Intratumoral Therapy Research Articles

IMMU-37. PRE-TREATMENT INNATE INFLAMMATION ASSOCIATES WITH SURVIVAL AFTER POLIO VIROTHERAPY AND CAN BE MODULATED TO SENSITIZE GLIOMAS TO IN SITU VACCINATION

Abstract Early-stage clinical trials in glioblastoma (GBM) have tested various virotherapy strategies, yet only a subset of patients appear to benefit. We previously demonstrated that a recombinant poliovirus (PVSRIPO) functions by inducing MDA5-dependent, type I interferon (IFN) dominant innate inflammation in myeloid cells to provoke durable and functional antitumor T cell responses in mice. Long-term survival after intratumoral PVSRIPO infusion was observed in a subset of patients with recurrent GBM (rGBM) in two clinical trials. Survival after PVSRIPO was associated with higher pre-treatment intratumoral neutrophil density and MHC-class II gene expression, but also higher pre-treatment inflammatory cytokines in blood. Post-treatment induction of type I/III IFNs detected in blood one day after PVSRIPO infusion also associated with survival of patients with rGBM, suggesting that the proficiency of IFN responses to PVSRIPO may dictate therapy outcome. In human ex vivo glioma tissue assays, only a subset of gliomas mounted type I IFN responses to PVSRIPO and STING agonist treatment, in a manner linked with higher pre-treatment IL-1b levels, and lower CXCL10 induction after treatment. Thus, baseline innate inflammation either influences or reflects the capacity of glioma tumors to respond to virotherapy. Strikingly, in glioma bearing mice, peripheral vaccination against tumor irrelevant vaccines in alum adjuvant (i.e., against antigens not expressed by the tumor) induced bone marrow, spleen and intratumoral innate inflammation; rescued the antitumor efficacy of PVSRIPO in an otherwise refractory glioma model; and improved survival after intratumor STING agonist therapy. Notably, peripheral vaccination induced intratumoral neutrophil and CD4+ T cell inflammation, and increased MHC-class II expression on myeloid cells. These data imply a role for pre-treatment systemic innate immune training in the antitumor efficacy of virotherapy and indicate that modulating either systemic innate inflammation and/or the inflammatory status of bone marrow myeloid progenitors may sensitize gliomas to virotherapy.

Leveraging long-acting IL-15 agonists for intratumoral delivery and enhanced antimetastatic activity

IntroductionIL-15 agonists hold promise as immunotherapeutics due to their ability to induce the proliferation and expansion of cytotoxic immune cells including natural killer (NK) and CD8+ T cells. However, they generally have short half-lives that necessitate frequent administration to achieve efficacy. To address this limitation, we have developed a half-life extension technology using hydrogel microspheres (MS). Here, the therapeutic is tethered to MSs by a releasable linker with pre-programed cleavage rates. We previously showed the MS conjugate of single-chain IL-15, MS~IL-15, effectively increased the half-life of IL-15 to approximately 1 week and enhanced the pharmacodynamics. We sought to determine whether the same would be true with a MS conjugate of the IL-15 agonist, receptor-linker IL-15 (RLI).MethodsWe prepared a long acting MS conjugate of RLI, MS~RLI. The pharmacokinetics and pharmacodynamics of MS~RLI were measured in C57BL/6J mice and compared to MS~IL-15. The antitumor efficacy of MS~RLI was measured when delivered subcutaneously or intratumorally in the CT26 tumor model and intratumorally in the orthotopic EO771 tumor model.ResultsMS~RLI exhibited a half-life of 30 h, longer than most IL-15 agonists but shorter than MS~IL-15. The shorter than expected half-life of MS~RLI was shown to be due to target-mediated-disposition caused by an IL-15 induced cytokine sink. MS~RLI resulted in very potent stimulation of NK and CD44hiCD8+ T cells, but also caused significant injection-site toxicity that may preclude subcutaneous administration. We thus pivoted our efforts toward studying the MS~RLI for long-acting intra-tumoral therapy, where some degree of necrosis might be beneficial. When delivered intra- tumorally, both MS~IL-15 and MS~RLI had modest anti-tumor efficacy, but high anti- metastatic activity.ConclusionIntra-tumoral MS~RLI and MS~RLI combined with systemic treatment with other agents could provide beneficial antitumor and anti-metastatic effects without the toxic effects of systemic IL-15 agonists. Our findings demonstrate that intra-tumorally administered long-acting IL-15 agonists counter two criticisms of loco-regional therapy: the necessity for frequent injections and the challenge of managing metastases.

