Search

Background and Rationale: Immune checkpoint inhibitor (ICI) therapy is an expanding therapeutic option for hepatocellular carcinoma (HCC). Antibiotics (ATB) taken prior to or early during ICI therapy can impact immunotherapy efficacy across indications; however, the effect of ATB is undefined in HCC. Methods: In a large international cohort of 450 ICI recipients from Europe, North America, and Asia, we categorized patients according to timing of ATB focusing on exposure within −30 to +30 days from ICI (early immunotherapy period [EIOP]). EIOP was evaluated in association with overall survival (OS), progression-free survival (PFS), and best radiologic response using RECIST 1.1 criteria. Results: Our study comprised mostly cirrhotic (329, 73.3%) males (355, 79.1%) with a Child-Turcotte Pugh class of A (332, 73.9%), receiving ICI after 1 therapy line (251, 55.9%) for HCC of Barcelona clinic liver cancer stage C (325, 72.4%). EIOP (n = 170, 37.9%) was independent of baseline clinicopathologic features of HCC and correlated with longer PFS (6.1 vs. 3.7 months, log-rank p = 0.0135). EIOP+ patients had similar OS, overall response, and disease control rates (DCRs) compared to EIOP. The effect of EIOP persisted in landmark time analyses and in multivariable models, confirming the independent predictive role of EIOP in influencing PFS following adjustment for covariates reflective of tumor burden, liver function, and ICI regimen administered. In patients receiving programmed cell death-1 receptor/ligand inhibitors monotherapy, EIOP was also associated with higher DCRs (61.4% vs. 50.9%, p = 0.0494). Conclusions: Unlike other oncological indications, ATB in the 30 days before or after ICI initiation is associated with improved benefit from immunotherapy, independent of disease and treatment-related features. Evaluation of the immune microbiologic determinants of response to ICI in HCC warrants further investigation.

Immune checkpoint inhibitor (ICI) therapy is an increasingly used treatment modality across the various stages of hepatocellular cancer (HCC). There is currently no standard peri-operative therapy for HCC, despite a high probability of recurrence. Emerging studies in a variety of tumors demonstrate significant pathologic and immune responses to neoadjuvant immunotherapy. Unlike kinase inhibitors and other targeted therapies, which demonstrated no benefit in the adjuvant setting and fail to induce significant responses, ICIs can induce radiologically appreciable reduction in disease burden, which make ICI combinations an appealing downstaging strategy in patients early or locally advanced disease. Additionally, induction of anti-tumor immunity in the pre-operative setting may induce protracted T-cell response that, in the post-operative phase, may be capable of eliminating micro-metastatic disease and prevent future recurrence. In this review, we discuss the rationale and clinical hurdles that underlie optimal integration of immunotherapy in the pre-operative setting, highlighting the positive impact on surgical and oncological outcomes in patients with early-stage HCC.

Several studies have revealed that the hyper-inflammatory response induced by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a major cause of disease severity and death. However, predictive biomarkers of pathogenic inflammation to help guide targetable immune pathways are critically lacking. We implemented a rapid multiplex cytokine assay to measure serum interleukin (IL)-6, IL-8, tumor necrosis factor (TNF)-α and IL-1β in hospitalized patients with coronavirus disease 2019 (COVID-19) upon admission to the Mount Sinai Health System in New York. Patients (n = 1,484) were followed up to 41 d after admission (median, 8 d), and clinical information, laboratory test results and patient outcomes were collected. We found that high serum IL-6, IL-8 and TNF-α levels at the time of hospitalization were strong and independent predictors of patient survival (P

Checkpoint blockade therapies have improved cancer treatment, but such immunotherapy regimens fail in a large subset of patients. Conventional type 1 dendritic cells (DC1s) control the response to checkpoint blockade in preclinical models and are associated with better overall survival in patients with cancer, reflecting the specialized ability of these cells to prime the responses of CD8+ T cells1–3. Paradoxically, however, DC1s can be found in tumours that resist checkpoint blockade, suggesting that the functions of these cells may be altered in some lesions. Here, using single-cell RNA sequencing in human and mouse non-small-cell lung cancers, we identify a cluster of dendritic cells (DCs) that we name ‘mature DCs enriched in immunoregulatory molecules’ (mregDCs), owing to their coexpression of immunoregulatory genes (Cd274, Pdcd1lg2 and Cd200) and maturation genes (Cd40, Ccr7 and Il12b). We find that the mregDC program is expressed by canonical DC1s and DC2s upon uptake of tumour antigens. We further find that upregulation of the programmed death ligand 1 protein—a key checkpoint molecule—in mregDCs is induced by the receptor tyrosine kinase AXL, while upregulation of interleukin (IL)-12 depends strictly on interferon-γ and is controlled negatively by IL-4 signalling. Blocking IL-4 enhances IL-12 production by tumour-antigen-bearing mregDC1s, expands the pool of tumour-infiltrating effector T cells and reduces tumour burden. We have therefore uncovered a regulatory module associated with tumour-antigen uptake that reduces DC1 functionality in human and mouse cancers. After taking up tumour-associated antigens, dendritic cells in mouse and human tumours upregulate a regulatory gene program that limits dendritic cell immunostimulatory function, and modulating this program can rescue antitumor immunity in mice.

Predicting COVID-19 severity is difficult, and the biological pathways involved are not fully understood. To approach this problem, we measured 4,701 circulating human protein abundances in two independent cohorts totaling 986 individuals. We then trained prediction models including protein abundances and clinical risk factors to predict adverse COVID-19 outcomes in 417 subjects and tested these models in a separate cohort of 569 individuals. For severe COVID-19, a baseline model including age and sex provided an area under the receiver operator curve (AUC) of 65% in the test cohort. Selecting 92 proteins from the 4,701 unique protein abundances improved the AUC to 88% in the training cohort, which remained relatively stable in the testing cohort at 86%, suggesting good generalizability. Proteins selected from different adverse COVID-19 outcomes were enriched for cytokine and cytokine receptors, but more than half of the enriched pathways were not immune-related. Taken together, these findings suggest that circulating proteins measured at early stages of disease progression are reasonably accurate predictors of adverse COVID-19 outcomes. Further research is needed to understand how to incorporate protein measurement into clinical care.

Macrophages have a key role in shaping the tumour microenvironment (TME), tumour immunity and response to immunotherapy, which makes them an important target for cancer treatment1,2. However, modulating macrophages has proved extremely difficult, as we still lack a complete understanding of the molecular and functional diversity of the tumour macrophage compartment. Macrophages arise from two distinct lineages. Tissue-resident macrophages self-renew locally, independent of adult haematopoiesis3–5, whereas short-lived monocyte-derived macrophages arise from adult haematopoietic stem cells, and accumulate mostly in inflamed lesions1. How these macrophage lineages contribute to the TME and cancer progression remains unclear. To explore the diversity of the macrophage compartment in human non-small cell lung carcinoma (NSCLC) lesions, here we performed single-cell RNA sequencing of tumour-associated leukocytes. We identified distinct populations of macrophages that were enriched in human and mouse lung tumours. Using lineage tracing, we discovered that these macrophage populations differ in origin and have a distinct temporal and spatial distribution in the TME. Tissue-resident macrophages accumulate close to tumour cells early during tumour formation to promote epithelial–mesenchymal transition and invasiveness in tumour cells, and they also induce a potent regulatory T cell response that protects tumour cells from adaptive immunity. Depletion of tissue-resident macrophages reduced the numbers and altered the phenotype of regulatory T cells, promoted the accumulation of CD8+ T cells and reduced tumour invasiveness and growth. During tumour growth, tissue-resident macrophages became redistributed at the periphery of the TME, which becomes dominated by monocyte-derived macrophages in both mouse and human NSCLC. This study identifies the contribution of tissue-resident macrophages to early lung cancer and establishes them as a target for the prevention and treatment of early lung cancer lesions. Single-cell RNA sequencing and imaging of macrophages in human non-small cell lung cancer and in a mouse model of lung adenocarcinoma show that tissue-resident macrophages have a key role in early tumour progression.

Indolent non-Hodgkin’s lymphomas (iNHLs) are incurable with standard therapy and are poorly responsive to checkpoint blockade. Although lymphoma cells are efficiently killed by primed T cells, in vivo priming of anti-lymphoma T cells has been elusive. Here, we demonstrate that lymphoma cells can directly prime T cells, but in vivo immunity still requires cross-presentation. To address this, we developed an in situ vaccine (ISV), combining Flt3L, radiotherapy, and a TLR3 agonist, which recruited, antigen-loaded and activated intratumoral, cross-presenting dendritic cells (DCs). ISV induced anti-tumor CD8+ T cell responses and systemic (abscopal) cancer remission in patients with advanced stage iNHL in an ongoing trial ( NCT01976585 ). Non-responding patients developed a population of PD1+CD8+ T cells after ISV, and murine tumors became newly responsive to PD1 blockade, prompting a follow-up trial of the combined therapy. Our data substantiate that recruiting and activating intratumoral, cross-priming DCs is achievable and critical to anti-tumor T cell responses and PD1-blockade efficacy. In situ vaccine recruits and activates cross-presenting dendritic cells and augments PD1 blockade efficacy in patients with indolent non-Hodgkin’s lymphoma.

