Evaluation of a Combinatorial Immunotherapy Regimen That Can Cure Mice Bearing MYCN-Driven High-Risk Neuroblastoma That Resists Current Clinical Therapy.

Background: Incorporating GD2-targeting monoclonal antibody into post-consolidation maintenance therapy has improved survival for children with high-risk neuroblastoma. However, ~50% of patients do not respond to, or relapse following, initial treatment. Here, we evaluated additional anti-GD2-based immunotherapy to better treat high-risk neuroblastoma in mice to develop a regimen for patients with therapy-resistant neuroblastoma. Methods: We determined the components of a combined regimen needed to cure mice of established MYCN-amplified, GD2-expressing, murine 9464D-GD2 neuroblastomas. Results: First, we demonstrate that 9464D-GD2 is nonresponsive to a preferred salvage regimen: anti-GD2 with temozolomide and irinotecan. Second, we have previously shown that adding agonist anti-CD40 mAb and CpG to a regimen of radiotherapy, anti-GD2/IL2 immunocytokine and anti-CTLA-4, cured a substantial fraction of mice bearing small 9464D-GD2 tumors; here, we further characterize this regimen by showing that radiotherapy and hu14.18-IL2 are necessary components, while anti-CTLA-4, anti-CD40, or CpG can individually be removed, and CpG and anti-CTLA-4 can be removed together, while maintaining efficacy. Conclusions: We have developed and characterized a regimen that can cure mice of a high-risk neuroblastoma that is refractory to the current clinical regimen for relapsed/refractory disease. Ongoing preclinical work is directed towards ways to potentially translate these findings to a regimen appropriate for clinical testing.

Histoplasty Modification of the Tumor Microenvironment in a Murine Preclinical Model of Breast Cancer.

PURPOSE: To develop a noninvasive therapeutic approach able to alter the biophysical organization and physiology of the extracellular matrix (ECM) in breast cancer. MATERIALS AND METHODS: In a 4T1 murine model of breast cancer, histoplasty treatment with a proprietary 700-kHz multielement therapy transducer using a coaxially aligned ultrasound (US) imaging probe was used to target the center of an ex vivo tumor and deliver subablative acoustic energy. Tumor collagen morphology was qualitatively evaluated before and after histoplasty with second harmonic generation. Separately, mice bearing bilateral 4T1 tumors (n = 4; total tumors = 8) were intravenously injected with liposomal doxorubicin. The right flank tumor was histoplasty-treated, and tumors were fluorescently imaged to detect doxorubicin uptake after histoplasty treatment. Next, 4T1 tumor-bearing mice were randomized into 2 treatment groups (sham vs histoplasty, n = 3 per group). Forty-eight hours after sham/histoplasty treatment, tumors were harvested and analyzed using flow cytometry. RESULTS: Histoplasty significantly increased (P = .0023) liposomal doxorubicin diffusion into 4T1 tumors compared with untreated tumors (2.12- vs 1.66-fold increase over control). Flow cytometry on histoplasty-treated tumors (n = 3) demonstrated a significant increase in tumor macrophage frequency (42% of CD45 vs 33%; P < .05) and a significant decrease in myeloid-derived suppressive cell frequency (7.1% of CD45 vs 10.3%; P < .05). Histoplasty-treated tumors demonstrated increased CD8+ (5.1% of CD45 vs 3.1%; P = .117) and CD4+ (14.1% of CD45 vs 11.8%; P = .075) T-cell frequency. CONCLUSIONS: Histoplasty is a nonablative focused US approach to noninvasively modify the tumor ECM, increase chemotherapeutic uptake, and alter the tumor immune microenvironment.

NK cells propagate T cell immunity following in situ tumor vaccination.

We report an in situ vaccination, adaptable to nearly any type of cancer, that combines radiotherapy targeting one tumor and intratumoral injection of this site with tumor-specific antibody and interleukin-2 (IL-2; 3xTx). In a phase I clinical trial, administration of 3xTx (with an immunocytokine fusion of tumor-specific antibody and IL-2, hu14.18-IL2) to subjects with metastatic melanoma increases peripheral CD8+ T cell effector polyfunctionality. This suggests the potential for 3xTx to promote antitumor immunity against metastatic tumors. In poorly immunogenic syngeneic murine melanoma or head and neck carcinoma models, 3xTx stimulates CD8+ T cell-mediated antitumor responses at targeted and non-targeted tumors. During 3xTx treatment, natural killer (NK) cells promote CTLA4+ regulatory T cell (Treg) apoptosis in non-targeted tumors. This is dependent on NK cell expression of CD86, which is upregulated downstream of KLRK1. NK cell depletion increases Treg infiltration, diminishing CD8+ T cell-dependent antitumor response. These findings demonstrate that NK cells sustain and propagate CD8+ T cell immunity following 3xTx.