Intratumoral lactic acid neutralization strategy for boosting chemoimmunotherapy using liposomal sodium bicarbonate

Glycolysis-related lactic acid overproduction creates an “ion-trapping” barrier and immunosuppressive tumor microenvironment that compromise effective intratumoral drug delivery and therapy. Therefore, normalization of tumor microenvironment via lactic acid neutralization can be a promising avenue for overcoming this therapeutic hurdle. In this study, the flexible liposomes loaded with sodium bicarbonate (NaHCO3@Flip) were used as a nano-adjuvant to boost chemoimmunotherapy. Their effects on assisting DOXIL and anti-PD-1 therapy were investigated. NaHCO3@Flip achieved deep tumor penetration, with the ability to neutralize lactic acid and normalize the acidic tumor microenvironment. NaHCO3@Flip is biosafe and can enhance cellular uptake efficiency of doxorubicin (DOX) by overcoming the ion-trapping barrier and amplify immunogenic cell death induced by DOX. The combination therapy of liposomal DOX and NaHCO3@Flip demonstrated enhanced inhibition of tumor growth. NaHCO3@Flip can also synergize with PD-1 antibody therapy. NaHCO3@Flip has the potential to serve as a therapeutic adjuvant for boosting chemoimmunotherapy by overcoming the ion-trapping effect and normalizing the tumor microenvironment.

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

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

An Abscopal Effect on Lung Metastases in Canine Mammary Cancer Patients Induced by Neoadjuvant Intratumoral Immunotherapy with Cowpea Mosaic Virus Nanoparticles and Anti-Canine PD-1.

Neoadjuvant intratumoral (IT) therapy could amplify the weak responses to checkpoint blockade therapy observed in breast cancer (BC). In this study, we administered neoadjuvant IT anti-canine PD-1 therapy (IT acPD-1) alone or combined with IT cowpea mosaic virus therapy (IT CPMV/acPD-1) to companion dogs diagnosed with canine mammary cancer (CMC), a spontaneous tumor resembling human BC. CMC patients treated weekly with acPD-1 (n = 3) or CPMV/acPD-1 (n = 3) for four weeks or with CPMV/acPD-1 (n = 3 patients not candidates for surgery) for up to 11 weeks did not experience immune-related adverse events. We found that acPD-1 and CPMV/acPD-1 injections resulted in tumor control and a reduction in injected tumors in all patients and in noninjected tumors located in the ipsilateral and contralateral mammary chains of treated dogs. In two metastatic CMC patients, CPMV/acPD-1 treatments resulted in the control and reduction of established lung metastases. CPMV/acPD-1 treatments were associated with altered gene expression related to TLR1-4 signaling and complement pathways. These novel therapies could be effective for CMC patients. Owing to the extensive similarities between CMC and human BC, IT CPMV combined with approved anti-PD-1 therapies could be a novel and effective immunotherapy to treat local BC and suppress metastatic BC.

Treatment of malignant melanoma with coxsackievirus A21 (V937): An emerging oncolytic virotherapy.

Despite rising melanoma incidence in recent decades, there is a trend towards overall decreased mortality, reflecting multiple factors including improved treatment options for metastatic disease. While local treatments are the mainstay for early-stage melanoma, metastatic disease necessitates systemic treatment, with oncolytic virotherapy emerging as a promising option. For this review, articles were retrieved from PubMed from 1964 through 2024. We conducted title, abstract and full-text screening in accordance with Preferred Reporting Items for Systematic Reviews and Meta-Analyses guidelines to identify articles describing the use of coxsackievirus A21 (V937), either as monotherapy or as part of combination therapy for malignant melanoma. Fifteen articles met inclusion criteria, offering preclinical and clinical data on V937's efficacy in reducing tumour burden. In addition to reporting manageable safety profiles, clinical trial data examining intratumoral V937 combination therapy with pembrolizumab and ipilimumab also endorsed favourable objective response rates compared to immune checkpoint inhibitor monotherapy (47% vs. 38% and 21% vs. 10%, respectively). In contrast, intravenous V937 monotherapy failed to yield additional benefit in a cohort of patients with Stage IIIC/IV melanoma (n = 3) despite achieving detectable levels in tumour tissue (1 × 109 TCID50). Although small subsets of patients experienced severe adverse effects and study design limitations imposed constraints on collected data, evidence for the efficacy of V937 remains encouraging. With few clinical trials evaluating V937 in melanoma, additional data is required before routine usage in standard treatment for metastatic lesions.

