Preclinical Tumor Models Research Articles

EXTH-35. REPURPOSING OF CLINICALLY APPROVED GLUTAMATE ANTAGONISTS TO TREAT GLIOBLASTOMA

Abstract The neurotransmitter glutamate plays a fundamental role in promoting epilepsy and tumor progression in glioblastoma. Glioblastoma cells secrete glutamate into the tumor microenvironment via the cystine-glutamate antiporter system Xc, leading to neuronal hyperexcitability, excitotoxicity, and tumor cell invasion. Additionally, glutamatergic neuroglioma synapses drive glioma progression in preclinical tumor models. To pharmacologically counteract effects of glutamate in glioblastoma, we employed a triple combination of anti-glutamatergic drugs: gabapentin, sulfasalazine, and memantine, which target glutamate synthesis, release, and post-synaptic signaling, respectively. In genetically engineered murine glioblastoma models, daily treatment with this triple combination prolonged median survival. This survival benefit was not observed with single or double drug combinations, nor with the standard antiepileptic drug levetiracetam, indicating the potential synergy and efficacy of this drug combination beyond merely treating epilepsy. Importantly, these preliminary results support the rationale for the ongoing phase Ib/II randomized clinical trial GLUGLIO, which explores the efficacy of combined gabapentin, sulfasalazine, and memantine in conjunction with standard chemoradiotherapy in patients with newly diagnosed glioblastoma (NCT05664464).

EXTH-09. GENE MODIFICATION OF HEMATOPOIETIC STEM AND PROGENITOR CELLS FOR IMPROVEMENT OF IMMUNOTHERAPIES FOR MALIGNANT GLIOMAS

Abstract Patients living with high-grade gliomas have a poor survival prognosis. Furthermore, standard of care treatment for these malignant gliomas has not significantly improved in the past decades. Our laboratory previously demonstrated that the combination of two different immunotherapies with HSPCs provided a significant survival benefit in preclinical brain tumor models, however, this therapeutic combination is still not 100% curative. We hypothesized that gene modification of HSPCs could add additional therapeutic benefit to immunotherapies for the treatment of resistant gliomas. Hematopoietic stem and progenitor cells (HSPCs) have been widely used in different clinical settings due to their ability to self-renew and differentiate into all immune cell lineages. In recent years, HSPCs have been the target of a variety of gene editing research efforts to decipher the most effective method for gene modification to use for therapeutic purposes. After comparison with previously established methods, we optimized a protocol for the successful editing of murine HSPCs and human CD34+ cells. Using Cas9 mRNA and sgRNAs in HSPCs, we achieved up to 80% reduction in gene expression. This method is superior to others due to its efficient, quick, inexpensive, and reliable nature. We also showed that, by knocking-down IL-6R in HSPCs, we can drive their differentiation into dendritic cells, while decreasing MDSCs and macrophages. Furthermore, gene-modified HSPCs are also capable of bone marrow reconstitution of lethally irradiated mice. Currently, we are testing whether the combination of gene-modified HSPCs with immunotherapy provides additional survival benefit to unmodified HSPCs with immunotherapy in a resistant murine glioma model.

Bayesian modeling for analyzing heterogeneous response in preclinical mouse tumor models.

In anticancer research, tumor growth measured in mouse models is important for assessing treatment efficacy for a treatment to progress to human clinical trials. Statistical analysis of time-to-event tumor volume data is complex because of heterogeneity in response and welfare-related data loss. Traditional statistical methods of testing the mean difference between groups are not robust because they assume common responses across a population. Heterogeneity in response is also seen in the clinic, and consequently, the assessment of the treatment considers the diversity through classification of the individual's response using the RECIST (Response Evaluation Criteria in Solid Tumors). To provide a comparable and translatable assessment of in vivo tumor response, we developed a statistical method called INSPECT (IN vivo reSPonsE Classification of Tumors) for analyzing heterogeneous responses through Bayesian modeling. This method can classify individual tumor behaviors into the categories of nonresponder, modest responder, stable responder, and regressing responder. Using both published and simulated data, we show that INSPECT methodology is more accurate and sensitive than existing methods with respect to balancing false-negative and false-positive rates. A case study demonstrates the value of INSPECT in drug projects for supporting the translation of drug efficacy from the preclinical phase into clinical trials. We also provide a package, "INSPECTumours," that launches a web interface to enable users to conduct the analysis and generate reports.

