lorukafusp alfa (APN301)

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.

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.

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.

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.

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.

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.

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.

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.

P1, N=0, Withdrawn, University of Wisconsin, Madison | Trial completion date: Sep 2025 --> Sep 2022 | Suspended --> Withdrawn | Trial primary completion date: Sep 2024 --> Sep 2022

Hu14.18-IL15 and Hu14.18-IL21 exhibit robust preclinical activity, warranting further consideration for clinical testing in patients with GD2-expressing NB.

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.

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.

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.

On days +14-16, mice were treated with the anti-GD2 immunocytokine hu14.18-IL2...Using allogeneic HSCT for NBL is a viable platform for immunocytokines and ex vivo 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.

P1, N=6, Suspended, University of Wisconsin, Madison | Trial completion date: Sep 2021 --> Sep 2025 | Trial primary completion date: Sep 2021 --> Sep 2024

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.

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.

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)...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.

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.

ISV augmented response to ICIs in murine melanoma at brain and extracranial tumor sites. Although baseline tumor-immune microenvironments were similar at brain and extracranial tumor sites, response to ISV+α-CTLA-4 was divergent with reduced infiltration and activation of immune cells in brain tumors. Additional therapies may be needed for effective antitumor immune response against melanoma brain metastases.

P1, N=6, Suspended, University of Wisconsin, Madison | Recruiting --> Suspended

P1/2, N=61, Recruiting, University of Wisconsin, Madison | Suspended --> Recruiting

We interpret these data to signify that both immunologic and tumoral cell processes, as measured by RNAseq 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.