Significant Tumor Growth Retardation Research Articles

GQIcombi application to subdue glioma via differentiation therapy.

Current therapy protocols fail to cure high-grade gliomas and prevent recurrence. Therefore, novel approaches need to be developed. A re-programing of glioma cell fate is an alternative attractive way to stop tumor growth. The two-step protocol applies the antiproliferative GQ bi-(AID-1-T) and small molecule inducers with BDNF to trigger neural differentiation into terminally differentiated cells, and it is very effective on GB cell cultures. This original approach is a successful example of the "differentiation therapy". To demonstrate a versatility of this approach, in this publication we have extended a palette of cell cultures to gliomas of II, III and IV Grades, and proved an applicability of that version of differential therapy for a variety of tumor cells. We have justified a sequential mode of adding of GQIcombi components to the glioma cells. We have shown a significant retardation of tumor growth after a direct injection of GQIcombi into the tumor in rat brain, model 101/8. Thus, the proposed strategy of influencing on cancer cell growth is applicable to be further translated for therapy use.

Transvascular delivery of talaporfin sodium to subcutaneous tumors in mice by nanosecond pulsed laser-induced photomechanical waves

BackgroundWe previously developed a site-specific transvascular drug delivery system (DDS) based on photomechanical waves (PMWs) or laser-induced stress/shock waves (LISWs). In this study, we investigated the validity of this method to deliver a clinical photosensitizer, talaporfin sodium (TS), to subcutaneous tumors in mice and to enhance the efficacy of photodynamic therapy (PDT). MethodsTS solution (2.5 mg/kg) was intravenously injected into mice. Immediately thereafter, PMWs were applied to the tumor by irradiating a laser target with a Q-switched ruby laser pulse (0.8 J/cm2). Five hours after TS administration, some tumors were excised to evaluate the depth distribution of the delivered TS under a fluorescence microscope. Other tumors were subjected to PDT by irradiating the tissues with a 665 nm continuous-wave laser diode (75 mW/cm2, 667 s) at this timepoint. The effects of PDT were evaluated on the basis of the two primary therapeutic mechanisms of TS-mediated PDT: i) damage to tumor cells and ii) damage to endothelial cells of tumor vessels, i.e., the vascular shutdown effect on tumors. ResultsPMW application significantly increased the accumulation of TS in the tumor parenchyma but not in the tumor vessel walls; the endothelial cell junctions of tumor vessels should be the route of TS delivery enhanced by PMWs. Thus, as a result of PMW application followed by PDT, while the vascular shutdown effect on the tumors was not enhanced, direct damage to the tumor cells was increased, resulting in significant tumor growth retardation without body weight loss for 7 days after treatment.

Tandem Isotope Therapy with 225Ac- and 177Lu-PSMA-617 in a Murine Model of Prostate Cancer.

Radionuclide therapy targeting prostate-specific membrane antigen (PSMA) is a promising option for metastatic castration-resistant prostate cancer. Clinical experience using 177Lu or 225Ac has demonstrated encouraging treatment responses; however, responses are not durable. Dual-isotope combinations, or "tandem" approaches, may improve tolerability while retaining a high tumor dose. In this study, we directly compared α- versus β-particle treatment, as well as a combination thereof, at different stages of disease in a murine model of disseminated prostate cancer. Methods: First, to determine comparable injected activities from 177Lu- and 225Ac-PSMA-617, ex vivo biodistribution studies were performed at 5 time points after treatment of C4-2 subcutaneous tumor-bearing NSG mice. To establish a more representative model of metastatic prostate cancer, NSG mice were inoculated with luciferase-expressing C4-2 cells in the left ventricle, leading to disseminated visceral and bone lesions. At either 3 or 5 wk after inoculation, the mice were treated with equivalent tumor dose-depositing activities of 177Lu- or 225Ac-PSMA-617 alone or in combination (35 MBq of 177Lu, 40 kBq of 225Ac, or 17 MBq of 177Lu + 20 kBq 225Ac; 10/group). Disease burden was assessed by weekly bioluminescence imaging. Treatment efficacy was evaluated using whole-body tumor burden and overall survival. Results: The ex vivo biodistribution studies revealed that 35 MBq of 177Lu and 40 kBq of 225Ac yield equivalent absorbed tumor doses in a subcutaneous C4-2 model. The disease burden of mice treated at 3 wk after inoculation (microscopic disease) with 177Lu was not significantly different from that of untreated mice. However, 225Ac-PSMA-617 both as a single agent and in combination with 177Lu (17 MBq of 177Lu + 20 kBq of 225Ac) were associated with significant whole-body tumor growth retardation and survival benefit (overall survival, 8.3 wk for nontreatment, 9.4 wk for 177Lu, 15.3 wk for 225Ac alone, and 14.1 wk for tandem therapy). When treated at 5 wk after inoculation (macroscopic disease), all treatment groups showed retarded tumor growth and improved survival, with no significant differences between 225Ac alone and administration of half the 225Ac activity in tandem with 177Lu (overall survival, 7.9 wk for nontreatment, 10.3 wk for 177Lu, 14.6 wk for 225Ac alone, and 13.2 wk for tandem therapy). Conclusion: Treatment of a disseminated model of prostate cancer with simultaneous 225Ac- and 177Lu-PSMA-617 results in significantly decreased tumor growth compared with 177Lu, which was ineffective as a single agent against microscopic lesions. Mice treated later in the disease progression and bearing macroscopic, millimeter-sized lesions experienced significant tumor growth retardation and survival benefit in both monoisotopic and tandem regimens of 177Lu and 225Ac. Although the greatest benefits were observed with the single agent 225Ac, the tandem arm experienced no significant difference in disease burden or survival benefit, suggesting that the reduced activity of 225Ac was adequately compensated in the tandem arm. The superior therapeutic efficacy of 225Ac in this model suggests a preference for α-emitters alone, or possibly in combination, in the microscopic disease setting.