Monte Carlo dosimetric analyses on the use of 90Y-IsoPet intratumoral therapy in canine subjects

Objective. To investigate different dosimetric aspects of 90Y-IsoPet™ intratumoral therapy in canine soft tissue sarcomas, model the spatial spread of the gel post-injection, evaluate absorbed dose to clinical target volumes, and assess dose distributions and treatment efficacy. Approach. Six canine cases treated with 90Y-IsoPet™ for soft tissue sarcoma at the Veterinary Health Center, University of Missouri are analyzed in this retrospective study. The dogs received intratumoral IsoPet™ injections, following a grid pattern to achieve a near-uniform dose distribution in the clinical target volume. Two dosimetry methods were performed retrospectively using the Monte Carlo toolkit OpenTOPAS: imaging-based dosimetry obtained from post-injection PET/CT scans, and stylized phantom-based dosimetry modeled from the planned injection points to the gross tumor volume. For the latter, a Gaussian parameter with variable sigma was introduced to reflect the spatial spread of IsoPet™. The two methods were compared using dose-volume histograms (DVHs) and dose homogeneity, allowing an approximation of the closest sigma for the spatial spread of the gel post-injection. In addition, we compared Monte Carlo-based dosimetry with voxel S-value (VSV)-based dosimetry to investigate the dosimetric differences. Main results. Imaging-based dosimetry showed differences between Monte Carlo and VSV calculations in tumor high-density areas with higher self-absorption. Stylized phantom-based dosimetry indicated a more homogeneous target dose with increasing sigma. The sigma approximation of the 90Y-IsoPet™ post-injection gel spread resulted in a median sigma of approximately 0.44 mm across all cases to reproduce the dose heterogeneity observed in Monte Carlo calculations. Significance. The results indicate that dose modeling based on planned injection points can serve as a first-order approximation for the delivered dose in 90Y-IsoPet™ therapy for canine soft tissue sarcomas. The dosimetry evaluation highlights the non-uniformity of absorbed doses despite the gel spread, emphasizing the importance of considering tumor dose heterogeneity in treatment evaluation. Our findings suggest that using Monte Carlo for dose calculation seems more suitable for this type of tumor where high-density areas might play an important role in dosimetry.

Intratumoral injection of mRNA encoding survivin in combination with STAT3 inhibitor stattic enhances antitumor effects

Intratumoral delivery of mRNA encoding immunostimulatory molecules can initiate a robust, global antitumor response with little side effects by enhancing local antigen presentation in the tumor and the tumor draining lymph node. Neoantigen-based mRNA nanovaccine can inhibit melanoma growth in mice by intratumoral injection. Myeloid-derived suppressor cells (MDSCs) suppress antitumor immune responses by secreting immunosuppressive agents, such as reactive oxygen species (ROS). Suppression of STAT3 activity by stattic may reduce MDSC-mediated immunosuppression in the TME and promote the antitumor immune responses. In this study, in vitro transcribed mRNA encoding tumor antigen survivin was prepared and injected intratumorally in BALB/c mice bearing subcutaneous colon cancer tumors. In vivo studies demonstrated that intratumoral survivin mRNA therapy could induce antitumor T cell response and inhibit tumor growth of colon cancer. Depletion of CD8+ T cells could significantly inhibit survivin mRNA-induced antitumor effects. RT-qPCR and ELISA analysis indicated that survivin mRNA treatment led to increased expression of receptor activator nuclear factor-κB ligand (RANKL). In vitro experiment showed that MDSCs could be induced from mouse bone marrow cells by RANKL and RANKL-induced MDSCs could produce high level of ROS. STAT3 inhibitor stattic suppressed activation of STAT3 and NF-κB signals, thereby inhibiting expansion of RANKL-induced MDSCs. Combination therapy of survivin mRNA and stattic could significantly enhance antitumor T cell response, improve long-term survival and reduce immunosuppressive tumor microenvironment compared to each monotherapy. In addition, combined therapy resulted in a significantly reduced level of tumor cell proliferation and an obviously increased level of tumor cell apoptosis in CT26 colon cancer-bearing mice, which could be conducive to inhibit the tumor growth and lead to immune responses to released tumor-associated antigens. These studies explored intratumoral mRNA therapy and mRNA-based combined therapy to treat colon cancer and provide a new idea for cancer therapy.

Non-viral-mediated gene transfer of OX40 ligand for tumor immunotherapy.