Development of a novel immunocompetent murine tumor model for urothelial carcinoma using in vivo electroporation

A lack of advanced preclinical mouse tumor models impedes the progress in urothelial carcinoma research. We present here a novel fast, robust, reliable, and highly reproducible model for the genetic induction of bladder cancer in immunocompetent mice. Different sets of oncogenic transposons (Cmyc, Kras) and Cre drivers were transfected into the murine bladder wall of two different genetic backgrounds (Trp53fl/fl and BrafV600E, Ptenfl/fl, Ctnnb1exon3-fl/fl). Transfection was carried out using in vivo electroporation of the bladder after surgical exploration and transmural or transurethral intravesical plasmid injection. Up to 100% of animals developed urothelial carcinomas of the bladder. Time to tumor onset ranged from 16 to 97 days with a median of approximately 23 days in the fastest groups. Histological examination identified orthotopic urothelial carcinomas in most cases, in some experimental groups up to 100%. The resulting tumors were highly invasive and often metastatic. Metastases were found in up to 100% of tumor bearing mice per group. Taken together, this study establishes the proof-of-principle that in vivo electroporation can be versatilely employed as a reliable, fast, and robust method for the highly reproducible induction of urothelial carcinomas in the murine bladder wall. This novel murine tumor model could pave the way towards more easily modelling subtype specific urothelial carcinomas in mice.

MUC16 Retention after Neoadjuvant Chemotherapy in Pancreatic Ductal Adenocarcinoma.

Background/Objectives: Pancreatic ductal adenocarcinoma (PDAC) has a poor prognosis. Currently, surgical resection is the only potentially curative treatment. Unfortunately, less than 20% of PDAC patients are eligible for surgical resection at diagnosis. In the past few decades, neoadjuvant chemotherapy treatment (NCT) has been investigated as a way to downstage PDAC tumors for surgical resection. Fluorescence-guided surgery (FGS) is a technique that can aid in increasing complete resection rates by enhancing the tumor through passive or active targeting of a contrast agent. In active targeting, a probe (e.g., antibody) binds a protein differentially upregulated in the tumor compared to normal tissue. Mucin 16 (MUC16), a transmembrane glycoprotein, has recently been explored as an FGS target in preclinical tumor models. However, the impact of chemotherapy on MUC16 expression is unknown. Methods: To investigate this issue, immunohistochemistry was performed on PDAC patient samples. Results: We found that MUC16 expression was retained after NCT in patient samples (mean expression = 5.7) with minimal change in expression between the matched diagnostic (mean expression = 3.66) and PDAC NCT patient samples (mean expression = 4.5). Conclusions: This study suggests that MUC16 is a promising target for FGS and other targeted therapies in PDAC patients treated with NCT.

Targeting NQO1 induces ferroptosis and triggers anti-tumor immunity in immunotherapy-resistant KEAP1-deficient cancers

Immunotherapy has revolutionized cancer treatment, yet the efficacy of immunotherapeutic approaches remains limited. Resistance to ferroptosis is one of the reasons for the poor therapeutic outcomes in tumors with Kelch-like ECH-associated protein 1 (KEAP1) mutations. However, the specific mechanisms by which KEAP1-mutant tumors resist immunotherapy are not fully understood. In this study, we showed that the loss of function in KEAP1 results in resistance to ferroptosis. We identified NAD(P)H Quinone Dehydrogenase 1 (NQO1) as a transcriptional target of nuclear factor erythroid 2–related factor 2 (NRF2) and revealed that inducing NQO1-mediated ferroptosis in KEAP1-deficient tumors triggers an antitumor immune cascade. Additionally, it was found that NQO1 protein levels could serve as a candidate biomarker for predicting sensitivity to immunotherapy in clinical tumor patients. We validated these findings in several preclinical tumor models. Overall, KEAP1 mutations define a unique disease phenotype, and targeting its key downstream molecule NQO1 offers new hope for patients with resistance to immunotherapy.

Novel H-2Db-restricted CD8 epitope derived from mouse MAGE-type antigen P1A mediates antitumor immunity in C57BL/6 mice

BackgroundMelanoma antigen gene (MAGE)-type antigens are promising targets for cancer immunotherapy as they are expressed in cancer cells but not in normal tissues, except for male germline cells. The mouse...