Engineering Alendronate-Composed Iron Nanochelator for Efficient Peritoneal Carcinomatosis Treatment.

Iron is an essential element for various cellular metabolism. Cancer cells also have high requirement of iron in their proliferation, invasion, and metastasis processes. Alendronate (ALN), a kind of FDA‐approved bisphosphonates with metal‐chelating capability, is initially certified to selectively bind to intracellular Fe3+ theoretically and experimentally in this study. Hence, CaALN iron nanochelator is rationally designed to kill cancer cells by synergism of Fe‐depletion and calcium accumulation. In vitro experiments and RNA sequencing analysis indicate that CaALN nanomedicine inhibits the proliferation of cancer cells by depleting Fe, interfering with DNA replication, and triggering intracellular reactive oxygen species (ROS). Meanwhile, released Ca2+ and ROS mutually promote and induce damage of cellular macromolecules, which leads to mitochondrial apoptosis of cancer cells. In an intraperitoneal disseminated mouse model with the human ovarian cancer cells SKOV3, CaALN nanoparticles selectively accumulate in tumor tissues and result in significant retardation of tumor growth and ascites formation. The mean survival time of SKOV3‐bearing mice in treatment group is prolonged from 33 to 90 d. These results indicate that the alendronate‐originated iron chelator can serve as an efficient strategy for the treatment of peritoneal carcinomatosis.

Eribulin improves tumor oxygenation demonstrated by 18F-DiFA hypoxia imaging, leading to radio-sensitization in human cancer xenograft models.

Eribulin, an inhibitor of microtubule dynamics, is known to show antitumor effects through its remodeling activity in the tumor vasculature. However, the extent to which the improvement of tumor hypoxia by eribulin affects radio-sensitivity remains unclear. We utilized 1-(2,2-dihydroxymethyl-3-18F-fluoropropyl)-2-nitroimidazole (18F-DiFA), a new PET probe for hypoxia, to investigate the effects of eribulin on tumor hypoxia and evaluate the radio-sensitivity during eribulin treatment. Mice bearing human breast cancer MDA-MB-231 cells or human lung cancer NCI-H1975 cells were administered a single dose of eribulin. After administration, mice were injected with18F-DiFA and pimonidazole, and tumor hypoxia regions were analyzed. For the group that received combined treatment with radiation, 18F-DiFA PET/CT imaging was performed before tumors were locally X-irradiated. Tumor size was measured every other day after irradiation. Eribulin significantly reduced18F-DiFA accumulation levels in a dose-dependent manner. Furthermore, the reduction in 18F-DiFA accumulation levels by eribulin was most significant 7days after treatment. These results were also supported by reduction of the pimonidazole-positive hypoxic region. The combined treatment showed significant retardation of tumor growth in comparison with the control, radiation-alone, and drug-alone groups. Importantly, tumor growth after irradiation was inversely correlated with 18F-DiFA accumulation. These results demonstrated that 18F-DiFA PET/CT clearly detected eribulin-induced tumor oxygenation and that eribulin efficiently enhanced the antitumor activity of radiation by improving tumor oxygenation.

A Conjugation Strategy to Modulate Antigen Binding and FcRn Interaction Leads to Improved Tumor Targeting and Radioimmunotherapy Efficacy with an Antibody Targeting Prostate-Specific Antigen.

Simple SummaryThe present study demonstrates that a radiolabeling strategy can positively modify hu5A10′s capacity to bind PSA and complex with the FcRn receptor, which results in a more homogenous activity distribution in tumors and enhanced therapy efficacy. These new innovative radiochemistry ideas could potentially decrease relapse of tumors in patients treated with internalizing antibodies.Background: The humanized monoclonal antibody (mAb) hu5A10 specifically targets and internalizes prostate cancer cells by binding to prostate specific antigen (PSA). Preclinical evaluations have shown that hu5A10 is an excellent vehicle for prostate cancer (PCa) radiotheranostics. We studied the impact of different chelates and conjugation ratios on hu5A10′s target affinity, neonatal fc-receptor interaction on in vivo targeting efficacy, and possible enhanced therapeutic efficacy. Methods: In our experiment, humanized 5A10 (hu5A10) was conjugated with DOTA or DTPA at a molar ratio of 3:1, 6:1, and 12:1. Surface plasmon resonance (SPR) was used to study antigen and FcRn binding to the antibody conjugates. [111In]hu5A10 radio-immunoconjugates were administered intravenously into BALB/c mice carrying subcutaneous LNCaP xenografts. Serial Single-photon emission computed tomography (SPECT) images were obtained during the first week. Tumors were harvested and radionuclide distribution was analyzed by autoradiography along with microanatomy and immunohistochemistry. Results: As seen by SPR, the binding to PSA was clearly affected by the chelate-to-antibody ratio. Similarly, FcRn (neonatal fc-receptor) interacted less with antibodies conjugated at high ratios of chelator, which was more pronounced for DOTA conjugates. The autoradiography data indicated a higher distribution of radioactivity to the rim of the tumor for lower ratios and a more homogenous distribution at higher ratios. Mice injected with ratio 3:1 111In-DOTA-hu5A10 showed no significant difference in tumor volume when compared to mice given vehicle over a time period of 3 weeks. Mice given a similar injection of ratio 6:1 111In-DOTA-hu5A10 or 6:1 111In-DTPA-hu5A10 or 12:1 111In-DTPA-hu5A10 showed significant tumor growth retardation. Conclusions: The present study demonstrated that the radiolabeling strategy could positively modify the hu5A10′s capacity to bind PSA and complex with the FcRn-receptor, which resulted in more homogenous activity distribution in tumors and enhanced therapy efficacy.

