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.

CD155 blockade enhances allogeneic natural killer cell-mediated antitumor response against osteosarcoma.

Background: Osteosarcoma (OS) patients that present with metastatic disease have a poor prognosis and no curative options. Allogeneic bone marrow transplant (alloBMT) is curative for hematologic malignancies through the graft-versus-tumor (GVT) effect, but to date has been ineffective for solid tumors like OS. CD155 is expressed on OS and interacts strongly with the inhibitory receptors TIGIT and CD96 but also binds to the activating receptor DNAM-1 on natural killer (NK) cells but has never been targeted after alloBMT. Combining adoptive transfer of allogeneic NK (alloNK) cells with CD155 checkpoint blockade after alloBMT may enhance a GVT effect against OS but could enhance toxicities like graft-versus-host-disease (GVHD). Methods: Ex vivo activated and expanded murine NK cells were generated with soluble IL-15/IL- 15Rα. AlloNK and syngeneic NK (synNK) cell phenotype, cytotoxicity, cytokine production, and degranulation against the CD155-expressing murine OS cell line K7M2 were assessed in vitro. Mice bearing pulmonary OS metastases underwent alloBMT followed by infusion of alloNK cells with combinations of anti-CD155 and anti-DNAM-1 blockade. Tumor growth, GVHD 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 to synNK cells, and this activity was further enhanced by CD155 blockade. CD155 blockade increased alloNK cell degranulation and interferon gamma production through DNAM-1, as these functions were abrogated during DNAM-1 blockade. In vivo, CD155 blockade after alloBMT increased EFS with no exacerbation of GVHD. Treatment with combination CD155 and DNAM-1 blockade ameliorated survival and tumor control benefits seen with CD155 blockade alone. In mice treated with CD155 blockade, genes related to NK cell cytotoxicity were upregulated. DNAM-1 blockade resulted in upregulation of NK cell inhibition. 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 will be critical to translating combination adoptive NK cell and immune checkpoint inhibition for patients with solid tumors treated with alloBMT. WHAT IS ALREADY KNOWN ON THIS TOPIC: Allogeneic bone marrow transplant (alloBMT) has yet to show efficacy in treating solid tumors, such as osteosarcoma (OS). CD155 is expressed on OS and interacts with natural killer (NK) cell receptors, such as activating receptor DNAM-1 and inhibitory receptors TIGIT and CD96 and has a dominant inhibitory effect on NK cell activity. Targeting CD155 interactions on allogeneic NK cells could enhance anti-OS responses, but this has not been tested after alloBMT. WHAT THIS STUDY ADDS: CD155 blockade enhances allogeneic natural killer cell-mediated cytotoxicity against osteosarcoma and improved event-free survival after alloBMT in an in vivo mouse model of metastatic pulmonary OS. Addition of DNAM-1 blockade abrogated CD155 blockade-enhanced allogeneic NK cell antitumor responses. HOW THIS STUDY MIGHT AFFECT RESEARCH PRACTICE OR POLICY: These results demonstrate efficacy of allogeneic NK cells combined with CD155 blockade to mount an antitumor response against CD155-expressing OS. Translation of combination adoptive NK cell and CD155 axis modulation offers a platform for alloBMT treatment approaches for pediatric patients with relapsed and refractory solid tumors.

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.

Antibody landscape of C57BL/6 mice cured of B78 melanoma via a combined radiation and immunocytokine immunotherapy regimen.