Serologic markers that predict severe COVID-19 disease trajectories could enable early medical interventions and reduce morbidity and mortality. We found that distinct features of IgG Fab and Fc domain structures were present within three days of a positive test that predicted two separate disease trajectories in a prospective cohort of patients with COVID-19. One trajectory was defined by early production of neutralizing antibodies and led to mild disease. A distinct trajectory, characterized by an initial period of mild symptoms followed by rapid progression to more severe outcomes, was predicted by the absence of early neutralizing antibody responses with concomitant production of afucosylated IgGs. Elevated frequencies of monocytes expressing the receptor for afucosylated IgGs, FcγRIIIa (CD16a), were an additional antecedent in patients with the more severe outcomes. In mechanistic studies, afucosylated immune complexes in the lung triggered an inflammatory infiltrate and cytokine production that was dependent on CD16a. Finally, in healthy subjects, mRNA SARS-CoV-2 vaccination elicited neutralizing antibodies that were enriched for Fc fucosylation and sialylation and distinct from both infection-induced trajectories. These data show the importance of combined Fab and Fc domain functions in the antiviral response, define an early antibody signature in people who progressed to more severe COVID-19 outcomes and have implications for novel therapeutic strategies targeting FcγRIIIa pathways.

Hepatocellular carcinoma (HCC) can have viral or non-viral causes1–5. Non-alcoholic steatohepatitis (NASH) is an important driver of HCC. Immunotherapy has been approved for treating HCC, but biomarker-based stratification of patients for optimal response to therapy is an unmet need6,7. Here we report the progressive accumulation of exhausted, unconventionally activated CD8+PD1+ T cells in NASH-affected livers. In preclinical models of NASH-induced HCC, therapeutic immunotherapy targeted at programmed death-1 (PD1) expanded activated CD8+PD1+ T cells within tumours but did not lead to tumour regression, which indicates that tumour immune surveillance was impaired. When given prophylactically, anti-PD1 treatment led to an increase in the incidence of NASH–HCC and in the number and size of tumour nodules, which correlated with increased hepatic CD8+PD1+CXCR6+, TOX+, and TNF+ T cells. The increase in HCC triggered by anti-PD1 treatment was prevented by depletion of CD8+ T cells or TNF neutralization, suggesting that CD8+ T cells help to induce NASH–HCC, rather than invigorating or executing immune surveillance. We found similar phenotypic and functional profiles in hepatic CD8+PD1+ T cells from humans with NAFLD or NASH. A meta-analysis of three randomized phase III clinical trials that tested inhibitors of PDL1 (programmed death-ligand 1) or PD1 in more than 1,600 patients with advanced HCC revealed that immune therapy did not improve survival in patients with non-viral HCC. In two additional cohorts, patients with NASH-driven HCC who received anti-PD1 or anti-PDL1 treatment showed reduced overall survival compared to patients with other aetiologies. Collectively, these data show that non-viral HCC, and particularly NASH–HCC, might be less responsive to immunotherapy, probably owing to NASH-related aberrant T cell activation causing tissue damage that leads to impaired immune surveillance. Our data provide a rationale for stratification of patients with HCC according to underlying aetiology in studies of immunotherapy as a primary or adjuvant treatment., In hepatocellular carcinoma driven by non-alcoholic steatohepatitis, aberrant T cell activation and impaired immune surveillance seem to make hepatocellular carcinoma less responsive to anti-PD1 or anti-PDL1 immunotherapy.

Macrophages (MΦ) form a highly heterogenous compartment within tumor lesions. Using lineage tracing, we recently established that advanced NSCLC lesions in both mice and humans are depleted of tissue-resident macrophages (TRM) and are mainly populated by monocyte-derived MΦ (MoMΦ). Here, we show that TREM2+ MoMΦ dominate murine and human NSCLC lesions. Importantly, using mice reconstituted with mixed TREM2-deficient and -proficient bone marrow cells, we identified a cell-intrinsic ability for TREM2 to drive an efferocytosis-associated molecular program that profoundly reduced the expression of immunogenic molecules in tumor-infiltrating MoMΦ. Trem2 deletion significantly dampened this program, reversed MoMΦ immunosuppression, and significantly enhanced the accumulation of activated NK cells and cDC1 in tumor lesions. Notably, Trem2 deletion reduced lung cancer progression in an NK cell-dependent manner. Taken together, our results underscore the potential for therapeutic inhibition of TREM2 to mount a potent anti-tumor immune response by unleashing cooperative NK/cDC1 cell function.

Background CDX-1140 is an agonist anti-CD40 mAb selected to optimize systemic exposure and hence tumor microenvironment (TME) ingress. CDX-1140 activity may be enhanced by combining with CDX-301 (recombinant Flt3L), a dendritic cell growth factor, or with pembrolizumab, an anti-PD-1 mAb. Methods Patients with advanced solid or hematologic (Part 1 only) tumors are enrolled. Part 1 dose-escalation results have been presented (SITC 2019). In Part 2, CDX-1140 dose-escalation (0.09–1.5 mg/kg q4w) is in combination with CDX-301 (75 mcg/kg sc QD x 5 for 2 cycles). In Part 3, CDX-1140 dose-escalation (0.72–1.5 mg/kg q3w) is in combination with pembrolizumab 200 mg q3w. Part 1 and 2 expansion cohorts are dosed at the CDX-1140 MTD, 1.5 mg/kg q4w. Part 3 expansion cohorts are planned. Peripheral blood and tumor biomarkers analysis are ongoing. Results 92 patients have been treated (Part 1 n=57, Part 2 n=31, Part 3 n=4). Part 1 expansion cohorts in SCCHN (n=7) and RCC (n=5) are fully enrolled. Part 2 dose-escalation completed to the highest CDX-1140 dose and a SCCHN expansion cohort is ongoing. Part 3 dose-escalation recently initiated. Safety data is available for 23 and 10 patients at the MTD in Part 1 and 2, respectively. In general, the safety profiles were similar, with arthralgia (52% vs. 50%), pyrexia (44% vs 50%), fatigue (30% vs. 50%), chills (39% vs. 40%), vomiting (30% vs. 20%), nausea (26% vs 40%), myalgia (22% vs. 30%), increased ALT (22% vs. 20%), and increased AST (22% vs. 30%) being the most common drug related AEs at the MTD in Part 1 and 2, respectively. Most AEs were low grade. Across all cohorts, cytokine release syndrome (CRS) (G2 n=4, G3 n=2) occurred in 6 (Part 1 n=2; Part 2 n=4) and pneumonitis (G3) occurred in 5 (Part 1 n=4; Part 2 n=1) patients. Immune activation in the TME consistent with CD40 agonism and increases serum inflammatory cytokines were observed. Evidence of anti-tumor activity/clinical benefit include SD (n=13), tumor cavitation (n=2) and a uPR in solid tumors. A patient with follicular lymphoma has an ongoing durable complete metabolic response. Conclusions The CDX-1140 MTD dose of 1.5 mg/kg, a dose level expected to provide good systemic exposure and TME penetration, is generally well tolerated alone and with CDX-301. Transaminitis and CRS have generally been low grade and infrequent. A cohort combining CDX-1140 with chemotherapy will be initiated in patients with previously untreated metastatic pancreatic adenocarcinoma. Trial Registration NCT03329950 Ethics Approval The study was approved by the following: Providence St. Joseph Health IRB, approval number MOD2020001128; WIRB, approval number 1188814 (Hauke, Gabrail, Bordoni & Gordon); University of Pennsylvania IRB, approval number UPCC 18917; Mount Sinai School of Medicine IRB, approval number 18-00202; Memorial Sloan Kettering Cancer Center IRB, approval number 18-225A; Houston Methodist IRB, approval number MOD00000836

Background The efficacy of T cell directed immunotherapies relies on adequate priming of T cells to tumor-specific neoantigens, which some studies have augmented with synthetic neoantigen vaccines. This is the first report of a personalized genomic vaccine (PGV-001) in multiple histologies in the adjuvant setting. Methods Tumor and germline RNA and DNA were sequenced, and neoantigen peptides were selected using our OpenVax custom computation pipeline that identifies and ranks mutant sequences by a combination of predicted MHC-I binding affinity and neoantigen abundance within tumor. Up to 10 peptides were synthesized per patient and were administered over the course of 27 weeks in combination with the poly-ICLC. Primary objectives were to determine 1) the safety and tolerability; 2) the feasibility of PGV-001 production and administration; and 3) the immunogenicity of PGV-001. Secondary objectives included immunophenotyping neoantigen-specific T cells in peripheral blood, and characterization of peripheral blood lymphoid, myeloid and humoral responses. We report here for the first time on the primary endpoints. Results Vaccine was synthesized for 15 patients. A mean of 1619 somatic variants (range 521–5106) were detected. Our pipeline identified a mean of 67.1 neoantigens/patient (range 8–193) and 9.7 peptides/patient were synthesized (range 7–10). 13 patients received PGV-001 (11 patients received all 10 doses and 2 patients received at least 8 doses) while 2 had progressive disease before vaccine initiation. Transient grade 1 injection site reactions were seen in 31% of patients, and one patient experienced grade 1 fever. There were no other significant adverse events. Ex vivo ELISpot analysis of patient blood demonstrated significant induction of T cell responses following receipt of 10 vaccines that were not present after the 6th vaccine, supporting the need for a prolonged dosing schedule. Robust responses were seen in both CD4 and CD8 T cells by intracellular cytokine staining for TNF-a, IFN-a, and IL-2 following in vitro expansion in the presence of vaccine antigens. Additional studies are ongoing to define the most immunogenic antigens. Conclusions A personalized neoantigen vaccine of synthetic mutant peptides and adjuvant poly-ICLC was successfully synthesized for 15 patients and administered successfully to 87% patients over the course of 27 weeks. The vaccine was well tolerated, and T cell expansion and reactivity to synthetic neoantigens confirms immunogenicity of neoantigens identified with OpenVax. Trial Registration NCT02721043 Ethics Approval This study was approved by the IRB of The Mount Sinai Hospital in accordance with Federal law. HSM #15-00841.