Integrin-Targeted, Short Interfering RNA Nanocomplexes for Neuroblastoma Tumor-Specific Delivery Achieve MYCN Silencing with Improved Survival.

The authors aim to develop siRNA therapeutics for cancer that can be administered systemically to target tumors and retard their growth. The efficacy of systemic delivery of siRNA to tumors with nanoparticles based on lipids or polymers is often compromised by their rapid clearance from the circulation by the liver. Here, multifunctional cationic and anionic siRNA nanoparticle formulations are described, termed receptor‐targeted nanocomplexes (RTNs), that comprise peptides for siRNA packaging into nanoparticles and receptor‐mediated cell uptake, together with lipids that confer nanoparticles with stealth properties to enhance stability in the circulation, and fusogenic properties to enhance endosomal release within the cell. Intravenous administration of RTNs in mice leads to predominant accumulation in xenograft tumors, with very little detected in the liver, lung, or spleen. Although non‐targeted RTNs also enter the tumor, cell uptake appears to be RGD peptide‐dependent indicating integrin‐mediated uptake. RTNs with siRNA against MYCN (a member of the Myc family of transcription factors) in mice with MYCN‐amplified neuroblastoma tumors show significant retardation of xenograft tumor growth and enhanced survival. This study shows that RTN formulations can achieve specific tumor‐targeting, with minimal clearance by the liver and so enable delivery of tumor‐targeted siRNA therapeutics.

Systemic administration of polymersomal oncolytic peptide LTX-315 combining with CpG adjuvant and anti-PD-1 antibody boosts immunotherapy of melanoma

Oncolytic peptide LTX-315 while showing clinical promise in treating solid tumors is limited to intratumoral administration, which is not applicable for inaccessible or metastatic tumors. The cationic and amphipathic nature of oncolytic peptides engenders formidable challenges to developing systems for their systemic delivery. Here, we describe cRGD-functionalized chimaeric polymersomes (cRGD-CPs) as a robust systemic delivery vehicle for LTX-315, which in combination with CpG adjuvant and anti-PD-1 boost immunotherapy of malignant B16F10 melanoma in mice. cRGD-CPs containing 14.9 wt% LTX-315 (cRGD-CPs-L) exhibited a size of 53 nm, excellent serum stability, and strong and selective killing of B16F10 cells (versus L929 fibroblasts) in vitro, which provoked similar immunogenic effects to free LTX-315 as revealed by release of danger-associated molecular pattern molecules. The systemic administration of cRGD-CPs-L gave a notable tumor accumulation of 4.8% ID/g and significant retardation of tumor growth. More interestingly, the treatment of B16F10 tumor-bearing mice was further boosted by co-administration of polymersomal CpG and anti-PD-1 antibody, in which two out of seven mice were cured as a result of strong immune response and long-term immune memory protection. The immunotherapeutic effect was evidenced by secretion of IL-6, IFN-γ and TNF-α, tumor infiltration of CD8+ CTLs and Th, and induction of TEM and TCM in spleen. This study opens a new avenue to oncolytic peptides, which enables durable immunotherapy of tumors via systemic administration.

Nanoparticle-enabled innate immune stimulation activates endogenous tumor-infiltrating T cells with broad antigen specificities

Tumors are often infiltrated by T lymphocytes recognizing either self- or mutated antigens but are generally inactive, although they often show signs of prior clonal expansion. Hypothesizing that this may be due to peripheral tolerance, we formulated nanoparticles containing innate immune stimulants that we found were sufficient to activate self-specific CD8+ T cells and injected them into two different mouse tumor models, B16F10 and MC38. These nanoparticles robustly activated and/or expanded antigen-specific CD8+ tumor-infiltrating T cells, along with a decrease in regulatory CD4+ T cells and an increase in Interleukin-17 producers, resulting in significant tumor growth retardation or elimination and the establishment of immune memory in surviving mice. Furthermore, nanoparticles with modification of stimulating human T cells enabled the robust activation of endogenous T cells in patient-derived tumor organoids. These results indicate that breaking peripheral tolerance without regard to the antigen specificity creates a promising pathway for cancer immunotherapy.

