Significant Tumor Growth Delay Research Articles

Matrix Metalloproteinase- and pH-Sensitive Nanoparticle System Enhances Drug Retention and Penetration in Glioblastoma.

Glioblastoma (GBM) is a primary malignant brain tumor with limited therapeutic options. One promising approach is local drug delivery, but the efficacy is hindered by limited diffusion and retention. To address this, we synthesized and developed a dual-sensitive nanoparticle (Dual-NP) system, formed between a dendrimer and dextran NPs, bound by a dual-sensitive [matrix metalloproteinase (MMP) and pH] linker designed to disassemble rapidly in the tumor microenvironment. The disassembly prompts the in situ formation of nanogels via a Schiff base reaction, prolonging Dual-NP retention and releasing small doxorubicin (Dox)-conjugated dendrimer NPs over time. The Dual-NPs were able to penetrate deep into 3D spheroid models and detected at the tumor site up to 6 days after a single intratumoral injection in an orthotopic mouse model of GBM. The prolonged presence of Dual-NPs in the tumor tissue resulted in a significant delay in tumor growth and an overall increase in survival compared to untreated or Dox-conjugated dendrimer NPs alone. This Dual-NP system has the potential to deliver a range of therapeutics for efficiently treating GBM and other solid tumors.

Exploiting Metabolic Defects in Glioma with Nanoparticle Encapsulated NAMPT Inhibitors.

The treatment of primary central nervous system (CNS) tumors is challenging due to the blood-brain barrier and complex mutational profiles, which is associated with low survival rates. However, recent studies have identified common mutations in gliomas (IDH-WT and mutant, WHO grades II-IV; with grade IV tumors referred to as glioblastomas; GBMs). These mutations drive epigenetic changes, leading to promoter methylation at the NAPRT gene locus, which encodes an enzyme involved in generating NAD+. Importantly, NAPRT-silencing introduces a therapeutic vulnerability to inhibitors targeting another NAD+ biogenesis enzyme, NAMPT, rationalizing a treatment for these malignancies. Multiple systemically-administered NAMPTis have been developed and tested in clinical trials, but dose-limiting toxicities-including bone marrow suppression and retinal toxicity-have limited their efficacy. Here, we report a novel approach for the treatment of NAPRT-silenced GBMs using nanoparticle-encapsulated (NP) NAMPT inhibitors (NAMPTis) administered by convection-enhanced delivery (CED). We demonstrate that GMX1778 (a NAMPTi) can be formulated in degradable polymer NPs with retention of potency for NAMPT inhibition and anticancer activity in vitro, plus sustained drug release in vitro and in vivo. Direct injection of these drugs via CED into the brain is associated with reduced retinal toxicity compared with systemic administration. Finally, we show that CED of NP-encapsulated GMX1778 to NAPRT-silenced intracranial GBM xenografts in mice exhibit significant tumor growth delay and extends survival. These data support an approach to treat gliomas harboring defects in NAD+ metabolism using CED of NP-encapsulated NAMPTis to greatly improve the therapeutic index and treatment efficacy for this class of drugs.

Efficacy of LCMV-based cancer immunotherapies is unleashed by intratumoral injections of polyI:C

BackgroundLymphocytic choriomeningitis virus (LCMV) belongs to the Arenavirus family known for inducing strong cytotoxic T-cell responses in both mice and humans. LCMV has been engineered for the development of cancer...

Abstract 6034: PSMA targeted alpha radiopharmaceutical [211At]PSAt-3 inhibits tumor growth in a preclinical model of prostate cancer

Abstract Introduction: Prostate cancer continues to be a highly prevalent cancer diagnosis among men and a leading cause of cancer related deaths worldwide. With the introduction[177Lu]Lu-PSMA-617, targeted radionuclide therapy has proven successful in increasing survival time for patients with metastasized castration resistant prostate cancer. However, despite encouraging results, such patients tend to develop progressive disease, and thus, enhanced efficacy is warranted. To this end, the use of radiopharmaceuticals harnessing alpha-emitters is a promising venue due to the potent energy deposition and short range of alpha particles in tissue. Here, we employ the PSMA targeting vector, [211At]PSAt-3, for targeted alpha radionuclide therapy in a preclinical prostate cancer model. Methods: [211At]PSAt-3, a structural derivative of PSMA-617, in which the L-2-naphtylalanine was replaced by L-3-[211At]astato-phenylalanine, was obtained by electrophilic [211At]astatodesilylation. Evaluation of [211At]PSAt-3 was conducted in male inbred nude mice of Balb/c background xenografted subcutaneously with human prostate cancer cells (LNCaP). Prior biodistribution studies were carried out with approximately 100 kBq intravenous injections, and subsequent biodistribution 2, 6 and 24h post injection (P.I.). For establishment of therapeutic effect, a single intravenous dose of [211At]PSAt-3 was administered. To asses hematological toxicity, blood cell composition was assessed weekly. Results: Favorable tumor uptake of 15.2±6 %ID/g was observed 2h after injection of [211At]PSAt-3 in LNCaP xenografted nude mice, with subsequent 9±5.1% ID/g and 6.4±2.4% ID/g 6 and 24h P.I., respectively. [211At]PSAt-3 cleared mainly by the kidneys, in which uptake was 72, 38 and 6% ID/g at 2, 6 and 24h P.I., respectively. Thyroid/throat uptake remained low at ~0.1% ID for all timepoints investigated, indicating a low degree of de-astatination. Initial efficacy studies using a single intravenous dose of 0.5 MBq (~20 MBq/kg) demonstrated significant growth delay and tumor volume reduction compared with control mice (saline). Blood platelet count was reduced after administration of [211At]PSAt-3 and subsequently recovered. No signs of severe toxicity, and no significant weight loss between control and treated mice was observed, suggesting that the treatment is well tolerated. Conclusion: 0.5 MBq [211At]PSAt-3 was well tolerated in mice and demonstrated tumor volume reduction and significant delay of tumor growth. These findings suggest [211At]PSAt-3 as a promising candidate for clinical translation. Citation Format: Lars Hvass, Marius Müller, Anne Clausen, Matthias Herth, Andreas Kjaer. PSMA targeted alpha radiopharmaceutical [211At]PSAt-3 inhibits tumor growth in a preclinical model of prostate cancer [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 6034.

