Amy K. Erbe

The current treatment regimen for neuroblastoma involves immunotherapy, including a monoclonal antibody that recognizes disialoganglioside (GD2), expressed at high levels on neuroblastoma. GD2 is not present on most normal tissues except for nerves. Thus, anti-GD2 antibody treatment causes substantial, dose-limiting neuropathic pain. B7-H3 is overexpressed on multiple tumor types, including neuroblastoma, and is largely absent on nerves and other normal tissues. We designed a bispecific antibody (bsAb) that requires simultaneous binding of these two tumor antigens to achieve tight binding of tumor cells. Our preclinical research shows that, compared with a monospecific anti-GD2 antibody, the GD2×B7-H3 bsAb has improved tumor specificity, comparable antitumor efficacy, and reduced nerve binding and pain-associated toxicity. Since this bsAb does not bind to nerves, it may permit more tolerable and sustained treatment schedules than are currently feasible with monospecific anti-GD2 antibodies. In addition, its enhanced tumor specificity may support future development as a targeted delivery platform for antibody-drug conjugates or other payload-based therapies, potentially improving both efficacy and quality of life for patients with neuroblastoma.

Rationale: Radium-223 dichloride (²²³RaCl₂) is an FDA-approved alpha-emitting radiopharmaceutical that targets bone metastases in metastatic castration-resistant prostate cancer (mCRPC). This study investigates the therapeutic and immunological effects of combining ²²³RaCl₂ with immune checkpoint inhibitors (ICIs) in a clinically relevant, immunocompetent murine model of prostate cancer bone metastasis. Methods: Luciferase-expressing MyC-CaP prostate cancer cells were implanted intratibially into FVB mice to establish bone metastases. Mice were treated with escalating doses of ²²³RaCl₂ (0.04–0.27 µCi) alone or a single dose combined with anti-CTLA-4 and anti-PD-L1 ICIs. Tumor growth was monitored using bioluminescence imaging. Micro-CT, alpha camera imaging, histology, and qPCR were used to assess bone remodeling, radiopharmaceutical distribution, immune infiltration, and gene expression. Ex vivo biodistribution and blood analyses quantified tissue uptake and toxicity. Results: Escalating doses of ²²³RaCl₂ did not significantly inhibit tumor growth or improve survival. Biodistribution and imaging showed preferential localization of ²²³RaCl₂ to tumor-adjacent bone, with minimal signal in isolated tumor tissue. Immunohistochemistry revealed increased CD4⁺ and CD8α⁺ T-cell infiltration in regions of high γH2AX expression, indicating localized immune modulation. However, combination therapy with ICIs did not enhance tumor control or immune infiltration beyond monotherapy. qPCR demonstrated significant upregulation of Mhc1 only in the combination group, suggesting localized immune activation. Toxicity profiles remained acceptable. Conclusions: ²²³RaCl₂ localizes primarily to bone surfaces, limiting direct cytotoxic and immunomodulatory effects within the tumor microenvironment. While combination with ICIs did not improve efficacy, these findings provide a platform for studying spatial dose distribution and support future development of tumor-targeted alpha therapies to potentiate immunotherapy in mCRPC.

Radiopharmaceutical therapy (RPT) synergises with immune checkpoint inhibitors (ICI), but comparison of immunomodulation by different radioisotopes is lacking. Here, we evaluate mechanisms of response and timing of ICI administration relative to α- (²²⁵Ac) and β-emitting (⁹⁰Y, ¹⁷⁷Lu) radioisotope therapy, coupled with alkylphosphocholine NM600, when combined with dual (anti-PD-L1 and anti-CTLA4) ICI, using syngeneic poorly immunogenic (B78 and Myc-CaP) and immunogenic (MC38) murine models. Regardless of the isotope, RPT delivering 2 Gy mean tumor dose promotes tumor regression and improves survival in B78 or MC38 tumor-bearing mice when combined with early ICI administration. Greatest anti-tumor responses are seen in MC38 to ⁹⁰Y-NM600 + ICI and in B78 and Myc-CaP to ²²⁵Ac-NM600 + ICI. Flow cytometry and single-cell RNA and T cell receptor sequencing reveal that, combined with ICI, β-emitting radioisotopes expand existing adaptive immunity, whereas α-emitting radiopharmaceuticals initiate immune priming. Thus, appropriate application of α- or β-emitting RPT in combination with ICI achieves distinct antitumor immune responses.

Summary: Despite advances in treatment, >50% of patients with advanced melanoma are unresponsive to current therapies. Using the B78-D14 melanoma model (GD2 ⁺ /MHC-I⁻/MHC-II ⁺ ), we can cure mice with a regimen that includes radiation therapy (RT) in combination with immunocytokine (IC; anti-GD2 monoclonal antibody linked to IL-2) while establishing immunological memory. We interrogated the role of T cells in the antitumor and memory responses following RT+IC. We show a requirement for CD4, but not CD8 T cells, to achieve both the initial and memory responses. Upon IC-induced cell-cell contact, subsets of CD4 T cells, including Foxp3⁺ T regulatory cells, trogocytose GD2 from tumor cells, acquire cytotoxic granules, and kill tumor cells. These results were confirmed using human tumor cell lines. These findings reveal that CD4⁺ T regulatory cells, upon immunologically-induced binding to tumor cells, can trogocytose tumor antigens and directly kill tumor cells, redefining their potential role in antitumor immunity.

