Publications by authors named "Paul M Sondel"

115 Publications

Short-course neoadjuvant in situ vaccination for murine melanoma.

J Immunother Cancer 2022 Jan;10(1)

Department of Human Oncology, University of Wisconsin, Madison, Wisconsin, USA

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.
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http://dx.doi.org/10.1136/jitc-2021-003586DOI Listing
January 2022

Interleukin-12 as an in situ cancer vaccine component: a review.

Cancer Immunol Immunother 2022 Jan 13. Epub 2022 Jan 13.

Departments of Human Oncology, University of Wisconsin, Madison, USA.

Interleukin-12 (IL-12) is a type I cytokine involved in both innate and adaptive immunity that stimulates T and natural killer cell activity and induces interferon gamma production. IL-12 has been identified as a potential immunotherapeutic component for combinatorial cancer treatments. While IL-12 has successfully been used to treat a variety of cancers in mice, it was associated with toxicity when administered systemically in cancer patients. In this review, we discuss the research findings and progress of IL-12 used in combination with other cancer treatment modalities. We describe different methods of IL-12 delivery, both systemic and local, and ultimately highlight the potential of an in situ vaccination approach for minimizing toxicities and providing antitumor efficacy. This review offers a basis for pursuing an in situ vaccine approach that may eventually allow IL-12 to be more readily integrated as an immunotherapy into the clinical treatment of cancers.
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http://dx.doi.org/10.1007/s00262-022-03144-1DOI Listing
January 2022

Mac Cheever (1944-2021): a tribute to a life of achievement and service.

J Immunother Cancer 2022 Jan;10(1)

Pediatrics, Human Oncology, and Genetics, and Carbone Cancer Center, University of Wisconsin-Madison, Madison, Wisconsin, USA.

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http://dx.doi.org/10.1136/jitc-2021-004433DOI Listing
January 2022

Improved Outcome in Children With Newly Diagnosed High-Risk Neuroblastoma Treated With Chemoimmunotherapy: Updated Results of a Phase II Study Using hu14.18K322A.

J Clin Oncol 2021 Dec 6:JCO2101375. Epub 2021 Dec 6.

Department of Oncology, St Jude Children's Research Hospital, Memphis, TN.

Purpose: We evaluated whether combining a humanized antidisialoganglioside monoclonal antibody (hu14.18K322A) throughout therapy improves early response and outcomes in children with newly diagnosed high-risk neuroblastoma.

Patients And Methods: We conducted a prospective, single-arm, three-stage, phase II clinical trial. Six cycles of induction chemotherapy were coadministered with hu14.18K322A, granulocyte-macrophage colony-stimulating factor (GM-CSF), and low-dose interleukin-2 (IL-2). The consolidation regimen included busulfan and melphalan. When available, an additional cycle of parent-derived natural killer cells with hu14.18K322A was administered during consolidation (n = 31). Radiation therapy was administered at the end of consolidation. Postconsolidation treatment included hu14.18K322A, GM-CSF, IL-2, and isotretinoin. Early response was assessed after the first two cycles of induction therapy. End-of-induction response, event-free survival (EFS), and overall survival (OS) were evaluated.

Results: Sixty-four patients received hu14.18K322A with induction chemotherapy. This regimen was well tolerated, with continuous infusion narcotics. Partial responses (PRs) or better after the first two chemoimmunotherapy cycles occurred in 42 of 63 evaluable patients (66.7%; 95% CI, 55.0 to 78.3). Primary tumor volume decreased by a median of 75% (range, 100% [complete disappearance]-5% growth). Median peak hu14.18K322A serum levels in cycle one correlated with early response to therapy ( = .0154, one-sided -test). Sixty of 62 patients (97%) had an end-of-induction partial response or better. No patients experienced progressive disease during induction. The 3-year EFS was 73.7% (95% CI, 60.0 to 83.4), and the OS was 86.0% (95% CI, 73.8 to 92.8), respectively.

Conclusion: Adding hu14.18K322A to induction chemotherapy improved early objective responses, significantly reduced tumor volumes in most patients, improved end-of-induction response rates, and yielded an encouraging 3-year EFS. These results, if validated in a larger study, may be practice changing.
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http://dx.doi.org/10.1200/JCO.21.01375DOI Listing
December 2021

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

Front Immunol 2021 15;12:763888. Epub 2021 Nov 15.

Department of Human Oncology, University of Wisconsin School of Medicine and Public Health, Madison, WI, United States.

Introduction: Combining CpG oligodeoxynucleotides with anti-OX40 agonist antibody (CpG+OX40) is able to generate an effective 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 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 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 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 vaccine CpG+OX40 in immunologically "cold", solid, murine tumor models where RT or CpG+OX40 alone fail to stimulate tumor regression.
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http://dx.doi.org/10.3389/fimmu.2021.763888DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8634717PMC
November 2021

Combining Immunocytokine and Activated NK Cells as a Platform for Enhancing Graft-Versus-Tumor Effects Against GD2 Murine Neuroblastoma.

Front Immunol 2021 19;12:668307. Epub 2021 Aug 19.

Department of Pediatrics, University of Wisconsin School of Medicine and Public Health, Madison, WI, United States.

