Significant Tumor Growth Delay Research Articles

Abstract B034: Myeloperoxidase limits tumor-associated neutrophils mediated immunosuppression in pancreatic cancer

Abstract Pancreatic ductal adenocarcinoma (PDAC) is a deadly disease with a survival rate under 12%. Treatments like immunotherapy and chemotherapy are ineffective in PDAC due to the immunosuppressive tumor microenvironment, in which tumor-associated neutrophils (TANs) are a major component. TAN immunosuppression has been shown to depend upon increased production of reactive oxygen species, with myeloperoxidase (MPO) being a major source of it. We investigated the contribution of MPO in mediating TAN immunosuppression using orthotopic PDAC models in wild-type and syngeneic MPO-deficient mice. We observed a significant delay in tumor growth and improved clinical scores when MPO was deficient. Additionally, treatment with the MPO-specific inhibitor Verdiperstat also delayed tumor growth. Flow cytometry analysis of the tumor microenvironment further confirmed a significant increase in CD8 T and NK cells, along with an increase in granzyme B level in both subsets, when MPO was deficient. Additionally, there was an increase in activation marker (CD25) and memory subset among both CD8 and CD4 T cells in MPO-deficient mice. Ex vivo studies with isolated TANs from tumor-bearing mice showed reduced CD8 T and NK cell suppression, and reactive oxygen species level in absence of MPO, supporting the contribution of MPO in TAN immunosuppression. These data suggest that loss of MPO in TANs has decreased immunosuppressive functions resulting in increased cytotoxic and activated T cells and NK cells with increased memory response within the tumor microenvironment. Our results demonstrate the contribution of MPO in regulating tumor progression, TAN immunosuppression, and immune cell infiltration using an orthotopic PDAC model. Citation Format: Angisha Basnet, Brain Boone, Tracy Liu. Myeloperoxidase limits tumor-associated neutrophils mediated immunosuppression in pancreatic cancer [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 B034.

Engineered PLGA Core-Lipid Shell Hybrid Nanocarriers Improve the Efficacy and Safety of Irinotecan to Combat Colon Cancer.

Poly(lactide-co-glycolide) (PLGA) is a biocompatible and biodegradable copolymer that has gained high acceptance in biomedical applications. In the present study, PLGA (Mw = 13,900) was synthesized by ring-opening polymerization in the presence of a biocompatible zinc-proline initiator through a green route. Irinotecan (Ir) loaded with efficient PLGA core-lipid shell hybrid nanocarriers (lipomers, LPs) were formulated with 1,2-distearoyl-sn-glycero-3-phosphoethanolamine and 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[amino (polyethylene glycol)-2000] (DSPE-PEG-2000), using soya lecithin, by a nanoprecipitation method, and the fabricated LPs were designated as P-DSPE-Ir and P-DSPE-PEG-Ir, respectively. The formulated LPs were further validated for their physicochemical properties and biological potential for colon cancer application. The potential delivery of a poorly water-soluble chemotherapeutic drug (Ir) was studied for the treatment of colon cancer. LPs were successfully prepared, providing controlled size (80-120 nm) and surface charge (∼ -35 mV), and the sustained release properties and cytotoxicity against CT-26 colon cancer cells were studied. The in vivo biodistribution and tumor site retention in CT-26 xenograft tumor-bearing Balb/C mice showed promising results for tumor uptake and retention for a prolonged time period. Unlike P-DSPE-Ir, the P-DSPE-PEG-Ir LP exhibited significant tumor growth delay as compared to untreated and blank formulation-treated groups in CT-26 (subcutaneous tumor model) after 4 treatments of 10 mg irinotecan/kg dose. The biocompatibility and safety of the LPs were confirmed by an acute toxicity study of the optimized formulation. Overall, this proof-of-concept study demonstrates that the PLGA-based LPs improve the efficacy and bioavailability and decrease neutropenia of Ir to combat colon cancer.

Performance of PSMA-targeted radiotheranostics in an experimental model of renal cell carcinoma.