Quantifying in vivo collagen reorganization during immunotherapy in murine melanoma with second harmonic generation imaging.

Significance: Increased collagen linearization and deposition during tumorigenesis can impede immune cell infiltration and lead to tumor metastasis. Although melanoma is well studied in immunotherapy research, studies that quantify collagen changes during melanoma progression and treatment are lacking. Aim: Image in vivo collagen in preclinical melanoma models during immunotherapy and quantify the collagen phenotype in treated and control mice. Approach: Second harmonic generation imaging of collagen was performed in mouse melanoma tumors in vivo over a treatment time-course. Animals were treated with a curative radiation and immunotherapy combination. Collagen morphology was quantified over time at an image and single fiber level using CurveAlign and CT-FIRE software. Results: In immunotherapy-treated mice, collagen reorganized toward a healthy phenotype, including shorter, wider, curlier collagen fibers, with modestly higher collagen density. Temporally, collagen fiber straightness and length changed late in treatment (Day 9 and 12) while width and density changed early (Day 6) compared to control mice. Single fiber level collagen analysis was most sensitive to the changes between treatment groups compared to image level analysis. Conclusions: Quantitative second harmonic generation imaging can provide insight into collagen dynamics in vivo during immunotherapy, with key implications in improving immunotherapy response in melanoma and other cancers.

Generation of Novel Immunocompetent Mouse Cell Lines to Model Experimental Metastasis of High-Risk Neuroblastoma.

NB, being a highly metastatic cancer, is one of the leading causes of cancer-related deaths in children. Increased disease recurrence and clinical resistance in patients with metastatic high-risk NBs (HR-NBs) result in poor outcomes and lower overall survival. However, the paucity of appropriate in vivo models for HR-NB metastasis has limited investigations into the underlying biology of HR-NB metastasis. This study was designed to address this limitation and develop suitable immunocompetent models for HR-NB metastasis. Here, we developed several highly metastatic immunocompetent murine HR-NB cell lines. Our newly developed cell lines show 100% efficiency in modeling experimental metastasis in C57BL6 mice and feature metastasis to the sites frequently observed in humans with HR-NB (liver and bone). In vivo validation demonstrated their specifically gained metastatic phenotype. The in vitro characterization of the cell lines showed increased cell invasion, acquired anchorage-independent growth ability, and resistance to MHC-I induction upon IFN-γ treatment. Furthermore, RNA-seq analysis of the newly developed cells identified a differentially regulated gene signature and an enrichment of processes consistent with their acquired metastatic phenotype, including extracellular matrix remodeling, angiogenesis, cell migration, and chemotaxis. The presented newly developed cell lines are, thus, suitable and promising tools for HR-NB metastasis and microenvironment studies in an immunocompetent system.

Cyclophosphamide augments the efficacy of in situ vaccination in a mouse melanoma model.

Introduction: We have previously shown that an intratumoral (IT) injection of the hu14.18-IL2 immunocytokine (IC), an anti-GD2 antibody linked to interleukin 2, can serve as an in situ vaccine and synergize with local radiotherapy (RT) to induce T cell-mediated antitumor effects. We hypothesized that cyclophosphamide (CY), a chemotherapeutic agent capable of depleting T regulatory cells (Tregs), would augment in situ vaccination. GD2+ B78 mouse melanoma cells were injected intradermally in syngeneic C57BL/6 mice. Methods: Treatments with RT (12Gy) and/or CY (100 mg/kg i.p.) started when tumors reached 100-300 mm3 (day 0 of treatment), followed by five daily injections of IT-IC (25 mcg) on days 5-9. Tumor growth and survival were followed. In addition, tumors were analyzed by flow cytometry. Results: Similar to RT, CY enhanced the antitumor effect of IC. The strongest antitumor effect was achieved when CY, RT and IC were combined, as compared to combinations of IC+RT or IC+CY. Flow cytometric analyses showed that the combined treatment with CY, RT and IC decreased Tregs and increased the ratio of CD8+ cells/Tregs within the tumors. Moreover, in mice bearing two separate tumors, the combination of RT and IT-IC delivered to one tumor, together with systemic CY, led to a systemic antitumor effect detected as shrinkage of the tumor not treated directly with RT and IT-IC. Cured mice developed immunological memory as they were able to reject B78 tumor rechallenge. Conclusion: Taken together, these preclinical results show that CY can augment the antitumor efficacy of IT- IC, given alone or in combination with local RT, suggesting potential benefit in clinical testing of these combinations.

