Treg function brought about by GX15. Taken together, these findings suggest that when a Bcl-2 inhibitor is combined with active immunotherapy in humans, such as the use of a vaccine or immune checkpoint inhibitor, immunotherapy should precede administration of the Bcl-2 inhibitor to allow T cells to become mature, and thus resistant to the cytotoxic effects of the Bcl-2 inhibitor.

Identification by digital immunohistochemistry of intratumoral changes of immune infiltrates after vaccine in the absence of modifications of PBMC immune cell subsets.

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.

Dual effects of a targeted small-molecule inhibitor (cabozantinib) on immune-mediated killing of tumor cells and immune tumor microenvironment permissiveness when combined with a cancer vaccine.

BACKGROUND: Growing awareness of the complexity of carcinogenesis has made multimodal therapies for cancer increasingly compelling and relevant. In recent years, immunotherapy has gained acceptance as an active therapeutic approach to cancer treatment, even though cancer is widely considered an immunosuppressive disease. Combining immunotherapy with targeted agents that have immunomodulatory capabilities could significantly improve its efficacy. METHODS: We evaluated the ability of cabozantinib, a receptor tyrosine kinase inhibitor, to modulate the immune system in vivo as well as alter the phenotype of tumor cells in vitro in order to determine if this inhibitor could act synergistically with a cancer vaccine. RESULTS: Our studies indicated that cabozantinib altered the phenotype of MC38-CEA murine tumor cells, rendering them more sensitive to immune-mediated killing. Cabozantinib also altered the frequency of immune sub-populations in the periphery as well as in the tumor microenvironment, which generated a more permissive immune environment. When cabozantinib was combined with a poxviral-based cancer vaccine targeting a self-antigen, the combination significantly reduced the function of regulatory T cells and increased cytokine production from effector T cells in response to the antigen. These alterations to the immune landscape, along with direct modification of tumor cells, led to markedly improved antitumor efficacy. CONCLUSIONS: These studies support the clinical combination of cabozantinib with immunotherapy for the treatment of cancer.

Soluble immune checkpoints: implications for cancer prognosis and response to immune checkpoint therapy and conventional therapies.

Longitudinal sampling of tumor tissue from patients with solid cancers, aside from melanoma and a few other cases, is often unfeasible, and thus may not capture the plasticity of interactions between the tumor and immune system under selective pressure of a given therapy. Peripheral blood analyses provide salient information about the human peripheral immunome while offering technical and practical advantages over traditional tumor biopsies, and should be utilized where possible alongside interrogation of the tumor. Some common blood-based biomarkers used to study the immune response include immune cell subsets, circulating tumor DNA, and protein analytes such as cytokines. With the recent explosion of immune checkpoint inhibitors (ICI) as a modality of treatment in multiple cancer types, soluble immune checkpoints have become a relevant area of investigation for peripheral immune-based biomarkers. However, the exact functions of soluble immune checkpoints and their roles in cancer for the most part remain unclear. This review discusses current literature on the production, function, and expression of nine soluble immune checkpoints - sPD-L1, sPD-1, sCTLA4, sCD80, sTIM3, sLAG3, sB7-H3, sBTLA, and sHVEM - in patients with solid tumors, and explores their role as biomarkers of response to ICI as well as to conventional therapies (chemotherapy, radiotherapy, targeted therapy, and surgery) in cancer patients.

Blood-based biomarkers in patients with non-small cell lung cancer treated with immune checkpoint blockade.

The paradigm of non-small cell lung cancer (NSCLC) treatment has been profoundly influenced by the development of immune checkpoint inhibitors (ICI), but the range of clinical responses observed among patients poses significant challenges. To date, analyses of tumor biopsies are the only parameter used to guide prognosis to ICI therapy. Tumor biopsies, however, are often difficult to obtain and tissue-based biomarkers are limited by intratumoral heterogeneity and temporal variability. In response, there has been a growing emphasis on the development of "liquid biopsy"‒ derived biomarkers, which offer a minimally invasive means to dynamically monitor the immune status of NSCLC patients either before and/or during the course of treatment. Here we review studies in which multiple blood-based biomarkers encompassing circulating soluble analytes, immune cell subsets, circulating tumor DNA, blood-based tumor mutational burden, and circulating tumor cells have shown promising associations with the clinical response of NSCLC patients to ICI therapy. These investigations have unveiled compelling correlations between the peripheral immune status of patients both before and during ICI therapy and patient outcomes, which include response rates, progression-free survival, and overall survival. There is need for rigorous validation and standardization of these blood-based assays for broader clinical application. Integration of multiple blood-based biomarkers into comprehensive panels or algorithms also has the potential to enhance predictive accuracy. Further research aimed at longitudinal monitoring of circulating biomarkers is also crucial to comprehend immune dynamics and resistance mechanisms and should be used alongside tissue-based methods that interrogate the tumor microenvironment to guide treatment decisions and may inform on the development of novel therapeutic strategies. The data reviewed here reinforce the opportunity to refine patient stratification, optimize treatments, and improve outcomes not only in NSCLC but also in the wider spectrum of solid tumors undergoing immunotherapy.

