OBJECTIVE: Multiple common cancers benefit from immunotherapy; however, less is known about efficacy in rare tumors. We report the results of the adrenocortical carcinoma cohort of NCI/SWOG S1609 Dual Anti-CTLA-4 and Anti-PD-1 blockade in Rare Tumors.
METHODS: A prospective, phase 2 clinical trial of ipilimumab plus nivolumab was conducted by the SWOG Early Therapeutics and Rare Cancers Committee for multiple rare tumor cohorts across >1,000 National Clinical Trial Network sites.
METHODS: 21 eligible patients were registered. Median age was 53 years (range 26-69); 16 (76%) were women.
METHODS: Ipilimumab 1 mg/kg intravenously every 6 weeks with nivolumab 240 mg intravenously every 2 weeks was administered until disease progression, symptomatic deterioration, treatment delay for any reason >56 days, unacceptable or immune-related toxicity with inability to decrease prednisone to <10 mg daily, or per patient request.
METHODS: The primary endpoint was the overall response rate (ORR) (RECIST V.1.1). Secondary endpoints include clinical benefit rate (CBR) (includes stable disease (SD)>6 months), progression-free survival (PFS), overall survival (OS), and toxicity. Immune-related outcomes included immune ORR (iORR), immune CBR (iCBR), and immune PFS (iPFS). A two-stage design was used assuming: null=5% alternative=30%, n=6 in the first stage, 16 max, one-sided alpha=13%.
RESULTS: The median number of prior therapy lines was 2 (range: 1-9). 3 of 21 patients attained confirmed partial response (PR) (ORR=14%). In addition, one patient had an unconfirmed PR; one, stable disease (SD)>6 months; one, immune-related RECIST (iRECIST) PR (iPR); and one patient attained iSD>6 months: clinical benefit rate (response or SD>6 months)=5/21 (24%), iORR=4/21 (19%), iCBR=7/21 (33%). The 6-month PFS was 24%; 6-month iPFS, 33%. The PFS for patients (N=7) with iRECIST clinical benefit were 57, 52, 18, 15, 13, 7, and 7 months. The 6-month OS was 76%; the median OS, was 15.8 months. The most common toxicities were fatigue (62%) and rash (38%), and the most common grade 3/4 immune-related adverse events were hepatic dysfunction (9.5%) and adrenal insufficiency (9.5%). Treatment-related adverse events leading to discontinuation of therapy in four patients (21%). There were no grade 5 adverse events.
CONCLUSIONS: Ipilimumab plus nivolumab is active in refractory metastatic adrenocortical cancer meeting the primary endpoint of the study, with a 19% iORR and 33% iCBR (includes SD/iSD>6 months) and with the longest PFS/iPFS of 52 and 57 months.
BACKGROUND: NCT02834013 (registered 15 July, 2016; https://clinicaltrials.gov/ct2/show/NCT02834013).