Management for high-risk neuroblastoma (NBL) has included autologous hematopoietic stem cell transplant (HSCT) and anti-GD2 immunotherapy, but survival remains around 50%. The aim of this study was to determine if allogeneic HSCT could serve as a platform for inducing a graft-versus-tumor (GVT) effect against NBL with combination immunocytokine and NK cells in a murine model. Lethally irradiated C57BL/6 (B6) x A/J recipients were transplanted with B6 bone marrow on Day +0. On day +10, allogeneic HSCT recipients were challenged with NXS2, a GD2 NBL. On days +14-16, mice were treated with the anti-GD2 immunocytokine hu14.18-IL2. In select groups, hu14.18-IL2 was combined with infusions of B6 NK cells activated with IL-15/IL-15Rα and CD137L . Allogeneic HSCT alone was insufficient to control NXS2 tumor growth, but the addition of hu14.18-IL2 controlled tumor growth and improved survival. Adoptive transfer of CD137L/IL-15/IL-15Rα activated NK cells with or without hu14.18-IL2 exacerbated lethality. CD137L/IL-15/IL-15Rα activated NK cells showed enhanced cytotoxicity and produced high levels of TNF-α , but induced cytokine release syndrome (CRS) . Infusing Perforin CD137L/IL-15/IL-15Rα activated NK cells had no impact on GVT, whereas TNF-α CD137L/IL-15/IL-15Rα activated NK cells improved GVT by decreasing peripheral effector cell subsets while preserving tumor-infiltrating lymphocytes. Depletion of Ly49H NK cells also improved GVT. Using allogeneic HSCT for NBL is a viable platform for immunocytokines and activated NK cell infusions, but must be balanced with induction of CRS. Regulation of TNFα or activating NK subsets may be needed to improve GVT effects.
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http://dx.doi.org/10.3389/fimmu.2021.668307DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8417312PMC
October 2021

Safety and feasibility of an in situ vaccination and immunomodulatory targeted radionuclide combination immuno-radiotherapy approach in a comparative (companion dog) setting.

PLoS One 2021 12;16(8):e0255798. Epub 2021 Aug 12.

Department of Medical Sciences, School of Veterinary Medicine, University of Wisconsin-Madison, Madison, Wisconsin, United States of America.

Rationale: Murine syngeneic tumor models have revealed efficacious systemic antitumor responses following primary tumor in situ vaccination combined with targeted radionuclide therapy to secondary or metastatic tumors. Here we present studies on the safety and feasibility of this approach in a relevant translational companion dog model (n = 17 dogs) with advanced cancer.

Methods: The three component of the combination immuno-radiotherapy approach were employed either separately or in combination in companion dogs with advanced stage cancer. In situ vaccination was achieved through the administration of hypofractionated external beam radiotherapy and intratumoral hu14.18-IL2 fusion immunocytokine injections to the index tumor. In situ vaccination was subsequently combined with targeted radionuclide therapy using a theranostic pairing of IV 86Y-NM600 (for PET imaging and subject-specific dosimetry) and IV 90Y-NM600 (therapeutic radionuclide) prescribed to deliver an immunomodulatory 2 Gy dose to all metastatic sites in companion dogs with metastatic melanoma or osteosarcoma. In a subset of dogs, immunologic parameters preliminarily assessed.

Results: The components of the immuno-radiotherapy combination were well tolerated either alone or in combination, resulting in only transient low grade (1 or 2) adverse events with no dose-limiting events observed. In subject-specific dosimetry analyses, we observed 86Y-NM600 tumor:bone marrow absorbed-dose differential uptakes ≥2 in 4 of 5 dogs receiving the combination, which allowed subsequent safe delivery of at least 2 Gy 90Y-NM600 TRT to tumors. NanoString gene expression profiling and immunohistochemistry from pre- and post-treatment biopsy specimens provide evidence of tumor microenvironment immunomodulation by 90Y-NM600 TRT.

Conclusions: The combination of external beam radiotherapy, intratumoral immunocytokine, and targeted radionuclide immuno-radiotherapy known to have activity against syngeneic melanoma in murine models is feasible and well tolerated in companion dogs with advanced stage, spontaneously arising melanoma or osteosarcoma and has immunomodulatory potential. Further studies evaluating the dose-dependent immunomodulatory effects of this immuno-radiotherapy combination are currently ongoing.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0255798PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8360580PMC
December 2021

Low-dose targeted radionuclide therapy renders immunologically cold tumors responsive to immune checkpoint blockade.

Sci Transl Med 2021 07;13(602)

Department of Human Oncology, University of Wisconsin-Madison, Madison, WI 53792, USA.

Molecular and cellular effects of radiotherapy on tumor microenvironment (TME) can help prime and propagate antitumor immunity. We hypothesized that delivering radiation to all tumor sites could augment response to immunotherapies. We tested an approach to enhance response to immune checkpoint inhibitors (ICIs) by using targeted radionuclide therapy (TRT) to deliver radiation semiselectively to tumors. NM600, an alkylphosphocholine analog that preferentially accumulates in most tumor types, chelates a radioisotope and semiselectively delivers it to the TME for therapeutic or diagnostic applications. Using serial Y-NM600 positron emission tomography (PET) imaging, we estimated the dosimetry of Y-NM600 in immunologically cold syngeneic murine models that do not respond to ICIs alone. We observed strong therapeutic efficacy and reported optimal dose (2.5 to 5 gray) and sequence for Y-NM600 in combination with ICIs. After combined treatment, 45 to 66% of mice exhibited complete response and tumor-specific T cell memory, compared to 0% with Y-NM600 or ICI alone. This required expression of STING in tumor cells. Combined TRT and ICI activated production of proinflammatory cytokines in the TME, promoted tumor infiltration by and clonal expansion of CD8 T cells, and reduced metastases. In mice bearing multiple tumors, combining TRT with moderate-dose (12 gray) external beam radiotherapy (EBRT) targeting a single tumor augmented response to ICIs compared to combination of ICIs with either TRT or EBRT alone. The safety of TRT was confirmed in a companion canine study. Low-dose TRT represents a translatable approach to promote response to ICIs for many tumor types, regardless of location.
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http://dx.doi.org/10.1126/scitranslmed.abb3631DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8449934PMC
July 2021

Optimizing Flow Cytometric Analysis of Immune Cells in Samples Requiring Cryopreservation from Tumor-Bearing Mice.

J Immunol 2021 07 14;207(2):720-734. Epub 2021 Jul 14.