Abstract 1353: Potentiating salvage radiotherapy in radiorecurrent prostate cancer through anti-CTLA4 therapy: Implications from a syngeneic model

Abstract Background: Advanced prostate cancer (PCa) is a leading cause in cancer death and can elicit significant morbidity and mortality. A common treatment modality for advanced PCa is radiation therapy (RT). Currently, salvage of local disease recurrence after RT is a major clinical problem. Emerging evidence indicates the importance of the immune system in governing RT response. In support of this, immune checkpoint inhibitors (ICIs), which enhance immune activation, have demonstrated clinical therapeutic promise in combination with RT in certain advanced cancers. Irradiation (IR) combined with ICIs may prime the immune system to recognize and target recurrent cancer. Purpose: Investigate the therapeutic efficacy of ICIs in combination with RT for radiorecurrent PCa in a syngeneic pre-clinical model. Methods: TRAMP-C2 cells were treated with 10 Gy of radiation over 5 fractions (similar to clinical hypofractionated (HF) schedule) to generate TRAMP-C2 HF cells. Immune-competent mice were transplanted subcutaneously with TRAMP-C2 HF cells. Once tumors reached palpable size, mice were administered either ICIs (anti-PDL1 or anti-CTLA4) alone or in combination with RT. Tumor volume was monitored to determine the treatment effect. Correlative studies on excised tumors and secondary lymphoid organs included flow cytometry and NanoString gene expression panel to evaluate immunological mechanisms that contribute to anti-tumor effects. Results: TRAMP-C2 HF cells were validated for radiation resistance and exhibited a more aggressive phenotype (reduction in senescence, increase in clonogenicity) similar to clinically recurrent PCa. Radiation resistance of TRAMP-C2 HF tumors was validated in vivo. Administration of anti-PDL1 and anti-CTLA4 as monotherapy or in combination did not achieve significant tumor growth delay compared to control. IR alone produced an observable tumor growth delay compared to ICIs alone. The combination of anti-PDL1 and IR did not yield additional growth delay compared to IR alone. Strikingly, significant tumor growth delay was seen with the combination of IR and anti-CTLA4 compared to IR alone, while also resulting in complete cure in a third of the mice. mice. Lymph nodes and tumors from mice treated with IR and anti-CTLA4 vs. IR and isotype control demonstrated differential expression of genes in T cell functions and enrichment in both CD4+ and CD8+ T cell populations. Conclusion: We generated the first syngeneic radiorecurrent PCa model and demonstrated that combining anti-CTLA4 and IR results in synergistic tumor response. Combined therapy resulted in augmented immune response, most notably enhancement of CD8 T cell activity. Significance: These findings contribute to our understanding of immunological events associated with RT and ICIs in the context of radiorecurrent PCa and support new avenues for salvage therapy in clinical trials. Citation Format: Hanzhi Wang, Linsey Gong, Xiaoyong Huang, Stephanie D. White, Hanz T. Chung, Danny Vesprini, Tera N. Petchiny, Emmanouil Fokas, Hansen He, Robert S. Kerbel, Stanley K. Liu. Potentiating salvage radiotherapy in radiorecurrent prostate cancer through anti-CTLA4 therapy: Implications from a syngeneic model [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 1353.

Abstract 6021: Lutetium-177 (177Lu) radiolabeled engineered antibody fragment against human prostate stem cell antigen (hPSCA) demonstrates targeting and antitumor effects in a syngeneic mouse model of pancreatic ductal adenocarcinoma (PDAC)

Abstract Introduction: hPSCA is a cell surface protein overexpressed in PDAC but minimally expressed in normal tissues, making it a promising therapeutic marker for targeted radiopharmaceutical therapy (TRT). Anti-hPSCA A2 scFv-Fc2DM antibody (A2DM) was engineered with a double mutation in the FcRn binding site for rapid blood clearance to the liver, reducing bone marrow and kidney radiotoxicity. The potential of using 177Lu radiolabeled A2DM antibody for targeted anti-hPSCA imaging and TRT was investigated in a syngeneic mouse model of PDAC. Methods: hPSCA knock-in mice were engrafted subcutaneously with KPC cells overexpressing hPSCA (KPC-hPSCA). ImmunoPET with zirconium-89 (89Zr) radiolabeled with A2DM was conducted to confirm in vivo targeting. For imaging, A2DM and an isotype control (anti-CD20) antibody were radiolabeled with 89Zr. Mice (n = 5; tumor sizes: 150 mm3 - 180 mm3) were i.v. injected with 89Zr-A2DM or 89Zr-isotype control (administered activity: 1.5 MBq/10 µg) or co-injected with excess cold antibody. ImmunoPET was acquired at 21 h post-injection. Ex vivo biodistribution was conducted and %ID/g was calculated based on decay-corrected standards. For therapy, KPC-hPSCA tumor bearing mice (n = 8 - 9, average tumor sizes: 20.1 ± 5.1 mm3) was i.v. injected with 177Lu-A2DM (administered activity: 17.8 MBq/10 µg or 9.25 MBq/10 µg) or saline. Tumor sizes and body weights measured every 2-3 days. Results: 89Zr immunoPET and biodistribution of A2DM demonstrated significantly higher tumor uptake (15.6 ± 0.6 %ID/g) than theisotype control (10.7 ± 1.0 %ID/g, p= 0.0001) or blocked control with 70-fold excess cold antibody (8.6 ± 1.3 %ID/g, p< 0.0001), suggesting A2DM binds to hPSCA-positive tumors in an antigen-specific manner. Therapy studies show tumor growth inhibition in a dose-dependent manner when treated with 17.8 MBq of 177Lu-A2DM (tumor size at day 25: 178 ± 119 mm3) or 9.25 MBq of 177Lu-A2DM (tumor size at day 25: 309 ± 182 mm3) (tumor size at day 25: 740 ± 317 mm3, p= 0.006, 0.04 respectively, compared to saline). Median survival was significantly improved in mice treated with high dose of 17.8 MBq 177Lu-A2DM compared to saline-treated group (48 vs. 25 days, p= 0.002). Median survival of mice treated with low dose 9.25 MBq of 177Lu-A2DM showed a trend towards improved survival but results were not significant (48 vs. 34 days, p= 0.25). Minimal weight loss observed in the 177Lu-A2DM and saline groups, suggesting the 177Lu therapy was well tolerated. Conclusions: 89Zr immunoPET studies confirmed specific targeting of A2DM to KPC-hPSCA tumors in vivo. 177Lu-A2DM therapeutic studies demonstrated significant tumor growth delay in a dose-dependent manner. Future studies will explore dose fractionation strategies, with the aim to enhance tumor growth control for PDAC. Citation Format: Bao Ying Chen, Masoud Farshbaf, Felix B. Salazar, Saad N. Ahmed, Sunidhi Jaiswal, Jennifer Chean, Anakim Sherman, Teresa Hong, Paul J. Yazaki, Tove Olafsen, Kirstin A. Zettlitz, Anna M. Wu. Lutetium-177 (177Lu) radiolabeled engineered antibody fragment against human prostate stem cell antigen (hPSCA) demonstrates targeting and antitumor effects in a syngeneic mouse model of pancreatic ductal adenocarcinoma (PDAC) [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 6021.

