Botensilimab, an Fc-enhanced anti-CTLA-4 antibody, is effective against tumors poorly responsive to conventional immunotherapy.

Conventional immune checkpoint inhibitors (ICI) targeting CTLA-4 elicit durable survival, but primarily in patients with immune-inflamed tumors. Although the mechanisms underlying response to anti-CTLA-4 remain poorly understood, Fc-gamma receptor (FcγR) IIIA co-engagement appears critical for activity, potentially explaining the modest clinical benefits of approved anti-CTLA-4 antibodies. We demonstrate that anti-CTLA-4 engineered for enhanced FcγR affinity leverages FcγR-dependent mechanisms to potentiate T cell responsiveness, reduce intratumoral Tregs, and enhance antigen presenting cell activation. Fc-enhanced anti-CTLA-4 promoted superior efficacy in mouse models and remodeled innate and adaptive immunity versus conventional anti-CTLA-4. These findings extend to patients treated with botensilimab, an Fc-enhanced anti-CTLA-4 antibody, with clinical activity across multiple poorly immunogenic and ICI treatment-refractory cancers. Efficacy was independent of tumor neoantigen burden or FcγRIIIA genotype. However, FcγRIIA and FcγRIIIA expression emerged as potential response biomarkers. These data highlight the therapeutic potential of Fc-enhanced anti-CTLA-4 antibodies in cancers unresponsive to conventional ICI therapy.

Pipeline to characterize antigen-specific TCR repertoires in tumors: Examples from an HPV16 tumor model.

Immunotherapies that improve T cell-based anti-tumor immunity have revolutionized cancer. However, the underlying mechanisms of cancer immune responsiveness are still not fully understood. Using immune competent mice for preclinical development of novel mono and combination therapies is a common strategy, and to monitor the T cell response inside tumors and in the periphery offers valuable insight. T cells recognize target cells by based on the binding between the T cell receptor (on T cells) and peptides presented on MHC-I (on tumor cells). As such, the T cell receptor can be used as a "barcode" for a specific T cell clone. Via TCR sequencing, the sequence of this "barcode" can be identified, and eventually, the TCR repertoire in a sample can be assessed as a whole. This information can be useful in multiple ways, including but not excluded to: (i) tracing specific clones in tissues and in blood, and (ii) determine clonal expansion of a specific clone in the tumor microenvironment which suggest anti-tumor activity of the clone in question. This protocol can be used as a guide from experimental design through TCR-sequencing to analysis of the repertoire. Instead of being specifically focused on one type of TCR-sequencing, this protocol can be used as a resource and contains links and references to useful information that has to be considered. Lastly, certain common metrics when analyzing the TCR repertoire are given and discussed.

Next-generation CTLA-4 targeting molecules and combination therapy: promising strategies for improving cancer immunotherapy.

Radiation therapy and anti-CTLA-4 combination therapy can induce meaningful responses in some patients. Adding CD40 may provide additional benefit. Next-generation anti-CTLA-4 antibodies, such as botensilimab, are showing promise in clinical trials. Combining botensilimab with RT and/or CD40 agonist may offer additional benefits for challenging tumor types.

Pipeline to identify neoantigens exposed by radiation.

Mutation-associated neoantigens are key targets of tumor-specific T cells and thus play a major role in driving responses to immune checkpoint blockade (ICB) therapy in tumors with high mutational burden. However, only a small number of mutated peptides are actually presented by MHC molecules and only a minority can induce T cell responses. In addition, the recognition of these neoantigens by T cells is limited by the level of expression of the mutated gene product in the tumor cells. Preclinical studies have shown that radiation can convert the irradiated tumor into an in situ vaccine, leading to the priming of tumor-specific T cells and to the rejection of otherwise ICB-resistant tumors. There is now preclinical and clinical evidence that radiation can upregulate the expression of genes containing immunogenic mutations and expose them to the immune system. Therefore, the identification of neoantigens upregulated by radiation could help to predict which patients might benefit from treatment with combinations of radiotherapy and ICB and could also be incorporated into personalized neoantigen vaccination strategies. In this chapter, we present the pipeline that we used to identify relevant radiation-upregulated neoantigens in a poorly immunogenic mouse model of metastatic breast cancer.

Immunotherapy targeting different immune compartments in combination with radiation therapy induces regression of resistant tumors.