Abstract 6649: Inhibiting adenosine signaling and KRAS enhances the effect of α-PD-1 therapy in a KRASG12C/TP53R172H/+ pancreatic cancer model

Abstract Introduction: High levels of adenosine (ADO) in the tumor microenvironment have been shown to suppress immune responses and curtail T cell activation in the presence of anti-PD-1/PD-L1 blocking antibodies. CD73 catalyzes the extracellular generation of ADO from adenosine monophosphate (AMP). KRAS mutations, of which 60% were derived from pancreatic ductal adenocarcinoma (PDAC) samples, were associated with significantly upregulated CD73 expression, which resulted in a worsening prognosis. These immunosuppressive effects can be counteracted by CD73 inhibitors or by a dual ADO receptor (A2aR/A2bR) antagonist. Method: Linear models were used to evaluate the ability of 299 pan-cancer consensus oncogenic drivers to predict CD73 expression independent of tumor type in the TCGA dataset. Changes in gene expression induced by KRAS inhibition were determined by RNAseq. Metabolic and proteomic alterations induced by KRAS inhibition were determined by LC-MS. C57BL/6J mice bearing established KP4662-G12C (KRASG12C/TP53R172H/+) tumors (at least 150 mm3) were treated as indicated: A1421 (CD73i; 30 mg/kg/day, s.c.), anti-PD1 (Clone RMP 1-14; 10 mg/kg; twice per week, i.p), and MRTX-1257 (KRASi, 100 mg/kg/day, p.o.). Treatment efficacy was monitored in a blinded manner using micro-computed tomography (mCT). A1412 and MRTX-1257 were provided by Arcus Biosciences. Data: Direct KRAS inhibition reduced but did not abolish CD73 and A2aR/A2bR expression in multiple PDAC models. Metabolic analyses indicated that KRAS inhibition increased ADO and AMP levels. KRAS inhibition in PDAC models induced gene expression changes consistent with increased tumor immunogenicity. In a murine model of pancreatic cancer-bearing the KRASG12C mutation, co-administration of a CD73 inhibitor with anti-PD-1 in established tumors resulted in significant tumor growth retardation, comparable to KRASG12C inhibition alone. Durable tumor regression was observed when mice were treated with the triple combination therapy. These data support the rationale for the clinical development of modulators of immunosuppressive adenosine signaling in pancreatic cancer. Conclusion: Here, we show that direct inhibition of mutant KRAS in pancreatic cancer models yields complex immunomodulatory effects. While antigen presentation pathways are transcriptionally upregulated and the expression of immunosuppressive chemokine is reduced, KRAS inhibition also reprograms nucleotide metabolism leading to elevated levels of ADO. These findings suggest that co-targeting mutant KRAS and adenosine signaling may enhance immunotherapy against pancreatic cancer and potentially other KRAS driven malignancies. Citation Format: THUC LE, Evan Abt, Woosuk Kim, Akshata Udyavar, Arthur Cho, Joe Capri, Jing Cui, Razmik Ghukasyan, Luyi Li, Daniel DiRenzo, Brandon Rosen, Timothy Donahue, Matt Walters, Caius Radu. Inhibiting adenosine signaling and KRAS enhances the effect of α-PD-1 therapy in a KRASG12C/TP53R172H/+ pancreatic cancer model [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 6649.

Abstract A09: Photoimmunotherapy (PIT) for treatment of bladder cancer

Abstract Introduction: The use of light as a means of therapy for bladder cancer (BC) has a long history but has been hampered by a lack of tumor specificity and therefore, damage to the normal bladder mucosa. Here, we describe a targeted form of phototherapy called photoimmunotherapy (PIT). To begin with, anti-EGFR antibody panitumumab (pan) was labeled with the photo-absorber (PA), IRDye 700Dx (IR700), to create pan IR700, an antibody-PA conjugate that is activated by near-infrared radiation (NIR). However, PIT may be less effective when a tumor lacks “overwhelming” expression of a single target such as EGFR. Hence, a combinatorial PIT approach for targeting BC expressing EGFR and HER2, using PA-labeled pan and trastuzumab (tra), respectively. Methods: Human BC tissue microarray (TMAs) and BC cell lines were analyzed for expression of EGFR and HER2. Mechanism of PIT-induced cell death was studied using proliferation assays, transmission electron microscopy (TEM), and production of reactive and singlet oxygen species (ROS/SOS). Finally, efficacy of PA-labeled antibodies singly and in combination was analyzed in vitro and in vivo. Results: Most BC tissues have some expression of EGFR, but squamous cell carcinoma (SCC) of bladder has maximum EGFR expression. About 45% of BC tissues stain for both EGFR and HER2. Pan IR700 activated by NIR light rapidly killed UMUC-5 cells, a bladder SCC line. Pan alone, pan IR700 without NIR, or NIR alone had no effect on cells. TEM demonstrated that cell death is due to necrosis. Singlet oxygen species (SOS) had a substantially larger contribution towards cell death than reactive oxygen species (ROS). NIR-PIT with pan IR700 demonstrated reduced growth compared to only pan IR700-treated UMUC-5 xenograft tumors. In non-SCC BCs we used a combinatorial PIT approach using pan IR700 and tra IR700 to target EGFR and HER2, respectively. In vitro, PIT in the presence of combination pan IR700 and tra IR700 was more efficacious than either agent alone with lethal dose 50 (LD50) of 28.66 J/cm2 for combination PIT compared to 71.55 J/cm2 for the PanIR700 alone. In vivo, tumor xenografts treated with combination PIT showed significant tumor growth retardation compared to single-agent therapy. Conclusion: PIT is a new targeted treatment for BC. Our data demonstrate that pan IR700-induced PIT selectively kills EGFR-expressing bladder cancer. Combination PIT is a promising approach for treating BC by selectively inducing death in BC cells with low/moderate expression of surface receptors. Citation Format: Reema Railkar, Mohammad Rashid Siddiqui, Thomas Sanford, Hisataka Kobayashi, Peter L. Choyke, Piyush K. Agarwal. Photoimmunotherapy (PIT) for treatment of bladder cancer [abstract]. In: Proceedings of the AACR Special Conference on Bladder Cancer: Transforming the Field; 2019 May 18-21; Denver, CO. Philadelphia (PA): AACR; Clin Cancer Res 2020;26(15_Suppl):Abstract nr A09.