Abstract 2656: The selective IKZF2 molecular glue degrader, PVTX-405, counters Treg immune suppression,shows significant tumor growth delay as single agent and synergistic response with immune checkpoint therapies (ICTs)

Abstract Immunosuppressive regulatory T-cells (Tregs) are key modulators of tumor immune evasion and resistance to immune checkpoint therapies (ICTs). IKZF2 (Helios) is a transcription factor that is selectively expressed by Treg and has been shown to be essential for maintaining function and stability of Tregs in the face of inflammatory conditions that include autoimmune disease as well as cancer. Here we describe the discovery of PVTX-405, a potent and selective IKZF2 molecular glue degrader (DC50=6.3 nM). Our molecular glue degrader promotes a productive ternary complex with the E3 ubiquitin ligase substrate receptor cereblon (CRBN) to selectively degrade IKZF2 while sparing other CRBN neosubstrates (IKZF1/3, GSPT1, CK1a). PVTX-405 demonstrates rapid and selective degradation of IKZF2 in Jurkat cells in vitro, human PBMC derived Tregs ex vivo, and in cynomolgus monkeys in vivo. Degradation of IKZF2 in Jurkat cells leads to dose dependent increase in production of inflammatory cytokine IL-2. In ex vivo co-cultures of Tregs and effector T-cells, PVTX-405 reduces the suppressive activity of Tregs. To determine whether the relief of Treg suppressive activity observed in vitro and ex vivo can translate to anti-tumor activity in vivo we utilized CRBNI391V(humanized CRBN) mice. We developed a novel MC38 syngeneic tumor model to test both monotherapy and combination efficacy in an immune-competent setting. Once daily oral administration of PVTX-405 as single agent significantly delays the growth of MC38 tumors. Further, in vivo treatment of PVTX-405 in combination with ICTs, anti-PD1 or anti-LAG3, significantly increased animal survival and led to durable tumor regressions compared to ICTs alone. Together, these results demonstrate a strong synergy between ICTs and Treg destabilization through pharmacological degradation of IKZF2 and represent a promising combination strategy for treatment of cancer. Citation Format: Harshil D. Dhruv, Donna McEachern, Longchuan Bai, Xuqing Zhang, Zhixiang Chen, Rakesh Nagilla, Elham Behshad, Bomie Han, Peter Orth, Rohan Rej, Paul Kirchhoff, Niu Shin, Larry Jolivette, Corey Strickland, Zhihua Sui, Scott Priestley, Shaomeng Wang, Helai Mohammad. The selective IKZF2 molecular glue degrader, PVTX-405, counters Treg immune suppression,shows significant tumor growth delay as single agent and synergistic response with immune checkpoint therapies (ICTs) [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 2656.

PROTAC-Mediated Dual Degradation of BCL-xL and BCL-2 Is a Highly Effective Therapeutic Strategy in Small-Cell Lung Cancer.

BCL-xL and BCL-2 are validated therapeutic targets in small-cell lung cancer (SCLC). Targeting these proteins with navitoclax (formerly ABT263, a dual BCL-xL/2 inhibitor) induces dose-limiting thrombocytopenia through on-target BCL-xL inhibition in platelets. Therefore, platelet toxicity poses a barrier in advancing the clinical translation of navitoclax. We have developed a strategy to selectively target BCL-xL in tumors, while sparing platelets, by utilizing proteolysis-targeting chimeras (PROTACs) that hijack the cellular ubiquitin proteasome system for target ubiquitination and subsequent degradation. In our previous study, the first-in-class BCL-xL PROTAC, called DT2216, was shown to have synergistic antitumor activities when combined with venetoclax (formerly ABT199, BCL-2-selective inhibitor) in a BCL-xL/2 co-dependent SCLC cell line, NCI-H146 (hereafter referred to as H146), in vitro and in a xenograft model. Guided by these findings, we evaluated our newly developed BCL-xL/2 dual degrader, called 753b, in three BCL-xL/2 co-dependent SCLC cell lines and the H146 xenograft models. 753b was found to degrade both BCL-xL and BCL-2 in these cell lines. Importantly, it was considerably more potent than DT2216, navitoclax, or DT2216 + venetoclax in reducing the viability of BCL-xL/2 co-dependent SCLC cell lines in cell culture. In vivo, 5 mg/kg weekly dosing of 753b was found to lead to significant tumor growth delay, similar to the DT2216 + venetoclax combination in H146 xenografts, by degrading both BCL-xL and BCL-2. Additionally, 753b administration at 5 mg/kg every four days induced tumor regressions. At this dosage, 753b was well tolerated in mice, without observable induction of severe thrombocytopenia as seen with navitoclax, and no evidence of significant changes in mouse body weights. These results suggest that the BCL-xL/2 dual degrader could be an effective and safe therapeutic for a subset of SCLC patients, warranting clinical trials in future.