Immune checkpoint blockade (ICB) is rapidly becoming a standard of care in the treatment of many cancer types. However, the subset of patients who respond to this type of therapy is limited. Another way to promote antitumoral immunity is the use of immunostimulatory molecules, such as cytokines or T cell co-stimulators. The systemic administration of immunotherapeutics leads to significant immune-related adverse events (irAEs), therefore, the localized antitumoral action is needed. One way to achieve this is intratumoral non-viral gene-immune therapy, which allows for prolonged and localized gene expression, and multiple drug administration. In this study, we combined the previously described non-viral gene delivery system, PEG-PEI-TAT copolymer, PPT, with murine OX40L-encoding plasmid DNA. The resulting OX40L/PPT nanoparticles were characterized via gel mobility assay, dynamic light scattering analysis and in vitro transfection efficiency evaluation. The antitumoral efficacy of intratumorally (i.t.) administered nanoparticles was estimated using subcutaneously (s.c.) implanted CT26 (colon cancer), B16F0 (melanoma) and 4T1 (breast cancer) tumor models. The dynamics of stromal immune cell populations was analyzed using flow cytometry. Weight loss and cachexia were used as irAE indicators. The effect of combination of i.t. OX40L/PPT with intraperitoneal PD-1 ICB was estimated in s.c. CT26 tumor model. The obtained OX40L/PPT nanoparticles had properties applicable for cell transfection and provided OX40L protein expression in vitro in all three investigated cancer models. We observed that OX40L/PPT treatment successfully inhibited tumor growth in B16F0 and CT26 tumor models and showed a tendency to inhibit 4T1 tumor growth. In B16F0 tumor model, OX40L/PPT treatment led to the increase in antitumoral effector NK and T killer cells and to the decrease in pro-tumoral myeloid cells populations within tumor stroma. No irAE signs were observed in all 3 tumor models, which indicates good treatment tolerability in mice. Combining OX40L/PPT with PD-1 ICB significantly improved treatment efficacy in the CT26 subcutaneous colon cancer model, providing protective immunity against CT26 colon cancer cells. Overall, the anti-tumor efficacy observed with OX40L non-viral gene therapy, whether administered alone or in combination with ICB, highlights its potential to revolutionize cancer gene therapy, thus paving the way for unprecedented advancements in the cancer therapy field.

A platform technology for ultra-long acting intratumoral therapy

Intratumoral (IT) therapy is a powerful method of controlling tumor growth, but a major unsolved problem is the rapidity that injected drugs exit tumors, limiting on-target exposure and efficacy. We have developed a generic long acting IT delivery system in which a drug is covalently tethered to hydrogel microspheres (MS) by a cleavable linker; upon injection the conjugate forms a depot that slowly releases the drug and “bathes” the tumor for long periods. We established technology to measure tissue pharmacokinetics and studied MSs attached to SN-38, a topoisomerase 1 inhibitor. When MS ~ SN-38 was injected locally, tissues showed high levels of SN-38 with a long half-life of ~ 1 week. IT MS ~ SN-38 was ~ tenfold more efficacious as an anti-tumor agent than systemic SN-38. We also propose and provide an example that long-acting IT therapy might enable safe use of two drugs with overlapping toxicities. Here, long-acting IT MS ~ SN-38 is delivered with concurrent systemic PARP inhibitor. The tumor is exposed to both drugs whereas other tissues are exposed only to the systemic drug; synergistic anti-tumor activity supported the validity of this approach. We propose use of this approach to increase efficacy and reduce toxicities of combinations of immune checkpoint inhibitors such as αCTLA-4 and αPD-1.

The efficacy of recombinant human adenovirus type 5 (H101) intra-tumor therapy in persistent/recurrent/metastatic gynecological cancer and the exploration of changes in tumor microenvironment.

The efficacy of recombinant human adenovirus type 5 (H101) intra-tumor therapy in persistent/recurrent/metastatic gynecological cancer and the exploration of changes in tumor microenvironment.

Phase 1b trial of IFx-Hu2.0, a novel in situ cancer vaccine, in checkpoint inhibitor-resistant Merkel cell carcinoma (MCC) and cutaneous squamous cell carcinoma (cSCC).