Peripheral immune biomarkers for immune checkpoint inhibition of solid tumours.

BACKGROUND: With the rapid adoption of immunotherapy for the treatment of cancer comes the pressing need for readily accessible biomarkers to guide immunotherapeutic strategies and offer insights into outcomes with specific treatments. Regular sampling of solid tumour tissues outside of melanoma for immune monitoring is not often feasible; conversely, routine, frequent interrogation of circulating immune biomarkers is entirely possible. As immunotherapies and immune checkpoint inhibitors, in particular, are more widely used in first-line, neoadjuvant, and metastatic settings, the discovery and validation of peripheral immune biomarkers are urgently needed across solid tumour types for improved prediction and prognostication of clinical outcomes in response to immunotherapy, as well as elucidation of mechanistic underpinnings of the intervention. Careful experimental design, encompassing both retrospective and prospective studies, is required in such biomarker identification studies, and concerted efforts are essential for their advancement into clinical settings. CONCLUSION: In this review, we summarize shared immune features between the tumour microenvironment and systemic circulation, evaluate exploratory peripheral immune biomarker studies, and discuss associations between candidate biomarkers with clinical outcomes. We also consider integration of multiple peripheral immune parameters for better prediction and prognostication and discuss considerations in study design to further evaluate the clinical utility of candidate peripheral immune biomarkers for immunotherapy of solid tumours. HIGHLIGHTS: Peripheral immune biomarkers are critical for improved prediction and prognostication of clinical outcomes for patients with solid tumours treated with immune checkpoint inhibition. Candidate peripheral biomarkers, such as cytokines, soluble factors, and immune cells, have potential as biomarkers to guide immunotherapy of solid tumours. Multiple peripheral immune parameters may be integrated to improve prediction and prognostication. The potential of peripheral immune biomarkers to guide immunotherapy of solid tumours requires critical work in biomarker discovery, validation, and standardization.

Hypothesis: the generation of T cells directed against neoepitopes employing immune-mediating agents other than neoepitope vaccines.

The development of vaccines, especially RNA-based, directed against patient-specific tumor neoepitopes is an active and productive area of cancer immunotherapy. Promising clinical results in melanoma and other solid tumor types are emerging. As with all cancer therapy modalities, neoepitope vaccine development and delivery also has some drawbacks, including the level of effort to develop a patient-specific product, accuracy of algorithms to predict neoepitopes, and with the exception of melanoma and some other tumor types, biopsies of metastatic lesions of solid tumors are often not available. We hypothesize that in some circumstances the use of rationally designed combinations of "off-the-shelf" agents may prove an additional path to enable the patient to produce his/her own "neoepitope vaccine" in situ. These combination therapies may consist of agents to activate a tumor-associated T-cell response, potentiate that response, reduce or eliminate immunosuppressive entities in the tumor microenvironment, and/or alter the phenotype of tumor cells to render them more susceptible to immune-mediated lysis. Examples are provided in both preclinical and clinical studies in which combinations of "off-the-shelf" agents lead to the generation of T cells directed against tumor-derived neoepitopes with consequent antitumor activity.

TRAIL-induced cytokine production via NFKB2 pathway promotes neutrophil chemotaxis and immune suppression in triple negative breast cancers.

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is a potential cancer therapeutic that induces apoptosis in cancer cells while sparing the non-malignant cells in preclinical models. However, its efficacy in clinical trials has been limited, suggesting unknown modulatory mechanisms responsible for the lack of TRAIL activity in patients. Here, we hypothesized that TRAIL treatment elicits transcriptional changes in triple negative breast cancer (TNBC) cells that alter the immune milieu. To test this, we performed an RNAseq analysis of MDA-MB-231 cells treated with TRAIL, followed by validation in additional TNBC cell lines. TRAIL significantly induces expression of multiple cytokines such as CXCLs 1, 2, 3, 8,11 and IL-6, which are known to modify neutrophil function. Mechanistically, the induction of these cytokines was predominantly mediated by death receptor 5, caspase 8 (but not caspase 8 enzymatic activity), and the non-canonical NFKB2 pathway. The cytokines produced by the TRAIL-treated TNBC cells enhanced chemotaxis of healthy human donor isolated neutrophils. In vivo , TRAIL treated TNBC murine xenograft tumors showed activation of the NFKB2 pathway, elevated production of CXCLs and IL-6, and increased neutrophil recruitment into the tumors. Moreover, donor isolated neutrophils preincubated in supernatants from TRAIL-treated TNBC cells exhibited impaired cytotoxic effect against TNBC cells. Transcriptomic analysis of neutrophils incubated with either TRAIL alone or supernatant of TRAIL-treated TNBC cells revealed increased expression of inflammatory cytokines, immune modulatory genes, immune checkpoint genes, and genes implicated in delayed neutrophil apoptosis. Functional studies with these neutrophils confirmed their suppressive effect on T cell proliferation and an increase in Treg suppressive phenotype. Collectively, our study demonstrates a novel role of TRAIL-induced NFKB2-dependent cytokine production that promotes neutrophil chemotaxis and immune suppression.