Background CD73 is present on subsets of B and T cells and is involved in lymphocyte activation. CPI-006 is a humanized IgG1, Fcγ receptor deficient anti-CD73 that has agonistic properties. In vitro studies and ongoing cancer clinical trials show that CPI-006 binds to B cells leading to expression of CD69, trafficking to lymph nodes, immunoglobulin class switching, transformation to plasmablasts and generation of memory B cells.1 Recently, a patient in the cancer trial with asymptomatic COVID-19 developed high titers of neutralizing anti-SARS-CoV-2 antibodies following administration of CPI-006. A phase 1 trial in COVID-19 was initiated to evaluate the use of CPI-006 to enhance anti-viral immune response (NCT04464395). Methods Single intravenous dose escalation with N=5 per cohort of 0.3, 1.0, 3.0 and 5.0 mg/kg. Pt eligibility included PCR positive nasal swab for COVID-19; hospitalized with O2 saturation of ≥92% on Results 10 pts have been treated in the first 2 cohorts; median age 64 (range 28–76) and all had comorbidities: diabetes (4), hypertension (2), obesity (7) and/or cancer (2). Median duration of symptoms prior to CPI-006 was 8 days (range 1–21 days). No treatment-related adverse events were reported. There was no correlation between duration of symptoms and baseline anti-viral titers. Kinetics of anti-SARS-CoV-2 response to spike protein are shown for 7 pts with follow-up ≥ 7 days post CPI-006 (figure 1). One pt with lymphopenia (600/mm3) had delayed response to CPI-006; all other pts generated antibody response by Day 7 post-CPI-006 to both spike and RBD. Increasing titers of IgG and IgM antibodies were observed out to 28 days post treatment. In one pt examined, memory B cells increased from 1.81% to 4.83% of B cells 28 days after treatment with serum IgG titers to spike and to RBD of >1:50,000. 2 of 2 pts had increase in both CD4 and CD8 T effector memory cells at day 28. All pts were discharged (median 4 days) with clinical improvement. Conclusions CPI-006 is well tolerated in COVID-19 pts. Low baseline titers of antibodies to virus were increased following CPI-006 in all treated pts. Immunomodulation with CPI-006 represents a novel therapy for COVID-19 aimed at stimulating more robust and prolonged anti-SARS-CoV-2 immunity potentially after infection or with vaccination. Trial Registration NCT04464395 Ethics Approval The study was approved by Temple University Hospital’s Ethics Board, Western IRB, approval number 1-1317457-1. Reference Luke J, Powderly J, Merchan J, Barve M, Hotson A, Mobasher M, Kwei L, Luciano G, Buggy J, Piccione E, Miller R. Immunobiology, preliminary safety, and efficacy of CPI-006, an anti-CD73 antibody with immune modulating activity, in a phase 1 trial in advanced cancers. J Clin Oncol 2019; 37:15 suppl, 2505.

COVID-19 is a global pandemic that has resulted in over 800,000 deaths. Robust humoral anti-viral immune responses have the potential to generate a diverse set of neutralizing antibodies to eliminate viruses and protect against re-infection, transmission, and the evolution of mutations that escape targeted therapeutics. CD73 is present on the majority of human B cells and a subset of T cells where it plays a role in lymphocyte activation and migration. CD73 also functions as an ectoenzyme that converts AMP into adenosine, which can be immunosuppressive. Here we report on CPI-006, a humanized FcγR binding-deficient IgG1 anti-CD73 antibody that blocks CD73 enzymatic activity and directly activates CD73POS B cells, inducing differentiation into plasmablasts, immunoglobulin class switching, and antibody secretion independent of adenosine. Immunophenotypic analysis of peripheral blood from advanced cancer patients receiving CPI-006 revealed evidence of B cell activation, clonal expansion, and development of memory B cells. These immune effects suggested that CPI-006 may be effective at enhancing the magnitude, diversity, and duration of humoral and cellular responses to viruses such as SARS-CoV-2. We have therefore initiated a Phase 1, single-dose, dose-escalation trial in hospitalized patients with mild to moderate COVID-19. The objectives of this trial are to evaluate the safety of CPI-006 in COVID-19 patients and to determine effects of CPI-006 on anti-SARS-CoV-2 antibody responses and the development of memory B cell and T cells. Ten patients have been enrolled in the trial receiving doses of 0.3 mg/kg or 1.0 mg/kg. All evaluable patients had low pre-treatment serum levels of anti-viral antibodies to the SARS-CoV-2 trimeric spike protein and its receptor binding domain, independent of the duration of their COVID-19 related symptoms prior to enrollment. Anti-viral antibody responses were induced 7 days after CPI-006 treatment and titers continued to rise past Day 56. Increases in the frequency of memory B cells and effector/memory T cells were observed 28 days after treatment. These preliminary results suggest that CPI-006 activates B cells and may enhance and prolong anti-SARS-CoV-2 antibody responses in patients with COVID-19. This approach may be useful for treating COVID-19 or as an adjuvant to enhance the efficacy of vaccines.

SUMMARYImmunotherapy is becoming a mainstay in the treatment of NSCLC. While tumor mutational burden (TMB) has been shown to correlate with response to immunotherapy, little is known about the relation of the baseline immune response with the tumor genotype. Here, we profiled 35 early stage NSCLC lesions using multiscale single cell sequencing. Unsupervised clustering identified in a subset of patients a key cellular module consisting of PDCD1+ CXCL13+ activated T cells, IgG+ plasma cells, and SPP1+ macrophages, referred to as the lung cancer activation module (LCAMhi). Transcriptional data from two NSCLC cohorts confirmed a subset of patients with LCAMhi enrichment, which was independent of overall immune cell content. The LCAMhi module strongly correlated with TMB, expression of cancer testis antigens, and with TP53 mutations in smokers and non-smokers. These data establish LCAM as a key mode of immune cell activation associated with high tumor antigen load and driver mutations.

BackgroundNivolumab is Food and Drug Administration approved in sorafenib-experienced, advanced hepatocellular carcinoma (HCC). Post-registration data of treatment in a real-world setting is lacking.Patients and methodsWe performed an international, multicenter observational study to confirm safety and efficacy of nivolumab in 233 patients treated outside clinical trials from eight centers in North America, Europe and Asia.ResultsPatients received nivolumab for Barcelona Clinic Liver Cancer stage C (n=191, 92.0%) and Child-Pugh (CP) A (n=158, 67.8%) or B (n=75, 32.2%) HCC as first (n=85, 36.5%) or second to fourth systemic therapy line (n=148, 63.5%). Objective response rate (ORR) was 22.4% and disease control rate was 52.1%. Median overall survival (OS) was 12.2 months (95% CI 8.4 to 16.0) and median progression-free survival was 10.1 months (95% CI 6.1 to 14.2). Treatment-related adverse events of grade >2 occurred in 26 patients (11.2%). Efficacy and safety were similar across CP classes and therapy line. OS was shorter in CP-B than A (7.3 months vs 16.3 months, p=0.05). Achievement of an objective response predicted for improved OS (25.4 months vs 13.2 months, pConclusionsThis study confirms safety and efficacy of nivolumab in advanced HCC across various lines of therapy and degrees of liver dysfunction. Despite equal ORR and toxicity to nivolumab, patients with CP-B functional class have shorter survival than the patients with CP-A.

Immune checkpoint inhibitors (ICI) have shown positive results in patients with hepatocellular carcinoma (HCC). As liver function contributes to prognosis, its precise assessment is necessary for the safe prescribing and clinical development of ICI in HCC. We tested the accuracy of the albumin-bilirubin (ALBI) grade as an alternative prognostic biomarker to the Child-Turcotte-Pugh (CTP). In a prospectively maintained multi-centre dataset of HCC patients, we assessed safety and efficacy of ICI across varying levels of liver dysfunction described by CTP (A to C) and ALBI grade and evaluated uni- and multi-variable predictors of overall (OS) and post-immunotherapy survival (PIOS). We studied 341 patients treated with programmed-death pathway inhibitors (n = 290, 85%). Pre-treatment ALBI independently predicted for OS, with median OS of 22.5, 9.6, and 4.6 months across grades (p &lt, 0.001). ALBI was superior to CTP in predicting 90-days mortality with area under the curve values of 0.65 (95% CI 0.57&ndash, 0.74) versus 0.63 (95% CI 0.54&ndash, 0.72). ALBI grade at ICI cessation independently predicted for PIOS (p &lt, 0.001). Following adjustment for ICI regimen, neither ALBI nor CTP predicted for overall response rates or treatment-emerging adverse events (p &gt, 0.05). ALBI grade identifies a subset of patients with prolonged survival prior to and after ICI therapy, lending itself as an optimal stratifying biomarker to optimise sequencing of systemic therapies in advanced HCC.