Anti-OX40 Antibody Combined with HBc VLPs Delays Tumor Growth in a Mouse Colon Cancer Model

ObjectiveCombination immunotherapy strategies targeting OX40, a co-stimulatory molecule that can enhance antitumor immunity by modulating the proliferation, differentiation, and effector function of tumor-infiltrating T cells, have attracted much attention for their excellent therapeutic effects. In this study, we aimed to evaluate the antitumor efficacy of combined anti-OX40 and hepatitis B core virus-like particles (HBc VLPs) therapy using a mouse colon cancer model. MethodsHumanized B-hOX40 mice were injected subcutaneously with MC38 colon tumor cells and treated with HBc VLPs+anti-hOX40 antibody. Tumor growth was monitored. Flow cytometric analysis was performed to evaluate the populations of T cell subsets in the tumors. ResultsThe combination of anti-OX40 with HBc VLPs resulted in a significant delay in tumor growth, suggesting that a potent antitumor immunity was induced by the combination therapy. Further studies revealed that HBc VLPs+anti-OX40 treatment induced a significant increase in effector T cells (Teffs) and a significant decrease in regulatory T cells (Tregs) in the tumor microenvironment (TME), which accounted for the synergistic antitumor effect of anti-OX40 in combination with HBc VLPs. ConclusionCombination therapy of anti-hOX40 and HBc VLPs provides synergistic antitumor activity in colon cancer-bearing mice, which may represent a potential design strategy for cancer immunotherapy.

Abstract C174: MerTK as a putative biomarker for treatment response in triple negative breast cancer

Abstract Triple Negative Breast Cancer (TNBC) is characterized by the lack of estrogen, progesterone, and HER2 receptors. TNBC is a highly invasive subtype of breast cancer, with limited targeted therapies. Current therapeutic options for TNBC patients include surgery, chemotherapy, and radiation but disease recurrence is common. Anti-PD-1 and anti-PD-L1 therapeutic antibodies have been approved for the treatment of TNBC in combination with chemotherapy, but biomarkers for response and new therapeutic targets are still needed. The receptor tyrosine kinase MerTK has been associated with many types of cancer and is highly expressed in TNBC. To investigate the role of MerTK in TNBC, we created a syngeneic cell line (4T1) overexpressing MerTK to evaluate tumor growth, the tumor immune microenvironment (TIME), and response to immune checkpoint inhibitors (ICIs) in vivo. First, we tested the effect of MerTK on tumor growth in vivo by inoculating the 4T1 cell line, which has very low MerTK expression, and two 4T1 MerTK-overexpressing clones into eight Balb/c mice per group subcutaneously on the flank. We measured tumor growth over 27 days and found that MerTK-overexpressing tumors exhibited a statistically significant delay in tumor growth when compared to the parental 4T1 tumors. This indicates that MerTK may be inducing a hotter TIME in MerTK overexpressing tumors, as this slowed growth was not observed in athymic nude mice. To test this hypothesis, we evaluated the TIME of these tumors by flow cytometry, which revealed robust expansion of macrophage, natural killer cell and T cell compartments, and reduction in tumor-infiltrating myeloid derived suppressor cells. These results suggest that MerTK is playing a yet unidentified role in promoting infiltration of anti-tumor immune cells into the TIME and excluding pro-tumor immune cells. Further, we saw robust changes in genes associated with cytokine signaling pathways including CD38, CCL2 and CCL5 by Nanostring nCounter analysis in MerTK-overexpressing 4T1 cells compared to parental 4T1 cells. Finally, there is evidence that hotter tumors respond well to ICI therapy, and we hypothesized that MerTK-overexpressing tumors would exhibit a significant response to ICI when compared to parental 4T1 tumors. Indeed, our data showed a robust and durable response marked by a significant reduction in tumor growth in the MerTK-overexpressing tumors compared to parental 4T1 tumors in Balb/c mice when treated with either anti-PD-L1 or anti-CTLA-4 antibodies. Together, these results suggest that MerTK overexpression could serve as a predictive biomarker for response to immune therapy in patients with TNBC. Citation Format: Bridget E Mehall, Mari Iida, Kourtney L Kostecki, Jillian M Adams, Deric L Wheeler. MerTK as a putative biomarker for treatment response in triple negative breast cancer [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr C174.