Abstract C023: PDAC tumors from patients treated with combination MEK and autophagy inhibition reveal a treatment sensitive epithelial subtype that is enriched in murine models

Abstract Combining MEK and autophagy inhibitors in preclinical PDAC mouse models resulted in tumor regressions and improved rodent survival. These findings provided the rationale for clinical investigation of this combination. The phase 1 MEKiAUTO trial evaluated combination cobimetinib, HCQ, with or without atezolizumab in 14 patients (pts) with KRAS-mutated pancreatic adenocarcinoma (PDAC). The study was closed due to a lack of preliminary efficacy and poor tolerability; however, three patients experienced progression free survival (PFS) beyond 12 weeks (two with KRAS G12R , one with KRAS G12D ). Correlative analyses on twenty-one biopsies (8-paired FFPE and 6-paired fresh frozen specimens) with multiplex immunofluorescence (mIF) demonstrated decreased phosphorylation of ERK and accumulation of p62 indicating a pharmacodynamic effect. Reverse phase protein array (RPPA) also confirmed MAPK pathway inhibition, with a decrease in pERK, and autophagy inhibition, with decreased autophagy signature score, within the tumor compartment of on-treatment biopsies compared to baseline. Single nucleus RNAseq with protein expression activity extrapolated using ARACNe (Algorithm for the Reconstruction of Accurate Cellular Networks) and VIPER (Virtual Inference of Protein-activity by Enriched Regulon) pipelines identified five distinct tumor subpopulations. Comparing prevalence of tumor clusters before and after treatment identified differential responses to treatment. Gene set enrichment analysis revealed that the most sensitive tumor cluster, tumor cluster 2, was enriched in autophagy and MAPK signaling. Pre- treatment frequency of tumor cluster 2 was higher in patients who experienced a clinical benefit and independently correlated with PFS. Among the non-tumor cell clusters identified, patients with clinical benefit also had increased immune cell infiltration. To explain the discordance in efficacy with this combination in preclinical models and humans, we interrogated the preclinical tumor model systems for tumor heterogeneity. Single cell RNA-Seq analysis of tumors from Kras LSL.G12D/+ ; p53 LSL.R172H/+ ; Pdx1-Cre tg/+ (KPC) mice treated with the combination confirmed presence of similar treatment-sensitive and treatment-resistant clusters, with the former being enriched for MAPK and autophagy signatures. Parallel analysis of cell lines previously shown to be sensitive to combination MEK and autophagy inhibition, including MiaPACA2 and PANC1, showed they were mostly enriched in the treatment-sensitive human cluster 2 phenotype but not treatment-resistant clusters. Our results suggest that some preclinical models may overrepresent subtypes which are sensitive to MEK and autophagy inhibitors and thus do not recapitulate the extent of intertumoral heterogeneity in human PDAC. Observed preclinical responses likely represented elimination of treatment-sensitive cluster(s), with the remaining clusters responsible for maintained resistance. Citation Format: Brian W Labadie, Aleksandar Z Obradovic, Sarah Sta Ana, Michael May, Naomi Sender, Isabelle V Ross, Monica Rodriguez-Mendez, Urszula Wasko, Lorenzo Tomassoni, Li Li, Manan Vij, Sinan Abuzaid, Julia Wulfkuhle, Winston Wong, Ilenia Pellicciotta, Benjamin Izar, Emanuel F Petricoin, Aik Choon Tan, Kenneth P Olive, Alex G Raufi, Gulam A Manji. PDAC tumors from patients treated with combination MEK and autophagy inhibition reveal a treatment sensitive epithelial subtype that is enriched in murine models [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Advances in Pancreatic Cancer Research; 2024 Sep 15-18; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2024;84(17 Suppl_2):Abstract nr C023.

An in situ depot for the sustained release of a TLR7/8 agonist in combination with a TGFβ inhibitor promotes anti-tumor immune responses

Cancer curing immune responses against heterogeneous solid cancers require that a coordinated immune activation is initiated in the antigen avid but immunosuppressive tumor microenvironment (TME). The plastic TME, and the poor systemic tolerability of immune activating drugs are, however, fundamental barriers to generating curative anticancer immune responses. Here, we introduce the CarboCell technology to overcome these barriers by forming an intratumoral sustained drug release depot that provides high payloads of immune stimulatory drugs selectively within the TME. The CarboCell thereby induces a hot spot for immune cell training and polarization and further drives and maintains the tumor-draining lymph nodes in an anticancer and immune activated state. Mechanistically, this transforms cancerous tissues, consequently generating systemic anticancer immunoreactivity. CarboCell can be injected through standard thin-needle technologies and has inherent imaging contrast which secure accurate intratumoral positioning. In particular, here we report the therapeutic performance for a dual-drug CarboCell providing sustained release of a Toll-like receptor 7/8 agonist and a transforming growth factor-β inhibitor in preclinical tumor models in female mice.