The COVID-19 pandemic caused by infection with Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) has led to more than 100,000 deaths in the United States. Several studies have revealed that the hyper-inflammatory response induced by SARS-CoV-2 is a major cause of disease severity and death in infected patients. However, predictive biomarkers of pathogenic inflammation to help guide targetable immune pathways are critically lacking. We implemented a rapid multiplex cytokine assay to measure serum IL-6, IL-8, TNF-α, and IL-1β in hospitalized COVID-19 patients upon admission to the Mount Sinai Health System in New York. Patients (n=1484) were followed up to 41 days (median 8 days) and clinical information, laboratory test results and patient outcomes were collected. In 244 patients, cytokine measurements were repeated over time, and effect of drugs could be assessed. Kaplan-Meier methods were used to compare survival by cytokine strata, followed by Cox regression models to evaluate the independent predictive value of baseline cytokines. We found that high serum IL-6, IL-8, and TNF-α levels at the time of hospitalization were strong and independent predictors of patient survival. Importantly, when adjusting for disease severity score, common laboratory inflammation markers, hypoxia and other vitals, demographics, and a range of comorbidities, IL-6 and TNF-α serum levels remained independent and significant predictors of disease severity and death. We propose that serum IL-6 and TNF-α levels should be considered in the management and treatment of COVID-19 patients to stratify prospective clinical trials, guide resource allocation and inform therapeutic options. We also propose that patients with high IL-6 and TNF-α levels should be assessed for combinatorial blockade of pathogenic inflammation in this disease.

Immunotherapy is a mainstay of non-small cell lung cancer (NSCLC) management. While tumor mutational burden (TMB) correlates with response to immunotherapy, little is known about the relationship between the baseline immune response and tumor genotype. Using single-cell RNA sequencing, we profiled 361,929 cells from 35 early-stage NSCLC lesions. We identified a cellular module consisting of PDCD1(+)CXCL13(+) activated T cells, IgG(+) plasma cells, and SPP1(+) macrophages, referred to as the lung cancer activation module (LCAM(hi)). We confirmed LCAM(hi) enrichment in multiple NSCLC cohorts, and paired CITE-seq established an antibody panel to identify LCAM(hi) lesions. LCAM presence was found to be independent of overall immune cell content and correlated with TMB, cancer testis antigens, and TP53 mutations. High baseline LCAM scores correlated with enhanced NSCLC response to immunotherapy even in patients with above median TMB, suggesting that immune cell composition, while correlated with TMB, may be a nonredundant biomarker of response to immunotherapy.

BackgroundMupadolimab (mupa) is a humanized FcγR binding-deficient IgG1 anti-CD73 antibody that has agonistic properties.1 CD73 is involved in production of adenosine and in cellular trafficking. Mupa reacts with the majority of circulating B cells leading to activation and expression of differentiation markers CD69, CD138 and CD38, and transformation into plasmablasts with secretion of IgM and IgG. B cell activation provided the rationale to develop mupa for immunotherapy of cancer and Covid-19. Intratumor HPV specific B cells have been reported in HNSCC.2 This report describes properties of mupa and the early signs of clinical activity in HPV+ HNSCC.MethodsELISA and flow cytometry were used to measure binding of anti-CD73. Humanized NSG-SGM3 mice were used to evaluate effects of Mupa on human anti-SARS CoV2 spike protein (SP) response. CD73 expression in biopsies was measured by immunohistochemistry. Mupa (IV q 3 weeks) with or without pembrolizumab is being evaluated in an ongoing phase 1 trial in patients with refractory cancers.ResultsMupa binding to CD73 was blocked by APCP, an analog of adenosine diphosphate that locks CD73 in the closed conformation, indicating mupa binding to the open conformation. Cross blocking and cellular internalization studies showed that mupa is distinct from other anti-CD73 antibodies such as MEDI9447 and AD2. NSG-SGM3 mice were immunized with 50 µg SP subcutaneously and treated with mupa 10mg/kg or control IgG IP. Mupa treated animals mounted an antigen specific human anti-SP response; no antibody responses were seen in controls (P=0.02). In the dose-escalation portion of the phase 1 trial, mupa doses of ≥12 mg/kg saturated CD73 sites on circulating B cells. High stromal CD73 expression was observed in HPV+ HNSCC biopsies from 5 evaluable patients with chemotherapy and anti-PD1 refractory disease, and tumor regression was seen in 2 of these patients receiving 7 and 16 cycles of ≥12 mg/kg mupa without pembrolizumab. Safety of mupa+pembrolizumab was evaluated in 16 patients with no MTD reached and no changes in serum immunoglobulins. Transient reductions in circulating CD73 B cells were observed consistent with redistribution to lymphoid tissues.ConclusionsCD73 plays a role in B cell activation and differentiation. Mupa is an antibody with agonistic activity that stimulates B cells and enhances antigen specific antibody production. This activity supports a strategy to combine mupa with pembrolizumab to enhance both humoral and cellular immunity in the treatment of viral associated cancers such as HPV+HNSCC, and viral infections.Trial RegistrationNCT03454451ReferencesWillingham S, Criner G, Hill C, Hu S, Rudnick J, Daine-Matsuoka B, Hsieh J, Mashhedi H, Hotson A, Brody J, Marron T, Piccione E, Buggy J, Mahabhashyam S, Jones W, Mobasher M, Miller R. Characterization and Phase 1 trial of a B cell activating anti-CD73 antibody for the immunotherapy of COVID-19. medRxiv, 2020; https://doi.org/10.1101/2020.09.10.20191486.Wieland A, Patel M, Cardenas M, Eberhardt C, Hudson W, Obeng R, Griffith C, Wang X, Chen Z, Kissick H, Saba N, Ahmed R. Defining HPV-specific B cell responses in patients with head and neck cancer. Nature 2020; https://doi.org/10.1038/s41586-020-2931-3.Ethics ApprovalThe study was approved by Western IRB, approval number 1-1066703-1. Participants gave informed consent before taking part.

Introduction: Hepatic resection is the accepted treatment of early-stage hepatocellular carcinoma (HCC), however, the majority of tumors recur despite surgery and no perioperative intervention has demonstrated a survival advantage. Neoadjuvant immunotherapy has shown success in inducing pathological responses alongside induction of immune memory in multiple tumor types and is being evaluated to determine its potential in HCC. Methods: We recently completed accrual into a trial of perioperative cemiplimab (anti-PD-1) for resectable HCC (NCT03916627, Cohort B). This trial enrolled 21 patients to receive 2 cycles of neoadjuvant cemiplimab (350 mg Q3W) followed by surgical resection and an additional 8 adjuvant cycles. The primary endpoint was significant tumor necrosis, defined as >70% necrosis of the resected tumor. Secondary endpoints included delay of surgery, overall response rate, adverse events (AEs), and change in lymphocyte infiltration. Patients underwent pre-treatment biopsies and regular blood collection throughout treatment to enable exploratory analyses including multiplex IHC and single-cell proteomic and transcriptomic analysis. The final patient enrolled underwent resection on December 28, 2020. Results: Over the course of 18 months, 21 patients were enrolled. All patients received 2 cycles of preoperative cemiplimab, and all but 1 patient underwent successful resection; 1 patient was found to have metastatic disease at the time of surgery and resection was aborted. Cemiplimab demonstrated an acceptable risk-benefit profile: 90.5% of patients experienced any treatment-emergent AE (TEAE) of any grade during the neoadjuvant treatment period through the perioperative period; 28.6% of patients experienced any Grade 3 TEAE. One patient experienced Grade 3 pneumonitis during neoadjuvant therapy and surgery was delayed by 2 weeks past protocol defined surgical window. Of the 20 patients with resected tumors, 4 (20%) met the predefined endpoint of >70% tumor necrosis and 7 (35%) had ≥50% tumor necrosis. Three (15%) of the 4 patients with >70% tumor necrosis had a pathological complete response, defined as 100% tumor necrosis. We observed similar patterns of change in degree of necrosis on pathological assessment and 3D MRI at baseline and following completion of immunotherapy. Initial pathological assessment suggests an association between immune cell infiltration and tumor response. Tissue analysis is ongoing and multiplex IHC relating myeloid and lymphoid infiltration patterns with response to cemiplimab will be presented at the meeting. Conclusion: This is the largest trial reported to date of neoadjuvant PD-1 targeted monotherapy in HCC. Pathological response data support larger trials to identify optimal clinical endpoints that correlate with improvement in survival, and to establish the utility and safety of perioperative PD-1 blockade. Citation Format: Thomas Urban Marron, Ganesh Gunasekaran, Parissa Tabrizian, Edward Kim, Maria Isabel Fiel, Stephen Ward, Zhen Zhao, Deborah Doroshow, Meredith Legg, Ashley Hammad, Paul Kennedy, Guray Akturk, Pauline Hamon, Antoinette Bonaccorso, Daniel Labow, Sacha Gnjatic, Bachir Taouli, Elizabeth Miller, Pradeep Thanigaimani, Nathalie Fiaschi, Jennifer Sims, Jayakumar Mani, Nicola James, Sara Hamon, Vladimir Jankovic, Siyu Li, Hung Kam Cheung, Gavin Thurston, Israel Lowy, Myron Schwartz, Miriam Merad. Neoadjuvant cemiplimab demonstrates complete pathological responses in hepatocellular carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr CT182.