Abstract A025: Combination of anti-CTLA4 and radiation induce synergistic tumor control in radiorecurrent prostate cancer

Abstract Background: Advanced prostate cancer (PCa) is a leading cause in cancer death and can elicit significant morbidity and mortality. A common treatment modality for advanced PCa is radiation therapy (RT). Currently, salvage of local disease recurrence after RT is a major clinical problem. Emerging evidence indicates the importance of the immune system in governing RT response. In support of this, immune checkpoint inhibitors (ICIs), which enhance immune activation, have demonstrated clinical therapeutic promise in combination with RT in certain advanced cancers. Irradiation (IR) combined with ICIs may prime the immune system to recognize and target recurrent cancer. Purpose: Investigate the therapeutic efficacy of ICIs in combination with RT for radiorecurrent PCa in a syngeneic pre-clinical model. Methods: TRAMP-C2 cells were treated with 10 Gy of radiation over 5 fractions (similar to clinical hypofractionated (HF) schedule) to generate TRAMP-C2 HF cells. Immune-competent mice were transplanted subcutaneously with TRAMP-C2 HF cells. Once tumors reached palpable size, mice were administered either ICIs (αPDL1 or αCTLA4) alone or in combination with RT. Tumor volume was monitored to determine the treatment effect. Correlative studies on excised tumors and secondary lymphoid organs included flow cytometry and NanoString gene expression panel to evaluate immunological mechanisms that contribute to anti-tumor effects[SL1]. Results: TRAMP-C2 HF cells were validated for radiation resistance and exhibited a more aggressive phenotype (reduction in senescence, increase in clonogenicity) similar to clinically recurrent PCa. Radiation resistance of TRAMP-C2 HF tumors was validated in vivo. Administration of αPDL1 and αCTLA4 as monotherapy or in combination did not achieve significant tumor growth delay compared to control. IR alone produced an observable tumor growth delay compared to ICIs alone. The combination of αPDL1 and IR did not yield additional growth delay compared to IR alone. Strikingly, significant tumor growth delay was seen with the combination of IR and αCTLA4 compared to IR alone, while also resulting in complete cure in a third of the mice. NanoString analysis of lymph nodes from mice treated with αCTLA4 and IR vs. IR alone demonstrated differential expression of genes regulating CD molecules, interleukins, and T-cell functions. Furthermore, enrichment of CD45 and CD8a RNA following αCTLA4 and IR was observed. Validation by flow cytometry is currently ongoing. Conclusion: We generated the first syngeneic radiorecurrent PCa model and demonstrated that combining αCTLA4 and IR results in synergistic tumor response. Combined therapy resulted in augmented immune response, most notably enhancement of CD8 T cell activity. Significance: These findings contribute to our understanding of immunological events associated with RT and ICIs in the context of radiorecurrent PCa and support new avenues for salvage therapy in clinical trials. Citation Format: Hanzhi Wang, Xiaoyong Huang, Stepahnie D White, Linsey Gong, Tera N Petchiny, Hans Chung, Emmanouil Fokas, Robert S Kerbel, Stanley K Liu. Combination of anti-CTLA4 and radiation induce synergistic tumor control in radiorecurrent prostate cancer [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Tumor Immunology and Immunotherapy; 2023 Oct 1-4; Toronto, Ontario, Canada. Philadelphia (PA): AACR; Cancer Immunol Res 2023;11(12 Suppl):Abstract nr A025.

Potentiating Local and Abscopal Antitumor Efficacy through Radiation with FAP-CD40 DARPin and Anti-PD1 Therapy

Although the advent of Immune Checkpoint Inhibitors (ICI) has changed the facet of oncology, about 70% of patients develop resistance. CD40 is a potent costimulatory molecule that drives dendritic cells (DCs) and tumor-associated macrophages (TAMs) to prime T-cells. Clinically, agonistic CD40 antibodies have demonstrated antitumor activity, but dose-limiting toxicities have impaired efficacy. Radiation (RT), especially at higher doses, activates DCs and TAMs, but also upregulates Fibroblast Activation Protein (FAP) in the tumor microenvironment (TME). The upregulated FAP expression can be harnessed for the design of targeted drug delivery to the TME. The FAP-CD40 DARPin is a molecule that utilizes FAP and CD40 binding domains to selectively cross-link and activate CD40 in the tumor, thus avoiding systemic CD40 activation. This study aimed to evaluate if RT combined with a murine FAP-CD40 (mFAP-CD40) and ICI could improve survival and local and abscopal responses in a pre-clinical lung adenocarcinoma model. Human FAPxCD40 (MP0317) is being evaluated in Phase I trials. The 344SQ-P tumor cells were bilaterally injected into the right and left hind legs of 129Sv/Ev mice to establish primary and secondary tumors respectively. When primary tumors reached ∼7mm in diameter, they were irradiated with 12 Gy x 3 fractions, while secondary tumors were monitored. mFAP-CD40 (5mg/kg) was injected intraperitoneally at 2, 7, and 11 days after the last fraction of RT. A backbone of α-PD1 ICI was given twice per week starting with RT for 5 shots total. Mice were euthanized when tumors reached 14mm in diameter. Lungs were collected at experimental endpoints, fixed, and enumerated for metastases. For FAP detection in the TME, we first conducted an IHC analysis on 344SQ-P tumors harvested 11 days post RT. Although 5 Gy x 3 was superior to 12 Gy x 3 in upregulating FAP expression compared to unirradiated controls (P = 0.0088), we chose the 12 Gy x 3 dose for subsequent experiments due to its superiority in releasing tumor antigens, priming T-cells, and promoting abscopal responses. In our bilaterally established murine groups, we have recorded the following median survival days: Control = 23; RT = 26; RT+α-PD1 = 33; mFAP-CD40+α-PD1 = 23; and RT+mFAP-CD40+α-PD1 (Triple therapy) = 44 days. These results were further buttressed by the difference in average tumor growth observed between groups; whereby there was a significant delay in tumor growth of both primary (P<0.0001) and secondary tumors (P<0.0001) of the Triple therapy group compared to either RT+α-PD1 or mFAP-CD40+α-PD1 dual therapies. In addition, the Triple therapy cohort had a significantly lower count of lung metastases vs. control (P = 0.0007) and vs. mFAP-CD40+α-PD1 (P = 0.0246) cohorts. mFAP-CD40 DARPin with RT and α-PD1 proved efficacious to control primary and secondary tumors in a murine lung carcinoma model with no detected toxicities.