Renal cell carcinoma (RCC) represents cancer originating from the renal epithelium and accounts for > 90% of cancers in the kidney. Prostate-specific membrane antigen (PSMA) is overexpressed in tumor-associated neovascular endothelial cells of many solid tumors, including metastatic RCC. Although studied in several small clinical studies, PSMA-based imaging and therapy have not been pursued rigorously in preclinical RCC. This study aimed to evaluate the preclinical performance of PSMA-based radiotheranostic agents in a relevant murine model. A PSMA-overexpressing murine cell line, PSMA+ RENCA, was developed by lentiviral transduction. PSMA-based theranostic agents, 68Ga-L1/177Lu-L1/225Ac-L1, were synthesized in high radiochemical yield and purity following our reported methods. Immunocompetent BALB/c mice were used for flank and orthotopic tumor inoculation. 68Ga-L1 was evaluated in small animal PET/CT imaging in flank and PET/MR imaging in orthotopic models. Cell viability studies were conducted for 177Lu-L1 and 225Ac-L1. Proof-of-concept treatment studies were performed using 225Ac-L1 (0, 37 kBq, 2 kBq × 37 kBq, 1 week apart) using PSMA+ RENCA in the flank model. Cellular uptake of 68Ga-L1, 177Lu-L1, and 225Ac-L1 confirmed the specificity of the agents to PSMA+ RENCA cells rather than to RENCA (wt) cells, which are low in PSMA expression. The uptake in PSMA+ RENCA cells at 1 h for 68Ga-L1 (49.0% incubated dose [ID] ± 3.6%ID/million cells), 177Lu-L1 (22.1%ID ± 0.5%ID)/million cells), and 225Ac-L1 (4.1% ± 0.2% ID)/million cells), respectively, were higher than the RENCA (wt) cells (~ 1%ID-2%ID/million cells). PET/CT images displayed > 7-fold higher accumulation of 68Ga-L1 in PSMA+ RENCA compared to RENCA (wt) in flank implantation at 1 h. A twofold higher accumulation of 68Ga-L1 was observed in orthotopic tumors than in normal kidneys during 1-3 h postinjection. High lung uptake was observed with 68Ga-L1 PET/MR imaging 3 weeks after orthotopic implantation of PSMA+ RENCA due to spontaneous lung metastases. The imaging data were further confirmed by immunohistochemical characterization. 225Ac-L1 (0-37 kBq) displayed a dose-dependent reduction of cell proliferation in the PSMA+ RENCA cells after 48 h incubation; ~ 40% reduction in the cells with treated 37 kBq compared to vehicle (p < 0.001); however, no effect was observed with 177Lu-L1 (0-3700 kBq) up to 144 h postinoculation, suggesting lower efficacy of β-particle-emitting radiations in cellular studies compared to α-particle-emitting 225Ac-L1. Animals treated with 225Ac-L1 at 1 week posttumor inoculation in flank models displayed significant tumor growth delay (p < 0.03) and longer median survival of 21 days and 24 days for the treatment groups 37 kBq and 2 kBq × 37 kBq, respectively, compared to the vehicle group (12 days). The results suggest that a theranostic strategy targeting PSMA, employing PET and α-emitting radiopharmaceuticals, enabled tumor growth control and enhanced survival in a relevant immunocompetent murine model of RCC. These studies provide the rationale for clinical studies of PSMA-targeted theranostic agents in patients with RCC.

Potentiating Salvage Radiotherapy in Radiorecurrent Prostate Cancer Through Anti-CTLA4 Therapy: Implications from a Syngeneic Model.

High-risk prostate cancer (PCa) is a leading cause in cancer death and can elicit significant morbidity and mortality. Currently, the salvage of local disease recurrence after radiation therapy (RT) is a major clinical problem. Immune checkpoint inhibitors (ICIs), which enhance immune activation, have demonstrated clinical therapeutic promise in combination with ionizing radiation (IR) in certain advanced cancers. We generated the TRAMP-C2 HF radiorecurrent syngeneic mouse model to evaluate the therapeutic efficacy of ICIs in combination with RT. The administration of anti-PDL1 and/or anti-CTLA4 did not achieve a significant tumor growth delay compared to the control. The combination of IR and anti-PDL1 did not yield additional a growth delay compared to IR and the isotype control. Strikingly, a significant tumor growth delay and complete cure in one-third of the mice were seen with the combination of IR and anti-CTLA4. Immune cells in tumor-draining lymph nodes and tumor-infiltrating lymphocytes from mice treated with IR and anti-CTLA4 demonstrated an upregulation of genes in T-cell functions and enrichment in both CD4+ and CD8+ T-cell populations compared to mice given IR and the isotype control. Taken together, these results indicate enhancement of T-cell response in radiorecurrent PCa by IR and anti-CTLA4.

Chemo-immunotherapy by nanoliposomal epacadostat and docetaxel combination to IDO1 inhibition and tumor microenvironment suppression

The over-activation of tryptophan (Trp) metabolism to kynurenine (Kyn) catalyzed by Indoleamine 2,3-dioxygenase-1 (IDO1) enzyme, is one of the main metabolic pathways involved in tumor microenvironment (TME) immune escape and cancer treatment failure. The most efficient of IDO1 inhibitors is Epacadostat (EPA). Since monotherapy with single-agent IDO1 inhibitor regimen has led to an insufficient anti-tumor activity, we examined the efficacy of simultaneous treatment by Liposomal epacadostat (Lip-EPA) as a potent IDO inhibitor, in combination with docetaxel (DTX) as a complement immunogenic cell death (ICD) agent against B16F10 model. First, the in vitro combination index (CI) of epacadostat (EPA) and DTX was investigated by using the unified theory. Then, the in vivo efficacy of the combination therapy was assessed. Results indicated the synergestic cytotoxic effect of the combination on B16F10 compared to normal fibroblast cells (NIH). The immune profiling demonstrated a significant increase in the percentage of infiltrated T lymphocytes and IFN-γ release, a significant decrease in the percentage of regulatory T cells (Treg) population and the subsequent low levels of IL-10 generation in mice treated with Lip-EPA + DTX. Further, a significant tumor growth delay (TGD = 69.15 %) and an increased life span (ILS > 47.83 %) was observed with the combination strategy. Histopathology analysis revealed a remarkable increase in the Trp concentration following combination treatment, while Kyn levels significantly decreased. Results showed that the nano-liposomal form of IDO1 inhibitor in combination with chemotherapy could significantly improve the imunity response and dominate the tumor immuno-suppressive micro-environment, which merits further investigations.

