lorukafusp alfa (APN301) / invIOs - LARVOL DELTA

In vitro detection of canine anti-human antibodies following intratumoral injection of the hu14.18-IL2 immunocytokine in spontaneous canine melanoma. (PubMed, PLoS One) - "This study advances CAHA detection strategies and reports the kinetics of CAHA following IT-IC in dogs with spontaneous melanoma."

Journal • Preclinical • Melanoma • Oncology • Solid Tumor • IL2

In vivo label-free imaging reveals distinct CD8 T cell metabolic changes during effective radio-immunotherapy in cold murine melanoma versus hot colon carcinoma (AACR 2025) - "This therapy includes external beam radiation, hu14.18-IL2 immunocytokine (anti-GD2 mAb fused to IL2) or free IL2, and anti-CTLA-4, leading to in situ vaccination and cure of established murine tumors...Combined with other traditional assays, we can elucidate key immune cell populations and the crucial timepoints during therapy where changes are occurring. This imaging platform may be leveraged to help develop new immunotherapy combinations and study other cancer types where cures have not been realized."

Preclinical • Colon Cancer • Colorectal Cancer • Melanoma • Oncology • Solid Tumor • CD8 • IL2

A534260: IT-hu14.18-IL2 With Radiation, Nivolumab and Ipilimumab for Melanoma (clinicaltrials.gov) - P1/2 | N=8 | Active, not recruiting | Sponsor: University of Wisconsin, Madison | Suspended ➔ Active, not recruiting | N=61 ➔ 8

Enrollment change • Enrollment closed • Melanoma • Oncology • Solid Tumor

In vitro detection of canine anti-human antibodies following intratumoral injection of the hu14.18-IL2 immunocytokine in spontaneous canine melanoma. (PubMed, bioRxiv) - "Normal canine sera did not mediate binding inhibition. This study advances CAHA detection strategies and reports the kinetics of CAHA following IT-IC in dogs with spontaneous melanoma."

Journal • Preclinical • Melanoma • Oncology • Solid Tumor • IL2

In vivo label-free imaging reveals distinct CD8 T cell metabolic changes and collagen reorganization during effective immunotherapy in murine melanoma versus colon tumors (SITC 2024) - "This therapy includes external beam radiation, intratumoral hu14.18-IL2 immunocytokine (anti-GD2 mAb fused to IL2) or free IL2, and intraperitoneal anti-CTLA-4, leading to in situ vaccination and cure of murine tumors...B) In vivo single-cell CD8 T cell fluorescence lifetime data. C) In vivo second harmonic generation images of B78 and MC38 tumor collagen"

Preclinical • Colon Cancer • Colorectal Cancer • Gastrointestinal Cancer • Melanoma • Oncology • Solid Tumor • CD8 • IL2

A534260: IT-hu14.18-IL2 With Radiation, Nivolumab and Ipilimumab for Melanoma (clinicaltrials.gov) - P1/2 | N=61 | Suspended | Sponsor: University of Wisconsin, Madison | Trial completion date: Aug 2025 ➔ Aug 2026 | Trial primary completion date: Aug 2024 ➔ Aug 2025

IO biomarker • Metastases • Trial completion date • Trial primary completion date • Melanoma • Oncology • Solid Tumor • IL2

Evaluation of a Combinatorial Immunotherapy Regimen That Can Cure Mice Bearing MYCN-Driven High-Risk Neuroblastoma That Resists Current Clinical Therapy. (PubMed, J Clin Med) - " 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. 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."

Journal • Preclinical • CNS Tumor • Neuroblastoma • Oncology • Solid Tumor • IL2 • MYCN

NK cells propagate T cell immunity following in situ tumor vaccination. (PubMed, Cell Rep) - "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...NK cell depletion increases T infiltration, diminishing CD8 T cell-dependent antitumor response. These findings demonstrate that NK cells sustain and propagate CD8 T cell immunity following 3xTx."