Abstract A46: CD73 inhibition enhances the effect of anti-PD-1 therapy on KRAS-mutated pancreatic cancer model

Abstract Background: CD73 catalyzes the extracellular generation of adenosine (ADO) from adenosine monophosphate (AMP). High levels of ADO, found in the tumor microenvironment (TME), have been shown to suppress immune responses and curtail T-cell activation in the presence of anti-PD-1/PD-L1 blocking antibodies. Oncogenic drivers resulting from activating mutations, such as KRAS, BRAF, and EGFR mutations, are commonly treated with tyrosine kinase inhibitors that result in robust but nondurable responses. We show here that KRAS mutations, of which 60% were derived from pancreatic adenocarcinoma (PDAC) samples, significantly upregulated CD73 expression and resulted in worsening prognosis. In a murine model of pancreatic cancer bearing KRASG12C mutation, coadministration of CD73 inhibitor with anti-PD-1 in established tumors resulted in significant tumor growth retardation, comparable to KRAS inhibitor alone. These data support the rationale for the clinical development of CD73 inhibitors in pancreatic cancer. Methods: Linear models were used to evaluate the ability of 299 pan-cancer consensus oncogenic drivers to predict CD73 expression independent of tumor type in the TCGA dataset. Immunohistochemistry (IHC) on formalin-fixed, paraffin-embedded (FFPE) samples was performed using CD73 (Cell Signaling, D7F9A) primary antibody and detected using anti-rabbit HRP. CD73 expressing cells were detected by DAB chromogen and quantified using QuPath Software. CD73 activity in fresh frozen tissues were detected using the Wachstein-Meisel method. C57BL/6J mice bearing established KP4662-G12C tumors (>150 mm3) were treated with A0001421 (CD73i), 30 mg/kg; once a day; s.c. and anti-PD1 (Clone RMP 1-14): 10 mg/kg; twice a week; i.p. Mice were treated for two weeks starting from day 10 post-tumor. Treatment efficacy was monitored using micro-computed tomography (Genisys PET/CT G8 (Sofie Biosciences). All small-molecule inhibitors were synthesized by Arcus Biosciences, Inc. Results: Out of the 299 oncogenes, alterations in KRAS, BRAF, and EGFR were the top 3 oncogenes upregulating CD73 expression. Increase in CD73 expression in KRAS-mutated tumors was further confirmed by IHC. Data extrapolated from Koyama et al. suggest that PD-1 nonresponsive mice express higher levels of adenosine pathway genes, including CD73 and CD39. Coadministration of CD73 inhibitor with anti-PD-1 was superior to anti-PD-1 alone in limiting tumor growth of an aggressive model of pancreatic cancer. Global changes in immune contexture and TME were also observed, consistent with the immune modulatory effects of inhibiting CD73 activity. Conclusion: CD73 is a highly efficient ecto-enzyme that catalyzes the hydrolysis of AMP to immune-suppressive ADO and is associated with worsening prognosis across multiple malignancies. AB680, a potent and selective CD73 inhibitor with excellent safely and pharmacokinetic profile, is currently undergoing clinical evaluation in 1L metastatic PDAC in combination with AB122 and chemotherapy. Citation Format: Thuc M. Le, Akshata Udyavar, Woosuk Kim, Arthur E. Cho, Luyi Li, Daniel DiRenzo, Brandon Rosen, Matthew J. Walters, Joanne B.L. Tan, Caius G. Radu. CD73 inhibition enhances the effect of anti-PD-1 therapy on KRAS-mutated pancreatic cancer model [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2019 Nov 17-20; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2020;8(3 Suppl):Abstract nr A46.

Therapeutic Efficacy and Dosimetry of Targeted Alpha Therapy using 225Ac-PSMA-617 in a Murine Model of Prostate Cancer

