Intratumoral radiation dose heterogeneity augments antitumor immunity in mice and primes responses to checkpoint blockade.

Radiation therapy (RT) activates multiple immunologic effects in the tumor microenvironment (TME), with diverse dose-response relationships observed. We hypothesized that, in contrast with homogeneous RT, a heterogeneous RT dose would simultaneously optimize activation of multiple immunogenic effects in a single TME, resulting in a more effective antitumor immune response. Using high-dose-rate brachytherapy, we treated mice bearing syngeneic tumors with a single fraction of heterogeneous RT at a dose ranging from 2 to 30 gray. When combined with dual immune checkpoint inhibition in murine models, heterogeneous RT generated more potent antitumor responses in distant, nonirradiated tumors compared with any homogeneous dose. The antitumor effect after heterogeneous RT required CD4 and CD8 T cells and low-dose RT to a portion of the tumor. At the 3-day post-RT time point, dose heterogeneity imprinted the targeted TME with spatial differences in immune-related gene expression, antigen presentation, and susceptibility of tumor cells to immune-mediated destruction. At a later 10-day post-RT time point, high-, moderate-, or low-RT-dose regions demonstrated distinct infiltrating immune cell populations. This was associated with an increase in the expression of effector-associated cytokines in circulating CD8 T cells. Consistent with enhanced adaptive immune priming, heterogeneous RT promoted clonal expansion of effector CD8 T cells. These findings illuminate the breadth of dose-dependent effects of RT on the TME and the capacity of heterogeneous RT to promote antitumor immunity when combined with immune checkpoint inhibitors.

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: We aim to 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 was 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 (days 9 and 12), while width and density changed early (day 6) compared with control mice. Single-fiber collagen features calculated in CT-FIRE were the most sensitive to the changes among treatment groups compared with bulk collagen features. 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.

A combined radio-immunotherapy regimen eradicates late-stage tumors in mice.

Background: The majority of experimental approaches for cancer immunotherapy are tested against relatively small tumors in tumor-bearing mice, because in most cases advanced cancers are resistant to the treatments. In this study, we asked if even late-stage mouse tumors can be eradicated by a rationally designed combined radio-immunotherapy (CRI) regimen. Methods: CRI consisted of local radiotherapy, intratumoral IL-12, slow-release systemic IL-2 and anti- CTLA-4 antibody. Therapeutic effects of CRI against several weakly immunogenic and immunogenic mouse tumors including B78 melanoma, MC38 and CT26 colon carcinomas and 9464D neuroblastoma were evaluated. Immune cell depletion and flow cytometric analysis were performed to determine the mechanisms of the antitumor effects. Results: Tumors with volumes of 2,000 mm3 or larger were eradicated by CRI. Flow analyses of the tumors revealed reduction of T regulatory (Treg) cells and increase of CD8/Treg ratios following CRI. Rapid shrinkage of the treated tumors did not require T cells, whereas T cells were involved in the systemic effect against the distant tumors. Cured mice developed immunological memory. Conclusions: These findings underscore that rationally designed combination immunotherapy regimens can be effective even against large, late-stage tumors.

Targeting both GD2 and B7-H3 using bispecific antibody improves tumor selectivity for GD2-positive tumors.