9592 Background: MCC and cSCC have limited treatment options when refractory to immune checkpoint inhibitor (ICI) therapy. IFx-Hu2.0 (IFx) is a plasmid DNA encoding for an immunogenic bacterial protein, Emm55, formulated with a transfection agent for direct intra-tumoral (IT) injection. In this Phase 1b study, we evaluated the safety and efficacy of different schedules of IT IFx in patients (pts) with advanced MCC or cSCC. Methods: In the first trial stage (n=9), IT IFx was administered in up to 3 lesions on 3 schedules following a 3+3 exposure escalation schema; weekly x 1, 2, or 3. In the second trial expansion stage (n=11), IT IFx was administered weekly x 3. The primary objective of the study was to establish safety and feasibility of repeated IT administrations of the investigational agent. ≥80% completion of planned study therapy was predefined as a successful feasibility outcome. Given the proposed potential for immune priming effects of IFx, we performed an unplanned exploratory analysis of post-protocol treatment efficacy to evaluate response to ICI rechallenge. Results: We treated 22 pts (MCC, 13; cSCC, 9). All pts had received prior anti-PD(L)1 based treatment with disease progression being the reason for ICI discontinuation in all patients but one. Two pts did not complete planned study therapy due to rapid clinical progression and were replaced but included in the safety analysis. IT IFx was well tolerated at all dose schedules evaluated with 1 high-grade toxicity deemed possibly treatment related (G3 hepatitis in a patient recently treated with ICI). The study therefore met predefined study endpoints for safety and efficacy. After protocol specified IT therapy, 11 MCC pts and 6 cSCC pts were treated with anti-PD(L)1 based therapy as the immediate post-protocol treatment. Five of 9 (56%) evaluable MCC patients and 1 of 7 (14%) cSCC patients experienced an objective response to this ICI rechallenge, with duration of response ongoing in 4 patients (6+, 19+, 21+, 23+ months) and the two other responses lasting 23 and 33 months. The two remaining MCC patients were not evaluable for response from IO rechallenge due to radiation administered to the only measurable disease site(s), but both remain progression free at 11+ and 13+ months with previously progressive disease. Conclusions: IT IFx-Hu2.0 is safe and feasible to administer at weekly dosing repeated up to 3 weeks. An unplanned exploratory analysis revealed frequent (56%) and durable responses in advanced MCC to ICI re-challenge despite prior resistance to this class of drug, suggesting that IFx induced an immune priming effect. Preliminary biomarker analyses to investigate this phenomenon are ongoing and will be presented. Clinical trial information: NCT04160065 .

Effect of P. gingivalis on osimertinib resistance in EGFR-mutant lung cancer.

e20511 Background: While most patients with EGFR-mutant lung cancers respond to osimertinib, resistance inevitably develops. Mechanisms of osimertinib resistance remain inadequately understood. Recent studies suggest a potential link between intra-tumoral bacteria and therapy resistance in cancer. Our previous studies found that the pre-conditioned medium (PCM) of certain bacteria in the Bacteroidetes phylum conferred resistance of all EGFR-TKIs tested. In this study, we specifically investigate the effect of Porphyromonas gingivalis, a common component of the human oral microbiome and the main pathogen responsible for periodontal disease, on osimertinib sensitivity in EGFR mutant lung cancer. Methods: P. gingivalis PCM (P-CM) was applied to EGFR-mutant lung cancer cell lines for 3 days and cell viability was measured. Western blot and receptor tyrosine kinase (RTKs) assay were used to analyze the changes of cell signaling pathways, and the contribution of identified signaling pathway to P. gingivalis-mediated resistance to osimertinib were further validated by rescue experiments. To investigate the existence of P. gingivalis in human lung cancer tissues, we used Fluorescence In Situ Hybridization (FISH) probes specific to P. gingivalis and hybridized probes to a tissue microarray (TMA) containing 3 mm punches of lung adenocarcinoma tissue from 30 patients in duplicate. The underlying mechanisms of osimertinib resistance conferred by P. gingivalis were explored by a combination of bacterial genomic and cellular proteomic approaches. Results: P-PCM led to osimertinib resistance in all EGFR-mutant lung cancer cell lines tested. Both Western blot and RTKs assay showed that phosphorylated insulin-like growth factor 1 receptor (pIGF1R) level significantly increased when P-PCM was applied to PC9 EGFR mutant lung cancer cells. We found that both IGF1R inhibitor (linsitinib) and IGF1R knockdown with siRNA eliminated the effect of P-PCM-mediated osimertinib resistance. We found that two cysteine proteases secreted by P. gingivalis, lysine-specific gingipain (Kgp) and arginine-specific gingipain A (RgpA) led to osimertinib, whereas arginine-specific gingipain B (RgpB), which lacks haemagglutinin/adhesin (HA) region of RgpA had no effect on osimertinib response. We also found that gingipains-mediated osimertinib resistance is independent of enzymatic activity. We detected intra-tumoral P. gingivalis in tumor tissue of 9 (30%) patients by using FISH probes specific to P. gingivalis. Conclusions: Our studies demonstrate that P. gingivalis can induce osimertinib resistance in EGFR-mutant lung cancer cells through secreted gingipains that activate the IGF1R pathway through an enzymatic- independent mechanism. P. gingivalis is frequently detected in human lung cancer tissues , suggesting that therapies targeting P. gingivalis may improve EGFR-TKI response in certain patients with EGFR-mutant lung cancer.

Development of Intratumoral Drug Delivery Based Strategies for Antitumor Therapy.