Targeted alpha therapy shows promise as a treatment in metastatic castration-resistant prostate cancer (mCRPC), owing to the high dose deposition and short range characteristics of alpha particle radiation. Ac-225 decays via multiple alpha particles – each capable of inducing irreparable DNA double-strand breaks in cell nuclei. PSMA-617 is a targeting ligand with specific affinity for prostate-specific membrane antigen (PSMA), a protein that is overexpressed in mCRPC but has low expression in normal tissues. Coupled with the lethal effects of alpha radiation, 225Ac-PSMA-617 is potentially highly cytotoxic to mCRPC while sparing normal tissues. The objective of this study was to evaluate the efficacy of alpha-particle radioligand therapy in a mouse model of mCRPC. Radiation dosimetry analysis was carried out to determine tumor dose as well as dose-limiting organs. NSG mice bearing subcutaneous PSMA-expressing C4-2 tumors were injected with escalating activities of 225Ac-PSMA-617: 20, 40, and 100 kBq/mouse (n=8/group). The tumor volumes were assessed over time by weekly low-dose microCT and compared with an untreated control group. In parallel, twenty-five NSG mice were used in a biodistribution study at five time points – 1, 4, 24, 48, and 168 hours post-administration of 40 kBq of the radiopharmaceutical. After sacrifice, percent tumor and organ radioactivity uptake was measured using gamma spectroscopy. Biexponential curve-fitting was used to fit the time-activity curves for the tumor and each organ, and integrated according to standard medical internal radiation dose methods to estimate the tumor and organ doses. Significant tumor growth retardation was observed in all treatment groups compared with the untreated group. Mice treated with 100 kBq exhibited some weight loss, while the mice treated with lower activities experienced only transient weight loss. There was a significant survival benefit conferred on tumor-bearing mice treated with 225Ac-PSMA617. The biodistribution over five time points showed high uptake and slow tumor activity clearance, and low uptake with fast clearance in non-target organs. The salivary glands, a dose-limiting organ in humans, did not show high uptake in mice, possibly due to lower PSMA expression. Estimates of the absorbed dose to the tumor and organs are reported. An administered activity of 40 kBq per mouse of the peptidomimetic 225AcPSMA-617 is well-tolerated and results in significant tumor growth retardation and improved survival. This work provides a preclinical foundation for further studies towards a more effective treatment option for advanced castration-resistant prostate cancer.

TMIC-51. GBP1 RECRUITS MACROPHAGES TO PROMOTE GLIOBLASTOMA GROWTH

Abstract Guanylate binding protein 1 (GBP1) is an interferon-inducible large GTPase which plays a key role in tumor development, but the molecular mechanism is poorly understood. Here we investigated whether GBP1 could influence the tumor microenvironment in glioblastoma, the most common and malignant brain tumor. We found that forced expression of GBP1 in glioblastoma cells induced macrophage polarization toward an M2 phenotype via upregulating Chemokine (C-C motif) ligand 2 (CCL2). CCL2 acted via its receptor C-C chemokine receptor 2 (CCR2) to enhance macrophage cell migration in vitro. The M2 macrophages in turn promoted glioblastoma cell proliferation and migration. The orthotopic mouse model showed that GBP1 recruited M2 macrophages into tumor to promote glioblastoma progression, and targeting CCL2/CCR2 signaling axis with a small molecule inhibitor RS504393 led to decreased macrophage attraction and M2 polarization and a significant tumor growth retardation and prolonged survival of tumor-bearing mice. Clinically, GBP1 expression positively correlated with M2 macrophage numbers and CCL2 expression in glioblastoma. Taken together, our results reveal that GBP1 modulates the tumor immune microenvironment through CCL2 induction to promote glioblastoma infiltrating growth, and targeting tumor-associated macrophages may represent a new therapeutic strategy against glioblastoma.

Apatinib Combined with Local Irradiation Leads to Systemic Tumor Control via Reversal of Immunosuppressive Tumor Microenvironment in Lung Cancer.

PurposeThis study aimed to investigate the potential systemic antitumor effects of stereotactic ablative radiotherapy (SABR) and apatinib (a novel vascular endothelial growth factor receptor 2 inhibitor) via reversing the immunosuppressive tumor microenvironment for lung carcinoma.Materials and MethodsLewis lung cancer cells were injected into C57BL/6 mice in the left hindlimb (primary tumor; irradiated) and in the right flank (secondary tumor; nonirradiated). When both tumors grew to the touchable size, mice were randomly divided into eight treatment groups. These groups received normal saline or three distinct doses of apatinib (50 mg/kg, 150 mg/kg, and 200 mg/kg) daily for 7 days, in combination with a single dose of 15 Gy radiotherapy or not to the primary tumor. The further tumor growth/regression of mice were followed and observed.ResultsFor the single 15 Gy modality, tumor growth delay could only be observed at the primary tumor. When combining SABR and apatinib 200 mg/kg, significant retardation of both primary and secondary tumor growth could be observed, indicated an abscopal effect was induced. Mechanism analysis suggested that programmed death-ligand 1 expression increased with SABR was counteract by additional apatinib therapy. Furthermore, when apatinib was combined with SABR, the composition of immune cells could be changed. More importantly, this two-pronged approach evoked tumor antigen–specific immune responses and the mice were resistant to another tumor rechallenge, finally, long-term survival was improved.ConclusionOur results suggested that the tumor microenvironment could be managed with apatinib, which was effective in eliciting an abscopal effect induced by SABR.