Factors impacting the efficacy of the in-situ vaccine with CpG and OX40 agonist.

BACKGROUND: The in-situ vaccine using CpG oligodeoxynucleotide combined with OX40 agonist antibody (CpG + OX40) has been shown to be an effective therapy activating an anti-tumor T cell response in certain settings. The roles of tumor volume, tumor model, and the addition of checkpoint blockade in the efficacy of CpG + OX40 in-situ vaccination remains unknown. METHODS: Mice bearing flank tumors (B78 melanoma or A20 lymphoma) were treated with combinations of CpG, OX40, and anti-CTLA-4. Tumor growth and survival were monitored. In vivo T cell depletion, tumor cell phenotype, and tumor infiltrating lymphocyte (TIL) studies were performed. Tumor cell sensitivity to CpG and macrophages were evaluated in vitro. RESULTS: As tumor volumes increased in the B78 (one-tumor) and A20 (one-tumor or two-tumor) models, the anti-tumor efficacy of the in-situ vaccine decreased. In vitro, CpG had a direct effect on A20 proliferation and phenotype and an indirect effect on B78 proliferation via macrophage activation. As A20 tumors progressed in vivo, tumor cell phenotype changed, and T cells became more involved in the local CpG + OX40 mediated anti-tumor response. In mice with larger tumors that were poorly responsive to CpG + OX40, the addition of anti-CTLA-4 enhanced the anti-tumor efficacy in the A20 but not B78 models. CONCLUSIONS: Increased tumor volume negatively impacts the anti-tumor capability of CpG + OX40 in-situ vaccine. The addition of checkpoint blockade augmented the efficacy of CpG + OX40 in the A20 but not B78 model. These results highlight the importance of considering multiple preclinical model conditions when assessing the efficacy of cancer immunotherapy regimens and their translation to clinical testing.

Single cell metabolic imaging of tumor and immune cells in vivo in melanoma bearing mice.

Introduction: Metabolic reprogramming of cancer and immune cells occurs during tumorigenesis and has a significant impact on cancer progression. Unfortunately, current techniques to measure tumor and immune cell metabolism require sample destruction and/or cell isolations that remove the spatial context. Two-photon fluorescence lifetime imaging microscopy (FLIM) of the autofluorescent metabolic coenzymes nicotinamide adenine dinucleotide (phosphate) (NAD(P)H) and flavin adenine dinucleotide (FAD) provides in vivo images of cell metabolism at a single cell level. Methods: Here, we report an immunocompetent mCherry reporter mouse model for immune cells that express CD4 either during differentiation or CD4 and/or CD8 in their mature state and perform in vivo imaging of immune and cancer cells within a syngeneic B78 melanoma model. We also report an algorithm for single cell segmentation of mCherry-expressing immune cells within in vivo images. Results: We found that immune cells within B78 tumors exhibited decreased FAD mean lifetime and an increased proportion of bound FAD compared to immune cells within spleens. Tumor infiltrating immune cell size also increased compared to immune cells from spleens. These changes are consistent with a shift towards increased activation and proliferation in tumor infiltrating immune cells compared to immune cells from spleens. Tumor infiltrating immune cells exhibited increased FAD mean lifetime and increased protein-bound FAD lifetime compared to B78 tumor cells within the same tumor. Single cell metabolic heterogeneity was observed in both immune and tumor cells in vivo. Discussion: This approach can be used to monitor single cell metabolic heterogeneity in tumor cells and immune cells to study promising treatments for cancer in the native in vivo context.

KIR/KIR-ligand genotypes and clinical outcomes following chemoimmunotherapy in patients with relapsed or refractory neuroblastoma: a report from the Children's Oncology Group.