Department of Human Oncology, Wisconsin Institutes for Medical Research, Carbone Cancer Center, University of Wisconsin-Madison, Madison, WI;

Most shared resource flow cytometry facilities do not permit analysis of radioactive samples. We are investigating low-dose molecular targeted radionuclide therapy (MTRT) as an immunomodulator in combination with in situ tumor vaccines and need to analyze radioactive samples from MTRT-treated mice using flow cytometry. Further, the sudden shutdown of core facilities in response to the COVID-19 pandemic has created an unprecedented work stoppage. In these and other research settings, a robust and reliable means of cryopreservation of immune samples is required. We evaluated different fixation and cryopreservation protocols of disaggregated tumor cells with the aim of identifying a protocol for subsequent flow cytometry of the thawed sample, which most accurately reflects the flow cytometric analysis of the tumor immune microenvironment of a freshly disaggregated and analyzed sample. Cohorts of C57BL/6 mice bearing B78 melanoma tumors were evaluated using dual lymphoid and myeloid immunophenotyping panels involving fixation and cryopreservation at three distinct points during the workflow. Results demonstrate that freezing samples after all staining and fixation are completed most accurately matches the results from noncryopreserved equivalent samples. We observed that cryopreservation of living, unfixed cells introduces a nonuniform alteration to PD1 expression. We confirm the utility of our cryopreservation protocol by comparing tumors treated with in situ tumor vaccines, analyzing both fresh and cryopreserved tumor samples with similar results. Last, we use this cryopreservation protocol with radioactive specimens to demonstrate potentially beneficial effector cell changes to the tumor immune microenvironment following administration of a novel MTRT in a dose- and time-dependent manner.
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http://dx.doi.org/10.4049/jimmunol.2000656DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8313010PMC
July 2021

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

J Immunother Cancer 2021 06;9(6)

Department of Human Oncology, University of Wisconsin Madison, Madison, Wisconsin, USA

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 mm 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 mm 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.
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http://dx.doi.org/10.1136/jitc-2021-002715DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8237721PMC
June 2021

Intravital Metabolic Autofluorescence Imaging Captures Macrophage Heterogeneity Across Normal and Cancerous Tissue.

Front Bioeng Biotechnol 2021 20;9:644648. Epub 2021 Apr 20.

Department of Biomedical Engineering, University of Wisconsin-Madison, Madison, WI, United States.

Macrophages are dynamic immune cells that govern both normal tissue function and disease progression. However, standard methods to measure heterogeneity in macrophage function within tissues require tissue excision and fixation, which limits our understanding of diverse macrophage function . Two-photon microscopy of the endogenous metabolic co-enzymes NAD(P)H and flavin adenine dinucleotide (FAD) (metabolic autofluorescence imaging) enables dynamic imaging of mouse models . Here, we demonstrate metabolic autofluorescence imaging to assess cell-level macrophage heterogeneity in response to normal and cancerous tissue microenvironments . NAD(P)H and FAD fluorescence intensities and lifetimes were measured for both tissue-resident macrophages in mouse ear dermis and tumor-associated macrophages in pancreatic flank tumors. Metabolic and spatial organization of macrophages were determined by performing metabolic autofluorescence imaging and single macrophage segmentation in mice engineered for macrophage-specific fluorescent protein expression. Tumor-associated macrophages exhibited decreased optical redox ratio [NAD(P)H divided by FAD intensity] compared to dermal macrophages, indicating that tumor-associated macrophages are more oxidized than dermal macrophages. The mean fluorescence lifetimes of NAD(P)H and FAD were longer in dermal macrophages than in tumor-associated macrophages, which reflects changes in NAD(P)H and FAD protein-binding activities. Dermal macrophages had greater heterogeneity in optical redox ratio, NAD(P)H mean lifetime, and FAD mean lifetime compared to tumor-associated macrophages. Similarly, standard markers of macrophage phenotype (CD206 and CD86) assessed by immunofluorescence revealed greater heterogeneity in dermal macrophages compared to tumor-associated macrophages. Ultimately, metabolic autofluorescence imaging provides a novel tool to assess tissue-specific macrophage behavior and cell-level heterogeneity in animal models.
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http://dx.doi.org/10.3389/fbioe.2021.644648DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8093439PMC
April 2021

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

J Immunother Cancer 2021 04;9(4)

Department of Human Oncology, University of Wisconsin-Madison, Madison, Wisconsin, USA

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 mm, 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.
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http://dx.doi.org/10.1136/jitc-2020-002107DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8055108PMC
April 2021

Long-Term Follow-up of a Phase III Study of ch14.18 (Dinutuximab) + Cytokine Immunotherapy in Children with High-Risk Neuroblastoma: COG Study ANBL0032.

Clin Cancer Res 2021 04 27;27(8):2179-2189. Epub 2021 Jan 27.

University of Wisconsin Carbone Cancer Center, Madison, Wisconsin.

Purpose: Previously our randomized phase III trial demonstrated that immunotherapy including dinutuximab, a chimeric anti-GD2 mAb, GM-CSF, and IL2 improved survival for children with high-risk neuroblastoma that had responded to induction and consolidation therapy. These results served as the basis for FDA approval of dinutuximab. We now present long-term follow-up results and evaluation of predictive biomarkers.

Patients And Methods: Patients recieved six cycles of isotretinoin with or without five cycles of immunotherapy which consists of dinutuximab with GM-CSF alternating with IL2. Accrual was discontinued early due to meeting the protocol-defined stopping rule for efficacy, as assessed by 2-year event-free survival (EFS). Plasma levels of dinutuximab, soluble IL2 receptor (sIL2R), and human anti-chimeric antibody (HACA) were assessed by ELISA. Fcγ receptor 2A and 3A genotypes were determined by PCR and direct sequencing.

Results: For 226 eligible randomized patients, 5-year EFS was 56.6 ± 4.7% for patients randomized to immunotherapy ( = 114) versus 46.1 ± 5.1% for those randomized to isotretinoin only ( = 112; = 0.042). Five-year overall survival (OS) was 73.2 ± 4.2% versus 56.6 ± 5.1% for immunotherapy and isotretinoin only patients, respectively ( = 0.045). Thirteen of 122 patients receiving dinutuximab developed HACA. Plasma levels of dinutuximab, HACA, and sIL2R did not correlate with EFS/OS, or clinically significant toxicity. Fcγ receptor 2A and 3A genotypes did not correlate with EFS/OS.