IO biomarker • Journal • Head and Neck Cancer • Melanoma • Oncology • Solid Tumor • CD8 • CD86 • CTLA4 • IL2 • NKG2D

Cyclophosphamide augments the efficacy of in situ vaccination in a mouse melanoma model. (PubMed, Front Oncol) - "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. Cured mice developed immunological memory as they were able to reject B78 tumor rechallenge. 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."

Journal • Preclinical • Melanoma • Oncology • Solid Tumor • CD8 • IL2

CD4 T cell-driven response to immunotherapy against mouse melanoma tumors (AACR 2023) - "Using an in situ vaccine (ISV) regimen that includes a combination therapy of radiation (given at D0) with hu14.18-IL2 immunocytokine [anti-GD2 linked to IL2, (Anyxis Immuno-Oncology GmbH (Austria)); given at D5-D9], we can cure mice of large B78 melanoma tumors (B78s)...MHCII expression on tumors can directly engage CD4 cytotoxic T cells, suggesting an important role in the response to immunotherapy for CD4 T cells in melanoma tumors that express MHCII. Understanding the cellular and molecular mechanisms involved in the ISV-induced immune recognition and destruction of B78 may guide future improvements of this clinically-relevant immunotherapy regimen."

IO biomarker • Preclinical • Melanoma • Oncology • Solid Tumor • CD4 • CD8 • IFNG • IL2 • PTPRC

In vivo multiphoton autofluorescence imaging is sensitive to CD8 T cell and tumor cell metabolic changes during immunotherapy in a murine melanoma model (AACR 2023) - "This therapy includes external beam radiation, intratumoral hu14.18-IL2 immunocytokine (anti-GD2 mAb fused to IL2, provided by Anyxis Immuno-Oncology GmbH of Vienna, Austria), and intraperitoneal anti-CTLA-4 leading to in situ vaccination and cure of GD2+ murine tumors... These results show that in vivo metabolic imaging enables single cell quantification of metabolic changes in tumor and immune cells during therapy. Combined with other traditional assays, we can elucidate key immune cell populations and the crucial timepoints during therapy where changes are occurring. With continued efforts, this imaging platform may be leveraged to develop new combinations of immunotherapies."

Preclinical • Tumor cell • Melanoma • Oncology • Solid Tumor • CD8 • IL2

Stimulation of natural killer cells with small molecule inhibitors of CD38 for the treatment of neuroblastoma. (PubMed, Chem Sci) - "Additionally, we have illustrated that NK cells exhibited enhanced cytotoxicity toward NB cells (14% reduction of NB cells over 90 minutes) when given a combination treatment of our inhibitor and the immunocytokine ch14.18-IL2. Herein we describe the synthesis and biological evaluation of small molecule CD38 inhibitors and demonstrate their potential utility as a novel approach to NB immunotherapy. These compounds represent the first examples of small molecules that stimulate immune function for the treatment of cancer."

IO biomarker • Journal • CNS Tumor • Immune Modulation • Neuroblastoma • Neuroendocrine Tumor • Oncology • Pediatrics • Solid Tumor • IFNG

Radiation to all macroscopic sites of tumor permits greater systemic antitumor response to in situ vaccination. (PubMed, J Immunother Cancer) - "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."

Journal • Immune Modulation • Inflammation • Melanoma • Oncology • Solid Tumor • CD8 • IL2

Optimizing a combination radio-immunotherapy regimen in a preclinical model of treatment-resistant, high-risk neuroblastoma (SITC 2022) - "Of these patients, ~40% are refractory to, or relapse following, initial treatment; there are currently no effective treatment options for these patients once they have failed salvage therapy of chemotherapy combined with αGD2 therapy (dinutuximab)...1,2 CAIR utilizes αGD2 immunocytokine (hu14.18-IL2), radiotherapy (RT), αCD40, CpG, and αCTLA4 but questions remain about the relative contribution of each component. In this study, we tested if our model of HR-NBL is resistant to salvage therapy of temozolomide and irinotecan (TEM+IRI) and αGD2-based therapy and if components of our effective CAIR therapy can be removed, in order to determine their necessity...In contrast, these tumors can be cured by both CAIR therapy and by several versions of a reduced CAIR therapy (CAIR subtracting αCD40, αCTLA4, or CpG). We are now exploring the role of each component of CAIR in anti-tumor immune responses in this immunologically cold model."