BACKGROUND: In the Children's Oncology Group ANBL1221 phase 2 trial for patients with first relapse/first declaration of refractory high-risk neuroblastoma, irinotecan and temozolomide (I/T) combined with either temsirolimus (TEMS) or immunotherapy (the anti-GD2 antibody dinutuximab (DIN) and granulocyte macrophage colony stimulating factory (GM-CSF)) was administered. The response rate among patients treated with I/T/DIN/GM-CSF in the initial cohort (n=17) was 53%; additional patients were enrolled to permit further evaluation of this chemoimmunotherapy regimen. Potential associations between immune-related biomarkers and clinical outcomes including response and survival were evaluated. METHODS: Patients were evaluated for specific immunogenotypes that influence natural killer (NK) cell activity, including killer immunoglobulin-like receptors (KIRs) and their ligands, Fc gamma receptors, and NCR3. Total white cells and leucocyte subsets were assessed via complete blood counts, and flow cytometry of peripheral blood mononuclear cells was performed to assess the potential association between immune cell subpopulations and surface marker expression and clinical outcomes. Appropriate statistical tests of association were performed. The Bonferroni correction for multiple comparisons was performed where indicated. RESULTS: Of the immunogenotypes assessed, the presence or absence of certain KIR and their ligands was associated with clinical outcomes in patients treated with chemoimmunotherapy rather than I/T/TEMS. While median values of CD161, CD56, and KIR differed in responders and non-responders, statistical significance was not maintained in logistic regression models. White cell and neutrophil counts were associated with differences in survival outcomes, however, increases in risk of event in patients assigned to chemoimmunotherapy were not clinically significant. CONCLUSIONS: These findings are consistent with those of prior studies showing that KIR/KIR-ligand genotypes are associated with clinical outcomes following anti-GD2 immunotherapy in children with neuroblastoma. The current study confirms the importance of KIR/KIR-ligand genotype in the context of I/T/DIN/GM-CSF chemoimmunotherapy administered to patients with relapsed or refractory disease in a clinical trial. These results are important because this regimen is now widely used for treatment of patients at time of first relapse/first declaration of refractory disease. Efforts to assess the role of NK cells and genes that influence their function in response to immunotherapy are ongoing. TRIAL REGISTRATION NUMBER: NCT01767194.

Radiation to all macroscopic sites of tumor permits greater systemic antitumor response to in situ vaccination.

BACKGROUND: The antitumor effects of external beam radiation therapy (EBRT) are mediated, in part, by an immune response. We have reported that a single fraction of 12 Gy EBRT combined with intratumoral anti-GD2 hu14.18-IL2 immunocytokine (IC) generates an effective in situ vaccine (ISV) against GD2-positive murine tumors. This ISV is effective in eradicating single tumors with sustained immune memory; however, it does not generate an adequate abscopal response against macroscopic distant tumors. Given the immune-stimulatory capacity of radiation therapy (RT), we hypothesized that delivering RT to all sites of disease would augment systemic antitumor responses to ISV. METHODS: We used a syngeneic B78 murine melanoma model consisting of a 'primary' flank tumor and a contralateral smaller 'secondary' flank tumor, treated with 12 Gy EBRT and intratumoral IC immunotherapy to the primary and additional EBRT to the secondary tumor. As a means of delivering RT to all sites of disease, both known and occult, we also used a novel alkylphosphocholine analog, NM600, conjugated to 90Y as a targeted radionuclide therapy (TRT). Tumor growth, overall survival, and cause of death were measured. Flow cytometry was used to evaluate immune population changes in both tumors. RESULTS: Abscopal effects of local ISV were amplified by delivering as little as 2-6 Gy of EBRT to the secondary tumor. When the primary tumor ISV regimen was delivered in mice receiving 12 Gy EBRT to the secondary tumor, we observed improved overall survival and more disease-free mice with immune memory compared with either ISV or 12 Gy EBRT alone. Similarly, TRT combined with ISV resulted in improved overall survival and a trend towards reduced tumor growth rates when compared with either treatment alone. Using flow cytometry, we identified an influx of CD8+ T cells with a less exhausted phenotype in both the ISV-targeted primary and the distant secondary tumor following the combination of secondary tumor EBRT or TRT with primary tumor ISV. CONCLUSIONS: We report a novel use for low-dose RT, not as a direct antitumor modality but as an immunomodulator capable of driving and expanding antitumor immunity against metastatic tumor sites following ISV.

Comparative Study of the Effect of Radiation Delivered by Lutetium-177 or Actinium-225 on Anti-GD2 Chimeric Antigen Receptor T Cell Viability and Functions.