Conclusions: Immunotherapy with dinutuximab improved outcome for patients with high-risk neuroblastoma. Early stoppage for efficacy resulted in a smaller sample size than originally planned, yet clinically significant long-term differences in survival were observed.
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http://dx.doi.org/10.1158/1078-0432.CCR-20-3909DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8046731PMC
April 2021

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

J Immunother Cancer 2020 10;8(2)

Department of Pediatrics, University of Wisconsin-Madison, Madison, Wisconsin, USA

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.
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http://dx.doi.org/10.1136/jitc-2020-001262DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7594540PMC
October 2020

Vaccine Plus Checkpoint Blockade Induces Memory Humoral Response.

Front Immunol 2020 24;11:1610. Epub 2020 Jul 24.

Department of Human Oncology, University of Wisconsin, Madison, WI, United States.

In a syngeneic murine melanoma (MEL) model, we recently reported an 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 × 10 GD2+ B78 MEL and treated at a target tumor size of ~200 mm 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 × 10 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.
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http://dx.doi.org/10.3389/fimmu.2020.01610DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7396490PMC
April 2021

A Phase 1 and pharmacokinetic study evaluating daily or weekly schedules of the humanized anti-GD2 antibody hu14.18K322A in recurrent/refractory solid tumors.

MAbs 2020 Jan-Dec;12(1):1773751

Department of Oncology, St. Jude Children's Research Hospital , Memphis, TN, USA.

Hu14.18K322A is a humanized anti-GD2 monoclonal antibody with a single point mutation that reduces complement-mediated cytotoxicity, with a maximum tolerated dose (MTD) of 60 mg/m daily for 4 days in children with recurrent/refractory neuroblastoma. We report additional results of a Phase 1 trial to determine the MTD and safety profile of hu14.18K322A in patients with osteosarcoma, and of an alternative schedule of weekly hu14.18K322A administration in patients with neuroblastoma or osteosarcoma. Eligible patients with recurrent/refractory osteosarcoma received hu14.13K22A daily x4 every 28 days in a Phase 1 traditional 3 + 3 dose escalation design. Additional patients with osteosarcoma were then enrolled to receive hu14.18K322A once weekly for 4 weeks per course. Patients with recurrent/refractory neuroblastoma were also enrolled on the weekly schedule at 50 mg/m/dose. Six patients with osteosarcoma treated on the daily schedule received a median of 2 (range 1-6) courses; the recommended daily dose was established as 60 mg/m. Three patients had stable disease (SD) as best overall response. Five patients (3 neuroblastoma, 2 osteosarcoma) enrolled on the weekly schedule received a median of 1 (1-3) course; 2 achieved SD as best overall response. Pain, fever, hematologic toxicities, hyponatremia, and ocular/visual abnormalities were common toxicities among both schedules. Dose-limiting toxicities attributed to hu14.18K322A included anorexia and fatigue (n = 1). Pharmacokinetic profiles were similar between daily and weekly schedules. The recommended dose for patients with osteosarcoma receiving daily hu14.18K322A x4 is 60 mg/m. Patients receiving the weekly schedule experienced similar pharmacokinetics and toxicity profile as the daily schedule.
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http://dx.doi.org/10.1080/19420862.2020.1773751DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7531516PMC
July 2021

Analysis of ex vivo expanded and activated clinical-grade human NK cells after cryopreservation.

Cytotherapy 2020 08 11;22(8):450-457. Epub 2020 Jun 11.

Department of Pediatrics, University of Wisconsin School of Medicine and Public Health, Madison, Wisconsin, USA; Carbone Cancer Center, University of Wisconsin, Madison, Wisconsin, USA. Electronic address:

Background Aims: Several methods to expand and activate (EA) NK cells ex vivo have been developed for the treatment of relapsed or refractory cancers. Infusion of fresh NK cells is generally preferred to the infusion of cryopreserved/thawed (C/T) NK cells because of concern that cryopreservation diminishes NK cell activity. However, there has been little head-to-head comparison of the functionality of fresh versus C/T NK cell products.

Methods: We evaluated activity of fresh and C/T EA NK cells generated by interleukin (IL)-15, IL-2 and CD137L expansion.

Results: Analysis of C/T NK cell products demonstrated decreased recovery of viable CD56 cells, but the proportion of NK cells in the C/T EA NK cell product did not decrease compared with the fresh EA NK cell product. Fresh and C/T EA NK cells demonstrated increased granzyme B compared with NK cells pre-expansion, but only fresh EA NK cells showed increased NKG2D. Compared with fresh EA NK cells, cytotoxic ability of C/T EA NK cells was reduced, but C/T EA NK cells remained potently cytotoxic against tumor cells via both antibody-independent and antibody-dependent mechanisms within 4 h post-thaw. Fresh EA NK cells generated high levels of gamma interferon (IFN-γ), which was abrogated by JAK1/JAK2 inhibition with ruxolitinib, but C/T EA NK cells showed lower IFN-γ unaffected by JAK1/JAK2 inhibition.

Discussion: Usage of C/T EA NK cells may be an option to provide serial "boost" NK cell infusions from a single apheresis to maximize NK cell persistence and potentially improve NK-induced responses to refractory cancer.
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http://dx.doi.org/10.1016/j.jcyt.2020.05.001DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7387178PMC
August 2020

Irinotecan, Temozolomide, and Dinutuximab With GM-CSF in Children With Refractory or Relapsed Neuroblastoma: A Report From the Children's Oncology Group.

J Clin Oncol 2020 07 28;38(19):2160-2169. Epub 2020 Apr 28.

Children's Hospital of Philadelphia and University of Pennsylvania, Philadelphia, PA.

Purpose: The combination of irinotecan, temozolomide, dintuximab, and granulocyte-macrophage colony-stimulating factor (I/T/DIN/GM-CSF) demonstrated activity in patients with relapsed/refractory neuroblastoma in the randomized Children's Oncology Group ANBL1221 trial. To more accurately assess response rate and toxicity, an expanded cohort was nonrandomly assigned to I/T/DIN/GM-CSF.