Therapeutic Efficacy and Dosimetry of Targeted Alpha Therapy using 225Ac-PSMA-617 in a Murine Model of Prostate Cancer

Targeted alpha therapy shows promise as a treatment in metastatic castration-resistant prostate cancer (mCRPC), owing to the high dose deposition and short range characteristics of alpha particle radiation. Ac-225 decays via multiple alpha particles – each capable of inducing irreparable DNA double-strand breaks in cell nuclei. PSMA-617 is a targeting ligand with specific affinity for prostate-specific membrane antigen (PSMA), a protein that is overexpressed in mCRPC but has low expression in normal tissues. Coupled with the lethal effects of alpha radiation, 225Ac-PSMA-617 is potentially highly cytotoxic to mCRPC while sparing normal tissues. The objective of this study was to evaluate the efficacy of alpha-particle radioligand therapy in a mouse model of mCRPC. Radiation dosimetry analysis was carried out to determine tumor dose as well as dose-limiting organs. NSG mice bearing subcutaneous PSMA-expressing C4-2 tumors were injected with escalating activities of 225Ac-PSMA-617: 20, 40, and 100 kBq/mouse (n=8/group). The tumor volumes were assessed over time by weekly low-dose microCT and compared with an untreated control group. In parallel, twenty-five NSG mice were used in a biodistribution study at five time points – 1, 4, 24, 48, and 168 hours post-administration of 40 kBq of the radiopharmaceutical. After sacrifice, percent tumor and organ radioactivity uptake was measured using gamma spectroscopy. Biexponential curve-fitting was used to fit the time-activity curves for the tumor and each organ, and integrated according to standard medical internal radiation dose methods to estimate the tumor and organ doses. Significant tumor growth retardation was observed in all treatment groups compared with the untreated group. Mice treated with 100 kBq exhibited some weight loss, while the mice treated with lower activities experienced only transient weight loss. There was a significant survival benefit conferred on tumor-bearing mice treated with 225Ac-PSMA617. The biodistribution over five time points showed high uptake and slow tumor activity clearance, and low uptake with fast clearance in non-target organs. The salivary glands, a dose-limiting organ in humans, did not show high uptake in mice, possibly due to lower PSMA expression. Estimates of the absorbed dose to the tumor and organs are reported. An administered activity of 40 kBq per mouse of the peptidomimetic 225AcPSMA-617 is well-tolerated and results in significant tumor growth retardation and improved survival. This work provides a preclinical foundation for further studies towards a more effective treatment option for advanced castration-resistant prostate cancer.

Targeting Epidermal Growth Factor Receptor (EGFR) and Human Epidermal Growth Factor Receptor 2 (HER2) Expressing Bladder Cancer Using Combination Photoimmunotherapy (PIT)

Bladder cancer (BC) is heterogeneous and expresses various cell surface targets. Photoimmunotherapy (PIT) involves monoclonal antibodies (MAbs) conjugated to a photoabsorber (PA), IR Dye 700Dx, and then activated by near infra-red light (NIR) to specifically target tumors. We have demonstrated that tumors expressing EGFR can be targeted with PIT. However, PIT may be less effective when a tumor lacks “overwhelming” expression of a single target such as EGFR. We present a combinatorial PIT approach for targeting BC expressing EGFR and HER2, using PA- labeled panitumumab (pan) and trastuzumab (tra), respectively. Human BC tissues and cell lines were analyzed for EGFR and HER2 expression. Efficacy of PA-labeled MAbs singly and in combination was analyzed. About 45% of BC tissues stain for both EGFR and HER2. In vitro, the combination of pan IR700 and tra IR700 with NIR was more efficacious than either agent alone. Tumor xenografts treated with combination PIT showed significant tumor growth retardation. Combination PIT is a promising approach for treating BC with low/moderate expression of surface receptors. In addition, given the molecular heterogeneity of bladder cancer, targeting more than one surface receptor may allow for more effective cell death across different bladder tumors.

Abstract 2766: Resistance to radiotherapy and PD-L1 blockade is mediated by TIM-3 upregulation in anti-PD-L1 refractory head and neck cancer

Abstract Background: Clinical trials targeting the programmed-death ligand axis (PD-1/PD-L1) show that a majority of head and neck squamous cell carcinoma (HNSCC) patients are resistant to PD-1/PD-L1 inhibition. Strategies aimed at sensitizing tumors to immunotherapy can provide durable response compared to conventional therapies. We previously reported that local radiation to the tumor can transform the immune landscape and render poorly immunogenic murine orthotopic HNSCC tumors sensitive to PD-L1 inhibition. However, the response to combined radiotherapy (RT) and PD-L1 inhibition was transient. In this work, we characterize the immune landscape of HNSCC tumors during RT and PD-L1 treatment and interrogate mechanisms of resistance. Methods: We employed time-of-flight mass cytometry (CyTOF) for characterizing the tumor immune microenvironment at early and late stages of response to RT and anti-PD-L1. For studying the effect of dual checkpoint blockade and RT, poorly immunogenic murine squamous cell carcinoma cells (LY2 and MOC2) were injected into the right buccal mucosa. Mice were randomized to IgG, anti-PD-L1, anti-TIM-3, RT or combinations of RT, PD-L1 and TIM-3. Mechanistic experiments were performed on tumors harvested 72 hours after treatment. Tumors were assessed for levels of activated T cells (CD44+IFNg+) and regulatory T cells (FoxP3+CD4+) by flow cytometry and T-cell infiltration by immunohistochemistry. Results: Tumors treated with RT and PD-L1 blockade significantly upregulated TIM-3 expression on CD4 and CD8 T cells. Furthermore, the proportion of regulatory T cells expressing TIM-3 was significantly increased during the tumor regrowth phase with RT and anti-PD-L1 treatment. Targeting TIM-3 concurrently with PD-L1 and RT led to significant tumor growth retardation, enhanced T-cell cytotoxicity and improved survival. In addition, dual targeting of PD-L1 and TIM-3 in combination with RT significantly decreased the proportion of regulatory T cells (4.1-fold decrease relative to IgG control and 2.8-fold decrease relative to RT+anti-PD-L1). However, the response to dual checkpoint blockade and RT was not durable and all tumors eventually relapsed. Analysis of relapsed tumors revealed decreased T-cell infiltration, re-emergence of regulatory T cells and decreased proportion of cytotoxic and helper T cells. Conclusion: Our study shows upregulation of TIM-3 in response to RT and PD-L1 inhibition. However, dual targeting of TIM-3 and PD-L1 with RT did not provide a durable response in two genetically distinct HNSSCC tumor models. In light of the increased number of clinical trials employing dual immune checkpoint blockade, our findings reveal the complexity of tumor immune evasion mechanisms and underscore the need for multimodality targeting of HNSCCs. Citation Format: Ayman J. Oweida, Mohammad Hararah, Andy Phan, Shilpa Bhatia, Shelby Lennon, David Binder, David Raben, Lynn Heasley, Eric Clambey, Raphael Nemenoff, Sana D. Karam. Resistance to radiotherapy and PD-L1 blockade is mediated by TIM-3 upregulation in anti-PD-L1 refractory head and neck cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2766.