BACKGROUND AND PURPOSE: Chimeric antigen receptor (CAR) T cells have been relatively ineffective against solid tumors. Low-dose radiation which can be delivered to multiple sites of metastases by targeted radionuclide therapy (TRT) can elicit immunostimulatory effects. However, TRT has never been combined with CAR T cells against solid tumors in a clinical setting. This study investigated the effects of radiation delivered by Lutetium-177 (177Lu) and Actinium-225 (225Ac) on the viability and effector function of CAR T cells in vitro to evaluate the feasibility of such therapeutic combinations. After the irradiation of anti-GD2 CAR T cells with various doses of radiation delivered by 177Lu or 225Ac, their viability and cytotoxic activity against GD2-expressing human CHLA-20 neuroblastoma and melanoma M21 cells were determined by flow cytometry. The expression of the exhaustion marker PD-1, activation marker CD69 and the activating receptor NKG2D was measured on the irradiated anti-GD2 CAR T cells. Both 177Lu and 225Ac displayed a dose-dependent toxicity on anti-GD2 CAR T cells. However, radiation enhanced the cytotoxic activity of these CAR T cells against CHLA-20 and M21 irrespective of the dose tested and the type of radionuclide. No significant changes in the expression of PD-1, CD69 and NKG2D was noted on the CAR T cells following irradiation. Given a lower CAR T cell viability at equal doses and an enhancement of cytotoxic activity irrespective of the radionuclide type, 177Lu-based TRT may be preferred over 225Ac-based TRT when evaluating a potential synergism between these therapies in vivo against solid tumors.

GPC2 antibody-drug conjugate reprograms the neuroblastoma immune milieu to enhance macrophage-driven therapies.

BACKGROUND: Antibody-drug conjugates (ADCs) that deliver cytotoxic drugs to tumor cells have emerged as an effective and safe anticancer therapy. ADCs may induce immunogenic cell death (ICD) to promote additional endogenous antitumor immune responses. Here, we characterized the immunomodulatory properties of D3-GPC2-PBD, a pyrrolobenzodiazepine (PBD) dimer-bearing ADC that targets glypican 2 (GPC2), a cell surface oncoprotein highly differentially expressed in neuroblastoma. METHODS: ADC-mediated induction of ICD was studied in GPC2-expressing murine neuroblastomas in vitro and in vivo. ADC reprogramming of the neuroblastoma tumor microenvironment was profiled by RNA sequencing, cytokine arrays, cytometry by time of flight and flow cytometry. ADC efficacy was tested in combination with macrophage-driven immunoregulators in neuroblastoma syngeneic allografts and human patient-derived xenografts. RESULTS: The D3-GPC2-PBD ADC induced biomarkers of ICD, including neuroblastoma cell membrane translocation of calreticulin and heat shock proteins (HSP70/90) and release of high-mobility group box 1 and ATP. Vaccination of immunocompetent mice with ADC-treated murine neuroblastoma cells promoted T cell-mediated immune responses that protected animals against tumor rechallenge. ADC treatment also reprogrammed the tumor immune microenvironment to a proinflammatory state in these syngeneic neuroblastoma models, with increased tumor trafficking of activated macrophages and T cells. In turn, macrophage or T-cell inhibition impaired ADC efficacy in vivo, which was alternatively enhanced by both CD40 agonist and CD47 antagonist antibodies. In human neuroblastomas, the D3-GPC2-PBD ADC also induced ICD and promoted tumor phagocytosis by macrophages, which was further enhanced when blocking CD47 signaling in vitro and in vivo. CONCLUSIONS: We elucidated the immunoregulatory properties of a GPC2-targeted ADC and showed robust efficacy of combination immunotherapies in diverse neuroblastoma preclinical models.

Local TLR4 stimulation augments in situ vaccination induced via local radiation and anti-CTLA-4 checkpoint blockade through induction of CD8 T-cell independent Th1 polarization.