Patients And Methods: Patients were eligible at first relapse or first designation of refractory disease. Oral T and intravenous (IV) irinotecan were administered on days 1 to 5 of 21-day cycles. DIN was administered IV (days 2-5), and GM-CSF was administered subcutaneously (days 6-12). The primary end point was objective response, analyzed on an intent-to-treat basis per the International Neuroblastoma Response Criteria.

Results: Seventeen eligible patients were randomly assigned to I/T/DIN/GM-CSF (February 2013 to March 2015); 36 additional patients were nonrandomly assigned to I/T/DIN/GM-CSF (August 2016 to May 2017). Objective (complete or partial) responses were observed in nine (52.9%) of 17 randomly assigned patients (95% CI, 29.2% to 76.7%) and 13 (36.1%) of 36 expansion patients (95% CI, 20.4% to 51.8%). Objective responses were seen in 22 (41.5%) of 53 patients overall (95% CI, 28.2% to 54.8%); stable disease was also observed in 22 of 53. One-year progression-free and overall survival for all patients receiving I/T/DIN/GM-CSF were 67.9% ± 6.4% (95% CI, 55.4% to 80.5%) and 84.9% ± 4.9% (95% CI, 75.3% to 94.6%), respectively. Two patients did not receive protocol therapy and were evaluable for response but not toxicity. Common grade ≥ 3 toxicities were fever/infection (18 [35.3%] of 51), neutropenia (17 [33.3%] of 51), pain (15 [29.4%] of 51), and diarrhea (10 [19.6%] of 51). One patient met protocol-defined criteria for unacceptable toxicity (grade 4 hypoxia). Higher DIN trough levels were associated with response.

Conclusion: I/T/DIN/GM-CSF has significant antitumor activity in patients with relapsed/refractory neuroblastoma. Study of chemoimmunotherapy in the frontline setting is indicated, as is further evaluation of predictive biomarkers.
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http://dx.doi.org/10.1200/JCO.20.00203DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7325366PMC
July 2020

Pre-existing antitherapeutic antibodies against the Fc region of the hu14.18K322A mAb are associated with outcome in patients with relapsed neuroblastoma.

J Immunother Cancer 2020 03;8(1)

Department of Human Oncology, University of Wisconsin, Madison, Wisconsin, USA

Purpose: Patients with cancer receiving tumor-reactive humanized monoclonal antibody (mAb) therapy can develop a human antihuman antibody (HAHA) response against the therapeutic mAb. We evaluated for HAHA in patients with neuroblastoma treated in a phase I study of humanized anti-GD2 mAb (immunoglobulin (Ig)G1 isotype), hu14.18K322A (NCT00743496). The pretreatment sera (collected prior to mAb treatment) from 9 of 38 patients contained antitherapeutic antibodies, even though they had no prior mAb exposure. We sought to characterize these pre-existing antitherapeutic antibodies (PATA).

Experimental Design: The PATA+ pretreatment samples were characterized via ELISA; clinical associations with PATA status were evaluated.

Results: Pretreatment sera from eight of nine PATA+ patients also bound rituximab and demonstrated preferential ELISA reactivity against the Fc portions of hu14.18K322A and rituximab as compared with the Fab portions of these mAbs. These PATA+ sera also recognized dinutuximab (human IgG1 isotype) and mouse IgG2a isotype mAbs, but not a mouse IgG1 isotype or the fully human panitumumab (IgG2 isotype) mAb. Of the 38 treated patients, only 4 patients (all in the PATA+ cohort) demonstrated no disease progression for 2.5 years without receiving further therapy (p=0.002).

Conclusions: This study demonstrates an association between clinical outcome and the presence of PATA against determinant(s) on the Fc component of the therapeutic mAb, suggesting that the PATA may be playing a role in augmenting mAb-based antitumor effects. Further analyses for the presence of PATA in a larger cohort of patients with relapsed neuroblastoma, analyses of their clinical correlates, identification of their immunological targets, and potential antitumor mechanisms are warranted.
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http://dx.doi.org/10.1136/jitc-2020-000590DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7069273PMC
March 2020

Outcome-Related Signatures Identified by Whole Transcriptome Sequencing of Resectable Stage III/IV Melanoma Evaluated after Starting Hu14.18-IL2.

Clin Cancer Res 2020 07 9;26(13):3296-3306. Epub 2020 Mar 9.

Department of Human Oncology, University of Wisconsin-Madison, Madison, Wisconsin.

Purpose: We analyzed whole transcriptome sequencing in tumors from 23 patients with stage III or IV melanoma from a pilot trial of the anti-GD2 immunocytokine, hu14.18-IL2, to identify predictive immune and/or tumor biomarkers in patients with melanoma at high risk for recurrence.

Experimental Design: Patients were randomly assigned to receive the first of three monthly courses of hu14.18-IL2 immunotherapy either before (Group A) or after (Group B) complete surgical resection of all known diseases. Tumors were evaluated by histology and whole transcriptome sequencing.

Results: Tumor-infiltrating lymphocyte (TIL) levels directly associated with relapse-free survival (RFS) and overall survival (OS) in resected tumors from Group A, where early responses to the immunotherapy agent could be assessed. TIL levels directly associated with a previously reported immune signature, which associated with RFS and OS, particularly in Group A tumors. In Group A tumors, there were decreased cell-cycling gene RNA transcripts, but increased RNA transcripts for repair and growth genes. We found that outcome (RFS and OS) was directly associated with several immune signatures and immune-related RNA transcripts and inversely associated with several tumor growth-associated transcripts, particularly in Group A tumors. Most of these associations were not seen in Group B tumors.

Conclusions: We interpret these data to signify that both immunologic and tumoral cell processes, as measured by RNA-sequencing analyses detected shortly after initiation of hu14.18-IL2 therapy, are associated with long-term survival and could potentially be used as prognostic biomarkers in tumor resection specimens obtained after initiating neoadjuvant immunotherapy.
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http://dx.doi.org/10.1158/1078-0432.CCR-19-3294DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7334053PMC
July 2020

Reduction in oral mucositis severity using a topical vasoconstrictor: A case report of three bone marrow transplant patients.