Sera of immune mice that were previously cured of their melanoma through a combined radiation and immunocytokine immunotherapy regimen consisting of 12 Gy of external beam radiation and the intratumoral administration of an immunocytokine (anti-GD2 mAb coupled to IL-2) with long-term immunological memory showed strong antibody-binding against melanoma tumor cell lines via flow cytometric analysis. Using a high-density whole-proteome peptide array (of 6.090.593 unique peptides), we assessed potential protein-targets for antibodies found in immune sera. Sera from 6 of these cured mice were analyzed with this high-density, whole-proteome peptide array to determine specific antibody-binding sites and their linear peptide sequence. We identified thousands of peptides that were targeted by these 6 mice and exhibited strong antibody binding only by immune (after successful cure and rechallenge), not naïve (before tumor implantation) sera and developed a robust method to detect these differentially targeted peptides. Confirmatory studies were done to validate these results using 2 separate systems, a peptide ELISA and a smaller scale peptide array utilizing a slightly different technology. To the best of our knowledge, this is the first study of the full set of germline encoded linear peptide-based proteome epitopes that are recognized by immune sera from mice cured of cancer via radio-immunotherapy. We furthermore found that although the generation of B-cell repertoire in immune development is vastly variable, and numerous epitopes are identified uniquely by immune serum from each of these 6 immune mice evaluated, there are still several epitopes and proteins that are commonly recognized by at least half of the mice studied. This suggests that every mouse has a unique set of antibodies produced in response to the curative therapy, creating an individual "fingerprint." Additionally, certain epitopes and proteins stand out as more immunogenic, as they are recognized by multiple mice in the immune group.

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.

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.

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.

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.

Short-course neoadjuvant in situ vaccination for murine melanoma.

BACKGROUND: Surgical resection remains an important component of multimodality treatment for most solid tumors. Neoadjuvant immunotherapy has several potential advantages, including in-situ tumor vaccination and pathologic assessment of response in the surgical specimen. We previously described an in-situ tumor vaccination strategy in melanoma using local radiation (RT) and an intratumoral injection of tumor-specific anti-GD2 immunocytokine (IT-IC). Here we tested whether neoadjuvant in-situ tumor vaccination using anti-GD2 immunocytokine and surgical resection, without RT, could generate immunologic memory capable of preventing recurrence or distant metastasis. METHODS: Mice bearing GD2 expressing B78 melanoma tumors were treated with neoadjuvant radiation, IT-IC, or combined RT + IT-IC. Surgical resection was performed following neoadjuvant immunotherapy. Immune infiltrate was assessed in the resection specimens. Mice were rechallenged with either B78 contralateral flank tumors or pulmonary seeding of non-GD2 expressing B16 melanoma metastasis induced experimentally. Rejection of rechallenge in mice treated with the various treatment regimens was considered evidence of immunologic memory. RESULTS: Neoadjuvant IT-IC and surgical resection resulted in increased CD8 T cell infiltration, a higher CD8:regulatory T cell ratio, and immunologic memory against contralateral flank rechallenge. The timing of resection did not significantly impact the development of memory, which was present as early as the day of surgery. There was less local wound toxicity with neoadjuvant IT-IC compared with neoadjuvant RT +IT IC. Neoadjuvant IT-IC and resection resulted in the rejection of B16 lung metastasis in a CD4 T cell dependent manner. CONCLUSIONS: Neoadjuvant IT-IC generates immunologic memory capable of preventing distant metastasis despite limited efficacy against large primary melanoma tumors. By combining neoadjuvant tumor vaccination and surgery, the toxicity of local RT was avoided. These preclinical data support further investigation regarding the use of neoadjuvant IT-IC in patients with melanoma at high risk for occult distant disease.

Radiation Augments the Local Anti-Tumor Effect of In Situ Vaccine With CpG-Oligodeoxynucleotides and Anti-OX40 in Immunologically Cold Tumor Models.