BACKGROUND: Natural killer (NK) cells are being extensively studied as a cell therapy for cancer. These cells are activated by recognition of ligands and antigens on tumor cells. Cytokine therapies, such as IL-15, are also broadly used to stimulate endogenous and adoptively transferred NK cells in patients with cancer. These stimuli activate the membrane protease ADAM17, which cleaves various cell-surface receptors on NK cells as a negative feedback loop to limit their cytolytic function. ADAM17 inhibition can enhance IL-15-mediated NK cell proliferation in vitro and in vivo. In this study, we investigated the underlying mechanism of this process.
METHODS: Peripheral blood mononuclear cells (PBMCs) or enriched NK cells from human peripheral blood, either unlabeled or labeled with a cell proliferation dye, were cultured for up to 7 days in the presence of rhIL-15±an ADAM17 function-blocking antibody. Different fully human versions of the antibody were generated; Medi-1 (IgG1), Medi-4 (IgG4), Medi-PGLALA, Medi-F(ab\')2, and TAB16 (anti-ADAM17 and anti-CD16 bispecific) to modulate CD16A binding. Flow cytometry was used to assess NK cell proliferation and phenotypic markers, immunoblotting to examine CD16A signaling, and IncuCyte-based live cell imaging to measure NK cell antitumor activity.
RESULTS: The ADAM17 function-blocking monoclonal antibody (mAb) Medi-1 markedly increased early NK cell activation by IL-15. By using different engineered versions of the antibody, we demonstrate involvement by CD16A, an activating Fcγ receptor and well-described ADAM17 substrate. Hence, Medi-1 when bound to ADAM17 on NK cells is engaged by CD16A and blocks its shedding, inducing and prolonging its signaling. This process did not promote evident NK cell fratricide or dysfunction. Synergistic signaling by Medi-1 and IL-15 enhanced the upregulation of CD137 on CD16A+ NK cells and augmented their proliferation in the presence of PBMC accessory cells or an anti-CD137 agonistic mAb.
CONCLUSIONS: Our data reveal for the first time that CD16A and CD137 underpin Medi-1 enhancement of IL-15-driven NK cell activation and proliferation, respectively, with the latter requiring PBMC accessory cells. The use of Medi-1 represents a novel strategy to enhance IL-15-driven NK cell proliferation, and it may be of therapeutic importance by increasing the antitumor activity of NK cells in patients with cancer.

BACKGROUND: Cervical cancer\'s lymphatic spread primarily begins from the sentinel lymph nodes (SLNs), underlining their pivotal role in disease metastasis. However, these nodes\' immune gene expression profiles and immunoregulation mechanisms have yet to be explored.
METHODS: Our study aimed to elucidate the immune cell populations and their roles in the immune gene expression profile of negative SLNs compared with positive SLNs and non-SLNs using Nanostring RNA seq analysis. We performed a principal component analysis on the log2 normalized expression of 685 endogenous genes in the nCounter PanCancer Immune Profiling Panel, followed by an assessment of the differential expression of genes and immune cell type abundance.
RESULTS: We found significant variations in gene expression among the groups, with negative SLNs displaying overexpression of genes related to tumor-infiltrating immune cells, specifically innate cell populations. They also demonstrated the upregulation of genes involved in antigen presentation and T-cell priming. In contrast, positive SLNs were enriched in regulatory networks, suggesting their potential role in immune evasion. A comparison of negative SLNs and non-SLNs revealed increased innate and adaptive immune cell types, underscoring the ongoing T cell response to tumor antigens.
CONCLUSIONS: Our findings underscore a specific immunogenetic phenotype profile in negative SLNs, emphasizing their crucial role in the initial anticancer response, immunosurveillance, and the propagation of immune tolerance from the primary cervical tumor. These results highlight the potential of SLNs as a novel target for immunotherapy strategies and underscore the importance of new imaging methods for accurately identifying SLN status without removal. Future investigations are needed to understand further the immunological interplay within SLNs and their influence on cervical cancer progression.

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.