Integr Cancer Sci Ther 2018 Dec 29;5(6). Epub 2018 Nov 29.

Wisconsin Institutes of Medical Research, University of Wisconsin-Madison, USA.

Background: Grade 3 oral mucositis (OM) is historically observed in >90% of bone marrow transplant patients who received the cyclophosphamide + total body irradiation (CY+TBI) conditioning regimen. It was previously shown that orotopically applied adrenergic vasoconstrictor prevented up to 100% of radiation-induced oral mucositis in two preclinical animal models.

Methods: drenergic vasoconstrictor (i.e., phenylephrine in an aqueous-alcohol NG11-1 formulation) was orotopically applied to three patients (ages 24-29) who received the CY+TBI conditioning regimen; they were compared to five matched controls who received no orotopical vasoconstrictor. All patients received the CY+TBI conditioning regimen for acute lymphoblastic leukemia within the University of Wisconsin Adult Bone Marrow Transplant Program. Over the seven-day Cy+TBI conditioning regimen, 20 min before each treatment, either radiation or chemotherapy, vasoconstrictor was applied topically to the oral cavity, and patients then received either 1.5 Gy whole-body radiation or IV cyclophosphamide.

Results: OM severity was scored over a three-week period using: i) physican assessments, ii) daily photos of the oral cavity, iii) oral pain and oral function score sheets, and iv) recorded narcotic consumption. Both "Grade 3 OM" duration and "any OM" duration in vasoconstrictor-treated patients were substantially lower than for the five control patients. Though nasogastric tube or total parenteral nutrition were used in 3 out of 5 control patients, there was no use of these supportive care measures in the three vasoconstrictor-treated patients.

Conclusion: Orotopically applied NG11-1 vasoconstrictor formulation substantially reduced the incidence and severity of "Grade 3" and "any" oral mucositis when compared to matched control patients, all of whom received the same CY+TBI conditioning regimen. The liquid orotopical formulation was easily tolerated by patients both in its ease of use and lack of side effects.
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http://dx.doi.org/10.15761/ICST.1000293DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6907163PMC
December 2018

Combined innate and adaptive immunotherapy overcomes resistance of immunologically cold syngeneic murine neuroblastoma to checkpoint inhibition.

J Immunother Cancer 2019 12 6;7(1):344. Epub 2019 Dec 6.

Department of Pediatrics, University of Wisconsin, 4159 WIMR Bldg., UWCCC, 1111 Highland Ave, Madison, WI, 53711, USA.

Background: Unlike some adult cancers, most pediatric cancers are considered immunologically cold and generally less responsive to immunotherapy. While immunotherapy has already been incorporated into standard of care treatment for pediatric patients with high-risk neuroblastoma, overall survival remains poor. In a mouse melanoma model, we found that radiation and tumor-specific immunocytokine generate an in situ vaccination response in syngeneic mice bearing large tumors. Here, we tested whether a novel immunotherapeutic approach utilizing radiation and immunocytokine together with innate immune stimulation could generate a potent antitumor response with immunologic memory against syngeneic murine neuroblastoma.

Methods: Mice bearing disialoganglioside (GD2)-expressing neuroblastoma tumors (either NXS2 or 9464D-GD2) were treated with radiation and immunotherapy (including anti-GD2 immunocytokine with or without anti-CTLA-4, CpG and anti-CD40 monoclonal antibody). Tumor growth, animal survival and immune cell infiltrate were analyzed in the tumor microenvironment in response to various treatment regimens.

Results: NXS2 had a moderate tumor mutation burden (TMB) while N-MYC driven 9464D-GD2 had a low TMB, therefore the latter served as a better model for high-risk neuroblastoma (an immunologically cold tumor). Radiation and immunocytokine induced a potent in situ vaccination response against NXS2 tumors, but not in the 9464D-GD2 tumor model. Addition of checkpoint blockade with anti-CTLA-4 was not effective alone against 9464D-GD2 tumors; inclusion of CpG and anti-CD40 achieved a potent antitumor response with decreased T regulatory cells within the tumors and induction of immunologic memory.

Conclusions: These data suggest that a combined innate and adaptive immunotherapeutic approach can be effective against immunologically cold syngeneic murine neuroblastoma. Further testing is needed to determine how these concepts might translate into development of more effective immunotherapeutic approaches for the treatment of clinically high-risk neuroblastoma.
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http://dx.doi.org/10.1186/s40425-019-0823-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6898936PMC
December 2019

A Phase II Trial of Hu14.18K322A in Combination with Induction Chemotherapy in Children with Newly Diagnosed High-Risk Neuroblastoma.

Clin Cancer Res 2019 11 10;25(21):6320-6328. Epub 2019 Oct 10.

Department of Oncology, St. Jude Children's Research Hospital, Memphis, Tennessee.

Purpose: We sought to evaluate whether combining a humanized antidisialoganglioside mAb (hu14.18K322A) with induction chemotherapy improves early responses and outcomes in children with newly diagnosed high-risk neuroblastoma.

Patients And Methods: We conducted a prospective nonrandomized, single-arm, two-stage, phase II clinical trial. Six courses of induction chemotherapy were coadministered with hu14.18K322A and followed with granulocyte-macrophage colony-stimulating factor (GM-CSF) and low-dose IL2. Consolidation was performed with a busulfan/melphalan preparative regimen. An additional course of hu14.18K322A was administered with parent-derived natural killer cells, when available, during consolidation. Hu14.18K322A, GM-CSF, IL2, and isotretinoin were then administered. Secondary outcomes included reduced tumor volume and semiquantitative I-metaiodobenzylguanidine scoring [i.e., Curie scores (CS)] at the end of induction.