Novel Arf1 Inhibitors Drive Cancer Stem Cell Aging and Potentiate Anti-Tumor Immunity.

The small G protein Arf1 has been identified as playing a selective role in supporting cancer stem cells (CSCs), making it an attractive target for cancer therapy. However, the current Arf1 inhibitors have limited translational potential due to their high toxicity and low specificity. In this study, two new potent small-molecule inhibitors of Arf1, identified as DU101 and DU102, for cancer therapy are introduced. Preclinical tumor models demonstrate that these inhibitors triggered a cascade of aging in CSCs and enhance anti-tumor immunity in mouse cancer and PDX models. Through single-cell sequencing, the remodeling of the tumor immune microenvironment induced by these new Arf1 inhibitors is analyzed and an increase in tumor-associated CD8+ CD4+ double-positive T (DPT) cells is identified. These DPT cells exhibit superior features of active CD8 single-positive T cells and a higher percentage of TCF1+PD-1+, characteristic of stem-like T cells. The frequency of tumor-infiltrating stem-like DPT cells correlates with better disease-free survival (DFS) in cancer patients, indicating that these inhibitors may offer a novel cancer immunotherapy strategy by converting the cold tumor immune microenvironment into a hot one, thus expanding the potential for immunotherapy in cancer patients.

Targeting proliferating cell nuclear antigen enhances ionizing radiation-induced cytotoxicity in prostate cancer cells.

Proliferating cell nuclear antigen (PCNA) is essential for DNA replication and repair, cell growth, and survival. PCNA also enhances androgen receptor (AR) signaling in prostate cancer (PC) cells. We identified a PCNA interaction protein (PIP) box at the N-terminal domain of AR and developed a small peptide PCNA inhibitor R9-AR-PIP containing AR PIP-box. We also identified a series of small molecule PCNA inhibitors (PCNA-Is) that bind directly to PCNA and interrupt PCNA functions. The present study investigated the effects of the PCNA inhibitors on the sensitivity of PC cells to X-ray radiation. The effects of targeting PCNA on radio sensitivity of PC cells were investigated in four lines of castration-resistant PC (CRPC) cells with different AR expression statuses. The cells were treated with the PCNA inhibitors and X-ray radiation alone or in combination. The effects of the treatment on expression of AR target genes, DNA damage response, DNA damage, homologous recombination repair (HRR), and cytotoxicity were evaluated. We found that the androgen response element (ARE) occupancy of the DNA damage response gene PARP1 by AR is significantly attenuated by PCNA-I1S or R9-AR-PIP combined with X-ray radiation, while X-ray radiation alone does not enhance the ARE occupancy. PCNA-I1S or R9-AR-PIP alone significantly inhibits occupancy of the AR-occupied regions (AROR) in PRKDC and XRCC2 genes. R9-AR-PIP and PCNA-I1S inhibit expression of AR-Vs target gene cyclin A2 and show the additive effects with radiation in AR-positive CRPC cells. Targeting PCNA by PCNA-I1S and R9-AR-PIP downregulates expression of DNA damage response genes EXO1, Rad54L, Rad51, and/or PARP1 and shows the additive effects with radiation as compared with their respective controls in AR-positive CRPC LNCaP-AI, 22Rv1, and R1-D567 cells, but not in AR-negative PC-3 cells. R9-AR-PIP and PCNA-I1S elevate the levels of phospho-DNA-PKcs(S2056) and γH2AX, indicating DNA damage in response to radiation in AR-positive cells. The HRR is significantly attenuated by PCNA inhibitors PCNA-I1S, R9-AR-PIP, and T2AA in all four CRPC cells examined, and inhibited by Enzalutamide (Enz) only in 22RV1 cells. The cytotoxicity induced by X-ray radiation in androgen-dependent LNCaP cells is enhanced by Enz and a lower concentration of R9-AR-PIP in the colony formation assay. R9-AR-PIP at higher concentration reduces the colony formation and has an additive effect with X-ray radiation in all AR expressing cells, regardless of AR-FL and AR-Vs, but does not significantly alter the colony formation in AR-negative PC-3 cells. PCNA-I1S attenuates colony formation and has an additive effect with ionizing radiation in all four CRPC cells, regardless of AR expression status. These data provide a strong rationale for the therapy studies using PCNA-I1S or R9-AR-PIP in combination with X-ray radiation against CRPC tumors in preclinical models.