Radiation therapy (RT) increases tumor response to CTLA-4 inhibition (CTLA4i) in mice and in some patients, yet deep responses are rare. To identify rational combinations of immunotherapy to improve responses we use models of triple negative breast cancer highly resistant to immunotherapy in female mice. We find that CTLA4i promotes the expansion of CD4+ T helper cells, whereas RT enhances T cell clonality and enriches for CD8+ T cells with an exhausted phenotype. Combination therapy decreases regulatory CD4+ T cells and increases effector memory, early activation and precursor exhausted CD8+ T cells. A combined gene signature comprising these three CD8+ T cell clusters is associated with survival in patients. Here we show that targeting additional immune checkpoints expressed by intratumoral T cells, including PD1, is not effective, whereas CD40 agonist therapy recruits resistant tumors into responding to the combination of RT and CTLA4i, indicating the need to target different immune compartments.

Age-related long-term response in rat thyroid tissue and plasma after internal low dose exposure to 131I.

131I is used clinically for therapy, and may be released during nuclear accidents. After the Chernobyl accident papillary thyroid carcinoma incidence increased in children, but not adults. The aims of this study were to compare 131I irradiation-dependent differences in RNA and protein expression in the thyroid and plasma of young and adult rats, and identify potential age-dependent biomarkers for 131I exposure. Twelve young (5 weeks) and twelve adult Sprague Dawley rats (17 weeks) were i.v. injected with 50 kBq 131I (absorbed dose to thyroid = 0.1 Gy), and sixteen unexposed age-matched rats were used as controls. The rats were killed 3-9 months after administration. Microarray analysis was performed using RNA from thyroid samples, while LC-MS/MS analysis was performed on proteins extracted from thyroid tissue and plasma. Canonical pathways, biological functions and upstream regulators were analysed for the identified transcripts and proteins. Distinct age-dependent differences in gene and protein expression were observed. Novel biomarkers for thyroid 131I exposure were identified: (PTH), age-dependent dose response (CA1, FTL1, PVALB (youngsters) and HSPB6 (adults)), thyroid function (Vegfb (adults)). Further validation using clinical samples are needed to explore the role of the identified biomarkers.

Hallmarks of Resistance to Immune-Checkpoint Inhibitors.

Immune-checkpoint inhibitors (ICI), although revolutionary in improving long-term survival outcomes, are mostly effective in patients with immune-responsive tumors. Most patients with cancer either do not respond to ICIs at all or experience disease progression after an initial period of response. Treatment resistance to ICIs remains a major challenge and defines the biggest unmet medical need in oncology worldwide. In a collaborative workshop, thought leaders from academic, biopharma, and nonprofit sectors convened to outline a resistance framework to support and guide future immune-resistance research. Here, we explore the initial part of our effort by collating seminal discoveries through the lens of known biological processes. We highlight eight biological processes and refer to them as immune resistance nodes. We examine the seminal discoveries that define each immune resistance node and pose critical questions, which, if answered, would greatly expand our notion of immune resistance. Ultimately, the expansion and application of this work calls for the integration of multiomic high-dimensional analyses from patient-level data to produce a map of resistance phenotypes that can be utilized to guide effective drug development and improved patient outcomes.

ATR-mediated CD47 and PD-L1 up-regulation restricts radiotherapy-induced immune priming and abscopal responses in colorectal cancer.

Radiotherapy (RT) of colorectal cancer (CRC) can prime adaptive immunity against tumor-associated antigen (TAA)-expressing CRC cells systemically. However, abscopal tumor remissions are extremely rare, and the postirradiation immune escape mechanisms in CRC remain elusive. Here, we found that irradiated CRC cells used ATR-mediated DNA repair signaling pathway to up-regulate both CD47 and PD-L1, which through engagement of SIRPα and PD-1, respectively, prevented phagocytosis by antigen-presenting cells and thereby limited TAA cross-presentation and innate immune activation. This postirradiation CD47 and PD-L1 up-regulation was observed across various human solid tumor cells. Concordantly, rectal cancer patients with poor responses to neoadjuvant RT exhibited significantly elevated postirradiation CD47 levels. The combination of RT, anti-SIRPα, and anti-PD-1 reversed adaptive immune resistance and drove efficient TAA cross-presentation, resulting in robust TAA-specific CD8 T cell priming, functional activation of T effectors, and increased T cell clonality and clonal diversity. We observed significantly higher complete response rates to RT/anti-SIRPα/anti-PD-1 in both irradiated and abscopal tumors and prolonged survival in three distinct murine CRC models, including a cecal orthotopic model. The efficacy of triple combination therapy was STING dependent as knockout animals lost most benefit of adding anti-SIRPα and anti-PD-1 to RT. Despite activation across the myeloid stroma, the enhanced dendritic cell function accounts for most improvements in CD8 T cell priming. These data suggest ATR-mediated CD47 and PD-L1 up-regulation as a key mechanism restraining radiation-induced immune priming. RT combined with SIRPα and PD-1 blockade promotes robust antitumor immune priming, leading to systemic tumor regressions.