Preclinical • CNS Tumor • Neuroblastoma • Oncology • Solid Tumor • IL2

Administration of intratumoral hu14.18-IL2 immunocytokine and local radiation therapy to activate immune rejection of spontaneous canine melanoma (SITC 2022) - "A 9-marker multi-color immunophenotyping panel for flow cytometry (CD3, CD5, CD4, CD8, CD14, CD21, CD25, FoxP3, and PD-1) was optimized using cryopreserved healthy canine PBMC and will be used to assay PBMC from pre and post-treatment timepoints for the protocol treatment dogs. Conclusions IT-IC in combination with local RT in canine melanoma is safe and has antitumor activity with potential to inform clinical development of IT-IC in melanoma patients."

IO biomarker • Hematological Malignancies • Melanoma • Oncology • Solid Tumor • CD14 • CD21 • CD5 • CD8 • FOXP3 • IL2 • IL2RA • PD-1

Intravital multiphoton autofluorescence imaging is sensitive to changes in CD8 T cell and tumor cell metabolism during immunotherapy in a murine melanoma model (SITC 2022) - "4 This therapy includes external beam radiation, intratumoral hu14.18-IL2 immunocytokine (anti-GD2 mAb fused to IL2), and intraperitoneal anti-CTLA-4 leading to in situ vaccination and cure of GD2+ murine tumors...Combined with other traditional assays, we can elucidate key immune cell populations and the crucial timepoints during therapy where changes are occurring. With continued efforts, this imaging platform may be leveraged to develop new combinations of immunotherapies."

Preclinical • Melanoma • Oncology • Solid Tumor • CD8 • IL2

Treatment of Relapsed or Refractory Neuroblastoma and Osteosarcoma With Expanded Haploidentical NK Cells and Hu14.18-IL2 (clinicaltrials.gov) - P1 | N=0 | Withdrawn | Sponsor: University of Wisconsin, Madison | Trial completion date: Sep 2025 ➔ Sep 2022 | Suspended ➔ Withdrawn | Trial primary completion date: Sep 2024 ➔ Sep 2022

Preclinical • Trial completion date • Trial primary completion date • Trial withdrawal • Neuroblastoma • Oncology • Osteosarcoma • Sarcoma • Solid Tumor • IL2

Anti-GD2 antibodies conjugated to IL15 and IL21 mediate potent anti-tumor cytotoxicity against neuroblastoma. (PubMed, Clin Cancer Res) - "Hu14.18-IL15 and Hu14.18-IL21 exhibit robust preclinical activity, warranting further consideration for clinical testing in patients with GD2-expressing NB."

Journal • Neuroblastoma • Oncology • Solid Tumor • Transplantation • CD8 • IL15 • IL21

Mechanism of effective combination radio-immunotherapy against 9464D-GD2, an immunologically cold murine neuroblastoma. (PubMed, J Immunother Cancer) - "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."

IO biomarker • Journal • Preclinical • Immune Modulation • Immunology • Inflammation • Neuroblastoma • Oncology • Pediatrics • Solid Tumor • IFNG • IL2

Combining Immunotherapies with Conventional Cancer Therapies in a Preclinical Model of Treatment-Resistant, High-Risk Neuroblastoma (IMMUNOLOGY 2022) - "Here we aimed to test if the clinically-approved salvage therapy of temozolomide and irinotecan (T/I) and αGD2-based therapy replicated human trials and to test if CAIR can be reduced to improve relevance. For chemo studies, mice were treated T/I and αGD2 immunocytokine (Hu14.18-IL2)...In contrast, CAIR can cure 9464D-GD2 bearing mice, and our data indicate that this regimen can be further reduced without decreasing efficacy. A reduced regimen enhances both clinical relevance and our ability to add additional agents to further improve survival."