Sulindac exhibits anti-proliferative and anti-invasive effects in uterine serous carcinoma cells

PurposeUterine serous carcinoma (USC) is a highly aggressive and frequently recurring subtype of endometrial cancer with limited treatment options for advanced or recurrent stages. Sulindac, a classic non-steroidal anti-inflammatory drug, has demonstrated anti-tumor activity in several pre-clinical tumor models. This study aims to evaluate the effect of sulindac on cell proliferation and invasion in USC cells.MethodsHuman USC cell lines ARK-1 and SPEC2 were treated with different concentrations of sulindac. Cell proliferation was assessed using MTT and colony formation assays. ELISA assays measured cellular stress, cleaved caspase 3 activity, antioxidant ability, and adhesion. Cell cycle arrest was evaluated by Cellometer. The invasive capability was detected by wound healing assay. Western blotting was used to analyze the changes in protein expression induced by sulindac.ResultsExposure to sulindac decreased cellular viability in a dose-dependent manner in ARK-1 and SPEC2 cells. Sulindac effectively inhibited cell cycle progression, increased cellular stress, caused apoptosis, and reduced cell adhesion and invasion in USC cells. Additionally, sulindac decreased the expression of COX-2 and blocked phosphorylation of NF-κB induced by TNF-α.ConclusionSulindac is a potential therapeutic agent for USC that deserves further exploration in pre-clinical studies and potentially future clinical trials.

Is Autophagy Targeting a Valid Adjuvant Strategy in Conjunction with Tyrosine Kinase Inhibitors?

Tyrosine kinase inhibitors (TKIs) represent a relatively large class of small-molecule inhibitors that compete with ATP for the catalytic binding site of tyrosine kinase proteins. While TKIs have demonstrated effectiveness in the treatment of multiple malignancies, including chronic myelogenous leukemia, gastrointestinal tumors, non-small cell lung cancers, and HER2-overexpressing breast cancers, as is almost always the case with anti-neoplastic agents, the development of resistance often imposes a limit on drug efficacy. One common survival response utilized by tumor cells to ensure their survival in response to different stressors, including anti-neoplastic drugs, is that of autophagy. The autophagic machinery in response to TKIs in multiple tumor models has largely been shown to be cytoprotective in nature, although there are a number of cases where autophagy has demonstrated a cytotoxic function. In this review, we provide an overview of the literature examining the role that autophagy plays in response to TKIs in different preclinical tumor model systems in an effort to determine whether autophagy suppression or modulation could be an effective adjuvant strategy to increase efficiency and/or overcome resistance to TKIs.

Nanoparticles targeting immune checkpoint protein VISTA induce potent antitumor immunity

BackgroundImmune checkpoint protein V-domain immunoglobulin suppressor of T cell activation (VISTA) controls antitumor immunity and is a valuable target for cancer immunotherapy. Previous mechanistic studies have indicated that VISTA impairs...

BP1003 Decreases STAT3 Expression and Its Pro-Tumorigenic Functions in Solid Tumors and the Tumor Microenvironment.

Overexpression and aberrant activation of signal transducer and activator of transcription 3 (STAT3) contribute to tumorigenesis, drug resistance, and tumor-immune evasion, making it a potential cancer therapeutic target. BP1003 is a neutral liposome incorporated with a nuclease-resistant P-ethoxy antisense oligodeoxynucleotide (ASO) targeting the STAT3 mRNA. Its unique design enhances BP1003 stability, cellular uptake, and target affinity. BP1003 efficiently reduces STAT3 expression and enhances the sensitivity of breast cancer cells (HER2+, triple negative) and ovarian cancer cells (late stage, invasive ovarian cancer) to paclitaxel and 5-fluorouracil (5-FU) in both 2D and 3D cell cultures. Similarly, ex vivo and in vivo patient-derived models of pancreatic ductal adenocarcinoma (PDAC) show reduced tissue viability and tumor volume with BP1003 and gemcitabine combination treatments. In addition to directly affecting tumor cells, BP1003 can modulate the tumor microenvironment. Unlike M1 differentiation, monocyte differentiation into anti-inflammatory M2 macrophages is suppressed by BP1003, indicating its potential contribution to immunotherapy. The broad anti-tumor effect of BP1003 in numerous preclinical solid tumor models, such as breast, ovarian, and pancreatic cancer models shown in this work, makes it a promising cancer therapeutic.