Investigation of Hydrogen Peroxide for 45MV 5-Aminolevulinic Acid-Mediated Radiodynamic Therapy

Cytotoxicity caused by the reactive oxygen species (ROS), such as singlet oxygen species (1O2), superoxide radical (O2-), and hydroxyl radical (HO), is a mechanism for treating cancer cells in radiation therapy. 5-aminolevulinic acid (5-ALA)-mediated radiodynamic therapy (RDT) is more effective in killing tumor cells than conventional radiation therapy. ROS is produced not only by ionizing radiation but also by Cherenkov light-activated protoporphyrin IX (PpIX), which is metabolized endogenously from 5-ALA. Moreover, PpIX also catalyzes hydrogen peroxide to generate 1O2, and an enhanced catalytic yield of 1O2 was observed in X-ray irradiation in vitro. Therefore, using an in-vivo mouse model, this study aimed to investigate the effect of hydrogen peroxide as a coenzyme catalyst on a novel 45MV 5-ALA-mediated RDT. A subcutaneous C57BL/6 mouse model of KP1 cell line was used. The tumors (n = 240) were randomized into six groups, consisting of untreated, conventional radiation treatment (RT), and RDT with or without hydrogen peroxide: 1. control (untreated), 2. hydrogen peroxide, 3. 45MV RT, 4. 45MV RT + hydrogen peroxide, 5. 45MV RDT, 6. 45MV RDT + hydrogen peroxide. For 45MV photon irradiation, a single fraction of 4 Gy was delivered to the tumors. 5-ALA was systemically injected at 100 mg/kg by tail-vein 4 hours before the treatment for endogenous PpIX accumulation in the tumor. Carbamide peroxide was used to deliver hydrogen peroxide to tissue and was administered at 60 mg/kg intratumorally into tumors ∼3-5 min before the treatment. The treatment effect of a single fraction of treatment was measured by calculating tumor growth, measured using a 1.5 T MR scanner on the day of treatment (prior to the treatment), 3 and 7 days post-treatment. Two-way repeated ANOVA with Bonferroni correction was used to compare each treatment group to determine the statistically significant difference in tumor growth. A total of 45MV RDT with hydrogen peroxide was shown to significantly delay the tumor growth for the mouse model and cell line investigated in this work. 45MV RDT with hydrogen peroxide group resulted in a decrease in tumor growth by 51.3 ± 4.1 % and 56.1 ± 5.1 % compared to the control group on 3 and 7 days post-treatment, respectively (P<0.001), and 43.4 ± 0.8 % and 50.9 ± 0.8 % compared to 45MV RT alone on 3 and 7 days post-treatment, respectively. Moreover, the enhancement effect of hydrogen peroxide on 45MV RDT was 2.2-4.7 times greater on 45MV RT alone (P<0.05). Hydrogen peroxide did not contribute to tumor growth when administered alone. A total of 45MV 5-ALA-mediated RDT with hydrogen peroxide resulted in the most significant tumor growth delay compared to the other groups. The catalytic effect of PpIX and hydrogen peroxide was observed in-vivo. These preliminary results demonstrate an effective cancer treatment modality.

Identification of a miRNA multi-targeting therapeutic strategy in glioblastoma

Glioblastoma (GBM) is a deadly and the most common primary brain tumor in adults. Due to their regulation of a high number of mRNA transcripts, microRNAs (miRNAs) are key molecules in the control of biological processes and are thereby promising therapeutic targets for GBM patients. In this regard, we recently reported miRNAs as strong modulators of GBM aggressiveness. Here, using an integrative and comprehensive analysis of the TCGA database and the transcriptome of GBM biopsies, we identified three critical and clinically relevant miRNAs for GBM, miR-17-3p, miR-222, and miR-340. In addition, we showed that the combinatorial modulation of three of these miRNAs efficiently inhibited several biological processes in patient-derived GBM cells of all these three GBM subtypes (Mesenchymal, Proneural, Classical), induced cell death, and delayed tumor growth in a mouse tumor model. Finally, in a doxycycline-inducible model, we observed a significant inhibition of GBM stem cell viability and a significant delay of orthotopic tumor growth. Collectively, our results reveal, for the first time, the potential of miR-17-3p, miR-222 and miR-340 multi-targeting as a promising therapeutic strategy for GBM patients.

β2-AR inhibition enhances EGFR antibody efficacy hampering the oxidative stress response machinery

The β2-Adrenergic receptor (β2-ARs) is a cell membrane-spanning G protein-coupled receptors (GPCRs) physiologically involved in stress-related response. In many cancers, the β2-ARs signaling drives the tumor development and transformation, also promoting the resistance to the treatments. In HNSCC cell lines, the β2-AR selective inhibition synergistically amplifies the cytotoxic effect of the MEK 1/2 by affecting the p38/NF-kB oncogenic pathway and contemporary reducing the NRF-2 mediated antioxidant cell response. In this study, we aimed to validate the anti-tumor effect of β2-AR blockade and the synergism with MEK/ERK and EGFR pathway inhibition in a pre-clinical orthotopic mouse model of HNSCC. Interestingly, we found a strong β2-ARs expression in the tumors that were significantly reduced after prolonged treatment with β2-Ars inhibitor (ICI) and EGFR mAb Cetuximab (CTX) in combination. The β2-ARs down-regulation correlated in mice with a significant tumor growth delay, together with the MAPK signaling switch-off caused by the blockade of the MEK/ERK phosphorylation. We also demonstrated that the administration of ICI and CTX in combination unbalanced the cell ROS homeostasis by blocking the NRF-2 nuclear translocation with the relative down-regulation of the antioxidant enzyme expression. Our findings highlighted for the first time, in a pre-clinical in vivo model, the efficacy of the β2-ARs inhibition in the treatment of the HNSCC, remarkably in combination with CTX, which is the standard of care for unresectable HNSCC.