BACKGROUND: Radiation therapy (RT) has been demonstrated to generate an in situ vaccination (ISV) effect in murine models and in patients with cancer; however, this has not routinely translated into enhanced clinical response to immune checkpoint inhibition (ICI). We investigated whether the commonly used vaccine adjuvant, monophosphoryl lipid A (MPL) could augment the ISV regimen consisting of combination RT and ICI. MATERIALS/METHODS: We used syngeneic murine models of melanoma (B78) and prostate cancer (Myc-CaP). Tumor-bearing mice received either RT (12 Gy, day 1), RT+anti-CTLA-4 (C4, day 3, 6, 9), MPL (20 µg IT injection days 5, 7, 9), RT+C4+MPL, or PBS control. To evaluate the effect of MPL on the irradiated tumor microenvironment, primary tumor with tumor draining lymph nodes were harvested for immune cell infiltration analysis and cytokine profiling, and serum was collected for analysis of antitumor antibody populations. RESULTS: Combination RT+C4+MPL significantly reduced tumor growth, increased survival and complete response rate compared with RT+C4 in both B78 and Myc-CaP models. MPL favorably reprogrammed the irradiated tumor-immune microenvironment toward M1 macrophage and Th1 TBET+CD4+ T cell polarization. Furthermore, MPL significantly increased intratumoral expression of several Th1-associated and M1-associated proinflammatory cytokines. In co-culture models, MPL-stimulated macrophages directly activated CD8 T cells and polarized CD4 cells toward Th1 phenotype. MPL treatment significantly increased production of Th1-associated, IgG2c antitumor antibodies, which were required for and predictive of antitumor response to RT+C4+MPL, and enabled macrophage-mediated antibody-dependent direct tumor cell killing by MPL-stimulated macrophages. Macrophage-mediated tumor cell killing was dependent on FcγR expression. In metastatic models, RT and MPL generated a systemic antitumor immune response that augmented response to ICIs. This was dependent on macrophages and CD4+ but not CD8+T cells. CONCLUSIONS: We report the potential for MPL to augment the ISV effect of combination RT+C4 through FcγR, macrophage, and TBET+CD4+ Th1 cell dependent mechanisms. To our knowledge, this is the first report describing generation of a CD8+ T cell-independent, Th1 polarized, systemic antitumor immune response with subsequent generation of immunologic memory. These findings support the potential for vaccine adjuvants to enhance the efficacy of in situ tumor vaccine approaches.

Roles of CD4+ T cells as mediators of antitumor immunity.

It has been well established that CD8+ T cells serve as effector cells of the adaptive immune response against tumors, whereas CD4+ T cells either help or suppress the generation of CD8+ cytotoxic T cells. However, in several experimental models as well as in cancer patients, it has been shown that CD4+ T cells can also mediate antitumor immunity either directly by killing tumor cells or indirectly by activating innate immune cells or by reducing tumor angiogenesis. In this review, we discuss the growing evidence of this underappreciated role of CD4+ T cells as mediators of antitumor immunity.

Multifunctional nanoparticle potentiates the in situ vaccination effect of radiation therapy and enhances response to immune checkpoint blockade.

Radiation therapy (RT) activates an in situ vaccine effect when combined with immune checkpoint blockade (ICB), yet this effect may be limited because RT does not fully optimize tumor antigen presentation or fully overcome suppressive mechanisms in the tumor-immune microenvironment. To overcome this, we develop a multifunctional nanoparticle composed of polylysine, iron oxide, and CpG (PIC) to increase tumor antigen presentation, increase the ratio of M1:M2 tumor-associated macrophages, and enhance stimulation of a type I interferon response in conjunction with RT. In syngeneic immunologically "cold" murine tumor models, the combination of RT, PIC, and ICB significantly improves tumor response and overall survival resulting in cure of many mice and consistent activation of tumor-specific immune memory. Combining RT with PIC to elicit a robust in situ vaccine effect presents a simple and readily translatable strategy to potentiate adaptive anti-tumor immunity and augment response to ICB or potentially other immunotherapies.

Phase 2 study of anti-disialoganglioside antibody, dinutuximab, in combination with GM-CSF in patients with recurrent osteosarcoma: A report from the Children's Oncology Group.

PURPOSE: Novel effective therapies are urgently needed in recurrent osteosarcoma. GD2 is expressed in human osteosarcoma tumours and cell lines. This study evaluated the disease control rate (DCR) in patients with recurrent osteosarcoma treated with the anti-GD2 antibody dinutuximab plus cytokine therapy as compared to historical outcomes. METHODS: AOST1421 was a single-arm Phase 2 study for patients with recurrent pulmonary osteosarcoma in complete surgical remission. Patients received up to five cycles of dinutuximab (70 mg/m2/cycle) with granulocyte-macrophage colony-stimulating factor (GM-CSF). Two different dinutuximab infusion schedules were studied: 35 mg/m2/day over 20 h (2 days) and 17.5 mg/m2/day over 10 h (4 days). Primary end point was DCR, defined as a proportion of patients event free at 12 months from enrolment. The historical benchmark was 12-month DCR of 20% (95% CI 10-34%). Dinutuximab would be considered effective if ≥ 16/39 patients remained event free. Secondary objectives included toxicity evaluation and pharmacokinetics. RESULTS: Thirty-nine eligible patients were included in the outcome analysis. Dinutuximab did not demonstrate evidence of efficacy as 11/39 patients remained event free for a DCR of 28.2% (95% CI 15-44.9%). One of 136 administered therapy cycles met criteria for unacceptable toxicity when a patient experienced sudden death of unknown cause. Other ≥ Grade 3 toxicities included pain, diarrhoea, hypoxia, and hypotension. Pharmacokinetic parameters were similar in the two schedules. CONCLUSIONS: The combination of dinutuximab with GM-CSF did not significantly improve DCR in recurrent osteosarcoma. Dinutuximab toxicity and pharmacokinetics in adolescent and young adult osteosarcoma patients were similar to younger patients. Other strategies for targeting GD2 in osteosarcoma are being developed.