Introduction: Combining CpG oligodeoxynucleotides with anti-OX40 agonist antibody (CpG+OX40) is able to generate an effective in situ vaccine in some tumor models, including the A20 lymphoma model. Immunologically "cold" tumors, which are typically less responsive to immunotherapy, are characterized by few tumor infiltrating lymphocytes (TILs), low mutation burden, and limited neoantigen expression. Radiation therapy (RT) can change the tumor microenvironment (TME) of an immunologically "cold" tumor. This study investigated the effect of combining RT with the in situ vaccine CpG+OX40 in immunologically "cold" tumor models. Methods: Mice bearing flank tumors (A20 lymphoma, B78 melanoma or 4T1 breast cancer) were treated with combinations of local RT, CpG, and/or OX40, and response to treatment was monitored. Flow cytometry and quantitative polymerase chain reaction (qPCR) experiments were conducted to study differences in the TME, secondary lymphoid organs, and immune activation after treatment. Results: An in situ vaccine regimen of CpG+OX40, which was effective in the A20 model, did not significantly improve tumor response or survival in the "cold" B78 and 4T1 models, as tested here. In both models, treatment with RT prior to CpG+OX40 enabled a local response to this in situ vaccine, significantly improving the anti-tumor response and survival compared to RT alone or CpG+OX40 alone. RT increased OX40 expression on tumor infiltrating CD4+ non-regulatory T cells. RT+CpG+OX40 increased the ratio of tumor-infiltrating effector T cells to T regulatory cells and significantly increased CD4+ and CD8+ T cell activation in the tumor draining lymph node (TDLN) and spleen. Conclusion: RT significantly improves the local anti-tumor effect of the in situ vaccine CpG+OX40 in immunologically "cold", solid, murine tumor models where RT or CpG+OX40 alone fail to stimulate tumor regression.

Combination of radiation therapy, bempegaldesleukin, and checkpoint blockade eradicates advanced solid tumors and metastases in mice.

BACKGROUND: Current clinical trials are using radiation therapy (RT) to enhance an antitumor response elicited by high-dose interleukin (IL)-2 therapy or immune checkpoint blockade (ICB). Bempegaldesleukin (BEMPEG) is an investigational CD122-preferential IL-2 pathway agonist with prolonged in vivo half-life and preferential intratumoral expansion of T effector cells over T regulatory cells. BEMPEG has shown encouraging safety and efficacy in clinical trials when used in combination with PD-1 checkpoint blockade. In this study, we investigated the antitumor effect of local RT combined with BEMPEG in multiple immunologically 'cold' tumor models. Additionally, we asked if ICB could further enhance the local and distant antitumor effect of RT+BEMPEG in the setting of advanced solid tumors or metastatic disease. METHODS: Mice bearing flank tumors (B78 melanoma, 4T1 breast cancer, or MOC2 head and neck squamous cell carcinoma) were treated with combinations of RT and immunotherapy (including BEMPEG, high-dose IL-2, anti(α)-CTLA-4, and α-PD-L1). Mice bearing B78 flank tumors were injected intravenously with B16 melanoma cells to mimic metastatic disease and were subsequently treated with RT and/or immunotherapy. Tumor growth and survival were monitored. Peripheral T cells and tumor-infiltrating lymphocytes were assessed via flow cytometry. RESULTS: A cooperative antitumor effect was observed in all models when RT was combined with BEMPEG, and RT increased IL-2 receptor expression on peripheral T cells. This cooperative interaction was associated with increased IL-2 receptor expression on peripheral T cells following RT. In the B78 melanoma model, RT+BEMPEG resulted in complete tumor regression in the majority of mice with a single ~400 mm3 tumor. This antitumor response was T-cell dependent and supported by long-lasting immune memory. Adding ICB to RT+BEMPEG strengthened the antitumor response and cured the majority of mice with a single ~1000 mm3 B78 tumor. In models with disseminated metastasis (B78 primary with B16 metastasis, 4T1, and MOC2), the triple combination of RT, BEMPEG, and ICB significantly improved primary tumor response and survival. CONCLUSION: The combination of local RT, BEMPEG, and ICB cured mice with advanced, immunologically cold tumors and distant metastasis in a T cell-dependent manner, suggesting this triple combination warrants clinical testing.

Depth of tumor implantation affects response to in situ vaccination in a syngeneic murine melanoma model.