Boron Neutron Capture Therapy Enhanced by Boronate Ester Polymer Micelles: Synthesis, Stability, and Tumor Inhibition Studies.

Boron neutron capture therapy (BNCT) targets invasive, radioresistant cancers but requires a selective and high B-10 loading boron drug. This manuscript investigates boron-rich poly(ethylene glycol)-block-(poly(4-vinylphenyl boronate ester)) polymer micelles synthesized via atom transfer radical polymerization for their potential application in BNCT. Transmission electron microscopy (TEM) revealed spherical micelles with a uniform size of 43 ± 10 nm, ideal for drug delivery. Additionally, probe sonication proved effective in maintaining the micelles' size and morphology postlyophilization and reconstitution. In vitro studies with B16-F10 melanoma cells demonstrated a 38-fold increase in boron accumulation compared to the borophenylalanine drug for BNCT. In vivo studies in a B16-F10 tumor-bearing mouse model confirmed enhanced tumor selectivity and accumulation, with a tumor-to-blood (T/B) ratio of 2.5, surpassing BPA's T/B ratio of 1.8. As a result, mice treated with these micelles experienced a significant delay in tumor growth, highlighting their potential for BNCT and warranting further research.

First-in-human study of 225actinium mti-201 (225Ac-MTI-201) in metastatic uveal melanoma (UM).

TPS9612 Background: Prognosis in metastatic uveal melanoma (UM) has historically been poor approximating 1 year from diagnosis. Tebentafusp is the only approved systemic agent for HLA-A*02:01 positive metastatic UM, and novel therapies are needed. The melanocortin-1 receptor (MC1R) receptor is highly expressed in UM with limited expression in normal tissues. Actinium-225 (225Ac) is an alpha particle emitting radionuclide ideal for targeted ligand binding due to high linear energy transfer and limited free path (&lt;100 µm) in tissue. We developed a novel MC1R targeted radiopharmaceutical 225Ac-MTI-201, and demonstrated high biostability, affinity, MC1R-specific cytotoxicity with defined dosimetry and pharmacokinetics in pre-clinical studies (1). Murine efficacy studies of UM showed significant delay of tumor growth and improved survival compared to controls. Methods: This is a single institution first-in-human phase I study (NCT05496686) of 225Ac-MTI-201 in metastatic UM. The primary objective is to determine the safety of a single intravenous dose of 225Ac-MTI-201 relative to radioactivity dose. Secondary endpoints include 1) pharmacokinetics (PK) and clearance of 225Ac-MTI-201, 2) objective response rate and duration, progression-free survival, and overall survival in metastatic UM. Salient eligibility criteria include: 1) age ≥18 years; 2) histologically confirmed metastatic UM with progression after at least 1 prior line of therapy; 3) measurable disease per Response Evaluation Criteria in Solid Tumors version 1.1; 4) Eastern Cooperative Oncology Group performance status of ≤1; 5) adequate marrow, renal and hepatic function; 6) no more than 25% of bone marrow treated with prior radiotherapy. Patients will receive a single dose of 225Ac-MTI-201 under appropriate guidelines and precautions for administration of radiopharmaceuticals. Pre-injection and post-injection PK sampling at defined intervals are undertaken. There are 12 dose levels for 225Ac-MTI-201 being investigated ranging from 4.7 microcurie (µCi) to 1327 µCi. Dose escalation is done using a modified continual re-assessment method with a cohort size of one based on dose limiting toxicity (DLT) assessment within 28 days of drug administration using CTCAE v5.0. Definitions of DLT include G4 neutropenia or febrile neutropenia, G4 thrombocytopenia or G3 thrombocytopenia with clinically significant bleeding, &gt; G3 non-hematological toxicity with selected exceptions, laboratory abnormalities that satisfy Hy’s law, or any death not related to underlying disease or extraneous cause. Response assessments are undertaken with cross-sectional imaging every 8 weeks in year 1, and every 12 weeks in year 2. This study is currently open for enrollment. Dose levels 1-4 have been completed without DLT. 1. Tafreshi, J Nucl Med 2019. Clinical trial information: NCT05496686 .

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 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 [isocitrate dehydrogenase (IDH)-wild-type and mutant, WHO grades II-IV; with grade IV tumors referred to as glioblastomas (GBM)]. These mutations drive epigenetic changes, leading to promoter methylation at the nicotinic acid phosphoribosyl transferase (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, nicotinamide phosphoribosyl transferase (NAMPT), rationalizing a treatment for these malignancies. Multiple systemically administered NAMPT inhibitors (NAMPTi) 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 (NP)-encapsulated 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&amp;lt; 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.