BACKGROUND: OX40 has been widely studied as a target for immunotherapy with agonist antibodies taken forward into clinical trials for cancer where they are yet to show substantial efficacy. Here, we investigated potential mechanisms of action of anti-mouse (m) OX40 and anti-human (h) OX40 antibodies, including a clinically relevant monoclonal antibody (mAb) (GSK3174998) and evaluated how isotype can alter those mechanisms with the aim to develop improved antibodies for use in rational combination treatments for cancer.
METHODS: Anti-mOX40 and anti-hOX40 mAbs were evaluated in a number of in vivo models, including an OT-I adoptive transfer immunization model in hOX40 knock-in (KI) mice and syngeneic tumor models. The impact of FcγR engagement was evaluated in hOX40 KI mice deficient for Fc gamma receptors (FcγR). Additionally, combination studies using anti-mouse programmed cell death protein-1 (mPD-1) were assessed. In vitro experiments using peripheral blood mononuclear cells (PBMCs) examining possible anti-hOX40 mAb mechanisms of action were also performed.
RESULTS: Isotype variants of the clinically relevant mAb GSK3174998 showed immunomodulatory effects that differed in mechanism; mIgG1 mediated direct T-cell agonism while mIgG2a acted indirectly, likely through depletion of regulatory T cells (Tregs) via activating FcγRs. In both the OT-I and EG.7-OVA models, hIgG1 was the most effective human isotype, capable of acting both directly and through Treg depletion. The anti-hOX40 hIgG1 synergized with anti-mPD-1 to improve therapeutic outcomes in the EG.7-OVA model. Finally, in vitro assays with human peripheral blood mononuclear cells (hPBMCs), anti-hOX40 hIgG1 also showed the potential for T-cell stimulation and Treg depletion.
CONCLUSIONS: These findings underline the importance of understanding the role of isotype in the mechanism of action of therapeutic mAbs. As an hIgG1, the anti-hOX40 mAb can elicit multiple mechanisms of action that could aid or hinder therapeutic outcomes, dependent on the microenvironment. This should be considered when designing potential combinatorial partners and their FcγR requirements to achieve maximal benefit and improvement of patient outcomes.

BACKGROUND: Lung metastasis is the primary cause of breast cancer-related mortality. Neutrophil extracellular traps (NETs) are involved in the progression of breast cancer. However, the mechanism of NET formation is not fully understood. This study posits that tumor cell-released autophagosomes (TRAPs) play a crucial role in this process.
METHODS: TRAPs were isolated from breast cancer cell lines to analyze their impact on NET formation in both human and mouse neutrophils. The study used both in vitro and in vivo models, including Toll-like receptor 4 (TLR4-/-) mice and engineered breast cancer cell lines. Immunofluorescence, ELISA, Western blotting, RNA sequencing, and flow cytometry were employed to dissect the signaling pathways leading to NET production and to explore their immunosuppressive effects, particularly focusing on the impact of NETs on T-cell function. The therapeutic potential of targeting TRAP-induced NETs and their immunosuppressive functions was evaluated using DNase I and αPD-L1 antibodies. Clinical relevance was assessed by correlating circulating levels of TRAPs and NETs with lung metastasis in patients with breast cancer.
RESULTS: This study showed that TRAPs induced the formation of NETs in both human and mouse neutrophils by using the high mobility group box 1 and activating the TLR4-Myd88-ERK/p38 signaling axis. More importantly, PD-L1 carried by TRAP-induced NETs inhibited T-cell function in vitro and in vivo, thereby contributing to the formation of lung premetastatic niche (PMN) immunosuppression. In contrast, Becn1 KD-4T1 breast tumors with decreased circulating TRAPs in vivo reduced the formation of NETs, which in turn attenuated the immunosuppressive effects in PMN and resulted in a reduction of breast cancer pulmonary metastasis in murine models. Moreover, treatment with αPD-L1 in combination with DNase I that degraded NETs restored T-cell function and significantly reduced tumor metastasis. TRAP levels in the peripheral blood positively correlated with NET levels and lung metastasis in patients with breast cancer.
CONCLUSIONS: Our results demonstrate a novel role of TRAPs in the formation of PD-L1-decorated NETs, which may provide a new strategy for early detection and treatment of pulmonary metastasis in patients with breast cancer.