Background: The majority of novel cancer immunotherapies rely on adequate priming of T cells to tumor-specific neoantigens, which is believed to be lacking in patients who do not respond to therapy. We developed a personalized genomic vaccine (PGV-001) in which patient-specific synthetic neoantigen peptides (25 mer),are formulated and administered to patients with multiple cancer types in the adjuvant setting (NCT02721043). Methods: This trial enrolled patients whom had undergone curative-intent surgery (solid tumor patients) or autologous stem cell transplant (multiple myeloma patients), and for whom there was >30% chance of recurrence. Sequencing of tumor and germline DNA and RNA was performed and the OpenVax custom computation pipeline was used to identify candidate neoantigens; this platform ranks transcribed mutations using predicted MHC-I binding affinity and neoantigen abundance. A maximum of 10 peptides were synthesized per patient. Peptides were administered over the course of 27 weeks with poly-ICLC and a tetanus helper peptide. Primary objectives were to determine the safety and tolerability of vaccination, feasibility of vaccine production and administration, and immunogenicity. Results: Within 15 patients enrolled, the OpenVax pipeline identified an average of 67.1 neoantigens/patient (range 8-193), only two patients did not have adequate number of neoantigens identified to synthesize 10 peptides. 13 of the 15 patients received PGV-001, including 10 patients with solid tumor diagnoses and 3 patients with multiple myeloma, 11 of whom received all 10 doses, while 1 experienced progression of disease while on treatment. The vaccine was well tolerated, with grade 1 injection site reactions in 31% of patients, and grade 1 fever in one patient; there were no other significant adverse events. While one patient was lost to follow-up, of the remaining 12 patients the median progression-free survival from the time of their surgery or transplant of 618 days. With a mean follow-up of 925 days, 4 patients remain without evidence of disease, 4 patients are receiving subsequent lines of therapy, and 4 patients have died, though notably only two with documented recurrence of their malignancy. Initial analysis of the patient samples analyzed confirms immunogenicity. T cell responses were measured using ex vivo ELISpot and intracellular cytokine staining following expansion with neoantigen peptide libraries, both demonstrating induction of IFN-gamma, TNF-alpha and IL-2. Notably, robust T cell reactivity was only seen at the completion of all 10 vaccines, supporting the need for a prolonged schedule. Conclusions: PGV-001 was successfully synthesized for 15 patients and administered successfully to 13 patients without significant adverse events. Immune monitoring of immunogenicity is ongoing, with initial analysis demonstrating induction of neoantigen-specific CD4 and CD8 T cell expansion. Citation Format: Thomas Urban Marron, Mansi Saxena, Nina Bhardwaj, Marcia Meseck, Alex Rubinsteyn, John Finnigan, Julia Kodysh, Ana Blazquez, Tim O'Donnel, Mathew Galsky, Deborah Doroshow, Brett Miles, Krysztof Misiukiewicz, Hanna Irie, Amy Tiersten, Samir Parekh, Marshall Posner, Andrea Wolf, John Mandeli, Rachel Brody, Sacha Gnjatic, Eric Schadt, Philip Friedlander, Jeffrey Hammerbacher. An adjuvant personalized neoantigen peptide vaccine for the treatment of malignancies (PGV-001) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr LB048.

Hepatocellular carcinoma (HCC) has a dismal prognosis, and though checkpoint blocking antibodies have significantly improved patient outcome, many patients remain left out highlighting the need to identify additional immune target to enhance therapeutic immunity. Macrophages (MΦ) are an abundant and heterogeneous population in the tumor microenvironment (TME), and are associated with a poor prognosis in multiple tumor types, including HCC, however, their molecular and functional diversity is still poorly understood. We analyzed the molecular and spatial organization patterns of immune cells within the TME and adjacent tissue of 23 resected HCC lesions using single-cell RNA sequencing and multiplex immunohistochemistry (IHC). Strikingly, we found that Kupffer cells (KC), the self-renewing tissue-resident macrophages in liver tissue, are lacking from the TME, which is dominated by monocyte-derived macrophages. ScRNAseq followed by high-resolution clustering identified distinct MΦ molecular programs within the monocyte-derived macrophage compartment. One MΦ subset expressed a shared signature with monocytes including FCN1 and S100A8 in addition to T cell activation genes like CXCL9 and IL32. Conversely, one subset of MΦ expressed FOLR2, SEPP1 and genes of the complement (C1Qs), a program shared with KC in the adjacent uninvolved tissue, and include another intratumoral subset enriched for the expression of TREM2 and GPNMB. Guided by these results, we are developing an IHC antibody panel that allows to visualize distinct MΦ localization in the TME. Intratumoral MΦ interface with, and potentially regulate, the T cell compartment within the TME. We are analyzing HCC tumor lesions in our treatment-naïve cohort and in patients treated with neoadjuvant anti-PD-1 therapy (NCT03916627) to study direct interaction of MΦ and T cells using an innovative technology of physically-interacting cell sequencing (PICseq). This analysis enables us to identify MΦ with direct cell-cell contact with T cells, and our preliminary analysis demonstrates an enrichment in MΦ with a highly immunosuppressive phenotype. We are also using PICseq to map the molecular program of MΦ that are interacting with T cells and that we will corroborated with additional patients. Taken together, our data provide a new understanding of intratumoral MΦ diversity and highlight the presence of specific immunoregulatory MΦ programs unique to tumor lesions, with subsets of these MΦ found to be directly interacting with T cells, potentially modulating anti-tumor responsiveness. Our analysis of resected tumor from anti-PD-1 treated patients, will allow us to correlate MΦ programs, and direct T cell interaction, with clinical response, and will inform therapeutic trials targeting specific MΦ populations so as to improve clinical efficacy of cancer immunotherapy. Citation Format: Pauline Hamon, Assaf Magen, Merav Cohen, Alexandra Tabachnikova, Christie Chang, Mark Buckup, Leanna Troncoso, Zhen Zhao, Joel Kim, Amir Giladi, Nausicaa Malissen, Fiona Desland, Jessica Le Berichel, Ido Amit, Ephraim Kenigsberg, Myron Schwartz, Thomas Marron, Miriam Merad. Characterization of molecular and spatial diversity of macrophages in hepatocellular carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 64.

Throughout the field of oncology, immunotherapy is moving further towards the first-line setting, and there is encouraging data for the use of these novel therapies in the management of lymphomas, utilizing treatments approved for both solid and hematologic malignancies. Herein, we review promising advances in this rapidly moving field from the past year.In the last year, we have seen promising clinical data on engineered antibody therapies for the treatment of lymphomas, as well as further optimization of engineered antibody fragments fused onto linkers or chimeric T cell receptors, both of the modalities capable of transforming non-specific T cells into tumor-specific, serial killer cells. Here we will review the promising data on these advances in antibody-based therapies, as well as some of the immunomodulators and checkpoint-blocking therapies that shown to have promising results in the treatment of lymphomas within the past year.

Given its ability to induce both humoral and cellular immune responses, NY-ESO-1 has been considered a suitable antigen for a cancer vaccine. Despite promising results from early-phase clinical studies in patients with melanoma, NY-ESO-1 vaccine immunotherapy has not been widely investigated in larger trials; consequently, many questions remain as to the optimal vaccine formulation, predictive biomarkers, and sequencing and timing of vaccines in melanoma treatment. We conducted an adjuvant phase I/II clinical trial in high-risk resected melanoma to optimize the delivery of poly-ICLC, a TLR-3/MDA-5 agonist, as a component of vaccine formulation. A phase I dose-escalation part was undertaken to identify the MTD of poly-ICLC administered in combination with NY-ESO-1 and montanide. This was followed by a randomized phase II part investigating the MTD of poly-ICLC with NY-ESO-1 with or without montanide. The vaccine regimens were generally well tolerated, with no treatment-related grade 3/4 adverse events. Both regimens induced integrated NY-ESO-1–specific CD4+ T-cell and humoral responses. CD8+ T-cell responses were mainly detected in patients receiving montanide. T-cell avidity toward NY-ESO-1 peptides was higher in patients vaccinated with montanide. In conclusion, NY-ESO-1 protein in combination with poly-ICLC is safe, well tolerated, and capable of inducing integrated antibody and CD4+ T-cell responses in most patients. Combination with montanide enhances antigen-specific T-cell avidity and CD8+ T-cell cross-priming in a fraction of patients, indicating that montanide contributes to the induction of specific CD8+ T-cell responses to NY-ESO-1.