Results: Forty-two patients received hu14.18K322A and induction chemotherapy. This regimen was well tolerated, with continuous-infusion narcotics adjusted to patient tolerance. Partial responses (PR) or better after the first two chemoimmunotherapy courses occurred in 32 patients [76.2%; 95% confidence interval (CI), 60.6-88.0]. This was accompanied by primary tumor volume reductions (median, -76%; range, -100% to 5%). Of 35 patients with stage IV disease who completed induction, 31 had end-of-induction CSs of 2 or less. No patients experienced progression during induction. Two-year event-free survival (EFS) was 85.7% (95% CI, 70.9-93.3).

Conclusions: Adding hu14.18K322A to induction chemotherapy produced early PR or better in most patients, reduced tumor volumes, improved CSs at the end of induction, and yielded an encouraging 2-year EFS. These results, if validated in a larger study, may change the standard of care for children with high-risk neuroblastoma.
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http://dx.doi.org/10.1158/1078-0432.CCR-19-1452DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6825564PMC
November 2019

Antitumor Activity and Tolerability of hu14.18-IL2 with GMCSF and Isotretinoin in Recurrent or Refractory Neuroblastoma: A Children's Oncology Group Phase II Study.

Clin Cancer Res 2019 10 29;25(20):6044-6051. Epub 2019 Jul 29.

Departments of Pediatrics, Human Oncology and Genetics and the University of Wisconsin, Madison, Wisconsin.

Purpose: Combining anti-GD2 (disialoganglioside) mAb with GM-CSF, IL2, and isotretinoin is now FDA-approved for high-risk neuroblastoma minimal residual disease (MRD) therapy. The humanized anti-GD2 antibody conjugated to IL2 (hu14.18-IL2) has clinical activity in neuroblastoma and is more effective in neuroblastoma-bearing mice than antibody and cytokine given separately. We therefore evaluated the safety, tolerability, and antitumor activity of hu14.18-IL2 given with GM-CSF and isotretinoin in a schedule similar to standard MRD therapy.

Patients And Methods: Hu14.18-IL2 was given at the recommended phase II dose of 12 mg/m/day on days 4-6 of a 28-day cycle with GM-CSF (250 mg/m/dose, days 1-2 and 8-14) and isotretinoin (160 mg/m/day, days 11-25). Tolerability was determined on the basis of the number of unacceptable toxicities observed. Response was evaluated separately for patients with disease measurable by standard radiologic criteria (stratum 1), and for patients with disease evaluable only by I-metaiodobenzylguanidine (I-MIBG) scan and/or bone marrow histology (stratum 2).

Results: Fifty-two patients with recurrent or refractory neuroblastoma were enrolled; 51 were evaluable for toxicity and 45 were evaluable for response. Four patients had unacceptable toxicities, well below the protocol-defined rule for tolerability. Other grade 3 and 4 nonhematologic toxicities were expected and reversible. No responses were seen in stratum 1 ( = 14). In stratum 2 ( = 31), 5 objective responses were confirmed by central review (3 complete, 2 partial).

Conclusions: Hu14.18-IL2 given in combination with GM-CSF and isotretinoin is safe and tolerable. Patients with MIBG and/or bone marrow-only disease had a 16.1% response rate, confirming activity of the combination.
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http://dx.doi.org/10.1158/1078-0432.CCR-19-0798DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6945765PMC
October 2019

Human and murine IL2 receptors differentially respond to the human-IL2 component of immunocytokines.

Oncoimmunology 2019;8(6):e1238538. Epub 2018 May 7.

Department of Human Oncology, University of Wisconsin, Madison, WI, USA.

The humanized immunocytokine, hu14.18-IL2 (ICp), leads to the immune cell-mediated destruction of GD2-expressing tumors in mouse models, resulting in potent antitumor effects with negligible IL2-related toxicity. In contrast, when ICp is used clinically, antitumor activity is accompanied by dose-limiting IL2-related toxicities. These species-specific differences in ICp toxicity may be linked to differential binding to mouse vs. human IL2 receptors (IL2Rs). We evaluated immunocytokines genetically engineered to preferentially bind either high-affinity αβγ-IL2Rs or intermediate-affinity βγ-IL2Rs. These ICs have the IL2 fused to the C-terminus of the IgG light chains rather than the heavy chains. We found that IC35, containing intact huIL2, maintained activation of human and mouse αβγ-IL2Rs but exhibited a 20-fold reduction in the ability to stimulate human βγ-IL2Rs, with no activation of mouse βγ-IL2Rs at the concentrations tested. The reduced ability of IC35 to stimulate human βγ-IL2Rs (associated with IL2-toxicities) makes it a potential candidate for clinical trials where higher clinical IC doses might enable better tumor targeting and increased antitumor effects with less toxicity. Contrastingly, ICSK (IC with an IL2 mutein that has enhanced binding to the IL2R β-chain) showed increased activation over ICp on mouse βγ-IL2Rs, with a dose-response curve similar to that seen with IC35 on human βγ-IL2Rs. Our data suggest that ICSK might be used in mouse models to simulate the anticipated effects of IC35 in clinical testing. Understanding the differences in species-dependent IL2R activation should facilitate the design of reagents and mouse models that better simulate the potential activity of IL2-based immunotherapy in patients.
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http://dx.doi.org/10.1080/2162402X.2016.1238538DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6492958PMC
May 2018

Advances in Anti-GD2 Immunotherapy for Treatment of High-risk Neuroblastoma.

J Pediatr Hematol Oncol 2019 04;41(3):163-169

Department of Pediatrics, Division of Hematology, Oncology and Bone Marrow Transplant.

Neuroblastoma (NBL) is the most common extracranial solid tumor in pediatrics, yet overall survival is poor for high-risk cases. Immunotherapy regimens using a tumor-selective antidisialoganglioside (anti-GD2) monoclonal antibody (mAb) have been studied for several decades now, but have only recently been incorporated into standard of care treatment for patients with high-risk NBL with clear benefit. Here we review a brief history of anti-GD2-based immunotherapy, current areas of neuroblastoma research targeting GD2, and potential diagnostic and therapeutic uses targeting GD2.
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http://dx.doi.org/10.1097/MPH.0000000000001369DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6430125PMC
April 2019

Follicular lymphoma patients with KIR2DL2 and KIR3DL1 and their ligands (HLA-C1 and HLA-Bw4) show improved outcome when receiving rituximab.