Abstract 3811: Targeted Interleukin 2 and synergy with immune check-point inhibitors

Abstract Systemic high-dose Interleukin 2 (IL2) is able to elicit complete responses in patients suffering from metastatic melanoma. However, the majority of patients cannot receive the whole treatment dose due to severe toxicities. Other approved drugs for the treatment of metastatic melanoma include chemotherapeutics (e.g. Dacarbazine), a protein kinase B-raf inhibitor (Vemurafenib) and an immune check-point inhibitor (Ipilimumab, an anti CTLA-4 antibody). These drugs provide a clear benefit to patients, but cancer cures are still rare. Targeting IL2 to the site of disease represents a promising strategy to circumvent the toxicity and maximize the efficacy of this immunomodulatory payload. Various formats of targeted IL2 fusion proteins are currently in clinical development. Furthermore, recent successful approaches in the field of immunotherapy focus on the inhibition of check-point pathways, aiming to release the immunosuppressive brakes of the immune system. To explore the possibility of improving therapeutic efficacy of the single agents, a targeted version of IL2 (namely F8-IL2, where the F8 antibody recognizes the alternatively-spliced extra-domain A of fibronectin) was investigated in combination with an anti PD-1, anti PD-L1, or anti CTLA-4 antibody in an immunocompetent mouse model of CT26 colon carcinoma. In a first study, the combination of F8-IL2 and CTLA-4 blockade led to significant tumor growth retardation (curing 3/ 5 mice). Increasing the dose of F8-IL2 from 30 to 45 µg led to a 100 % cure rate with no signs of toxicity (body weight loss <5 %). Moreover, all cured mice acquired protective long-term immunity, which prevented tumor growth upon subsequent rechallenge with CT26 colon carcinoma cells. Furthermore, high dose F8-IL2 combined with the anti PD-1 antibody showed better therapeutic results than the respective monotherapies. In contrast, combination of F8-IL2 with the anti PD-L1 antibody did not improve the therapeutic outcome. Taken together, these data provide a strong rationale for the clinical use of targeted IL2 in combination with Ipilimumab. Citation Format: Cornelia Deborah Hutmacher-Berndt, Dario Neri. Targeted Interleukin 2 and synergy with immune check-point inhibitors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3811.

Upregulation of cell cycle genes in head and neck cancer patients may be antagonized by erufosine's down regulation of cell cycle processes in OSCC cells.

The TCGA database was analyzed to identify deregulation of cell cycle genes across 24 cancer types and ensuing effects on patient survival. Pan-cancer analysis showed that head and neck squamous cell carcinoma (HNSCC) ranks amongst the top four cancers showing deregulated cell cycle genes. Also, the median gene expression of all CDKs and cyclins in HNSCC patient samples was higher than that of the global gene expression. This was verified by IHC staining of CCND1 from HNSCC patients. When evaluating the quartiles with highest and lowest expression, increased CCND1/CDK6 levels had negative implication on patient survival. In search for a drug, which may antagonize this tumor profile, the potential of the alkylphosphocholine erufosine was evaluated against cell lines of the HNSCC subtype, oral squamous cell carcinoma (OSCC) using in-vitro and in-vivo assays. Erufosine inhibited growth of OSCC cell lines concentration dependently. Initial microarray findings revealed that cyclins and CDKs were down-regulated concentration dependently upon exposure to erufosine and participated in negative enrichment of cell cycle processes. These findings, indicating a pan-cdk/cyclin inhibition by erufosine, were verified at both, mRNA and protein levels. Erufosine caused a G2/M block and inhibition of colony formation. Significant tumor growth retardation was seen upon treatment with erufosine in a xenograft model. For the decreased cyclin D1 and CDK 4/6 levels found in tumor tissue, these proteins can serve as biomarker for erufosine intervention. The findings demonstrate the potential of erufosine as cell cycle inhibitor in HNSCC treatment, alone or in combination with current therapeutic agents.