T-cell transfer into lymphodepleted recipients induces homeostatic activation and potentiates antitumor efficacy. In contrast to canonical T-cell receptor–induced activation, homeostatic activation yields a distinct phenotype and memory state whose regulatory mechanisms are poorly understood. Here, we show in patients and murine models that, following transfer into lymphodepleted bone marrow transplant (BMT) recipients, CD8+ T cells undergo activation but also simultaneous homeostatic inhibition manifested by upregulation of immune-checkpoint molecules and functional suppression. T cells transferred into BMT recipients were protected from homeostatic inhibition by PD-1/CTLA4 dual checkpoint blockade (dCB). This combination of dCB and BMT—”immunotransplant”—increased T-cell homeostatic activation and antitumor T-cell responses by an order of magnitude. Like homeostatic activation, homeostatic inhibition is IL7/IL15-dependent, revealing mechanistic coupling of these two processes. Marked similarity in ex vivo modulation of post-BMT T cells in mice and patients is promising for the clinical translation of immunotransplant (NCT03305445) and for addressing homeostatic inhibition in T-cell therapies. Significance: For optimal anticancer effect, T-cell therapies including chimeric antigen receptor T-cell, tumor-infiltrating lymphocyte, and transgenic T-cell therapies require transfer into lymphodepleted recipients and homeostatic activation; however, concomitant homeostatic inhibition mitigates T-cell therapies' efficacy. Checkpoint blockade uncouples homeostatic inhibition from activation, amplifying T-cell responses. Conversely, tumors nonresponsive to checkpoint blockade or BMT are treatable with immunotransplant. See related commentary by Ansell, p. 1487. This article is highlighted in the In This Issue feature, p. 1469

Immune checkpoint inhibitors (ICIs) have improved overall survival for cancer patients, however, optimal duration of ICI therapy has yet to be defined. Given ICIs were first used to treat patients with metastatic melanoma, a condition that at the time was incurable, little attention was initially paid to how much therapy would be needed for a durable response. As the early immunotherapy trials have matured past 10 years, a significant per cent of patients have demonstrated durable responses; it is now time to determine whether patients have been overtreated, and if durable remissions can still be achieved with less therapy, limiting the physical and financial toxicity associated with years of treatment. Well-designed trials are needed to identify optimal duration of therapy, and to define biomarkers to predict who would benefit from shorter courses of immunotherapy. Here, we outline key questions related to health, financial and societal toxicities of over treating with ICI and present four unique clinical trials aimed at exposing criteria for early cessation of ICI. Taken together, there is a serious liability to overtreating patients with ICI and future work is warranted to determine when it is safe to stop ICI.

Background: Antibiotic exposure has been associated with worse outcomes with immune checkpoint inhibitors (ICIs) in cancer patients, likely due to disruption of the gut microbiome. Other commonly prescribed medications, such as proton pump inhibitors (PPIs) and histamine-2-receptor antagonists (H2RAs), are also known to disrupt the microbiome, but data on their association with ICI outcomes are conflicting. Methods: We conducted a retrospective, multicenter, international cohort study including 314 hepatocellular carcinoma (HCC) patients treated with ICIs from 2017 to 2019 to assess the association between PPI or H2RA exposure (up to 30 days before ICI) and overall survival. Secondary outcomes included overall response rate (ORR) and development of any treatment-related adverse events (AEs). Results: Baseline PPI/H2RA exposure was not associated with overall survival in univariable (HR 1.01, 95% CI 0.75–1.35) or multivariable analysis (HR 0.98, 95% CI 0.71–1.36). Baseline PPI/H2RA exposure was not associated with either ORR (OR 1.32, 95% CI 0.66–2.65) or AEs (OR 1.07, 95% CI 0.54–2.12) in multivariable analysis. Conclusions: Our results suggest that exposure to PPI/H2RA prior to ICIs does not adversely affect outcomes in HCC patients.

The clonal B-cell immunoglobulin idiotype found on the surface of lymphomas was the first targeted tumor-specific antigen, and combinations of idiotype with classical and novel adjuvants were shown to stimulate robust humoral and cellular responses, though clinical efficacy was more variable. Cellular and in situ vaccination to help target a wider array of tumor-specific antigens have also been able to stimulate tumor-specific cellular responses, though their clinical success has also been limited. Our growing understanding of the role of regulatory cells and the immunosuppressive tumor microenvironment, along with a wide variety of immunomodulatory agents developed as of late, offer promising adjuvants to potentiate the immune responses elicited by these vaccine protocols and to achieve durable remissions.

Background: Mutation-derived tumor antigens (MTAs) arise as a result of somatic mutations, such as nucleotide substitutions and small insertions/deletions. MTAs can serve as specific targets for antitumor therapy, including neoantigen vaccines. The goal of neoantigen vaccination is to help prime T cells to recognize such tumor-specific mutations. Here we describe a phase I trial testing a personalized genomic vaccine (PGV-001) in multiple histologies in the adjuvant setting (NCT02721043). Methods: This trial included patients with histologic diagnosis of solid malignancies or multiple myeloma with a >30% risk of recurrence but no measurable disease at the time of first vaccination. For each patient, HLA typing was performed, and the patient's tumor and germline DNA and tumor RNA were sequenced. Mutated peptides containing predicted neoantigens were selected using the OpenVax computational pipeline, which prioritizes somatic mutations by expression of the mutant allele in the tumor RNA and predicted MHC class I epitope binding for the patient's HLA type. A maximum of 10 peptides was included in each patient's personalized vaccine. The vaccine was administered over the course of 6 months, given in combination with poly-ICLC as the adjuvant. The primary objectives were to determine safety and tolerability, feasibility, and immunogenicity of the PGV-001 neoantigen vaccine. Results: The neoantigen vaccine was successfully administered to 13 patients, spanning 5 different tumor types. For each patient, an average of 1730 somatic mutations were identified (range 521-5106), of which 349 were coding variants (range 68-1493), 88 were coding and expressed in the tumor RNA (range 9-233), and 71 were coding, expressed and resulted in predicted MHC class I ligands (range 8-193). The urothelial, head & neck, and lung tumors resulted in larger numbers of predicted neoantigens than in the cases of multiple myeloma or breast tumors. Despite this difference, each of the 13 patient tumor samples showed enough of a neoantigen load to enable vaccination. We have also initiated analysis of immunogenicity, and early reports demonstrate neoantigen-specific CD4 and CD8 T-cell responses. Conclusions: The PGV-001 personalized neoantigen vaccine was successfully administered to 13 patients, where each patient's neoantigen load was adequate for vaccine synthesis. While we have demonstrated the computational feasibility of identification of neoantigens for inclusion in the PGV-001 genomic vaccine, clinical outcomes will be reported separately. Citation Format: Julia Kodysh, Thomas Marron, Alex Rubinsteyn, Tim O'Donnell, John Finnigan, Ana Blazquez, Mansi Saxena, Marcia Meseck, Philip Friedlander, Nina Bhardwaj. PGV-001: A phase I trial of a multipeptide personalized neoantigen vaccine in the adjuvant setting [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr CT173.

The past decade has seen clinical cancer research turn away from refining cytotoxic therapies to identifying immunomodulatory therapies that can harness the immune system to do the work of fighting cancer for us. The first revolution in cancer therapy brought us potent but toxic chemotherapies that were often initially good at controlling tumors; however, in the metastatic setting, recurrence was typically inevitable and more difficult to control. Immunotherapy has now emerged as among the most promising class of therapeutics for the treatment of cancer. These therapies harness the potent tumoricidal potential of cytotoxic effector cells of the patient’s own immune system. Although checkpoint-blocking antibodies approved in the past decade offer great promise, they non-specifically release the brakes on immune cells, and the majority of cancer patients are, so far, non-responders, while some patients experience toxic autoimmune adverse effects. One approach to improve on these limitations is with localized, in situ therapies, inducing a vaccinal response within the tumor microenvironment. These approaches alter the tumor microenvironment by inducing local immunogenic cell death to release tumor antigen, recruiting inflammatory leukocytes, priming of the adaptive immune response, and inhibiting tolerogenic mechanisms through which tumors dampen the immune response. Through a combined approach using some or all of these features, we are working to develop an in situ vaccine strategy that will result in systemic elimination of tumor cells as well as long-term remissions.

e14307 Background: Mutation-derived tumor antigens (MTAs) arise as a direct result of somatic variations that occur during carcinogenesis and can be characterized via genetic sequencing and used to identify MTAs. We developed a platform for a fully-personalized MTA-based vaccine in the adjuvant treatment of solid and hematological malignancies. Methods: This is a single-arm, open label, proof-of-concept phase I study designed to test the safety and immunogenicity of Personalized Genomic Vaccine 001 (PGV001) that targets up to 10 predicted personal tumor neoantigens based on patient’s HLA profile (ClinicalTrials.gov: NCT02721043). Results: Patients who completed vaccination with PGV001_002 (head and neck squamous cell cancer) received 10 doses of vaccine comprising 10 long peptides (LP) combined with poly-ICLC (toll-like receptor-3 agonist) intradermally. Vaccine-induced T-cell responses were determined at weeks 0 and 27 (before and after treatment, respectively), ex vivo by interferon (IFN)-g enzyme-linked immunospot assay and after expansion by intracellular cytokine staining. Overlapping 15-mer peptides (OLPs) spanning the entirety of each LP and 9-10-mer peptides corresponding to each predicted class I epitope (Min) were pooled. Ex vivo responses to these peptide pools were undetectable at week 0 but were evident at week 27 against 2 OLPs out of 10 (20%) and in 5 Min out of 10 (50%). After in vitro expansion, neoantigen-specific CD4+ and CD8+ T-cell responses were found in 5 out of 10 pooled peptides (50%). 7 out of 10 (70%) epitopes elicited polyfunctional T-cell responses (secretion of INF-g, TNF-a, and/or IL-2) from either CD4+ or CD8+ T cells. Conclusions: The PGV001 vaccine in our first patient showed both safety and immunogenicity, eliciting CD4+ and CD8+ responses to the vaccine peptides. As we enroll additional patients in this clinical trial, and perform deeper phenotyping of their tumor-reactive T cells, we will learn the determinants necessary for the successful generation of MTA-based vaccines, while informing future immunotherapeutic approaches and rational combinations. Clinical trial information: NCT02721043.