J Immunother Cancer 2019 03 12;7(1):70. Epub 2019 Mar 12.

Department of Human Oncology, University of Wisconsin, Madison, WI, USA.

Background: The ECOG-ACRIN Cancer Research Group evaluated rituximab treatment schedules for patients with newly-diagnosed low-tumor-burden follicular-lymphoma (FL). All patients received 4-weekly rituximab treatments as induction therapy. Clinically-responding patients were randomized to receive rituximab every 13 weeks ("maintenance") vs. no additional rituximab until progression ("non-maintenance"). Based on "time-to-rituximab-failure (TTRF)", the study-committee reported there was no overall-benefit for maintenance rituximab in this setting. Tumor-reactive mAbs, like rituximab, trigger natural killer (NK) cells. NK-cell responses are regulated, in part, by interactions between killer immunoglobulin-like receptors (KIRs) on NK cells and their interactions with KIR-ligands. In a separate study of children with neuroblastoma treated with a different mAb, we found certain KIR/KIR-ligand genotypes associated with improved outcome. Here, we assessed whether a subset of FL patients show improved outcome from the maintenance rituximab based on these same KIR/KIR-ligand genotypes.

Methods: Genotypes for KIR/KIR-ligand were determined and assessed for associations with outcome [duration of response, TTRF and % tumor shrinkage] as a post-hoc analysis of this phase III trial. Our primary objective was to assess specific KIR/KIR-ligand genotype associations, followed by separate prespecified KIR/KIR-ligand genotype associations in follow-up analyses. Statistical analyses for association of genotype with clinical outcome included: Log-rank tests and Cox proportional hazards regression models to assess duration of response and TTRF; analysis of variance (ANOVA) was used for assessment of % tumor shrinkage.

Results: We found that patients inheriting KIR2DL2 and its ligand (HLA-C1) along with KIR3DL1 and its ligand (HLA-Bw4) had improved outcome over patients without this genotype. In addition, patients with KIR2DL2 and HLA-C1 along with KIR3DL1 and HLA-Bw4 also showed improved duration of response and tumor shrinkage if they received maintenance, while patients without this genotype showed no such improvement when receiving maintenance.

Conclusions: The data presented here indicate that a subset of FL patients, identified by certain KIRs/KIR-ligands, have improved outcome and may benefit from additional rituximab treatment. Taken together, this suggests that the efficacy of tumor-reactive mAb treatment for some patients is influenced by KIRs on NK cells. However, prior to considering these genotypes in a clinically-actionable manner, these findings need independent validation in other studies.
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http://dx.doi.org/10.1186/s40425-019-0538-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6419437PMC
March 2019

Y-NM600 targeted radionuclide therapy induces immunologic memory in syngeneic models of T-cell Non-Hodgkin's Lymphoma.

Commun Biol 2019 26;2:79. Epub 2019 Feb 26.

Department of Radiology, University of Wisconsin School of Medicine and Public Health, Madison, WI, USA.

Finding improved therapeutic strategies against T-cell Non-Hodgkin's Lymphoma (NHL) remains an unmet clinical need. We implemented a theranostic approach employing a tumor-targeting alkylphosphocholine (NM600) radiolabeled with Y for positron emission tomography (PET) imaging and Y for targeted radionuclide therapy (TRT) of T-cell NHL. PET imaging and biodistribution performed in mouse models of T-cell NHL showed in vivo selective tumor uptake and retention of Y-NM600. An initial toxicity assessment examining complete blood counts, blood chemistry, and histopathology of major organs established Y-NM600 safety. Mice bearing T-cell NHL tumors treated with Y-NM600 experienced tumor growth inhibition, extended survival, and a high degree of cure with immune memory toward tumor reestablishment. Y-NM600 treatment was also effective against disseminated tumors, improving survival and cure rates. Finally, we observed a key role for the adaptive immune system in potentiating a durable anti-tumor response to TRT, especially in the presence of microscopic disease.
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http://dx.doi.org/10.1038/s42003-019-0327-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6391402PMC
April 2020

MUC16 suppresses human and murine innate immune responses.

Gynecol Oncol 2019 03 6;152(3):618-628. Epub 2019 Jan 6.

Department of Obstetrics and Gynecology, University of Wisconsin, Madison, WI, USA. Electronic address:

Objective: MUC16, the mucin that contains the CA125 epitopes, suppresses the cytolytic responses of human NK cells and inhibits the efficacy of therapeutic antibodies. Here, we provide further evidence of the regulatory role of MUC16 on human and murine NK cells and macrophages.

Methods: Target cell cytolysis and doublet formation assays were performed to assess effects of MUC16 on human NK cells. The effect of MUC16 on ovarian tumor growth was determined in a mouse model by monitoring survival and ascites formation. Innate immune cells from spleens and peritoneal cavities of mice were isolated and stimulated in vitro with anti-CD40 antibody, lipopolysaccharide and IFN-γ and their ability to cytolyse MUC16 expressing and non-expressing cells was determined.

Results: We confirm that MUC16 inhibits cytolysis by human NK cells as well as the formation of NK-tumor conjugates. Mice implanted with MUC16-knockdown OVCAR-3 show >2-fold increase in survival compared to controls. Murine NK cells and macrophages are more efficient at lysing MUC16-knockdown cells. In vitro cytotoxicity assays with NK cells and macrophages isolated from mice stimulated with anti-CD40 antibody showed 2-3-fold increased activity against the MUC16-knockdown cells as compared to matching target cells expressing this mucin. Finally, knockdown of MUC16 increased the susceptibility of cancer cells to ADCC by murine splenocytes.

Conclusions: For the first time, we demonstrate the immunoregulatory effects of MUC16 on murine NK cells and macrophages. Our study implies that the immunoregulatory role of MUC16 on murine NK cells and macrophages should be considered when examining the biology of MUC16 in mouse models.
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http://dx.doi.org/10.1016/j.ygyno.2018.12.023DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8327469PMC
March 2019
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