Preclinical • Immune Modulation • Immunology • Inflammation • Neuroblastoma • Oncology • Solid Tumor • IL2

Discovery and development of NK cell immunostimulatory small molecule CD38 inhibitors for the treatment of neuroblastoma (ACS-Sp 2022) - "Additionally, we have illustrated that NK cells exhibited enhanced cytotoxicity toward NB cells (14% reduction of NB cells over 90 minutes) when given a combination treatment of our inhibitor and the immunocytokine ch14.18-IL2. In this presentation, the synthesis and biological evaluation of small molecule inhibitors specifically for use in NB immunotherapy will be described."

IO biomarker • Immune Modulation • Inflammation • Neuroblastoma • Oncology • Pediatrics • Solid Tumor • IFNG

Intravital multiphoton imaging of infiltrating CD8 T cell and tumor cell metabolism during immunotherapy in a murine melanoma model (SITC 2021) - "The metabolism of tumor and immune cells is closely associated with cancer progression and tissue site,1–4 so we aim to study metabolic trends during administration of an effective, triple-combination immunotherapy within murine melanoma tumors.5 This therapy includes external beam radiation, intratumoral hu14.18-IL2 immunocytokine (anti-GD2 mAb fused to IL2), and intraperitoneal anti-CTLA-4 leading to in situ vaccination and cure of GD2+ murine tumors.5 Previous work has shown that a T cell response is critical to the efficacy of this therapy,5–6 so we created an mCherry-labeled T cell mouse model to study this response...Specifically, OMI enabled us to probe single cell metabolic changes occurring during our immunotherapy regimen. With continued work, this imaging platform may be leveraged to develop new combinations of immunotherapies."

Preclinical • Melanoma • Oncology • Solid Tumor • CD8 • IL2

Cyclophosphamide augments the efficacy of an in situ vaccine in a mouse melanoma model (SITC 2021) - "Background We have previously shown that a direct 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...Conclusions CY can augment the antitumor efficacy of IT- IC given alone or in combination with local RT in tumor-bearing mice. These preclinical results suggest the value of initiating clinical testing of the combination of CY, RT and IT-IC as an in situ vaccine."

Preclinical • Melanoma • Oncology • Solid Tumor • CD8 • IL2

Intravital multiphoton imaging of infiltrating CD8 T cell and tumor cell metabolism during immunotherapy in a murine melanoma model (SITC 2021) - "5 This therapy includes external beam radiation, intratumoral hu14.18-IL2 immunocytokine (anti-GD2 mAb fused to IL2), and intraperitoneal anti-CTLA-4 leading to in situ vaccination and cure of GD2+ murine tumors...Specifically, OMI enabled us to probe single cell metabolic changes occurring during our immunotherapy regimen. With continued work, this imaging platform may be leveraged to develop new combinations of immunotherapies."

Preclinical • Melanoma • Oncology • Solid Tumor • CD8 • IL2

[VIRTUAL] Phase I/II trial of intratumoral administration of hu14.18-IL2, with local radiation, nivolumab, and ipilimumab in subjects with advanced melanoma. (ASCO 2021) - P1/2 | "Key inclusion criteria: 1) histologically proven, malignant melanoma that is advanced (Stage IV) or surgically incurable; 2) at least 1 (preferably 2) sites of disease amenable to safe repeated IT injections; and 3) must have received or declined at least one FDA approved therapy, either in the adjuvant setting or for metastatic disease, with an impact on survival . Two subjects have been accrued into Phase IA as of 2-4-2021."

Clinical • P1/2 data • Immune Modulation • Immunology • Inflammation • Melanoma • Oncology • Solid Tumor • IL2