Radiopharmaceutical therapy (RPT) offers tumor-selective radiation delivery and represents a promising platform for combination with immune checkpoint inhibitors (ICIs). While prior studies suggest that RPT can stimulate antitumor immunity, synergy with ICIs may depend on radionuclide properties, absorbed dose, and radiation distribution within the tumor microenvironment. This study evaluated how radionuclide selection and dose influence immune stimulation and therapeutic efficacy of GD2-targeted antibody-based RPT combined with ICIs. Dinutuximab, an anti-GD2 monoclonal antibody, was radiolabeled with β⁻-emitters (⁹⁰Y, ¹⁷⁷Lu) or an α-emitter (²²⁵Ac). C57Bl6 mice bearing GD2⁺ tumors received 4 or 15 Gy tumor-absorbed doses, determined by individualized dosimetry, with or without dual ICIs (anti-CTLA-4 and anti-PD-L1). In vivo imaging, ex vivo biodistribution, survival, histological, and gene expression analyses were performed to assess therapeutic and immunological outcomes. All radiolabeled constructs demonstrated preferential uptake in GD2⁺ tumors. Combination therapy improved survival in a radionuclide- and dose-dependent manner, with the greatest benefit in the ²²⁵Ac + ICI group at 15 Gy. Treatment activated type I interferon signaling and increased MHC-I and PD-L1 expression. Notably, ⁹⁰Y reduced regulatory T cells, enhancing CD8⁺/Treg ratios, while ²²⁵Ac induced robust interferon-driven activation. Radionuclide selection and absorbed dose critically shape immune and therapeutic outcomes of antibody-based RPT combined with ICIs, underscoring the importance of delivery mechanism and dose optimization in combination therapy strategies.

Children and adults diagnosed with malignancies that are not curable in 2025 with surgery alone require multimodal therapy. For decades, this has included radiation therapy and/or chemotherapy. This approach can be curative for many patients with certain cancers, such as acute lymphoblastic leukemia, Wilms tumor, Hodgkin’s disease, and testicular cancer. For those who are not being cured, additional therapies are needed. The last 15 years have shown that those previously incurable patients are now having a chance for a cure using immunotherapy in the form of immune checkpoint inhibition (ICI). However, with the exception of rare patients with mutations in their DNA repair pathways associated with very high neoantigen loads, most patients receiving ICI therapy are not cured by it. This pertains to virtually all patients with some tumors, such as prostate or gastrointestinal (GI) cancers and even for a majority of those patients with advanced cancers classified as “immunogenic”, such as melanoma, renal cell cancer, and lung cancer. One feature of these many patients not responding to ICI is that their tumors are immunologically cold. Namely, they are characterized by low actionable neoantigen load, low or absent major histocompatibility complex expression, and/or an immune-hostile tumor microenvironment. These features are typical of high-risk neuroblastoma (HR-NBL). Despite its cold features, substantial progress has been made in advancing the treatment of HR-NBL using different forms of immunotherapy. This manuscript describes a clinical goal for HR-NBL for 20 years from now. In addition, it extrapolates from this experience and anticipated progress for HR-NBL to how these concepts might apply to improving treatments for patients with more common, yet cold, cancers of adults.

Immunotherapy has potential for impactful cancer cures by empowering patients’ own immune cells. We developed a radio-immunotherapy regimen that can cure large immunologically hot and cold murine tumors. Here, we explored the divergent role of CD8 T cells during this radio-immunotherapy in contrasting hot colon carcinoma versus cold melanoma. We introduced an immunocompetent mouse model with mCherry-expressing CD8 T cells to provide cell tracking in vivo . We investigated single-cell function, metabolism, and gene expression temporal changes using flow cytometry, in vivo multiphoton imaging, single-cell RNA sequencing, and multiplexed immunofluorescence to determine the underlying mechanisms. We found that in contrast to the hot colon carcinoma model, CD8 T cells from the cold melanoma model do not drive tumor cures, despite getting activated, possibly due to a static oxidative metabolism and exhausted phenotype plus down regulation of tumor MHC-I expression. These findings have implications for improving immunotherapy response in immunologically cold cancers.