An important component of research using animal models is ensuring rigor and reproducibility. This study was prompted after two experimenters performing virtually identical studies obtained different results when syngeneic B78 murine melanoma cells were implanted into the skin overlying the flank and treated with an in situ vaccine (ISV) immunotherapy. Although both experimenters thought they were using identical technique, we determined that one was implanting the tumors intradermally (ID) and the other was implanting them subcutaneously (SC). Though the baseline in vivo immunogenicity of tumors can depend on depth of their implantation, the response to immunotherapy as a function of tumor depth, particularly in immunologically 'cold' tumors, has not been well studied. The goal of this study was to evaluate the difference in growth kinetics and response to immunotherapy between identically sized melanoma tumors following ID versus SC implantation. We injected C57BL/6 mice with syngeneic B78 melanoma cells either ID or SC in the flank. When tumors reached 190-230 mm3, they were grouped into a 'wave' and treated with our previously published ISV regimen (12 Gy local external beam radiation and intratumoral hu14.18-IL2 immunocytokine). Physical examination demonstrated that ID-implanted tumors were mobile on palpation, while SC-implanted tumors became fixed to the underlying fascia. Histologic examination identified a critical fascial layer, the panniculus carnosus, which separated ID and SC tumors. SC tumors reached the target tumor volume significantly faster compared with ID tumors. Most ID tumors exhibited either partial or complete response to this immunotherapy, whereas most SC tumors did not. Further, the 'mobile' or 'fixed' phenotype of tumors predicted response to therapy, regardless of intended implantation depth. These findings were then extended to additional immunotherapy regimens in four separate tumor models. These data indicate that the physical 'fixed' versus 'mobile' characterization of the tumors may be one simple method of ensuring homogeneity among implanted tumors prior to initiation of treatment. Overall, this short report demonstrates that small differences in depth of tumor implantation can translate to differences in response to immunotherapy, and proposes a simple physical examination technique to ensure consistent tumor depth when conducting implantable tumor immunotherapy experiments.

Tumor-Specific Antibody, Cetuximab, Enhances the In Situ Vaccine Effect of Radiation in Immunologically Cold Head and Neck Squamous Cell Carcinoma.

In head and neck squamous cell carcinoma (HNSCC) tumors that over-expresses huEGFR, the anti-EGFR antibody, cetuximab, antagonizes tumor cell viability and sensitizes to radiation therapy. However, the immunologic interactions between cetuximab and radiation therapy are not well understood. We transduced two syngeneic murine HNSCC tumor cell lines to express human EGFR (MOC1- and MOC2-huEGFR) in order to facilitate evaluation of the immunologic interactions between radiation and cetuximab. Cetuximab was capable of inducing antibody-dependent cellular cytotoxicity (ADCC) in MOC1- and MOC2-huEGFR cells but showed no effect on the viability or radiosensitivity of these tumor cells, which also express muEGFR that is not targeted by cetuximab. Radiation enhanced the susceptibility of MOC1- and MOC2-huEGFR to ADCC, eliciting a type I interferon response and increasing expression of NKG2D ligands on these tumor cells. Co-culture of splenocytes with cetuximab and MOC2-huEGFR cells resulted in increased expression of IFNγ in not only NK cells but also in CD8+ T cells, and this was dependent upon splenocyte expression of FcγR. In MOC2-huEGFR tumors, combining radiation and cetuximab induced tumor growth delay that required NK cells, EGFR expression, and FcγR on host immune cells. Combination of radiation and cetuximab increased tumor infiltration with NK and CD8+ T cells but not regulatory T cells. Expression of PD-L1 was increased in MOC2-huEGFR tumors following treatment with radiation and cetuximab. Delivering anti-PD-L1 antibody with radiation and cetuximab improved survival and resulted in durable tumor regression in some mice. Notably, these cured mice showed evidence of an adaptive memory response that was not specifically directed against huEGFR. These findings suggest an opportunity to improve the treatment of HNSCC by combining radiation and cetuximab to engage an innate anti-tumor immune response that may prime an effective adaptive immune response when combined with immune checkpoint blockade. It is possible that this approach could be extended to any immunologically cold tumor that does not respond to immune checkpoint blockade alone and for which a tumor-specific antibody exists or could be developed.

Intratumoral injection reduces toxicity and antibody-mediated neutralization of immunocytokine in a mouse melanoma model.