OMIP-105: A 30-color full-spectrum flow cytometry panel to characterize the immune cell landscape in spleen and tumor within a syngeneic MC-38 murine colon carcinoma model.

This panel was designed to characterize the immune cell landscape in the mouse tumor microenvironment as well as mouse lymphoid tissues (e.g., spleen). As an example, using the MC-38 mouse syngeneic tumor model, we demonstrated that we could measure the frequency and characterize the functional status of CD4 T cells, CD8 T cells, regulatory T cells, NK cells, B cells, macrophages, granulocytes, monocytes, and dendritic cells. This panel is especially useful for understanding the immune landscape in "cold" preclinical tumor models with very low immune cell infiltration and for investigating how therapeutic treatments may modulate the immune landscape.

40 Preclinical evaluation of a novel dendritic cell vaccine for kidney cancer

Abstract Background Dendritic cell (DC)-based vaccines have been previously shown to promote therapeutic T cell responses in both preclinical tumor models and in cancer patients, where extended patient overall survival has been noted in many cases. The goal of the present study is to develop a novel DC-based vaccine as an effective immuno-oncology (IO) agent for the prevention/treatment of kidney cancer. We previously demonstrated that actin-binding protein profilin1 (Pfn1) is overexpressed in tumor-associated vascular endothelial cells (ECs), where it may serve as a prognostic factor in human clear cell renal cancer (ccRCC). We further established a direct causal relationship between EC Pfn1 dysregulation, immune microenvironment alterations, and tumor progression in mouse models of RCC. The current work specifically explores whether Pfn1-targeted DC-based vaccines are effective in preventing orthotopic RCC in mice. Methods To generate Pfn1-targeted DC vaccines, we harvested and matured DCs from Balb/C mouse bone marrow-derived precursor cells in cultures containing rmIL4 and rmGM-CSF. DCs loaded with individual Pfn1 synthetic peptides (these sequences were identified using three MHC class I/II peptide-binding algorithms) were injected into syngeneic mice on days 0, 7 and 14. Spleens were harvested from immunized mice on day 21 to isolate CD4+ and CD8+ T cells, with T cells then stimulated with unmanipulated DCs or DCs pulsed with individual immunizing peptides to detect specific T cell activation, as measured by IFN-gamma release quantitated by ELISA. Tissue histology was performed to assess immune-related adverse events (iRAE) in vaccinated mice as a safety index. For cancer studies, Balb/c mice were immunized with DCs loaded with pooled Pfn1 peptides a few days prior to establishing either subcutaneous or orthotopic RCC tumors. Tumor-bearing mice were subjected to an identical booster vaccine prior one week later in advance of euthanasia and study end-point analyses. Results Our studies show that vaccination of Balb/c mice with DCs pulsed with Pfn1 peptides elicit specific CD4+ and/or CD8+ T cell responses without promoting irAEs. When applied in the prophylactic setting, DC-Pfn1 peptide vaccines substantially slow the growth of subcutaneous RCC tumors. In support of these findings, we have also developed preliminary data supporting the therapeutic effects of DC/Pfn1-peptide vaccines in a murine orthotopic model of RCC. Conclusions In summary, our studies provide first evidence for Pfn1-targeted IO agent in the cancer setting. Given that the survival benefit for dual immune-check point inhibitors (ICI)/anti-angiogenic therapy remains modest and is limited to a small minority of ccRCC patients, Pfn1-targeted vaccines could represent a novel therapeutic agent for improved patient outcomes when applied alone or in combination with in-clinic ICI agents. DOD CDMRP Funding: yes

IL-2 based cancer immunotherapies: an evolving paradigm.