Research for tumor treatment with significant therapy effects and minimal side-effects has been widely carried over the past few decades. Different drug forms have received a lot of attention. However, systemic biodistribution induces efficacy and safety issues. Intratumoral delivery of agents might overcome these problems because of its abundant tumor accumulation and retention, thereby reducing side effects. Delivering hydrogels, nanoparticles, microneedles, and microspheres drug carriers directly to tumors can realize not only targeted tumor therapy but also low side-effects. Furthermore, intratumoral administration has been integrated with treatment strategies such as chemotherapy, enhancing radiotherapy, immunotherapy, phototherapy, magnetic fluid hyperthermia, and multimodal therapy. Some of these strategies are ongoing clinical trials or applied clinically. However, many barriers hinder it from being an ideal and widely used option, such as decreased drug penetration impeded by collagen fibers of a tumor, drug squeezed out by high density and high pressure, mature intratumoral injection technique. In this review, we systematically discuss intratumoral delivery of different drug carriers and current development of intratumoral therapy strategies.

CRACD loss induces neuroendocrine cell plasticity of lung adenocarcinoma

Tumor cell plasticity contributes to intratumoral heterogeneity and therapy resistance. Through cell plasticity, some lung adenocarcinoma (LUAD) cells transform into neuroendocrine (NE) tumor cells. However, the mechanisms of NE cell plasticity remain unclear. CRACD (capping protein inhibiting regulator of actin dynamics), a capping protein inhibitor, is frequently inactivated in cancers. CRACD knockout (KO) is sufficient to de-repress NE-related gene expression in the pulmonary epithelium and LUAD cells. In LUAD mouse models, Cracd KO increases intratumoral heterogeneity with NE gene expression. Single-cell transcriptomic analysis showed that Cracd KO-induced NE cell plasticity is associated with cell de-differentiation and stemness-related pathway activation. The single-cell transcriptomic analysis of LUAD patient tumors recapitulates that the distinct LUAD NE cell cluster expressing NE genes is co-enriched with impaired actin remodeling. This study reveals the crucial role of CRACD in restricting NE cell plasticity that induces cell de-differentiation of LUAD.

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

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

Abstract LT05: METTL8-MEDIATED EPITRANSCRIPTOMIC REGULATION OF GBM CYCLING STATE IS DEPENDENT ON RTK/PI3K/AKT SIGNALING

Abstract Glioblastoma (GBM) is a lethal brain tumor with limited treatment options. Treatment failures are often attributed to the existence of glioma stem cells (GSCs), which confer intratumoral heterogeneity and therapy resistance. Contrary to common belief that cancer cells employ aerobic glycolysis (Warburg effect) to sustain their metabolic requirements, GSCs rely on oxidative phosphorylation (OXPHOS) for energy production. As a result, GSCs are uniquely sensitive to OXPHOS inhibitors (Gboxin), as well as mitochondrial translation inhibitors which disrupt OXPHOS. METTL8 is a newly discovered mitochondrial methyltransferase which catalyzes m3C32 modifications in mitochondrial tRNAsThr/Ser(UCN) (mt-tRNAs). This post-transcriptional modification ensures the optimal structure of mt-tRNAs to facilitate efficient translation of mitochondrial proteins involved in electron transport chain (ETC). Although the epitranscriptomic functions of METTL8 have been well characterized, its roles in cancers (i.e. glioblastoma) remain largely unexplored. In this study, we showed that METTL8 is overexpressed in GBM and high METTL8 expression portends poor patient prognosis. In GSC, METTL8 is under the transcriptional regulation of HIF-1α, and the chromatin accessibility at METTL8 promoter is facilitated by the deposition of histone variant H2AZ. METTL8 depletion downregulated m3C32 modifications in mt-tRNAsThr/Ser(UCN) in GSC, leading to impaired mitochondrial translation and OXPHOS defects. Phenotypically, METTL8 knockdown (KD) compromised GSCs’ self-renewal, proliferation, invasiveness and tumorigenicity. Interestingly, METTL8-KD GSC acquired a slow cycling state which is linked to reduced receptor tyrosine kinase (RTK) signaling. Consistently, METTL8-KD GSC showed lower Akt phosphorylation and exhibited increased sensitivity to mTOR/Akt inhibitors. Finally, our mechanistic findings led to the identification of a novel HIF1-α/Akt inhibitor drug combination which faithfully recapitulates the phenotypes of METTL8-KD GSCs. In conclusion, our findings provide the first in-depth study of METTL8 roles in GBM pathogenesis, and highlights a novel drug combination which could potentially be used to treat this dreadful disease. Citation Format: Bernice Woon Li Lee, You Heng Chuah, Jeehyun Yoon, Oleg V. Grinchuk, Jia Feng, Yajing Liang, Jayshree L Hirpara, Ya Ting Shen, Loo Chien Wang, Yan Ting Lim, Tianyun Zhao, Radoslaw M Sobota, Tan Boon Toh, Shazib Pervaiz, Zhewang Lin, Derrick Sek Tong Ong. METTL8-MEDIATED EPITRANSCRIPTOMIC REGULATION OF GBM CYCLING STATE IS DEPENDENT ON RTK/PI3K/AKT SIGNALING [abstract]. In: Proceedings of Frontiers in Cancer Science; 2023 Nov 6-8; Singapore. Philadelphia (PA): AACR; Cancer Res 2024;84(8_Suppl):Abstract nr LT05.