Objectives: Disialoganglioside 2 (GD2), overexpressed by cancers such as melanoma and neuroblastoma, is a tumor antigen for targeted therapy. The delivery of conventional IgG antibody technologies targeting GD2 is limited clinically by its co-expression on nerves that contributes to toxicity presenting as severe neuropathic pain. To improve the tumor selectivity of current GD2-targeting approaches, a next-generation bispecific antibody targeting GD2 and B7-H3 (CD276) was generated. Methods: Differential expression of human B7-H3 (hB7-H3) was transduced into GD2+ B78 murine melanoma cells and confirmed by flow cytometry. We assessed the avidity and selectivity of our GD2-B7-H3 targeting bispecific antibodies (INV34-6, INV33-2, and INV36-6) towards GD2+/hB7-H3- B78 cells relative to GD2+/hB7-H3+ B78 cells using flow cytometry and competition binding assays, comparing results an anti-GD2 antibody (dinutuximab, DINU). The bispecific antibodies, DINU, and a non-targeted bispecific control (bsAb CTRL) were conjugated with deferoxamine for radiolabeling with Zr-89 (t1/2 = 78.4 h). Using positron emission tomography (PET) studies, we evaluated the in vivo avidity and selectivity of the GD2-B7-H3 targeting bispecific compared to bsAb CTRL and DINU using GD2+/hB7-H3+ and GD2+/hB7-H3- B78 tumor models. Results: Flow cytometry and competition binding assays showed that INV34-6 bound with high avidity to GD2+/hB7-H3+ B78 cells with high avidity but not GD2+/hB7-H3+ B78 cells. In comparison, no selectivity between cell types was observed for DINU. PET in mice bearing the GD2+/hB7-H3- and GD2+/hB7-H3+ B78 murine tumor showed similar biodistribution in normal tissues for [89Zr]Zr-Df-INV34-6, [89Zr]Zr-Df-bsAb CTRL, and [89Zr]Zr-Df-DINU. Importantly, [89Zr]Zr-Df-INV34-6 tumor uptake was selective to GD2+/hB7-H3+ B78 over GD2+/hB7-H3- B78 tumors, and substantially higher to GD2+/hB7-H3+ B78 than the non-targeted [89Zr]Zr-Df-bsAb CTRL control. [89Zr]Zr-Df-DINU displayed similar uptake in both GD2+ tumor models, with uptake comparable to [89Zr]Zr-Df-INV34-6 in the GD2+/hB7-H3+ B78 model. Conclusion: The GD2-B7-H3 targeting bispecific antibodies successfully improved selectivity to cells expressing both antigens. This approach should address the severe toxicities associated with GD2-targeting therapies by reducing off-tumor GD2 binding in nerves. Continued improvements in bispecific antibody technologies will continue to transform the therapeutic biologics landscape.

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.

Administration of intratumoral GD2-directed interleukin-2 immunocytokine and local radiation therapy to activate immune rejection of spontaneous canine melanoma.

Canine malignant melanoma provides a clinically relevant, large animal parallel patient population to study the GD2-reactive hu14.18-IL-2 immunocytokine as it is similar to human melanoma and expresses GD2. The objectives of this study were to evaluate safety, radiation fractionation, and identify informative biomarkers of an in-situ tumor vaccine involving local radiation therapy plus intratumoral-immunocytokine in melanoma tumor-bearing dogs. Twelve dogs (six dogs/arm) with locally advanced or metastatic melanoma were randomized to receive a single 8 Gy fraction (arm A) or three 8 Gy fractions over 1 week (arm B) to the primary site and regional lymph nodes (when clinically involved) with the single or last fraction 5 days before intratumoral-immunocytokine at 12 mg/m 2 on 3 consecutive days. Serial tumor biopsies were obtained. All 12 dogs completed protocol treatment, and none experienced significant or unexpected adverse events. Evidence of antitumor activity includes one dog with a complete response at day 60, one dog with a partial response at day 60, and four dogs with mixed responses. Histology of serial biopsies shows a variably timed increase in intratumoral lymphocytic inflammation in some dogs. Canine NanoString analyses of serial biopsies identified changes in gene signatures of innate and adaptive cell types versus baseline. There were no significant differences in NanoString results between arm A and arm B. We conclude that intratumoral-immunocytokine in combination with local radiation therapy in canine melanoma is well tolerated and has antitumor activity with the potential to inform clinical development in melanoma patients.

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 = .002) 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 = .022) and a significant decrease in myeloid-derived suppressive cell frequency (7.1% of CD45 vs 10.3%; P = .044). 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.

Ranking Antibody Binding Epitopes and Proteins Across Samples from Whole Proteome Tiled Linear Peptides.

Ultradense peptide binding arrays that can probe millions of linear peptides comprising the entire proteomes or immunomes of human or mouse, or numerous microbes, are powerful tools for studying the abundance of different antibody repertoire in serum samples to understand adaptive immune responses. There are few statistical analysis tools for exploring high-dimensional, significant and reproducible antibody targets for ultradense peptide binding arrays at the linear peptide, epitope (grouping of adjacent peptides), and protein level across multiple samples/subjects (I.e. epitope spread or immunogenic regions within each protein) for understanding the heterogeneity of immune responses. We developed HERON (Hierarchical antibody binding Epitopes and pROteins from liNear peptides), an R package, which allows users to identify immunogenic epitopes using meta-analyses and spatial clustering techniques to explore antibody targets at various resolution and confidence levels, that can be found consistently across a specified number of samples through the entire proteome to study antibody responses for diagnostics or treatment. Our approach estimates significance values at the linear peptide (probe), epitope, and protein level to identify top candidates for validation. We test the performance of predictions on all three levels using correlation between technical replicates and comparison of epitope calls on 2 datasets, which shows HERON's competitiveness in estimating false discovery rates and finding general and sample-level regions of interest for antibody binding. The code is available as an R package downloadable from http://github.com/Ong-Research/HERON.

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.

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.

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.