BACKGROUND: The majority of anti-programmed cell-death 1 (PD-1) monoclonal antibodies (mAbs) use S228P mutation IgG4 as the structural basis to avoid the activation of immune cells or complement. However, little attention has been paid to the Fc-Fc interactions between IgG4 and other IgG Fc fragments that could result in adverse effects. Fc-null IgG1 framework is a potential safer alternative to avoid the undesirable Fc-Fc interactions and Fc receptor binding derived effects observed with IgG4. This study provides a comprehensive evaluation of anti-PD-1 mAbs of these two frameworks.
METHODS: Trastuzumab and rituximab (both IgG1), wildtype IgG1 and IgG4 were immobilized on nitrocellulose membranes, coated to microplates and biosensor chips, and bound to tumor cells as targets for Fc-Fc interactions. Wildtype IgG1 and IgG4, anti-PD-1 mAb nivolumab (IgG4 S228P), penpulimab (Fc-null IgG1), and tislelizumab (Fc-null IgG4 S228P-R409K) were assessed for their binding reactions to the immobilized IgG proteins and quantitative kinetic data were obtained. To evaluate the effects of the two anti-PD-1 mAbs on immune responses mediated by trastuzumab and rituximab in the context of combination therapy, we employed classic immune models for antibody-dependent cellular cytotoxicity, antibody-dependent cellular phagocytosis, and complement dependent cytotoxicity. Tumor-bearing mouse models, both wildtype and humanized, were used for in vivo investigation. Furthermore, we also examined the effects of IgG1 and IgG4 on diverse immune cell populations RESULTS: Experiments demonstrated that wildtype IgG4 and nivolumab bound to immobilized IgG through Fc-Fc interactions, diminishing antibody-dependent cell-mediated cytotoxicity and phagocytosis reactions. Quantitative analysis of kinetic parameters suggests that nivolumab and wildtype IgG4 exhibit comparable binding affinities to immobilized IgG1 in both non-denatured and denatured states. IgG4 exerted inhibitory effects on various immune cell types. Wildtype IgG4 and nivolumab both promoted tumor growth in wildtype mouse models. Conversely, wildtype IgG1, penpulimab, and tislelizumab did not show similar adverse effects.
CONCLUSIONS: Fc-null IgG1 represents a safer choice for anti-PD-1 immunotherapies by avoiding both the adverse Fc-Fc interactions and Fc-related immune inhibitory effects of IgG4. Fc-null IgG4 S228P-R409K and Fc-null IgG1 displayed similar structural properties and benefits. This study contributes to the understanding of immunotherapy resistance and the advancement of safer immune therapies for cancer.

Cancer immunotherapy has flourished over the last 10-15 years, transforming the practice of oncology and providing long-term clinical benefit to some patients. During this time, three distinct classes of immune checkpoint inhibitors, chimeric antigen receptor-T cell therapies specific for two targets, and two distinct classes of bispecific T cell engagers, a vaccine, and an oncolytic virus have joined cytokines as a standard of cancer care. At the same time, scientific progress has delivered vast amounts of new knowledge. For example, advances in technologies such as single-cell sequencing and spatial transcriptomics have provided deep insights into the immunobiology of the tumor microenvironment. With this rapid clinical and scientific progress, the field of cancer immunotherapy is currently at a critical inflection point, with potential for exponential growth over the next decade. Recognizing this, the Society for Immunotherapy of Cancer convened a diverse group of experts in cancer immunotherapy representing academia, the pharmaceutical and biotechnology industries, patient advocacy, and the regulatory community to identify current opportunities and challenges with the goal of prioritizing areas with the highest potential for clinical impact. The consensus group identified seven high-priority areas of current opportunity for the field: mechanisms of antitumor activity and toxicity; mechanisms of drug resistance; biomarkers and biospecimens; unique aspects of novel therapeutics; host and environmental interactions; premalignant immunity, immune interception, and immunoprevention; and clinical trial design, endpoints, and conduct. Additionally, potential roadblocks to progress were discussed, and several topics were identified as cross-cutting tools for optimization, each with potential to impact multiple scientific priority areas. These cross-cutting tools include preclinical models, data curation and sharing, biopsies and biospecimens, diversification of funding sources, definitions and standards, and patient engagement. Finally, three key guiding principles were identified that will both optimize and maximize progress in the field. These include engaging the patient community; cultivating diversity, equity, inclusion, and accessibility; and leveraging the power of artificial intelligence to accelerate progress. Here, we present the outcomes of these discussions as a strategic vision to galvanize the field for the next decade of exponential progress in cancer immunotherapy.