Outcomes Following GD2-Directed Postconsolidation Therapy for Neuroblastoma After Cessation of Random Assignment on ANBL0032: A Report From the Children's Oncology Group.

PURPOSE: Postconsolidation immunotherapy including dinutuximab, granulocyte-macrophage colony-stimulating factor, and interleukin-2 improved outcomes for patients with high-risk neuroblastoma enrolled on the randomized portion of Children's Oncology Group study ANBL0032. After random assignment ended, all patients were assigned to immunotherapy. Survival and toxicities were assessed. PATIENTS AND METHODS: Patients with a pre-autologous stem cell transplant (ASCT) response (excluding bone marrow) of partial response or better were eligible. Demographics, stage, tumor biology, pre-ASCT response, and adverse events were summarized using descriptive statistics. Event-free survival (EFS) and overall survival (OS) from time of enrollment (up to day +200 from last ASCT) were evaluated. RESULTS: From 2009 to 2015, 1,183 patients were treated. Five-year EFS and OS for the entire cohort were 61.1 ± 1.9% and 71.9 ± 1.7%, respectively. For patients ≥ 18 months old at diagnosis with International Neuroblastoma Staging System stage 4 disease (n = 662) 5-year EFS and OS were 57.0 ± 2.4% and 70.9 ± 2.2%, respectively. EFS was superior for patients with complete response/very good partial response pre-ASCT compared with those with PR (5-year EFS: 64.2 ± 2.2% v 55.4 ± 3.2%, P = .0133); however, OS was not significantly different. Allergic reactions, capillary leak, fever, and hypotension were more frequent during interleukin-2-containing cycles than granulocyte-macrophage colony-stimulating factor-containing cycles (P < .0001). EFS was superior in patients with higher peak dinutuximab levels during cycle 1 (P = .034) and those with a high affinity FCGR3A genotype (P = .0418). Human antichimeric antibody status did not correlate with survival. CONCLUSION: Analysis of a cohort assigned to immunotherapy after cessation of random assignment on ANBL0032 confirmed previously described survival and toxicity outcomes. EFS was highest among patients with end-induction complete response/very good partial response. Among patients with available data, higher dinutuximab levels and FCGR3A genotype were associated with superior EFS. These may be predictive biomarkers for dinutuximab therapy.

Stress Keratin 17 Expression in Head and Neck Cancer Contributes to Immune Evasion and Resistance to Immune-Checkpoint Blockade.

PURPOSE: We investigated whether in human head and neck squamous cell carcinoma (HNSCC) high levels of expression of stress keratin 17 (K17) are associated with poor survival and resistance to immunotherapy. EXPERIMENTAL DESIGN: We investigated the role of K17 in regulating both the tumor microenvironment and immune responsiveness of HNSCC using a syngeneic mouse HNSCC model, MOC2. MOC2 gives rise to immunologically cold tumors that are resistant to immune-checkpoint blockade (ICB). We engineered multiple, independent K17 knockout (KO) MOC2 cell lines and monitored their growth and response to ICB. We also measured K17 expression in human HNSCC of patients undergoing ICB. RESULTS: MOC2 tumors were found to express K17 at high levels. When knocked out for K17 (K17KO MOC2), these cells formed tumors that grew slowly or spontaneously regressed and had a high CD8+ T-cell infiltrate in immunocompetent syngeneic C57BL/6 mice compared with parental MOC2 tumors. This phenotype was reversed when we depleted mice for T cells. Whereas parental MOC2 tumors were resistant to ICB treatment, K17KO MOC2 tumors that did not spontaneously regress were eliminated upon ICB treatment. In a cohort of patients with HNSCC receiving pembrolizumab, high K17 expression correlated with poor response. Single-cell RNA-sequencing analysis revealed broad differences in the immune landscape of K17KO MOC2 tumors compared with parental MOC2 tumors, including differences in multiple lymphoid and myeloid cell types. CONCLUSIONS: We demonstrate that K17 expression in HNSCC contributes to immune evasion and resistance to ICB treatment by broadly altering immune landscapes of tumors.