Introduction: Mutation-derived tumor antigens (MTAs) arise as a direct result of somatic variations, including nucleotide substitutions, insertions, and deletions that occur during carcinogenesis. These somatic variations can be characterized via genetic sequencing and used to identify MTAs. We developed a platform for a fully-personalized MTA-based vaccine in the adjuvant treatment of solid and hematologic malignanicies. Methods: This is a single-arm, open label, proof-of-concept phase I study designed to test the safety and immunogenicity of Personalized Genomic Vaccine 001 (PGV001) that targets up to 10 predicted personal tumor neoantigens. The single-center study will enroll 20 eligible subjects with histologic diagnosis of solid and hematologic malignancies. Subjects must have no measurable disease at time of first vaccine administration, and 5-year disease recurrence risk of > 30%. Toxicity will be defined by CTCAE v5.0. Blood samples will be collected at various time points for immune response monitoring. Each patient’s vaccine peptides are selected by identifying somatic mutations from comparison of tumor and normal exome sequencing data, phasing somatic variants with co-occurring germline variants using tumor RNA sequencing data, and ranking mutated peptide sequences ”Openvax pipeline.” The process for determining somatic variants hews closely to the Broad Institute’s “Best Practices” for cancer SNVs and indels. The phasing of somatic and germline variants is implemented in a custom bioinformatics tool called Isovar. Mutated protein sequences containing phased variants are ranked according to two criteria: expression of the mutant allele in tumor RNA and aggregated predicted affinity to the patient’s Class I MHCs. Both quantities are normalized and multiplied together to create single ranked ordering of the candidate mutant sequences. Results: PGV001_002 (head and neck squamous cell cancer), who has completed vaccination, received 10 doses of vaccine comprising 10 long peptides (25 amino acid length) combined with poly-ICLC (toll-like receptor-3 agonist) intradermally. Vaccine-induced blood T-cell responses were determined, at weeks 0 (before-treatment) and 27 (after-treatment), ex vivo by interferon (IFN)-g enzyme-linked immunospot (ELISPOT) assay and after in vitro expansion by intracellular cytokine staining (ICS). Overlapping 15-16-mer assays peptides (OLPs) spanning the entirety of each ILP and 9-10-mer peptides corresponding to each predicted class I epitope (Min) were pooled and used to monitor immunogenicity. Ex vivo responses to these peptide pools were undetectable at week 0 but were evident at week 27 against 2 OLPs out of 10 (20%) and in 5 Min out of 10 (50%). After in vitro expansion, neoantigen-specific CD4+ and CD8+ T-cell responses were found in 5 out of 10 pooled peptides (50%). 7 out of 10 (70%) epitopes elicited polyfunctional T-cell responses (secretion of INF-α, TNF-α, and/or IL-2) from either CD4+ or CD8+ T-cells. Conclusion: To identify which predicted epitopes within the peptides pools stimulated the T-cell responses, we deconvoluted all the pools by either ex vivo and in vitro expansion. Ex vivo IFN-α production was detected in 1 (15-mer) peptide out of 15 (6.7%) and in 4 (9-10-mer) peptides out of 22 (18.2%). After expansion with single peptides, of 22 (9-10-mer) peptides tested, CD8+ T-cells were reactive against 13 peptides (59%), while CD4+ responses were seen in response to 11 of 15 (15-16-mer) peptides tested. Both CD4+ and CD8+ T-cell responses were polyfunctional. The PGV001 vaccine in our first patient showed both safety and immunogenicity, eliciting both CD4+ and CD8+ responses to the vaccine peptides. As we are enrolling additional patients, the information learned from this clinical trial will instruct the next generation of MTA-based vaccines, future development of immunotherapeutic approaches and rational combinations. Citation Format: Ana B. Blazquez, Alex Rubinsteyn, Julia Kodysh, John P. Finnigan, Thomas Marron, Rachel L. Sabado, Marcia Meseck, Timothy J. O'Donnell, Jeffrey Hammerbacher, Michael Donovan, John Holt, Milind Mahajan, John Mandeli, Krysztof Misiukiewicz, Eric M. Genden, Brett A. Milles, Hooman Khorasani, Peter R. Dottino, Hanna Irie, Amy B. Tiersten, Elisa R. Port, Andrea S. Wolf, Hern J. Cho, Ashutosh Tewari, Samir S. Parekh, Sujit Nair, Matthew D. Galsky, William K. Oh, Sacha Gnjatic, Eric E. Schadt, Phillip A. Friedlander, Nina Bhardwaj. A phase I study of the safety and immunogenicity of a multipeptide personalized genomic vaccine in the adjuvant treatment of solid cancers [abstract]. In: Proceedings of the Fourth CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; Sept 30-Oct 3, 2018; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2019;7(2 Suppl):Abstract nr A005.

O1 IL-15 primes an mTOR-regulated gene-expression program to prolong anti-tumor capacity of human natural killer cells #### Andreas Lundqvist1, Vincent van Hoef1, Xiaonan Zhang1, Erik Wennerberg2, Julie Lorent1, Kristina Witt1, Laia Masvidal Sanz1, Shuo Liang1, Shannon Murray3, Ola Larsson1

Lymphomas, with an estimated 80,000 new cases, are the 5th most common cancer in the United States and the majority of lymphoma subtypes are incurable and in need of novel, mechanistically distinct therapies. Two approaches to potently increase the anti-tumor potential of T cells are: checkpoint blockade (e.g. anti-CTLA-4 and anti-PD-1 antibodies) and adoptive T-cell transfer into lympho-depleted recipients. We have developed a novel therapy combining these approaches into ‘checkpoint-blockade-primed immunotransplant’ comprised of: -treatment of tumor-bearing host with anti-CTLA-4 and/or anti-PD-1 antibodies -splenocyte harvest and transfer to lympho-depleted recipient Whereas checkpoint blockade alone has minimal anti-tumor effect in the pre-clinical model, our results show that the combined therapy results in superior anti-tumor immunity as seen by increased production of tumor-reactive IFN γ positive T-cells. Treatment of both tumor-bearing donor and recipient with checkpoint antibodies induces cure of the majority of recipients, in a CD8, NK, and IFNγ-dependent manner. Furthermore, we have demonstrated that CD8 T cells exposed to checkpoint blockade and transfer into the lymphopenic host demonstrate greater: -in vivo serum levels of IL-15 and IL-7 -surface expression levels of IL-15R and IL-7R -in vitro STAT5 phosphorylation in response to common γ-chain cytokines -in vivo proliferation in response to exposure to cognate tumor antigen Ongoing studies will seek to assess the dependence of the above observations (cytokine production, proliferation, anti-tumor effect) on specific common γ-chain cytokines, the role of lymphopenia in inducing T cell trafficking to tumor versus healthy tissue, and guide development of the immunotransplant model to optimize the amplification of anti-tumor immunity for near-term clinical translation. Citation Format: Netonia Marshall, Thomas Marron, Judith Agudo, Brian Brown, Joshua Brody. Immunotransplant: Merging checkpoint blockade and T-cell transfer into lymphodepleted recipients [abstract]. In: Proceedings of the Second CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; 2016 Sept 25-28; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(11 Suppl):Abstract nr B107.