Cytokines are small proteins that regulate the growth and functional activity of immune cells, and several have been approved for cancer therapy. Oncolytic viruses are agents that mediate antitumor activity by directly killing tumor cells and inducing immune responses. Talimogene laherparepvec is an oncolytic herpes simplex virus type 1 (oHSV), approved for the treatment of recurrent melanoma, and the virus encodes the human cytokine, granulocyte-macrophage colony-stimulating factor (GM-CSF). A significant advantage of oncolytic viruses is the ability to deliver therapeutic payloads to the tumor site that can help drive antitumor immunity. While cytokines are especially interesting as payloads, the optimal cytokine(s) used in oncolytic viruses remains controversial. In this review, we highlight preliminary data with several cytokines and chemokines, including GM-CSF, interleukin 12, FMS-like tyrosine kinase 3 ligand, tumor necrosis factor α, interleukin 2, interleukin 15, interleukin 18, chemokine (C-C motif) ligand 2, chemokine (C-C motif) ligand 5, chemokine (C-X-C motif) ligand 4, or their combinations, and show how these payloads can further enhance the antitumor immunity of oHSV. A better understanding of cytokine delivery by oHSV can help improve clinical benefit from oncolytic virus immunotherapy in patients with cancer.

BACKGROUND: Tumor-targeted therapy causes impressive tumor regression, but the emergence of resistance limits long-term survival benefits in patients. Little information is available on the role of the myeloid cell network, especially dendritic cells (DC) during tumor-targeted therapy.
METHODS: Here, we investigated therapy-mediated immunological alterations in the tumor microenvironment (TME) and tumor-draining lymph nodes (LN) in the D4M.3A preclinical melanoma mouse model (harboring the V-Raf murine sarcoma viral oncogene homolog B (BRAF)V600E mutation) by using high-dimensional multicolor flow cytometry in combination with multiplex immunohistochemistry. This was complemented with RNA sequencing and cytokine quantification to characterize the immune status of the tumors. The importance of T cells during tumor-targeted therapy was investigated by depleting CD4+ or CD8+ T cells in tumor-bearing mice. Tumor antigen-specific T-cell responses were characterized by performing in vivo T-cell proliferation assays and the contribution of conventional type 1 DC (cDC1) to T-cell immunity during tumor-targeted therapy was assessed using Batf3-/- mice lacking cDC1.
RESULTS: Our findings reveal that BRAF-inhibitor therapy increased tumor immunogenicity, reflected by an upregulation of genes associated with immune activation. The T cell-inflamed TME contained higher numbers of activated cDC1 and cDC2 but also inflammatory CCR2-expressing monocytes. At the same time, tumor-targeted therapy enhanced the frequency of migratory, activated DC subsets in tumor-draining LN. Even more, we identified a cDC2 population expressing the Fc gamma receptor I (FcγRI)/CD64 in tumors and LN that displayed high levels of CD40 and CCR7 indicating involvement in T cell-mediated tumor immunity. The importance of cDC2 is underlined by just a partial loss of therapy response in a cDC1-deficient mouse model. Both CD4+ and CD8+ T cells were essential for therapy response as their respective depletion impaired therapy success. On resistance development, the tumors reverted to an immunologically inert state with a loss of DC and inflammatory monocytes together with the accumulation of regulatory T cells. Moreover, tumor antigen-specific CD8+ T cells were compromised in proliferation and interferon-γ-production.
CONCLUSIONS: Our results give novel insights into the remodeling of the myeloid landscape by tumor-targeted therapy. We demonstrate that the transient immunogenic tumor milieu contains more activated DC. This knowledge has important implications for the development of future combinatorial therapies.