Discovered over 4 decades ago in the supernatants of activated T cells, interleukin-2 (IL-2) is a potent pleiotropic cytokine involved in the regulation of immune responses. It is required for effector T cell expansion and differentiation as well as for peripheral tolerance induced by regulatory T cells. High-dose IL-2 treatment was the first FDA-approved immunotherapy for renal cell carcinoma and melanoma, achieving single agent complete and durable responses, albeit only in a small proportion of patients. The therapeutic potential of wild type IL-2 is clinically limited by its short half-life and severe vascular toxicity. Moreover, the activation of regulatory T cells and the terminal differentiation of effector T cells on IL-2 pose additional restrictions. To overcome the toxicity of IL-2 in order to realize its full potential for patients, several novel engineering strategies are being developed and IL-2 based immunotherapy for cancer has emerged as a burgeoning field of clinical and experimental research. In addition, combination of IL-2 with PD-1/L1 pathway blockade shows vastly improved anti-tumor efficacy over either monotherapy in preclinical tumor models. In this review we discuss the biological characteristics of IL-2 and its receptors, as well as its efficacy and treatment limiting toxicities in cancer patients. We also explore the efforts aimed at developing novel and safer IL-2 therapies to harness the full therapeutic potential of this cytokine.

Adeno-associated virus delivered CXCL9 sensitizes glioblastoma to anti-PD-1 immune checkpoint blockade

There are numerous mechanisms by which glioblastoma cells evade immunological detection, underscoring the need for strategic combinatorial treatments to achieve appreciable therapeutic effects. However, developing combination therapies is difficult due to dose-limiting toxicities, blood-brain-barrier, and suppressive tumor microenvironment. Glioblastoma is notoriously devoid of lymphocytes driven in part by a paucity of lymphocyte trafficking factors necessary to prompt their recruitment and activation. Herein, we develop a recombinant adeno-associated virus (AAV) gene therapy that enables focal and stable reconstitution of the tumor microenvironment with C-X-C motif ligand 9 (CXCL9), a powerful call-and-receive chemokine for lymphocytes. By manipulating local chemokine directional guidance, AAV-CXCL9 increases tumor infiltration by cytotoxic lymphocytes, sensitizing glioblastoma to anti-PD-1 immune checkpoint blockade in female preclinical tumor models. These effects are accompanied by immunologic signatures evocative of an inflamed tumor microenvironment. These findings support AAV gene therapy as an adjuvant for reconditioning glioblastoma immunogenicity given its safety profile, tropism, modularity, and off-the-shelf capability.

Open-Label, Multicenter, Phase I/II, First-in-Human Trial of TK216: A First-Generation EWS::FLI1 Fusion Protein Antagonist in Ewing Sarcoma.

Ewing Sarcoma (ES), a rare cancer with a pathognomonic translocation resulting in the Ewing sarcoma gene (EWS)::FLI1 oncoprotein, has a poor prognosis in the relapsed/refractory (R/R) setting. Tokalas (TK)216 was designed to bind EWS::FLI1 proteins directly, disrupt protein-protein interactions, and inhibit transcription factor function. TK216 plus vincristine showed synergistic activity in preclinical tumor models. To our knowledge, we report the results of a first-in-class, first-in-human phase I/II trial of TK216 in R/R ES. TK216 was administered intravenously as a continuous infusion to patients with R/R ES in 11 cohorts. The dosing duration of 7 days was later extended to 10, 14, and 28 days. Vincristine could be added on day 1 after cycle 2, per investigators' choice. The trial used a 3 + 3 design with an expansion cohort at the recommended phase II dose (RP2D). A total of 85 patients with a median age of 27 years (range, 11-77) were enrolled. The maximum tolerated dose for the 14-day infusion of TK216, 200 mg/m2 once daily, was determined in cohort 9 and selected as the RP2D. The median previous number of systemic therapies regimens was three (range, 1-10). The most frequent-related adverse events in patients treated at the RP2D included neutropenia (44.7%), anemia (29.4%), leukopenia (29.4%), febrile neutropenia (15.3%), thrombocytopenia (11.8%), and infections (17.6%). In cohorts 9 and 10, two patients had a complete response, one had a partial response, and 14 had stable disease; the 6-month progression-free survival was 11.9%. There were no responses among the eight patients in cohort 11. TK216 administered as 14-day continuous infusion with or without vincristine was well tolerated and showed limited activity at the RP2D in R/R ES.