Expression of the mono-ADP-ribosyltransferase ART1 by tumor cells mediates immune resistance in non-small cell lung cancer.

Most patients with non-small cell lung cancer (NSCLC) do not achieve durable clinical responses from immune checkpoint inhibitors, suggesting the existence of additional resistance mechanisms. Nicotinamide adenine dinucleotide (NAD)-induced cell death (NICD) of P2X7 receptor (P2X7R)-expressing T cells regulates immune homeostasis in inflamed tissues. This process is mediated by mono-adenosine 5'-diphosphate (ADP)-ribosyltransferases (ARTs). We found an association between membranous expression of ART1 on tumor cells and reduced CD8 T cell infiltration. Specifically, we observed a reduction in the P2X7R+ CD8 T cell subset in human lung adenocarcinomas. In vitro, P2X7R+ CD8 T cells were susceptible to ART1-mediated ADP-ribosylation and NICD, which was exacerbated upon blockade of the NAD+-degrading ADP-ribosyl cyclase CD38. Last, in murine NSCLC and melanoma models, we demonstrate that genetic and antibody-mediated ART1 inhibition slowed tumor growth in a CD8 T cell-dependent manner. This was associated with increased infiltration of activated P2X7R+CD8 T cells into tumors. In conclusion, we describe ART1-mediated NICD as a mechanism of immune resistance in NSCLC and provide preclinical evidence that antibody-mediated targeting of ART1 can improve tumor control, supporting pursuit of this approach in clinical studies.

T-Cell Receptor Profiling and Prognosis After Stereotactic Body Radiation Therapy For Stage I Non-Small-Cell Lung Cancer.

Radiotherapy is known to influence immune function, including T cell receptor (TCR) repertoire. We evaluated the TCR repertoire before and after stereotactic body radiotherapy (SBRT) for stage I non-small-cell lung cancer (NSCLC) and explored correlations between TCR indexes and distant failure after SBRT. TCR repertoires were analyzed in peripheral blood mononuclear cells (PBMCs) collected before and after SBRT from 19 patients. TCR combinational diversity in V and J genes was assessed with multiplex PCR of genomic DNA from PBMCs and tested for associations with clinical response. All patients received definitive SBRT to a biologically effective dose of >=100 Gy. The number of unique TCR clones was decreased after SBRT versus before, but clonality and the Shannon Entropy did not change. Four patients (21%) developed distant metastases after SBRT (median 7 months); those patients had lower Shannon Entropy in post-SBRT samples than patients without metastasis. Patients with a low change in Shannon Entropy from before to after SBRT [(post-SBRT Shannon Entropy minus baseline Shannon)/(baseline Shannon) * 100] had poorer metastasis-free survival than those with high change in Shannon Entropy (P<0.001). Frequencies in V/J gene fragment expression in the TCR ß chain were also different for patients with or without metastases (two V fragments in baseline samples and 2 J and 9 V fragments in post-treatment samples). This comprehensive analysis of immune status before and after SBRT showed that quantitative assessments of TCRs can help evaluate prognosis in early-stage NSCLC.

Radiotherapy-exposed CD8+ and CD4+ neoantigens enhance tumor control.

Neoantigens generated by somatic nonsynonymous mutations are key targets of tumor-specific T cells, but only a small number of mutations predicted to be immunogenic are presented by MHC molecules on cancer cells. Vaccination studies in mice and patients have shown that the majority of neoepitopes that elicit T cell responses fail to induce significant antitumor activity, for incompletely understood reasons. We report that radiotherapy upregulates the expression of genes containing immunogenic mutations in a poorly immunogenic mouse model of triple-negative breast cancer. Vaccination with neoepitopes encoded by these genes elicited CD8+ and CD4+ T cells that, whereas ineffective in preventing tumor growth, improved the therapeutic efficacy of radiotherapy. Mechanistically, neoantigen-specific CD8+ T cells preferentially killed irradiated tumor cells. Neoantigen-specific CD4+ T cells were required for the therapeutic efficacy of vaccination and acted by producing Th1 cytokines, killing irradiated tumor cells, and promoting epitope spread. Such a cytotoxic activity relied on the ability of radiation to upregulate class II MHC molecules as well as the death receptors FAS/CD95 and DR5 on the surface of tumor cells. These results provide proof-of-principle evidence that radiotherapy works in concert with neoantigen vaccination to improve tumor control.