Multi-Omics Identification of Genetic Alterations in Head and Neck Squamous Cell Carcinoma and Therapeutic Efficacy of HNC018 as a Novel Multi-Target Agent for c-MET/STAT3/AKT Signaling Axis.

Amongst the most prevalent malignancies worldwide, head and neck squamous cell carcinoma (HNSCC) is characterized by high morbidity and mortality. The failure of standard treatment modalities, such as surgery, radiotherapy, and chemotherapy, demands the need for in-depth understanding of the complex signaling networks involved in the development of treatment resistance. A tumor's invasive growth and high levels of intrinsic or acquired treatment resistance are the primary causes of treatment failure. This may be a result of the presence of HNSCC's cancer stem cells, which are known to have self-renewing capabilities that result in therapeutic resistance. Using bioinformatics methods, we discovered that elevated expressions of MET, STAT3, and AKT were associated with poor overall survival in HNSCC patients. We then evaluated the therapeutic potential of our newly synthesized small molecule HNC018 towards its potential as a novel anticancer drug. Our computer-aided structure characterization and target identification study predicted that HNC018 could target these oncogenic markers implicated in HNSCC. Subsequently, the HNC018 has demonstrated its anti-proliferative and anticancer activities towards the head and neck squamous cell carcinoma cell lines, along with displaying the stronger binding affinities towards the MET, STAT3, and AKT than the standard drug cisplatin. Reduction in the clonogenic and tumor-sphere-forming ability displays HNC018's role in decreasing the tumorigenicity. Importantly, an vivo study has shown a significant delay in tumor growth in HNC018 alone or in combination with cisplatin-treated xenograft mice model. Collectively with our findings, HNC018 highlights the desirable properties of a drug-like candidate and could be considered as a novel small molecule for treating head and neck squamous cell carcinoma.

Injectable thermo-sensitive hydrogel loaded hollow copper sulfide nanoparticles for ROS burst in TME and effective tumor treatment.

Introduction: Lung cancer the most prevalent cause of cancer-related deaths, and current therapies lack sufficient specificity and efficacy. This study developed an injectable thermosensitive hydrogel harboring hollow copper sulfide nanoparticles and β-lapachone (Lap) (CLH) for lung tumor treatment. Methods: The hydrogel-encapsulated CLH system can remotely control the release of copper ions (Cu2+) and drugs using photothermal effects for non-invasive controlled-release drug delivery in tumor therapy. The released Cu2+ consumes the overexpressed GSH in TME and the generated Cu+ further exploits the TME characteristics to initiate nanocatalytic reactions for generating highly toxic hydroxyl radicals. In addition, in cancer cells overexpressing Nicotinamide adenine dinucleotide (phosphate): quinone oxidoreductase 1 (NQO1), Lap can catalyze the generation of hydrogen peroxide (H2O2) through futile redox cycles. H2O2 is further converted into highly toxic hydroxyl radicals via the Fenton-like reaction, leading to a burst of reactive oxygen species in TME, which further enhances the therapeutic effect of chemokines. Results: Analysis of the antitumor efficacy in a subcutaneous A549 lung tumor model mice showed a significant delay in tumor growth and no systemic toxicity was detected. Discussion: In conclusion, we have established a CLH nanodrug platform that enables efficient lung tumor therapy through combined photothermal/chemodynamic therapy (CDT) treatment and self-supplying H2O2 to achieve cascade catalysis, leading to explosive amplification of oxidative stress.

Synergy of heterodimeric IL-15 immunotherapy and a Fatty Acid Metabolism Modulator enhances intratumoral lymphocyte metabolism and eradicates EO771 murine breast tumors

Abstract Tumor-infiltrating cytotoxic CD8 +T cells frequently acquire an altered state of differentiation referred to as “exhaustion”, which prevents control of tumor growth. We study hetIL-15, a cytokine that stimulates the generation, proliferation and cytotoxic function of CD8 +T and NK cells. The objectives of this study was to assess the effects of hetIL-15 immunotherapy in TNBC tumors, to evaluate the metabolic profile of the tumor-infiltrating T cells and to study any potential synergy with a Fatty Acid Metabolism Modulator (FAMM). Using the EO771 orthotopic breast cancer model we monitored the efficacy of locoregional administration of hetIL-15 in combination with a FAMM. We monitored the effect of treatment on tumor growth, survival of mice and the status of tumor-infiltrating immune cells using Seahorse and flow cytometry. hetIL-15 locoregional administration resulted in complete tumor regression in 40% of the treated animals and increased survival. The infiltrating cytotoxic CD8 +T and NK cells were increased in hetIL-15 treated tumors and showed enhanced activation and proliferation. Their metabolic flux analysis confirmed a robust rise in Oxygen Consumption Rate (OCR) with substantial increase of spare respiratory capacity. Combined treatment of hetIL-15 immunotherapy and FAMM resulted in increased mitochondrial function, FA uptake and OCR. In addition, combination therapy resulted in statistically significant tumor growth delay and complete eradication of the tumors in 85% of mice. Our results indicate that metabolic reprogramming of tumor-specific CD8 +T cells might represent a strategy to promote survival in the metabolically hostile Tumor Microenvironment (TME), enhancing the clinical efficacy of immunotherapy.