Abstract LB400: Evolution of anti-viral and anti-tumor immunity in tumors in response to oncolytic virus therapy

Abstract Preclinical and clinical studies have shown that intratumoral oncolytic viruses (OVs) can convert “cold” tumors to “hot” and overcome resistance to immune checkpoint blockade. However, their modest clinical efficacy to date highlights major gaps in our understanding of how OVs interact with the immune system, and very limited information is available regarding the specificity of the T cell repertoires that emerge within tumors in response to OV therapy. To address these gaps, we interrogated the evolution of immune response to OV therapy in mice using oncolytic Newcastle Disease Virus (NDV). Leveraging a bilateral flank melanoma mouse model, we identified and phenotypically characterized anti-viral and anti-tumor T cells in local and abscopal tumors with single cell (sc) RNA and T cell receptor (TCR) sequencing. Intratumoral NDV therapy to a single flank tumor resulted in increased infiltration of CD4+ and CD8+ T cells in both the injected and distant tumors, higher TCR overlap between treated and distant tumors, and higher frequency of convergent TCR clonotypes, suggesting their non-random expansion, albeit without specificity information. To identify the specificity of the emergent TCR clones in an unbiased fashion, we developed an approach based on adoptive transfer of T cells from tumor-bearing control or NDV-treated donor mice into congenically marked recipients with either B16 or NDV antigen stimulation. This allowed the isolation and identification of expanded B16- or virus-specific TCR clones that were then linked to donor tumors to define their relative frequencies, their distribution across the treated and distant tumors, and their associated phenotypic states. We observed significantly greater expansion of B16-reactive T cells adoptively transferred from NDV-treated donors (compared to control donors), suggesting that NDV therapy leads to potentiation of tumor-specific immune response. Intriguingly, virus-specific and tumor-specific T cells appeared to distribute to distinct phenotypic clusters, with virus-specific and tumor-specific T cells found predominantly in the activated-dysfunctional and proliferating clusters, respectively. When looking at the distribution of the TCR clones, virus-specific clones dominated the majority of the response in both treated and distant tumors, despite the lack of virus injection in distant tumors. These results provide insights into understanding the balance between virus- and tumor-directed immune responses elicited by OV therapy and highlight that even in the presence of significant abscopal immune effect, the significant proportion of T cells in “hot” tumors represent anti-viral rather than anti-tumor response. These findings provide tools for tracking of immune response specificity and generate rationale for development of OV engineering strategies and combinations that favor anti-tumor rather than anti-viral immunity. Citation Format: Olga Lyudovyk, Bharat Burman, Nicholas Ceglia, Benjamin Greenbaum, Yuval Elhanati, Dmitriy Zamarin. Evolution of anti-viral and anti-tumor immunity in tumors in response to oncolytic virus therapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr LB400.

Abstract LB439: Intra-tumoral administration of releasable hydrogel microsphere drug conjugates for long acting therapeutics

Abstract Background. Intra-tumoral (IT) injections of therapeutics hold promise for the treatment of solid tumors. However, drugs administered directly in the tumor have short IT residence times potentially limiting their exposure and therefore efficacy. We are developing a platform of novel hydrogel microsphere (MS) conjugates that enables long-acting tumor residence after a single injection of the conjugate. We demonstrate this approach not only solves the problem of short acting intra-tumoral therapy, but also allows for the safe administration of two therapies with overlapping toxicities such as SN-38 and a PARP inhibitor (PARPi). Methods. Hydrogel microsphere (MS) conjugates were prepared and characterized. Using MS conjugates labeled with rhodamine and fluorescein, we established methods to directly measure the IT concentration and half-life of the released drug after IT administration into CT26 tumor bearing mice. We next measured the tissue pharmacokinetics and efficacy of MSs attached to SN-38, a potent topoisomerase 1 inhibitor and immunomodulator, using NSG mice bearing 22Rv1 ATM-/- xenografts. Finally, we measured the anti-tumor efficacy of MS~SN-38 in combination with an oral PARPi, talazoparib, in 22Rv1 ATM-/- xenografts. Results. We successfully established methods to quantify the concentration of released drug and MS bound drug from tumor biopsies using fluorescent surrogates. When applied to MS~SN-38 conjugates, locally injected tissues showed high levels of SN-38 with a long half-life of about one week. In contrast, the systemic half-life of free SN-38 is ~30 minutes. IT MS~SN-38 was ~10-fold more efficacious as an anti- tumor agent than systemic SN-38 from subcutaneous administration of MS~SN-38. Finally, we show high anti-tumor synergy from the SN-38 localized in a tumor by IT MS~SN-38 in combination with systemic administration of the PARPi talazoparib. Conclusions. This microsphere technology allows for sustained delivery of high local concentrations of drugs administered intratumorally. Here, IT MS~SN-38 resulted in an intra-tumoral half-life of ~1 week and marked anti-tumor efficacy in 22Rv1 ATM-/- xenografts. We also demonstrated that long-acting IT injections enables safe use of drug combinations with well-described overlapping toxicities such as SN-38 and PARPi. The microsphere technology may offer an alternative strategy for localizing and sustaining drugs in tumors and may mitigate toxicities from combination therapy where two agents have a similar toxicological profile. Other combinations, including small molecules and immuno-oncology agents, are being explored. Citation Format: John A. Hangasky, Jeff Henise, Deborah Charych, Christopher W. Carreras, Gary Ashley, Daniel V. Santi. Intra-tumoral administration of releasable hydrogel microsphere drug conjugates for long acting therapeutics [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr LB439.