Supporting the Next Generation of Scientists to Lead Cancer Immunology Research.

Recent success in the use of immunotherapy for a broad range of cancers has propelled the field of cancer immunology to the forefront of cancer research. As more and more young investigators join the community of cancer immunologists, the Arthur L. Irving Family Foundation Cancer Immunology Symposium provided a platform to bring this expanding and vibrant community together and support the development of the future leaders in the field. This commentary outlines the lessons that emerged from the inaugural symposium highlighting the areas of scientific and career development that are essential for professional growth in the field of cancer immunology and beyond. Leading scientists and clinicians in the field provided their experience on the topics of scientific trajectory, career trajectory, publishing, fundraising, leadership, mentoring, and collaboration. Herein, we provide a conceptual and practical framework for career development to the broader scientific community.

Isolation of DNA from exosomes.

Exosomes are small extracellular vesicles released by prokaryotic and eukaryotic cells with a crucial role in cell-to-cell communication in both physiological and pathological conditions. Exosomes contain and transfer active biomolecules, including nucleic acids, proteins and lipids to target recipient cells. In the last decade, many methodologies have been developed for isolating specific exosomal components. In this chapter, we will detail methods to isolate exosomal DNA, considering the crucial role of exosomal DNA in regulating the behavior of recipient cells in multiple settings, including the response of malignant cells to chemo-, radio- and immunotherapy.

Characterization of conventional dendritic cell populations in preclinical tumor models using flow cytometry.

Tumor infiltration of conventional dendritic cells has been shown to be essential for triggering efficient antitumor immune responses. These findings have generated an increasing demand for reliable methods to accurately identify and quantify specific DC-subpopulations, both in immune monitoring of clinical trial samples as well as in preclinical mouse tumor models. Here, we describe a flow cytometric approach to assess percentages and absolute counts of conventional dendritic cells in solid mouse tumors.

Exercise reduces immune suppression and breast cancer progression in a preclinical model.

Exercise is associated with favorable changes in circulating immune cells and improved survival in early-stage breast cancer patients, but the mechansims remain to be fully elucidated. Preclinical studies indicate that physical activity started before tumor injection reduces tumor incidence and progression. Here we tested whether exercise has anti-tumor effects in mice with established 4T1 mammary carcinoma, a mouse model of triple negative breast cancer. Exercise slowed tumor progression and reduced the tumor-induced accumulation of myeloid-derived suppressor cells (MDSCs). The reduction in MDSCs was accompanied by a relative increase in natural killer and CD8 T cell activation, suggesting that exercise restores a favorable immune environment. Consistently, exercise improved responses to a combination of programmed cell death protein 1 (PD-1) blockade and focal radiotherapy. These data support further investigations of exercise in breast cancer patients treated with combinations of immunotherapy and cytotoxic agents to improve cancer outcomes.

CD73 Blockade Promotes Dendritic Cell Infiltration of Irradiated Tumors and Tumor Rejection.

The ability of focal radiotherapy to promote priming of tumor-specific CD8+ T cells and increase responses to immunotherapy is dependent on infiltration of the tumor by Batf3-dependent conventional dendritic cell type 1 (cDC1) cells. Such infiltration is driven by radiotherapy-induced IFN type I (IFN-I). Other signals may also modulate cDC1 infiltration of irradiated tumors. Here we found increased expression of adenosine-generating enzymes CD38 and CD73 in irradiated mouse and human breast cancer cells and increased adenosine in mouse tumors following radiotherapy. CD73 blockade alone had no effect. CD73 blockade with radiotherapy restored radiotherapy-induced cDC1 infiltration of tumors in settings where radiotherapy induction of IFN-I was suboptimal. In the absence of radiotherapy-induced IFN-I, blockade of CD73 was required for rejection of the irradiated tumor and for systemic tumor control (abscopal effect) in the context of cytotoxic T-lymphocyte-associated protein 4 blockade. These results suggest that CD73 may be a radiation-induced checkpoint, and that CD73 blockade in combination with radiotherapy and immune checkpoint blockade might improve patient response to therapy.

Mutational and Antigenic Landscape in Tumor Progression and Cancer Immunotherapy.