Background: Adoptive cell therapies are emerging as a promising therapeutic option against hematological and solid malignancies. Memory-like natural killer (mlNK) cells are a specific subtype of NK cells generated after cytokine preactivation that have shown enhanced in vivo persistence after infusion into patients, an issue that has hindered traditional NK cell immunotherapy. However, the quality and variability of mlNK cell products remains poorly defined. Methods: In this study, we evaluated heterogeneity across critical functional and molecular aspects of mlNK cells generated from independent donors, including mlNK cytotoxicity, cluster formation, motility, mitochondria morphology, and gene expression. Results: We observed a correlation between changes in gene expression associated with glycolysis and key NK cell functions such as cytotoxicity and motility. For further characterization, we blocked glycolysis and oxidative phosphorylation (OXPHOS) and observed an impaired mlNK functional response, suggesting the importance of metabolism. Conclusions: Our findings provide insights into discriminating between mlNK cell products and how the predictive markers can identify optimal mlNK cell products for adoptive cell therapy of cancer.

PURPOSE: We conducted a phase I trial to determine the safety, tolerability, and preliminary antitumor activity of ¹³¹ I-metaiodobenzylguanidine (MIBG) combined with the anti-GD2 antibody dinutuximab with or without the histone deacetylase inhibitor vorinostat in patients with relapsed/refractory neuroblastoma (rNBL). METHODS: In part A, patients with MIBG-avid rNBL received MIBG intravenously (IV) on day 1 at 12, 15, or 18 mCi/kg per the rolling six design and dinutuximab (17.5 mg/m ² once daily) IV on days 8-11 and 29-32 and granulocyte-macrophage colony-stimulating factor (250 mcg/m ² once daily) subcutaneously on days 8-17 and 29-38. Autologous stem cells were infused on day 15. In part B, vorinostat at 180 mg/m ² once daily was given orally on days 0-13 in combination with the part A recommended phase II dose (RP2D). Patients could receive two courses. RESULTS: Forty-five eligible patients enrolled, of whom 31 were evaluable. The median age was 7.5 (range, 2.9-24.1) years. For part A (n = 19), no dose-limiting toxicities (DLTs) occurred across all dose levels and courses, establishing the RP2D of MIBG to be 18 mCi/kg. In part B (n = 12), 1 DLT (grade 3 hypokalemia) occurred during course 1, and 3 of 11 patients who received a second course experienced DLT: grade 3 ALT increase, grade 4 hypoxia and grade 5 pneumonitis, and grade 3 fatigue. The best overall response rate (BORR; complete response [CR] + partial response [PR]) on part A was 42% with a CR/PR/minor response (MR) rate of 46%, and 19% progressive disease (PD) rate. For part B, the BORR was 42% with a CR/PR/MR rate of 75% and 0% PD rate. CONCLUSION: MIBG combined with dinutuximab was well tolerated with encouraging antitumor activity. Vorinostat added to this combination may augment responses in this heavily pretreated patient population.

Background Allogeneic bone marrow transplant (alloBMT) is curative for hematologic malignancies through the graft-versus-tumor (GVT) effect but has been ineffective for solid tumors like osteosarcoma (OS). OS expresses CD155 which interacts strongly with inhibitory receptors TIGIT and CD96 but also binds to activating receptor DNAM-1 on natural killer (NK) cells. CD155 has never been targeted after alloBMT. Combining adoptively transferred allogeneic NK (alloNK) cells with CD155 blockade after alloBMT may enhance a GVT effect against OS. Methods Murine NK cells were activated and expanded ex vivo with superagonist interleukin (IL)-15/IL-15Rα. AlloNK and syngeneic NK (synNK) cell phenotype, cytotoxicity, cytokine production, and degranulation against CD155-expressing murine OS cell line K7M2 were assessed in vitro. Mice bearing pulmonary OS metastases underwent alloBMT and alloNK cell infusion with anti-CD155 either before or after tumor induction, with select groups receiving anti-DNAM-1 pretreated alloNK cells. Tumor growth, graft-versus-host disease and survival were monitored, and differential gene expression of lung tissue was assessed by RNA microarray. Results AlloNK cells exhibited superior cytotoxicity against CD155-expressing OS compared with synNK cells, and this activity was enhanced by CD155 blockade. CD155 blockade increased alloNK cell degranulation and interferon gamma production through DNAM-1. In vivo, CD155 blockade with alloNK infusion increased survival when treating OS that relapsed after alloBMT. No benefit was seen for treating established OS before alloBMT. Combining CD155 and anti-DNAM-1 pretreated alloNK did not affect survival and tumor control benefits seen with CD155 blockade alone. RNA microarray showed mice treated with alloNK and CD155 blockade had increased expression of cytotoxicity genes and the NKG2D ligand H60a, whereas mice treated with anti-DNAM-1 pretreated alloNK cells resulted in upregulation of NK cell inhibitory receptor genes. Whereas blocking DNAM-1 on alloNK abrogated cytotoxicity, blocking NKG2D had no effect, implying DNAM-1:CD155 engagement drives alloNK activation against OS. Conclusions These results demonstrate the safety and efficacy of infusing alloNK cells with CD155 blockade to mount a GVT effect against OS and show benefits are in part through DNAM-1. Defining the hierarchy of receptors that govern alloNK responses is critical to translating alloNK cell infusions and immune checkpoint inhibition for solid tumors treated with alloBMT.