Abstract 5034: Targeting CXCR4 and thioredoxin reductase in high grade neuroendocrine tumors and neuroendocrine carcinomas

Abstract Introduction/Background: Atypical lung carcinoids and lung neuroendocrine carcinomas (NECs) are currently incurable. Most of these cancers express the C-X-C chemokine receptor 4 (CXCR4), making CXCR4 an attractive target for cancer diagnosis and treatment using radioligand therapy (RLT) with nuclides such as alpha emitter Pb212 conjugated to Pentixather (Pent). Alpha particles produce dense ionization tracks and damage cytosolic structures such as mitochondria, leading to the generation of reactive oxygen species such as superoxide (O2 ●−) and hydroperoxides (H2O2 and ROOH) in targeted cancer cells. The FDA approved thioredoxin reductase inhibitor, auranofin (Aur), inhibits peroxiredoxin-mediated hydroperoxide metabolism increasing cell killing in targeted cancer cells. Hypothesis: Inhibition of hydroperoxide metabolism using Aur can enhance therapeutic efficacy of alpha particle emitting 212Pb-Pent in preclinical models of lung carcinoids and NECs. Results: Clinically, IHC revealed greater than 75% of atypical carcinoids and NECs tested had moderate to high CXCR4. qRT-PCR, flow cytometry and immunohistochemistry performed on different lung carcinoid and NEC lung cell lines, determined that the majority moderately or highly express CXCR4. The minimal non-toxic dose of Aur that inhibits 50-70% of thioredoxin reductase activity was 4 mg/kg (intraperitoneal once a day) in DMS273 (small cell lung cancer) xenografts 212Pb-Pent given IV (1 and 3 µCu/g) was effective at increasing the survival of mice with DMS273 xenografts. Mice were then treated with 1.5, 3 or 6 µCu/g 212Pb-Pent w/wo Aur 4 mg/kg/day. A trend test showed that both increasing dose of 212Pb-Pent and the addition of Aur, caused a significant (p&amp;lt;0.01) delay in tumor growth. There were no significant changes in body weights, complete blood counts, and serum alanine aminotransferase indicating the combination treatments were well tolerated. Radiation dosimetry estimates using quantitative SPECT/CT imaging with Pb203-Pent demonstrated average absorbed doses of 0.133 Gy/µCi (liver), 0.098 Gy/µCi (kidneys), 0.013 Gy/µCi (H292 xenografts), 0.028 Gy/µCi (DMS53 xenografts), 0.057 Gy/µCi (DMS273 xenografts), and 0.096 Gy/µCi (H69AR xenografts). Xenograft absorbed dose levels were positively correlated with CXCR4 expression. Conclusion: The use of 212Pb-Pent therapy to target CXCR4 on NECs showed pre-clinical efficacy in a dose dependent manner that was enhanced by Aur without significant normal tissue toxicity supporting the hypothesis that CXCR4 can be successfully targeted with the radionuclide theragnostic pair, Pb212/Pb203-pentixather. Citation Format: Melissa A. Fath, Dijie Liu, Claudia Robles-Planells, Jordan T. Ewald, Keegan A. Christensen, Spenser S. Johnson, Stephen A. Graves, Douglas R. Spitz, Yusuf Menda, M. Sue O’Dorisio. Targeting CXCR4 and thioredoxin reductase in high grade neuroendocrine tumors and neuroendocrine carcinomas. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5034.

Abstract 5082: Heterodimeric IL-15 (hetIL-15) immunotherapy synergizes with Fatty Acid Metabolism Modulator (FAMM) to eradicate TNBC EO771 murine tumors

Abstract Introduction: Metabolic fitness and T cell survival are crucial in anti-tumor responses because nutrients are often scarce and other regulatory molecules may be unfavorable in the tumor microenvironment leading to T cell dysfunction, stress, and apoptosis. Tumor-infiltrating cytotoxic CD8+T cells frequently acquire an altered state of differentiation referred to as “exhaustion” and, as a result, they fail to control tumor outgrowth. IL-15 cytokine stimulates the generation, proliferation and cytotoxic function of tumor specific CD8+ T cells and NK cells. The objective of this study was to assess the effects of hetIL-15 immunotherapy in triple negative breast cancer tumors (TNBC), to evaluate the metabolic profile of the tumor-infiltrating T cells and to study any potential synergy using a Fatty Acid Metabolism Modulator (FAMM) to enhance T cell metabolism. Study design and methods: We used the murine EO771 orthotopic breast cancer model to study the efficacy of locoregional administration of hetIL-15 immunotherapy in combination with a FAMM. We monitored the effect of treatment on number, metabolism and mitochondrial function of the tumor-infiltrating immune cells by flow cytometry, Seahorse flux analysis and Mitotracker, 2-NBDG and/or Bodipy staining. Results: hetIL-15 locoregional administration, as as single agent, resulted in complete regression in 40% of the treated animals and increased survival. Tumor-infiltrating cytotoxic CD8+T and NK cells increased in hetIL-15 treated tumors and showed enhanced activation and proliferation. Metabolic flux analysis of the tumor-infiltrating cytotoxic CD8+T cells from hetIL-15-treated mice confirmed a rise in oxygen consumption rate (OCR) with substantial increase of spare respiratory capacity, which supports an activated/non exhausted phenotype of these hetIL-15 treated effector cells. Since, further promoting fatty acid (FA) catabolism improves the tumor-infiltrated CD8+T cells’s ability to slow tumor progression, we combined hetIL-15 immunotherapy with a FAMM. Combination therapy resulted in increased mitochondrial function, FA uptake and OCR, revealing a more metabolically active phenotype compared to the tumor-infiltrating CD8+T cells from hetIL-15 monotherapy group. In addition, combined treatment of IL-15 immunotherapy and FAMM resulted in statistically significant EO771 tumor growth delay and complete eradication of the tumors in 85% of mice. Conclusions: Our results indicate that hetIL-15 synergizes with metabolic reprogramming of T cells to achieve superior antitumor efficacy and complete cures. We suggest that metabolic reprogramming of tumor-specific CD8+Tcells might represent a strategy to promote survival in the metabolically hostile TME as part of an approach to enhance the clinical efficacy of immunotherapy. Citation Format: Sevasti Karaliota, Dimitris Stellas, Vasiliki Stravokefalou, Breana Myers, Barbara K. Felber, George N. Pavlakis. Heterodimeric IL-15 (hetIL-15) immunotherapy synergizes with Fatty Acid Metabolism Modulator (FAMM) to eradicate TNBC EO771 murine tumors. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5082.