BACKGROUND: Some patients with cancer treated with anticancer monoclonal antibodies (mAbs) develop antidrug antibodies (ADAs) that recognize and bind the therapeutic antibody. This response may neutralize the therapeutic mAb, interfere with mAb effector function or cause toxicities. We investigated the potential influence of ADA to modify the tumor-binding capability of a tumor-reactive 'immunocytokine' (IC), namely, a fusion protein (hu14.18-IL2) consisting of a humanized, tumor-reactive, anti-GD2 mAb genetically linked to interleukin 2. We characterize the role of treatment delivery of IC (intravenous vs intratumoral) on the impact of ADA on therapeutic outcome following IC treatments in an established antimelanoma (MEL) regimen involving radiotherapy (RT) +IC. METHODS: C57BL/6 mice were injected with human IgG or the hu14.18-IL2 IC to develop a mouse anti-human antibody (MAHA) response (MAHA+). In vitro assays were performed to assess ADA binding to IC using sera from MAHA+ and MAHA- mice. In vivo experiments assessed the levels of IC bound to tumor in MAHA+ and MAHA- mice, and the influence of IC route of delivery on its ability to bind to B78 (GD2+) MEL tumors. RESULTS: MAHA is inducible in C57BL/6 mice. In vitro assays show that MAHA is capable of inhibiting the binding of IC to GD2 antigen on B78 cells, resulting in impaired ADCC mediated by IC. When B78-bearing mice are injected intravenously with IC, less IC binds to B78-MEL tumors in MAHA+ mice than in MAHA- mice. In contrast, when IC is injected intratumorally in tumor-bearing mice, the presence of MAHA does not detectibly impact IC binding to the tumor. Combination therapy with RT+IT-IC showed improved tumor regression compared with RT alone in MAHA+ mice. If given intratumorally, IC could be safely readministered in tumor-bearing MAHA+ mice, while intravenous injections of IC in MAHA+ mice caused severe toxicity. Histamine levels were elevated in MAHA+ mice compared with MAHA- mice after reintroduction of IC. CONCLUSIONS: Intratumoral injection may be a means of overcoming ADA neutralization of therapeutic activity of tumor-reactive mAbs or ICs and may reduce systemic toxicity, which could have significant translational relevance.

In situ Vaccine Plus Checkpoint Blockade Induces Memory Humoral Response.

In a syngeneic murine melanoma (MEL) model, we recently reported an in situ vaccination response to combined radiation (RT) and intra-tumoral (IT) injection of anti-GD2 hu14. 18-IL2 immunocytokine (IC). This combined treatment resulted in 71% complete and durable regression of 5-week tumors, a tumor-specific memory T cell response, and augmented response to systemic anti-CTLA-4 antibody checkpoint blockade. While the ability of radiation to diversify anti-tumor T cell response has been reported, we hypothesize that mice rendered disease-free (DF) by a RT-based ISV might also exhibit a heightened B cell response. C57BL/6 mice were engrafted with 2 × 106 GD2+ B78 MEL and treated at a target tumor size of ~200 mm3 with 12 Gy RT, IT-IC on day (D)6-D10, and anti-CTLA-4 on D3, 6, and 9. Serum was collected via facial vein before tumor injection, before treatment, during treatment, after becoming DF, and following rejection of subcutaneous 2 × 106 B78 MEL re-challenge on D90. Flow cytometry demonstrated the presence of tumor-specific IgG in sera from mice rendered DF and rejecting re-challenge with B78 MEL at D90 after starting treatment. Consistent with an adaptive endogenous anti-tumor humoral memory response, these anti-tumor antibodies bound to B78 cells and parental B16 cells (GD2-), but not to the unrelated syngeneic Panc02 or Panc02 GD2+ cell lines. We evaluated the kinetics of this response and observed that tumor-specific IgG was consistently detected by D22 after initiation of treatment, corresponding to a time of rapid tumor regression. The amount of tumor-specific antibody binding to tumor cells (as measured by flow MFI) did not correlate with host animal prognosis. Incubation of B16 MEL cells in DF serum, vs. naïve serum, prior to IV injection, did not delay engraftment of B16 metastases and showed similar overall survival rates. B cell depletion using anti-CD20 or anti-CD19 and anti-B220 did not impact the efficacy of ISV treatment. Thus, treatment with RT + IC + anti-CTLA-4 results in adaptive anti-tumor humoral memory response. This endogenous tumor-specific antibody response does not appear to have therapeutic efficacy but may serve as a biomarker for an anti-tumor T cell response.