Background: Lymphomas are the 5th most common cancer in the United States, 40% of these are cases are indolent non-Hodgkin's lymphoma (iNHL) and are incurable with standard therapy. A previous trial of in situ vaccination in iNHLs, in which intratumoral CpG, the TLR9 agonist, was combined with low dose radiation to induce a systemic immune response against tumor. In this trial induction of tumor-specific CD8 T cell responses and durable clinical remissions of patients’ untreated sites of disease was seen in some patients. One limitation in this previous trial may have been the scarcity of intratumoral dendritic cells (DC) and the suppressive tumor microenvironment. DC are uniquely able to endocytose dying (e.g. irradiated) tumor cells for cross-presentation to anti-tumor CD8 T cells. In this new iteration of in situ vaccine, Flt3L is added as a priming step to increase the presence of intratumoral DCs ahead of vaccination. Fms-like tyrosin kinase 3 ligand (Flt3L) induces tumor leukocyte infiltration and regression in lymphoma tumors in pre-clinical trials, and CDX-301- a formulation of Flt3L - was shown to mobilize BDCA-1 and BDCA-3 myeloid DC subsets in an early phase trial. These DC subsets respond to several TLR agonists and cross-present antigens more effectively than plasmacytoid DCs. While pDCs are high expressors of TLR9, responsive to CpG, myeloid dendritic cells express a wider array of TLRs, including high levels of TLR3. Methods: This Phase I/II trial tests the hypothesis that this novel in situ vaccination will induce clinical remissions at distant (untreated) tumor sites in two cohorts of patients with either previously untreated or relapsed/refractory iNHL (n = 15 per group). Intratumoral CDX-301 25ug/kg is injected into a palpable lymph node for 9 days, followed 2Gy local radiotherapy on day 9 and 10 to the target lymph node. On day 10, following radiation therapy, intratumoral poly-ICLC 2mg is injected to activate local DCs. Poly-ICLC 2mg is then injected on day 14, day 17 (1 week after initial dose) and weekly thereafter for a total of 9 treatments over 8 weeks. Response is assessed with CT scans every three months as per the Revised Response Criteria for Malignant Lymphoma, also known as the Cheson criteria, and leukemic phase of lymphoma monitored by peripheral blood flow cytometry. The trial started accrual in January 2014 and is ongoing, 11 have been enrolled including 8 untreated patients and 3 with refractory disease. To perform correlative studies during the clinical trial we perform excisional biopsy of a distal lymph node before starting trial to assess baseline immunomodulatory or immunosuppressive characteristics of a patients tumor microenvironment. Fine needle aspirate (FNA) of the target tumor (site of Flt3L, Radiation and poly-ICLC administration) treatment at weeks 0, 2, 4 and 6, after which target tumor is significantly smaller and biopsy not typically possible. Blood is collected for immune monitoring at weeks 0, 2, 4, 6, 8, 12 to assess for transient enrichment of DC populations, and monitoring for tumor-specific effector T cell populations. Citation Format: Thomas U. Marron, Nina Bhardwaj, Linda Hammerich, Seunghee Kim-Schulze, Tibor Keler, Tom Davis, Elizabeth Crowley, Andres M. Salazar, Joshua Brody. In situ vaccination of low-grade lymphoma with intratumoral Flt3L and poly-ICLC with low-dose radiotherapy. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr LB-081.

Background Bruton tyrosine kinase (BTK) is a component of signaling pathways downstream from Toll-like receptors (TLRs) 2, 4, 7, 8, and 9. Previous work in BTK-deficient mice, cell lines, and cultured cells from patients with X-linked agammaglobulinemia (XLA) suggested defective TLR-driven cytokine production. Objective We sought to compare TLR-4–, TLR-7–, and TLR-8–induced cytokine production of primary cells from patients with XLA with that seen in control cells. Methods PBMCs from patients with XLA, freshly isolated plasmacytoid dendritic cells, monocytes, and monocytoid dendritic cells were activated with TLR-4, TLR-7, and TLR-8 agonists. Signaling intermediates and intracellular and secreted cytokine levels were compared with those seen in control cells. Results Although TLR-4, TLR-7, and TLR-8 activation of nuclear factor κB and mitogen-activated protein kinase pathways in cells from patients with XLA and control cells were comparable, TLR-activated freshly isolated monocytes and monocytoid dendritic cells from patients with XLA produced significantly more TNF-α, IL-6, and IL-10 than control cells. TLR-7/8–activated plasmacytoid dendritic cells produced normal amounts of IFN-α. In murine models BTK regulates the degradation of Toll–IL-1 receptor domain–containing adaptor protein, terminating TLR-4–induced cytokine production. Although this might explain the heightened TLR-4–driven cytokine production we observed, Toll–IL-1 receptor domain–containing adaptor protein degradation is intact in cells from patients with XLA, excluding this explanation. Conclusion In contrast to previous studies with BTK-deficient mice, cell lines, and cultured cells from patients with XLA suggesting impaired TLR-driven cytokine production, these data suggest that BTK inhibits TLR-induced cytokine production in primary human cells.

Background: Lymphomas are the 5th most common cancer in the U.S. and most are incurable with standard therapy. Previously, we completed three trials of ‘in situ vaccination’ - combining low-dose radiotherapy (XRT) with intratumoral administration of TLR9 agonist (CpG). We demonstrated induction of anti-tumor CD8 T cell responses and clinical remissions of patients' non-irradiated sites of disease, lasting up to 4+ years. One limitation may have been the paucity of intratumoral dendritic cells (DC). DC are uniquely able to endocytose dying (e.g. irradiated) tumor cells for cross-presentation to anti-tumor CD8 T cells. Methods: Flt3L – the predominant DC differentiation factor– induces tumor leukocyte infiltration and regression of lymphoma tumors pre-clinically and a new formulation of this cytokine -CDX-301- was shown to mobilize BDCA-1 and BDCA-3 DC subsets in an early phase trial. These DC subsets respond to several TLR agonists and cross-present antigens more effectively than plasmacytoid DC (the CpG-responsive DC subset). We initiated a phase I/II study of a new iteration of the in situ vaccine, adding Flt3L-priming and replacing the prior TLR9 agonist with the TLR3 agonist poly-ICLC (Fig 1A). The vaccine consists of: intratumoral Flt3L administration to increase DC within the tumorlow-dose XRT to induce immunogenic tumor cell death and release tumor-associated antigens, andintratumoral poly-ICLC administration to activate tumor antigen-loaded DC. Results: Six patients have been enrolled, two patients have completed therapy. Treated patients had 2-200-fold increases in BDCA1 and BDCA3 intratumoral DC after Flt3L administration and marked DC activation after XRT and poly-ICLC. Both treated patients have had partial remissions of untreated sites per Cheson criteria, persisting or improving for >6 months after vaccination. These include regressions of bulky lymph nodes (Fig 1B), as well as peripheral blood (Fig1C) and bone marrow disease. A patient with significant peripheral blood tumor burden experienced >10-fold decrease in malignant B cells with concurrent increase in non-tumor B cells, suggesting a degree of cell specificity in the tumor-killing mechanism. Conclusions: Preliminary results suggest that the Flt3L-primed in situ vaccine is feasible, safe and immunologically and clinically effective, warranting further study. Research supported by: Damon Runyon Cancer Research Foundation Clinical Investigator Award Lymphoma Research Foundation Clinical Research Mentorship Program Citation Format: Thomas Marron, Nina Bhardwaj, Elizabeth Crowley, Tibor Keler, Thomas Davis, Andres Salazar, Joshua Brody. Turning a tumor into a vaccine factory: In situ vaccination for low-grade lymphoma. [abstract]. In: Proceedings of the AACR Special Conference: Tumor Immunology and Immunotherapy: A New Chapter; December 1-4, 2014; Orlando, FL. Philadelphia (PA): AACR; Cancer Immunol Res 2015;3(10 Suppl):Abstract nr IA03.

BACKGROUND: Lymphomas are the 5th most common cancer in the U.S. and most are incurable with standard therapy. Previously, we completed three trials of ‘in situ vaccination’ - combining low-dose radiotherapy (XRT) with intratumoral administration of TLR9 agonist (CpG). We demonstrated induction of anti-tumor CD8 T cell responses and clinical remissions of patients’ non-irradiated sites of disease, lasting up to 4+ years. One limitation may have been the paucity of intratumoral dendritic cells (DC). DC are uniquely able to endocytose dying (e.g. irradiated) tumor cells for cross-presentation to anti-tumor CD8 T cells. METHODS: Flt3L– the predominant DC differentiation factor– induces tumor leukocyte infiltration and regression of lymphoma tumors pre-clinically and a new formulation of this cytokine -CDX-301- was shown to mobilize BDCA-1 and BDCA-3 DC subsets in an early phase trial. These DC subsets respond to several TLR agonists and cross-present antigens more effectively than plasmacytoid DC (the CpG-responsive DC subset). We initiated a phase I/II study of a new iteration of the in situ vaccine, adding Flt3L-priming and replacing the prior TLR9 agonist with the TLR3 agonist poly-ICLC (Fig 1A). The vaccine consists of: -intratumoral Flt3L administration to increase DC within the tumor -low-dose XRT to induce immunogenic tumor cell death and release tumor-associated antigens, and -intratumoral poly-ICLC administration to activate tumor antigen-loaded DC. RESULTS: Six patients have been enrolled, two patients have completed therapy. Treated patients had 2-200-fold increases in BDCA1 and BDCA3 intratumoral DC after Flt3L administration and marked DC activation after XRT and poly-ICLC. Both treated patients have had partial remissions of untreated sites per Cheson criteria, persisting or improving for >6 months after vaccination. These include regressions of bulky lymph nodes (Fig 1B), as well as peripheral blood (Fig1C) and bone marrow disease. A patient with significant peripheral blood tumor burden experienced >10-fold decrease in malignant B cells with concurrent increase in non-tumor B cells, suggesting a degree of cell specificity in the tumor-killing mechanism. Adverse effects have been mild. CONCLUSIONS: Preliminary results suggest that the Flt3L-primed in situ vaccine is feasible, safe and immunologically and clinically effective, warranting further study. Figure 1 Figure 1. Disclosures Off Label Use: CDX-301 - the purpose of which is to mobilize dendritic cells to an injected tumor site.. Crowley:Celldex Therapeutics Inc: Employment. Keler:Celldex Therapeutics, Inc.: Employment. Davis:Celldex Therapeutics Inc: Employment.