Mechanism of effective combination radio-immunotherapy against 9464D-GD2, an immunologically cold murine neuroblastoma.

BACKGROUND: Most pediatric cancers are considered immunologically cold with relatively few responding to immune checkpoint inhibition. We recently described an effective combination radio-immunotherapy treatment regimen ( c ombination a daptive- i nnate immunotherapy r egimen (CAIR)) targeting adaptive and innate immunity in 9464D-GD2, an immunologically cold model of neuroblastoma. Here, we characterize the mechanism of CAIR and the role of major histocompatibility complex class I (MHC-I) in the treatment response. METHODS: Mice bearing GD2-expressing 9464D-GD2 tumors were treated with CAIR (external beam radiotherapy, hu14.18-IL2 immunocytokine, CpG, anti-CD40, and anti-CTLA4) and tumor growth and survival were tracked. Depletion of specific immune cell lineages, as well as testing in immunodeficient R2G2 mice, were used to determine the populations necessary for treatment efficacy. Induction of MHC-I expression in 9464D-GD2 cells in response to interferon-γ (IFN-γ) and CAIR was measured in vitro and in vivo, respectively, by flow cytometry and quantitative real-time PCR. A cell line with IFN-γ-inducible MHC-I expression (9464D-GD2-I) was generated by transfecting a subclone of the parental cell line capable of expressing MHC-I with GD2 synthase and was used in vivo to assess the impact of MHC-I expression on responsiveness to CAIR. RESULTS: CAIR cures some mice bearing small (50 mm3) but not larger (100 mm3) 9464D-GD2 tumors and these cured mice develop weak memory responses against tumor rechallenge. Early suppression of 9464D-GD2 tumors by CAIR does not require T or natural killer (NK) cells, but eventual tumor cures are NK cell dependent. Unlike the parental 9464D cell line, 9464D-GD2 cells have uniformly very low MHC-I expression at baseline and fail to upregulate expression in response to IFN-γ. In contrast, 9464D-GD2-I upregulates MHC-I in response to IFN-γ and is less responsive to CAIR. CONCLUSION: Treatment with CAIR cures 9464D-GD2 tumors in a NK cell dependent manner and induction of MHC-I by tumors cells was associated with decreased efficacy. These results demonstrate that the early tumor response to this regimen is T and NK cell independent, but that NK cells have a role in generating lasting cures in the absence of MHC-I expression by tumor cells. Further strategies to better inhibit tumor outgrowth in this setting may require further NK activation or the ability to engage alternative immune effector cells.

Expression of neuroblastoma-related genes in bone marrow at end of high-risk neuroblastoma therapy.

BACKGROUND: Minimal disease quantification may predict event-free survival (EFS) and overall survival (OS). METHODS: We evaluated mRNA expression of five neuroblastoma-associated genes (NB5 assay) in bone marrows (BM) of patients with newly diagnosed high-risk neuroblastoma who received consistent immunotherapy. mRNA expression of CHGA, DCX, DDC, PHOX2B, and TH genes in BM of 479 patients enrolled on the immunotherapy arm of Children's Oncology Group trials ANBL0032 and ANBL0931 was evaluated using real-time polymerase chain reaction (PCR)-based TaqMan low-density array. Results from end-consolidation and end-therapy were analyzed for association with five-year EFS/OS and patient and tumor characteristics. Tests of statistical significance were two-sided. RESULTS: NB5 assay detected neuroblastoma-related mRNA in 222 of 286 (77.6%) of BMs obtained at end-consolidation and 188 of 304 (61.8%) at end-therapy. Any mRNA level detected in end-therapy BM correlated with significantly worse EFS (57% [49.6%-63.7%] vs 73.0% [63.5%-80.4%]; P = 0.005), but not OS. Analysis limited to patients in complete response at end-therapy still found a significant difference in EFS with detectable versus not detectable NB5 assay results (58.9% [49.5%-67.1%] vs 76.6% [66.1%-84.2%]; P = 0.01). End-consolidation results did not correlate with EFS or OS. Multivariable analysis determined end-therapy NB5 assay BM results (P = 0.02), age at diagnosis (P = 0.002), and preconsolidation response (P = 0.02) were significantly associated with EFS independent of other clinical and biological parameters evaluated, including end-therapy response. CONCLUSIONS: If further validated in additional patient cohorts, the NB5 assay's ability to independently predict EFS from end-therapy could improve patient stratification for novel maintenance therapy trials after current end-therapy to improve outcome.