Abstract 5247: Reprogramming tumor microenvironment with intratumor macrophage adoptive cell therapy in melanoma

Abstract The interplay between tumor cells and immune cells within the tumor microenvironment (TME) dictates protumor or antitumor immune responses. Tumor-associated macrophages (TAMs) respond to signals of the TME and exhibit a spectrum of phenotypes ranging between M1 (antitumor) and M2 (protumor) macrophages. In most tumor types, including melanoma, the balance between M1 and M2 macrophages is critical with a higher M1/M2 ratio favoring antitumor immunity. Therefore, strategies enhancing the M1/M2 ratio can significantly alter the TME towards antitumor immunity. In this study, we administered luciferase and GFP-expressing M1 macrophages as an adoptive cell therapy (ACT) through intravenous (i.v) and intratumor routes respectively, into mice bearing SM1 murine melanoma tumors to determine the effective treatment modality. We reprogrammed antitumor M1 macrophages ex-vivo with HDAC6 inhibitors to lock into the M1 phenotype and administered intratumorally as ACT in the syngeneic SM1 murine melanoma and humanized NSG-SGM3 melanoma models. We performed histological analysis of tumors for macrophage markers, immune phenotyping of infiltrated immune cells, single-cell secretome analysis of tumor macrophages, and single-cell RNA-seq analysis of CD45+ immune cell populations to demonstrate the benefit of macrophage ACT. Tail vein injected, luciferase-expressing M1 macrophages localized in the lungs and spleen, failed to reach the tumor as visualized by IVIS imaging, suggesting that i.v administration is ineffective for macrophage therapy. On the contrary, intratumor M1 macrophage ACT resulted in diminished tumor growth. Single-cell RNA-seq analysis of the CD45+ sorted tumor-associated immune cell population revealed distinct macrophage subsets and a significant M1/M2 macrophage ratio increase. NicheNet cell-cell interaction analysis indicated that M1-like TAMs activated infiltrating T-cells and monocytes through ligand-receptor interactions. Trajectory analysis of infiltrated monocytes indicated differentiation toward inflammatory macrophages in ACT tumors. Flow cytometry analysis corroborated that ACT increased the M1/M2 macrophage ratio and an increase in CD8 effector T-cells. Furthermore, HDAC6i-treated macrophages increased antigen cross-presentation. Single-cell secretome analysis of F4/80+ TAMs by the Isoplexis platform revealed polyfunctionality of HDAC6-treated M1 macrophages secreting inflammatory cytokine Tnfa and T-cell recruiting chemokine Cxcl10. Histological examination of tumor sections for macrophage phenotypic markers suggested that transplanted macrophages retained the M1 phenotype post-ACT in both SM1 murine and NSG-SGM3 melanoma tumor models. We demonstrated the potential of reprogramming macrophages ex vivo with HDAC6 inhibitors as a feasible macrophage cell therapy to treat solid tumors. Citation Format: Satish Kumar Reddy Noonepalle, Nithya Gajendran, Manasa Suresh, Xintang Li, Maria D. Hernandez, Christian Zevallos Delgado, Nima Aghdam, Michael Berrigen, Tessa Knox, Karen Tan, Marie Durr, Eduardo Sotomayor, Katherine B. Chiappinelli, Duncan Wardrop, Anelia Horvath, Brett A. Shook, Norman H. Lee, Anatoly Dritschilo, Rohan Fernandes, Karthik Musunuri, Maho Shibata, Alejandro Villagra. Reprogramming tumor microenvironment with intratumor macrophage adoptive cell therapy in melanoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5247.