Evolving neoplasms accumulate non-synonymous mutations at a high rate, potentially enabling the expression of antigenic epitopes that can be recognized by the immune system. Since they are not covered by central tolerance, such tumor neoantigens (TNAs) should be under robust immune control as they surge. However, genetic defects that impair cancer cell eradication by the immune system coupled with the establishment of local immunosuppression can enable TNA accumulation, which is generally associated with improved clinical sensitivity to various immunotherapies. Here, we explore how tumor-intrinsic factors and immunological processes shape the mutational and antigenic landscape of evolving neoplasms to influence clinical responses to immunotherapy, and propose strategies to achieve robust immunological control of the disease despite disabled immunosurveillance.

Radiation therapy and anti-tumor immunity: exposing immunogenic mutations to the immune system.

The expression of antigens that are recognized by self-reactive T cells is essential for immune-mediated tumor rejection by immune checkpoint blockade (ICB) therapy. Growing evidence suggests that mutation-associated neoantigens drive ICB responses in tumors with high mutational burden. In most patients, only a few of the mutations in the cancer exome that are predicted to be immunogenic are recognized by T cells. One factor that limits this recognition is the level of expression of the mutated gene product in cancer cells. Substantial preclinical data show that radiation can convert the irradiated tumor into a site for priming of tumor-specific T cells, that is, an in situ vaccine, and can induce responses in otherwise ICB-resistant tumors. Critical for radiation-elicited T-cell activation is the induction of viral mimicry, which is mediated by the accumulation of cytosolic DNA in the irradiated cells, with consequent activation of the cyclic GMP-AMP synthase (cGAS)/stimulator of interferon (IFN) genes (STING) pathway and downstream production of type I IFN and other pro-inflammatory cytokines. Recent data suggest that radiation can also enhance cancer cell antigenicity by upregulating the expression of a large number of genes that are involved in the response to DNA damage and cellular stress, thus potentially exposing immunogenic mutations to the immune system. Here, we discuss how the principles of antigen presentation favor the presentation of peptides that are derived from newly synthesized proteins in irradiated cells. These concepts support a model that incorporates the presence of immunogenic mutations in genes that are upregulated by radiation to predict which patients might benefit from treatment with combinations of radiotherapy and ICB.

Transcriptional effects of 177Lu-octreotate therapy using a priming treatment schedule on GOT1 tumor in nude mice.

BACKGROUND: 177Lu-octreotate is used for therapy of somatostatin receptor expressing neuroendocrine tumors with promising results, although complete tumor remission is rarely seen. Previous studies on nude mice bearing the human small intestine neuroendocrine tumor, GOT1, have shown that a priming injection of 177Lu-octreotate 24 h before the main injection of 177Lu-octreotate resulted in higher 177Lu concentration in tumor, resulting in increased absorbed dose, volume reduction, and time to regrowth. To our knowledge, the cellular effects of a priming treatment schedule have not yet been studied. The aim of this study was to identify transcriptional changes contributing to the enhanced therapeutic response of GOT1 tumors in nude mice to 177Lu-octreotate therapy with priming, compared with non-curative monotherapy. RESULTS: RNA microarray analysis was performed on tumor samples from GOT1-bearing BALB/c nude mice treated with a 5 MBq priming injection of 177Lu-octreotate followed by a second injection of 10 MBq of 177Lu-octreotate after 24 h and killed after 1, 3, 7, and 41 days after the last injection. Administered activity amounts were chosen to be non-curative, in order to facilitate the study of tumor regression and regrowth. Differentially regulated transcripts (RNA samples from treated vs. untreated animals) were identified (change ≥ 1.5-fold; adjusted p value < 0.01) using Nexus Expression 3.0. Analysis of the biological effects of transcriptional regulation was performed using the Gene Ontology database and Ingenuity Pathway Analysis. Transcriptional analysis of the tumors revealed two stages of pathway regulation for the priming schedule (up to 1 week and around 1 month) which differed distinctly from cellular responses observed after monotherapy. Induction of cell cycle arrest and apoptotic pathways (intrinsic and extrinsic) was found at early time points after treatment start, while downregulation of pro-proliferative genes were found at a late time point. CONCLUSIONS: The present study indicates increased cellular stress responses in the tumors treated with a priming treatment schedule compared with those seen after conventional 177Lu-octreotate monotherapy, resulting in a more profound initiation of cell cycle arrest followed by apoptosis, as well as effects on PI3K/AKT-signaling and unfolded protein response.

Correction to: Toward a comprehensive view of cancer immune responsiveness: a synopsis from the SITC workshop.

Following publication of the original article [1], the author reported that an author name, Roberta Zappasodi, was missed in the authorship list.